key: cord-0031595-y368u2ek authors: athanassiou, panagiotis; mavragani, clio; athanassiou, lambros; kostoglou-athanassiou, ifigenia; koutsilieris, michael title: vitamin d deficiency in primary sjögren’s syndrome: association with clinical manifestations and immune activation markers date: 2022-03-31 journal: mediterr j rheumatol doi: 10.31138/mjr.33.1.106 sha: b8b9b9ff05e9b16f694d1b74da9af7fb7565bb3d doc_id: 31595 cord_uid: y368u2ek vitamin d is an agent involved in bone and mineral homeostasis. it has been recognized as a potent immunomodulator. it has immune-enhancing properties, and it induces immune tolerance. vitamin d deficiency has been shown to be related to the development of autoimmune disorders. vitamin d deficiency has been observed in patients with rheumatoid arthritis (ra) and it has been shown to be related with disease activity. vitamin d deficiency has also been found in patients with systemic lupus erythematosus (sle) and it was shown to be related to disease activity and renal involvement. vitamin d deficiency has also been observed in multiple sclerosis. vitamin d has been found to act as a supplemental therapeutic agent in multiple sclerosis. sjögren’s syndrome is a systemic autoimmune disease affecting the exocrine glands, known as an autoimmune epithelitis. the disease has a complex pathogenesis, requiring a genetic background, immune cell activation, and autoantibody production. the disease affects the exocrine glands, lacrimal, and salivary glands leading to ocular and oral dryness. vitamin d levels have been measured in patients with sjögren’s syndrome and an association was observed between low vitamin d levels, peripheral neuropathy and the presence of lymphoma. in other cohorts, such as a turkish cohort, vitamin d deficiency was observed in patients with sjögren’s syndrome. the aim is to measure serum vitamin d levels in consecutive patients with primary sjögren’s syndrome and investigate the relationship between vitamin d levels and the presence of immunologic markers, clinical, serological, and histopathological characteristics. leading to generation of oral and ocular dryness known as "sicca syndrome" 5, 6 , while systemic manifestations are not uncommon. hashimoto's thyroiditis is commonly observed in patients with sjögren's syndrome. 5 loviselli et al. studied thyroid function parameters and thyroid antibodies in a cohort of patients with primary and secondary sjögren's syndrome. they found higher prevalence of thyroid antibodies in patients with sjögren's syndrome, which was more pronounced in primary sjögren syndrome. 7 it has been suggested that sjögren's syndrome and autoimmune thyroid disease may be the two sides of the same coin. 8 vitamin d is an agent involved in the regulation of bone/mineral metabolism 9 as well as in immune pathways. [10] [11] [12] it has immune-enhancing properties, 13, 14 and is also known to induce immune tolerance. [15] [16] [17] vitamin d deficiency has been related to the development and disease activity of autoimmune disorders, 18, 19 including rheumatoid arthritis, 20,21 systemic lupus erythematosus, 22 and multiple sclerosis. 23 in the latter group, vitamin d supplementation may act as a supplemental therapeutic agent. 24 agmon-levin et al. 25 measured vitamin d levels in a cohort of patients with sjögren's syndrome, and they found an association between low vitamin d levels, peripheral neuropathy, and the presence of lymphoma, while in a cohort of turkish patients with sjögren's, lower vitamin d levels were observed 26 and in a cohort of indian patients low vitamin d levels were related to a high risk for high lip grading and rheumatoid factor positivity. 27 the aim was to measure serum vitamin d levels in consecutive patients with sjögren's syndrome and investigate the relationship between vitamin d levels and the presence of immunologic markers, clinical, serological, and histopathological characteristics. for our study, 25(oh)d3 levels will be retrospectively measured in stored sera from consecutive patients with primary sjögren's syndrome (ss) in the "molecular physiology-clinical application unit", department of physiology, national and kapodistrian university of athens as well as in prospectively collected sera from the department of rheumatology st. paul's hospital, thessaloniki, greece and department of rheumatology, asclepeion hospital, voula, athens, greece following written consent to participate in the study. all primary ss subjects participating in the study fulfil the revised international criteria for the classification of primary ss. 28 healthy individuals of similar age and sex distribution to the patients with primary ss will be also included. exclusion criteria for all participants include pregnancy, age <18 years, and renal dysfunction (serum creatinine levels >3 mg/dl, creatinine clearance <30 ml/minute). demographic data, clinical features, and therapeutic regimens will be recorded in all participating patients and controls. demographic data including age, sex, and bmi will be recorded. clinical manifestations including the presence of subjective and objective oral and ocular dryness (documented by unstimulated salivary flow rates and schirmer's test/rose bengal staining, respectively); dry cough; dyspareunia; fever; arthralgias; arthritis; carpal tunnel syndrome (documented by physical examination and nerve conduction studies); raynaud's phenomenon; lymphadenopathy; splenomegaly; purpura; pulmonary involvement (small airway disease or interstitial lung disease documented by pulmonary function tests and high resolution computed tomography scans); pleuritis; pericarditis; renal involvement including interstitial nephritis (documented by urine-specific gravity <1.010 or ph >5.5 on at least two consecutive measurements after fluid restriction) and glomerulonephritis documented by renal biopsy; liver involvement (documented by liver biopsy showing changes compatible with primary biliary cirrhosis in the setting of increased liver enzymes or anti-mitochondrial antibodies); peri-epithelial disease (defined as peribronchial, interstitial nephritis, autoimmune cholangitis); myositis (documented by muscle biopsy in the setting of increased aldolase or creatinophosphokinase); peripheral neuropathy (documented by nerve conduction studies in patients with clinical symptoms or signs suggestive of neuropathy); central nervous involvement; lymphoma (documented by biopsy) will be recorded. sjögren's syndrome disease activity index (essdai) will be determined. 29 25(oh)d3 will be measured by an electrochemiluminescence binding assay (elecsys vitamin d total, for cobas e 411 analyser, roche diagnostics gmbh, mannheim, germany). the assay is based on a competition principle. in summary, after a first incubation, so that the bound 25(oh)d could be released from the vitamin d binding protein, a second incubation is performed. during the second incubation, the pre-treated sample is incubated with a ruthenium labelled vitamin d binding protein. by a photomultiplier. the sensitivity of the assay is 10.03 nmol/l. the within run cv of the assay ranges from 3.1% at 70.0 nmol/l to 7.8% at 16.9 ng/ml. statistical analysis will be performed by spss v.21 package. two-group comparisons of continuous data will be assessed using t-tests, or the mann-whitney test, when the data do not have a normal distribution. comparisons between groups will be performed by fisher's exact two tailed test and mann whitney test. difference is considered statistically significant if p<0.05. should vitamin d levels found to be associated with the presence of specific immunologic markers in patients with sjögren's syndrome, vitamin d substitution may be considered, and underlying mechanisms will be further explored. new advances in the classification, pathogenesis and treatment of sjögren's syndrome sjögren's syndrome: old and new therapeutic targets autoimmune epithelitis and chronic inflammation in sjögren's syndrome-related dry eye disease maladaptive autophagy in the pathogenesis of autoimmune epithelitis in sjӧgren's syndrome. arthritis rheumatol increased prevalence of antibodies to thyroid peroxidase in dry eyes and mouth syndrome or sicca asthenia polyalgia syndrome sicca syndrome following immune checkpoint inhibition development of thyroid disease in patients with primary and secondary sjögren's syndrome sjögren's syndrome and autoimmune thyroid disease: two sides of the same coin sunlight and vitamin d for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease vitamin d and immune function vitamin d and the immune system vitamin d: nutrient, hormone, and immunomodulator mini-review on the roles of vitamin c, vitamin d, and selenium in the immune system against covid-19 putative roles of vitamin d in modulating immune response and immunopathology associated with covid-19 vitamin d, immune tolerance, and prevention of type 1 diabetes the role of vitamin d towards immune tolerance in white adipose tissue (wat) mechanisms underlying the regulation of innate and adaptive immunity by vitamin d vitamin d, autoimmune disease and rheumatoid arthritis vitamin d and autoimmune diseases vitamin d deficiency and rheumatoid arthritis vitamin d and rheumatoid arthritis vitamin d and systemic lupus erythematosus -the hype and the hope multiple sclerosis and vitamin d: an update effect of high-dose vitamin d3 intake on ambulation, muscular pain and bone mineral density in a woman with multiple sclerosis: a 10-year longitudinal case report low levels of vitamin-d are associated with neuropathy and lymphoma among patients with sjögren's syndrome comparison of plasma vitamin d levels in patients with sjögren's syndrome and healthy subjects vitamin d levels and associations in indian patients with primary sjögren's syndrome american college of rheumatology/ european league against rheumatism classification criteria for primary sjögren's syndrome: a consensus and data-driven methodology involving three international patient cohorts eular sjögren's syndrome disease activity index: development of a consensus systemic disease activity index for primary sjögren's syndrome the authors declare no conflict of interest. key: cord-0015995-eb2plovv authors: keyßer, gernot title: sicherheitsaspekte der therapie mit glukokortikoiden bei rheumatoider arthritis date: 2021-03-11 journal: z rheumatol doi: 10.1007/s00393-021-00972-x sha: e8360cd15076232138521a5cc80bf427758af5ca doc_id: 15995 cord_uid: eb2plovv glucocorticoids (gc) are still the recommended initial treatment for rheumatoid arthritis, although the treatment should be temporary and confined to the administration of low doses. the complex mechanism of action is accompanied by side effects that particularly occur in long-term treatment exceeding 5 mg prednisolone per day. in this dosage range they promote osteoporosis, diabetes and hyperglycemia as well as cardiovascular events and infections, thereby contributing to an excess mortality. the risks of gc treatment are dependent on patient-related parameters, such as age, comorbidity and additional medication. a negative influence of very low steroid doses on overall survival is possibly due to high cumulative steroid doses; however, the data in this respect are contradictory. recently, a validated index was developed to monitor gc-related toxicity. in the future, this index should help to describe the advantages of steroid-sparing treatment strategies. in the future, more selectively acting substances could achieve an uncoupling of desired and adverse effects. glukokortikoide (gc) gehören zu den medikamenten, mit deren umgang viele ärztliche fachdisziplinen vertraut sind. sie sind nicht selten die ersten medikamente, die bei einer entzündlichen gelenkerkrankung verschrieben werdenhäufig schon vom hausarzt. die enorme vielfältigkeit und potenz ihrer biologischen effekte kann sowohl segensreich als auch hochgefährlich für die behandelten patienten sein. daher schwankte die haltung von ärzten und patienten zu den gc lange zwischen ablehnung und positiver akzeptanz. mittlerweile hat sich die erkenntnis durchgesetzt, dass sehr niedrige dosierungen unter 5 mg prednisolon vertretbar sicher und effektiv sind [15] , sodass dieser dosisbereich sowohl für skeptiker als auch befürworter einer gc-therapie akzeptabel ist [14] . die wirkungen von gc sind genetisch fixiert. zahlreiche gene weisen promotorsequenzen auf, die für eine hemmung odersteigerungdergenexpressiondurch steroide verantwortlich sind (sog. "steroid response elements") [31] . daher lassen sich in der konventionellen gc-therapie erwünschter effekt und nebenwirkung schwer voneinander trennen, da sie oft 2 seiten eines identischen wirkmechanismus darstellen (. tab. 1). in einer konsensuskonferenz der eular (european league against rheumatism) wurden von ärztlicher seite osteoporose, diabetes/hyperglykämie, kardiovaskuläre ereignisse und infektion zu den 4 relevantesten problemen der gc-therapie gezählt [15] . interessant ist, dass der blick des patienten andere schwerpunkte setzt: die als besorgniserregend empfundenen nebenwirkungen von gc werden zwischen rheumatologen und patienten z. t. unterschiedlich gewichtet. zwar stimmten beide gruppen in der kritischen bewertung von osteoporose, diabetes und kardiovaskulären erkrankungen überein. rheumatologen bewerteten jedoch hypertonie, infektionen und arteriosklerose stärker als besorgniserregend, während der blick von patienten intensiver auf subjektive faktoren gerichtet war: fatigue, cushingoide fazies, schlaflosigkeit, palpitationen [73] . dies deckt sich mit ergebnissen einer postalischen umfrage, an der über 2400 patienten teilnahmen, die unter einer gc-medikation standen: gewichtszunahme, schlafstörungen und emotionale instabilität wurden als wesentlich stärker beeinträchtigend empfunden als erhöhte blutzucker-und blutdruckwerte [22] , dabei war eine klare dosisabhängigkeit erkennbar. dessen ungeachtet war in der vorbiologikaära die compliance der patienten mit rheumatoider arthritis (ra) zu gc deutlich höher als zu nsar (nichtsteroidale antirheumatika) und basistherapeutika [77] . die nebenwirkungen einer steroidtherapie hängen von mehreren faktoren ab. [72] . diese kofaktoren können v. a. im dosisbereich zwischen 5 und 10 mg prednisolon maßgeblich zu unerwünschten gc-effekten beitragen [72] . auch die inzidenz und die schwere der gc-induzierten osteoporose werden stark von faktoren außerhalb der gc-behandlung beeinflusst (s. unten). der terminus "steroidinduzierter diabetes" suggeriert eine enge assoziation zwischen gc-applikation und diabetesrisiko. in der tat erhöhen gc den blutzuckerspiegel im tagesverlauf kardiovaskuläres risiko · osteoporose · prognose · infektionen · nebenwirkungen safety aspects of the treatment with glucocorticoids for rheumatoid arthritis abstract glucocorticoids (gc) are still the recommended initial treatment for rheumatoid arthritis, although the treatment should be temporary and confined to the administration of low doses. the complex mechanism of action is accompanied by side effects that particularly occur in long-term treatment exceeding 5 mg prednisolone per day. in this dosage range they promote osteoporosis, diabetes and hyperglycemia as well as cardiovascular events and infections, thereby contributing to an excess mortality. the risks of gc treatment are dependent on patient-related parameters, such as age, comorbidity and additional medication. a negative influence of very low steroid doses on overall survival is possibly due to high cumulative steroid doses; however, the data in this respect are contradictory. recently, a validated index was developed to monitor gc-related toxicity. in the future, this index should help to describe the advantages of steroid-sparing treatment strategies. in the future, more selectively acting substances could achieve an uncoupling of desired and adverse effects. cardiovascular risk · osteoporosis · prognosis · infections · side effects ge prednisolon-gaben in der dauertherapie das frakturrisiko erhöhen. ältere studien berichteten, dass schon 2,5 mg pro tag zu einer messbar erhöhen rate an wirbelfrakturen führen, im dosisbereich zwischen 5 und 7,5 mg nahm auch die gefahr von schenkelhalsfrakturen deutlich zu [76] . allerdings konnte eine metaanalyse von 7 studien gerade in der frühphase einer ra keine unterschiede in der knochendichte zwischen patienten mit und ohne gc-therapie feststellen [ die infektbegünstigenden effekte der gc sind von ihren erwünschten immunsuppressiven eigenschaften nicht zu trennen. ihre hemmung von lymphozytenproliferation und-aktivierung, die antieosinophile wirkung und die starke unterdrückung der produktion inflammatorischer zytokine führen zwangsläufig zu einer vermehrten infektneigung. diese übertrifft bei dosierungen über 10 mg/tag die auswirkungen der üblichen basistherapeutika und biologika auf das infektionsrisiko [82] . niedrig dosierte gc erhöhen zwar das gesamtcholesterin, verbessern jedoch in einigen studien an ra-patienten das lipidprofil [9, 30]. kohortenanalysen haben eine assoziation zwischen der häufigkeit kardiovaskulärer ereignisse mit der einnahme mittlerer (maximal 7,5 mg/tag) und hoher (über 7,5 mg/tag) gc-dosen nachgewiesen. bei hoher dosis lag das risiko bei 2,56 im vergleich zu personen ohne prednisolon-medikation [78] . im einzelnen betrug das relative risiko für eine herzinsuffizienz 3,7, für einen herzinfarkt 3,3 und für einen schlaganfall 1,7. allerdings konnte auch diese analyse nicht überzeugend den einfluss der grunderkrankung auf das kardiovaskuläre risiko herausfiltern. in der randomisierten barfot-studie erhielten patienten mit ra methotrexat entweder gemeinsam mit placebo oder mit einer prednisolon-therapie von 7,5 mg/tag über 2 jahre. die langzeitergebnisse offenbarten, dass im 10-jahres-zeitraum 5 (1,1-2,0) , bei über 15 mg auf 3,6 (2,1-6,1). übereinstimmend damit zeigte sich in einer kohorte von 16.762 ra-patienten, dass eine dosis unter 5 mg nicht mit vermehrter mortalität assoziiert war, höhere dosen steigerten die sterblichkeit jedoch dosisabhängig [59] . eine prospektive kohortenstudie von 602 patienten mit früher ra, von denen 386 im mittel 3,1 mg prednisolon pro tag erhielten, ergab ebenfalls keine unterschiede in der inzidenz von todesfällen, kardiovaskulären ereignissen, infektionen oder osteoporotischen frakturen zwischen patienten mit oder ohne gc [66] . allerdings ist eine strikte sparsamkeit für gc auch im niedrigdosisbereich angebracht, da auch die kumulative dosis von gc einfluss auf die sterblichkeit hat. ein erhöhtes mortalitätsrisiko besteht ab einer kumulativen dosis von mindestens 40 g (hr 1,78, konfidenzintervall 1,2-2,6) [23]. derartige dosen können mit einer low-dose-therapie nach jahrzehnten erreicht werden. in zahlreichen klinischen studien zu synthetischen und biologischen dmard wurden die auswirkungen einer komedikation mit gc auf sicherheit und wirksamkeit der behandlung unzureichend dokumentiert und ausgewertet [2], sodass erst spätere sichtungen der datensätze positive oder negative effekte von gc zutage fördern [67] . bereits 2010 wurde vonseiten der eular auf den mangel an wissenschaftlicher evidenz verwiesen, der in diesem punkt herrscht [74] . zugleich wurden anstrengungen unternommen, um die gc-toxizität in der praxis und in klinischen studien systematisch zu erfassen und daraus handlungsempfehlungen abzuleiten. für die low-dose-therapie in der klinischen praxis empfahl die eular-initiative die ausweiterung des monitorings auf die osteoporoseüberwachung, die dokumentation von risikofaktoren für ein glaukom sowie ein screening auf erhöhte blutdruck-und blutzuckerwerte sowie das vorhandensein von knöchelödemen [74] . für klinische studien wurde vorgeschlagen, mit einem monitoringprogramm gezielt nach gc-bedingten unerwünschten arzneimittelwirkungen (uaw) zu fahnden. nachfolgend wurde daher der gc-toxizitäts-index (gti) entwickelt. dieser zusammengesetzte index enthält 9 parameter, die unter anderem veränderungen in bmi (body-mass-index), blutdruck, lipiden und knochendichte, aber auch infektionen erfassen [55] . diese parameter werden durch eine liste ergänzt, die spezifische toxizitätsaspekte der gc berücksichtigt wie glaukomentwicklung, avaskuläre knochennekrosen oder sehnenrisse. der gti quantifiziert spezifische einflüsse einer steroidtherapie, erlaubt aber auch die einschätzung des wertes einer gc-sparenden therapie [55] . dieser index ist bereits bestandteil von aktuell laufenden klinischen studien zur ra [75] , zur anca(antineutrophile zytoplasmatische antikörper)-assoziierten vaskulitis [52] und zum asthma [51] . ra-patienten profitieren von einer gc-therapie, wenn deren gefahrenpotenzial ausreichend berücksichtigung findet, die dosis engmaschig an die aktuelle krankheitsaktivität angepasst und die anwendung möglichst befristet wird. dabei dient die aktuelle s2e-leitlinie der deutschen gesellschaft für rheumatologie als richtschnur [29] . diese leitlinie empfiehlt, bei erreichen einer stabilen remission gc als erste präparate zu reduzieren und die therapiedauer auf 3 bis 6 monate zu begrenzen. die relevantesten uaw von gc -osteoporose, diabetes/hyperglykämie, kardiovaskuläre ereignisse und infektion -sind im low-dose-bereich unter 5 mg prednisolon beherrschbar. ein negativer einfluss sehr niedriger tagesdosen auf das gesamtüberleben wurde bei hoher kumulativer dosis in einer kohortenstudie beschrieben. die datenlage ist aber hier als nicht eindeutig zu bezeichnen, da mehrere analysen großer patientenkollektive keinen zusammenhang zwischen niedrig dosierter prednisolon-behandlung und überleben feststellen konnten (s. oben). das monitoring einer gc-vermittelten toxizität mithilfe eines validierten index sollte in zukunft dazu beitragen, die vorteile einer steroidsparenden behandlung besser zu beschreiben. es bleibt abzuwarten, inwieweit neuere pharmakologische entwicklungen eine bessere trennung von erwünschten und unerwünschten gc-wirkungen ermöglichen können. dazu gehören innovative substanzen, die selektiver auf die transrepressiven effekte der gc (unterdrückung der aktivierung proinflammatorischer zytokingene) einwirken als auf die transaktivierenden gc-wirkungen (förderung der genexpression). letztere werden für unerwünschte metabolische gc-effekte, insbesondere in bezug auf den glukosemetabolismus verantwortlich gemacht [ high-dose but not low-dose dexamethasone impairs glucose tolerance by inducing compensatory failureofpancreaticbeta-cellsinnormalmen quantification of glucocorticoid-associated morbidity in severe asthma using the glucocorticoid toxicity index evaluation of the safety and efficacy of avacopan, a c5a receptor inhibitor, in patients with antineutrophil cytoplasmicantibody-associatedvasculitistreated concomitantly with rituximab or cyclophosphamide/azathioprine: protocol for a randomized, double-blind, active-controlled rheumatoid arthritis, cardiovascular disease and physical exercise: a systematic review perioperative infection in the patient with rheumatic disease development of a glucocorticoid toxicity index (gti) using multicriteria decision analysis does mtx-induced osteoporosis exist? periprosthetic joint infection in patients with inflammatory joint disease: a review of risk factors and current approaches to diagnosis and management risk of incident diabetes mellitus associated with the dosage and duration of oral glucocorticoid therapy in patients with rheumatoid arthritis oral glucocorticoid therapy and all-cause and causespecific mortality in patients with rheumatoid arthritis: a retrospective cohort study medications associated with fracture risk in patients with rheumatoid arthritis long-termexposuretomediumdose glucocorticoid therapy associates with hypertension in patients with rheumatoid arthritis glucocorticoid-induced myopathy effects of prednisolone on energy and fat metabolism in patients with rheumatoid arthritis: tissue-specific insulin resistance with commonly used prednisolone doses osteoporosis in patients with rheumatoid arthritis: an update in epidemiology, pathogenesis, and fracture prevention low dose prednisolone therapy (ldpt) retards radiographically detectable destruction in early rheumatoid arthritis-preliminary results of a multicenter, randomized, parallel, double blind study seven-year tolerability profile of glucocorticoids use in early rheumatoid arthritis: data from the espoir cohort effect on efficacy and safety trial outcomes of also enrolling patients on ongoing glucocorticoid therapy in rheumatoid arthritis clinical trials of tocilizumab or adalimumab or methotrexate monotherapy long-term outcome is better when a methotrexate-based treatment strategy is combined with 10 mg prednisone daily: follow-up after the second computerassisted management in early rheumatoid arthritis trial glucocorticoid therapy-induced skin atrophy metaanalysis of tumor necrosis factor inhibitors and glucocorticoids on bone density in rheumatoid arthritis and ankylosing spondylitis trials oral and inhaled glucocorticoid use and risk of achilles or biceps tendon rupture: a population-based casecontrol study defining conditions where long-term glucocorticoid treatment has an acceptably low level of harm to facilitate implementation of existing recommendations: viewpoints from an eular task force patient and rheumatologist perspectives on glucocorticoids: an exercise to improve the implementation of the european league against rheumatism (eular) recommendations on the managementofsystemicglucocorticoidtherapyin rheumatic diseases monitoring adverse events of low-dose glucocorticoid therapy: eular recommendations for clinical trials and daily practice effectiveness of tocilizumab in comparison to prednisone in rheumatoid arthritis patients with insufficient response to disease-modifying antirheumatic drugs (topira): study protocol for a pragmatic trial use of oral corticosteroids and risk of fractures compliance to drug treatment of patients with rheumatoid arthritis: a 3 year longitudinal study taking glucocorticoids by prescription is associated with subsequent cardiovascular disease glucocorticoid-induced osteoporosis and osteonecrosis glucocorticoid resorption and influence on the hypothalamic-pituitary-adrenal axis after intra-articular treatment of the knee in resting and mobile patients incidence and risk of glucocorticoid-associated adverse effects in patients with rheumatoid arthritis infections and biologic therapy in rheumatoid arthritis: our changing understanding of risk and prevention glucocorticoids. mood, memory, andmechanisms key: cord-0024768-9fyrlsc5 authors: meißner, yvette; strangfeld, anja title: neues aus dem rabbit-register date: 2021-12-23 journal: z rheumatol doi: 10.1007/s00393-021-01139-4 sha: abce5103349659835e6455976bcf9a53f598208f doc_id: 24768 cord_uid: 9fyrlsc5 since 2001 rheumatologists throughout germany have been recruiting patients with rheumatoid arthritis into the biologics register (rheumatoid arthritis: observation of biologics treatment, rabbit) to investigate the long-term safety and efficacy of modern antirheumatic treatment. over the past 20 years more than 20,000 patients have been enrolled in the prospective cohort study. this article summarizes the research findings published in 2020/2021, focusing on safety aspects, factors influencing treatment efficacy and patient-reported outcomes. with herpes zoster, facial nerve palsy and psoriasis, several adverse events were investigated that were either reported as a safety signal from clinical trials or through the eudravigilance database or occurred as a paradoxical reaction under drug treatment. for these events, the influence of biological disease-modifying antirheumatic drug (dmard) treatment was analyzed. in the publication on herpes zoster, we also considered drug treatment with janus kinase inhibitors. severe overweight can influence the success of treatment. there are gender-specific differences and the mode of action of a treatment also determines whether obesity reduces the response to treatment. the majority of patients observed in rabbit were satisfied with the treatment they have received after 1 year. we were able to show which factors either favor or negatively influence satisfaction with the effectiveness and safety of the treatment. this review article shows that long-term observational studies such as the rabbit register contribute to the understanding of treatment risks and can identify factors that influence the effects of treatment even after two decades of data collection. herpes-zoster-infektionen · psoriasis · fazialisparese -therapiewirksamkeit -patient:innen im fokus -neue arzneimittelzulassungen: biosimilars und januskinaseinhibitoren -rabbit-risikoscore für schwerwiegende infektionen -ausblick seit 2001 rekrutieren rheumatolog:innen deutschlandweit patient:innen mit rheumatoider arthritis in das biologikaregister rabbit, um die langzeitsicherheit und -wirksamkeit moderner antirheumatischer therapien zu untersuchen. in den vergangenen 20 jahren wurden mehr als 20.000 patient:innen in die prospektive kohortenstudie eingeschlossen. in diesem beitrag fassen wir aktuelle forschungsergebnisse der jahre 2020 und 2021 zusammen; dabei stehen sicherheitsaspekte, einflussfaktoren auf die therapiewirksamkeit und patientenberichtete outcomes im fokus. mit herpes zoster, fazialisparese und psoriasis wurden verschiedene unerwünschte ereignisse untersucht, die entweder als sicherheitssignal aus klinischen studien oder durch die eudravigilance-datenbank gemeldet wurden oder als paradoxe reaktion unter medikamentöser behandlung aufgetreten sind. für diese ereignisse wurde der einfluss der biologischen dmard("disease-modifying antirheumatic drug")-therapie analysiert. in der publikation zu herpes zoster berücksichtigten wir auch die medikamentöse behandlung mit januskinaseinhibitoren. starkes übergewicht kann den erfolg einer therapie beeinflussen. hier gibt es geschlechtsspezifische unterschiede, und auch das wirkprinzip einer therapie entscheidet, ob eine adipositas das therapieansprechen reduziert. die mehrheit der in rabbit beobachteten patient:innen ist nach 1 jahr behandlung mit der erhaltenen therapie zufrieden. wir konnten zeigen, welche faktoren die zufriedenheit mit der wirksamkeit und der sicherheit der behandlung begünstigen oder aber negativ beeinflussen. diese übersichtsarbeit zeigt, dass langzeitbeobachtungsstudien wie das rabbit-register auch nach 2 dekaden der datenerhebung zum verständnis von therapierisiken beiträgt sowie faktoren identifiziert, die die wirkung der therapien beeinflussen können. pharmakoepidemiologie · pharmakovigilanz · beobachtungsstudie · kohortenstudie · arzneimitteltherapiesicherheit seit nunmehr 2 jahrzehnten stehen für die behandlung der rheumatoiden arthritis (ra) therapien zur verfügung, die durch neue und gezieltere wirkprinzipen über die konventionelle basistherapie ("conventional synthetic disease-modifying antirheumatic drug" [csdmard]) und insbesondere das methotrexat als "ankermedikament" hinausgehen. mit der marktzulassung der ersten tumornekrosefaktorinhibitoren (tnfi), die originalpräparate autoimmunreaktionen befürchtet, die es zu überwachen galt [1] . zu einigen dieser forschungsfragen der therapiesicherheit, aber auch zu anderen aspekten der (langzeit-)therapie mit biologika, gibt es inzwischen evidenzbasierte antworten. dennoch bleiben in der rheumatologie viele offene fragen, die insbesondere durch beobachtungsdaten geklärt werden können. sie reichen von (sehr) selten auftretenden ereignissen und sicherheitssignalen, für die es einer ausreichend großen anzahl an dokumentierten patient:innen mit hinreichend langer beobachtungsdauer bedarf, über behandlungsabfolgen und therapiekombinationen ohne zulassungsempfehlung bis hin zu einflussfaktoren auf die medikamentenwirksamkeit und auf patientenberichtete parameter. nicht zu vergessen sind natürlich die neuen therapien zur behandlung der ra, v. a. die januskinaseinhibitoren (jaki), die sich seit ihrer marktzulassung zwar sehr schnell in der rheumatologischen routineversorgung etabliert haben, für die es aber bislang nur wenige daten außerhalb klinischer studien gibt. zwanzig jahre nach dem einschluss des ersten teilnehmers in das rabbit-register werden weiterhin erwachsene patient:innen mit ra rekrutiert, um ihre rheumatologisch-klinische versorgung im alltag für mindestens 5 und bis zu 10 jahre zu beobachten. inzwischen wurden mehr als 20.000 patient:innen von rund 350 rheumatologischen einrichtungen in rabbit eingeschlossen (. abb. 1). im folgenden besprechen wir aktuelle forschungsergebnisse, die in den jahren 2020/21 aus dem rabbit-register publiziert wurden. sie umfassen verschiedene sicherheitsaspekte, den einfluss des gewichts auf die wirksamkeit von therapien sowie die zufriedenheit von patient:innen. herpes-zoster-infektionen im vergleich zur allgemeinbevölkerung haben patient:innen mit ra ein höheres risiko für das auftreten eines herpes zoster (hz) [2] . in einer unserer frühen rabbit-publikationen zeigten wir, dass auch bestimmte therapien das auftreten eines hz begünstigen können. wir fanden höhere inzidenzen unter der behandlung mit monoklonalen und löslichen tnfi im vergleich zu kontrollpatient:innen unter konventionellen antirheumatika mit inzidenzraten (ir) von 11,1 (monoklonale tnfi),8,9 (löslichetnfi) und 5,6 (csdmard) ereignissen pro 1000 patientenjahre (pj) [3] . aus klinischen studien ist ein höheres hz-risiko unter jaki berichtet worden [4] , das wir nun mit ersten deutschen beobachtungsdaten aus rabbit bestätigen konnten [5] . diese auswertung umfasste adipöse patient:innen mit ra erreichen seltener eine remission und berichten häufiger von einer eingeschränkten funktionsfähigkeit sowie einer verminderten lebensqualität [11, 12] . adipositas, nach der weltgesundheitsorganisation (who) definiert als body mass index (bmi) von mindestens 30 kg/m 2 , ist ein global zunehmendes problem [13] . rund ein viertel der deutschen allgemeinbevölkerung ist laut robert koch-institut adipös (24 % der frauen, 23 % der männer) [14] . im rabbit-register sind dies sogar 28 patientenberichtete outcomes sind inzwischen ein fester bestandteil klinischer studien [16] . auch im rabbit-register fragen wir die patient:innen nach ihrem körperlichen (wohl-)befinden, insbesondere zum derzeitigen gesundheitszustand, zu erschöpfung und müdigkeit, schmerzen und schlafstörungen. die funktionskapazität wird zu jedem messzeitpunkt mit dem funktionsfragebogen hannover (ffbh) erfasst und die gesundheitsbezogene lebensqualität jährlich mit dem krankheitsunabhängigen fragebogen short form 36 (sf-36). der die körperliche funktionsfähigkeit messende ffbh wird regelmäßig als kovariable in den statistischen analysen des rabbit-registers berücksichtigt, zum sf-36 gibt es eine eigene publikation [17] . in einer kürzlich erschienenen auswertung haben wir uns der patientenzufriedenheit gewidmet. sowohl zu einschluss in das register und damit zu beginn einer neuen therapie als auch nach 1 jahr beobachtung fragen wir die patient:innen, wie zufrieden sie mit dem erfolg und der verträglichkeit ihrer bisherigen rheumamedikation sind. von den 10.646 in die analyse eingeschlossenen patient:innen waren 55 % bei einschluss und 85 % nach 1 jahr mit dem therapierfolg zufrieden [18] . für die zufriedenheit mit der verträglichkeit der therapie konnten wir eine steigerung von 68 % auf 90 % innerhalb des ersten beobachtungsjahres zeigen. für beide outcomes wurde ein positiver zusammenhang mit der zufriedenheit zu baseline, der reduktion von das28-bsg und schmerzen, der verbesserung der körperlichen funktion sowie seropositivität festgestellt (. abb. 4) . negativ wirkten sich eine vom arzt angegebene psychische erkrankung bzw. depression, eine höhere zahl von biologikatherapien vor einschluss in das register, ein therapiewechsel während der beobachtungszeit sowie tägliche glukokortikoiddosen von 5 mg pro tag oder mehr aus. diese ergebnisse legen nahe, dass die patientenzufriedenheit durch eine wirksame, gut verträgliche therapie und die dadurch mögliche einsparung von glukokortikoiden positiv beeinflusst werden kann. dass seropositivität die zufriedenheit mit therapieerfolg und -wirksamkeit erhöht, könnte daran liegen, dass beim vorhandensein von rheumafaktor oder antikörpern gegen citrullinierte proteine früher und aggressiver behandelt wird. im jahr 2015 kamen in der rheumatologie mit den infliximab-biosimilars erstmals nachahmerpräparate von biologika auf den deutschen markt (. abb. 1). ein jahr später folgte das erste etanercept-biosimilar, noch 1 jahr darauf ein rituximab-biosimilar und im jahr 2018 eine vielzahl von adalimumab-nachfolgepräparaten. im rabbit-register zählten 2021 insgesamt 13 biosimilars zu den einschlusstherapien. eine publikation aus rabbit zu biosimilartherapien und beispielsweise den wechsel vom patentfreien original-auf ein nachahmerpräparat gibt es derzeit noch nicht. einige kennzahlen zu den biosimilars wurden aber im "kompendium biosimilars" zusammengestellt [19] . im remit den jaki wurden durch die europäische arzneimittelbehörde ema im jahr 2017 erstmals oral applizierbare präparate mit einem gezielten wirkmechanismus für die behandlung der ra zugelassen. in der zwischenzeit stehen 4 verschiedene präparate zur auswahl, deren hersteller sich an der finanzierung des rabbit-registers beteiligen. hierdurch können wichtige daten zum einsatz der jaki im rheumatologischen alltag generiert werden. die neue wirkstoffgruppe der jaki wurde bereits in der publikation zum herpes-zoster-risiko berücksichtigt [5] . dafür standen informationen von 713 patient:innen mit einem jaki als einschlusstherapie zur verfügung. die expositionszeit unter therapie summierte sich auf knapp 2700 pj. diese zahlen zeigen, dass es nach der markteinführung eines arzneimittels bzw. einer arzneimittelgruppe einer gewissen zeit bedarf, um mit einer ausreichend großen menge an daten stabile und valide informationen aus den durchgeführten analysen erzielen zu können. zusätzlich ist es wichtig, im register auch bereits etablierte und gut untersuchte präparate weiterzuführen, um neuere therapien mit einer zeitlich adäquaten kontrollgruppe vergleichen zu können. die abwägung des infektionsrisikos bei patient:innen mit ra ist noch immer ein zeitschrift für rheumatologie 5 für rheumatologen wichtiges thema [20] . der rabbit-risikoscore für schwerwiegende infektionen wurde 2011 mit daten aus rabbit entwickelt, um das 12-monats-risiko für schwerwiegende infektionen abzuschätzen [21] . er der fokus künftiger auswertungen wird zum einen auf den neueren therapien lie-gen und neben deren wirksamkeit auch deren risiken untersuchen. die von den zulassungsbehörden geäußerten sicherheitsbedenken zu jaki, insbesondere das auftreten thromboembolischer und kardiovaskulärer ereignisse sowie maligner erkrankungen [28, 29] , werden wir mit unseren daten genauer unter die lupe nehmen. dabei geht es um die frage, ob die in klinischen studien gesehenen signale auch unter alltagsbedingungen verifiziert werden können. weitere analysen werden zu seltenen, aber klinisch relevanten komorbiditäten durchgeführt werden oder zu solchen mit einer schlechten prognose. bei den interstitiellen lungenerkrankungen beispielsweise gibt es noch immer wenige daten und informationen darüber, wie sich eine dmard("disease-modifying antirheumatic drugs")-therapie auf den verlauf dieser begleiterkrankung auswirkt. das rabbit-register ist an mehreren europäischen und internationalen kooperationen beteiligt mit dem ziel, daten verschiedener kohorten gemeinsam zu analysieren. es geht dabei unter anderem um sicherheitsaspekte einer abatacept-therapie [30] , um die charakterisierung von patient:innen unter jaki [31] since 2001 rheumatologists throughout germany have been recruiting patients with rheumatoid arthritis into the biologics register (rheumatoid arthritis: observation of biologics treatment, rabbit) to investigate the long-term safety and efficacy of modern antirheumatic treatment. over the past 20 years more than 20,000 patients have been enrolled in the prospective cohort study. this article summarizes the research findings published in 2020/2021, focusing on safety aspects, factors influencing treatment efficacy and patient-reported outcomes. with herpes zoster, facial nerve palsy and psoriasis, several adverse events were investigated that were either reported as a safety signal from clinical trials or through the eudravigilance database or occurred as a paradoxical reaction under drug treatment. for these events, the influence of biological disease-modifying antirheumatic drug (dmard) treatment was analyzed. in the publication on herpes zoster, we also considered drug treatment with janus kinase inhibitors. severe overweight can influence the success of treatment. there are genderspecific differences and the mode of action of a treatment also determines whether obesity reduces the response to treatment. the majority of patients observed in rabbit were satisfied with the treatment they have received after 1 year. we were able to show which factors either favor or negatively influence satisfaction with the effectiveness and safety of the treatment. this review article shows that long-term observational studies such as the rabbit register contribute to the understanding of treatment risks and can identify factors that influence the effects of treatment even after two decades of data collection. pharmacoepidemiology · pharmacovigilance · observational studies · cohort studies · drug treatment safety european biologicals registers: methodology, selected results and perspectives increased risk of opportunistic infection in early rheumatoid arthritis risk of herpes zoster in patients with rheumatoid arthritis treated with anti-tnf-alpha agents jak inhibitors andinfectionsrisk:focusonherpeszoster risk of herpes zoster (shingles) in patients with rheumatoid arthritis under biologic, targeted synthetic and conventional synthetic dmard treatment: data from the german rabbit register bijlmetal(2020)2019updateofeular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases psoriasis induced by anti-tumor necrosis factor therapy: a paradoxical adverse reaction different risk profiles of biologic agents for new-onset psoriasis in patients with rheumatoid arthritis minutes of the meeting on 01-04 incidence of facial nerve palsies stratified by dmard treatment in patients with rheumatoid arthritis: data from the rabbit register obesity and rates of clinical remission and low mri inflammation in rheumatoid arthritis the association of obesity with disease activity, functional ability and quality of life in early rheumatoid arthritis: data from the early rheumatoid arthritis study/ early rheumatoid arthritis network uk prospective cohorts global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2016: a systematic analysis for the global burden of disease study 2016 obesity reduces the real-world effectiveness of cytokine-targeted but not cell-targeted disease-modifying agents in rheumatoid arthritis use and detailed metric properties of patient-reported outcome measures for rheumatoid arthritis: a systematic review covering two decades healthrelatedqualityoflifeinpatientswithlong-standing rheumatoid arthritis in the era of biologics: data from the german biologics register rabbit factors associated with treatment satisfaction in patients with rheumatoid arthritis: data from the biological registerrabbit infections in the era of targeted therapies: mapping the road ahead treatment benefit or survival of the fittest: what drives the time-dependent decrease in serious infection rates under tnf inhibition and what does this imply for the individual patient evaluation of the rabbit riskscoreforseriousinfections repaaetal(2021)incidence,riskfactors and validation of the rabbitscore for serious infections in a cohort of 1557 patients with rheumatoid arthritis performance of the rabbit infection risk score in an argentinian rheumatoid arthritis cohort rabbit risk score and icu admission due to infection in patients with rheumatoid arthritis impact of treatment with biologic dmards on the risk of sepsis or mortality after serious infection in patients with rheumatoid arthritis factors associated with covid-19-related death in people withrheumaticdiseases:resultsfromthecovid-19 global rheumatology alliance physician-reported registry pharmacovigilance risk assessment committee initial clinical trial resultsofincreasedriskofmajoradversecardiovascular events and malignancies (excluding nmsc) with use of tofacitinib relative to tnf-alpha inhibitors ema confirms xeljanz to be used with caution in patients at high risk of blood clots solid tumour outcomes in patients with ra treated with abatacept and other dmards: results from a 10-year international post-approval study. eular, madrid op0231 comparative effectiveness of jak-inhibitors, tnf-inhibitors, abatacept and il-6 inhibitors in an international collaboration of registers of register and cohort studies : overview of the most important data sources at the german rheumatism research center key: cord-0006731-gf3idl98 authors: kwong, yok-lam; au, wing-yan; leung, anskar y. h.; tse, eric w. c. title: t-cell large granular lymphocyte leukemia: an asian perspective date: 2010-01-19 journal: ann hematol doi: 10.1007/s00277-009-0895-3 sha: 94fbf11e9ee544d7824d3aeb92b420b54abef601 doc_id: 6731 cord_uid: gf3idl98 to characterize t-cell large granular leukemia in asia, 22 chinese patients from a single institute were reported, together with an analysis of 88 asian and 272 western patients identified from the literature. in our cohort, anemia due to pure red cell aplasia (prca) occurred in 15/22 (68%) of cases, being the most common indication for treatment. neutropenia was only found in 8/22 (36%) cases, and recurrent infections, the most important clinical problem in western patients, were not observed. none of our cases presented with rheumatoid arthritis. these clinical features were consistently observed when compared with the 88 other asian patients. combined data from our cohort and other asian cases showed that asian patients, compared with western patients, had more frequent anemia (66/110, 60% versus 113/240, 47%; p = 0.044), attributable to a much higher incidence of prca (52/110, 47% versus 6/143, 4%; p < 0.001). however, western patients presented more frequently than asian patients with neutropenia (146/235, 62% versus 33/110, 30%; p < 0.001) and splenomegaly (99/246, 40% versus 16/110, 15%; p < 0.001). notably, western patients were about eight to ten times more likely than asian patients to have rheumatoid arthritis (73/272, 27% versus 4/106, 4%; p < 0.001) and recurrent infections (81/272, 30% versus 3/107, 3%; p < 0.001). these clinicopathologic differences have important implications on disease pathogenesis and treatment. electronic supplementary material: the online version of this article (doi:10.1007/s00277-009-0895-3) contains supplementary material, which is available to authorized users. introduction t-cell large granular lymphocyte (t-lgl) leukemia is a rare chronic lymphoproliferative disorder, characterized by a cd2 + cd3 + cd4 -cd8 + large granular lymphocytosis (>2× 10 9 /l) [1] . the t-cell receptor (tcr) gene is clonally rearranged, which helps to establish the diagnosis when the large granular lymphocytosis is less than 2×10 9 /l [1] . the clinical features of t-lgl leukemia have been described mainly in western patients [2] [3] [4] . relatively few asian patients with t-lgl leukemia have been reported [5] , so that the behavior of the disease in this population remains unclear. owing to global population migration, the features and optimal treatment of t-lgl leukemia in asian patients are becoming important to physicians worldwide. in order to delineate the clinicopathologic characteristics of t-lgl leukemia in asia, a cohort of chinese patients seen at a single center was studied. asian patients with t-lgl leukemia were also identified from a literature search and reviewed. the combined results were then compared and contrasted with those of western patients, so as to define similarities and differences of t-lgl leukemia in these two populations. case definitions t-lgl leukemia was defined according to the world health organization (who) classification criteria, which stipulated that lgl lymphocytosis should at least be 2×10 9 /l, lasting for 6 months or longer [1] . however, it has been proposed that for cases with morphologic and immunophenotypic features typical of t-lgl leukemia, even if the lgl lymphocytosis is less than 2×10 9 /l and lasts less than 6 months, the diagnosis can still be made if clonal tcr gene rearrangement is present [3] . such cases were also included in this study. patients all patients with t-lgl leukemia diagnosed consecutively from 1996 to 2009 at queen mary hospital, hong kong were examined. there were no exclusion criteria. review strategy english articles with the term "large granular lymphocyte leukemia" were searched for in pubmed. all returned articles were screened. as the ethnicities of the patients were rarely, if ever, described in published articles, reports from western countries were regarded to have described western patients and those from asian countries asian patients. the bibliographies of these articles were also examined in order to ensure that all reported cases were covered. only cases that fit the who diagnostic criteria of t-lgl leukemia were included. cases described as "large granular lymphocyte leukemia," but were negative for cd3 and showed germline tcr gene, which likely corresponded to neoplastic proliferation of natural killer cells [6] , were excluded. data analysis for asian patients, since there were relatively few cases described, all articles from single case reports to patient series were studied. for western patients, to ensure that representative cases were analyzed, only reports containing at least 20 patients were reviewed. statistical analyses of data were performed by t test or χ 2 test where appropriate (spss, chicago, il, usa). patients a total of 22 ethnic chinese (14 males, eight females) patients at a median age of 46.5 (21-78) years were diagnosed with t-lgl leukemia during a 14-year period. their relevant demographic and clinicopathologic features were shown in table 1 . t-lgl leukemia was the primary hematologic disease in 20 patients. in two patients, t-lgl leukemia developed after an antecedent blood disorder. in case 15, t-lgl leukemia of donor cell origin developed after allogeneic hematopoietic stem cell transplantation for chronic myeloid leukemia [7] . in case 16, t-lgl leukemia presented 2 years after successful treatment of idiopathic thrombocytopenia purpura. the most frequent presenting feature was anemia (hemoglobin <10 g/dl; 17/ 22 cases, 77%). marrow examination showed pure red cell aplasia (prca) in 15 cases (68%). no patient had a previous history of use of erythropoietin. neutropenia (absolute neutrophil count <1.5×10 9 /l) was found in eight cases (36%) and none of the patients presented with infections. granulocyte colony stimulation factor was not used in any of the cases, so that the low frequency of neutropenia was genuine. lgl lymphocytosis (>2×10 9 /l) was found in 14 cases (64%). no patient presented with autoimmune phenomena at the time of diagnosis. case 3 was the only patient who presented with an aggressive course, characterized by fever, organ infiltration, and adult respiratory distress syndrome. all other patients pursued an indolent clinical course predominated by transfusiondependent anemia. treatment nineteen patients were given treatment. the treatment indications were anemia (n=15), lymphocytosis (n=2), leucopenia (n=1), and severe systemic illness (n=1). two patients with lgl lymphocytosis but otherwise normal blood counts have not received any therapy. before 1999, single-agent treatment with cyclophosphamide, chlorambucil, and cyclosporine was used. no patients treated with cyclophosphamide (n=6) and chlorambucil (n=2) responded. cyclosporine was used in 15 patients. there were four complete remissions, two partial remissions, and nine non-remissions, giving an overall response rate of 6/15 (40%). fludarabine, 25 mg/m 2 /day×3 days; mitoxantrone, 12 mg/day×1 day; dexamethasone 20 mg/day×5 days (fnd; monthly for a planned 6 months) was used in thirteen patients, for a median of six (one to six) courses. the outcome had been briefly described previously [8, 9] . there were eight complete remissions. five patients showed a partial remission with improvement in cytopenias. at a median follow-up of 88 (12-110) months, four patients were still in complete remission, lasting a median of 90 (39-110) months, without any maintenance medication. two patients had died of unrelated diseases while still in fnd-induced remission. outcome there were five deaths. three cases died from unrelated causes (cerebrovascular accident, bladder cancer, graft-versus-host disease). two cases died from the leukemia [10] . asian patients identified from the literature twenty articles were evaluable based on the availability of clinicopathologic features and treatment outcome. eighty-eight patients in reviewing patients in this study, some assumptions were made that might have limitations. while asian studies were unlikely to have reported non-asian patients, it might be possible for western studies to have reported some asian patients. however, the significant differences observed between asian and western studies suggest that different patient populations were involved. therefore, if asian patients had been included in western studies, they would have been the minority. because of the small number of asian patients reported in the literature, we elected to include even single case reports in this study so that a reasonable number of patients could be reviewed. a selection bias toward including t-lgl leukemia patients with complications might therefore be introduced. with these limitations, our study still showed a number of interesting observations. this study described the largest single-center series of t-lgl leukemia in chinese patients. t-lgl leukemia is actually a very rare disease. it has been estimated that no more than 450 cases have ever been reported worldwide [34] . our cohort therefore adds substantially to the understanding of this disorder, particularly in asian patients. our series of chinese t-lgl leukemia patients showed a number of interesting features. anemia was the principal presenting problem attributed to prca in the majority of cases. on the other hand, neutropenia was rare, so that recurrent infections were hardly a clinical problem. consequently, our case series has a low mortality, with only two patients dying directly from t-lgl leukemia. when our cases were compared with other asian patients reported in the literature, there was an apparent lower frequency and count of lgl lymphocytosis, but a higher frequency of prca. these disparities might reflect differences in diagnostic approaches. when patients present with lgl lymphocytosis, the diagnosis of t-lgl leukemia is usually not problematic. however, when patients present with anemia without obvious lgl lymphocytosis, t-lgl leukemia may not be immediately obvious. we routinely performed flow cytometry and analysis for tcr gene rearrangement for patients presenting with unexplained cytopenia(s), which enabled us to detect more subtle cases of t-lgl leukemia. in studies where routine investigation for clonal tcr gene rearrangement was not performed, the diagnosis of t-lgl leukemia would understandably be based on lgl lymphocytosis. therefore, the difference of lgl levels might be related to diagnostic approaches. these differences notwithstanding, our cases and other asian patients showed a very high frequency of prca. this complication was originally reported at a high frequency in japanese t-lgl leukemia patients [5] . subsequently, prca has been observed in both chinese and western patients [35] [36] [37] . with recognition of the importance of prca as a cause of anemia in t-lgl leukemia, increasing numbers of cases were reported worldwide. by 2001, prca has been established as a consistent albeit infrequent complication of t-lgl leukemia in western patients [38] . in fact, t-lgl leukemia is now regarded to surpass all other pathologies as the commonest cause of prca [3, 38] . the high frequency of prca in our cases (68%) and other asian patients (42%) is in sharp contrast to the very low frequency of prca at 4% in the western patients reviewed here. the rarity of prca was not due to under-reporting, as in a recent western series where this complication was purposefully looked for, the incidence was only 7% (15/203 cases) [38] . on the other hand, neutropenia was less frequent in asian patients, as opposed to the high frequency of 62% in western patients analyzed here and up to 85% in other reviews [4] . therefore, infections were uncommon in asian patients, but are the foremost challenge in the treatment of western patients. death due to recurrent infections is also uncommon in asian patients, which is contrary to western patients, where death due to recurrent infections can be considered a direct consequence of the leukemia. the observations of these clinical differences may have important implications on disease pathogenesis. although the cause of neutropenia in t-lgl leukemia remains obscure, it has been postulated that lgl leukemic cells expressed fasl, tumor necrosis factor (tnf)-α and interferon (ifn)-γ [39] . tnf-α and ifn-γ up-regulated fas expression on myeloid progenitors, which were then induced into apoptosis by the fasl-expressing lgl leukemic cells [39] . interestingly, these mechanisms overlap with those involved in the neutropenia typically found in rheumatoid arthritis complicated by felty syndrome. in the latter disease, humoral mediated mechanisms also contribute to shortened neutrophil survival. the shared mechanisms of neutrophil suppression in t-lgl leukemia and felty syndrome have led to the premise that both disorders might be part of the spectrum of a similar disease process [39] . this proposition may explain the frequent association between t-lgl leukemia and rheumatoid arthritis in western patients. in the western patients reviewed here, splenomegaly occurred in 40% of cases. although the causes of the splenomegaly were not specified, it could conceivably represent either leukemic infiltration or a felty syndrome in patients with rheumatoid arthritis. in asian t-lgl leukemia patients, as splenomegaly happens less frequently, and rheumatoid arthritis is very rare, it can be deduced that felty syndrome is highly unlikely. this finding may also explain in part why neutropenia is relatively infrequent in asian t-lgl leukemia patients. the above observations suggest that different disease mechanisms might be involved in t-lgl leukemia in different populations. t-lgl leukemia has been proposed to arise from chronic activation of t cells due to endogenous or exogenous antigens [40, 41] . the chronic antigenic stimulation then leads to extreme expansion of a clone of cd8 + cytotoxic t-cells [42] . rheumatoid arthritis and other autoimmune diseases might provide the antigenic stimuli in western patients [43] . in asian patients, however, prca is the most common complication. lgl leukemic cells with cytotoxic activity against red cell precursors has been postulated to occur in asian patients [44] . therefore, it is conceivable that in asian patients, antigenic stimuli related to erythropoiesis could be involved in t-lgl leukemia associated with prca. it is interesting to note that in western t-lgl leukemia patients, prca and rheumatoid arthritis/neutropenia have been observed to be mutually exclusive, again suggesting that they involve different pathogenetic mechanisms [38] . the most important indication for treatment in western t-lgl leukemia patients is neutropenia. low-dose cyclophosphamide and methotrexate with or without corticosteroids have been the mainstay of treatment in this population [3] . amelioration of the neutropenia and suppression of the lgl leukemic clone leads to a high rate of complete remission. however, disease relapse often occurs once treatment is stopped, so that it has been suggested that treatment should continue indefinitely once a response is obtained [3] . treatment with cyclosporine also improves the cytopenia in t-lgl leukemia, but the leukemic clone is usually unaffected [3, 45] . cyclosporine treatment is also indefinite [3] . conversely, the main indication for treatment in asian patients is prca. the efficacy of cyclophosphamide in t-lgl leukemia related prca is uncertain [28] . in the asian patients reviewed here, cyclophosphamide and cyclosporine therapy was effective in some patients. we have previously reported that the purine analogue fludarabine together with mitoxantrone and dexamethasone resulted in high remission rates in chinese t-lgl leukemia patients [9] . updated results presented here showed that complete remission was maintained in five of 13 fludarabine-treated patients for a median follow-up exceeding 4 years. the advantages of this approach include a finite duration of treatment and the obviation of maintenance therapy. fludarabine was combined with mitoxantrone and dexamethasone as it is less active as a single agent than in combination regimens. furthermore, fludarabine as a single agent had been reported to only improve the cytopenias in t-lgl leukemia without eradicating the leukemic clone [46] and fludarabine combined with cyclophosphamide only induced partial remissions in western t-lgl leukemia patients [32] . fludarabine combination regimens offers the possibility of a durable remission without maintenance, as opposed to the daunting prospect of indefinite treatment with cyclophosphamide, methotrexate, and cyclosporine. finally, the monoclonal anti-cd52 antibody alemtuzumab has also been used in t-lgl leukemia with variable success [31, 47, 48] . it remains to be determined if further clarification of the pathogenetic pathways in different patient populations might enable these different therapeutic approaches to be used more rationally. vardiman jw (eds) who classification of tumours of haematopoietic and lymphoid tissues clonal diseases of large granular lymphocytes large granular lymphocyte leukemia t-cell large granular lymphocyte leukemia and related disorders laboratory findings and clinical courses of 33 patients with granular lymphocyte-proliferative disorders tumours of haematopoietic and lymphoid tissues. world health organization classification of tumours t-cell large granular lymphocyte leukemia of donor origin after allogeneic bone marrow transplantation fludarabine, mitoxantrone and dexamethasone in the treatment of indolent b-and t-cell lymphoid malignancies in chinese patients fludarabine, mitoxantrone and dexamethasone as firstline treatment for t-cell large granular lymphocyte leukemia protein-losing enteropathy due to t-cell large granular lymphocyte leukemia ti (wt31)-negative, cd3-positive, large granular lymphocyte leukemia with nonspecific cytotoxicity acute transformation of chronic large granular lymphocyte leukemia associated with additional chromosome abnormality functional analysis of clonally expanded cd8, tcr gamma delta t cells in a patient with chronic t-gamma lymphoproliferative disease large granular lymphocyte leukemia. a study of nine cases in a chinese population large granular lymphocyte leukemia with pure red cell aplasia in a renal transplant recipient large granular lymphocytic leukaemia with a mixed tcell/b-cell phenotype spontaneous remission of large granular lymphocyte t cell leukemia improvement of extrathymic t cell type of large granular lymphocyte (lgl) leukemia by cyclosporin a: the serum level of fas ligand is a marker of lgl leukemia activity pure red cell aplasia associated with parvovirus b19 infection in t-large granular lymphocyte leukemia establishment of the t-cell large granular lymphocyte leukemia cell line motn-1 carrying natural killer-cell antigens pathologic quiz case: chronic anemia with red cell aplasia and lymphocytosis in a middle-aged man. tcell large granular lymphocyte leukemia t-cell large granular lymphocyte leukaemia: association with cd4 single and cd4/cd8 double positive phenotypes cytomegalovirus infection associated with clonal proliferation of t-cell large granular lymphocytes: causal or casual? cd3+ tcrgammadelta+cd4+ cd8-t-cell large granular lymphocyte leukaemia showing skin infiltrations t-cell large granular lymphocytic leukemia occurring after autologous peripheral blood stem cell transplantation gammadelta t-cell large granular lymphocyte (lgl) leukemia with spontaneous remission t-gamma delta large granular lymphocyte leukemia preceded by pure red cell aplasia and complicated with hemophagocytic syndrome caused by epstein-barr virus infection cyclophosphamide therapy for pure red cell aplasia associated with granular lymphocyte-proliferative disorders clinical analysis of 52 patients with granular lymphocyte proliferative disorder (glpd) showed frequent anemia in indolent t-cell glpd in japan clinical spectrum of clonal proliferations of t-large granular lymphocytes: a t-cell clonopathy of undetermined significance t-cell large granular lymphocyte leukemia: a report on the treatment of 29 patients and a review of the literature t-cell large granular lymphocytic (t-lgl) leukemia: experience in a single institution over 8 years clinical spectrum of gammadelta+t cell lgl leukemia: analysis of 20 cases clinical features of large granular lymphocyte leukemia pure red cell aplasia: clinical features and treatment results in 16 cases pure red cell aplasia: association with large granular lymphocyte leukemia and the prognostic value of cytogenetic abnormalities association of pure red cell aplasia with t large granular lymphocyte leukaemia acquired pure red cell aplasia associated with lymphoproliferative disease of granular t lymphocytes pathogenesis of neutropenia in large granular lymphocyte leukemia and felty syndrome survival signals in leukemic large granular lymphocytes analysis of the t cell receptor in the lymphoproliferative disease of granular lymphocytes: superantigen activation of clonal cd3+ granular lymphocytes pathologic clonal cytotoxic t-cell responses: nonrandom nature of the t-cell-receptor restriction in large granular lymphocyte leukemia t-cell large granular leukemia and related proliferations pure red cell aplasia associated with expansion of cd3+ cd8+ granular lymphocytes expressing cytotoxicity against hla-e+cells neutropenia associated with t-cell large granular lymphocyte leukemia: long-term response to cyclosporine therapy despite persistence of abnormal cells neutropenia and anaemia associated with t-cell large granular lymphocyte leukaemia responds to fludarabine with minimal toxicity alemtuzumab induced complete remission of therapy-resistant pure red cell aplasia remission of pure red cell aplasia in t-cell receptor gammadelta-large granular lymphocyte leukemia after therapy with low-dose alemtuzumab key: cord-0006675-2dah82sw authors: gause, a.; arbach, o.; lamprecht, p. title: der einsatz von tnfα-antagonisten bei primär systemischen vaskulitiden date: 2003 journal: z rheumatol doi: 10.1007/s00393-003-0527-6 sha: f3139411171a749a54be8343c520e64a36295d75 doc_id: 6675 cord_uid: 2dah82sw after the introduction of the tnfα blocking drugs etanercept and infliximab for the standard therapy of rheumatoid arthritis these effective substances have also been used successfully in many patients with primary systemic vasculitides, who were unresponsive to standard therapy. from pathophysiologic findings their use is justified by the prominent role of tnfα in the inflammation of small and large vessels. so far only open studies with a maximum of 20 patients and case reports are published. in summary there are reports of the successful treatment of 7 patients with rheumatoid vasculitis, 39 patients with wegener's granulomatosis, 3 patients each with microscopic polyangiitis, and 5 patients each with temporal arteritis or takayasu disease with etanercept as well as with infliximab. in addition, infliximab was also used with a good response in severe polymyalgia rheumatica in 4 cases and in one case of a cryoglobulinemic vasculitis. more than 60 patients with panuveitis and other manifestations of behçet's disease were treated with infliximab or etanercept according to preliminary reports. because of the overall positive reports with tnfα collected in this review controlled investigations for their use in primary systemic vasculitis are necessary. n zusammenfassung nach der einführung der tnfa blockierenden medikamente etanercept und infliximab in die standardtherapie der rheumatoiden arthritis wurden diese hochwirksamen substanzen bei zahlreichen patienten mit systemischen vaskulitiden, die auf die immunsuppressive standardtherapie nicht ansprachen, mit erfolg eingesetzt. pathophysiologisch ist ihr einsatz durch die prominente rolle von tnfa bei der entzündung kleiner und großer gefäße begründet. in der literatur finden sich bisher lediglich offene studien bis zu einer maximalen zahl von 20 patienten und fallsammlungen, hierunter insgesamt berichte über 7 patienten mit rheumatoider vaskulitis, 39 patienten mit wegener'scher granulomatose, je 3 patienten mit mikroskopischer polyangiitis und churg-strauss-syndrom und je 5 patienten mit arteritis temporalis und takayasu arteritis, bei denen etanercept oder infliximab mit erfolg eingesetzt wurden. darüber hinaus wurde infliximab auch bei 4 patienten mit polymyalgia rheumatica und bei einem patienten mit kryoglobulinämischer vaskulitis erfolgreich verwendet. mehr als 60 patienten mit panuveitis und anderen manifestationen der behçet-erkrankung wurden nach vorläufigen berichten ebenfalls mit infliximab oder etanercept behandelt. aufgrund der in dieser übersicht zusammengestellten überwiegend positiven erfahrungen mit tnfa-antagonisten sind kontrollierte studien zu ihrer anwendung bei primär systemischen vaskulitiden dringend erforderlich. n summary after the introduction of the tnfa blocking drugs etanercept and infliximab for the standard therapy of rheumatoid arthritis these effective substances have also been used successfully in many patients with primary systemic vasculitides, who were unresponsive to standard therapy. from pathophysiologic findings their use is justified by the prominent role of tnfa in the inflammation of small and large vessels. so far only open studies with a maximum of 20 patients and case reports are published. in summary there are reports of the successful treatment of 7 patients with rheumatoid vasculitis, 39 patients with wegener's granulomatosis, 3 patients each with microscopic polyangiitis, and 5 patients each with temporal arteritis or takayasu disease with etanercept as well as with infliximab. in addition, infliximab was also used with a good response in severe polymyalgia rheumatica in 4 cases and in one case of a cryoglobulinemic vasculitis. more than 60 patients with panuveitis and other manifestations of behçet's disease were treated with infliximab or etanercept according to preliminary reports. because of the overall positive re-ports with tnfa collected in this review controlled investigations for their use in primary systemic vasculitis are necessary. n schlüsselwörter primär systemische vaskulitis -psv-tnfa-antagonisten -infliximab -etanercept n key words primary systemic vasculitis -psv -tnfa antagonists -infliximab -etanercept das erkennen und die behandlung primär systemischer vaskulitiden hat sich in den letzten jahren durch neue erkenntnisse in der pathogenese und die fortschritte erstens in der klassifikation, zweitens in der diagnostik und drittens vor allem in der therapie erheblich gebessert (11) (12) (13) . dennoch können primär systemische vaskulitiden auch heute noch, vor allem wenn sie zu spät erkannt werden, deletär verlaufen und rasch durch versagen lebenswichtiger organe oder komplizierende infektiöse komplikationen zum tode führen. des weiteren gibt es einen anteil von etwa 10% der patienten, die trotz einer optimalen und intensiven therapie nach dem standardschemata nicht auf die therapie ansprechen und anhaltend vaskulitische aktivität haben, die nur durch hohe glucocorticosteroiddosen eingedämmt werden kann. dies sind die sogenannten therapierefraktären verläufe. eine weitere patientengruppe leidet an sekundären komplikationen einer immunsuppressiven therapie, z. b. mit cyclophosphamid in form einer hämorrhagischen cystitis oder sogar einer neoplasie des urothels oder aber einem toxischen knochenmarkschaden, der sogar in eine myelodysplasie münden kann (18) . bei diesen patienten kann cyclophosphamid als standardmedikation nicht mehr eingesetzt werden. darüber hinaus sind heute die schweren komplikationen durch eine länger dauernde hoch dosierte glucocorticosteroidbehandlung, vor allem infektionen, aber auch hautatrophie, osteoporose, diabetes mellitus, hypertonus und akzelerierte arteriosklerose bekannt (15, 17) . diese verschiedenen probleme machen die weitersuche nach effektiven und möglichst wenig toxischen medikamenten erforderlich. nach einführung der tnfa-antagonisten etanercept und infliximab für die therapie der rheumatoiden arthritis stellte sich schnell die frage, ob diese substanzen auch für die therapie primär systemischer vaskulitiden in frage kommen. bei den meisten primär systemischen vaskulitiden finden sich in der pathogenese hinweise auf eine rolle von tnfa, die bei den einzelnen entitäten kurz wiedergegeben werden. ansonsten führte natürlich die rheumatoide arthritis selbst, die ebenfalls als komplikation in eine vaskulitis münden kann, zu der überlegung, tnfa-antagonisten für die therapie primär systemischer vaskulitiden einzusetzen. da es sich bei den vaskulitiden jedoch um seltene erkrankungen han-delt, ist es schwer, für diese krankheitsbilder große studien zusammenzustellen, so dass sich die bisherige datenlage überwiegend auf berichte über einzelfälle, sammlung von fällen und kleine pilotstudien stützen kann. eine übersicht über die bisher publizierten studien ist in tabelle 1 wiedergegeben. im folgenden werden kurz die ergebnisse zu einzelnen krankheitsentitäten wiedergegeben, um abschließend mögliche perspektiven aufzuzeichnen. bei der rheumatoiden vaskulitis als der schwersten extraartikulären komplikation der rheumatoiden arthritis wurde in einem ersten fallbericht im rahmen einer therapiestudie zur behandlung der rheumatoiden arthritis mit dem rekombinanten p55-tnf-rezeptorfusionsprotein lenercept, das verschwinden digitaler nekrosen nach infusion des medikaments (50 mg alle 4 wochen i. v.) und wiederauftreten bei wirkungsabfall nach ca. 3 wochen beschrieben (9) . zwei weitere fallberichte mit besserung kutaner ulzera und mononeuritis multiplex durch 5 mg/kg infliximab finden sich bei bartolucci (5). unger et al. therapierten 3 patienten mit schwerer therapierefraktärer rheumatoider vaskulitis mit 3 mg/kg infliximab mit gutem erfolg, auch hier heilten bei 2 patienten hautulzera ab, bei dem dritten patienten bildete sich ein hämodynamisch wirksamer perikarderguss zurück (25) . eine mononeuritis multiplex als manifestation der rheumatoiden vaskulitis wurde von richter et al. erfolgreich durch additive gabe von etanercept zur 3fach-therapie nach o'dell behandelt (19) . für die anca-assoziierten vaskulitiden, die wegener'sche granulomatose (wg) und die mikroskopische polyangiitis (mpa), liegen umfangreichere erfahrungen vor. die anwendung bei der wg gründet sich darauf, dass bei der generalisierten vaskulitis überwiegend ein th1-cytokinprofil nachweisbar ist, das sich unter der standardtherapie mit cyclophosphamid und prednisolon normalisiert, es wurden erhöhte tnfa-spiegel im blut bei wg, außerdem ist eine prominente rolle von tnfa im granulom bekannt (zusammengefasst in (16, 23) ). bei der nekrotisierenden vaskulitis kleiner gefäße und von kapillaren spielt tumor-nekrose-faktor-a einmal eine rolle bei der aktivierung der neutrophilen, monozyten und endothelzellen unter anderem durch vermehrte expression von adhäsionsmolekülen und die aktivierung von t-lymphozyten und makrophagen. dies wiederum kann dann im weiteren zu weiterer bereitstellung von tnfa und damit zu einem circulus vitiosus einer sich herauf-schaukelnden entzündung mit ischämien und nekrosen der betroffenen organe führen. die erste publizierte studien zum einsatz von etanercept in kombination mit konventioneller behandlung bei 20 patienten mit wg, die therapierefraktär gegenüber der maximal möglichen standardtherapie waren, zeigte in einer offenen 6 monate dauernden studie, dass die therapie sicher und effektiv durchzuführen ist (23) . der vaskulitisaktivitätsindex bvas/ wg, der in dieser studie verwendet wurde, nahm signifikant ab, ebenso der mittlere tägliche prednisolonin einer bislang noch unveröffentlichten pilotstudie mit infliximab zur remissionsinduktion wurden 10 patienten mit generalisierter, aktiver wg (bvas1 > 7, dei > 4) mit infliximabinfusionen (5 mg/kg kg) in woche 0 -2 -6 -12 -18 -24 additiv zu 2 mg/ kg/d cyc und 0,5 mg/kg prednisolon initial mit nachfolgender reduktion auf < 6 mg/d prednisolon in woche 12 behandelt. die tägliche gc-dosis und die dauer ihrer gabe wurde verglichen mit dem "nih-standard"-protokoll auf die hälfte reduziert. primäre endpunkte waren 50% reduktion von bvas und dei, prednisolonreduktion entsprechend protokoll und das auftreten von unerwünschten ereignissen. 10 patienten wurden in die studie eingeschlossen (5 männer mit einem altersmedian von 50 jahren, 5 frauen mit einem altersmedian von 32), die alle die acr-und chc-kriterien erfüllten. alle waren canca/pr3-anca positiv (medianer titer 1 : 1024) und aktiv (medianer bvas1: 21; dei: 10). alle patienten reagierten mit einer prompten reduktion der entzündungsparameter und des bvas1 nach den ersten infliximabinfusionen. ein patient wurde bei zunächst gutem ansprechen im weiteren verlauf wegen erneuter krankheitsprogression ausgeschlossen. ein weiterer patient erhielt nur eine infliximabinfusion (studienausschluss wegen mangelnder compliance) und erlitt 6 wochen danach eine sepsis unter laufender nih-standardtherapie. ein patient erkrankte an einer pneumocystis carinii pneumonie (pcp) bei gleichzeitiger lymphopenie 6 wochen nach der 3. infliximabinfusion. er verstarb an einem ards mit postmortalem nachweis von cytomegalievirus (cmv) in der lunge. wegen schwerer infektionen bei 2 von 10 patienten mit einem todesfall wurde die studie abgebrochen. bis zu dem zeitpunkt hatten von den verbliebenen patienten je zwei zwei infusionen und vier vier infusionen sowie je einer drei oder fünf infliximabinfusionen erhalten. mit ausnahme von zwei patienten waren alle bereits in kompletter remission. nach umstellung auf eine remissionserhaltende therapie nach 3 bis 6 monaten sind bisher keine rezidive aufgetreten (abb. 1 und 2). nach den vorläufigen ergebnissen ist die sehr effektive therapie mit infliximab zur remissionsinduktion der wg mit schweren infektiösen komplikationen belastet. zur ermittlung eines therapieregimes für weitere kontrollierte studien kommen wir aufgrund unserer erfahrungen zu folgenden modifikationen: einsatz einer geringeren dosis von infliximab (3 mg/kg kg), kombination mit "milderer" immunsuppression (z. b. cyc bolus-therapie), imperative pcp-prophylaxe mit cotrimoxazol oder pentamidine, therapiepause bei einer lymphopenie von 500/ll und gezielte cmv-antikörpertiter bzw. antigen-kontrollen ggf. nach den erfahrungen der transplantationsmedizin mit pre-emptiver antiviraler therapie. das churg-strauss-syndrom zeichnet sich durch eine nekrotisierende vaskulitis mit gleichzeitiger gewebsinfiltration durch eosinophile und extravaskuläre 231 a. gause et al. abb. 1 übersicht über das prompte ansprechen auf die induktionstherapie der wegener'schen granulomatose mit infliximab additiv zu cyclophosphamid und reduzierter prednisolondosis gemessen am bvas (birmingham vasculitis activity score). alle patienten fielen prompt mit dem bvas ab. bei 3 patienten zeigte sich ein wiederanstieg bei rezidiv bzw. infektion granulome aus. bei allen drei pathologischen faktoren wirkt tnfa stimulierend, so dass auch bei dieser erkrankung der einsatz einer tnfa-blockade gerechtfertigt sein kann. allerdings kommt das churg-strauss-syndrom wesentlich seltener als die wegener'sche granulomatose vor, dementsprechend sind patienten mit schweren krankheitsverläufen noch seltener. drei fälle mit langjährigen komplizierten verläufen sind beschrieben, die letztlich nur durch die additive gabe einmal von etanercept zusätzlich zu cyclophosphamid und in zwei fällen durch infliximab zusätzlich zu cyclophosphamid-dauertherapie beherrscht werden konnten (4). bei der kryoglobulinämischen vaskulitis steht die immunkomplexkrankheit im vordergrund, so dass hier nicht so leicht ein ansatz für tnfa-inhibition gesehen werden kann. allerdings gibt es in der arbeit von bartolucci einen fallbericht mit einer erfolgreichen behandlung einer kryoglobulinämischen vaskulitis durch infliximabinfusionen in der dosis von 5 mg/kg körpergewicht mit ansprechen der hautulzerationen und nervenschmerzen (5). die behçet-erkrankung wird als chronisch rezidivierende entzündliche erkrankung mit multiorganbefall den vaskulitiden zugerechnet. während in milden fällen wiederholte mukokutane manifestationen die einzigen krankheitszeichen darstellen können, kommt es in 50-70% zu augenbeteiligung, in ca. 50% zu gelenkbeteiligung, in 25% zu vaskulären und in jeweils 5-10% zu gastrointestinalen oder zentralnervösen manifestationen. die bei dieser erkrankung häufig schwer beherrschbaren uveitiden führen in 20 bis 25% zur erblindung. infliximab wurde bei auf konventionelle therapie refraktären patienten in dosierungen von 3 mg/kg, 5 mg/kg und 10 mg/kg erfolgreich eingesetzt, in einem zeitraum von 6 monaten erfolgten eine einzige bis zu 4 infusionen. offenbar ist bei einem rezidiv einer panuveitis eine remission des krankheitsschubs mit wiederherstellung der sehkraft durch eine einzige infliximabinfusion zu erreichen (5 mg/kg bei 5 patienten) (22) . nach berichten über 11 weitere erfolgreich mit infliximab behandelte patienten werden zur zeit studien in griechenland, der türkei und japan zum langzeiteffekt der infliximabtherapie auf die okulären und sonstigen systemischen manifestationen durchgeführt (21) . über schwere komplikationen bei der behandlung der behçet-erkrankung wurde nicht berichtet. die anwendung von etanercept bei refraktärer uveitis ebenso wie die durchführung einer randomisierten placebokontrollierten studie mit 40 patienten mit mukokutanen manifestationen durch hassan yazici wird als kongressreport ebenfalls von sfikakis berichtet (21) . die etanercept behandelten patienten waren zu 40% gegenüber 5% in der placebogruppe frei von ulzerationen und nodulären oder papulopustulösen hauteffloreszenzen. nach absetzten des etanercept kam es jedoch zu prompten rezidiven. bei der temporal-arteritis ist in experimentellen untersuchungen der nachweis von t-zell-und makrophagenaktivierung u. a. durch tnfa gelungen, von einer entzündung im bereich der adventitia kommt es zum mediaödem und zur fortschreitenden entzündung mit sekundärer granulombildung, so dass auch hier vom pathogenetischen hintergrund der einsatz eines tnfa-antagonisten erfolgversprechend sein sollte (14) . zum einsatz in der klinik liegen ein fallbericht sowie eine fallsammlung mit 4 patienten vor mit gutem effekt einer infliximabtherapie (3, 8) . gerade bei einer arteritis temporalis mit anhaltender entzündungsaktivität und hohem gc-bedarf, der in den beschriebenen fällen erfolgreich reduziert werden konnte, erscheint wegen der risiken der glucocorticosteroide in hohem alter ein intensiver therapieansatz mit tnfa-antagonisten sinnvoll. unter diesem aspekt sind auch 4 fallberichte mit erfolgreicher anwendung von infliximab bei therapierefraktärer polymyalgia rheumatica publiziert (20) . allerdings wird die verwendung der tnfa-antagonisten möglicherweise bei bereits vorhandenen komorbiditäten in einem älteren patientenkollektiv wie z. b. diabetes mellitus und herzinsuffizienz eingeschränkt. für die zu den riesenzellarteritiden gehörende und mit ihrer durch aktivierte t-lymphozyten, nk-zellen und makrophagen vermittelte gefäßläsion pathogenetisch verwandte takayasu-arteritis liegt ein erfahrungsbericht an 5 patienten in einem buchkapitel vor (14) . in einem abstract auf dem letzten kongress des american college of rheumatology (1) zeigten die gruppe von steven et al. aus birmingham die ergebnisse von 3 patienten mit primär systemischer vaskulitis, bei denen sich die mittels laser-doppler-flussmessung geprüfte endotheldysfunktion unter infliximabinfusion reproduzierbar kurzzeitig besserte, was unter cyclophosphamid und methylprednisolon nicht der fall war. hierdurch wird die relevanz von tnfa für die diffuse endotheliale dysfunktion bei primären systemischen nekrotisierenden vaskulitiden, die für verschiedene krankheitsentitäten gezeigt wurde (10), belegt. dies ist ein weiterer experimenteller nachweis für die bedeutung von tnfa in der vaskulitispathogenese. die aufnahme von tnfa-antagonisten in die therapieregimes für primär systemische vaskulitiden wird durch die befunde unterstützt. das konzept einer möglichen intensiven remissionsinduktion, um dann eine remission mit milder basistherapie oder sogar therapiefreiheit zu erreichen ist ein besonders sinnvoller ansatz für junge patienten mit primär systemischen vaskulitiden gerade in hinsicht auf die bereits erwähnten spätfolgen der bisherigen standardtherapie. unter berücksichtigung eigener und publizierter erfahrungen mit infektiösen komplikationen unter der therapie mit tnfa-antagonisten wird es notwendig sein, infektionsprophylaxen in die therapieprotokolle aufzunehmen (7, 24) . wegen der bereits anfangs geschilderten seltenheit der primär systemischen vaskulitiden sind hierfür jedoch multizentrische anstrengungen erforderlich, die auf den vorhandenen strukturen des kompetenznetzes rheuma und der euvas aufgebaut werden können. infection complications associated with the use of biologic agents treatment of longstanding active giant cell arteritis with infliximab: report of four cases isolated digital vasculitis in a patient with rheumatoid arthritis: good response to tumour necrosis factor alpha blocking treatment diffuse endothelial dysfunction is common to anca associated systemic vasculitis and polyarteritis nodosa primär systemische vaskulitiden. teil i. allgemeine übersicht primär systemische vaskulitiden primär systemische vaskulititden. teil iii. pathogenese und vasculitis. in: smolen js, lipsky pe (hrsg) targeted therapies in rheumatology premature atherosclerosis in systemic lupus erythematosus effectiveness of tnfalpha blockade with infliximab in refractory wegener's granulomatosis suppression of inflammation in primary systemic vasculitis restores vascular endothelial function: lessons for atherosclerotic disease an interdisciplinary approach to the care of patients with wegener's granulomatosis: long-term outcome in 155 patients mononeuritis secondary to rheumatoid arthritis responds to etanercept treatment of refractory polymyalgia rheumatica with infliximab: a pilot study behçet's disease: a new target for anti-tumour necrosis factor treatment effect of infliximab on sight-threatening panuveitis in behet's disease etanercept combined with conventional treatment in wegener's granulomatosis: a six-month open-label trial to evaluate safety treatment of severe systemic lupus erythematosus with high-dose chemotherapy and haemopoietic stem-cell transplantation: a phase i study successful treatment of severe rheumatoid vasculitis by infliximab key: cord-0009744-90kt3406 authors: schumacher, h. ralph; newton, charles; halliwell, richard e. w. title: synovial pathologic changes in spontaneous canine rheumatoid‐like arthritis date: 2005-12-11 journal: arthritis rheum doi: 10.1002/art.1780230404 sha: 5c36354a79e5f5d9eb1fb6a88276dadb7069a426 doc_id: 9744 cord_uid: 90kt3406 the synovial fluid and membrane were studied in 10 dogs meeting the american rheumatism association criteria for classic human rheumatoid arthritis (ra). light microscopic pathologic features were consistent with those found in the human disease. neutrophilic infiltration of synovium was somewhat more prominent than in chronic human ra, and activated lymphocytes in fluid or membrane were less frequent. the proliferative and plasma cell reaction seemed identical. electron microscopy (em) suggested microvascular injury with findings which included electron dense deposits in the vessel walls of 2 dogs. seven dogs had meshworks of 20‐25 mm tubules in tubuloreticular structures (trs) similar to those seen in human systemic lupus erythematosus and only occasionally in human ra. there were also crystalline arrays of tubules, a configuration previously reported in tumors and virus infections and possibly suggestive of a cellular reaction to virus infection. to date no initiating agent has been identified, but this spontaneous canine disease which is very similar to human ra can provide a valuable model in which to examine pathogenesis of chronic arthritis. a spontaneous, chronic, erosive polyarthritis similar to human rheumatoid arthritis (ra) occurs in dogs (1) (2) (3) . this arthritis is of importance to rheumatologists because it may provide the first spontaneously occurring model for the human disease. a preliminary study suggests that canine ra can be responsive to gold salt therapy (4). it is also intriguing that epidemiologic studies (5,6) have shown a greater exposure of human ra patients to dogs and other pets during the 5 years prior to onset of disease than for osteoarthritics and patients with other miscellaneous minor musculoskeletal problems. examples of coincidental arthritis in pets and owners have been found. household dogs of patients with systemic lupus erythematosus (7) have also recently been suggested to be clinically and serologically involved. this report describes the gross and the light and electron microscopic (em) synovial changes in 10 dogs with canine arthritis that fulfilled the american rheumatism association (ara) criteria for classic ra (8). all dogs presented to the university of pennsylvania veterinary hospital (upvh) were screened for symptoms of arthritis. after eliminating from the study dogs with degenerative joint disease, septic arthritis, and polyarthritis associated with systemic lupus, 10 dogs were further studied by x-ray, rose-waaler tests for rheumatoid factor, clinical examination, and biopsies to identify those with a definite diagnosis of rheumatoid arthritis. all dogs diagnosed as having canine rheumatoid-like arthritis met at least 7 of the ara criteria for a diagnosis of human rheumatoid arthritis (8). canine ra-like arthritis affected both pure and mixed breeds. the age range at initial presentation was from 13 months to 8 years, and age at onset was from 5 months to 7 years, with 1 male and 9 females affected. seven dogs had rheumatoid factor titers according to the joints primarily affected with the disease were the large peripheral joints, i.e., carpus, tarsus, knee, and elbow. the metacarpophalangeal, metatarsophalangeal, and interphalangeal joints were less prominently affected. erosions of multiple joints were seen in all 10 dogs reported here. gross changes in synovial membrane of rheumatoidlike joints were noted at surgical synovial biopsy in 7 animals or on gross pathologic examination at necropsy in the remaining 3 animals. histopathologic examination was performed on multiple specimens from the affected joints of the 10 rheumatoid dogs. specimens were collected in 10% formalin or bouin's solution and processed through routine paraffin embedding and sectioning. hematoxylin and eosin staining was performed on all synovial membranes. electron microscopy was performed on specimens obtained simultaneously with those collected for light microscopy and immediately placed in + strength karnovsky's paraformaldehyde-glutaraldelhyde fixative (10). specimens were diced into 1 x 1 mm pieces, fixed for a total of 4 hours at room temperature, washed in 0.1m sodium cacodylate buffer at 4"c, post fixed in cold palade's osmium-veronal for 2 hours, and then dehydrated in alcohol. they were then placed in 50% propylene oxide and epon mixtures over 2 hours and embedded in epon 812. thick sections (lp) were cut on an lkb-2 ultramicrotome with a glass knife and stained with toluidine blue for orientation. thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and then examined on a zeiss em-i0 electron microscope with a 60 kv beam. in 2 dogs, em histochemical staining for acid phosphatase was performed by using a modified gomori technique (1 1). synovial fluid was collected from affected joints of 8 dogs and smeared and stained with wright's and sudan stains ( table 1 ). in most instances there was insufficient fluid to pro gross findings. gross changes included striking synovial proliferation with bony and cartilaginous destruction. all 10 dogs demonstrated proliferative, brown, discolored synovium that in most instances was markedly thickened 1-3 mm. there were large papillae of synovium and long fingers of fibrin present in 4 animals. mild invasion of synovial tissue into subchondral bone was present in 3 dogs, and massive invading pannus was present in the other 7. light microscopy. table 2 delineates the light microscopy findings. histologically, synovium dernonstrated marked lining cell proliferation in 8 of the dogs examined (figures 1 and 2 ). in most instances the underlying connective tissue was equally proliferative with increased numbers of fixed tissue cells. infiltration of the underlying connective tissue with plasma cells occurred in all 10 dogs (figure 2) . the plasma cells were found to be scattered diffusely in most dogs, although focal aggregations were found as well. more than rare lymphocytes were also found in the synovium of 5 dogs. the lymphocytes were diffusely scattered throughout two of the synovial membranes and were present in more focal aggregates in two. infiltration with polymorphonuclear neutrophils (pmn) was seen in 5 dogs; in these tissues, the pmn were diffuse in 3 dogs and focal in the other 2. deposits of acellular fibrin were easily demonstrated on the synovial surface in 7 dogs ( figure 1 ). hemosiderin was recognizable in deep macrophages in 6 dogs. congested capillaries were seen in 3 tissues and edema in 2. electron microscopy. table 3 shows the em results. ultrastructural studies allowed some extension of the light microscopic findings. finely granular material was frequently mixed in with the prominent surface fibrin. lining cells could be characterized as including types a (phagocytic), b (synthetic), and c (intermediate) cells (13) with b and c cells ( predominant plasma cells ( figure 5 ) was similar to that seen by light microscopy. note that despite examining multiple blocks by em, sampling still was limited so that inflammatory cells, for example, were not seen by em in all dogs. interstitial polymorphonuclear cells were degenerating (figure 4 ) or degranulating in 3 synovia. plasma cells often had dramatically dilated rough endoplasmic reticulum. activated lymphocytes with polyribosomes were not identified. vessels showed occasional gaps ( figure 6 ) between endothelial cells and multilaminated basement membranes, but no vessel wall necrosis, fibrinoid, or evidence of virus-like particles was observed. electrondense deposits were seen in vessel walls of 2 dogs (figures 6 and 7) , and degranulating polymorphonuclear cells and platelet clumps ( figure 6 ) were present in vessel lumens of 2 dogs each. occasional extravasated erythrocytes and iron in deep macrophages were consistent with the light microscopic finding of hemosiderin deposits in some dogs. seven synovia had prominent lipid droplets in deep synovial cells or lining cells. no identifiable bacterial or other organisms were found. tubuloreticular structures (trs) as typically seen in human sle (14-16) were seen in 7 dogs (figures 3, 8, and 9 ). these were seen in endoplasmic reticutubules of similar 20-25 nm diameter were also seen in crystalline arrays ( figure 10 ) in 9 dogs including all 7 who had identifiable trs. crystalline arrays were documented in cross and longitudinal sections. some were in the er, but others were in the cytoplasm or in membrane-bound dense bodies. acid phosphatase em histochemistry confirmed the lysosomal nature of these dense bodies with crystalline arrays of tubules in 2 dogs (figure 11 ). trs were never seen associated with acid phosphatase positive areas. cells with the crystal-like arrangement of tubules in dense bodies also frequently contained other unidentified, highly dense, lipid-like or finely granular material or ferritin granules. in 3 dogs some round vesicle-like bodies mixed with various types of cell debris were seen in occasional vacuoles or in interstitium ( figure 5 ). none strongly suggested any specific virus or other organism. twenty control dog synovial membranes were reviewed. no trs were found in 7 normal mongrels used for experimental injection of urates (17), 10 normal mongrels used in study of joint trauma, or 3 osteo-arthritic dogs. in two of the supposedly normal mongrels, small crystal-like arrays of membrane bound tubules were found in lining cells. synovial fluid analysis (table 1) . total cell counts on the 3 synovial fluids with sufficient volume for leukocyte counts ranged from 5,800 to 54,000/mm3. the widely varying differential counts found are shown in table 1 . pmn predominated in 3 fluids. there were no large mononuclear cells that had phagocytized pmn ("reiters cells"), le cells, or other unusual features. since first being reported in the last 10 years (i-3), canine rheumatoid-like arthritis has begun to receive attention as a potential animal model of human disease. major studies of the disease have been reported from the university of california at davis ( 2 ) and the university of pennsylvania ( i ,4,9). case reports account for an additional 15 cases present in the literature (3, 18) . all reports agree on the major gross and histopathologic findings. these are described as villous proliferation, hyperplastic synovium, fibrin deposition over the proliferative synovium, bony erosions, and infiltration with mononuclear cells including plasma cells. pederson ( 2 ) did not mention the pmn which were also seen in some , magnification x 19,000.) of our dogs, but scott did (1 8). otherwise, our light microscopic findings differ little from previous reports. these findings meet the ara criteria as characteristic of human ra synovium (8). necrosis also mentioned in the ara criteria was not usually appreciated by light microscopy but was seen prominently by em in 3 dogs. polymorphonuclear cell infiltration in synovium in our series and in the dog studied by scott (1 8) appeared to be slightly more common than in human ra. however, pmn can be seen in some chronic human ra synovial membranes and are even more common in early ra (19) . the prominence of hemosiderin deposits was more marked than in most reports of human ra, possibly because dogs abused actively inflamed joints more than humans. iron was localized, as it is in human ra synovial membranes, predominantly in deep phagocytic cells we previously reported a single case of only limited em findings in canine rheumatoid-like arthritis (9); (20) . no other report appears in the literature. the absence of activated lymphoblasts in our synovial biopsies appears to be different from human ra (21), but this may represent in part a sampling problem since activated lymphocytes were seen in some synovial fluids. microvascular changes have not been as prominent in human ra of chronic duration as in these dogs, but these changes have been emphasized in early human ra of up to 6 weeks duration (19, 22) . the presence of large electron-dense deposits in vessel walls of 2 dogs resembled findings in some early human ra (19) which suggests the possibility of deposition of immune complexes in these vessels. these dense deposits in the dogs have not yet been further characterized. platelet plugging, degranulating intraluminal cells, gaps between endothelial cells, and multilaminated vascular basement membranes were also evidences of some microvascular injury. the tubuloreticular structures found in synovial membranes in 7 of our rheumatoid dogs are familiar to rheumatologists because of their frequent occurrence in human systemic lupus erythematosus (14-16). they have also occasionally been described in human ra (14,23). to our knowledge, em studies to search for trs in synovial membrane or other tissues in canine sle have not been performed. tubuloreticular structures have also been identified in various other tissues (in addition to synovium) of patients with systemic lupus erythematosus (14-16), in muscle tissue of patients with dermatomyositis (14, 29, 30) , and in a variety of sites in other diseases. they have been found in infectious mononucleosis (3 1 ), human lymphoma cell lines growing herpes simplex (32), human herpes encephalitis cerebral tissue (33) , dog intestine in coronavirus infection (34) , and a variety of tumors (35) . thus there seems to be a suggestion of association of trs with rheumatic disease, virus infection, and neoplasm. the nature and cause of trs are not known. tubuloreticular structures differ from paramyxoviruses, including canine distemper virus, by the regular association of trs with the endoplasmic reticulum and the smaller (15-17 nm) diameter fibrillar nucleocapsids of the virus (24, 25) . although nucleic acid is apparently not demonstrable in trs (36), pincus et a1 (37) have suggested that they might be a cellular reaction to virus infection. the crystalline arrays of tubules seen in 9 rheumatoid-like dogs have not been reported in human ra. these inclusions appear virtually identical to clumps of cytoplasmic tubular arrays without binding membranes found in the cytoplasm of endothelial cells in placentas have found crystalline arrays of similar tubules with light cores that are an estimated 25 nm (22-28 nm) in diameter in er of rabbit myxosarcomas. there were also identical size tubules in er arranged more like the typical tubuloreticular structures in the same tumors. schaff (36) and others (43) have suggested that there may be a relationship between the two types of tubules. some relationship is also supported by our studies showing the 2 kinds of structures together in 7 of the ra dogs and some densely packed, but not crystalline, trs (figure 8 ) that might suggest a transition phase between these and crystalline arrays seen in rough endoplasmic reticulum. no type c particles or coronavirus-like particles have been found in our dogs. they have occasionally been noted in human rheumatoid synovial membrane only with very early disease (19) or with cocultivation (44). the wide variety of types of debris in the dog synovium as well as human ra could certainly harbor material from an infectious agent that is no longer morphologically identifiable. there have been a few light microscopic studies of dog synovial membrane in other states that should be compared with our rheumatoid-like dog findings. dog synovial membrane in culture-positive chlamydia arthritis has been described by young et a1 (45) as showing large amounts of superficial fibrin, hyperplastic hing cells, and infiltration of neutrophils and plasma cells. a group of culture-negative dogs (46) with non-ings reported included increased numbers of intermediate type (13) synovial lining cells, some of which included large vacuoles containing particles of various sizes and electron density. they did not report trs or crystalline arrays or evidence of infectious agents. as noted above, we have observed occasional crystalline arrays but not trs in apparently normal dog synovium. the synovial fluid findings in our dogs with ralike disease are quite consistent with the generally accepted findings in human ra (49). polymorphonuclear neutrophils or mononuclear cells predominated in different fluids. prominence of activated lymphocytes, which we recently reported in human ra effusions (12), was seen in only 2 dogs. we recognize that dogs with systemic lupus erythematosus can also have polyarthritis (50,5 1). systemic lupus was excluded in our dogs by absence of any of the characteristic clinical or laboratory findings. we are not aware of em studies on lupus synovium in dogs. a light microscopy study (50) on synovium of a dog with feaerosive arthritis, including some with sle and some undiagnosed, were reported to show superficial fibrin, a paucity of synovial lining cells, and predominantly neutrophilic infiltration with mononuclear cells. there was no villous hyperplasia or pannus. dogs whose knees were experimentally injected with cartilage homogenate (47) developed synovial round cell infiltration after several months. as with human disease, most synovial membrane findings are not diagnostic. ra findings can be mimicked by other diseases, but synovial findings in conjunction with other features can be helpful in diagnosis (8). previous electron microscopic studies on dog synovium in other diseases include those of huxtable and davis (48) who studied of young greyhounds with erosive inflammatory polyanhritis and negative tests for rheumatoid factor. electron microscopic findtures of systemic lupus, as well as a destructive arthritis, demonstrated surface fibrin, increased villi, clumps of infiltrating lymphocytes, plasma cells, a n d perivascular neu trophils. this model, which we consider t o very closely mimic h u m a n ra, nevertheless does have some differences as documented above. we believe that rheumatoid-like dogs are potentially useful for studies o f therapy. w e a r e studying pathogenetic mechanisms in hopes that they will provide clues t o mechanisms in human disease. rheumatoid arthritis in dogs non-infectious canine arthritis rheumatoid arthritis in a dog effectiveness of gold therapy in canine rheumatoid arthritis pets and rheumatoid arthritis: an epidemiologic survey unpublished studies familial lupus erythematosus: antibodies to dna in household dogs revision of diagnostic criteria for rheumatoid arthritis a formaldehyde glutaraldehyde fixative of high osmolality for use in electron microscopy (abstract) lytic activities in protein absorption droplets mononuclear cells in human synovial fluid: identification of lymphoblasts in rheumatoid arthritis fine structure and cytochemistry of the rheumatoid synovial membrane with special reference to lysosomes a morphologic study on the occurrence and distribution of structures resembling viral nucleocapsids in collagen diseases tubular paramyxovirus-like structures in synovial vascular endothelium tubular inclusions of systemic lupus erythematosus urate crystal induced inflammation in dog joints: sequence of synovial changes rheumatoid arthritis in a dog synovial membrane and fluid morphologic alterations in early rheumatoid arthritis: microvascular injury and virus-like particles iron in the synovial membrane in rheumatoid arthritis and other joint disease electron microscopic observations of immunoreactive cells in the rheumatoid synovial membrane early joint lesions of rheumatoid arthritis lymphocyte tubular structures in rheumatoid arthritis et a 1 ultrastructural features of canine distemper virus infection in a dog kidney cell line comparison of canine distemper viral strains: an electron microscopic study the occurrence and frequency of type c virus-like particles in placentas from patients with systemic lupus erythematosus and from normal subjects new cytoplasmic components in vascular endothelia rayns dg: a new inclusion body in rheumatoid synovia endothelial inclusions in dermatomyositis dermatomyositis: electron microscopic, immunologic and tissue culture studies of paramyxovirus-like inclusions infectious mononucleosis: detection of herpes-like virus and reticular aggregates of small cytoplasmic particles in continuous lymphoid cell lines derived from peripheral blood growth in vitro of herpes simplex in human lymphoma cell lines tubular aggregates in endoplasmic reticulum in herpes simplex encephalitis electron microscopic study of experimental enteric infection in neonatal dogs with a canine coronavirus i97 1 endoplasmic reticulum in human tumor cells ultramorphological and ultracytochemical studies on tubuloreticular structures in lymphoid cells glomerular microtubules of systemic lupus erythematosus ultrastructure of meningeal tumors induced in dogs with rous sarcoma virus electron microscopy of monkey kidney cell cultures infected with rubella virus electron microscopic observations of crystalline arrays of virus-like particles in tissues of mink with aleutian disease brief communication: spontaneous myxosarcoma in a cottontail rabbit (sulilogusfloridunus): observation of tubular structures in the endoplasmic reticulum of tumor cells poliovirus crystals within the endoplasmic reticulum of endothelial and mononuclear cells in the monkey spinal cord viral-like particles in co-cultivated rheumatoid synovial cells (abstract) pathologic features of experimentally induced chlamydia1 infection in dogs non-infectious canine arthritis: the inflammatory, nonerosive arthritides production of synovitis and marginal articular exostoses in knee joints of dogs the pathology of polyarthritis in young greyhounds synovial fluid changes in joint disease. cambridge canine systemic lupus erythematosus: presenting with symmetrical polyarthritis canine systemic lupus erythematosus the authors gratefully acknowledge the technical assistance of mrs. susan rothfuss, ms gilda clayburne, and ms marie sieck, and the secretarial assistance of ms mary ellen maguire. mrs. eileen donaldson, and ms joanne loguidice. key: cord-0002549-8xndud0h authors: lin, bo; liu, kun; wang, wenting; li, wei; dong, xu; chen, yi; lu, yan; guo, junli; zhu, mingyue; li, mengsen title: expression and bioactivity of human α-fetoprotein in a bac-to-bac system date: 2017-01-17 journal: biosci rep doi: 10.1042/bsr20160161 sha: cc43e3bf906ec492da56895a85e1a85d87bf59b2 doc_id: 2549 cord_uid: 8xndud0h α-fetoprotein (afp) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. a growing number of investigations of afp as a tumour-specific biomarker have concluded that afp is an important target for cancer treatment. afp also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. in an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human afp in a bac-to-bac system. two key factors affecting the expression of recombinant human afp (r-afp), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. we achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72–96 h incubation period after infection and an inoculum volume ratio of 1:100. we also assessed the role of r-afp in the proliferation of the human liver cancer cell line bel 7402, and the results indicated that r-afp promoted the growth of hepatoma cells. we concluded that this method can produce high yields of r-afp, which can be used for studies related to afp. high levels of serum α-fetoprotein (afp) are associated with embryonic development and cancer growth [1] [2] [3] . the afp gene is a member of the family of albuminoid genes, including serum albumin (sa), vitamin d-binding protein (vtdb) and α-albumin (afamin). the albuminoid genes evolved from a common ancestor and exhibit considerable similarity in their primary structure. for example, human afp and human serum albumin (hsa) share 40% identity with highly conserved cysteine residues. human afp consists of 609 amino acids, has a molecular mass of 69 kda and contains only one glycosylation site (n233). however, the glycosylation site may link various carbohydrate moieties, and the structure of the carbohydrate moieties varies in different tissues and diseases [3] . studies have found that afp can regulate hepatocellular growth, differentiation, regeneration and transformation in oncogenic growth processes [4] [5] [6] [7] [8] . afp is also an immunomodulatory molecule, as transfer of foetal afp through the placenta into the mother's circulation is correlated with remission of rheumatoid arthritis, multiple sclerosis and other autoimmune disorders [9] . recombinant expression of human afp is under development as a biopharmaceutical for the treatment of autoimmune diseases [9] , and human afp is also being used as a bioactivated molecule in drug discovery studies for cancer treatment [4] [5] [6] [7] [8] . recombinant expression of human afp has been described in escherichia coli expression systems [10, 11] , in yeast [12] and in the milk of transgenic goats [13] , but human afp expression is unsatisfying in these systems. for example, human afp production in e. coli expression systems yields inclusion bodies, and refolding of this material is not practical for commercial production [13] . in addition, human afp produced in the milk of transgenic goats is not easily purified. the present study is the first to report a high yield of recombinant human afp (r-afp) in a bac-to-bac baculovirus expression system. we also detected the bioactivity of r-afp in the human liver cancer cell line bel 7402 and found that r-afp promotes hepatoma cell growth. the present study established a reliable, convenient method for expressing and producing a high yield of r-afp, which could be used for drug screening and for structural and functional studies. human afp (ncbi: nm 001134) with a c-terminal 6× his-tag was cloned into the pfastbac 1 vector (invitrogen inc, u.s.a.). after the fusion sequences and the reading frames were confirmed by sequencing, this pfastbac 1 vector construct was transformed into bacterial dh10 cells, and the extracted bacmid was then transfected into sf9 cells using cellfectin ii reagent (invitrogen inc, u.s.a.) to obtain passage 1 baculoviruses (p1 baculoviruses) [14] . r-afp was expressed using the bac-to-bac baculovirus expression system (invitrogen inc, u.s.a.). the process was as follows: (1) sf9 insect cells were cultured in insect-xpress protein-free medium (lonza group ltd., switzerland) without serum at a density of 2 × 10 6 cells/ml. (2) the p1 baculoviruses were harvested after the transfected sf9 cells were incubated at 27 • c for 7 days. (3) one hundred microlitres of p1 baculovirus were added to 8 ml of sf9 cells and harvested at 72 h after infection. the baculoviruses were amplified for two rounds to obtain p3 baculoviruses. (4) ten millilitres of p3 baculoviruses was added to 1 litre of sf9 cells, and secreted r-afp was harvested in the medium at 72 h after infection, as described previously [14] [15] [16] [17] [18] . the insect medium (approximately 2 ml) containing secreted r-afp was collected and centrifuged at 6000 rpm for 15 min. the supernatant was added to 200 μl of 10× hbs buffer (10 mm hepes [ph 7.2] and 150 mm nacl) and 80 μl of nickel (ni)-charged resin (ge healthcare company, u.s.a.). after the sample was mixed and shaken for 2 h, the 6× his-tag r-afp in the supernatant was captured by ni-charged resin and was eluted with 100 μl of 300 mm imidazole in hbs buffer. the eluted r-afp was analysed by sds/page. the reduced protein sds/page sample contained a reducing buffer, such as dtt, and was boiled for 3 min. the non-reduced protein sample did not contain a reducing buffer and was not heated [18] . cytoplasmic r-afp was analysed as follows: after 72 h incubation, the media were harvested and centrifuged at 3000 rpm for 15 min. cell pellets were resuspended in 40 ml of hbs buffer, sonicated on ice for 15 min with 3 s/9 s intervals and then centrifuged at 13000 rpm for 60 min. the supernatant was collected, and supernatant containing r-afp protein was further purified and analysed to determine the expression level of secreted r-afp as described recently [18] . after 72 h incubation, the media (approximately 1 litre) containing the secreted r-afp were harvested and centrifuged at 4000 rpm for 10 min, the supernatant was collected and filtered with a 0.45 μm filter membrane (millipore corp., u.s.a). the supernatant was then concentrated to 100 ml by cross-flow filtration (millipore corp., u.s.a.), and the buffer was changed to hbs buffer. the concentrate was centrifuged at 10000 rpm for 30 min, and the supernatant was collected and passed through ni-charged resin (ge healthcare). r-afp was captured by the resin and eluted with 300 mm imidazole in hbs buffer. the eluted buffer was concentrated to 1 ml using a 10 kda filter tube (millipore corp., u.s.a.) and then further purified by gel filtration chromatography using a superdex 200 column (ge healthcare company, u.s.a.). the detection wavelength was 280 nm. elution was performed with an hbs buffer at a flow rate of 1 ml/min [18] . human cord blood afp was precipitated by ammonium sulfate and passed through an anti-afp affinity chromatography column. afp-positive fractions were collected and concentrated. the purity of prepared afp was 92.7% as determined by sds/page. the protein was stored at −80 • c until use [19] . the target protein was electrophoretically separated by sds/page. electrotransfer: proteins were transferred from the gel to a pvdf membrane in a constant voltage of 60 v for 2 h at 4 • c. immunodetection: blocking buffer (3%) was added to the pvdf membrane, and the membrane was rocked gently for 2 h. then, the membrane was rinsed with tbst buffer three times. the appropriate concentration of anti-afp antibody was added, and the membrane was rocked gently for 12 h at 4 • c. the appropriate concentration of horseradish peroxidase-conjugated anti-rabbit antibody was added, and the membrane was rocked gently for 1 h at 37 • c. the membrane was then washed with tbst buffer, developing reagent was added, and development was monitored. the antibody against afp and the horseradish peroxidase-conjugated anti-rabbit antibody were purchased from sangon biotech co., ltd. (shanghai) and jackson immunores lab, inc., (u.s.a.) respectively [5, 7, 8] . the optimization experiments were designed by comparing the effects of two major factors (the infectious baculovirus inoculum volume and the post-infection time) on the production of r-afp. these experiments were performed by infecting 1-litre cell cultures with baculovirus and incubating the cells at a constant temperature of 27 • c under shaking conditions (110 rpm) [14] [15] [16] [17] [18] . we harvested the medium after infection at each monitoring time, and the medium was replaced with hbs buffer. the proteins were captured by ni-charged resin (ge healthcare company, u.s.a.) and eluted with 300 mm imidazole in hbs buffer. the eluted 6× his-tagged proteins were concentrated and purified by gel filtration chromatography using a superdex 200 column. finally, the proteins were analysed with a nanodrop 2000 spectrophotometer (at 280 nm) and sds/page as described recently [14] . the monoclonal antibody against human afp (anti-afp) was prepared according to standard procedures [20] . briefly, balb/c mice were immunized with purified human afp (sigma-aldrich) in complete freund's adjuvant at 2-to 3-week intervals. spleen cells were removed from the immunized mice. myeloma cells in the exponential phase were mixed with the spleen cells in a certain proportion and fused by 50% peg3000 buffer. both cell types were cultured in hat (h-hypoxanthine, a-aminopterin, t-thymidine) medium supplemented with feeder cells to form a hybridoma cell line. the hybridoma cells that secreted antibody were screened by elisa and continually cloned by the limited dilution method; thus, a stable anti-afp antibody producing hybridoma cell line was obtained. balb/c mice were inoculated with positive clones. anti-afp antibody was harvested from ascites fluid and purified by affinity chromatography. the specificity of the monoclonal antibody (with a titre higher than 5000 for afp) was ascertained by elisa and western blot assay to prevent interference from human albumin, which has a structure similar to that of human afp. the results of the elisa and western blot assay showed the specific binding of the monoclonal antibody to human afp and a lack of reaction to human albumin [19] . expression of the human afp receptor (afpr) in the bel 7402 cell line was examined using a laser confocal microscope as described previously [8] . briefly, bel 7402 cells were incubated with a mouse anti-human afpr antibody (abcam biotech company, cambridge, u.k.) for 12 h, followed by incubation with secondary goat anti-mouse antibodies conjugated to fitc (zhongshan boil tech co., beijing) for 2 h. then, 10 μl dapi (1 mg/ml) was added to the mixture. localization and expression of afpr in cells were observed and captured by laser confocal microscopy (leica tcs-nt sp2, germany). bel 7402 cells were suspended in rpmi-1640 medium containing 10% fbs and were added to a 24-well plate at 1 ml per well followed by incubation at 37 • c in a humidified atmosphere of 5% co 2 for 48 h. the supernatant was removed and replaced with 150 μl of fresh medium without fbs for another 24 h. different concentrations of extracted human afp (e-afp), r-afp (0-80 mg/l), hsa or anti-afp were added to each well for 24 h and then pulsed with 1 mci of 3 h-tdr. the cells were harvested on to a glass microfibre filter 4 h later using a multiple sample harvester. the incorporation of 3 h-tdr was measured using an lkb 1209 rackbeta liquid scintillation counter. we used e-afp as a positive control. to determine whether the influence of human afp on proliferation was specific, the blockage of anti-afp and hsa as structural analogues was also assessed, and a control group without afp or hsa was also paralleled to be done, according to the previously described procedure [19] . a total of 1.5 × 10 4 bel 7402 cells per well were plated in 96-well plates and cultured in rpmi-1640 medium supplemented with 10% fbs at 37 • c in a humidified atmosphere of 5% co 2 for 48 h. the cultured cell medium was replaced with medium without fbs for another 24 h, and the cells were treated with human afp (20 mg/l) for 48 h. the effects of e-afp and r-afp on cell growth were measured by mtt assay as described in a previous study [19] . the growth ratio = (control group a 490 − treated group a 490 )/control group a 490 × 100%. the expression vectors were confirmed by agarose gel electrophoresis. figure 1 (lane 2) shows that the pfastbac 1-afp vector could be digested with bamhi and hindiii restriction enzymes to release a band measuring approximately 2000 bp, confirming the presence of the recombinant human afp gene. dna sequencing of recombinant pfastbac 1-afp confirmed the existence of the coding sequence in the correct frame of the vector, without any mutation or alteration in the afp gene sequences. figure 1 (lane 4) shows that the recombinant bacmid contained the pfastbac 1-afp vector and confirmed that the correct homologous recombination occurred in the bacmid. secreted r-afp (in the insect medium) and cytoplasmic r-afp (in the lysed insect cells) were assessed by sds/page, and the results are shown in figure 2 (a). the non-reduced or native secreted r-afp band was tilted at approximately 67 kda, which indicated that the protein was in a non-linear form (lane 1). the reduced protein band (lane 2) was at approximately 69 kda, consistent with the size of the afp gene. the molecular mass of the cytoplasmic r-afp bands (lanes 3 and 4) was similar to that of the secreted r-afp bands, but the bands were faint and impure. these results indicated that the cytoplasmic r-afp concentration was lower than the extracellular r-afp concentration in the medium and that cytoplasmic r-afp may bind with other proteins. the r-afp protein was further evaluated by western blotting ( figure 2b ). the medium containing expressed r-afp was amplified and used for large-scale expression of the recombinant protein. purification of the secreted r-afp in the medium is straightforward. first, the medium was concentrated by buffer exchange using hbs buffer. in the second step, r-afp in the medium was captured on ni-charged resin, eluted with 300 mm imidazole and purified by gel filtration chromatography. the eluted chromatography position of r-afp is shown in figure 3 (a) at a elution volume 15.0 ml. the position indicated a molecular mass of 65-70 kda according to the superdex 200 column (ge healthcare) [13] . r-afp fractions from gel filtration chromatography were collected for further sds/page analysis. the highest peak showed a high concentration of r-afp ( figure 3b, lane c) , indicating that the purification process was successful. the purified r-afp is soluble and stable, we test its isoelectric point (ip) is ph 4.5. in the process of analysing r-afp expression, it was found that different conditions led to different expression levels. figure 4(a) shows the r-afp expression yield in samples at different times. after 72 h, the expression yield increased slowly. to optimize the yield of r-afp protein, the parameters were evaluated systematically under the same virus titres (2 × 10 7 pfu/ml) and the same sf9 cell density (2 × 10 6 cells/ml). the infectious baculovirus inoculum volumes (1:50, 1:100 and 1:500) and r-afp production were monitored at different post-infection times (48, 72 and 96 h). r-afp protein expression reached the highest level (approximately 1.5 mg/l of medium) at a 1:100 volume of the infectious baculovirus inocula when harvested at 96 h post-infection, the expression of r-afp in 1:100 volume of the infectious baculovirus inocula has statistical difference compared with 1:500 volume groups (p<0.05) ( figure 4b , red line). to detect the bioactivity of r-afp, in the present study, we selected the human liver cell line bel 7402 to examine the effects of r-afp on cellular proliferation. first, we observed the expression of afpr, and the results revealed that bel 7402 expressed afpr and that the receptor was located in the cell membrane ( figure 5a ). second, bel 7402 cells were treated with r-afp or e-afp (20 mg/l) for 24 h, and the 3 h-tdr incorporation results indicated that after treatment with r-afp or e-afp, the content of 3 h-tdr in bel 7402 cells was markedly enhanced ( figure 5b , groups 2 and 3 respectively). we also selected r-afp or e-afp (20 mg/l) co-treated with a human afp antibody (anti-afp). the results showed that anti-afp (40 mg/l) antagonized the effects of r-afp or e-afp ( figure 5c , groups 4 and 5 respectively). when we changed the concentration of r-afp or e-afp (10-80 mg/l), the results indicated that the 3 h-tdr content in the bel 7402 cells was markedly enhanced in a dose-dependent manner. the peak of the increase reached 123.0% compared with the control group ( figure 5c , the red and blue lines). anti-afp (40 mg/l) antagonized the effects of r-afp and e-afp, but as the concentration of r-afp or e-afp increased, the antagonizing effect decreased ( figure 5c , green and purple lines). further, we used the mtt method to analyse the effects of r-afp or e-afp (20 mg/l) on the proliferation of bel 7402 cells, and the results also demonstrated that r-afp or e-afp stimulated the proliferation of bel 7402 cells ( figure 5d ). in addition, anti-afp reversed the effects of r-afp or e-afp ( figure 5e ). these results demonstrated that the effects of r-afp on cellular proliferation are similar to those of e-afp. hsa did not show an obvious influence on the proliferation of bel 7402 cells. r-afp expressed in the bac-to-bac system is soluble and stable as a monomer, and its bioactivity is similar to human afp extracted from blood. its ip is ph 4.5. to extract human afp, a great deal of human cord blood is needed, which is difficult to purify. expressing r-afp in the bac-to-bac system allows a large amount of purified bioactive protein to be obtained without the need for human cord blood. r-afp may allow the study of the biochemical features of human afp and drug screening. human afp expression was previously reported in e. coli expression systems, in yeast and in the milk of transgenic goats, but these expression systems produced low amounts of human afp. for example, human afp expressed in e. coli was deposited in bacterial inclusion bodies and subjected to harsh denaturants [10, 11, 21] . human afp expressed in yeast was indistinguishable immunologically from authentic human afp, and the human afp produced in yeast contains seven extra amino acid residues at the n-terminus that are not present in mature human afp [12] . the purification of human afp expressed in the milk of transgenic goats was difficult and time consuming [13] . there is no such problem when human afp produced in the bac-to-bac system. the bac-to-bac system had achieved successful protein expression levels for potential drug applications [16] . the r-afp produced in the bac-to-bac system is highly expressed and easily purified, and its bioactivity is similar to that of the native human afp. this finding will aid clinical research on the immunomodulatory function of this protein. to optimize the yield of the r-afp protein, the post-infection time was considered. recombinant protein was expressed intracellularly and needed time to be modified and transported to the extracellular space. thus, the best harvest time depends on the protein and the experiment [14] . in the optimization of r-afp expression, we found that many cells died and were lysed after 96 h. therefore, the best harvest time was approximately 72-96 h. the effects of extracellular human afp on cellular proliferation are mainly achieved through signal transduction mediated by afpr. previously, we found that bel 7402 cells express afpr [8] . in the present study, we found that afpr was highly expressed in the membrane of bel 7402 cells. this result provided further evidence that the effects of extracellular human afp on the proliferation of bel 7402 cells were mediated by membrane-bound afpr. previously, we found that human afp bound to afpr might activate camp and ca 2+ to stimulate the transduction of cellular signals to stimulate n-ras and c-myc expression, leading to promote proliferation of nih 3t3 cells and bel 7402 cells [19, 22] . in the present study, r-afp and e-afp played similar roles in the proliferation of bel 7402 cells, suggesting that r-afp harbours biological characteristics identical with those of e-afp. human afp is a protein that is specifically expressed in human hepatocellular carcinoma (hcc) cells. we previously found that cytoplasmic human afp could interact with signal molecules, such as caspase-3 [7] , retinoic acid receptor-β (rar-β) [23] and phosphatase and tensin homologue (pten) [5] . these interactions inhibited the transduction of apoptotic signals and activated growth signals, which led to the promotion of proliferation or drug resistance in liver cancer cells. these results suggested that human afp may interact with these signalling molecules. in the present study, we successfully expressed and purified human afp protein. we also found that the bioactivity of human afp protein is similar to that of cytoplasmic human afp. recombinantly expressed human afp is soluble, is stable and can be used to identify signalling molecules or potential drug interactions by surface plasmon resonance (spr) [24, 25] . in conclusion, the present study demonstrated the successful expression and purification of r-afp, which paves the way for future structural and functional studies of human afp. review of the third domain receptor binding fragment of alpha-fetoprotein (afp): plausible binding of afp to lysophospholipid receptor targets the alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor targets alpha-fetoprotein: a renaissance molecular analysis of afp and hsa interactions with pten protein alpha-fetoprotein: a new member of intracellular signal molecules in regulation of the pi3k/akt signaling in human hepatoma cell lines nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. an update and commentary alpha fetoprotein is a novel protein-binding partner for caspase-3 and blocks the apoptotic signaling pathway in human hepatoma cells alpha-fetoprotein receptor as an early indicator of hbx-driven hepatocarcinogenesis and its applications in tracing cancer cell metastasis mm-093, a recombinant human alpha-fetoprotein for the potential treatment of rheumatoid arthritis and other autoimmune diseases purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in escherichia coli the refolding of different alpha-fetoprotein variants expression of human alpha-fetoprotein in yeast purification and characterization of a recombinant version of human alpha-fetoprotein expressed in the milk of transgenic goats expression of functional recombinant human tissue transglutaminase (tg2) using the bac-to-bac baculovirus expression system expression of winged bean basic agglutinin in spodoptera frugiperda insect cell expression system engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells expression, purification and characterization of peanut (arachis hypogaea) agglutinin (pna) from baculovirus infected insect cells efficient expression of acetylcholine-binding protein from aplysia californica in bac-to-bac system the intracellular mechanism of alpha-fetoprotein promoting the proliferation of nih 3t3 cells human alfa-fetoprotein: isolation and production of monoclonal antibodies a simplified bioprocess for human alpha-fetoprotein production from inclusion bodies the promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma bel7402 cell line cytoplasmic alpha-fetoprotein functions as a co-repressor in ra-rar signaling to promote the growth of human hepatoma bel 7402 cells structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases this is an open access the authors declare that there are no competing interests associated with the manuscript. ml and bl conceived and designed the experiments. bl, kl, ww, wl, xd, yc, yl and mz performed the expriments. ml, bl, kl, ww and jg analyzed the data. kl, ww and bl contributed reagents materials analysis tools. ml, mz and bl wrote the manuscript. all the authors read and approved the final manuscript. key: cord-0017076-eenxr5dj authors: solipuram, vinod; mohan, akhila; patel, roshniben; ni, ruoning title: effect of janus kinase inhibitors and methotrexate combination on malignancy in patients with rheumatoid arthritis: a systematic review and meta-analysis of randomized controlled trials date: 2021-04-28 journal: auto immun highlights doi: 10.1186/s13317-021-00153-5 sha: 844deee0448e20c4d4905a73fccf9411a562c4ec doc_id: 17076 cord_uid: eenxr5dj background: rheumatoid arthritis (ra) is a systemic autoimmune disease. the combination therapy of methotrexate (mtx) and janus kinase inhibitor (jaki) is commonly used. patients with ra are at increased risk of malignancy, however, it remains unclear whether the combination therapy is associated with a higher risk. objective: to assess the malignancy risk among patients with ra receiving combination therapy of jaki and mtx compared to mtx alone. methods: pubmed, cochrane and embase were thoroughly searched for randomized controlled trials (rcts) in patients with ra receiving jaki and mtx, from inception to july 2020. primary endpoints were malignancy events, non melanomatous skin cancer (nmsc) and malignancy excluding nmsc and secondary endpoints were serious adverse events (sae), deaths. risk ratio (rr) and 95% ci were calculated using the mantel–haenszel random-effect method. results: 659 publications were screened and 13 rcts with a total of 6911 patients were included in the analysis. there was no statistically significant difference in malignancy [rr = 1.42; 95% ci (0.59, 3.41)], neither nmsc [rr = 1.44 (0.36, 5.76)] nor malignancies excluding nmsc [rr = 1.12 (0.40, 3.13)]. no statistically significant difference between the two groups for sae [rr = 1.15 (0.90, 1.47)] and deaths [rr = 1.99 (0.75, 5.27)] was found. conclusion: the adjunction of jaki to mtx is not associated with an increased risk of malignancy when compared to mtx alone. there is no increased risk of sae and deaths when compared to mtx alone in patients with ra. supplementary information: the online version contains supplementary material available at 10.1186/s13317-021-00153-5. rheumatoid arthritis (ra) is a systemic autoimmune disease, estimated to affect approximately 0.5% to 1% of population [1] . patients with ra are predisposed to an increased risk for malignancy, especially malignant lymphomas [2] [3] [4] [5] [6] [7] , lung cancers [5, 6] and non-melanoma skin cancer [7] . higher mortality was associated with the presence of cancer, varied by stage of malignancy [8] . persistent inflammation triggers the development and progression of cancer [9, 10] . it has been well-established that severity of inflammation in ra is positively correlated with the risk of lymphoma [11] . taking the inflammation into control may reduce the risk of developing malignancy. janus kinase (jak) can initiate lymphocyte activation, function and proliferation via tyrosine phosphorylation of the receptors and downstream signal transducer and activator of transcription (stat) signaling [12] . jak-stat signaling pathways mediate a double-edged sword effect on both antitumor defense and tumor progression [13] . inhibiting jak-stat pathways raises the concern of losing immune cell function to malignancy, as well as proposes the possibility of suppressing tumor formation. jak can be divided into 4 types: jak1, jak2, jak3 and tyrosine kinase 2 (tyk2), responsive to myriad cytokines. various janus kinase inhibitors (jaki) are being widely investigated in randomized controlled trials (rcts) and proved efficacy in patients with ra [14] . jaki consist of tofacitinib, selectively inhibiting jak1 and jak3, baricitinib, blocking jak1 and jak2, peficitinib, acting on all types of jak, decernotinib, highly selective for jak3, upadacitinib and filgotinib, selectively targeting jak1. limited evidence demonstrated no statistical difference in malignancy incidences in patients receiving tofacitinib compared to the general population [15] . since most patients with ra are treated with combinations of traditional disease-modifying antirheumatic drugs (dmards), especially methotrexate, it is important to explore the safety profile of these therapies. the role of different jaki in the risk of malignancy remains undetermined. even scarce data exists, to see the malignancy outcomes of jaki when used in combination with methotrexate. in consideration of the current unclear malignancy risk of jaki and mtx combination, we sought to explore a potential association between jaki and mtx combination and malignancies in patients with ra. a systematic search was performed in pubmed, embase and cochrane library without language limitations from inception to july 30, 2020. search terms included "rheumatoid arthritis", "tofacitinib", "baricitinib", "upadacitinib", "filgotinib", "peficitinib", "decernotinib", "jak inhibitors", "methotrexate". references of the retrieved articles were searched to identify further relevant studies suitable for this meta-analysis. search strategy is listed in table 1 . two independent authors (vs and am) screened all titles and abstracts for potential inclusion. any discrepancy among the selected studies were resolved by a third author (rn). double-blind rcts that reported malignancy events in adult patients with ra receiving the combination of methotrexate with any jaki, with methotrexate in the control arm. studies that did not report malignancy outcomes were excluded. exclusion criteria included reviews, editorials, letters, observational studies, non human studies. long term extension studies with single arms and trials involving other biologic dmards were excluded as well. two independent investigators (vs and am) performed the search and relevant studies were selected based on the inclusion and exclusion criteria. vs and am extracted the data using a predefined data abstraction form: age, sex, duration of rheumatoid arthritis, mean number of tender and swollen joints, length of follow-up. patient-years were calculated based on the extracted data. any discordance between these two authors was resolved by the third author (rn). the cochrane quality assessment tool for rcts was used to assess risk of bias [16] . the primary endpoints of interest were the incidence of malignancy, non melanomatous skin cancers (nmsc) and malignancies excluding nmsc. the secondary endpoints were incidence of serious adverse events (sae) and deaths. extracted data were combined using review manager (revman) software (cochrane collaboration) version 5.4. risk ratio (rr) of malignancies, nmsc, malignancies excluding nmsc, sae, death was calculated with 95% confidence intervals (cis) based on mantel-haenszel random-effect method. i 2 was used to evaluate heterogeneity among the studies (< 25% considered low heterogeneity and > 50% considered significant heterogeneity). publication bias was assessed via the funnel plot for the primary endpoint. sensitivity analysis was performed by leave-one-out method. results were reported according to the preferred reporting items for systematic reviews and meta-analyses protocol (prisma-p) 2015 statement [17] . the protocol for this systematic review and meta-analysis was registered with prospero (crd42020201473). a total of thirteen rcts comprising 6911 patients met the inclusion criteria [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] , as summarised in fig. 1 , with 2377 patients in the mtx group and 4534 patients in the combination of mtx and jaki group. total patient-years in the mtx group and jaki and mtx combination group were 988 and 3684, respectively. among all the trials included, 3 trials used tofacitinib, 3 trials for baricitinib, 3 trials for upadacitinib, 2 trials for peficitinib and 1 trial for filgotinib in combination with mtx (additional files 1, 2, 3, 4, 5, and 6). the baseline characteristics of the patients with ra included in the studies were comparable among the two groups, as summarised in table 2 . the length of followup of the trials ranged from 12 weeks to 24 months. the mean age for the jaki and mtx combination group was 53 ± 11.7 years and 53 ± 11.8 years in the mtx alone group. the average duration of ra was 7.6 years in the mtx alone group and 8.2 years in the jaki and mtx combination group. the crude incidence rates of malignancies (nmsc/non nmsc) in jaki and mtx combination group and mtx group were, respectively, 1.086 (0.497/0.651) and 0.709 (0.202/0.409) per 100 patient years. a total of forty patients suffering from malignancies were reported in the jaki and mtx group among 3684 patient-years and seven malignancy events in the mtx group among 988 patient-years. there was no statistically significant difference in malignancy events (rr = 1.42; 95% ci 0.59 to 3.41, p = 0.44) between the combination group and the control group, seen in fig. 2 . we found a relatively low level of heterogeneity across all included rcts (χ 2 = 5.35, df = 7, p = 0.62, i 2 = 0%). for the mantel-haenszel random methods, funnel plot showed no evidence of publication bias in all comparisons in fig. 3 . considered separately, statistical differences remained undetectable for non melanomatous skin cancers (nmsc) (rr = 1.44; 95% ci 0.36 to 5.76, p = 0.61) (fig. 4 ) and malignancies excluding nmsc (rr = 1.12; 95% ci 0.40 to 3.13, p = 0.82) (fig. 5 ). among 40 malignancy events in the jaki and mtx combination group, 15 (37.5%) were nmsc and 24 (60%) were malignancies excluding nmsc. among 7 malignancy events reported in mtx along group, 2 (28.5%) were nmsc and 4 (57%) were malignancies excluding nmsc. one malignancy in each group was not reported in detail. among solid tumors for the mtx and jaki combination group, the most common types were cervical cancer in 6 patients (25%), lung cancer in 5 patients (21%), breast cancer in 4 patients (17%) and ovarian cancer in 1 patient. two cases of non-hodgkin's lymphoma and two cases of melanoma were reported. although more serious adverse events and deaths were reported in the combination therapy group, there was no statistically significant difference between the two groups with rr = 1.15 (95% ci 0.90 to 1.47, p = 0.26) in sae (fig. 6) and rr = 1.99 (95% ci 0.75 to 5.27, p = 0.17) in deaths (fig. 7) . the sensitivity analysis was performed for malignancy outcomes with leave one out method and did not show evidence of bias, as summarised in table 3 . all rcts (100%) adequately reported the generation of random sequence and 7 rcts (53.8%) had adequate descriptions of concealed allocation. blinding of participants, personnel, and outcome assessor was performed in all rcts. only 1 rcts was unable to report the complete outcome. no selective reporting was discovered, seen in fig. 8 . patients with ra are predisposed to a higher risk for malignancy [2] [3] [4] [5] [6] [7] . it remains not entirely clear if the increased risk of malignancy is primarily related to the pathogenesis of the disease or due to the immunosuppressive therapy. currently, the combination of jaki and mtx is widely used in patients with ra. however, whether biologic therapy increases the risk of malignancy has not been well addressed. according to our results, combination therapy of jaki and mtx did not show any statistically significant increase in the number of malignancies as compared to mtx alone, neither in nmsc nor in malignancies excluding nmsc. moreover, there are no statistical significant differences that were discovered in the incidence of sae and mortality among these two groups. the majority of sae are associated with infections including herpes zoster virus (hzv), urinary tract infection (uti) and upper respiratory tract infections (urti). to be noticed, the exposure time of patients with ra to jaki and mtx is positively correlated to sae as evidenced by the trials with longer follow up [19, 25, 26] . among all the trials included, the mortality incidences were similar between patients under jaki and mtx combination therapy and mtx monotherapy except oral scan trial [25] , in which the combination group had a higher proportion of deaths compared to mtx alone. the deaths were predominantly contributed to multiorgan dysfunction, acute respiratory distress syndrome (ards) and major cardiovascular events (maces). to our knowledge, this is the first systematic review exclusively to explore the safety profile, especially from the malignancy perspective, of jaki and mtx combination therapy. according to observational studies, patients with ra have an increased risk of malignancy [3, 4, 31] . this is well studied in non-hodgkin's lymphoma (nhl), with diffuse large b-cell lymphoma (dlbcl) as the most common type of nhl among patients with ra. these patients are also at increased risk of non-hematologic malignancy such as lungs, kidney, nasopharyngeal carcinoma [32] . the pathophysiology is associated with organ damage from chronic inflammation, genetic mutations, and autoimmune b lymphocyte activation [3, 32] . viral infection, such as epstein-barr virus (ebv) infection may also play a role in the development of b cell lymphoma [33] . it was estimated that patients with ra have approximately 12-fold risk of developing lymphoma and twofold for lung cancer [34] . the risk of developing malignancy is closely related to disease activity [11] . overtime, the stimulated immune cells from chronic inflammation may undergo malignant transformation, eventually leading to lymphoma. interestingly, the chronic use of non-steroid anti-inflammatory drugs (nsaids) in these patients was associated with a decreased risk of developing colorectal cancer [34] . in terms of treatment, safety profiles, especially the effects on malignancy, should be carefully addressed. previous studies have shown association between specific therapies and their corresponding risk of malignancy. the role of mtx on the incidence of malignancies remains controversial. several population based studies did not show any increased risk of malignancies with mtx use compared to baseline risk among patients with ra [35, 36] . mtx may even decrease the risk of lymphoma by suppressing immunologic activation in ra activity but at the same time can increase the risk of other lymphomas [37] . the effects of jaki on the risk of malignancies still remain unclear. although genetic variation of jak-stat pathway is a known risk factor for malignancy [38] , it is unclear whether the use of jaki would lead to a decreased incidence of malignancies. us corrona ra registry, a 5-year prospective observational study, did not detect any statistically significant difference in developing malignancy between tofacitinib and other biologic dmards [39] . there was no increased risk of malignancy for tofacitinib in patients with ra, when compared to conventional synthetic dmards or tumor necrosis factor-α inhibitors per a meta-analysis of observational studies [40] . with the widespread use of jaki, not only tofacitinib, and mtx among patients with ra, there is accumulating data from multiple rcts and a definite necessity to explore the safety profile of the combination therapy. it is important that the effect of dose and exposure time of jaki and mtx combination on malignancy should be interpreted with caution. among all the trials included in our study, oral scan [25] study using tofacitinib, reported the largest number of malignancies. this trial is also distinguished for the longest follow up period of 24 months. however, a definitive conclusion should not be drawn from this single study and more data from long term extension studies is warranted to establish the association between exposure length and incidence of malignancy. moreover, there is no significant difference in risk of malignancy between high dose and low dose jaki and mtx. the results of our secondary analysis are consistent with previous studies, while still with few differences [41] . a previously published meta-analysis also showed no significant increase in the number of malignancies with jaki treatment but it did not specifically account for the effect of mtx [41] . safety profile of jaki and mtx combination is comparable to mtx alone as reported in previous studies. incidence of sae and deaths in our analysis were similar to prior published studies. it was reported that higher doses of jaki were associated with increased sae, but studies included in our analysis had comparable sae across different dose ranges of jaki without large differences [41] . although not included in our analysis, cardiovascular mortality was also not significantly high in patients receiving jaki when compared to placebo [42] . overall jaki has an acceptable safety profile and combination with mtx did not change it. there are several limitations in our study and the results should be carefully interpreted. first, the patientyear exposure in the control group was much less compared to the combination group. the increased number of malignancies in the combination group, may be partly related to longer inflammation exposure in the setting of ra. secondly, long term extension studies were excluded as they were mostly single arm studies without a control group, which could result in potential bias. last, the majority of malignancies have a latency period and develop over the course of months to years and some trials included in our analysis had relatively limited follow up duration which may underestimate the actual malignancy rate. the strengths of our study is that it included large patient-years to detect any differences among the groups. there was no heterogeneity among the studies included in our analysis and the results of sensitivity analyses are consistent with overall results, proving the robustness of the study. our meta-analysis showed combination therapy of jaki and mtx did not increase the malignancies in rheumatoid arthritis patients when compared to mtx alone. sae and deaths are also not significantly different among the two groups. these results have been consistent among all the studies included in the analysis suggesting overall acceptable safety profile of jaki and mtx combination. prevalence of rheumatoid arthritis in the united states adult population in healthcare claims databases risk of malignant lymphomas in patients with rheumatoid arthritis and in their first-degree relatives the risk of cancer in patients with rheumatoid arthritis: a nationwide cohort study in taiwan cancer risk in hospitalized rheumatoid arthritis patients risk of cancer among rheumatoid arthritis patients in california incidence and patterns of malignancies in a multi-ethnic cohort of rheumatoid arthritis patients haematopoietic malignancies in rheumatoid arthritis: lymphoma risk and characteristics after exposure to tumour necrosis factor antagonists what is the impact of chronic systemic inflammation such as rheumatoid arthritis on mortality following cancer? nf-kappab: linking inflammation and immunity to cancer development and progression inflammation, a key event in cancer development association of chronic inflammation, not its treatment, with increased lymphoma risk in rheumatoid arthritis jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins jak-stat signaling: a doubleedged sword of immune regulation and cancer progression efficacy and safety of tofacitinib, baricitinib, and upadacitinib for rheumatoid arthritis: a systematic review and meta-analysis an oral janus kinase inhibitor: analysis of malignancies across the rheumatoid arthritis clinical development programme the cochrane collaboration's tool for assessing risk of bias in randomised trials preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015: elaboration and explanation cp-690,550) in combination with methotrexate in patients with active rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitors: a randomised phase 3 trial baricitinib, methotrexate, or combination in patients with rheumatoid arthritis and no or limited prior disease-modifying antirheumatic drug treatment upadacitinib versus placebo or adalimumab in patients with rheumatoid arthritis and an inadequate response to methotrexate: results of a phase iii, double-blind, randomized controlled trial efficacy and safety of abt-494, a selective jak-1 inhibitor, in a phase iib study in patients with rheumatoid arthritis and an inadequate response to methotrexate peficitinib, a jak inhibitor, in the treatment of moderate-to-severe rheumatoid arthritis in patients with an inadequate response to methotrexate a phase iib study of abt-494, a selective jak-1 inhibitor, in patients with rheumatoid arthritis and an inadequate response to anti-tumor necrosis factor therapy baricitinib in patients with rheumatoid arthritis with inadequate response to methotrexate: results from a phase 3 study tofacitinib in combination with methotrexate in patients with rheumatoid arthritis: clinical efficacy, radiographic, and safety outcomes from a twenty-four-month, phase iii study efficacy and safety of peficitinib (asp015k) in patients with rheumatoid arthritis and an inadequate response to methotrexate: results of a phase iii randomised, double-blind efficacy and safety of baricitinib in japanese patients with active rheumatoid arthritis receiving background methotrexate therapy: a 12-week, double-blind, randomized placebo-controlled study baricitinib versus placebo or adalimumab in rheumatoid arthritis tofacitinib or adalimumab versus placebo in rheumatoid arthritis effect of baseline serum crp levels on clinical efficacy in rheumatoid arthritis patients treated with filgotinib: post-hoc analysis from two phase 2b studies incidence of melanoma and other malignancies among rheumatoid arthritis patients treated with methotrexate risk of lymphoma and solid cancer among patients with rheumatoid arthritis in a primary care setting biologic treatment of rheumatoid arthritis and the risk of malignancy: analyses from a large us observational study incidence of malignancy in adult patients with rheumatoid arthritis: a metaanalysis convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? lymphoma and luekemia in rheumatoid arthritis: are they associated with azathioprine, cyclophosphamide, or methotrexate? lymphoma in rheumatoid arthritis: the effect of methotrexate and anti-tumor necrosis factor therapy in 18,572 patients malignancy incidence, management, and prevention in patients with rheumatoid arthritis janus kinase inhibitors: a therapeutic strategy for cancer and autoimmune diseases comparison of malignancy and mortality rates between tofacitinib and biologic dmards in clinical practice: five-year results from a us-based rheumatoid arthritis registry. acr meeting abstracts risk of malignancy with non-tnfi biologic or tofacitinib therapy in rheumatoid arthritis: a meta-analysis of observational studies malig nanci es-and-serio us-infec tions-in-rando mized-contr olled-trials-of-janus-kinase-inhib itors-in-patie nts-with-rheum atoid-arthr itis-a-syste matic-review-and-meta-analy sis impact of janus kinase inhibitors on risk of cardiovascular events in patients with rheumatoid arthritis: systematic review and meta-analysis of randomised controlled trials publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. the online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13317-021-00153-5.additional file 1: figure s1 . rrs of all malignancy in patients with ra treated with jaki and mtx compared to mtx alone in rcts using the m-h random-effect method, subgroup by jaki and dose.additional file 2: figure s2 . rrs of all malignancy in patients with ra treated with tofacitinib and mtx compared to mtx alone in rcts using the m-h random-effect method.additional file 3: figure s3 . rrs of all malignancy in patients with ra treated with baricitinib and mtx compared to mtx alone in rcts using the m-h random-effect method.additional file 4: figure s4 . rrs of all malignancy in patients with ra treated with updacitinib and mtx compared to mtx alone in rcts using the m-h random-effect method.additional file 5: figure s5 . rrs of all malignancy in patients with ra treated with filgotinib and mtx compared to mtx alone in rcts using the m-h random-effect method.additional file 6: figure s6 . rrs of all malignancy in patients with ra treated with peficitinib and mtx compared to mtx alone in rcts using the m-h random-effect method.authors' contributions vs participated in the selection of title, key words, selection papers, data extraction, analysis, and writing. am participated in the selection papers, data extraction, and writing. rp participated in the writing. rn participated in the searching of papers, and writing. all authors read and approved the final manuscript. none. all relevant data are within the paper. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.received: 23 january 2021 accepted: 25 march 2021 key: cord-0011587-tgmh0mn7 authors: schmalz, gerhard; patschan, susann; patschan, daniel; ziebolz, dirk title: oral-health-related quality of life in adult patients with rheumatic diseases—a systematic review date: 2020-04-19 journal: j clin med doi: 10.3390/jcm9041172 sha: 0f5aae142a50085785fda206ea1bd577bd60d06a doc_id: 11587 cord_uid: tgmh0mn7 objectives: the aim of this systematic review was to assess the oral-health-related quality of life (ohrqol) of adult patients with rheumatic diseases. material and methods: a systematic literature search was performed, including clinical studies on adults (aged at least 18 years) with a verified diagnosis of rheumatic disease. results: 26 out of 41 clinical studies including rheumatoid arthritis (ra, seven studies), systemic sclerosis (ssc, five), sjögren syndrome (ss, eight), behcet disease (bd, four), systemic lupus erythematosus (sle, one) and ankylosing spondylitis (as, one) were found. in 15 studies, a healthy control group was recruited. the short form of the oral health impact profile (ohip 14) was most frequently applied. the majority of studies (14/15) reported worse ohrqol in patients with rheumatic disease compared to healthy individuals. in particular, patients with ss (salivary flow and composition) or bd (oral ulcers) showed a relation between ohrqol and disease-specific oral manifestations. most studies investigating subscales of ohrqol (5/6) found the subscale physical disability to be predominantly affected in patients with rheumatic diseases. about half of the studies reported impaired psychosocial aspects. conclusion: patients with rheumatic diseases exhibit reduced ohrqol, especially in diseases with oral manifestations like ss and bd. physical affections due to oral diseases and psychosocial impairments caused by disease-related parameters must be recognized within patient-centered dental care. autoimmune rheumatic diseases are a heterogeneous group of disorders causing high morbidity in affected patients. therefore, regardless of their rare occurrence, the clinical relevance of these diseases appears to be high [1] . while the progress in therapeutic management of rheumatic disorders has been enormous in recent decades, one focus in the care of these patients is their quality of life [2] . while quality of life is a broad area, the oral-health-related quality of life (ohrqol) represents a partial aspect of the general health-related quality of life (hrqol) [3] . this ohrqol reflects the individual, subjective perception of the oral health status of a patient, and is primarily related to the physical oral conditions, but also to general hrqol issues [4] . thereby, different dimensions of ohrqol exist, including psychosocial and functional issues, whereby many patient-reported outcome measures are available, which differ regarding their response format, number of items, context of use, or target group [5] . oral diseases, especially periodontitis and tooth loss, are highly prevalent in patients with rheumatic diseases [6] . this could be related to patients' ohrqol. thereby, oral manifestations of based on the heterogeneity of the available data, the existing literature regarding ohrqol of patients with different systemic rheumatic diseases was reviewed. the following primary question was formed: is there a difference between different entities of systemic rheumatic diseases and healthy controls? a systematic search was performed by two independent reviewers based on the pubmed, scopus, and cochrane databases in january 2020 using the following search terms: "rheumatic diseases" or "rheumatoid arthritis" or "lupus" or "systemic sclerosis" or "sjögren syndrome" or "ankylosing spondylitis" or "vasculitis" or "behcet's disease" and "oral-health-related quality of life." based on the findings, an additional manual search considering the references of the included studies was executed. the search results were screened and checked for eligibility with regard to previously formed criteria. only full-text articles in english that were published after the year 1999 were considered. mandatory condition for inclusion included the recruitment of patients suffering from a systemic rheumatic disease with a verified diagnosis by clearly defined, disease-specific criteria within a clinical study. furthermore, only studies explicitly reporting on any ohrqol outcome were included. only adult patients were examined. therefore, studies with patients <18 years of age were excluded. following the prisma guidelines [18] , duplicate findings were checked and removed as a first step. second, abstracts of the findings from the systematic search were screened. after screening, full-text articles were carefully reviewed regarding the in-and exclusion criteria, and included in the further data extraction procedure if they fulfilled these criteria. for the qualitative analysis, the following major information was extracted from the included investigations: • form of disease, year of publication, number of participants, study setting, age, sex, and disease duration of the diseased group; • recruitment of a healthy control group for comparison of ohrqol findings; • form of ohrqol assessment, results of ohrqol and its potential relationship to general hrqol, oral-health-and/or disease-specific parameters; • if applicable, results for subscales of the ohrqol measurements and whether they were worse than for healthy control individuals; • if applicable, information regarding the validity of the applied measurements. all clinical studies were screened for this information. if studies included patients who were part of previously published investigations, it was checked whether there were repetitious results. only if other in-and exclusion criteria, other examination procedures, other ohrqol assessment measures, or additional information was available were both studies included. the whole systematic review process, including literature search, abstract/title/full-text screening, and extraction of data, was accomplished by two independent individuals. the systematic search revealed 41 articles, which were found by the applied search terms complemented by manual search. two articles were reviews and thus were excluded from further process. a total of 39 full-text articles were screened with regard to the defined in-and exclusion criteria. accordingly, 13 articles were excluded for the following reasons: one article did not assess ohrqol in rheumatic diseases at all; one questionnaire-based examination did not verify the diagnosis of rheumatic disease; and seven studies were performed in patients with temporomandibular disorders, including craniomandibular dysfunction or osteoarthritis restricted to the temporomandibular joint. furthermore, four studies included patients with juvenile idiopathic arthritis and were excluded because of their inclusion of patients with an age below 18 years (supplementary table s1 ). consequently, a total of 26 eligible studies were included in the qualitative analysis of this systematic review ( figure 1 ). the following rheumatic diseases were subject of the included investigations: rheumatoid arthritis (ra, seven studies), systemic sclerosis (ssc, five studies), sjögren syndrome (ss, eight studies), behcet's disease (bd, four studies), systemic lupus erythematosus (sle, one study), and ankylosing spondylitis (as, one study). regarding study type, the majority of examinations were cross-sectional studies, wherein 4 were multi-centric and 18 monocentric. two studies were randomized clinical trials and two investigations were observational with a follow-up of two weeks or three years, respectively. the number of included patients differed between 20 and 675 the following rheumatic diseases were subject of the included investigations: rheumatoid arthritis (ra, seven studies), systemic sclerosis (ssc, five studies), sjögren syndrome (ss, eight studies), behcet's disease (bd, four studies), systemic lupus erythematosus (sle, one study), and ankylosing spondylitis (as, one study). regarding study type, the majority of examinations were cross-sectional studies, wherein 4 were multi-centric and 18 monocentric. two studies were randomized clinical trials and two investigations were observational with a follow-up of two weeks or three years, respectively. the number of included patients differed between 20 and 675 participants with rheumatic diseases. in 17 studies, a control group was recruited (15 healthy controls, 2 other controls). the main study characteristics are presented in table 1 . the main findings of the included studies are presented in table 2 . the measurement reported most often was the short form of the oral health impact profile (ohip 14), which was applied in 15 table 2 . of the 15 investigations that compared the ohrqol of a patient group suffering from rheumatic disease with healthy controls, 14 studies reported worse ohrqol in rheumatic-diseased individuals. the relationship between ohrqol and general hrqol has rarely been examined. results regarding potential associations and/or correlations between ohrqol and oral health, as well as rheumatic-disease-specific parameters, were heterogeneous (supplementary table s2 ). patients with sjögren syndrome (salivary flow and composition) or behcet's disease (oral ulcers) showed a particular relationship between ohrqol and disease-specific oral manifestations (table 2) . a total of eleven studies reported subscales of the ohrqol measurement, of which five reported ohip 14 subscales, five reported ohip 49 subscales, and one study examined the subscales of xeqol. within six studies, the subscales were compared between rheumatic-diseased individuals and a healthy control group. the majority of these studies (5/6) found the ohrqol associated with physical disability to be lower in rheumatic-diseased patients. about half of the studies reported impaired psychosocial aspects (tables 3 and 4) . the study that applied the xeqol found significantly worse results for the subscales related to physical pain, psychosocial discomfort, physical disability, and social disability in individuals with ss compared to healthy controls [8] . a total of eleven studies reported subscales of the ohrqol measurement, of which five reported ohip 14 subscales, five reported ohip 49 subscales, and one study examined the subscales of xeqol. within six studies, the subscales were compared between rheumatic-diseased individuals and a healthy control group. the majority of these studies (5/6) found the ohrqol associated with physical disability to be lower in rheumatic-diseased patients. about half of the studies reported impaired psychosocial aspects (table 3 and 4) . the study that applied the xeqol found significantly worse results for the subscales related to physical pain, psychosocial discomfort, physical disability, seven of the included investigations provided information about the validity of the applied measurement. the intraclass correlation coefficient fell between 0.61 to 0.94, and the cronbach's α was determined to be between 0.89 and 0.99 (table 5 ). however, one study that explicitly focused on the reliability of ohip 49 in ssc reported a large standard error of measurement and concluded ohip 49 to be not precise and sensitive for these patients [19] . this is the first systematic review of ohrqol in patients with different rheumatic diseases. the qualitative analysis revealed 26 clinical studies, from which six disorders, i.e., ra, ssc, ss, sle, as, and bd, were included in the review. in accordance to the world dental federation (fdi) definition of oral health as a synthesis of physical and psychological well-being with regard to the orofacial system [35] , the ohrqol plays an indispensable role in dental care. the concept of ohrqol enables a shift from the pure physical consideration of the patients with a focus restricted to the haptic presence of oral diseases to a comprehensive patient-centered assessment [4, 36] . thereby, ohrqol must be seen as a mandatory sub-aspect of general hrqol, including the possible interrelationship between orofacial health and general hrqol in a complex interaction [3, 4, 36] . accordingly, the assessment of ohrqol in the context of oral-health-and disease-related parameters in patients with rheumatic diseases appears to be contemporary and reasonable. however, rheumatic diseases constitute a heterogeneous group of different disorders with manifold therapeutic strategies [1] , making comparison and interpretation difficult. moreover, the inconsistency of included studies regarding their study design, patient selection criteria, methodology, and geographical specifics has an impact on their comparability. nevertheless, the results of this systematic review can provide some interesting information for the interdisciplinary dental care of patients with rheumatic diseases. moreover, recommendations for future research in the field can be formulated. generally a reduced ohrqol is apparent in patients with rheumatic disease. a total of 15 studies reported the ohrqol in comparison with a healthy control group, of which 14 studies found worse results in patients suffering from rheumatic diseases [8, [11] [12] [13] 15, 16, 21, 22, 24, 26, 30, 31, 33, 34] . the valid interpretation of further results is difficult. reference values for generally healthy individuals can help to provide a statement as to whether results represent a deficit in the ohrqol of the patients. due to cultural differences as well as a large influence of age, sex, and the presence of teeth/prosthodontic treatment on ohrqol outcome [37, 38] , consistent international reference values are not available. for instance, the german dentate general population exhibits a sum score of ohip 14 of 0-4 or ohip 49 between 5 and 15 points depending on full or partial dentition [38, 39] . the comparability is limited due to different countries with particularities potentially affecting ohrqol [37] . irrespective of this fact, the ohip values of all included studies with patients suffering from rheumatic diseases were higher than these reference values. within the studies that applied the ohip questionnaire to patients with rheumatic diseases, ss and bd in particular showed the highest ohip scores, representing a reduced ohrqol (figures 2 and 3) . ss is a chronic inflammatory disorder primarily affecting the exocrine glands, what leads to a dryness of mouth and eyes due to involvement of salivary and lacrimal glands, respectively [40] . the resulting xerostomia is a complex condition, leading to remarkable impairment of ohrqol [41] . accordingly, majority of included studies dealing with ss showed associations between worse ohrqol and salivary flow or composition [8, [24] [25] [26] 28] . one study that examined ra also investigated the influence of xerostomia on ohrqol and was also able to show a negative impact [14] . the disease-related xerostomia in patients with ss might therefore be a major reason for their impaired ohrqol. similarly, bd represents a chronic multisystem disease with a special disease-related condition affecting the oral cavity: oral ulcers [42] . this painful efflorescence of oral soft tissues constitutes a logical reason for their impaired ohrqol. this was confirmed by the negative effect of active ulcers on ohrqol in the included studies [30] [31] [32] [33] . therefore, rheumatic diseases leading to a primarily oral manifestation appear to have the largest impact on ohrqol among the included investigations. in this context, the relationship between periodontaitis and rheumatic diseases, especially ra, needs to be mentioned [43] . although ra is related to a higher prevalence and increased severity of periodontitis [6] , this appears to have no remarkable effect on patients' ohrqol, because none of the included studies reported associations between periodontitis and ohrqol explicitly ( table 2 ). within the oral conditions, only the presence of missing/extracted teeth and/or wearing dentures was repeatedly reported to affect ohrqol [9, 14, 17, 30, 32, 34] . it appears evident that tooth loss can be seen as a factor influential on ohrqol, independently of the applied instrument for measurement or included patients [44] . accordingly, this finding appears not to be specific for rheumatic diseases. however, the higher risk for tooth loss in rheumatic diseases, especially ra, due to the relationship to periodontal burden should be recognized in this context [45] . considering both the influence of tooth loss on ohrqol in general [44] and the high prevalence of tooth loss in rheumatic disease [6, 45] , this is an important issue. on one hand, the avoidance of tooth loss might be of increasing relevance for these patients to reduce the impairment of their ohrqol. on the other hand, tooth loss or denture wearing should be assessed and considered in studies dealing with the ohrqol of patients suffering from rheumatic diseases. another issue touched on by this systematic review is the occurrence of associations between general hrqol and/or rheumatic-disease-related parameters with ohrqol. as presented in table 2 , results regarding this issue are heterogeneous and only a small number of studies also examined hrqol. for generally healthy individuals, a relationship between ohrqol and general hrqol is documented in literature, whereby ohrqol is more strongly related to physical oral health than to general hrqol [4, 36] . however, patients with rheumatic diseases often show reduced hrqol due to their disease burden, pain, anxiety, or depression [2] . therefore, the interrelation between ohrqol and general hrqol is of interest in these patient groups. the majority of included studies (5/6) that examined this relationship found associations, whereby different measurements for hrqol were applied [8, 9, 15, 21, 22] . two potential explanations might be conceivable for this finding. on one hand, the oral conditions related to rheumatic diseases might have an influence on both ohrqol and general hrqol in these patients. based on the literature, this seems conceivable [4, 36] , but because none of the studies explicitly examined this issue, this remains speculative. the second potential explanation could be the effect of rheumatic disease on whole hrqol, including ohrqol, due to the physical and psychosocial impairment of patients caused by the underlying disease. accordingly, the impaired ohrqol of these patients would be more strongly affected by rheumatic disease burden than by their oral conditions. to support this hypothesis, the associations between ohrqol and rheumatic-disease-related parameters were examined. the majority of studies that examined this issue (16/21) were able to confirm a relationship between ohrqol and disease-related parameters [8, [12] [13] [14] 16, 17, 22, [24] [25] [26] 28, [30] [31] [32] [33] [34] . these parameters were based on the heterogeneity of different diverse rheumatic diseases, including age, disease duration, disease activity, or disease severity. thus, disease-specific oral manifestations in ss (xerostomia) and bd (oral ulcers) that primarily affect oral cavity were of particular relevance, as described above [8, [24] [25] [26] 28, [30] [31] [32] [33] . the general hrqol of patients with rheumatic diseases is affected by disease-related parameters like age, disease duration, and disease severity [46, 47] . the relationship between ohrqol and disease-related parameters as assessed in the majority of studies might thus imply that this is similar for ohrqol and for general hrqol in these patients. for a deeper understanding, the subscales of ohrqol can be considered additionally. because five studies did not compare subscales to a healthy control [14, 17, 28, 29, 32] , and different subscales were applied, the comparability is limited. however, some interesting information can be derived from the findings: in the vast majority of studies with a control group (5/6), a significant effect of the subscale "physical disability" was confirmed without any predominant manifestation in one of the included diseases [8, 11, 20, 30, 34] . furthermore, half of examinations (3/6) also confirmed a significant impairment of the psychosocial and/or psychological sub-aspects of ohrqol [8, 20, 31] . moreover, the potential relevance of psychosocial patterns is highlighted by two special results. in a randomized clinical trial, the effect of malic acid on ohrqol of patients with ss was tested. the largest improvement was recorded for psychological pattern [29] . another study in patients with ra found correlations between disease activity and the pattern of psychosocial impact [17] . rheumatic diseases constitute an important psychosocial impairment [48] . accordingly, the relevance of psychological and/or psychosocial issues in the context of ohrqol might be of clinical relevance. it is therefore conceivable that beside impaired physical oral health due to related manifestations like xerostomia or oral ulcers, a psychosocial impairment caused by rheumatic disease burden might be responsible for the impaired ohrqol of these patients. this condition might be of particular relevance, because a solely physical understanding might be inadequate for their dental care. in fact, this rather argues for a patient-centered, individual, and multidisciplinary care model [49] , which must also be implemented in the dental care of patients with rheumatic diseases. another issue of relevance is the methodology applied in the included studies. a total of six different questionnaires, including ohip 49, ohip 14, gohai, mhiss, xeqol, and a specific ohrqol questionnaire, were used. the ohip and gohai questionnaires appear to be valid and reliable assessment measures for ohrqol [5] . the available examinations showed appropriate validity for the applied questionnaires, and the majority of available information was provided for ohip questionnaire [19, [28] [29] [30] [31] [32] . only one study concluded ohip 49 to be not precise and sensitive for patients with ssc and disease-specific questionnaires to be needed [19] . while a strong conclusion as to the most appropriate measurement tool is not possible based on the available studies, several consideration can be highlighted. the use of the ohip questionnaire can be especially recommended for rheumatic diseases with oral manifestations because of its appropriate psychometric properties, especially in case of xerostomia [50] . this was confirmed by the results for ss and bd in the included studies. for rheumatic diseases like ssc with a lower number of oral manifestations, disease-specific questionnaires like the mhiss might be more informative and suitable [10, 21] . however, for these diseases, the ohip questionnaire, reporting mean value, standard deviation, and median are also recommended to ensure comparability with available literature. furthermore, reference values for interpretation of ohrqol results are recommended [38] . therefore, future studies might focus on the evaluation of these references for rheumatic-diseased individuals. furthermore, the clinical relevance of detected differences like ohip scores must be considered. interventional studies especially focus on minimal important difference as a reference for a noticeable clinical effect [51] . these values are available for systemically healthy but not rheumatic-diseased individuals, making investigations regarding this issue recommendable. additionally, studies assessing ohrqol of patients with rheumatic diseases should also evaluate hrqol, oral conditions (especially missing teeth and denture wearing), and disease-related parameters to draw robust conclusions. strengths and limitations: this systematic review followed the prisma guidelines [18, 52] and was executed according to previously defined in-and exclusion criteria for the studies. furthermore, this is the first systematic evaluation of ohrqol in patients with different rheumatic diseases. some limitations need to be recognized. rheumatic diseases are heterogeneous [1] , making comparison difficult. furthermore, although in-and exclusion criteria were formulated, very different study designs with specific patient groups were included. however, the review aimed to provide a comprehensive insight of the ohrqol in rheumatic-diseased individuals. due to the different questionnaires and the inconsistent reporting of the results, a quantitative analysis was not possible. patients suffering from rheumatic diseases exhibit reduced ohrqol, especially in diseases with oral manifestations like ss and bd. physical affections due to oral diseases and psychosocial impairments caused by disease-related parameters must be recognized. therefore, multidisciplinary, patient-centered dental care is recommendable for patients with rheumatic diseases. future examinations should include general and disease-specific ohrqol measurements and should apply standardized methodology and reporting of the results. the evaluation of disease-related reference values for ohrqol and minimal important differences for the interpretation of clinical relevance should be part of future studies in the field. the following are available online at http://www.mdpi.com/2077-0383/9/4/1172/s1, table s1 : excluded studies during the evaluation process with reasons for exclusion, table s2 . oral health parameters that were examined related to ohrqol in the included studies. clinical aspects of autoimmune rheumatic diseases managing rheumatic and musculoskeletal diseasespast, present and future association between perceived oral and general health individuals' perception of oral health and its impact on the health-related quality of life oral health and quality of life: current concepts main oral manifestations in immune-mediated and inflammatory rheumatic diseases new therapeutic opportunities for covid-19 patients with tocilizumab: possible correlation of interleukin-6 receptor inhibitors with osteonecrosis of the jaws oral health-related quality of life in primary sjögren's syndrome oral health-related quality of life among outpatients with rheumatoid arthritis the italian version of the mouth handicap in systemic sclerosis scale (mhiss) is valid, reliable and useful in assessing oral health-related quality of life (ohrqol) in systemic sclerosis (ssc) patients oral health-related quality of life among individuals with rheumatoid arthritis oral health-related quality of life is associated with disease specific parameters in patients with ankylosing spondylitis oral health-related quality of life depending on oral health in patients with rheumatoid arthritis assessment of relationship between xerostomia and oral health-related quality of life in patients with rheumatoid arthritis association of oral-health related quality of life and general health assessment in patients with rheumatoid arthritis ghaleb, i. oral health-related quality of life in patients with rheumatoid arthritis. open access rheumatol disease activity, morning stiffness and missing teeth are associated with oral health-related quality of life in individuals with rheumatoid arthritis the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration test-retest reliability of oral health impact profile (ohip-49) in adults with systemic sclerosis. spec. care dent the canadian systemic sclerosis oral health study: orofacial manifestations and oral health-related quality of life in systemic sclerosis compared with the general population the canadian systemic sclerosis oral health study ii: the relationship between oral and global health-related quality of life in systemic sclerosis association of low socioeconomic status and physician assessment of disease severity with oral health-related quality of life in patients with systemic sclerosis: a pilot study from croatia, a country in transition impact of sjogren's syndrome on oral health-related quality of life in southern chinese correlation between salivary epidermal growth factor levels and refractory intraoral manifestations in patients with sjögren's syndrome rapid decrease in salivary epidermal growth factor levels in patients with sjögren's syndrome: a 3-year follow-up study oral disorders, saliva secretion, and oral health-related quality of life in patients with primary sjögren's syndrome dietary intake, body composition, and oral health parameters among female patients with primary sjögren's syndrome validation of oral health impact profile-14 and its association with hypossialia in a sjögren syndrome portuguese population patient-related outcomes in sjögren syndrome treated with stimulants of salivary secretion: randomized clinical trial oral health related quality of life is affected by disease activity in behcet's disease the assessment of oral health-related quality of life by factor analysis in patients with behcet's disease and recurrent aphthous stomatitis oral health and related quality of life status in patients from uk and turkey: a comparative study in behcet's disease one-year period prevalence of oral aphthous ulcers and oral health-related quality of life in patients with behçet's disease impact of systemic lupus erythematosus on oral health-related quality of life a new definition for oral health developed by the fdi world dental federation opens the door to a universal definition of oral health oral conditions and health-related quality of life: a systematic review oral health-related quality of life: what, why, how, and future implications biffar, r. reference values in oral health-related quality of life for the abbreviated version of the oral health impact profile oral health-related quality of life in germany sjögren's syndrome the impact of xerostomia on oral-health-related quality of life among younger adults behçet's disease-a contemporary review the case for periodontitis in the pathogenesis of rheumatoid arthritis tooth loss and oral health-related quality of life: a systematic review and meta-analysis association of periodontal disease and tooth loss with rheumatoid arthritis in the us population health-related quality of life of rheumatic disease patients treated in a specialized ips in medellin, colombia clinical significance of fibromyalgia syndrome in different rheumatic diseases: relation to disease activity and quality of life burden of disease in treated rheumatoid arthritis patients: going beyond the joint the personal impact of rheumatoid arthritis on patients' identity: a qualitative study utility of two oral health-related quality-of-life measures in patients with xerostomia assessing the responsiveness of measures of oral health-related quality of life reprint-preferred reporting items for systematic reviews and meta-analyses: the prisma statement the authors acknowledge support from the german research foundation (dfg) and leipzig university within the program of open access publishing. the authors declare no conflict of interest. key: cord-0009011-b05qpb7d authors: riemann, dagmar; schwachula, annette; hentschel, michael; langner, jürgen title: demonstration of cd13/aminopeptidase n on synovial fluid t cells from patients with different forms of joint effusions date: 2011-11-02 journal: immunobiology doi: 10.1016/s0171-2985(11)80243-3 sha: 89ca18e68a23445d3c5165c565809fb0cd966528 doc_id: 9011 cord_uid: b05qpb7d cytofluorometric analysis was performed to characterize the immunophenotype of lymphocytes of the synovial fluid (sf) and the peripheral blood (pb) from patients suffering from juvenile chronic arthritis (jca) or rheumatoid arthritis (ra). the most obvious difference could be found in expression of the surface protease aminopeptidase n (ap n/cd13). whereas monoclonal antibodies specific to cd13 failed to reveal surface expression on lymphocytes of the pb; 63 ± 15% of sf t cells gave positive staining for cd 13 using leu-m7. no correlation between cd13 expression and joint disease could be found in patients who had different types of inflammatory joint effusions. cd13 expression of t cells was also found in synovial tissue and inflammatory serous cavity effusions. fixation of t cells revealed the presence of intracellular cd13 antigen already located in the pb t cells of healthy individuals. induction of cd13 expression on pb t cells could be demonstrated after incubation with con a/il-2 or sf from patients with ra. our findings suggest a role for ap n as a new activation-associated molecule of t lymphocytes. although recent studies have failed to find arthritis-specific t cell phenotypes in the pb of patients with varying forms of joint diseases, the synovial lymphocytes exhibit a number of peculiarities. besides the heavy preponderance of t cells -which are composed almost entirely of cells of the memory phenotype expressing cd45ro (1), -there is a known discrepancy between the increased expression of hla-dr, but not of cd25 on t cells (2, 3) . synovial t cells are described as expressing specific molecules -such as um4d4 (4) -and as representing the th1-phenotype (5) . functional abnormalities include a reduced response to mitogenic lectins (6, 7) , but comparable or increased responses to recall antigens (7, 8) . in order to further characterize the functional state of sf t cells, we compared the immunophenotype of pb and sf lymphocytes (paired samples) of 10 patients having either ra or lca. particular attention was paid to the surface proteases ap n/cd13 and dp iv/cd26. in recent years, the interest in these and other proteases has increased remarkably because, among other things, peptidase inhibitors indicate roles of ectopeptidases in regulating biologically active peptides, such as cytokines and growth factors. thus, a great number of studies have been published with respect to the participation of the constitutively expressed transmembrane protease dp iv in t cell activation and memory (9) (10) (11) . however, contradictory results have been published concerning the occurrence of the ubiquitous zinc-containing exopeptidase ap n in lymphocytes. whereas ap n-like enzymatic activities are detectable using spectrophotometric methods (9, (12) (13) (14) ; the available monoclonal antibodies against cd13 recognize monocytes and granulocytes, but not lymphocytes of the pb (15, 16) . ap n catalyzes the removal from peptides of n-terminal, preferentially neutral, amino acid residues and is suggested to be involved in the metabolism of regulatory peptides, e.g., through small intestinal and renal tubular epithelial cells and through synaptic membranes (17) . using two-color flow cytometry, we were able to detect the occurrence of cd26 on both pb and sf lymphocytes. surprisingly, sf t cells -but not pb lymphocytes -showed a varying surface-labelling with different monoclonal antibodies specific for cd13. we subsequently studied the immunophenotype of sf t cells from patients with a variety of inflammatory arthropathies in order to investigate possible correlations between the expression of cd13 and the type of joint disease. in addition, lymphocytes of synovectomy tissue, spleen and serous cavity effusions were studied to see whether they displayed surface expression of cd13. incubation experiments with sf and pb lymphocytes were carried out to obtain first insights into the regulation of cd13 on t lymphocytes. patients with different types of joint effusions were studied. the diagnoses were defined by the clinicians who supplied the sf that was drawn for therapeutic reasons. the 32 arthritis patients were categorized into four clinical groups: jca, ra, traumatic arthropathy (ta) and degenerative joint disease (djd). patients were treated with non-steroidal anti-inflammatory drugs and/or steroids as well as disease-modifying drugs such as sulfasalazine. none of the patients had received intra-articular steroid therapy during the last month before sampling. sf and, in ten patients with ra or lca, paired blood samples, were collected via sterile heparinized tubes, and mnc were isolated by ficoll-hypaque density gradient centrifugation. blood-stained sf specimens were excluded from the study. the cell-free sf was stored at -80°c. blood from healthy staff members of our laboratory served as the control group. synovial tissues were obtained from 3 patients with jca who were undergoing synovectomy. mnc were eluted as described by abrahamsen (18) . human spleen tissues were kindly provided by the hla laboratory of our medical faculty. mnc of the spleen tissues were separated using ficoll-hypaque. during therapeutic procedures, peritoneal fluids were aspirated from 2 patients suffering from ethanolic liver cirrhosis. pericardial fluid was obtained from ten patients undergoing heart valve replacement. cells were collected by centrifugation and used for double-labelling after two washings. the monoclonal antibodies used were tal (cd26, coulter, krefeld, germany), ioti4 (cd25, immunotech/dianova, hamburg, germany), leu-m7 (cd13, becton dickinson, heidelberg, germany), wm-47 (cd13, dako, hamburg, germany), 5]'101 (cd13, immunotech/ dianova). all other monoclonal antibodies used were purchased from becton dickinson. optimal dilutions for use in flow cytometry were established in preliminary experiments. double-staining of mnc was performed at 20 0 e for 20 minutes. after two cold washes with pbs containing 0.1 % sodium azide, the cells were fixed using 1 % paraformaldehyde. for comparison of different cd13 specific monoclonal antibodies, indirect immunofluorescence staining was used. cells were incubated with either monoclonal antibody or purified normal mouse igg. after two washings, cells were labelled with the pe-conjugated secondary antibody and washed and fixed as mentioned above. cytofluorometric analyses were performed on a becton dickinson facscan. non-lymphoid cells and dead cells were excluded by the setting of appropriate forward and 90° light scatter gates. 5000 cells per sample were counted. the threshold was defined in such a manner that positive-staining included no more than 1 % of the relevant negative control. in order to detect intracellular cd13 in cd3+ lymphocytes, cells were gently permeabilized following the method of schmid (19) . in brief, after blocking non-specific binding with a i-min pre-incubation with human ab serum, cells were labelled with the first antibody (leu-4/fitc). after two washes, the pelleted cells were fixed with 0.25 % paraformaldehyde solution at 4 °e for i h . after centrifugation, pelleted cells were resuspended in 0.2 % tween 20 in pbs at room temperature, washed and centrifuged again. staining of the internal antigen (negative control/pe or leu-m7/pe) followed a further blocking of non-specific binding with human ab serum. after two washes in 0.2 % tween 20 in pbs, cells could be analyzed. aliquots of pb mnc at 1 x 10 6 cells/ml were incubated for various time periods between 30 min and 18 h at 37"e with an equal amount of autologous cell-free sf. the cells were washed twice in pbs and investigated for the presence of cd13 on cd3+ cells using doublelabelling. mnc from the pb of healthy subjects were incubated at a concentration of 1 x 10 6 cells/ ml in rpm i containing antibiotics, l-glutamine and 10 % fetal calf serum (fcs) under the following conditions : (a) with no additives or (b) with con a (5 i-lg/ml) and il-2 (10 v lml) or (c) with sf (dilution 1:10) from patients with ra. after 1, 2 and 3 days, respectively, lymphocytes were investigated for surface molecules. in some cases, cells were incubated in the presence of cycloheximide (1.5 i-lg/ ml). data are expressed as mean ± standard deviation. student's t-test for paired differences was used to compare paired samples of blood and sf. 1 n the other cases, statistical differences were analyzed using a t-test for unpaired data with welch's approximation. figure 1 compares the expression of five selected surface molecules on cd3+ lymphocytes from patients suffering from ra or lca (paired samples of sf and pb). we found a significant preponderance of the expression of hla-dr and cd45ro on sf versus pb cd3+ cells (76 ± 15 % versus 7 ± 3 %, and 90 ± 7 % versus 42 ± 15 %, respectively) as already described by several authors (1) (2) (3) (4) . no clear difference could be demonstrated in the case of cd25 expression (14 ± 9 % versus 16 ± 13 %). surprisingly, the most obvious difference between surface molecule expression in sf and pb was found for cd13. as in the pb of healthy controls (data not shown), only a negligible amount of surface expression was demonstrable on cd3+ cells in the pb of all the patients studied. by contrast, 63 ± 16 % of sf cd3+ cells were cd13+, using the monoclonal antibody leu-m7. expression of cd13 was not an all-or-none phenomenon. instead, the fluorescence histograms were typically rather broad (fig. 2) . both cd4+ and cd8+ sf t cells expressed cd13, as shown in figure 3 . furthermore, a slightlybut significantly -decreased expression of the exopeptidase cd26 was demonstrable on sf versus pb cd3+ cells (45 ± 13 % versus 59 ± 13 %) (fig. 1) . using the pe-conjugated leu-m7, the fitc-conjugated wm-47 and the fitc-conjugated sj.id1 in one sample of sf cells, we observed a staining of cells in the ratio: 6:2:1. we found no obvious difference in staining when comparing the 3 above-mentioned antibodies in a non-conjugated form and using an indirect method with a pe-conjugated second antibody (data not shown). in the following experiments, pe-conjugated leu-m7 was used (unless otherwise indicated) for the demonstration of cd13, to investigate a possible correlation between the type of arthritis and a special pattern of expression on sf t cells of activation molecules and cd13 and cd26, double-staining experiments were performed with sf :' .. ::,' : .... figure. 3. two-color immunofluorescence analysis of sf t cells from a representative patient with jca. cells of different inflammatory joint effusions. we could find no significant difference in the expression on cd3+ cells of all the surface molecules studied, although cd13 seemed to be increased in patients with a highlyactive }ca ( table 1) . to answer the question whether cd13 occurs on t cells exclusively within sf, we investigated the immunophenotype of lymphocytes from different sources. on t cells prepared from synovectomy tissue of 3 patients with }ca, we were able to demonstrate a cd13 expression comparable to sf lymphocytes (32, 52 and 67 % of cd3+ cells, respectively). next, spleen tissue of 3 kidney donors was investigated. the expression of cd13 on lymphocytes was as negligible as in pb. t cells prepared from non-inflammatory peritoneal fluid were also cdlr. in contrast, t cells from the pericardial fluid of ten patients undergoing heart valve replacement showed varying levels of cd13 expression (27 ± 14 % of cd3+ cells). immunophenotyping of blast cells from acute myeloid leukaemia has shown that the cd13 antigen may be detected in the cytoplasm of myeloblasts before its membrane expression (20) . for this reason, we (table 2) . interestingly, as many as 4-14 % of pb t cells from healthy persons were stained positively for cd13. 8-22 % of fixed pb lymphocytes of lca patients stained cd13+, a result that needs further investigation. permeabilized sf cells also exhibited a greater amount of cd13-expression than native sf cells. in order to examine the effect of autologous sf on the immunophenotype of pb mnc, we incubated mnc of the pb of 5 patients with lca at 37°c between 30 min and 18 h in the presence of an equal amount of cell-free sf. we observed negligible staining for cd13 after an incubation of lymphocytes ranging from 30 min to 4 h (2-5 % of cd3+ cells were cdh') only an 18-h incubation resulted in a distinct expression of cd13 on pb t cells (6-14 % of cd3+ cells became cd13+). this can be taken as an argument against cd13 expression only occurring as a result of an alteration of the surface of lymphocytes by synovial enzymes. due to the fact that several studies describing an increase of aminopeptidase activities after mitogen stimulation of lymphocytes already exist (13, 14) , we investigated cd13 expression on cd3+ cells after stimulation of pb mnc in control subjects with cona/il-2. in addition, the cells were cultivated with sf from a patient with ra. figure 4 reaction. furthermore, we observed a tight co-expression of all 3 surface molecules studied after stimulation with con a/il-2, though there were differences over the course of time and between individuals. on the other hand, incubation of mnc with sf from a patient with ra resulted in a clear increase of cd13 and of hla-dr, but in a slight decrease of cd25. after addition of cycloheximide to sf, only approximately 10 % of cd3+ cells stained positively for cd13 (fig. 5 ). this could be the result of the distribution of internal «storage places», because staining for cd13 of fixed pb lymphocytes also revealed approximately 10 % cd13+ t cells (table 2) . possible conclusions are as follows: (1) cd13 expression on pb lymphocytes is inducible by mitogens -though not to the extent reached in sf; (2) cd25 and cd13 are regulated independently of each other; (3) the cell-free sf contains cd13-and hla-dr-but not cd25-augmenting mediators and (4) expression of cd13 on more than approximately 10 % of cd3+ cells needs pathways that may be inhibited by cycloheximide. the effects of sf on pb lymphocytes have not been uniformly discussed in literature. crout (21) reported that the addition of autologous cell-free sf resulted in an increased incorporation of tritiated thymidine by pb lymphocytes. on the other hand, hain (22) demonstrated that sf by itself had no significant stimulatory effect on pb lymphocytes. further experiments are necessary to obtain a clearer picture of the influence of cell-free sf on lymphocytes. to the best of our knowledge, in this paper we are discussing the occurrence of cd13 on human sf t lymphocytes using different monoclonal antibodies specific to this antigen for the first time. the same antibodies could not detect cd13 on the surface of pb lymphocytes of healthy persons and patients with ra and lca, but permeabilization of t cells revealed the presence of intracellular cd13. our observations confirm results of enzymatic evidence of ap n in lymphocytes (9, 12, 13) . it seems unlikely that t cells acquire the surface aminopeptidase as a consequence of the migration process through a tissue compartment, since lymphocytes prepared from spleen tissue show no detectable expression of cd13 by use of different monoclonal antibodies. in addition, also t cells of the peritoneal fluid of patients who had ethanolic liver cirrhosis displayed no detectable staining for cd13. we lean towards the assumption that t cells could acquire this phenotype within the synovium. it is known that cd13 antibodies can be subgrouped according to their ability to bind at least 3 different epitopes of the ap n glycoprotein (23) . furthermore, it has been suggested that at least five subpopulations of ap n exist, owing to differential utilization of glycosylation sites or to subtle differences in oligosaccharide composition (24) . this raises the question as to whether an unmasking or alteration of certain cd13 epitopes by synovial enzymes is the reason for the binding of monoclonal antibodies to sf lymphocytes. this seems not to be the case, because an incubation of pb lymphocytes of patients with ra in autologous sf lasting 4 h at 3rc induces no subsequent labelling of lymphocytes with cd13 specific antibodies. furthermore, in the presence of con a/il-2, a 3-day incubation of pb mnc from control persons not only induced an increase on t cells of the activation markers hla-dr and cd25, but also of cd13 (fig. 4) . this data strongly supports the hypothesis that cd13 is a new activationassociated antigen of lymphocytes. results from literature concerning the function of cd13 on other blood cells are not uniform. griffin (25) investigated myeloid precursor cells with the cd13 specific monoclonal antibody anti-my7. he suggested that my7-antigen, though not recognized by the antibody, could be expressed at low antigen-density throughout myeloid differentiation. detectable my7-expression identified an actively proliferating fraction of myeloid colony forming cells (25) . while investigating cd13 on monocytes and rheumatoid synovial tissue macrophages using the monoclonal antibody 3ds, koch (26) observed an augmentation of cd13 when monocytes were exposed to interferon-gamma and lipopolysaccharide. the cd13 antigen increased parallel with the mhc class ii antigen expression and also when monocytes in culture were maturing to macrophages. the author suggested that as monocytes enter the synovial tissue from the blood, they rapidly migrate to the synovial lining layer, where they bear both mhc class ii and some activation antigens, such as cd13 (26) . werfel (27) observed variable enhancement of cd13 expression on monocytes and granulocytes within minutes after these cells had been stimulated with c5a. though these examples are not simply transferable to the situation in lymphocytes, they show the complexity of involvement of cd13 in the activation processes of blood cells. the rare occurrence of cd13 on lymphatic leukaemia cellsinterpreted as myeloic co-expression with a worse prognosis (2s) -is a further example of the scarcely understood integration of cd13 in prolifer-ation or differentiation processes. in addition, the observation that cd13 represents a receptor for coronaviruses could be of major pathological importance (29, 30) . as a membrane protease that cleaves n -terminal amino acid residues from small polypeptides, ap n seems suitable for regulation of biologically active peptide hormones. this has been already shown for enkephalins and endorphins, degraded by ap n in synaptic membranes of the central nervous system (31) . in this case, the enzyme is accompanied by another zinc-dependent membrane protease, the endopeptidase 24.11 (calla/ cd10). we were unable to detect this differentiation antigen of lymphoid precursor cells on synovial lymphocytes « 3 %, data not shown). however, the majority of t cells expressed cd13 together with cd26, a membrane protease expressed on activated t cells irrespective of cell cycle (32) . this result makes it tempting to speculate about a functional cooperation of both the peptidases. in summary, we have described the occurrence of cd13 as a new activation-associated molecule on synovial t cells of patients with different forms of joint effusions. the estimation of cd13+ t cells is not useful in establishing a diagnosis in patients with different forms of arthritis, as we had expected it to be. however, experiments are in preparation to investigate a possible association between inflammatory activity and cd13 expression on t cells, not only from sf, but also from other inflammatory effusion fluid. abnormal helperinducer/suppressor-inducer t-cell subset distribution and t-cell activation status are common to all types of chronic synovitis synovial fluid lymphocytes differ from peripheral blood lymphocytes in patients with rheumatoid arthritis expression of hla-dr, dq and dp antigens and interleukin-2 receptor on synovial fluid t lymphocyte subsets in rheumatoid arthritis: evidence for «frustrated» activation a novel pathway of human t lymphocyte activation. identification by a monoclonal antibody generated against a rheumatoid synovial t cell line t cells cloned from human rheumatoid synovial membrane functionally represent the th1 subset in vitro mitogen stimulation of synovial' fluid lymphocytes from rheumatoid arthritis and juvenile rheumatoid arthritis patients: dissociation between the response to antigens and polyclonal mitogens immunological and functional characteristics of peripheral blood and synovial fluid lymphocytes from patients with rheumatoid arthritis peripheral blood and synovial fluid t cells differ in their response to alloantigens and recall antigens presented by dendritic cells membrane-bound peptidases of lymphocytes: functional implications the t cell triggering molecule tp103 is associated with dipeptidyl aminopeptidase iv activity coassociation of cd26 (dipeptidyl peptidase iv) with cd45 on the surface of human t lymphocytes serum leucine aminopeptidase for monitoring viral infections with plasmocytoid reaction surface aminopeptidase activity of human lymphocytes. i. biochemical and biologic properties of intact cells properties and activities of aminopeptidases in normal and mitogen-stimulated human lymphocytes an immunohistochemical study of endopeptidase-24.11 and aminopeptidase n in lymphoid tissues metalloprotease activity of cd13/aminopeptidase n on the surface of human myeloid cells mammalian proteases: a glossary and bibliography elution and characterization of lymphocytes from rheumatoid inflammatory tissue a gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in dna quantification early expression of mcs2 (cd13) in the cytoplasm of blast cells from acute myeloid leukaemia induction of peripheral blood lymphocyte transformation by autologous synovial fluid lymphocytes and synovial fluid stimulation of rheumatoid synovial and blood t cells and lines by synovial fluid and interleukin-2: characterization of clones and recognition of a co-stimulatory effect report on the cd13 (aminopeptidase n) cluster workshop variable o-glycosylation of cd13 (aminopeptidase n) expression of my7 antigen on myeloid precursor cells aminopeptidase n/cd13 on synovial t cells . 3s monoclonal antibodies detect monocyte/macrophage activation and differentiation antigens and identify functionally distinct subpopulations of human rheumatoid synovial tissue macrophages rapid increases in the membrane expression of neutral endopeptidase (cdi0), aminopeptidase n (cd13), tyrosine phosphatase (cd4s), and fey-rill (cd16) upon stimulation of human peripheral leucocytes with human csa clinical importance of myeloid antigen expression in adult acute lymphoblastic leukemia. n. eng! aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev human aminopeptidase n is a receptor for human coronavirus 229e endopeptidase-24.11 and aminopeptidase activity in brain synaptic membranes are jointly responsible for the hydrolysis of cholecystokinin octapeptide (cck-8) tab a novel los kd human t cell activation antigen defined by a monoclonal antibody we gratefully acknowledge the expert secretarial assistance of mrs. christel w alcker and thank mrs. rosemarie meinicke for her excellent assistance in these experiments. key: cord-0006833-1t031aoy authors: heller, axel; koch, thea; schmeck, joachim; van ackern, klaus title: lipid mediators in inflammatory disorders date: 2012-11-30 journal: drugs doi: 10.2165/00003495-199855040-00001 sha: ce4bde08cb7e899ebf6061f3d369e81e94087113 doc_id: 6833 cord_uid: 1t031aoy during the past few decades, intensive collaborative research in the fields of chronic and acute inflammatory disorders has resulted in a better understanding of the pathophysiology and diagnosis of these diseases. modern therapeutic approaches are still not satisfactory and shock, sepsis and multiple organ failure remain the great challenge in intensive care medicine. however, the treatment of inflammatory diseases like rheumatoid arthritis, ulcerative colitis or psoriasis also represents an unresolved problem. many factors contribute to the complex course of inflammatory reactions. microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. in the early phase of inflammation, excessive amounts of interleukins and lipid-mediators are released and play a crucial role in the pathogenesis of organ dysfunction. arachidonic acid (aa), the mother substance of the pro-inflammatory eicosanoids, is released from membrane phospholipids in the course of inflammatory activation and is metabolised to prostaglandins and leukotrienes. various strategies have been evaluated to control the excessive production of lipid mediators on different levels of biochemical pathways, such as inhibition of phospholipase a(2), the trigger enzyme for release of aa, blockade of cyclooxygenase and lipoxygenase pathways and the development of receptor antagonists against platelet activating factor and leukotrienes. some of these agents exert protective effects in different inflammatory disorders such as septic organ failure, rheumatoid arthritis or asthma, whereas others fail to do so. encouraging results have been obtained by dietary supplementation with long chain ω-3 fatty acids like eicosapentaenoic acid (epa). in states of inflammation, epa is released to compete with aa for enzymatic metabolism inducing the production of less inflammatory and chemotactic derivatives. inflammatory processes are the physiological response of the organism to different stimuli such as trauma, infections or immunological mechanisms. the inflammatory reaction is characterised by the stimulation of humoural and cellular medi-ator systems and the release of a great variety of inflammatory mediators which result in alterations in microvascular tone and permeability. the products of the classical cascade systems and various other mediators are capable of activating the lipid mediator syntheses which are essentially involved in the regulation of the complex inflammatory reaction. [1] prostaglandins such as prostaglandin (pg) e 2 and pgi 2 (prostacyclin) induce oedema formation (tumour) and vasodilation (rubor), and contribute to the development of hyperalgesia at the site of inflammation (dolor). fatty acid peroxides and leukotrienes additionally increase local permeability and are potent chemoattractants for neutrophil granulocytes, resulting in a further accumulation of phagocytes in the microcirculation. the inflammatory activity is further maintained by platelet activating factor (paf), which is another important lipid mediator stimulating neutrophils and partially influencing the vascular tone and permeability. moreover, paf is a chemotactic agent, which con-tributes to leucocyte-mediated injury in inflammatory processes. the aim of this article is to give a brief update of the pathophysiological impact of lipid mediators in inflammatory disorders and to summarise the recent development of therapeutic approaches. the precursors of eicosanoids are lipid compounds of cellular membranes. the activation of the phospholipase a 2 (pla 2 ) family induces the mobilisation of fatty acids, particularly arachidonic acid (aa), from the membrane lipid pool for the synthesis of lipid mediators at the site of cellular damage or inflammation. in individuals without relevant dietary intake of ω-3 polyunsaturated fatty acids (ω-3-pufas, contained in fish oils), aa is predominantly released from the phospholipid pool of cellular membranes, which is metabolised by 2 major pathways to pro-inflammatory mediators [2] (fig. 1 ). the vaso-and bronchoconstrictive metabolites thromboxane a 2 (txa 2 ) and pgf 2α are produced via the cyclooxygenase (cox) pathway. the txa 2 -induced vaso-and bronchospasm predominates over the relaxing effects of simultaneously generated pgi 2 and pge 2 on smooth muscle cells of vessels and bronchioli. the pattern of eicosanoids formed depends on the enzyme content of the particular cells [3] (fig. 2) . alternatively, aa is metabolised by 5-lipoxygenase, thereby forming chemotactic leukotriene (lt) b 4 and the slow reacting substance of anaphylaxis (srs-a) identified in 1980 as ltc 4 , ltd 4 and lte 4 [4] , which increase capillary permeability. in the case of increased membrane lipid content of ω-3 fatty acids, eicosapentaenoic acid (epa) competes with aa for metabolism via the cox and lipoxygenase pathways. [5] the epa-derived metabolites have lower biological activity [6] than the analogous aa derivatives. in comparison with the aa-derived txa 2 , the epa-derived cox-product of the 3-series txa 3 has considerably reduced proaggregatory and vasoconstrictive properties, while pgi 3 possesses similar antiaggregatory and vasodilative effects to those of pgi 2 . enzymatic conversion of epa generates the 5series leukotrienes (ltb 5 , c 5 , d 5 , e 5 ) which have partially antagonistic biological effects compared with aa derivatives. [7] the vasoconstrictive and chemotactic potency of ltb 5 is 2 orders of magnitude lower than the activity of ltb 4 . [8] in addition, the generation of proinflammatory paf is reduced by epa via interference with the paf precursor pool. [9] figure 1 provides an overview of the experimentally and clinically relevant possibilities of eicosanoid pathway modulation. activation of the pla 2 family is a key step in the production of precursors for the biosynthesis of inflammatory lipid mediators. inhibition of this enzyme could result in the suppression of 3 important classes of inflammatory lipids (pgs, lts and paf), which offers an attractive therapeutic approach to the design of novel agents for the treatment of inflammation and tissue injury. the pla 2 family is a series of enzymes involved in phospholipid catabolism. more than 150 pla 2 amino acid sequences are currently available in protein sequence databases. pla 2 exists in both extracellular and intracellular forms. secretory pla 2 (spla 2 ) are ca ++dependent enzymes (molecular weight 14 kd, regulated by millimolar amounts of ca ++ ) which are classified in 2 groups on the basis of their primary structures. [10] secretory nonpancreatic pla 2 has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic pla 2 contributes to the tissue damage associated with acute pancreatitis. the cytosolic pla 2 (cpla 2 ) is an 85.2 kd enzyme with substrate specificity for aa. it is found in most cells and tissues and is regulated by ca ++ in the micromolar range. [11] cpla 2 plays an important role in both the rapid and the prolonged cellular responses occurring during inflammatory processes. accordingly, in the past few years it has been proposed that pla 2 inhibitors be applied to the control of inflammatory diseases. a tremendous number of inhibitors have been claimed to be inhibitors of pla 2 activity. pla 2 inhibitors are currently being investigated, using classical inflammatory models. [12] table i shows selected inhibitory agents with marked in vivo activity. for adequate treatment it has been found necessary to apply a cell-impermeable pla 2 inhibitor which affects cell activity at the membrane but does not enter the cell. yedgar et al. [13] demonstrated improvement in bleomycin-induced lung injury in hamsters after treatment with carmellose (carboxymethylcellulose) -linked phosphatidylethanolamine. to date, only a few interventional studies with pla 2 antagonists have been performed. baboons which were immunised with a neutralising monoclonal anti pla 2 -ii antibody showed significantly attenuated hypotension after treatment with viable escherichia coli. [14] moreover, lung vascular leakage in rats due to gut ischaemia and reperfusion was blunted. [15] in discussing pla 2 inhibition, it must be kept in mind that not only the formation of subsequent proinflammatory lipid mediators is prevented but also the generation of thromboregulatory, vasodilative and antiaggregatory pgi 2 and pge 2 ( fig. 1 ). to what extent pla 2 inhibitors will reach therapeutic significance in inflammatory diseases will need to be elucidated in further clinical studies. paf, a pla 2 -dependent phospholipid, is an extraordinarily potent mediator of shock [16] and inflammation. it exerts its biological effects by activating the paf receptor, consequently stimulating protein kinase c and increasing intracellular ca ++ . paf is found in most biological fluids and is synthesised in most cell types ( fig. 2 ). microvascular permeability is markedly increased by paf, allowing fluids to leak out of the circulation. in the area of haemodynamic effects, paf is negatively inotropic and lowers arterial blood pressure. it is a potent platelet aggregator and leucocyte activator, and it strongly promotes aa metabolism. it has been proposed that it plays a crucial role in the pathogenesis of rheumatoid arthritis, asthma, endotoxin shock and acute renal transplant rejection. in animal models, paf caused bronchoconstriction and increased airway resistance, pulmonary hypertension and gastrointestinal ulceration. hence, selective paf inhibition was investigated with a variety of paf receptor antagonists, which were used in different animal models of sepsis and gave promising results [17, 18] (table ii) . anti-inflammatory effects of paf antagonists on experimental models of arthritis have been published [19] indicating the modulation of different proinflammatory cytokines. data obtained in a model of immune complex arthritis demonstrated controversial regulatory effects of the paf antagonist bepafant (web-2170) on eicosanoid release. whereas pge 2 and tumour necrosis factor (tnf) levels dropped, ltb 4 release was increased. [20] a clinical trial carried out by hilliquin and co-workers [21] revealed improvement of rheumatoid arthritis after administration of another paf receptor antagonist, ginkgolide b (bn-50730) . dhainhaut et al. [22] performed a placebocontrolled, double-blind study including 262 patients with sepsis syndrome. besides their standard supportive therapy, patients received the paf receptor antagonist bn-52021 twice daily (120mg) over 4 days, or placebo. the overall 28-day mortality in the bn-52021 group did not differ significantly from the placebo group but in the subgroup of patients with gram-negative sepsis bn-52021 significantly reduced mortality (57% placebo vs 33% bn-52021). subsequent studies (n = 608) did not confirm any benefit from paf receptor antagonism on the overall mortality. [23] controversial results have been obtained from asthmatic patients [24, 25] and individuals with psoriasis. [26] decreased serum levels of interleukin (il)-8 and il-6 were detected after treatment with the paf antagonist lexipafant in human acute pancreatitis. [27] after the encouraging animal studies with paf antagonists, the few existing clinical trials in fact demonstrated improvement of rheumatic conditions but did not show beneficial effects on survival rates in sepsis. hence, it remains to be elucidated whether or not paf-receptor antagonists can reach significance in the therapy of the critically ill patient. a variety of substances that inhibit the cox pathway have been investigated, including nonsteroidal anti-inflammatory drugs (nsaids). these are commonly used for the treatment of inflammation, pain and fever. more than 25 nsaids are currently available. these compounds are believed to act via inhibition of the cox enzyme, which catalyses the conversion of aa to the prostaglandins and thromboxane ( fig. 1) . although available nsaids are efficacious anti-inflammatory agents, serious adverse effects limit their use. two forms of cox have been identified, a constitutively expressed cox-1 and a cytokineinducible cox-2. it has been suggested that nsaid toxicity is due to inhibition of cox-1, whereas therapeutic properties derive from cox-2 inhibition at the site of inflammation. [28, 29] therefore, selective cox-2 inhibitors such as sc-58125 may exert anti-inflammatory effects without gastrointestinal toxicity [30] (table iii) . recent clinical trials have evaluated the efficacy of cox-2 inhibitors in the therapy of arthritis, pointing towards sufficient symptom relief with an improved gastrointestinal tolerability. [31] moreover, evidence has accumulated that cox-2 inhibition could suppress the growth of colorectal cancer. [32] studies investigating the effect of cox inhibitors in septic conditions demonstrated that ibuprofen was able to attenuate most of the adverse consequences of endotoxin administration and to prolong survival in an animal model. [33] bernhard et al. [34] recently reported reduced levels of pgi 2 and thromboxane in septic patients after administration of ibuprofen. fever, tachycardia, oxygen consumption and lactic acidosis were blunted, whereas the development of shock, acute respiratory distress syndrome (ards) and mortality rate were unaltered. [34] while ibuprofen not only inhibits inflammatory cox products such as thromboxane but also the vasodilator pgi 2 , another interesting approach is the inhalational application of pgi 2 in the therapy of acute respiratory distress to improve the ventilation-perfusion ratio (v/q) in the lung. [35] aerosolised pgi 2 selectively induces pulmonary vasodilation and redistribution of blood flow from shunt areas to well ventilated regions, thereby increasing oxygenation in severe ards. [35] it appears that pgi 2 is a valuable tool in the therapy of v/q mismatch. the beneficial and adverse effects of cox inhibition suggest the combination of systemic cox-2 inhibition and additional supplementation with aerosolised pgi 2 supplementation in acute lung failure, to control inflammation. a further approach to the selective reduction of cox-induced vasoconstriction and platelet aggregation without blockade of pgi 2 is to antagonise txa 2 synthesis. in a model of coronary thrombosis, guth and müller [36] demonstrated antithrombotic properties of samixogrel (dttx-30se), a combined txa 2 receptor antagonist and synthase inhibitor. [36] thus, exclusive inhibition of txa 2 generation might be a helpful tool in preventing vasoconstriction and platelet aggregation. lts are potent proinflammatory agents involved in the pathophysiology of various inflammatory diseases such as asthma, psoriasis, rheumatoid arthritis and ulcerative colitis. [37] therefore, many anti-lt compounds have been developed which selectively block either lt receptor sites or enzymes related to their biosynthesis. table iv gives a synopsis of these drugs and their effects. both categories of agents have been shown to decrease bronchial obstruction in asthma, [38, 39] suggesting that lts and the 5-lipoxygenase products of aa are involved in sustaining the acute airway response that occurs after bronchial provocation in patients with mild to moderate asthma. lt antagonists and 5-lipoxygenase inhibitors seem to be promising therapeutic tools but they are unlikely to dominate the further therapy of asthma. one of the main advantages for their use is that they can be given orally. [40] nickerson-nutter and medvedeff [41] demonstrated the efficacy of the combination of lt synthesis inhibitors and cyclooxygenase inhibitors in models of rheuma-toid arthritis. with regard to the chemotactic properties of ltb 4 , specific receptor antagonists have been designed to control ltb 4 effects. it has been demonstrated that ly-293111 reduces upregulation of the macrophage and neutrophil adhesion molecule cd11b/cd18. [42] moreover, this ltb 4 receptor antagonist decreased the number of neutrophils in bronchoalveolar lavage fluid of patients with allergen-sensitive asthma. [43] ltb 4 antagonism could be a therapeutic approach in ltb 4dependent disorders, such as ulcerative colitis or psoriasis. to date, no data are available concerning the use of these agents in septic patients. whether the inhibition of lts might be useful as a strategy for controlling proinflammatory lipid mediators will need to be assessed in future clinical studies. after the epidemiological studies of dyerberg et al., [44] in which it was shown that greenland eskimos have a lower incidence of thrombosis, coronary heart disease and myocardial infarction, interest was focused on ω-3-pufas, which are contained in fish oils. numerous studies have been carried out, which showed beneficial properties of ω-3-pufas in various diseases (table v) . [45] in our own studies we demonstrated that parenteral ω-3-pufas were rapidly incorporated into lung tissue and reduced both vascular resistance and endothelial permeability in the pulmonary circulation, thus blunting oedema formation. [46] the beneficial effects of ω-3-pufas seem to be related to the uptake of epa into cellular membranes after dietary or parenteral application, and its subsequent metabolism. in states of inflammation, epa is released to compete with aa for metabolism at the cyclooxygenase and 5-lipoxygenase levels ( fig. 1 ). the metabolites of epa (pentaene-lts, triene-pgs and txa 3 ) have less proinflammatory and chemotactic potency than the substances derived from aa (tetraene-lts, diene-pgs and txa 2 ). considering the massive synthesis of lipid mediators during inflammatory reactions, supplementation of parenteral or enteral feeding with ω-3 fatty acids seems to be a promising therapeutic approach in inflammatory disorders, acting by modulating the lipid mediator spectrum. lipid mediators are 'local mediators' which act in the intercellular microenvironments, where they reach considerable levels. they are released from various tissues and cells and usually develop their effects at the site of production because of their short half-life and rapid enzymatic inactivation. numerous mediators stimulate the lipid mediator cascade generating pro-inflammatory eicosanoids which increase their own synthesis via positive feedback loops. as a consequence, cascade systems which are essential for host defence may become self-perpetuating and independent of the original stimuli, and finally cause tissue damage. research efforts of the past few decades have resulted in the development of therapeutic interventions at different sites of the inflammatory reaction. to date, a broad spectrum of different enzyme inhibitors and receptor antagonists has been investigated, showing a variety of effects on the course of diseases. animal studies and in vitro investigations have revealed important beneficial anti-inflamma-tory and immune modulating properties of lipid mediator antagonism. inflammation and critical illness is a multifactorial process. thus, it seems unlikely that pharmacological intervention to modulate the release of lipid mediators will be the magic bullet to improve patient outcome. the research efforts of recent years, however, contribute to a better understanding of the pathophysiological impact of lipid mediators in inflammatory disorders and offer new therapeutic approaches. differential regulation of cytokine receptor genes by paf, ltb4 and pge2 fish oil fatty acids and cardiovascular function: epidemiology and biochemical mechanisms activation of the pulmonary arachidonic acid system and its consequences for hemodynamics and fluid balance pt a: pathophysiological role of mediators and mediator inhibitors in shock slow-reacting substance of anaphylaxis: identification of leukotriene c-1 and d from human and rat sources prostaglandin i is formed in vivo in man after dietary eicosapentaenoic acid characterization of leukotriene b3: comparison of its biological activities with leukotriene b4 and leukotriene b5 in complement receptor enhancement, lysozyme release and chemotaxis of human neutrophils effects of omega-3 fatty acids on the generation of products of the 5-lipoxygenase pathway human neutrophil chemotactic and degranulating activities of leukotriene b (ltb) derived from eicosapentaenoic acid the effects of n-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes phospholipase a2: a structural review recent insight into the structure, function and biology of cpla2 cellular and topical in vivo inflammatory murine models in the evaluation of inhibitors of phospholipase a2 control of inflammatory processes by cell impermeable inhibitors of phospholipase a2 neutralising monoclonal antibody to human secretory phospholipase a2 is anti-hypotensive in a baboon e. coli induced septic shock model phospholipase a2 inhibition decouples lung injury from gut ischemia-reperfusion the potential role of platelet activating factor in shock and ischemia in vivo inhibition of plasma protein leakage and salmonella enteritidis-induced mortality in the rat by a specific platelet activating factor acether antagonist, bn52021 effect of platelet activating factor receptor antagonism on endotoxin-induced lung dysfunction in awake sheep anti-inflammatory effect of a paf receptor antagonist and a new molecule with antiproteinase activity in an experimental model of acute urate crystal arthritis paf modulates eicosanoids and tnf release in immune complex arthritis in rats treatment of rheumatoid arthritis with platelet activating factor antagonist bn50730 platelet activating factor receptor antagonist bn52021 in the treatment of severe sepsis: a randomized, double blind, placebo controlled, multicenter clinical trial treatment with the platelet activating factor antagonist tcv-309 in patients with severe systemic inflammatory response syndrome: a prospective, multi-center, double-blind, randomized phase ii trial effects of a potent platelet activating factor antagonist, sr27417a, on allergen-induced asthmatic responses effects of a paf antagonist, y-24180, on bronchial hyperresponsiveness in patients with asthma the effect of topical application of the platelet activating factor antagonist ro24-0238, in psoriasis vulgaris: a clinical and immunohistochemical study randomized, double blind phase ii trial of lexipafant, a platelet activating factor antagonist, in human acute pancreatitis cox-2 ho no! cyclooxygenase-2, hemoxygenase and nitric oxide synthase: their role and interactions in inflammation analgesic activity of the novel cox-2 preferring nsaid, meloxicam in monoarthritic rats: central and peripheral components gastrointestinal damage associated with the use of non-steroidal antiinflammatory drugs meloxicam: selective cox-2 inhibition in clinical practice a selective cyclooxygenase 2 inhibitor suppresses the growth of h-ras-transformed rat intestinal epithelial cells ibuprofen in animal models of septic shock the effects of ibuprofen on the physiology and survival of patients with sepsis inhaled prostacyclin (pgi2) versus inhaled nitric oxide in adult respiratory distress syndrome dttx30, a combined thromboxane receptor antagonist and thromboxane synthetase inhibitor, prevents coronary thrombosis in anesthetized dogs clinical activity of leukotriene inhibitors the pivotal role of 5-lipoxygenase products in the reaction of aspirin-sensitive asthmatics to aspirin effect of cysteinyl-leukotriene receptor antagonist ici204-219 on allergen-induced bronchoconstriction and airway hyperreactivity in atopic subject obstructive airway disease: unmet needs. meeting report the effect of leukotriene synthesis inhibitors in models of acute and chronic inflammation inhibition of ex vivo neutrophil activation by oral ly293111, a novel leukotriene b4 receptor antagonist effect of a leukotriene b4 antagonist, ly293111, on allergen induced responses in asthma fatty acid composition of the plasma lipids in greenland eskimos omega-3 fatty acids as adjuvant therapy in inflammatory diseases alteration of n-3 fatty acid composition in lung tissue after short-term infusion of fish oil emulsion attenuates inflammatory vascular reaction key: cord-0029277-1epj9xy2 authors: gray, anna l.; pun, nabina; ridley, amanda j. l.; dyer, douglas p. title: role of extracellular matrix proteoglycans in immune cell recruitment date: 2022-01-25 journal: int j exp pathol doi: 10.1111/iep.12428 sha: def5d8f17dc0c5b0fe216c7c54cf8e6dd0c05174 doc_id: 29277 cord_uid: 1epj9xy2 leucocyte recruitment is a critical component of the immune response and is central to our ability to fight infection. paradoxically, leucocyte recruitment is also a central component of inflammatory‐based diseases such as rheumatoid arthritis, atherosclerosis and cancer. the role of the extracellular matrix, in particular proteoglycans, in this process has been largely overlooked. proteoglycans consist of protein cores with glycosaminoglycan sugar side chains attached. proteoglycans have been shown to bind and regulate the function of a number of proteins, for example chemokines, and also play a key structural role in the local tissue environment/niche. whilst they have been implicated in leucocyte recruitment and inflammatory disease, their mechanistic function has yet to be fully understood, precluding therapeutic targeting. this review summarizes what is currently known about the role of proteoglycans in the different stages of leucocyte recruitment and proposes a number of areas where more research is needed. a better understanding of the mechanistic role of proteoglycans during inflammatory disease will inform the development of next‐generation therapeutics. response. 3, 4 once leucocytes have been recruited from the vasculature, they then undergo further migration to achieve distinct positions within the tissue. this process occurs during inflammation to facilitate the removal of invasive pathogens and infected cells by the innate and adaptive immune response. generally speaking, eosinophils, basophils, neutrophils and monocytes are recruited early in the immune response followed later by t and b cells. leucocytes (primarily monocytes/macrophages) are also key during the resolution of inflammation and associated tissue damage. whilst the process of leucocyte recruitment and positioning via transendothelial migration has been well studied, the role of the extracellular matrix, in particular proteoglycans, is often overlooked. in this review, we will summarize what is known about the mechanistic and functional roles of proteoglycans in the process of leucocyte recruitment and also highlight gaps in our knowledge where further research is needed. proteoglycans consist of proteins cores, either embedded in a cell membrane or soluble with sulphated glycosaminoglycan sugar side chains attached ( figure 1 ). 1, 5 membrane-embedded protein cores consist of the syndecan and glypican families, whilst the soluble proteoglycans consist of serglycin, agrin, perlecan and collagen xviii. these proteoglycan structures are found within the extracellular matrix of tissues, on the surface of the majority of mammalian cells, and are particularly prevalent on the surface of the endothelium lining blood vessels as part of the glycocalyx, and also within the basement membrane. 6 we currently do not have much insight into the potential differential function of proteoglycans in these two distinct environments, that is luminal glycocalyx vs basement membrane. the glycosaminoglycan (gag) sugars that are present as side chains on the protein cores of proteoglycans are made up of repeating disaccharide units that repeat to form long unbranched (linear) chains. 7 both heparan sulphate (hs) and chondroitin sulphate (cs) gags play critical roles in leucocyte recruitment and positioning. 2 whilst less studied in this context, dermatan sulphate (ds) may also contribute to this process via its ability to bind to relevant proteins, for example chemokines, albeit much weaker when compared to hs. 8 heparan sulphate is made up of repeating units of glucuronic acid (glca) [which can be epimerized to iduronic acid (idoa) during assembly] and n-acetylglucosamine (glcnac). 7 the cs chain sequence is slightly different and is made up of repeating units of glucuronic acid and n-acetylgalactosamine (galnac). 9 the ds sequence backbone is the same as cs but with some glucuronic acid residues epimerized to become iduronic acid. gag chains are acidic and are also hydrophilic; thus, when present together they form a hydrated and 'soft' gel-like structure ( figure 1b) . 5 this is particularly the case on the endothelial glycocalyx that f i g u r e 1 proteoglycans regulate leucocyte recruitment. (a) proteoglycans are composed of a protein core, depicted here embedded in a cell membrane as is the case with the syndecan family. proteoglycans have glycosaminoglycan (gag) side chains that can bind to a number of proteins, for example chemokines. (b) proteoglycans on the surface of both endothelial cells and leucocytes regulate interactions between the two by masking adhesion molecules. created with biore nder.com lines blood vessels and regulates leucocyte recruitment and inflammation by the mechanisms described below. glycosaminoglycan chains are sulphated at various points, in the case of hs (the dominant proteoglycan gag chain in leucocyte recruitment), n-and 6-o sulphation on the glucosamine, 2-o sulphation on the iduronic acid and more rarely 3-o sulphation on the glucosamine. 7,10 in contrast, cs can be 2-o-sulphated on the glucuronic acid and 4-o-and/or 6-o-sulphated on the n-acetylgalactosamine residue. 9 ds is more commonly 2-o-sulphated than cs, due to the epimerized iduronic acid residue. these sulphation points are critical to the ability of hs and cs gag chains to interact with a range of different ligands, many of which are important in the leucocyte migration process (discussed below). a range of literature has dissected the roles of gag sulphation points in interactions with different ligands; however, there is still much to learn about the potential specificity of these interactions and the effect this may have during leucocyte recruitment in vivo. new tools to analyse the contribution of specific sulphation points to gag interactions in a cellular context combined with increasing ability to sequence gags purified from biological contexts will significantly develop this field in the coming years. [11] [12] [13] 3 | endothelial the vascular endothelium constitutes a monolayer of endothelial cells, which comprise the innermost cellular lining of blood vessels. these cells serve a variety of important functions, which include the coordination of inflammation and immune responses. 14 the vascular endothelial surface is coated with a carbohydrate-rich matrix called the glycocalyx, which in the past has been somewhat overlooked, in the context of disease. the endothelial glycocalyx is a negatively charged, membrane-bound layer of proteoglycans and glycoproteins, which line the luminal surface of blood vessels. this protective structure was first visualized following the invention of transmission electron microscopy in the 1960s. 15 at the luminal surface of the endothelium, syndecans and glypicans (membrane-bound) and serglycin and agrin (soluble) are thought to be the dominant proteoglycans. 5 the endothelial glycocalyx also contains hyaluronan, which is non-sulphated and is anchored by the cd44 receptor, and the hyaluronic acid synthase enzyme. some additional glycocalyx glycoproteins have a sialic acid or a fucose cap, which serve important functions in coagulation (eg, selectins, integrins and immunoglobulins) or act as endothelial adhesion molecules. additionally, there are both endothelial-and plasma-derived soluble components incorporated into this matrix. due to the physical properties of its components, the glycocalyx forms a thick and hydrated barrier. the intact glycocalyx is too thick to allow interaction between adhesion molecules on leucocytes and the endothelium ( figure 1b) , a key component of leucocyte migration. [16] [17] [18] [19] even when projected onto endothelial protrusions, the mass of a leucocyte is insufficient to facilitate penetration of the glycocalyx. 20 the inhibition of the first step in leucocyte recruitment by glycocalyx proteoglycans means there is missing mechanistic understanding at the heart of leucocyte recruitment that has yet to be resolved, that is how do leucocytes interact with the endothelium in the context of the glycocalyx? a number of studies have demonstrated mechanisms whereby the glycocalyx can be remodelled to facilitate leucocyte:endothelial interactions. 5 for example, injection of the chemoattractant formylmethionyl-leucylphenylalanine (fmlp) was shown to mediate alterations in glycocalyx structure that promoted leucocyte rolling on the endothelium. 21 further studies demonstrated that this was by induction of heparinase leading to shedding of hs gags from proteoglycans facilitating access to the endothelial adhesion molecules, for example intercellular adhesion molecule 1 (icam-1). 22 more recent studies have demonstrated a similar mechanism whereby the inflammatory cytokine tumour necrosis factor (tnf) can induce heparinase-mediated glycocalyx shedding during sepsis. 18, 23 this was shown to facilitate recruitment of neutrophils and also induce side effects in the brains of sepsis patients by the gag-binding protein brain-derived neurotrophic factor (bdnf). 24 in recent years, the integral role of the glycocalyx in determining cardiovascular health and disease has been established. 25 in chronic conditions such as rheumatoid arthritis, diabetes, sepsis, atherosclerosis and ischaemia/ reperfusion injury, leucocyte recruitment can be excessive and detrimental. the presence of the glycocalyx constituents within the blood plasma can act as a biomarker for disease, since glycocalyx shedding occurs in numerous disease pathologies. specifically shedding of syndecans (major glycocalyx components), for example syndecan-1 by matrix metalloproteinases, into the circulation has been proposed as a marker of inflammation. 26 importantly, changes to the resulting composition of the glycocalyx can also drive disease through the exposure of adhesion sites, and thus the facilitation of leucocyte transmigration. whilst these studies, and others, have begun to develop our understanding of changes to proteoglycans during inflammation and leucocyte recruitment, a number of outstanding questions remain. for example how does the shedding of the glycocalyx fit with the studies demonstrating that proteoglycans are also essential for leucocyte recruitment (detailed later)? presumably during inflammation, there is a balance between shedding and retention of proteoglycans that must exist to remove sufficient glycocalyx to not only allow leucocyte:endothelial interaction but also allow the pro-migratory functions of proteoglycans. one issue that affects our understanding of the role of proteoglycans in leucocyte recruitment is their distribution and heterogeneity within the glycocalyx across different vascular beds, that is arterial vs venous blood vessels. it has been suggested that glycocalyx thickness increases with vessel diameter, at least in the arterial system where the matrix is more substantial. 27 the estimation of glycocalyx thickness is variable due to its sensitivity to processing and the differential methods used in analysis. 28 however, it seems likely that glycocalyx morphology is different between vessel types, with the thinnest measurements recorded in capillaries and venules, ranging from 0.2 to 0.5 μm. 27 whereas a glycocalyx of small arteries extends 2-3 μm, a glycocalyx of larger arteries extends up to 4.5 μm. furthermore, evidence suggests that the glycocalyx differs between the same vessel types of different organs. for instance, capillaries in the brain, heart and lung are all considered continuous with complete glycocalyx covering. however, the glycocalyx of cerebral capillaries is thicker than that of cardiac and pulmonary capillaries, likely due to its important contribution to the blood-brain barrier (bbb). 27 since the majority of leucocyte recruitment from the vasculature and into tissues is thought to occur within post-capillary venules, it is possible that the glycocalyx is thinner and thus more permissive to leucocyte:endothelial interactions at this site. 3, 5 it seems highly likely that the proteoglycan content and structure of the glycocalyx will differ across vascular beds and also across different tissues. future studies are needed to specifically define proteoglycan content and structure of the glycocalyx at different vascular beds within different tissues, before and after inflammation and leucocyte recruitment, to comprehensively understand this process. in addition to overall proteoglycan structure and content of the vascular system, the specific mechanistic role, and geographical location, of gag sulphation is also likely to be important. 7, 29 gags can be modified to have the sulphation points described above, and specific patterns likely mediate specific interactions within different ligands involved in leucocyte recruitment (discussed below). whilst the sulphation of gags across tissues and species has been shown to be specific, 30 we still have little information of how this varies across vascular beds and in response to different inflammatory stimuli. thus, future studies will also need to better address the specifics in changes to gag sulphation in defined geographical and inflammatory contexts. such approaches now seem increasingly feasible following recent technological advances in gag analysis. 12, 13 as mentioned above, a number of studies have demonstrated that the application of factors to induce shedding of proteoglycans and their gag chains has been shown to mediate increased rolling and migration of leucocytes from the vasculature. 5 in contrast, other studies have shown that proteoglycans are required for leucocyte migration, where their removal actually reduces leucocyte recruitment. 31, 32 there are likely a number of ways in which proteoglycans promote leucocyte recruitment. one of the most well-studied functions of proteoglycans during leucocyte recruitment is their ability to interact with chemokines, whose primary function is to facilitate firm adhesion of leucocytes to the endothelium. 33 chemokine:proteoglycan interactions within the basement membrane are likely also key in leucocyte recruitment and trafficking. chemokines bind to their receptors on circulating leucocytes to induce signalling events that result in integrin activation and thus firm adhesion to the endothelium. a number of years ago, it was shown that mutation of ccl2, ccl4 and ccl5, so that they could no longer bind to gags, ablated their ability to mediate leucocyte recruitment to the peritoneum of mice. 34 subsequently, a range of studies have demonstrated a similar function for other chemokines and have demonstrated a clear hierarchy in the ability of chemokines to bind to gags. [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] a number of studies have also explored the potential specificity in binding to different sulphation patterns on gags. 45, 46 indeed given the therapeutic potential in targeting chemokines, the chemokine:gag interaction is an ongoing focus for potential new therapeutics to target inflammatory disease. 47 despite this range of research, the mechanistic importance of chemokine:gag interactions has yet to be fully understood. 48 it seems likely that interaction with gags within the luminal glycocalyx is important to retain chemokines at inflammatory sites in the presence of blood flow. however, there is very limited evidence for the common assertion that gags facilitate formation of chemokine gradients within the vasculature, 49 where gradient formation has more commonly been observed within the lymphatic system or tissues in the absence of blood flow. 50 ccl19 and ccl21 have been shown to have specific functions, by their differential interactions with hs and cs proteoglycans, in trafficking of dendritic cells within the lymphatic system. [50] [51] [52] [53] furthermore, given recent discoveries on the importance of self-generated gradients there remains the exciting possibility that gags are important in this process both within tissues and the vasculature. 54 the other mechanistic role of chemokine:gag interactions is in protection from proteolysis, 55,56 this again seems likely to be important in chemokine-mediated leucocyte recruitment. given that these interactions will result in both bound and non-bound chemokine at any given time, the cloud hypothesis has recently been proposed. 48, 57 this states that gags mediate retention of a local cloud of soluble chemokine that is available to bind to circulating leucocytes within the vasculature to facilitate their firm adhesion. more recently, a number of studies, including our own, have demonstrated that as well as binding to gags certain chemokines, for example cxcl4 and cxcl12, can remodel the structure of hs. 39, 44 this involved cross-linking of individual gag chains to render them less mobile within a biophysical model of the cell membrane lipid bilayer. cross-linking also resulted in a reduced thickness of the glycocalyx-like structures formed by hs gag chains. work is now ongoing in our laboratory to determine the biological function of these remodelling events; for example, can chemokines bind and alter the endothelial glycocalyx structure on blood vessels to increase its permeability and enable leucocyte recruitment in vivo? as with the other areas of proteoglycan function in leucocyte recruitment, we are only at the beginning of our understanding of this complex biological problem. exciting technological developments will be at the heart of future studies to understand the mechanistic function of chemokine:gag interactions. for example why do chemokines exhibit such a wide range of affinities for gags, why can certain chemokines modify gag structure and what is the role of specific gag sulphation patterns in chemokine function? 48 numerous studies have highlighted the importance of sialyl-lewis x (sle x ) as a selectin ligand in the interactions between leucocytes and endothelial cells during leucocyte migration. [58] [59] [60] another direct function of proteoglycans, by their gag side chains, in facilitating leucocyte recruitment is their interaction with leucocyte adhesion molecules, for example selectins. the ability of l (leucocyte)-and p (platelet)-selectins but not e (endothelial)-selectins, to bind gags, has been demonstrated. 58 p-selectin has also been shown to bind to cs, in a model of metastatic breast cancer, where it is involved in facilitating tumour cell adhesion to platelet and endothelial cells, promoting tumour metastasis. 59 this interaction has been shown to play a role in selectin-dependent cell adhesion, for example neutrophil and monocyte rolling on the endothelium. 60-64 whilst much less appreciated than specific chemokine interactions, there is a wide range of literature demonstrating that proteoglycans, via gags, can also bind to, and modulate the function of, cytokines more generally. given that many of these cytokines are pro-inflammatory, these interactions again play an important, if less direct, role in leucocyte recruitment. glycosaminoglycans (gags) have been shown to bind to interferon-γ (ifn-γ) with high affinity, comparable to higher affinity chemokine:gag interactions. 65 ifn-γ is a cytokine that plays a key role in the complex immune response to infection, in particular by viruses, and as such plays an important role in leucocyte recruitment, for example by inducing production of the chemokines cxcl9, cxcl10 and cxcl11. 66 the ifn-γ:gag interaction has been shown to reduce signalling of this cytokine through its receptor, 65 suggesting overlapping binding sites on ifn-γ for its receptor and gags. thus, it seems likely the function of this interaction is independent of signalling, in contrast to the fibroblast growth factor (fgf) system. 65 however, gags can also promote ifn-γmediated outcomes, suggesting that this interaction may facilitate the function of this cytokine through a currently undefined mechanism. furthermore, a number of cytokines may bind to gags, for example il-2, il-5, il-6, il-7, il-12 and il-27, within the tissue extracellular matrix. 2 various members of the transforming growth factor-β (tgf-β) cytokine superfamily have been shown to contain heparin binding sites; for example, tgf-β1 and tgf-β2 are described to bind hs pgs. 67 although the effect of hs:tgf-β interactions on cytokine activity has not been fully elucidated, it could be speculated that as these cytokines are comparatively small, hs binding may interfere with tgf-β receptor signalling. 67 the function of proteoglycan:cytokine interactions remains unclear; however, it seems likely that they are important in cytokine localization, in protection from proteolysis and in regulation of signalling through their receptors. the effects of these interactions on leucocyte recruitment are indirect in that these cytokines are involved in the inflammatory process that results eventually in recruitment of leucocytes. this again highlights that further mechanistic work is needed to understand the role of proteoglycans in regulation of cytokine function and immunology more widely. toll-like receptors (tlrs) are transmembrane receptors with a critical role in the activation of the innate immune response through recognition of pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps). 68 this process is key in signalling to the immune system to recruit leucocytes to sites of infection to fight pathogenic agents. hs and cs gags have been shown to act as damps by signalling through tlr4. 69, 70 heparan sulphate can activate tlr4 on dendritic cells, in vitro, producing dendritic cell (dc) activation and alloreactive t-cell responses. 71 tlr4-dependent hs signalling has also been shown to mediate recruitment of neutrophils to the pancreas. 72 given the presence of hs gags on the endothelial surface and within the tissue extracellular matrix, it would make sense that their shedding during disease would be an important signal to the immune system that pathogens are present within the vasculature and surrounding tissues. there are still a number of questions around the role of proteoglycans as damps and in facilitating leucocyte recruitment that can now be explored using the advancing tools in the area. whilst the majority of the research understanding the role of proteoglycans in leucocyte recruitment is within the context of the endothelial luminal glycocalyx, we are now beginning to understand the function of proteoglycans on leucocytes themselves. a number of studies have demonstrated the presence of proteoglycans, either directly or indirectly, on the surface of neutrophils, monocytes, macrophages, mast cells and t cells. indeed, surface proteoglycans are required for entry of viruses into cells such as leucocytes, including sars-cov-2. [73] [74] [75] the proteoglycan syndecan-1 has been found to be expressed at higher levels on leucocytes during inflammation and disease. for instance, neutrophils and plasma cells from patients with type 2 diabetes or systemic lupus erythematosus (sle) have been shown to enhance their expression of syndecan-1, relative to healthy controls. 76, 77 genetic ablation of the proteoglycan syndecan-1 in monocytes and neutrophils reduces their ability to adhere to endothelial cells in vitro. 78 an early study demonstrated that acidic mucopolysaccharides, resembling chondroitin sulphate, could be isolated from human leucocytes. 79 further studies have then gone on to report that human leucocytes can indeed synthesize and secrete glycosaminoglycans, 80 with chondroitin 4-sulphate being thought to represent the major component. 81 furthermore, there is indirect evidence for proteoglycans on the surface of t cells; the entry of human t-cell leukaemia virus (htlv) into cd4 + t cells 74 serglycin has been demonstrated to facilitate storage granule formation in mast cells 82 and t cells. 83 proteoglycans have also been detected on b cells where they go through structural changes during development and play a role in the survival of long-lived plasma cells. [84] [85] [86] eosinophils have been shown to have cell surface proteoglycans that change in response to cytokine stimulation. 87 cxcl8, which can bind to gags, has been shown to bind to the surface of neutrophils, where gag-mediated inhibition of this interaction reduced in vitro chemotaxis of neutrophils and reactive oxygen species (ros) production. 88 additionally, enzymatic removal of gags has also been shown to ablate migration of neutrophils, in vitro. 31 whilst the mechanism underlying these observations is unclear, the authors propose that interactions between the chemokines cxcl8 and proteoglycans on the leucocyte surface promotes locomotion by creating local stores of ligand. this mechanism would provide a currently overlooked understanding of chemokine function in addition to binding to proteoglycans on the endothelial surface. indirect evidence from a number of papers has suggested that proteoglycans are on the surface of monocytes and may be functional. the gag-binding chemokines cxcl4 and ccl5 can both bind to the monocyte surface. 89 the wider presence and function of proteoglycans on the leucocyte surface is an exciting research topic with huge potential for a better understanding of leucocyte recruitment and potential therapeutic targeting of inflammatory-based disease. it seems highly likely that proteoglycans on the leucocyte surface have a key role in fighting infection, given that the glycocalyx represents the first part of a cell that any intracellular pathogens encounter. the proteoglycans, within the glycocalyx, play an important role during inflammation and represent a potential therapeutic target during disease. together, these studies emphasize that proteoglycans are key modulators of leucocyte recruitment and positioning, as well as the wider immune response. however, this review also highlights that much more research is needed to improve our current understanding regarding the architecture, expression patterns and functional role of proteoglycans in inflammation and disease. greater understanding will facilitate better targeted therapeutic interventions, such as gag mimetics, in inflammatory diseases such as rheumatoid arthritis. we are now at an exciting time in this field where development of new technologies, particularly gag analytics, will facilitate new and exciting discoveries in the near future. we would like to acknowledge the british society for matrix biology and the estate of professor john scott for the bsmb early career research award to dpd. douglas p. dyer https://orcid.org/0000-0001-5567-6241 an introduction to proteoglycans and their localization heparan sulfate as a regulator of inflammation and immunity leukocyte migration into inflamed tissues neutrophils cascading their way to inflammation role of the endothelial surface layer in neutrophil recruitment proteomic definitions of basement membrane composition in health and disease demystifying heparan sulfate-protein interactions interaction of chondroitin sulfate and dermatan sulfate from various biological sources with heparin-binding growth factors and cytokines biosynthesis and function of chondroitin sulfate heparan sulfate 3-o-sulfation: a rare modification in search of a function the gagome: a cell-based library of displayed glycosaminoglycans shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides dissecting structurefunction of 3-o-sulfated heparin and engineered heparan sulfates vascular endothelial cell biology: an update fine structures of capillary and endocapillary layer as revealed by ruthenium red role of the glycocalyx as a barrier to leukocyteendothelium adhesion the endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases the pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis endothelial cell glycocalyx modulates immobilization of leukocytes at the endothelial surface biomechanics of leukocyte rolling inflammation-and ischemiainduced shedding of venular glycocalyx role of glycocalyx in leukocyteendothelial cell adhesion structural behavior of the endothelial glycocalyx is associated with pathophysiologic status in septic mice: an integrated approach to analyzing the behavior and function of the glycocalyx using both electron and fluorescence intravital microscopy circulating heparan sulfate fragments mediate septic cognitive dysfunction the endothelial glycocalyx as a double-edged sword in microvascular homeostasis and pathogenesis syndecans in inflammation at a glance brain-specific ultrastructure of capillary endothelial glycocalyx and its possible contribution for blood brain barrier the endothelial glycocalyx: composition, functions, and visualization understanding the mechanisms that facilitate specificity, not redundancy, of chemokine mediated leukocyte recruitment isolation and characterization of heparan sulfate from various murine tissues a requirement for neutrophil glycosaminoglycans in chemokine: receptor interactions is revealed by the streptococcal protease spycep inflammatory chemokines direct and restrict leukocyte migration within live tissues as glycan-bound gradients a guide to chemokines and their receptors glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines a non-glycosaminoglycan-binding variant of cc chemokine ligand 7 (monocyte chemoattractant protein-3) antagonizes chemokine-mediated inflammation identification and characterization of a glycosaminoglycan recognition element of the c chemokine lymphotactin the monomer-dimer equilibrium and glycosaminoglycan interactions of chemokine cxcl8 regulate tissue-specific neutrophil recruitment oligomerization of cxcl10 is necessary for endothelial cell presentation and in vivo activity differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization multiple glycosaminoglycan-binding epitopes of monocyte chemoattractant protein-3/ccl7 enable it to function as a non-oligomerizing chemokine the anti-inflammatory protein tsg-6 regulates chemokine function by inhibiting chemokine/glycosaminoglycan interactions the dependence of chemokine-glycosaminoglycan interactions on chemokine oligomerization tsg-6 inhibits neutrophil migration via direct interaction with the chemokine cxcl8 cytokines and growth factors cross-link heparan sulfate enrichment of two isomeric heparin oligosaccharides exhibiting different affinities toward monocyte chemoattractant protein-1 synthetic heparan sulfate dodecasaccharides reveal single sulfation site interconverts cxcl8 and cxcl12 chemokine biology targeting chemokineglycosaminoglycan interactions to inhibit inflammation perspectives on the biological role of chemokine: glycosaminoglycan interactions a chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils interstitial dendritic cell guidance by haptotactic chemokine gradients immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells blood vessels pattern heparan sulfate gradients between their apical and basolateral aspects shape and function of interstitial chemokine ccl21 gradients are independent of heparan sulfates produced by lymphatic endothelium seeing around corners: cells solve mazes and respond at a distance using attractant breakdown structures of human ccl18, ccl3, and ccl4 reveal molecular determinants for quaternary structures and sensitivity to insulin degrading enzyme glycosaminoglycans regulate cxcr3 ligands at distinct levels: protection against processing by dipeptidyl peptidase iv/cd26 and interference with receptor signaling leukocyte adhesion: reconceptualizing chemokine presentation by glycosaminoglycans differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. implications for the use of unfractionated and low molecular weight heparins as therapeutic agents chondroitin sulfate glycosaminoglycans as major p-selectin ligands on metastatic breast cancer cell lines heparin oligosaccharides bind l-and p-selectin and inhibit acute inflammation endothelial heparan sulfate deficiency impairs l-selectin-and chemokine-mediated neutrophil trafficking during inflammatory responses heparan sulfate on activated glomerular endothelial cells and exogenous heparinoids influence the rolling and adhesion of leucocytes monocyte adhesion to activated aortic endothelium: role of l-selectin and heparan sulfate proteoglycans transendothelial migration of effector t cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans the heparan sulfate binding sequence of interferon-gamma increased the on rate of the interferon-gamma-interferon-gamma receptor complex formation cxcr3 ligands: redundant, collaborative and antagonistic functions heparin, heparan sulphate and the tgf-β cytokine superfamily toll-like receptors in the vascular system: sensing the dangers within heparan sulfate proteoglycans as relays of neuroinflammation glycosaminoglycans reduced inflammatory response by modulating toll-like receptor-4 in lps-stimulated chondrocytes heparan sulfate, an endogenous tlr4 agonist, promotes acute gvhd after allogeneic stem cell transplantation tlr4 dependent heparan sulphate-induced pancreatic inflammatory response is irf3-mediated human herpesvirus 8 interaction with target cells involves heparan sulfate heparan sulfate proteoglycans mediate attachment and entry of human t-cell leukemia virus type 1 virions into cd4+ t cells sars-cov-2 infection depends on cellular heparan sulfate and ace2 elevated serum level of circulating syndecan-1 (cd138) in active systemic lupus erythematosus enhanced syndecan-1 expression on neutrophils in patients with type 2 diabetes mellitus increased leukocyteendothelial interactions in syndecan-1-deficient mice involve heparan sulfate-dependent and -independent steps the occurrence of acid mucopolysaccharides in human leukocytes and urine biosynthesis of glycosaminoglycans by thymic lymphocytes. effects of mitogenic activation isolation and characterization of glycosaminoglycans from human leukocytes and platelets serglycin is essential for maturation of mast cell secretory granule serglycin-deficient cytotoxic t lymphocytes display defective secretory granule maturation and granzyme b storage modulated glycosylation of proteoglycans during differentiation of human b lymphocytes heparan sulfate proteoglycans in the control of b cell development and the pathogenesis of multiple myeloma syndecan-1 and stromal heparan sulfate proteoglycans: key moderators of plasma cell biology and myeloma pathogenesis characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor the influence of glycosaminoglycans on il-8-mediated functions of neutrophils heterophilic interactions of platelet factor 4 and rantes promote monocyte arrest on endothelium key: cord-0002419-5h6monh3 authors: lee, seung hoon; park, jin-sil; byun, jae-kyung; jhun, jooyeon; jung, kyungah; seo, hyeon-beom; moon, young-mee; kim, ho-youn; park, sung-hwan; cho, mi-la title: pten ameliorates autoimmune arthritis through down-regulating stat3 activation with reciprocal balance of th17 and tregs date: 2016-10-06 journal: sci rep doi: 10.1038/srep34617 sha: 131e6a035170ed5bd2dba6bec02649e9508a0c55 doc_id: 2419 cord_uid: 5h6monh3 pten is a tyrosine phosphatase with significant function in inhibiting stat3 activation. recently, inactivation of stat3 has been demonstrated as a therapeutic candidate for autoimmune arthritis. the expression of pten controlled by p53 regulates autoimmune arthritis through modulating the balance between th17 and treg. we hypothesized that pten regulated by p53 might reduce cia severity and inflammatory response via inhibiting stat3 activation. our results revealed that pten could ameliorate experimental autoimmune arthritis by reducing stat3 activity and th17 differentiation. systemic infusion of pten overexpression downregulated cia severity. in addition, pten overexpression decreased the activation of t cells and modulated reciprocal differentiation of th17 and treg cells. we observed that pten expression downregulated by p53 deficiency induced the activation of stat3. loss of p53 exacerbated autoimmune arthritis and dysregulated the population of th17 and treg. these data suggest that induction of stat3-modulatory activity of pten may be a therapeutic target for rheumatoid arthritis therapy. spontaneous inflammatory disorder 14 . pten production is associated with tumor protein p53 involving in the reduction of autoimmune inflammatory response 15 . it has been demonstrated that transcription of pten is controlled by p53 16 . we hypothesized that pten could attenuate the development of autoimmune arthritis by reducing stat3 activation and th17 cells differentiation. previously, we have reported that p53 could control autoimmune arthritis through stat3 mediated balance between th17 and treg 17 . the present study was conducted to identify whether pten had therapeutic potential related to p53 in autoimmune arthritis. thus, we evaluated the therapeutic efficacy of pten in experimental autoimmune arthritis. overexpression of pten ameliorates cia development. to determine whether pten had anti-arthritic effect, cia induced mice were injected with either pten overexpression or mock vector once weekly. pten overexpression significantly downregulated the severity of arthritis in cia induced mice (fig. 1a) . the concentrations of total igg, igg1, and igg2a in the serum were significantly decreased in mice injected with pten overexpression compared to mock group (fig. 1b) . pten overexpression significantly reduced the degree of inflammation, bone damage, and cartilage damage (fig. 1c) . immunohistochemical analysis revealed that injection with pten overexpression vector significantly suppressed the expression of proinflammatory cytokines and osteoclastogenesis related factor such as rankl and trap in joints compared to cia mice treated with mock vector (fig. 1d,e) . our results suggested that pten overexpression could suppress cia severity, thus reducing inflammatory response and osteoclastogenesis in joint. to determine whether pten overexpression could attenuate dysregulated balance between th17 and treg, we examined the differentiation of th17 and treg in cia mice. the overexpression of pten reduced th17 differentiation in the spleen tissues of cia induced mice. however, the differentiation of treg cell was promoted in cia induced mice injected with pten overexpression vector ( fig. 2a) . it is well documented that t cell activation is involved in the pathogenesis of ra 18 . in mixed lymphocyte reaction (mlr), the alloreactive t cell response was decreased in lbrm transfected with pten overexpression compared to that in the control (fig. 2b) . additionally, pten overexpression decreased the number of il-17 producing cd4 + p-stat705 + or p-stat727 + t cells. however, the number of cd4 + p-foxp3 + t cells in the spleen tissues of cia induced mice was significantly upregulated compared to that in the spleen tissues of mice treated with mock vector based on immunofluorescence confocal microscopy ( fig. 2c,d) . these data suggested that pten overexpression ameliorated the imbalance between th17 and treg in cia. loss of p53 induces stat3 activation. previously, p53 has been known as a modulator of stat3 activation through significant reducing stat3 phosphorylation and stat3 dna binding activity 19 . the phosphorylation levels of stat3 tyr705 and ser727 in p53 deficient mice splenocytes were increased compared to those of wt mice splenocytes (fig. 3a,b) . cells isolated from wt or p53 deficient mice were cultured under the condition of th0 or th17. il-17 production in p53 −/− mice was significantly increased compared to that in wt mice (fig. 3c,d) . gene expression of il-17 and ccl20 which causes recruiting of il-17 expressing cells 20 in wt mice splenocytes was increased after stimulation with tgf-β and il-6. but, mrna expression of il-17 and ccl20 was reduced significantly by p53 activator, nutlin-3a. in contrast, bpv(hopic), the inhibitor of pten, promoted gene expression of il-17 and ccl20 (fig. 3e ). gene expression of pten was enhanced significantly (c) spleens of cia mice were subjected to immunostaining for cd4 + il-17 or cd4 + cd25 + foxp3 + cells. (original magnification, 40× ) (d) spleens of cia mice were subjected to confocal staining for cd4 + pstat3y705 + or cd4 + pstat3s727 + cells (original magnification, 40× ). the number of cells was counted in four independent quadrants. data are presented as mean ± sd of three independent experiments (*p < 0.05, ***p < 0.01, n = 6). by nutlin-3a. however, pifithrin-α , the inhibitor of p53, significantly decreased the mrna level of pten in mice splenocytes (fig. 3f ). these results suggested that the inhibition of p53 under inflammatory milieu of ra might have enhanced inflammation. additionally, pten expression could be regulated by p53 dependent manner. recently, p53 has been demonstrated as a mediator of balance between th17 and treg in ra 17 . cells of wt or p53 deficient mice in normal state were cultured under th0 or th17 condition. the expression of il-17 was significantly increased (fig. 4a ). gene expression of il-17 in splenocytes isolated from p53 deficient mice was also significantly increased compare to that in wt mice (fig. 4b ). t cell activation was profoundly upregulated in cells isolated from p53 deficient mice compared to that in wt mice (fig. 4c ). th17 differentiation in p53 deficient mice was significantly increased whereas treg differentiation was significantly reduced compared to that in wt mice (fig. 4d ). these data suggested that p53 deficiency could lead to t cell activation and imbalance between th17 and treg. p53 deficiency exacerbates cia severity by upregulating inflammation. based on arthritis scores, it was found that p53 deficiency exacerbated cia progression in vivo (fig. 5a ). serum levels of total igg, igg1, and igg2a were significantly increased in p53 deficiency mice compared to those in wt mice (fig. 5b) . moreover, histological analysis showed that paws and ankles of p53 deficiency arthritis mice had higher degree of inflammation with bone damage and cartilage damage (fig. 5c ). the expression of proinflammatory cytokines in joints were significantly upregulated in p53 deficiency arthritis mice compared to that in wt mice (fig. 5d ). our results suggested that p53 deficiency might have failed to regulate inflammatory response, thus worsening local inflammatory milieu. treg. since pten expression is regulated by p53 16 , we investigated pten expression in p53 deficiency mice with cia. gene expression levels of pten in splenic cd4 + t cells, splenocytes, and draining lymph nodes isolated from p53 deficiency mice with cia were decreased significantly compared to those in wt mice with cia (fig. 6a) . since ra can result in dysregulated balance of th17/treg 1 , we also measured the balance between th17 and treg. the mrna level of th17 related molecules including il-17 was increased, whereas the mrna expression of treg cell-related molecules such as foxp3 was enhanced in p53 deficiency mice with cia (fig. 6b ). in addition, the number of cd4 + p-stat5 + t cells in the spleen tissues of p53 deficiency mice with cia was significantly downregulated compared to that in the spleen tissues of wt mice with cia based on immunofluorescence confocal microscopy (fig. 6c) . however, loss of p53 promoted the number of il-17 producing cd4 + p-stat705 + or p-stat727 + t cells in the spleen tissues based on immunofluorescence confocal microscopy (fig. 6d) . moreover, pten expression was significantly downregulated in p53 deficient mice compared to that in wt mice (fig. 6e) . these results demonstrated that p53 deficiency could accelerate the imbalance between th17 and treg in cia. until now, pten has been investigated extensively as a tumor suppressor with a role in cell metabolism, motility, and tumor microenvironment 21 . recently, pten has been observed to be associated with cell differentiation as a phosphatase 22 . in addition, pten is revealed to have therapeutic effect in rat with cia 23 . however, little is known about the process of pten function associated with p53 in ra. here, we studied the therapeutic activity of pten in ra and identified a new mechanism of ra regulation. the most notable observation of this investigation is that pten can attenuate ra via reciprocal differentiation of th17/treg. to our knowledge, this is the first research to provide evidence that pten could be used for ra therapy through regulating th17/treg balance. previously, a number of documents have demonstrated that imbalance between th17 and treg can contribute to ra 1,24 . modulation of th17 and treg cells has an important role in ra therapy 10, 25 . the activation of stat5 prolongs foxp3 production in treg, while stat3 activation increases the differentiation of th17 cells 26 . moreover, binding of p-stat3 and p-stat5 can competitively regulate il-17 transcription 27 . our results demonstrated that foxp3 + t cells were induced by pten overexpression while p53 deficiency significantly induced p-stat3 + t cells. this could be due to the diminishment of t cell transcriptional regulators such as foxp3 and socs3 and the enhancement of rorγ t, runx1, and batf. in addition, pten decreased the number of treg cells while th17 differentiation was promoted in ra mice model. hence, pten might be another efficient mechanism regulated by which p53 through controlling th17/treg balance. tumor protein 53 (p53), a tumor suppressor factor, is essential for cellular response to dna damage. it plays a key role in several gene expression as a resourceful transcription factor under stressful conditions. it has been demonstrated that p53 is involved in a variety of cellular signal pathways, cell proliferation, and apoptosis [28] [29] [30] . although p53 function has been recognized mainly in the cell cycle, emerging evidence has suggested that p53 has important role not only in apoptosis, cell differentiation, and dna repair, but also in the modulation of stat-mediated th17 cells 17 . loss of p53 can promote the progression of antigen-induced arthritis and increase activated t cell differentiation 31 . in this study, pten expression was found to be regulated in p53 dependent manner. additionally, p53 deficiency aggravated cia severity and reduced pten expression. these results suggested that pten could have therapeutic effect in autoimmune arthritis through p53. il-17 is a typical proinflammatory cytokine inducing the expression of il-6, -21 and tnf-α 32-34 . it has been documented that il-17 can upregulate proinflammatory cytokines including il-6 and il-8 and aggravate joint inflammation of ra through activating cd4 + t cells 35, 36 . th17 secreting il-17 performs a key role in the pathogenesis of ra. th17 frequency and il-17 level are strikingly correlated with ra development. it has been documented that th17 can result in excessive inflammation in patients with ra 3 . our data revealed that pten overexpression reduced the activation of t cells and that the loss of p53 enhanced the proliferation of th17 cells. since il-17 expression is well known to induce ra development, suggesting a novel therapeutic strategy to modulate ra via pten expression. (a,b) relative mrna levels of pten and factors such as il-17, rorγ t, and foxp3 involved in the differentiation of th17 and treg in splenic cd4 + t cells, splenocytes, and draining lymph nodes from cia induced wt or p53 −/− mice were assessed by real-time pcr. data are presented as mean ± sd of three independent experiments (*p < 0.05, **p < 0.03). (c) spleens of cia mice were subjected to immunostaining for cd4 + il-17 or cd4 + cd25 + foxp3 + cells. (d) spleens of cia mice were subjected to confocal staining for cd4 + pstat3y705 + or cd4 + pstat3s727 + cells. the number of cells was counted in four independent quadrants. data are presented as mean ± sd of three independent experiments (*p < 0.05, ***p < 0.01, n = 6). scientific reports | 6:34617 | doi: 10.1038/srep34617 granulocyte-macrophage colony-stimulating factor (gm-csf), an immune modulatory cytokine, performs a significant role in immune tolerance and attenuates autoimmune disorder 37 . it has been suggested that gm-csf suppressed progression of autoimmune disease via induction of tregs 38 . previously, immune tolerance can be a good strategy for cia therapy. indeed, immune tolerance induction using cii showed therapeutic effect in vivo and in vitro 39 . cd8 + tregs can reveal therapeutic implications in cii involved-disease inducing immune tolerance 40 . the therapeutic effect of pten in cia may be involved in upregulation of gm-csf and immune tolerance. thus, further study will be needed to confirm therapeutic activity of pten related with gm-csf expression and immune tolerance. we analyzed the gene expression of p53 and stat3 in cd4 + t cells of healthy individuals and ra patients from the national center for biotechnology information gene expression omnibus database (gse4588). the gse4588 database contains 10 healthy subjects and 8 ra patients with clinic and pathological information. we observed that the relative mrna level of stat3 of ra patients was promoted significantly compared to that of healthy individuals in this database. the mrna expression of p53 in ra patients was downregulated significantly compared to that in healthy individuals. the lack of p53 might have aggravated ra progression and induced stat3 activation. in this study, we demonstrated that pten overexpression could reduce cia progression and that the loss of p53 enhanced the expression of stat3. the function of pten and p53 in stat3 activation has already been studied in previous investigations 13, 19 . recently, p53 deficiency has revealed correlation with ra severity inducing th17 differentiation 17 . however, our study demonstrated significant mechanism of pten associated with p53 in the development of cia via reciprocally regulating th17 and treg. this preliminary evidence suggested that upregulating pten could be a strong therapeutic strategy for the treatment of ra. ethics statement. the animal care committee of the catholic university of korea approved the experimental protocol, and all the experimental procedures were carried out in accordance with the protocols approved by the animal research ethics committee at the catholic university of korea. all procedures performed followed the ethical guidelines for animal studies. animals. male dba1/j mice and c57bl/6 mice at 6-8 weeks old (orient, korea) were maintained in groups of five in polycarbonate cages in a specific pathogen-free environment. they were provided free access to standard mouse chow (ralston purina, gray summit, mo) and water ad libitum. mice harboring the p53-null allele with a c57bl/6 mice background (b6.129s2-trp53tm1tyj/j) were obtained from the jackson laboratory. collagen-induced arthritis (cia) was induced in dba1/j mice (each group: n = 10). mice were immunized with 100 μ g of chicken cii (chondrex inc., redmond, wa, usa) dissolved overnight in 0.1n acetic acid (4 mg/ml) in complete freund's adjuvant or incomplete freund's adjuvant (chondrex inc). the immunization was performed intradermally into the base of the tail. cia was induced in the p53 −/− strain mice as described previously 41 . eight days after immunization, mice were injected intravenously with 100 μ g of pten or mock vector in 2 ml of saline over a 10-second period. after 8 days, the same mice received intramuscular injection of 50 μ g of pten or mock vector in the left leg with electrical stimulation (electroporation) using a 31-gauge needle insulin syringe for hydrodynamic-based procedures. two days later, mice received an intramuscular injection of 50 μ g of pten or mock in the right leg through electroporation. clinical scoring and histological assessment of arthritis. arthritis score was measured visually twice per week based on the appearance of arthritis in the joints and graded according to williams et al. 42 . the joints of each mouse were fixed in 10% formalin, decalcified in 10% edta, and embedded in paraffin wax for histological analysis. hematoxylin-eosin (h&e) stained sections were scored for inflammation, destruction of cartilage, and bone damage according to published criteria 43,44 . real-time polymerase chain reaction (pcr). total flow cytometry. flow cytometry was conducted as described previously 45, 46 . cells were immunostained with various combinations of fluorescent antibodies against cd4, cd25, foxp3, ifn-γ , il-4, and il-17 (ebioscience, san diego, ca, usa). prior to intracellular staining, cells were restimulated with phorbol myristate acetate (pma; 25 ng/ml) and ionomycin (250 ng/ml) for 4 hours in the presence of golgistop (bd biosciences). for analysis of treg cells, cells were surface labeled with cd4 and cd25, followed by fixation, permeabilization and intracellular staining with foxp3 was perfirmed per the manufaturer's protocol. flow cytometry was performed on a facscalibur flow cytometer (bd biosciences). the data was analyzed using the flowjo software (tree star, ashland, or,usa). elisa. enzyme-linked immunosorbent assay (elisa) was conducted as described previously 47, 48 . briefly, blood was obtained from the orbital sinus of mice. serum levels of igg antibodies were measured using a commercially available elisa kit (bethyl laboratories, montgomery, tx, usa). horseradish peroxidase (hrp) activity was measured using tetramethyl benzidine as substrate (ebioscience, san diego, ca, usa). scientific reports | 6:34617 | doi: 10.1038/srep34617 staining for confocal microscopy analysis. tissue cryosections (7 μ m thick) were fixed with acetone and stained with fitc-, pe-, percp-cy5.5-, or apc-conjugated monoclonal antibodies against mouse cd4, pstat3 (tyr 705, ser 727), pstat5, il-17, and foxp3 (ebioscience). after incubation at 4 °c overnight, stained sections were visualized through confocal microscopy (lsm 510 meta; zeiss, göttingen, germany). immunohistochemistry. immunohistochemistry was performed using the vectastain abc kit. tissues were first incubated with primary anti-c-jun and anti-c-fos antibodies overnight at 4 °c. the primary antibody was detected with a biotinylated secondary antibody followed by incubation with a streptavidin-peroxidase complex for 1 h. dab chromogen was added to obtain colored product. transfection. pten vector purchased from addgene (plasmid#22231) was used to generate the overexpression of pten. mock and pten vector constructs were transfected into lbrm (mice t lymphoma cell line) cells using amaxa 4d-nucleofector x unit according to the manufacturer's recommendations with program dn-100 (lonza). splenocytes were harvested in ack lysis buffer, washed, and resuspended in complete culture medium (rpmi 1640 supplemented with 10% [v/v] heat-inactivated fetal calf serum). to purify splenic cd4 + t cells, splenocytes were incubated with anti-cd4-coated magnetic beads, and cd4 + t cells were isolated using magnetic-activated cell sorting (macs) separation columns (miltenyi biotec). the cells were pretreated with pifithrin-α , nutlin-3a (cayman chemical) or bpv(hopic) (santa cruz biotechnology) and then stimulated under the required polarizing conditions. aliquots of 2 × 10 5 cd4 + t cells (responders) were cultured with 2 × 10 5 irradiated (2,500 cgy) apcs in 96-well plates containing 200 μ l/well of complete medium, at 37 °c in a humidified 5% (v/v) co 2 /air atmosphere. cells were pulsed with 1 μ ci of tritiated thymidine ( western blot. western blot was performed as described previously 48, 49 . proteins were loaded onto 10% polyacrylamide gels and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transferring to nitrocellulose membranes (invitrogen life technologies, carlsbad, ca, usa). membranes were blocked with 5% (w/v) non-fat milk in tris-buffered saline containing 0.1% tween-20 for 1 h followed by incubation with antibodies against p-stat3 y705, p-stat3 s727, t-stat3 (cell signaling), and β -actin (santa cruz biotechnology) overnight at 4 °c. immunoreactivity was determined using enhanced chemiluminescence reagents (amersham biosciences, piscataway, nj, usa). statistical analysis. data were presented as means ± standard deviations (sd). statistical analysis was performed with nonparametric mann-whitney u test using graphpad prism v.5.01. one-way analysis of variance (anova) and bonferroni's post hoc test were used for multiple comparisons. statistical significance was considered when p value was less than 0.05. disturbed th17/treg balance in patients with rheumatoid arthritis pro-inflammatory cytokines in rheumatoid arthritis: pathogenetic and therapeutic aspects th17 cells, but not th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17a production high levels of il-17 in rheumatoid arthritis patients: il-15 triggers in vitro il-17 production via cyclosporin a-sensitive mechanism stat3 and nf-kappab signal pathway is required for il-23-mediated il-17 production in spontaneous arthritis animal model il-1 receptor antagonist-deficient mice stat3 and stat4 direct development of il-17-secreting th cells stat3 regulates cytokine-mediated generation of inflammatory helper t cells mechanisms underlying lineage commitment and plasticity of helper cd4 + t cells metformin attenuates experimental autoimmune arthritis through reciprocal regulation of th17/treg balance and osteoclastogenesis sta-21, a promising stat-3 inhibitor that reciprocally regulates th17 and treg cells, inhibits osteoclastogenesis in mice and humans and alleviates autoimmune inflammation in an experimental model of rheumatoid arthritis perturbations of the akt signaling pathway in human cancer activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor pten at sites of invasive growth and destruction pten is a negative regulator of stat3 activation in human papillomavirus-infected cells treg cells require the phosphatase pten to restrain th1 and tfh cell responses tumor suppressor p53 inhibits autoimmune inflammation and macrophage function regulation of pten transcription by p53 p53 controls autoimmune arthritis via stat-mediated regulation of the th17 cell/treg cell balance in mice the central role of t cells in rheumatoid arthritis p53 regulates stat3 phosphorylation and dna binding activity in human prostate cancer cells expressing constitutively active stat3 th17 cytokines stimulate ccl20 expression in keratinocytes in vitro and in vivo: implications for psoriasis pathogenesis the functions and regulation of the pten tumour suppressor the protein phosphatase activity of pten is essential for regulating neural stem cell differentiation amelioration of collagen-induced arthritis in rats by adenovirus-mediated pten gene transfer the t(reg)/th17 cell balance: a new paradigm for autoimmunity rebamipide suppresses collagen-induced arthritis through reciprocal regulation of th17/treg cell differentiation and heme oxygenase 1 induction human immunodeficiency: connecting stat3, th17 and human mucosal immunity opposing regulation of the locus encoding il-17 through direct, reciprocal actions of stat3 and stat5 the complexity of p53 modulation: emerging patterns from divergent signals wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6 p53 regulation by post-translational modification and nuclear retention in response to diverse stresses the tumour suppressor gene p53 modulates the severity of antigen-induced arthritis and the systemic immune response il-21 is produced by th17 cells and drives il-17 production in a stat3-dependent manner potential new targets in arthritis therapy: interleukin (il)-17 and its relation to tumour necrosis factor and il-1 in experimental arthritis il-17 can promote tumor growth through an il-6-stat3 signaling pathway il-17 induces production of il-6 and il-8 in rheumatoid arthritis synovial fibroblasts via nf-kappab-and pi3-kinase/akt-dependent pathways il-17 induces the production of il-16 in rheumatoid arthritis gm-csf: an immune modulatory cytokine that can suppress autoimmunity granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory t cells eye-mediated immune tolerance to type ii collagen in arthritis-prone strains of mice type ii collagen induces peripheral tolerance in balb/c mice via the generation of cd8 + t regulatory cells protocol for the induction of arthritis in c57bl/6 mice anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis trance/rankl knockout mice are protected from bone erosion in a serum transfer model of arthritis blockade of pi3kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis a novel pancreatic beta-cell targeting bispecific-antibody (bsab) can prevent the development of type 1 diabetes in nod mice the novel role of il-7 ligation to il-7 receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing identification of a novel toll-like receptor 7 endogenous ligand in rheumatoid arthritis synovial fluid that can provoke arthritic joint inflammation identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay conceived and designed the study, interpreted the data, and made critical revisions of the manuscript for important intellectual content supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord-0015761-00s5jfs1 authors: hsu, hui-ching; chang, yu-sheng; hou, tsung-yun; chen, lung-fang; hu, li-fang; lin, tzu-min; chiou, chi-sheng; tsai, kai-len; lin, sheng-hong; kuo, pei-i; chen, wei-sheng; lin, yi-chun; chen, jin-hua; chang, chi-ching title: pneumocystis jirovecii pneumonia in autoimmune rheumatic diseases: a nationwide population-based study date: 2021-03-01 journal: clin rheumatol doi: 10.1007/s10067-021-05660-4 sha: ca108728b207f161174ac5dce8b99628686076f9 doc_id: 15761 cord_uid: 00s5jfs1 objective: to compare pneumocystis jirovecii pneumonia (pjp) risk between patients with autoimmune rheumatic diseases (ard) and the general population methods: we identified patients with ard recorded in the national health insurance research database of taiwan from 2002 to 2015 and randomly selected a comparison cohort from the general population matched for age and sex. we analyzed pjp risk stratified by sex, age, comorbidities, and medications using cox proportional hazard model. results: we enrolled 103,117 patients with ard. pjp risk significantly increased in patients with any ard and with each individual ard like rheumatoid arthritis (ra), systemic lupus erythematosus (sle), sjogren’s syndrome (sjs), polymyositis and dermatomyositis (pm/dm), systemic sclerosis (ssc), and systemic vasculitis. patients with pm/dm showed prominent risk with incidence rate of 12.47/100,000 patient year (95% confidence interval (ci), 32.16–86.70). in a time-dependent cox proportional hazard model with comorbidities and medications as covariates, pm/dm, ssc, sle, and sjs significantly increased adjusted hazard ratios (ahr) of 5.40, 5.12, 4.09, and 3.64, respectively (95% ci of 2.82–10.35, 2.16–12.13, 2.41–6.95, and 2.06–6.42, respectively). ahr after adjusting for male sex, cancer, human immunodeficiency virus infection (hiv), and interstitial lung disease also significantly increased. use of daily oral steroid dose of >10 mg conferred the highest risk followed by mycophenolate. use of injected steroids, cyclophosphamide, biological agents, methotrexate, and cyclosporine conferred a significantly higher risk. conclusion: underlying ard significantly predisposes patients to pjp, with pm/dm posing the highest threat. in addition to underlying disease, comorbidities and concomitant immunosuppressants are major risks. the strongest risk is recent daily steroid dose of >10 mg. mycophenolate seems to be a more prominent risk factor than cyclophosphamide. pneumocystis jirovecii is an opportunistic organism that can cause high mortality among immunosuppressed patients including those with human immunodeficiency virus infection (hiv), malignancy, and underlying autoimmune rheumatic diseases (ard) [1, 2] . in recent years, due to the progress of organ transplantation, tumor chemotherapy, and ard treatment, the number of non-hiv patients infected by pneumocystis jirovecii has gradually increased [3] . patients with underlying ard may be exposed to immunosuppressive drugs, corticosteroid, biological agents, and cyclophosphamide, rendering them more vulnerable to pneumocystis jirovecii pneumonia (pjp) [4, 5] . mortality rates among non-hiv patients diagnosed with pjp are high (estimated to be between 39.4 and 59.1%), making this an important population to target for prophylaxis [6] . trimethoprim/ sulfamethoxazole prophylaxis in patients with ard can significantly reduce incidence of pjp [5, 7] . therefore, identifying patients with ard and a high risk of pjp is crucial for adequate initiation of pneumocystis jirovecii prophylaxis, which may improve patient outcomes as has proven effective for hiv patients [8] . in previous reports of ard, the most common underlying condition for development of pjp is wegener's granulomatosis (8-12%), followed by polyarteritis nodosa (pan) (6.5%), polymyositis and dermatomyositis (pm/dm) (2.7%), systemic lupus erythematosus (sle) (2%), and rheumatoid arthritis (ra) (0.1-0.3%) [9] . however, data on the prevalence of pjp in ard is relatively limited. several comorbidities other than hiv, malignancy, or ard may also increase risks of pjp, such as renal transplantation [10, 11] , end-stage renal disease (esrd) [11] , or interstitial lung disease (ild) [12] , which adds to the complexity of risk evaluation and management. in addition to the patient's underlying diseases, high-dose steroid use and immunosuppressants were found to increase the risk of pjp in patients with ard [10, 13] . patients with ard receiving an initial steroid dose of ≥60 mg/day prednisolone equivalent were also found to have a higher risk for pjp [5] . significant risk factors for pjp included higher age, male sex, underlying lung disease, use of corticosteroids, use of methotrexate, and use of biologics (anti-tnf alpha, anti-il-6 receptor antibody, or anti-t-cell costimulatory pathway agents) in patients with ra [8, 14] . anti-cd20 and cyclophosphamide use were also related to increased risk of pjp in patients with sle or malignancy [15, 16] . to summarize, the host's underlying disease and comorbidity, which directs different combination of immunosuppressant use including different dosage and duration, contribute greatly to the variability and complexity of pjp in clinical practice. due to the rare occurrence but high mortality of pjp infection in patients with ard, it is crucial to determine the risk of pjp infection individually when considering the initiation of prophylaxis. this study aims to determine (1) the risk of pjp with underlying ard as a whole or individual ard, (2) comorbidities that pose additional risks, and (3) effects of immunosuppressants, steroid, biologics, and cyclophosphamide on the risk of pjp. using nationwide data on pjp in patients with ard, we aimed to better understand this condition under complex clinical settings. the national health insurance (nhi) program in taiwan was instituted in 1995 to provide comprehensive health care for nearly all taiwanese citizens and has a coverage rate of 99%. for research purposes, the bureau of nhi in taiwan released the national health insurance research database (nhird), which provides detailed claim data for outpatient clinics, admission, and drug prescriptions of patients enrolled in the nhi. for this study, we collected data from the nhird from january 1, 2002, to december 31, 2015. since the data sets used in this study were comprised of de-identified secondary data for research purposes, patient consent was not required for this study. this study was approved by the taipei medical university institutional review board (approval numbers n201509007 and n201908055). the diagnostic codes used in this data set were based on the international classification of diseases, ninth revision, clinical modification (icd-9-cm). we conducted a retrospective matched cohort study using the nhird of taiwan to investigate the risk of pjp of patients with and without ard (fig. 1) . the nhird established a registry system for catastrophic illnesses. in taiwan, after a rheumatologist makes a diagnosis based on a patient's clinical features and laboratory and image results, patients who [25] , and giant cell arteritis (icd-9-cm: 446.5) [26] . patients with their first new catastrophic illness registration of each ard between 2002 and 2015 were enrolled, including those with ra, sle, sjs, pm/dm, ssc, and systemic vasculitis (sv). the sv group comprised patients with pan, hypersensitivity angiitis, wegener's granulomatosis, giant cell arteritis, and buerger's disease. the primary endpoint was incidence of pjp infection defined as pneumocystosis (icd-9-cm: 136.3) in the discharge diagnosis. patients with unknown sex or age or with pjp prior to enrollment were excluded. patients with overlapping ards were excluded. each patient with ard was matched with four non-ard subjects for age and sex based on the same exclusion criteria. all patients were followed until the development of primary endpoint, end of the study period, or discontinuance of insurance. for continuous variables, we employed student's t tests to evaluate the differences between the case group and the comparison group, and for categorical variables in our baseline analysis, we evaluated differences between the two groups using pearson's chi-squared tests. an incidence rate ratio (irr) was constructed to compare the incidence between the groups, and the confidence interval (ci) of irr was calculated using normal approximation. the cumulative incidence rate and gray's test for equality of cumulative incidence functions were employed to compare the risk of pjp infection between the two cohorts. a cox proportional hazard model was applied to evaluate the factors associated with pjp infection. factors analyzed in the model included age, sex, type of ard, comorbidities, and medications leading to immunosuppression status. comorbidities within 1 year before the index date that may lead to immunosuppression were identified using the corresponding icd-9-cm, including cancer, hiv, renal transplant, esrd, ild, congestive heart failure, diabetes mellitus, hypertension, cirrhosis, and stroke. medications used during the study period were analyzed as time-dependent covariates, such as hydroxychloroquine, methotrexate, mycophenolate, azathioprine, cyclosporine, cyclophosphamide, steroid, and biologics. biologics consisted of etanercept, adalimumab, golimumab, rituximab, tocilizumab, and abatacept. the follow-up period of each patient was retrospectively divided into successive 120-day blocks; the prescription status of each medication in each 120-day block and its association with the occurrence of the event at the end of each time block were analyzed. oral steroid use was analyzed according to the average daily dose in the time block, with a cutoff point of 5 and 10 mg prednisolone equivalent. all factors with p < 0.1 in the univariate analysis were selected for cox multivariate forward stepwise analysis. the results of all statistical tests were considered significant if p < 0.05. there were 103,117 patients with ard and 412,468 matched controls enrolled in this study ( table 1 ). the majority of patients in both cohorts were female (81.58%) and the mean age was 50.4 years in both groups. the ard group had significantly increased comorbidities and medication use. the detailed use of each medication is shown in supplementary material table 1 . the ard group had a significantly higher incidence of pjp development than the comparison group during the observation period (0.18% vs. 0.01%, p < 0.0001) ( table 1 ). the incidence rates (ir) and irr of pjp during the follow-up period revealed a significantly higher risk in patients with each individual ard ( table 2 . the incidence rate analyses for individual ard and overall ard also revealed a significantly higher risk of pjp infection (fig. 2, supplementary material figure 1 ). to analyze risk factors associated with pjp infection, a multivariate cox proportional hazard analysis was applied to all study participants (table 3) . age, male sex, and comorbidities including malignancy, hiv, and ild significantly increased the risk of pjp. after adjusting for sex, age, and comorbidities, several medications analyzed as time-dependent covariates in the model significantly related to pjp infection. the medication with the highest risk was daily oral steroid dose of >10 mg prednisolone equivalent (ahr 9.15, 95% ci 6.23-13.45), followed by (in order of risk) mycophenolate (ahr 7.14, 95% ci 4. previous studies of pjp in autoimmune diseases are mostly case series and single-center experiences of one or a few ard, with a relatively low incidence of pjp. to the best of our knowledge, this is the first study to attempt a broad, population-based perspective to better understand pjp in patients with ard, especially in patients with ssc and sjs. patients with ard had a significantly higher risk of pjp. in our multivariate cox proportional hazard analysis, patients with pm/dm had the highest risk, followed by those with ssc, sle, and sjs. the factors associated with pjp included comorbidity-related secondary immunosuppression and immunosuppressant use such as steroid, mycophenolate, biologic agents, cyclophosphamide, methotrexate, and cyclosporine. patients with different ard were at differing risks of pjp. in previous studies of pjp, the most common ard were inflammatory myopathy, sle, anca-associated vasculitis, and ra [3, 27, 28] . ssc and sjs have not been previously reported to be associated with higher risk of pjp infection, but in this study, patients with ssc or sjs had a significantly higher risk of pjp, and the risk in patients with ssc was even higher than that in patients with sle. in addition, the ard with the highest pjp risk was neither sle nor sv; patients with pm/dm had the highest risk followed by sv and ssc. in our multivariate cox proportional hazard analysis, most ard still had a risk of pjp after adjusting for all related factors. this was not true for ra or sv, which means the increased risk of pjp in patients with ra and sv may come mostly from the immunosuppressants and related comorbidities such as ild; therefore, the risk was no longer significant after adjusting for these factors. however, we believe that there were other reasons that lead to our failure to demonstrate the significant risk in patients with sv. the wide 95% ci implied that the patient year of sv was too low to have sufficient statistical power to show significance and only a trend toward higher risk remained in the model. the relatively lower patient year in the sv cohort might be due to the low prevalence and high mortality rate of this disease. among immunosuppressants, the risk of cyclophosphamide is well-documented and related prophylactic strategies were established in patients with hematological malignancies [29] . of note, several medications had higher risk than cyclophosphamide in this study. the highest was daily oral steroid use over 10 mg prednisolone equivalent. injected steroid therapy also had an increased risk. this result is consistent with previous reports emphasizing the risk of opportunistic infections such as pneumocystis jirovecii in patients with high-dose steroid use, and this effect seems to be dose-dependent, regardless of underlying disease [5, 30, 31] . cumulative steroid dose, duration of steroid use, and daily steroid dose all had prominent impacts on the risk of infection. it was demonstrated that current and recent doses of steroid have the greatest impact on infection risk, but the cumulative impact of doses taken in the past few years still affects risk in patients with ra [32] . use of steroids, mycophenolate, biological agents, cyclophosphamide, methotrexate, and cyclosporine had significantly increased risks of pjp infection. it is noteworthy that mycophenolate is believed to be equally effective but better tolerated than cyclophosphamide, but in our model, the risk of pjp with mycophenolate was even higher than that with cyclophosphamide. there has been a single-center report of increased mortality of pjp in patients with ard receiving mycophenolate [33] . moreover, in taiwan, biological agents endorsed by nhi were mostly used for treating ra. there have been reports of pjp infection in patients with ra under etanercept, adalimumab, golimumab, rituximab, tocilizumab, or abatacept treatment [8, 14, 34] . our results also showed a significantly higher risk of pjp infection in patients using biological agents. due to the paucity of evidence regarding primary prophylaxis of pjp in ard, the initiation of prophylaxis in patients with ard is still based on expert opinion with no clear recommendations [35] . the initiation and duration of trimethoprim/sulfamethoxazole prophylaxis should be considered in high risk patients in a patient-based manner [35] . this study provided data on the individual risk of each immunosuppressant, which may add insight to evaluating the overall risk of pjp under different clinical settings and assist decision-making on prophylaxis of this serious disease. several basic characteristics were associated with pjp risk. similar to a previous report in ra, older age and male sex were related to a higher risk of pjp [14] . our study showed that underlying ard, malignancy, hiv, and ild contribute to pjp infection. ild was associated with increased pulmonary colonization by pneumocystis jirovecii, which may explain this increased risk [12] . our study had several strengths including the long followup and the use of population-based, nationwide cohorts and the large study sample that may increase statistical precision. in addition, we validated ard diagnosis by catastrophic illness registration. in the nhi system, patients with the catastrophic illness registration for ard can be exempted from related medical expense. however, the verification requires fulfillment of the classification criteria of each ard, as supported by medical records and examination reports. these features made the ard diagnosis more reliable. second, we analyzed immunosuppressants as time-dependent variables in a multivariate cox-regression model, which could fit in with the temporal relationship between medication and the subsequent increased risk of pjp infection. our study had several limitations. first, because the nhi database does not include clinical data or culture results, we could not analyze information such as patients' lymphocyte count, cd4 count, or serum ldh level, which might have an additional predictive value of pjp [6, 36] . however, the main goal of this study was to investigate the epidemiology of pjp in ard, especially ard that have been less studied in this context such as pm/dm, ssc, and sjs and the risk of individual medications. second, different biologics were analyzed together despite the fact that different biologics might have different risks of pjp infection. we decided to simplify the analysis by combining different biologics as one variable because there were too many variables in the model given the low incidence of pjp events and the biologics were mainly used in patients with ra. we will focus on this issue in patients with ra in the future. the situation was similar in sv; we included different types of vasculitis as a single cohort due to relatively low case numbers of each type of vasculitis. however, we believe that the risk differed with different types of vasculitis. this study revealed the increased risk and epidemiologic data of pjp infection in several ard including ard with few reports on, such as ssc and sjs. the risk was highest in patients with pm/dm followed by sv, ssc, sle, sjs, and ra. this study also included risk factors for pjp infection such as male gender, older age, and comorbidities like cancer, hiv, or ild, or immunosuppressants including steroid, mycophenolate, cyclophosphamide, biologics, methotrexate, and cyclosporine. these data provide fundamental insights for further studies on prophylactic antibiotics for pjp for these populations. author contributions all authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. hui-ching hsu and yu-sheng chang contributed to study conception and design, article drafting, data interpretation, critical article revision for crucial intellectual content, and the final approval of the submitted version. tsung-yun hou, lung-fang chen, li-fang hu, tzu-min lin, chi-sheng chiou, kai-len tsai, sheng-hong lin, pei-i kuo, wei-sheng chen, and yi-chun lin contributed to data analysis, critical article revision for crucial intellectual content, and the final approval of the submitted version. jin-hua chen and chi-ching chang were responsible for study conception and design, complete data analysis, critical article revision for crucial intellectual content, and correspondence regarding the final approval of the submitted version. the manuscript does not contain clinical studies or patient data. disclosures none. pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illness and prior corticosteroid therapy pneumocystis jirovecci pneumonia in connective tissue diseases: comparison with other immunocompromised patients pneumocystis pneumonia suspected cases in 604 non-hiv and hiv patients use of glucocorticoids and risk of infections prophylactic effect of trimethoprim-sulfamethoxazole for pneumocystis pneumonia in patients with rheumatic diseases exposed to prolonged high-dose glucocorticoids pneumocystis jirovecii pneumonia in systemic autoimmune rheumatic disease: a case-control study prophylaxis of pneumocystis pneumonia in immunocompromised non-hivinfected patients: systematic review and meta-analysis of randomized controlled trials pneumocystis jirovecii pneumonia in rheumatoid arthritis patients: risks and prophylaxis recommendations update on pulmonary pneumocystis jirovecii infection in non-hiv patients pneumocystis jirovecii infection: an emerging threat to patients with rheumatoid arthritis pneumocystis jirovecii pneumonia in patients with end-stage renal disease: a comparison with the general population pneumocystis jirovecii colonisation in patients with interstitial lung disease a retrospective study of patients with systemic lupus erythematosus combined with pneumocystis jiroveci pneumonia treated with caspofungin and trimethoprim/sulfamethoxazole incidence and risk factors for infections requiring hospitalization, including pneumocystis pneumonia pneumocystis jiroveci pneumonia in patients with systemic lupus erythematosus after rituximab therapy pneumocystis jirovecii pneumonia: still a concern in patients with haematological malignancies and stem cell transplant recipients updating the american college of rheumatology revised criteria for the classification of systemic lupus erythematosus the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis classification criteria for sjogren's syndrome: a revised version of the european criteria proposed by the american-european consensus group subcommittee for scleroderma criteria of the american rheumatism association diagnostic and therapeutic criteria committee (1980) preliminary criteria for classification of systemic sclerosis (scleroderma) polymyositis and dermatomyositis (first of two parts) polymyositis and dermatomyositis (second of two parts) nomenclature of systemic vasculitides. proposal of an international consensus conference thromboangiitis obliterans the american college of rheumatology 1990 criteria for the classification of wegener's granulomatosis the american college of rheumatology 1990 criteria for the classification of giant cell arteritis frequency, risk factors and prophylaxis of infection in anca-associated vasculitis pneumocystis jiroveci pneumonia in rheumatic disease: a 20-year single-centre experience ecil guidelines for preventing pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients infection risk and safety of corticosteroid use pneumocystis and glucocorticoid use: to prophylax or not to prophylax (and when?); that is the question immediate and delayed impact of oral glucocorticoid therapy on risk of serious infection in older patients with rheumatoid arthritis: a nested case-control analysis pneumocystis jirovecii pneumonia in mycophenolate mofetil-treated patients with connective tissue disease: analysis of 17 cases comparison of the clinical characteristics of pneumocystis pneumonia between patients with rheumatoid arthritis being treated with biologics and those being treated without biologics risk factors and prevention of pneumocystis jirovecii pneumonia in patients with autoimmune and inflammatory diseases pneumocystis carinii pneumonia in patients with connective tissue disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-0012036-smfpzdpe authors: kanai, tomotake; kondo, naoki; okada, masayasu; sano, hiroshige; okumura, go; kijima, yasufumi; ogose, akira; kawashima, hiroyuki; endo, naoto title: the jnk pathway represents a novel target in the treatment of rheumatoid arthritis through the suppression of mmp-3 date: 2020-07-17 journal: j orthop surg res doi: 10.1186/s13018-020-01595-9 sha: a02dfce327159899d4256cf60c95f4884c0ad170 doc_id: 12036 cord_uid: smfpzdpe background and aim: the pathophysiology of rheumatoid arthritis (ra) is characterized by excess production of pro-inflammatory cytokines, including tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), and interleukin-6 (il-6) by neutrophils and macrophages in synovium. additionally, these cytokines promote the production of reactive oxygen species (ros), and increased production of matrix metalloproteinases (mmps), including mmp-3, in synoviocytes that result in joint destruction. there is limited information on how proteolytic enzymes such as mmp-3 can be regulated. we evaluated the effect of the antioxidant n-acetylcysteine (nac) on ra and identified the relationship between the c-jun n terminal kinase (jnk) pathway and mmp-3. we hypothesized that elucidating this relationship would lead to novel therapeutic approaches to ra treatment and management. methods: we investigated the effect of administering a low dose (1000 μm or less) of an antioxidant (nac) to human rheumatoid fibroblast-like synoviocytes (mh7a cells). we also investigated the response of antioxidant genes such as nuclear factor erythroid -derived 2-related factor 2 (nrf2) and sequestosome 1 (p62). the influence of mmp-3 expression on the jnk pathway leading to joint destruction and the mechanisms underlying this relationship were investigated through primary dispersion culture cells collected from the synovial membranes of ra patients, consisting of rheumatoid arthritis-fibroblast-like synoviocytes (ra-fls). results: low-dose nac (1000 μm) increased the expression of nrf2 and phospho-p62 in mh7a cells, activating antioxidant genes, suppressing the expression of mmp-3, and inhibiting the phosphorylation of jnk. ros, mmp-3 expression, and il-6 was suppressed by administering 30 μm of sp600125 (a jnk inhibitor) in mh7a cells. furthermore, the administration of sp600125 (30 μm) to ra-fls suppressed mmp-3. conclusions: we demonstrated the existence of an mmp-3 suppression mechanism that utilizes the jnk pathway in ra-fls. we consider that the jnk pathway could be a target for future ra therapies. studies on the treatment of rheumatoid arthritis (ra) have focused on biological agents that suppress proinflammatory cytokines, including tnfα, il-1β, and il-6 [1] [2] [3] . however, the suppression of oxidative stress, which causes inflammation, has not been sufficiently investigated [3] . antioxidants, including n-acetylcysteine (nac) that eliminate reactive oxidative species (ros), are used to suppress oxidative stress [4] [5] [6] . nac directly suppresses and eliminates ros [4, 7] . ros promotes the production of matrix metalloproteinase-(mmp-) 3, a proteolytic enzyme that induces joint destruction and suppressing ros might suppress mmp-3 [1-4, 8, 9] . in human synoviocytes stimulated with tnfα, high dose of nac (30 mm) administration suppressed nuclear factor-kappa b (nf-κb ) activation and production of tnfα and il-6 proteins [10] . high doses of nac have an anti-inflammatory effect; however, sadowska et al. indicated that doses of 10 mm and higher are cytotoxic. zafarrullah et al. found that low doses of nac (0.1-1 mm) regulated the redox state but doses higher than 10 mm resulted in structural alterations of tgf-β [4, 11] . in a clinical setting, caution is required when determining the appropriate nac dose. the mitogen-activated kinase (mapk) signal transduction pathway is associated with ros activity and a p38 inhibitor suppressed ros in hela cells treated with h 2 o 2 [12] . jnk pathway stimulates inflammatory activity. it was prominently suppressed by administering nac after hepatic ischemia-reperfusion injury in mice [7] . nac treatment eliminates ros and suppresses the jnk pathway and thereby protects granulosa cells from h 2 o 2 -induced apoptosis [13] . nac affects the activity of mapks (mainly jnk); however, human synoviocytes display large individual differences in the expression of interleukins and mmps. owing to these differences, human synoviocytes were considered unsuitable for clarifying the mechanisms, including signal transduction and influence of the jnk pathway. for this reason, experiments were carried out on cell lines with less individual variation in the expression of interleukins and mmps, particularly mh7a cells, which are human fibroblast-like synoviocytes. our primary objective was to investigate the effect of low-dose nac on ra. to achieve this, we attempted to confirm that mmp-3 expression is linked to anti-oxidative effects, anti-inflammatory activity, and joint destruction and determine its underlying mapk signal transduction pathway. our ultimate goal was to utilize the knowledge of signal transduction pathways to establish novel ra therapies using specific inhibitors. the mh7a rheumatoid fibroblast-like synoviocyte cell line was obtained from the institute of physical and chemical research (riken, tsukuba, ibaraki, japan). the pelleted mh7a cells were stored at − 80°c and cultivated in rpmi 1640 medium (gibco, grand island, ny, usa) supplemented with 10% fetal bovine serum (fbs) (biowest, nuaillé, france) and 1% antibiotic/antimycotic solution (invitrogen, carlsbad, ca, usa) in a humidified incubator with 95% air and 5% co 2 at 37°c. after the fifth passage, mh7a cells were seeded into 3.5 cm dishes at a concentration of 3 × 10 5 cells/well and cultured for three days until 80-90% confluency was achieved. these cells were then examined. synovial tissues were obtained from two ra patients undergoing total knee arthroplasty and one patient undergoing synovectomy of the wrist. these patients had been diagnosed with ra according to the revised criteria of the american college of rheumatology [14] and had been treated with biologics, methotrexate, prednisolone, immunosuppressants, disease-modifying antirheumatic drugs (dmards), and nonsteroidal antiinflammatory drugs (nsaids) ( table 1) . written informed consent was obtained from each patient before the specimens were collected in accordance with the protocols of the niigata university medical and dental hospital ethics committee. ra-fls were isolated using the methods of rosengren et al. [15] and sano et al. [16] . briefly, synovial tissues were cut into small pieces and digested with rpmi 1640 medium mixing collagenase (1 mg/ml) (worthington biochemical corporation, lakewood, nj, usa) for 3 h. the tissue was then filtered using a 70 μm nylon cell strainer, washed, and suspended in rpmi 1640 medium. dissociated cells were then centrifuged at 1500×g for 3 min twice and resuspended in rpmi 1640 medium supplemented with 10% fbs and 1% antibiotic/antimycotic solution. cells were cultured overnight, the non-adherent cells were removed, and the adherent cells were cultivated in rpmi 1640 medium supplemented with 10% fbs and 1% antibiotic/antimycotic solution. after the fifth passage, ra-fls were seeded into 3.5 cm dishes at a concentration of 3 × 10 5 cells/well and cultured for 3 days until 80-90% confluency was achieved. these cells were then examined. nac (sigma-aldrich, st. louis, usa), a specific jnk inhibitor, sp600125 (sigma-aldrich), h 2 o 2 (wako, osaka, japan), and dimethyl sulfoxide (dmso; meso scale discovery, rockville, md, usa) were used. primary antibodies specifically recognizing il-6 (cell signaling technology, danvers, ma, usa), mmp-3 (cell signaling technology), nrf2 (abcam, cambridge, uk), β-actin (sigma-aldrich), and phosphorylated antibody specifically recognizing phosphorylated forms of p62 (mbl, nagoya, japan), and jnk (cell signaling technology) were also used. the effect of nac on cell viability was determined using the xtt assay (cell proliferation kit ii, roche diagnostics, basel, switzerland), which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cells. cells were seeded into a 96-well plate at a density of 5 × 10 4 cells/ml and treated with different concentrations of nac ranging from 10 μm to 10 mm for 24 h at 37°c in 5% co 2 . then, 50 μl of xtt stock solution (0.3 mg/ml) was added to each well to attain a total volume of 150 μl. after incubation for 18 h, the optical density (od) 450-500 was read on a scanning multi-well spectrophotometer (model 680, bio-rad, hercules, ca, usa). mh7a cells in 3.5 cm dishes were incubated with medium containing nac or sp600125 for 3 and 24 h. ra-fls in 3.5 cm dishes were incubated with medium containing sp600125 for 3 and 24 h. treated cells were washed with phosphate-buffered saline (pbs) (non-ca and mg) and harvested with a cell scraper. to prepare whole cell lysates, cell pellets were extracted with lysis buffer containing 1× laemmli/urea (62.5 mm tris, ph 6.8, 2% sodium dodecyl sulfate, 5% glycerol, and 6 m urea) and proteinase inhibitor (4 μl). after measuring the protein concentration in the supernatant using the pierce tm bca protein assay kit (thermo fisher scientific, waltham, ma, usa), the supernatants were mixed with 5% (v/v) 1 m dithiothreitol and 5% (v/v) bromophenol blue and heated at 98°c for 5 min. equal amounts (50 μg/lane) of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and then electro-transferred onto nitrocellulose membranes. the membranes were incubated with the indicated primary antibodies (il-6, mmp-3, nrf2, phosphorylated p62, phosphorylated jnk) and further incubated with secondary g-horseradish peroxidase conjugates (amersham tm , ge healthcare, little chalfont, uk). protein bands were visualized by western blotting detection solution using an enhanced chemiluminescence western blotting detection solution (hi-rendol®, hi-renfix®, fujifilm, tokyo, japan) and exposing the membranes to xray film or the protein signals were detected with an ecl system (biorad, hercules, ca, usa) and visualized using a charge-coupled device (ccd) cooled camera (gene genome; syngene, cambrigde, uk). these x-ray film data were digitalized and graphs were individually created using public domain image processing software, imagej (u. s. national institutes of health, bethesda, maryland, usa, http://imagej.nih.gov/ij/). mh7a cells were cultured in chamber slides (super-frost®, matsunami glass, japan) for 24 h following pre-incubation for 3 and 24 h after the administering of nac (1000 μm). cells were again cultured for 1 h after the administering of h 2 o 2 (100 μm). the cells were then fixed with 4% paraformaldehyde and permeabilized with tris-buffered saline (tbs) containing 0.1% triton x-100. nonspecific binding was blocked with 5% bovine serum albumin (bsa) dissolved in dulbecco's phosphate-buffered saline (dpbs) for 30 min. the slides were incubated for 1 h at 25°c with primary antibody (anti-nrf2, ab53019, abcam). after three washes for 10 min each, the slides were incubated for 1 h with alexa fluor™ 568 phalloidin supplemented with goat antirabbit igg-alexa fluor 488(thermo fisher scientific) secondary antibodies. nuclei were stained with 4′,6-diamidino-2-phenylindole (dapi; thermo fisher scientific). after three washes for 10 min each, the slides were covered with mounting medium (dako, glostrup, denmark) and analyzed with a confocal laser scanning microscope (fluoview 1200, olympus, tokyo, japan). quantitative ros measurements for mh7a cells incubated in medium containing nac or sp600125 for 3 and 24 h was performed using the muse™ oxidative stress kit (emd millipore bioscience, billerica, ma, usa). this provided the relative percentages of cells that are ros-negative and ros-positive. after treatment with nac or sp600125, mh7a cells were harvested, incubated with oxidative stress reagent (dihydroethidium), and analyzed on the muse cell analyzer according to the manufacturer's protocol. to measure ros, a reflection of oxidative stress, mh7a cells were incubated in medium containing nac or sp600125 for 3 and 24 h each, and h 2 o 2 was added to the medium for 1 h because the intracellular ros level was highest 1 h after the addition of h 2 o 2 [17, 18] . the concentrations of il-6 in the mh7a cell culture supernatants were measured using a fully automated chemiluminescent enzyme immunoassay system (lumi-pulse® g1200, fujirebio, inc., tokyo, japan). il-6 in all measurements were replicated three or four times, and all values are expressed as the means ± the standard error of the mean (sem). statistical analyses were performed with one-way analysis of variance to analyze (anova) followed by turkey's multiple comparisons test, and twoway anova followed by dunnett's, turkey's, or bonferroni's multiple comparisons test using graphpad prism software (graphpad, inc., san diego, ca, usa). p value < .05 was considered statistically significance. to determine the appropriate experimental concentrations of nac and h 2 o 2 for the experiment, we determined the cytotoxicity of these solutions. at a concentration of 10 mm (10,000 μm) nac, cell viability of mh7a was 30% after 24 h of treatment ( fig. 1a(1) ). at a concentration of 1000 μm or lower nac, cell viability of mh7a was greater than 90%. we, therefore, chose 1000 μm or lower nac as our working concentration ( fig. 1.a(2) ). on the other hand, h 2 o 2 is typically used at concentrations from 100 to 1500 μm [13, 17, 18] . mh7a cell lived about only 40% by administering 100 μm of h 2 o 2 for 24 h. we decided to use 100 μm of h 2 o 2 for a brief time (fig. 1a(3) ). low-dose nac increases nrf2 and p62 (antioxidantrelated proteins) and suppresses ros elevation following nrf2 protein expression showed dose-and timedependent increases at 3 and 24 h after the administration of nac (10-1000 μm). its expression significantly increased at 24 h after administering nac (1000 μm) compared with that at 24 h without treatment of nac (1.33 vs. 1.08, p = .036) ( fig. 1b(1, 2) ). phosphorylation of p62 (serine 403-phosphorylated p62) also showed dose-and time-dependent increases at 3 and 24 h after the administration of nac (10-1000 μm). its expression significantly increased at 24 h after administering nac (1000 μm) compared with (2)). all values are expressed as the mean ± sem (n = 3). the significance levels are shown as *p value < .05, **p value < .01, ***p value < .001, ****p value < .0001 that at 24 h without treatment of nac (1.14 vs. 1.51, p = .037) (fig. 1c(1, 2) ). nrf2 expression was higher in cytoplasm than in nucleus after 24 h in untreated mh7a cells (fig. 1d(1) ). at 24 h after administering nac (1000 μm), nrf2 was translocated from cytoplasm to nucleus reflecting antioxidant response (fig. 1d(2) ). this phenomenon is known to be always caused by oxidative stress. the mean positive percentage of ros in mh7a cells was 26.2% at 3 h, and 34.5% at 24 h, without nac treatment. compared with these control data, there was a significant increase in ros (68.4%) 1 h after the administering h 2 o 2 (100 μm) (p < .0001). pre-incubation with nac (1000 μm) for 3 and 24 h followed by 1 h of h 2 o 2 (100 μm) treatment resulted in significant decreases in ros levels from 68.4 to 38.7% at 3 h (p < .0001), and from 68.4 to 47.2% at 24 h (p = .019), respectively ( fig. 1e(1, 2) ). these results demonstrated that low-dose nac (1000 μm) suppressed the ros elevation following h 2 o 2 treatment (100 μm) in mh7a cells. expression of mmp-3 protein was not altered 3 h after administering nac (10, 100, and 1000 μm); however, it decreased significantly after 24 h of nac administration (1000 μm) compared with that without nac treatment (0.45 vs. 0.61 in the band expression intensity, p = .03) (fig. 2a(1, 2) ). phosphorylation of jnk (54 kd) did not demonstrate significant dosedependent decrease at 3 h following low-dose nac treatment. however, phosphorylation of jnk (46 kd) decreased significantly 3 h after nac administration (1000 μm) compared with those at 3 h without nac treatment and 10 μm nac treatment (1.0 vs. 0.51in the band expression intensity, p = .049, and 1.04 vs. 0.51, p = 0.033, respectively) ( fig. 2b(1, 2, 3) ). to investigate the change of il-6 expression under treatment of nac in mh7a cells, we performed the same experiments as mmp-3 and jnk proteins. expression of il-6 protein was not significantly changed at 3 and 24 h after the administering of nac (10, 100, and 1000 μm) compared with the condition without nac treatment ( fig. 2c(1, 2) ). h 2 o 2 (100 μm) administration for 24 h significantly increased il-6 concentration in supernatant of mh-7a cells compared with untreated condition (743 vs. 601 pg/ml, p = .0047). nac treatment (1000 μm) for 24 h slightly decreased il-6 concentration compared with the condition under h 2 o 2 (100 μm) but no significant difference was detected (699 vs. 743 pg/ml). these findings indicated that nac was not able to reduce il-6 (fig. 2d) . jnk inhibitor (sp600125) concentrations of 15 and 30 μm were selected based on the previous research [19, 20] . sp600125 was dissolved in dmso and used at a final concentration of 0.1% or less. phosphorylation of jnk (54 kd) expression showed dose-dependent decrease by sp600125 treatment. the intensity of band expression of p-jnk (54kd) under 3 h treatment of 30 μm sp600125 was significantly decreased than those of untreated and 15 μm sp600125 (0.53 vs. 0.87, p = .0026 and 0.53 vs. 1.0, p = 0.0005, respectively) ( fig. 3a(1, 2) ). phosphorylation of jnk (46 kd) expression also showed dose-dependent decrease by sp600125 treatment. the intensity of band expression of p-jnk (46kd) under 3 h treatment of 30 μm sp600125 was significantly decreased than those of untreated and 15 μm sp600125 (0.53 vs. 0.75, p = .004 and 0.53 vs. 1.0, p < .0001, respectively) ( fig. 3a(1, 3) ). in addition, the intensity of band pattern of p-jnk (46kd) under 3 h treatment of 15 μm sp600125 was also significantly decreased than that of untreated condition (0.75 vs. 1.0, p = 0.0025) (fig. 3a(1, 3) ). there was no significant difference in phosphorylation of jnk (54 and 46 kd) at 24 h compared with 0.1% dmso-treated cells (fig. 3a(1) ). next, we measured the rate of ros positive cells to clarify whether jnk inhibitor has an antioxidative effect like nac. compared to untreated mh7a cells, 1 h treatment of h 2 o 2 (100 μm) significantly increased ros to 66.4% (p < .0001). sp600125 (15 μm) administration for 3 h significantly decreased h 2 o 2 -induced ros to 48.3% (p < .0005). however, sp600125 (30 μm) administration for 3 h did not significantly decrease h 2 o 2 -induced ros (fig. 3b(1) ). compared to untreated mh7a cells, 1 h treatment of h 2 o 2 (100 μm) significantly increased ros to 66.4% (p < .0001). sp600125 (15 μm) administration for 24 h significantly decreased h 2 o 2 -induced ros to 41.6% (p < .0001). sp600125 (30 μm) administration for 24 h also significantly decrease h 2 o 2 -induced ros to 38.7% (p < .0001) (fig. 3b(2) ). these findings were confirmed by western blotting of jnk proteins. under identical conditions, phosphorylation of jnk (46 kd) in mh7a cells increased significantly 1 h after administering h 2 o 2 (100 μm). however, phosphorylation of jnk decreased significantly 1 h after the significance levels are shown as *p value < .05, **p value < .01, ***p value < .001, ****p value < .0001 the administering of h 2 o 2 (100 μm) and 3 and 24 h after administering sp600125 (15 or 30 μm) (supplementary information: fig. s1 ). sp600125 treatment (30 μm) for 24 h significantly decreased il-6 concentration compared with the condition under h 2 o 2 (100 μm) for 24 h without sp600125 (417 vs. 743 pg/ml, p < .0001). these findings indicated that sp100625 was able to reduce il-6 concentration in supernatants in mh-7a cells (fig. 3c) . in mh7a cells, protein expression of mmp-3 showed a dose-dependent decrease at 3 and 24 h. sp600125 (30 μm) treatment for 24 h significantly decreased mmp-3 expression compared with untreated cells (0.51 vs. 0.93, p = .0457), but not in the condition of sp600125 (30 μm) treatment for 3 h (fig. 4a(1, 2) ). in ra-fls, protein expression of mmp-3 showed a dose-dependent decrease at 3 and 24 h. sp600125 (30 μm) treatment for both 3 and 24 h significantly decreased mmp-3 expression compared with untreated cells (0.52 vs. 1.0, p = .0359 at 3 h and 0.36 vs. 0.8, p = .0042, respectively) ( fig. 4b(1, 2) ). we found that low doses of nac (1000 μm) and sp600125 (15 and 30 μm) were effective in suppressing the production of the proteolytic enzyme mmp-3, which, through suppression of jnk pathway component phosphorylation, causes joint destruction. the concentration of nac required to suppress all proinflammatory cytokines and nf-κb is at least 5 mm. we determined that after 24 h of treatment, 10 mm nac is cytotoxic to mh7a cells with only 30% of mh7a cells remaining viable. we utilized a 1000 μm concentration of nac, which was cytotoxic to 10% or less of the mh7a cells in our sample. low-dose nac can reduce h 2 o 2 -induced ros and increases in nrf2 and p62 expression, which have antioxidant effects, and induce antioxidant genes (fig. 1b , c, d) [21] [22] [23] [24] [25] [26] . the expression of nf-κb is related to il-6 production. fujisawa et al. reported that low-dose nac (1000 μm) is unable to suppress the transcriptional activity of nf-κb, which was consistent with our findings (fig. 2c, d) [10] . however, we found that low-dose nac (1000 μm) could significantly suppress the phosphorylation of jnk and downstream mmp-3 protein expression (fig. 2a, b) . these findings confirm that low-dose nac is linked to the suppression of jnk phosphorylation and mmp-3 expression. we conducted experiments using sp600125, a jnk inhibitor, to confirm that the suppression of jnk was directly inhibited mmp-3 expression. in mh7a cells, phosphorylation of jnk pathway components was significantly suppressed, dependent on dose, 3 h after sp600125 treatment (15 and 30 μm) , and mmp-3 expression was significantly suppressed 24 h after administering sp600125 (30 μm; figs. 3a and 4a) . ros, which significantly increased 1 h after administering h 2 o 2 (100 μm), was significantly suppressed 3 h after sp600125 treatment (15 μm). at 24 h after administering sp600125 (15 and 30 μm) , ros was also significantly suppressed (fig. 3b) . the production of il-6 in the cell supernatant was significantly inhibited 24 h after sp600125 treatment (30 μm; fig. 3c ). findings acquired in our study demonstrated that low-dose nac suppressed mmp-3 and ros, but did not inhibit il-6 and that sp600125 suppressed all the three mmp-3, ros, and il-6 in mh-7a cells. in particular, we confirmed that sp600125 (30 μm) had an antioxidant, anti-inflammatory effect that suppressed ros and inhibited il-6 production (fig. 3b, c, and s1 ). experiments using ra-fls obtained through the primary dispersion culture of synoviocytes taken from ra patients found that mmp-3 was significantly suppressed at 3 and 24 h following the administering of sp600125 (30 μm; fig. 4b) , which was similar results in the case of mh7a cells. we believe that this indicates the presence of an mmp-3 suppression mechanism that utilizes the jnk pathway in ra-fls. ra treatment must control both joint inflammation and joint destruction [1] [2] [3] [4] [8] [9] [10] . shen h et al. indicated that jnk inhibitor (sp600125) suppressed the increase of activator protein-1 (ap-1) transcription factor unstream of mmps production and the increase of nf-κb (p65) transcription factor unstream of il-6 production due to paraquat injury in human lung basal epitherial calls. [27] . using mh7a cells, ra synovial model cells, we demonstrated that a jnk inhibitor suppresses inflammation and joint destruction. we finally considered the explainable mechanism of mmp-3 suppression via jnk pathway by low-dose nac and jnk inhibitor (fig. 5 ). mmp-3 expression is regulated by jnk pathway. once ros activate jnk-pathway, phosphorylated jnk activates nuclear transcription factor ap-1 and mmp-3 protein production is promoted. low-dose nac or jnk inhibitor (sp600125) inhibits ros production itself and specifically inhibits phosphorylation of jnk protein so mmp-3 protein production is prominently suppressed. mmp-3, which causes il-6 production and joint destruction in ra patients, is produced by mh7a cells. in mh7a cells, nac reduces ros and mmp-3. a jnk inhibitor reduces ros production, decreases il-6, and downregulates mmp-3. mmp-3 is also reduced in human synoviocytes collected from ra patients following the jnk inhibitor treatment. we believe that jnk pathway will be a novel therapeutic target for the treatment of ra. supplementary information accompanies this paper at https://doi.org/10. 1186/s13018-020-01595-9. redox signaling and the inflammatory response in rheumatoid arthritis role of interleukin-1 and tumor necrosis factor α in matrix degradation of human osteoarthritic cartilage oxidative stress relevance in the pathogenesis of the rheumatoid arthritis: a systematic review molecular mechanisms of nacetylcysteine actions genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/akt/nuclear factor κb and adenosine monophosphateactivated protein kinase signal pathways in human synoviocyte mh7a cells biological impacts of resveratrol, quercetin, and n-acetylcysteine on oxidative stress in human gingival fibroblasts nacetylcysteine attenuates ischemia-reperfusion induced apoptosis and autophagy in mouse liver via regulation of the ros / jnk / bcl-2 pathway il-4 inhibition of il-1 induced matrix metalloproteinase-3 (mmp-3) expression in human fibroblasts involvers decreased ap-1 activation via negative crosstalk involving of jun n-terminal kinase (jnk) a tale of two joints: the role of matrix metalloproteases in cartilage biology activation of transcription factor nf-κ b in human synovial cells in response to tumor necrosis factor α antioxidant and antiinflammatory efficacy of nac in the treatment of copd: discordant in vitro and in vivo dose-effects: a review n-acetylcysteine attenuates tnf-α-induced p38 map kinase activation and p38 map kinasemediated il-8 production by human pulmonary vascular endothelial cells oxidative stress-induced apoptosis in granulosa cells involves jnk, p53 and puma the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis acquisition, culture, and phenotyping of synovial fibroblasts secretion of vascular endothelial growth factor is inhibited by the antioxidant of n-acetylcysteine in fibroblast-like synoviocytes from rheumatoid arthritis antioxidants inhibit tumor necrosis factor-α mediated stimulation of interleukin-8, monocyte chemoattractant protein-1, and collagenase expression in cultured human synovial cells protective effects and mechanisms of terminalia catappa l. methenolic extract on hydrogenperoxide-induced oxidative stress in human skin fibroblasts c-jun n-terminal kinase is requited for metalloproteinase expression and joint destruction in inflammatory arthritis inhibition of cell proliferation and cell cycle progression by specific inhibition of basal jnk activity the selective autophagy substrate p62 activates the stress responsive transcription factor nrf2 through inactivation of keap1 serine 403 phosphorylation of p62/sqstm1 regulates selective autophagic clearance of ubiquitinated proteins molecular and chemical regulation of the keap1-nrf2 signaling pathway protective effect of n-acetlcysteine (nac) on renal ischemia/reperfusion injury through nrf2 signaling pathway emerging functional cross-talk between the keap1-nrf2 system and mitochondria nrf2, the master regulator of anti-oxidative responses jnk inhibitor sp600125 attenuates paraquat-induced acute lung injury: an in vivo and in vitro study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank mrs. keiko tanaka for providing technical assistance with qpcr and western blotting and mr. takashi ariizumi (m.d., ph.d.) for providing technical assistance in various experiments. this study was approved by institutional review board of niigata university medical and dental hospital (#2018-0377). written informed consent was obtained from each patient before the specimen was taken. 3 department of orthopedic surgery, uonuma institute of community medicine, niigata university medical and dental hospital, minami-uonuma, niigata, japan. authors' contributions tk, mo, and nk designed the study. tk and mo performed experiment. hs, go, yk, ao, and hk. treated patients and collected the synovial tissues. tk, mo and nk interpreted data and wrote the manuscript. n.e. supervised this study. all authors read and approved the final manuscript. we thank the ministry of education, culture, sports, and science technology who helped fund this research (18k09057 to n.k., 17k17739 to m.o., 18k09098 to a.o., and 17k10960 to h. k.). the datasets used during the current study are available from the corresponding author on reasonable request. written informed consent was obtained from each patient for publication. the authors declare that they have no competing interest. chuo-ku, niigata, niigata, japan . 2 department of neurosurgery, brain research institute, niigata university, chuo-ku, niigata, niigata, japan. key: cord-0032217-lkowb3hk authors: tardella, marika; di carlo, marco; carotti, marina; ceccarelli, luca; giovagnoni, andrea; salaffi, fausto title: a retrospective study of the efficacy of jak inhibitors or abatacept on rheumatoid arthritis-interstitial lung disease date: 2022-04-24 journal: inflammopharmacology doi: 10.1007/s10787-022-00936-w sha: 3d0c1e5ff496c52993bb48785b318b8591b138de doc_id: 32217 cord_uid: lkowb3hk objectives: to examine the effectiveness of janus-kinase inhibitors (jakis) or abatacept (aba) in patients with rheumatoid arthritis-interstitial lung disease (ra-ild). methods: patients with ra-ild receiving jakis or aba were retrospectively evaluated at baseline and after 18 months of treatment. a computer-aided method (cam) was used to assess the extent of high-resolution computed tomography (hrct) fibrosis percentage. according to hrct fibrosis changes, patients were classified as “worsened” (progression of 15% or more), “stable” (changes within 15%) or “improved” (reduction of 15% or more). correlations between ra characteristics and jakis or aba responses were studied using a multivariate regression model. results: seventy-five patients (69.3% women) were evaluated, 31 received a jaki while 44 received aba. in the jakis group, five patients (16.1%) showed ra-ild progression, 20 patients (64.5%) were considered stable, and six patients (19.4%) demonstrated ra-ild improvement. in the aba group, five patients (11.3%) showed ra-ild progression, 32 patients (72.7%) were stable, and seven patients (16.0%) demonstrated ra-ild improvement. in both groups, the percentage of current smokers was different between those classified as "worsened" and those classified as "improved/stable" (p = 0.01). in multivariate regression analysis, current smoking habit (p = 0.0051) and concomitant methotrexate treatment (p = 0.0078) were the two variables related to ra-ild progression in aba-treated patients, whereas in jakis-treated patients, the only ra-ild progression-related variable was disease duration of ra (p < 0.001). conclusions: treatment with jakis or aba was related to stability or improvement of ra-ild in 83.9% and 88.6% of patients, respectively. ra duration is the only variable associated with worsening ra-ild in jakis-treated patients. rheumatoid arthritis (ra) is a chronic autoimmune disease characterized by joint inflammation and destruction, with an incidence of 0.5% of the adult population in western countries (salaffi et al. 2005) . in addition to joint involvement, extra-articular manifestations of ra can affect several organs and systems. pulmonary involvement in ra has been the subject of increasing interest in recent years, particularly interstitial lung disease (ild) as one of the most severe extra-articular manifestation, leading to progressive respiratory failure . ild occurs in 1.8% to 67% of ra patients, determining a worse prognosis and mortality (fazeli et al. 2021; spagnolo et al. 2018; raimundo et al. 2019) . the management and treatment of ra-ild are challenging because there is still little information available and there are no clinical trials dedicated on the topic, although this aspect is increasingly considered by guidelines (holroyd et al. 2019) . current therapies for ra treatment are worldwide extensively evaluated to determine their effect on lung and ra-ild patients. since few years the efficacy of anti-fibrotic drugs used in idiopathic pulmonary fibrosis has been evaluated in ra-ild patients (redente et al. 2018) . many authors discuss the lung toxicity of disease modifying anti-rheumatic drugs (dmards), particularly methotrexate (england and hershberger 2020; roubille and haraoui 2014) . if until recently methotrexate was thought to be toxic for patients with ra-ild because it could lead to pulmonary toxicity, now the most recent data show that it could slow down the progression of lung damage (wells 2021; rojas-serrano et al. 2017) . the introduction of biotechnological dmards (bdmards) and, more recently, of janus-kinase inhibitors (jakis) has changed the course of ra, markedly improving the control of synovitis and, consequently, reducing joint destruction and physical disability (nash et al. 2021; fraenkel et al. 2021) . we have recently underlined the "protective" effect of abatacept (aba), a t lymphocyte co-stimulation antagonist, in patients with ra-ild, showing reasonable efficacy in slowing the progression of ra-ild in about 88% of cases (tardella et al. 2021) . other bdmards are suggested for ra-ild therapy, such as rituximab and tocilizumab, but in addition to their higher infectious risk compared with aba, cases of onset or worsening of ild have been reported in patients taking these drugs soubrier et al. 2008; wendling et al. 2013; hadjinicolaou et al. 2011) . jakis is a group of drugs classified as "small molecules" or "targeted synthetic dmards" to differentiate themselves from bdmards by the different mechanism of action. they are medications that are taken orally every day and have demonstrated efficacy in the treatment of ra, either alone or in combination with methotrexate (fleischmann et al. 2017; lee and bae 2016) . little is known about the safety and tolerability of jakis in patients with ra-ild (khoo et al. 2020; salvarani et al. 2021; citera et al. 2021) . new data are emerging in favor of tofacitinib (tfn), the longest used jaki, to slowing the progression of ild associated to connective tissue diseases (romero-bueno et al. 2020; chen et al. 2019; pineton de chambrun et al. 2020) . sendo and colleagues showed the effect of tfn on mice that developed arthritis and ild, revealing a significant slowdown in ild progression (sendo et al. 2019 ). lescoat and colleagues tested the effect of ruxolitinib, a jaki used in the treatment of myelofibrosis, on mice with scleroderma-like ild, revealing improved skin and lung involvement (lescoat et al. 2020 ). these preliminary data prompt testing of the combined antiinflammatory and anti-fibrotic properties of jakis also in patients with ra-ild. the primary outcome of interest of this study is the evaluation of the evolution of pulmonary fibrosis in patients receiving jakis compared to those receiving aba, using computerized hrct assessment. a secondary objective is to identify predictors of an unfavorable treatment outcome of ra-ild during jakis therapy. study data were extracted from a database dedicated to ra patients referred to the rheumatology clinic, università politecnica delle marche (italy). patients were included if aged > 18 years, met the american college of rheumatology (acr)/european league against rheumatism (eular) classification criteria for ra diagnosis and, concomitantly, the american thoracic society/ats/ers 2015 criteria for ild diagnosis (aletaha et al. 2010; travis et al. 2013) . moreover, patients included in the study were: ra-ild patients who took continuous jaki therapy (tfn at an oral dose of 5 mg bid or baricitinib at an oral dose of 4 mg daily) or aba therapy at a standard dose of 125 mg/ week subcutaneously for at least 18 months, who performed a rheumatologic evaluation [tender joint count (tjc) and swollen joint count (sjc), clinical disease activity index (cdai), health assessment questionnaire-disability index (haq-di)] and a pulmonary investigation [high-resolution computed tomography of the lung (hrct), pulmonary function test (pft), single-breath diffusion lung capacity of carbon monoxide (dlco, % predicted, corrected for hemoglobin), borg's dyspnea index (bdi)] within two weeks the beginning of a jaki or aba (time 0) and after 18 months (time 18). we included patients with ≥ 10% extent of fibrosis on hrct. we considered the group of patients treated with aba as a control group because it is one of the recommended treatments in ra-ild. patients concomitantly taking methotrexate (mtx) or other conventional synthetic dmards (csdmards) and/or glucocorticoids at a dose of less than 10 mg daily prednisone or equivalent were included. patients with a history of lung diseases, other than ild, and/or nyha stage ii-iv heart failure and patients who had been previously treated with aba or jakis were excluded. baseline data included demographic variables, smoking habits, duration of disease (defined as time elapsed since diagnosis), and the presence or absence of rheumatoid factor (rf) and anti-citrullinated peptide antibodies (acpa). the evaluation of the ild extension was performed with a computer-aided method (cam) able to estimate the percentage of fibrosis at hrct, based on what has been described in detail in previous works (salaffi et al. 2020a; ariani et al. 2014; salaffi et al. 2020b ). the cam was performed with osirix md 7, a dicom visualization software (osirix md version 7, 64-bit format) on a mac mini (2.8 ghz intel core 2 duo desktop computer, 16 gb random access memory; apple computer, cupertino, ca, usa) with mac osx 10.12.2 operating system. two independent radiologists, blind to the clinical data, evaluated hrct lung abnormalities and the cam quantification process. according to hrct results, performed at time 0 and time 18, patients were divided into three groups based on hrct-cam comparison: the "worsened" group included patients with ≥ 15% progression of pulmonary fibrosis, the "improved" group included patients with ≥ 15% reduction of pulmonary fibrosis, and the "stable" group included patients with progression or reduction of fibrosis < 15%. the 15% cam-change threshold derived from the standard deviation of the change in mean value after 18 months of follow-up. data were processed with medcalc 19.0.6 (statistical software packages for windows xp). normal distribution was tested using the kolmogorov-smirnov test. the median and interquartile ranges (iqr) as well as the mean and standard deviations (sd) were presented wherever appropriate. continuous variables were compared using the parametric two-sample t test and the one-way analysis of variance (anova) test, while categorical variables among patients were compared using the χ 2 test. values at time 0 and time 18 were compared with the two-sided coupled t test and the non-parametric wilcoxon signed rank test. corrected multivariate regression analysis was used to assess the strength of the association between ra characteristics and hrct response to aba or jakis, considering cam quantification as a dependent variable. age, sex, disease duration, disease onset, smoking habit, presence of rf, presence of acpa, cdai, and haq-di were the covariates. results were expressed as multivariate regression coefficient (r) and adjusted quadratic regression coefficient (r2) for the number of variables included in the analysis. significance was set at p < 0.05. we enrolled 75 patients (69.3% women, mean age 59.5 ± 7.77 and mean disease duration 7.44 ± 3.25 years), of whom 31 received a jaki (18 patients took baricitinib, 13 patients took tfn) and 44 were treated with aba. both treatment groups had similar demographic and clinical features (table 1 ). as shown, at time 0 the included patients had on average a mild to moderate lung function impairment (forced vital capacity 81.7% and dlco 59.2%) and the cam assessment showed a mean fibrosis rate of 19.4 and 18.5% in patients treated with aba or jakis, respectively. a fibrosis rate greater than 20% at cam evaluation occurred in 14/31 (45%) and 18/44 (41%) of patients in the jakis and aba groups, respectively. in aba group 23 (52.3%) patients were acpa positive and 28 (63.6%) rf positive, all patients were concomitantly treated with csdmards, 16 (36.4%) patients were previously treated with a bdmard and 31 (70.4%) patients took steroids at a mean dose of 3.7 (range 1.25-8.5) mg prednisolone/day equivalent. in jakis group 16 (51.6%) patients were acpa positive and 19 (61.3%) rf positive, no patients were simultaneously taking csdmards, 11 (35.5%) patients were previously treated with a bdmard and 21 (67.7%) patients took steroids at a mean dose of 3.3 (range 1.11-8.22) mg prednisolone/day equivalent. at time 18 the ra articular manifestations had a statistically significant improvement (p < 0.001) both as an estimate of disease activity and as of patients-outcome in both treatment groups (fig. 1) . in contrast, the pulmonary component did not show a statistically significant reduction for both patients-outcomes and instrumental evaluation (fig. 2) . in aba group 5 (11.4%) patients showed a hrct deterioration, 32 (72.6%) were considered stable, 7 (16.0%) patients showed an hrct improvement, while in jakis group 5 (16.1%) patients showed a hrct deterioration, 20 (64.5%) were considered stable, 6 (19.4%) patients showed an hrct improvement at time 18. considering the whole cohort, the percentage of current smokers was different between those classified as "worsened" and those classified as "improved/stable" (p = 0.01). in multivariate regression analysis, current smoking habit (p = 0.005) and concomitant methotrexate treatment (p = 0.0078) were the two variables related to ra-ild progression in aba-treated patients. in jakis-treated patients, the only variable related to ra-ild deterioration was disease duration (p < 0.001), whereas smoking exposure, immunological profile, lung function, percentage of lung fibrosis at time 0, and disease activity did not show a statistical correlation (table 2 ). ild is an insidious condition in terms of morbidity and quality of life in patients with connective tissue diseases. our study highlights the efficacy of jakis in patients with ra-ild, in relation also to the results obtained in a similar cohort treated with aba. to the best of our knowledge, this is the first study evaluating the efficacy of jakis treatment in patients with ra-ild by assessing the evolution of lung fibrosis rate with a hrct-cam. in 83.9% of ra-ild patients on jakis therapy there was no deterioration of the lung disease. this finding is like that obtained in ra-ild patients treated with aba (88.6%), representing another step in the knowledge of the complex ra-ild therapy. as mentioned, there are encouraging efficacy data of jakis in studies performed in ra-ild mice and, especially, in patients with ra-ild-related myositis (chen et (vacchi et al. 2021 ). this aspect is not minor because respiratory tract infections in patients treated with jakis are frequent and insidious. we were unable to study safety data because there was a lack of available data. salvarani and colleagues recently estimated the incidence of infectious and non-infectious ild in baricitinib-treated ra patients. they performed a review of 3770 patients with ra from eight randomized clinical trials and one long-term extension study on baricitinib, concluding that ra patients treated with baricitinib were at low risk of developing ild (salvarani et al. 2021) . a similar analysis was performed on 21 tofacitinib clinical trials, in which 42 (0.16%) ild events were identified among 7061 ra patients, confirming the low risk of developing ild in jakis-treated patients. the authors found also that age 65 years or older, current smokers, and a high disease activity index are the risk factors associated with the development of ra-ild (citera et al. 2021) . in our analysis, the only variable related to ild progression in jakis group was ra disease duration while the remaining covariates did not show correlation, and this could be due to the small number of patients enrolled or a bias in recruitment. this aspect is of non-unique interpretation and in many studies on the topic the results are not always concordant (dawson et al. 2002; zamora-legoff et al. 2017 ). however, disease duration is one of the already known risk factors to be considered in the management of ra patients. in 2014 two new anti-fibrotic drugs (nintedanib and pirfenidone) were licensed to treat idiopathic pulmonary fibrosis. data on their efficacy in the treatment of nonidiopathic ild are emerging. data from the inbuild trial show the efficacy of nintedanib, a small molecule protein kinase inhibitor, in patients with ild in connective tissue diseases. eighty-nine ra patients, 44 patients with systemic sclerosis and 20 patients with mixed connective tissue disease were recruited in the study (wells et al. 2020) . the drug reduced the annual rate of decline in forced vital capacity by 57% compared to placebo, promoting drug approval by the us food and drug administration for progressive-ild, including connective tissue disease-ild. additional data are emerging from real-life studies (vacchi et al. 2020) . narvaez and colleagues used nintedanib in combination with immunosuppressants for at least 6 months in six ra-ild patients. they showed a reversal of the decline in lung function parameters, achieving a stabilization of forced vital capacity and dlco (narváez et al. 2020) . in ra-ild mice models pirfenidone has demonstrated an inhibitory effect on transition from fibroblast to myofibroblast in the lungs (wu et al. 2019) . at present a trial on safety and efficacy of pirfenidone on ra-ild patients is ongoing . certainly, anti-fibrotic drugs offer a very important therapeutic opportunity in progressive ra-ild as adjunctive therapy to dmards. however, it could lead to additional side effects (liver toxicity and diarrhea) reducing adherence to treatment. therefore, it would be advisable using a drug that can be effective on both articular and extra-articular manifestations and jakis might be the best choice. in our study the progression of pulmonary fibrosis at hrct is used to assess response to treatment. in literature, the most widely used method for this purpose is the trend of pft parameters, particularly forced vital capacity, and of dlco. we use hrct-cam in daily practice because it is a reliable, reproducible and easily comparable parameter, so it might also be used in clinical research (tardella et al. 2021 , salaffi et al. 2020b . pft and dlco could be influenced by many factors, both pulmonary and extrapulmonary, and are operator-and patient-dependent. this aspect is a distinctive feature of our research and is a hot topic in rheumatology investigation because of the clear advantages demonstrated, particularly the rapidity of estimating fibrosis percentage (salaffi et al. 2020b) . this study has some limitations. it is a retrospective, single-center study with a small number of patients enrolled. we do not know how many patients had severe infectious complications that had to be permanently discontinued jakis or aba treatment. furthermore, we do not know the onset of ild so we do not know if they are progressive ilds and the rate at which they are advancing. in addition, lung fibrosis in our cohort is mild to moderate with less than 20% fibrosis in 43/75 (57.3%) cases, so our results cannot be generalized to patients with severe forms of ra-ild, which are the most difficult to manage in clinical practice, as they can lead to respiratory failure or death. our study showed that jakis-treated patients worsened in only 16% of cases. therefore, it can be stated that jakis are effective in slowing down fibrosis in ra-ild, and they should be considered as a first-choice therapy in ra patients with active synovitis and ild before a stage of extensive fibrosis. from these data, prospective studies with a larger cohort are mandatory to consolidate these promising results. author contributions fs conceived and designed the study and the protocol. mc and ag performed the hrct examinations and their relative interpretation and were involved in revising the paper for important intellectual content. fs, mt, and mdc carried out data interpretation and analysis. fs, mt, mdc, and mc wrote the paper. mt, ag and mdc were involved in drafting the article or revising it critically for important intellectual content. all authors approved the final version to be submitted for publication. funding no specific funding was received from any funding bodies in the public, commercial or not-for-profit sectors to carry out the work described in this manuscript. the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. ethical approval the local ethics committee (comitato unico regionale-asur marche) approved the protocol. the study was conducted in accordance with the helsinki declaration in its fifth edition (2000). all patients signed an informed consent for inclusion of personal records in the local database and for use to scientific research purposes. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative utility of an open-source dicom viewer software (osirix) to assess pulmonary fibrosis in systemic sclerosis: preliminary results tofacitinib in amyopathic dermatomyositis-associated interstitial lung disease incidence rates of interstitial lung disease events in tofacitinib-treated rheumatoid arthritis patients: post hoc analysis from 21 clinical trials efficacy of baricitinib in treating rheumatoid arthritis: modulatory effects on fibrotic and inflammatory biomarkers in a real-life setting predictors of progression of hrct diagnosed fibrosing alveolitis in patients with rheumatoid arthritis management issues in rheumatoid arthritis-associated interstitial lung disease rheumatoid arthritis-associated interstitial lung disease: epidemiology, risk/prognostic factors, and treatment landscape efficacy and safety of tofacitinib monotherapy, tofacitinib with methotrexate, and adalimumab with methotrexate in patients with rheumatoid arthritis (oral strategy): a phase 3b/4, double-blind, head-to-head, randomised controlled trial american college of rheumatology guideline for the treatment of rheumatoid arthritis non-infectious pulmonary complications of newer biological agents for rheumatic diseases-a systematic literature review the british society for rheumatology biologic dmard safety guidelines in inflammatory arthritis pulmonary adverse events of small molecule jak inhibitors in autoimmune disease: systematic review and meta-analysis comparative efficacy and safety of tocilizumab, rituximab, abatacept and tofacitinib in patients with active rheumatoid arthritis that inadequately responds to tumor necrosis factor inhibitors: a bayesian network meta-analysis of randomized controlled trials combined anti-fibrotic and anti-inflammatory properties of jak-inhibitors on macrophages in vitro and in vivo: perspectives for scleroderma-associated interstitial lung disease tocilizumab therapy in rheumatoid arthritis with interstitial lung disease: a multicentre retrospective study nintedanib for the treatment of refractory progressive rheumatoid arthritis-related interstitial lung disease: a real-life case series points to consider for the treatment of immune-mediated inflammatory diseases with janus kinase inhibitors: a consensus statement tofacitinib in antisynthetase syndrome-related rapidly progressive interstitial lung disease rheumatoid arthritisinterstitial lung disease in the united states: prevalence, incidence, and healthcare costs and mortality nintedanib reduces pulmonary fibrosis in a model of rheumatoid arthritis-associated interstitial lung disease rheumatoid arthritis-related interstitial lung disease (ra-ild): methotrexate and the severity of lung disease are associated to prognosis recommendations for the treatment of anti-melanoma differentiation-associated gene 5-positive dermatomyositis-associated rapidly progressive interstitial lung disease interstitial lung diseases induced or exacerbated by dmards and biologic agents in rheumatoid arthritis: a systematic literature review prevalence of musculoskeletal conditions in an italian population sample: results of a regional community-based study i high-resolution computed tomography of the lung in patients with rheumatoid arthritis: prevalence of interstitial lung disease involvement and determinants of abnormalities the role of a chest computed tomography severity score in coronavirus disease computed tomography assessment of evolution of interstitial lung disease in systemic sclerosis: comparison of two scoring systems janus kinase inhibitors as a therapeutic option in rheumatoid arthritis and associated interstitial lung disease. report of four cases baricitinib and the risk of incident interstitial lung disease: a descriptive clinical case report from clinical trials tofacitinib facilitates the expansion of myeloid-derived suppressor cells and ameliorates interstitial lung disease in skg mice the design and rationale of the trail1 trial: a randomized double-blind phase 2 clinical trial of pirfenidone in rheumatoid arthritis-associated interstitial lung disease organizing pneumonia after rituximab therapy: two cases the lung in rheumatoid arthritis: focus on interstitial lung disease abatacept in rheumatoid arthritis-associated interstitial lung disease: short-term outcomes and predictors of progression an official american thoracic society/european respiratory society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias combination therapy with nintedanib and sarilumab for the management of rheumatoid arthritis related interstitial lung disease to facitinib for the treatment of severe interstitial lung disease related to rheumatoid arthritis new insights into the treatment of ctd-ild nintedanib in patients with progressive fibrosing interstitial lung diseases-subgroup analyses by interstitial lung disease diagnosis in the inbuild trial: a randomised, double-blind, placebo-controlled, parallelgroup trial exacerbation of combined pulmonary fibrosis and emphysema syndrome during tocilizumab therapy for rheumatoid arthritis inhibitory effects of pirfenidone on fibroblast to myofibroblast transition in rheumatoid arthritis-associated interstitial lung disease via the downregulation of activating transcription factor 3 (atf3) progressive decline of lung function in rheumatoid arthritis-associated interstitial lung disease key: cord-0024898-h156n6jj authors: mucke, johanna; knitza, johannes; muehlensiepen, felix; grahammer, manuel; stenzel, ramona; simon, david; kleyer, arnd; krönke, gerhard; sharp, charlotte; bendzuck, gerlinde; korinth, marianne; elling-audersch, corinna; vuillerme, nicolas; schett, georg; pecher, ann-christin; krusche, martin title: telera—asynchronous telemedicine for patients with rheumatoid arthritis: study protocol for a prospective, multi-center, randomized controlled trial date: 2021-12-13 journal: front med (lausanne) doi: 10.3389/fmed.2021.791715 sha: a889bf6e05d25c89972f7e4f41c916eee5eae407 doc_id: 24898 cord_uid: h156n6jj innovative strategies are needed to adequately assess and monitor disease activity of patients with rheumatoid arthritis (ra) in times of scarce appointments. the aim of the telera study is to evaluate the feasibility and performance of asynchronous telemedicine visits based on patient-generated data and patient's drug history. ra patients use a medical app, abaton, that captures the results of a self-performed quick crp-test, joint-count, and electronic patient-reported outcomes in between visits. this is a prospective, multi-center, randomized controlled trial performed in four german university centers. the estimated sample size is 120 patients. the main outcome is the agreement of rheumatologists' treatment decisions based on asynchronous telemedicine patient-generated data with traditional in-person rheumatology clinic-based decisions and with patient suggestions. the telera trial will provide evidence regarding the implementation of remote care in rheumatology. clinical trial registration: this clinical trial was registered at german registry for clinical trials (drks). http://www.drks.de/drks00016350, identifier: drks00024928. the treat-to-target concept has been established as a treatment principle for rheumatoid arthritis (ra) (1) . the aim of this strategy is to define a treatment target at therapy initiation and to closely monitor treatment response in order to identify insufficient treatment success and modify the therapeutic strategy as needed. this approach represents a challenge for rheumatologists as frequent, one to three-monthly assessments are recommended in patients with active disease and resources are limited (1) (2) (3) . in reality, therapies are not assessed frequently enough and, therefore, are all too often not adjusted to the current state of the disease (4) . at least two important reasons for the currently poor disease management are (a) the limited access to rheumatology specialists and (b) an increasing follow-up appointment deficit for already diagnosed patients. this situation is likely to worsen as the current shortage of rheumatologists in germany and other nations will probably increase even further in the future (5, 6) . digitalization promises new ways to improve patient care and shape a more efficient and transparent health care environment (7, 8) and the large majority of german patients with rheumatic and musculoskeletal diseases (rmd) regularly uses a smartphone (9) . electronic patient reported outcomes (epros) and wearables facilitate continuous digital patient monitoring ("tight control") (9, 10) . by using telemedicine, patient care could be individually adapted according to disease activity and follow-up preferences ("virtual treat-to-target") (11) . by actively involving the patient in disease monitoring, self-efficacy and activation ("patient empowerment") can be increased (12) and ultimately remission reached earlier (13) . furthermore, the use of telemedicine could enable the rheumatologist to work more flexibly and efficiently. the aim of this study is to evaluate the feasibility and performance of asynchronous telemedicine visits based solely on patient-generated data in ra patients. this is a prospective, multi-center, randomized controlled trial. this study has received ethical approval by the ethical committee of the university clinic of tuebingen (# 4442020). we aim to include 120 patients with ra and written informed consent, treated at the rheumatology outpatient units of the university hospitals duesseldorf, erlangen, hamburg and tuebingen (germany). the overall study design is summarized in figure 1 . prior to a regular face-to-face appointment (t0), patients with ra will be supported to carry out a structured self-examination of joints by a video and instructions from the study personnel and to perform a quick self c-reactive protein (crp)-test. the patients will then enter the results in a study app. furthermore, patients will be asked to answer epros on a weekly basis in between the regular next face-to-face appointment 3 months later (t1), when the self-examination will be repeated. at each self-examination, the patient also enters his/her therapeutic suggestion: (a) escalation of therapy; (b) no therapeutic change needed; and (c) de-escalation of therapy. based on patient history, the patient-generated data from t0, t1 and in-between visits, two independent randomized rheumatologists from other university centers will be asked to enter their therapeutic suggestion solely based on the digital information. the patient and tele-rheumatologists' suggestions are then compared to the gold standard which is the traditional therapeutic decision based on the face-to-face visit for t0 and t1. after t1, patients complete an evaluation questionnaire and semi-structured telephone interviews will be conducted with 10 patients with ra and the four main study investigators to capture qualitative feedback concerning patient acceptance, app compliance, changes in self-management and self-efficacy. inclusion criteria are (1) the established diagnosis of ra according to the 2010 acr/eular classification criteria; (2) > 18 years of age; (3) sufficient german language skills; (4) confidence in using a smartphone; and (5) written informed consent. patients with newly diagnosed ra, unable to use a smartphone/tablet or to give written informed consent will be excluded from the trial. eligible outpatient ra patients will be informed about the study by their local rheumatologist. if consent is given, they will be contacted by the respective trial physician (jk, jm, acp, mk), invited to discuss the study, undergo screening for eligibility and sign the informed consent form. prior to the routine clinical visits, patients are instructed to carry out a structured self-examination. this includes a self-assessed joint count, capillary-based crp self-sampling, completion of epros and data entry into the study app. the duration of the study visit will be ∼1 h. the local study team teaches participants how to self-examine for tender and swollen joints using an instructional video (14) . the video was co-developed with patients for the remora study (see figure 2 ) (15) . the video was translated to german by jk and dubbed using artificial intelligence by the commercial dubbing company papercup r . the introduction provides an overview of how to correctly identify the joints included in the das28 score, including the differences between tender and swollen joints in rheumatoid arthritis vs. other conditions, and how to self-examine. in the video a patient is coached through self-examination by a nurse consultant, who then answers the patients' questions. the video is available open-access online in german (bit.ly/3rlyztq) and in english (bit.ly/3rlyztq), with optional subtitles. any questions will be answered by the local study team, if needed. patients receive written instructions on how to perform a semi-quantitative capillary-based crp-test (crp-check-1,vedalab, alencon, france). any questions will be answered by the local study team, if needed, however the test should be carried out independently by patients and study personnel will only intervene to prevent harm to patients. the immunochromatographic rapid test allows semi-quantitative detection of crp with three possible outcomes: (a) negative; <5 mg/l; (b) borderline; 5 to 10 mg/l or (c) positive; >10 mg/l. the test is currently only licensed for professional use, however we hypothesized that the test could also be carried out successfully by patients after appropriate education. the abaton web-app is a medical device developed and maintained by abaton gmbh (berlin, germany). this app provides a study module which has been adopted to this trial. all digital administered questionnaires, forms and monitoring instruments were pre-configured. each study center has access to the web-based study module via their own study account. patients get invited by the respective clinical site study personnel via a short messaging system (sms) invite which contains the personalized link for the patient. all questionnaires were administered on the patient's own phone ("bring your own device"). in addition to questionnaires completed by patients, all study relevant documentation (for example therapeutic suggestions of tele-rheumatologists) is performed digitally by the study personnel and patients. in addition to forms and questionnaires listed in table 1 , abaton provides documentation for other parameters (lab crp, tenderand swollen joint count (tjc/sjc), medication information) and allows to add extra notes. in between visits, the patients are being asked to complete the rapid-3 (routine assessment of patient index data 3) questionnaire and to state if they experienced a disease flare. if the flare question is answered with yes ("did you experience a flare of your disease in the past 7 days?"), the patients are being asked to specify the duration (1-7 days) on a numeric rating scale with anchors ("on how many days did you experience the disease flare (in days)"). a reminder logic is implemented to remind the patients 3 consecutive days if they have not filled out the questionnaires and stop as soon as the due questionnaire is completed. results are immediately available to the patient and the study site via the web-based dashboard. a graphical dashboard for the frequently entered questionnaires/scores is available to the patient all the time (see figure 4) . after having completed the t1 visit, patients will receive questionnaires to evaluate the app and the study experience via the study app after being manually triggered by the study personnel. local rheumatologists perform the routine patient visit including das-28 joint count, standard venous blood withdrawal for crp analysis, recording of patient-perceived disease activity (vas 0-10) and physician global assessment (pga) (vas 0-10). data is collected using the abaton platform with the study module ( figure 5 ) and the web-based patient app, which allow intuitive and rapid data entry. in addition to the patient-entered data via the application, general patient-related clinical data such as disease duration, previous and current medication is added manually to the abaton platform by the local study personnel ( table 1) . furthermore, all rheumatologists (local rheumatologist and two tele-rheumatologists) enter their therapeutic decision for each patient and visit (t0 and t1) via the web-based application. abaton allows a quick assessment of all study patients and the completeness status of related data points (monitoring, entries of health care professional, etc.). the four participating rheumatologists (jm, jk, mk and acp) are randomized to the respective cases t0 and t1 at the four centers, so that, for each patient visit (t0 and t1), therapeutic suggestions (escalate, no-change, deescalate) are available from two tele-rheumatologists by computer-generated block randomization with a block size of 2. primary outcome will be the agreement of therapeutic suggestions of patients and tele-rheumatologists compared to the gold-standard, the therapeutic decision by the local rheumatologist. secondary endpoints will include: the overall usability and acceptance of the study app [measured by the system-usability scale by patients and physicians (sus) and net-promoter score (nps)]; the agreement of patient-vs. physician-performed joint count; agreement of venous and capillary based crp; willingness to perform crp quick test among patients (nps); travel time to rheumatologist (hours); agreement of tele-rheumatologist and local rheumatologist-based clinical disease activity index (cdai); agreement of patient-based disease activity score (auto-das28-crp) and local rheumatologist-based das-28 crp; change in patient's confidence for self-management (patient activation measure, pam) over time; willingness to substitute routine appointments for telemedicine appointments among patients (nps); change of perceived assessment accuracy of rheumatologists and patients concerning patient disease activity since the last appointment (vas 0-10). due to the exploratory character of the trial, no formal sample size calculation was performed. a number of n = 120 patients was estimated to be sufficient to assess the primary outcome. with only two study visits 3 months apart, the estimated dropout will be low. however, even a moderate dropout rate of 20% would lead to a number of 96 patients to be analyzed. to assess disease activity, the crp-based das-28 will used (16) . the das28-crp is a composite score comprising the acute phase parameters crp, the patient reported disease activity in terms of the patient global assessment (ptga) as well as the tjc and the swollen joint count sjc. the ptga is assessed by a verbally administered numeric rating scale ranging from 0 to 100, asking for the patient's disease activity in the past 7 days. tjc and sjc comprise 28 predefined joints and are usually performed by a trained physician (16) . in this study, an auto-das28-crp will be performed: the das-28 subdomains tjc and sjc will be assessed by the patient himself and then entered into the app, together with the ptga at t0 and t1 and the result of the semi quantitative test (positive/borderline/negative). for calculation purposes we will use a pragmatic approach and enter crp values of 0 µg/ml for negative results, 6 µg/ml for marginal values and 20 µg/ml for positive values. the values were chosen considering the manufacturers thresholds and a meaningful impact on the das28-crp calculation. as an alternative ra specific disease activity score, the cdai was chosen as a crp-independent and purely clinical index. the cdai includes the 28 joint count examination, the pga and the ptga and is used both in clinical practice and in clinical trials. it has been shown to be valid an sensitive for the assessment of ra activity and treatment response (17) . the routine assessment of patient index (rapid-3) is a validated patient-reported outcome measure (prom) for ra patients (18) , evaluating the patient's physical function, pain, and global health. it's electronic version is validated (19) . the pam13 is a 13-item measure that assesses patient activation, including their knowledge, skills and confidence to manage their own health and well-being (20) . the 13 items are answered on a likert scale from 1 to 4 (strongly disagree to strongly agree). this questionnaire reflects the four stages of activation. level 1: patients believe that their role is important (item 1 and 2); level 2: patients have the confidence and knowledge to take an active part (items 3-8); level 3: patients take action and participate actively (items 9-11); level 4: patients have integrated their active role into their everyday life and hold on to it in stressful situations (items [12] [13] . higher pam scores indicate higher patient activation. the validated german version pam13-d will be used (21) . usability and acceptance of the abaton app will be tested using the sus (22) . the sus consists of a 10-item questionnaire answered on a scale from 1 to 5 (strongly disagree to strongly agree) (22) . the scores for each question are added and multiplied by 2.5 to transform to a 0-100 scale. scores above 68 are considered above average (22) . the nps measures the willingness of the participants to recommend the abaton app to another patient (23) . participants answer using a 11-point numeric rating scale (0 not at all likely, 10 extremely likely). answers between 0 and 6 are summarized as detractors, 7-8 as passives and 9-10 as promoters. the nps is calculated by subtracting the percentage of detractors from the percentage of promoters (23). to explore patient acceptance, app compliance, change in patient's confidence for self-management and change in patient's perceived self-efficacy, complementary qualitative interviews are conducted with patients (n = 10) and providers (n = 4). the interviews are held via telephone, using a semi-open interview guide. the interviews are audio-recorded, transcribed verbatim and analyzed using qualitative content analysis (24) . to achieve intersubjectivity and consistency, the analytical work is carried out within the group for qualitative research of the centre for health services research brandenburg using the analysis software maxqda. interrater reliability will be calculated by cohen's kappa. for this exploratory study, 120 patients with ra will be recruited. correlation analyses will be used to assess the agreement of patient-vs. physician-based das-28 and the clinical decisions of patient vs. tele-rheumatologists and face-to-face rheumatologist. multivariable regression analyses will be performed to assess changes in self-management and self-efficacy and their influential factors over time. analyses will be adjusted for potential confounders such as age, disease duration, disease activity and the number and type (conventional disease modifying antirheumatic drugs (dmard), biological dmard or targetedsynthetic dmard) of previous treatments. enrolment of participants started in august 2021. recruitment of 120 patients and follow-up assessments are expected to be completed by june 2022. thus far, patient feedback has been positive. patients seem to appreciate the ability to receive empowering in-depth information about disease evaluation and to independently assess their disease activity. telera is an exploratory study to assess the feasibility and performance of asynchronous telemedical assessments of patients with ra. the overall aim is to move current rheumatology care toward a needs-based approach with the help of remote care. currently, the number of new rheumatologists is declining (3) and it still takes multiple months for patients to see a rheumatologist (25) . however, we could demonstrate that the majority of patients and rheumatologists agreed that patients in remission do not need to be seen in person (26) . the recently published eular guidelines highlight the importance of self-management skills and potential of mobile apps to improve the clinical care of people with ra (27) . importantly, thurah et al. could previously demonstrate in a rct that a remote care approach was safe and not inferior to conventional management in ra (28) . in this rct, 294 patients with ra were requested to complete epros and a crp measurement each 3-4 months. the crp measurement was however not performed by patients themselves, no joint-count was carried out and epro questionnaires were completed before tele-health follow-ups, which were synchronous. based on our previous work (9), we chose a 1 week epro frequency, providing rheumatologists and patients with continuous disease activity data and including selfsampling (26, 29) . furthermore, we wanted to see if our study confirms previous results showing a significant increase in patient activation (pam) after using an ra monitoring app (12) . the benefits of an intensive ra telemonitoring strategy have also been demonstrated by salaffi et al. (13) , who could show that patients with early arthritis reach the state of remission more often and faster when receiving frequent telemonitoring and treatment adjustments according to a standardized treatment protocol rather than standard of care. in our opinion, the simple shift from physical in-person visits to virtual visits will not result in a significant liberation of resources and in particular time of rheumatologists. nevertheless, asynchronous visits give patients and rheumatologists the freedom of time and place. by using a combination of objective and subjective markers for disease-activity self-assessment, patients can inform their rheumatologists in detail about their health status. based on this remote patient-generated information, rheumatologists should continuously adapt the follow-up strategy incorporating remote care strategies (30) : in case a patient achieves remission after therapy induction, the subsequent epro frequency could be extended and a remote follow-up (i.e., telephone call) after 3 months could be scheduled instead of a face-to-face meeting. web based asynchronous home telemonitoring is already an integral part in the care of other chronic diseases such as diabetes and heart disease (31), as well as inflammatory bowel disease (32) and has great potential in rheumatology (33) . to the best of our knowledge, this is the first trial evaluating the potential of asynchronous telemedicine visits based on patient-based joint counts, self-performed crp and epros in ra patients. furthermore, it is the first study comparing treatment decisions of patients, rheumatologists and tele-rheumatologists. a major strength of this study is the early patient involvement. the study design and protocol was designed in close cooperation with three official patient research partners of the german league against rheumatism (deutsche rheuma-liga bundesverband e.v.) who commented on and edited the draft version and approved the final study protocol. we deliberately chose a risk-adverse study setting, where participation has only a minimal impact on clinical care (rheumatologists have access to epros in-between visits). this study design has some limitations. patients eligible for the study need to be in possession of a smartphone and need to have a sufficient level of understanding and dexterity to use the app, resulting in exclusion of some elderly or cognitively impaired, as well as underprivileged patients without smartphone or network access. this is a general limitation in app-based remote health care monitoring and will most probably change within the next decades. similary, the chose time-frame limits the analysis to two appointments per patient. due to the exploratory study character, patients will not be stratified by disease duration, activity or previous treatments. analyses will however be adjusted for these possible confounders. furthermore subgroup analyses will be performed to assess potential differences between patients in remission or with active disease. also, remote evaluation can be difficult in patients with recent treatment changes who have experienced some improvement but still have active disease. for these cases, remote rheumatologists receive information on previous therapies including recent changes. the usage of semi-quantitative crp tests provides only a certain degree of accuracy. however, this is a simple testing system that could be performed at home without the need of extensive equipment. further, after discussing the options with patients, it seemed more important to receive approximate results in a short period of time than exact results after multiple days. anticipating an aggravation of the current shortage of rheumatologists, we are convinced that it offers even greater value to empower patients to assess their disease activity more professionally and independently use quick crp tests, epros and a self-performed joint count. ultimately, this patient empowerment in combination with technological innovation could result in more need-adapted visits and increase of remote care. the studies involving human participants were reviewed and approved by ethical committee of the university clinic of tuebingen (# 4442020). the patients/participants will be required to provide written informed consent to participate in this study. mk and jk developed the concept of the trial. jm ran the statistical analyses. mg developed, maintained, and adapted the abaton study module and web-based application. cs developed the self-examination video, adapted for use in german by jk and a-cp. jm, jk, and a-cp drafted the manuscript which was revised by mk and jk. all authors contributed to the design of the trial. all authors contributed to the article and approved the submitted version. this project was supported by sanofi-aventis as the winner of the sanofi generation program. sanofi had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results. this work was supported by the deutsche forschungsgemeinschaft (dfg-for 2886 pandora-b01/a03/z01 to gk, gs, and ak). eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update american college of rheumatology workforce study: supply and demand projections of adult rheumatology workforce facharztweiterbildung quo vadis? treat-to-target concept implementation for evaluating rheumatoid arthritis patients in daily practice rural health issues in rheumatology: a review addressing the rheumatology workforce shortage: a multifaceted approach digital health transition in rheumatology: a qualitative study positionspapier der kommission digitale rheumatologie der deutschen gesellschaft für rheumatologie e. v: aufgaben, ziele und perspektiven für eine moderne rheumatologie mobile health usage, preferences, barriers, and ehealth literacy in rheumatology: patient survey study acceptance, usage, and barriers of electronic patient-reported outcomes among german rheumatologists: survey study what will be the job of the rheumatologist in 2030? a mobile app with optical imaging for the selfmanagement of hand rheumatoid arthritis: pilot study effectiveness of a telemonitoring intensive strategy in early rheumatoid arthritis: comparison with the conventional management approach co-op0076 development of a training video for people with rheumatoid arthritis providing 'the bigger picture': benefits and feasibility of integrating remote monitoring from smartphones into the electronic health record modified disease activity scores that include twentyeight-joint counts development and validation in a prospective longitudinal study of patients with rheumatoid arthritis acute phase reactants add little to composite disease activity indices for rheumatoid arthritis: validation of a clinical activity score rapid3 (routine assessment of patient index data 3), a rheumatoid arthritis index without formal joint counts for routine care: proposed severity categories compared to disease activity score and clinical disease activity index categories improving the measurement of disease activity for patients with rheumatoid arthritis: validation of an electronic version of the routine assessment of patient index data 3 development and testing of a short form of the patient activation measure measuring patient activation in italy: translation, adaptation and validation of the italian version of the patient activation measure sus: a quick and dirty usability scale the one number you need to grow qualitative inhaltsanalyse: methoden, praxis, computerunterstützung delays in assessment of patients with rheumatoid arthritis: variations across europe digital rheumatology in the era of covid-19: results of a national patient and physician survey 2021 eular recommendations for the implementation of self-management strategies in patients with inflammatory arthritis tele-health followup strategy for tight control of disease activity in rheumatoid arthritis: results of a randomized controlled trial patient self-sampling: a cornerstone of future rheumatology care? telerheumatology: a technology appropriate for virtually all home-based telehealth: a review and metaanalysis telemedicine for management of inflammatory bowel disease (myibdcoach): a pragmatic, multicentre, randomised controlled trial. the lancet asynchronous mhealth interventions in rheumatoid arthritis: systematic scoping review the authors thank all partners and collaborators for their support of this project. this study is part of the phd thesis of jk (ageis, université grenoble alpes, grenoble, france) and was performed to fulfill the requirements for obtaining the degree dr. med. for rs. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2021.791715/full#supplementary-material key: cord-0001353-0zsn4lu3 authors: lee, yu-ching; tsai, keng-chang; leu, sy-jye; wang, tuan-jen; liu, chia-yu; yang, yi-yuan title: isolation, characterization, and molecular modeling of a rheumatoid factor from a hepatitis c virus infected patient with sjögren's syndrome date: 2013-12-30 journal: scientificworldjournal doi: 10.1155/2013/516516 sha: 30556242d064131f0f2b645a727d42c55dc1c71e doc_id: 1353 cord_uid: 0zsn4lu3 we have previously isolated several igg rheumatoid factors (rfs) from patients with both rheumatoid arthritis and idiopathic thrombocytopenia purpura using phage display system. to study igg rfs in patients with other autoimmune diseases, phage display antibody libraries from a hepatitis c virus infected patient with sjögren's syndrome were constructed. after panning, a specific clone rfl11 was isolated for characterization in advance. the binding activity and specificity of rfl11 to igg fc fragment were comparable to those of rfs previously isolated. the analysis with existed rf-fc complex structures indicated the homology model of rfl11 is similar to igm rf61 complex with high binding affinity of about 6 × 10(−8) m. this effect resulted from longer complementarity-determining region (cdr) combining key somatic mutations. in the rfl11-fc interfaces, the cdr-h3 loop forms a finger-like structure extending into the bottom of fc pocket and resulting in strong ion and cation-pi interactions. moreover, a process of antigen-driven maturation was proven by somatically mutated vh residues on h2 and h3 cdr loops in the interfaces. taken together, these results suggested that high affinity igg rfs can be generated in patients with sjögren's syndrome and may play an important role in the pathogenesis of this autoimmune disease. sjögren's syndrome (ss) is an autoimmune disorder that mainly affects the exocrine glands and usually presents as persistent dryness of the mouth and eyes due to functional impairment of the salivary and lachrymal glands [1] . ss occurs in a primary form not associated with other diseases and in a secondary form that complicates other rheumatic conditions, with the most common being rheumatoid arthritis. positive rf was found in 96% of the patients with main extraglandular ss [2] . on the other hand, circulating monoclonal immunoglobulins (igm kappa or igg lambda) were detected in a significant higher frequency (43%) of ss-hcv patients as compared with the primary ss patients [3] . 2 the scientific world journal hepatitis c virus (hcv) has been demonstrated to be one of the most likely candidates as a potential pathogenic agent causing ss in a subset of patients [2, 4, 5] . many rheumatologic manifestations associated with chronic hcv infection include arthralgia, myalgia, arthritis, vasculitis, and sicca syndrome [6] . clinical studies suggest the possibility of a close relationship among ss, hcv, and b-cell lymphoproliferative disorders [2, 4] . this triple association suggests an important role of associated autoimmune and/or chronic viral diseases in the pathogenesis of b-cell lymphoproliferative disorders and reinforces the hypothesis of a link among autoimmunity, infection, and cancer [4] . rheumatoid factors (rfs) are antibodies directed against the fc part of autologous igg and are the most characteristic marker in rheumatoid arthritis (ra), a chronic joint inflammation with unknown etiopathogenesis [7, 8] . complex formation between rf and igg may lead to activation of complement and other inflammatory mediators directly [9] . physiological rf mainly belongs to igm isotype. it serves a beneficial role in host defense which facilitates the clearance of antigen by enhancing complement activation and phagocytosis. oppositely, pathological rf is associated with ra and other systemic autoimmune diseases [10, 11] . monospecific igg rfs are implicated in causing inflammation and tissue damage in the rheumatoid synovium [1, 7] . corper et al. were the first group to visualize rf binding by crystal structure directly showing an epitope spanning the junction of the c 2 and c 3 domains on fc fragment [12] . in another study, duquerroy et al. determined a different epitope recognition site using their igm rf [13] . their definite c 3-c 3 interface, including the c-terminal region of both heavy chains, is distant from the previously identified c 2-c 3 interface [12] . both complexes were generated from igm rf origin and indicated the involvement of somatic mutations in the interaction interface. human monoclonal antibodies were in great demand for the therapeutic and diagnostic purposes. the combinatorial methodologies have been improved rapidly and dramatically [14, 15] . particularly, the expression of combinatorial antibody fab fragments on phage surfaces allows for rapid enrichment of the desired fab fragments against target antigens. this technique has been used to study pathogenic autoantibodies in various human autoimmune disorders [16] [17] [18] . to study igg rfs in sjögren's syndrome patients with hcv infection, we used the combinatorial antibody technique and characterized isolated rf fab molecules. the results allow us to further define the pathogenic role of rfs played in autoimmune diseases complicated with viral infection. in addition, from the insight on the genetic level, clues are expected to reveal the association of the cause of autoimmune disorders. in order to comprehensively explain the interactions between an antibody and its target antigen, molecular modeling has been employed as a powerful and reliable approach for rational model prediction of an antibody-antigen complex. by analyzing and comparing the x-ray crystal structures of antibody-antigen complexes retrieved from the protein data bank (pdb), many researches have demonstrated that sufficient information is available for analysis to indicate critical residues of great influence on the binding sites from antibody-antigen recognition interfaces [19] . in this study, we demonstrated that the isolated igg rf has a better binding affinity than the existing complex generated by the igm rf molecule. we managed to construct antibody models via homology modeling and used structural alignment and loop minimization to illustrate the possible role of somatic mutation induced in the cdr areas during the interactions. the logical and reasonable predicted results provide a probable explanation and further insights into the diversity and origin of these igg rfs. 2.1. antibody library. two antibody libraries were constructed to express antibody binding fragment (fab) from patients with sjögren's syndrome associated with hepatitis c infection. library sizes for and light chain were 1.3 × 10 7 and 2.1 × 10 6 , respectively. equal amount of phage particles was taken from two libraries and mixed evenly for subsequent panning cycles. the antigen-binding clones in the prepared library were enriched by panning on antigen-coated surface of elisa plates (costar) as reported previously [20, 21] . briefly, human fc fragment protein (sigma) was coated as target protein with 0.5 ug/well at 4 ∘ c overnight. after blocking with 5% skim milk, 10 11 pfu of recombinant phages were added to each well and incubated at 37 ∘ c for 1 hr. unbound phages were removed and the wells were washed vigorously with tris-buffered saline containing 0.05% tween-20 (tbst) for ten times. next, bound phages were eluted with 0.1 m hcl/glycine (ph 2.2) and neutralized with 2 m trisbase. eluted phages were used to infect e. coli xl1-blue strain growing in log phase. phagemid particles were rescued from infected e. coli cells with 10 11 pfu of vcs-m13 helper phage (stratagene). after culture amplification, 4% peg-8000 and 3% nacl were used to precipitate recombinant phage particles. finally, the phages were resuspended in pbs and used for the next round of panning. panning processing against human fc fragment was repeated four times. thereafter, total phagemid dna was prepared and digested with nhe i and spe i (neb biolab) to remove the phage protein iii gene. the digested dna with compatible cohesive ends was self-ligated and electroporated into e. coli xl1-blue cells. individual clone was grown overnight in the presence of 0.5 mm isopropyl b-d-thiogalactopyranoside (iptg) for fab protein induction. the supernatants containing expressed fab molecules were harvested for elisa and western blotting assay. briefly, microtiter plates were coated with 0.5 ug/well with human fc fragment protein at 4 ∘ c overnight. the wells were blocked with 5% skim milk for 1 hr at room temperature. the expressed fab molecules prepared as described above were distributed to wells in duplicate and incubated for 1 hr at room temperature. after washing with pbs containing 0.05% tween-20 (pbst) six times, the wells were reacted with the scientific world journal 3 horseradish-peroxidase-(hrp-) conjugated goat anti-human or light chain antibodies (jackson immunoresearch laboratories) for 1 hr at room temperature. the wells were then washed with pbst six times and 3,5,5-tetramethubezidine dihydrochloride (tmb) substrate was added for color development. reaction was stopped by addition of 1 n hcl and absorbance was taken at 450 nm. for characterization of binding specificity, the selected fc-binding fab was studied their reactivities with type vi collagen (sigma), lipopolysaccharide (lps; sigma), keyhole limpet haemocyanin (klh; sigma), thyroglobulin (sigma), and calf thymus single-stranded dna (ssdna; sigma) simultaneously. the elisa analysis was carried out as described above. the amount of these antigens used for coating was 0.5 ug/well. to examine the fab expression, the iptg-induced bacterial lysates were subjected to sodium dodecyl sulfate polyacrylamide gel for electrophoresis (sds-page) and transferred onto nitrocellulose membrane (ge biosciences, uk), followed by blocking with 5% skim milk in pbst for 1 hr at room temperature with gently shaking. after washing, hrp-conjugated goat anti-human or light chain antibodies were added and incubated for one additional hour at room temperature with gently shaking. after washing with pbst, the membranes were developed with diaminobenzidine (dab) substrate until desired intensity was reached. to examine their binding ability, the cellular lysates containing recombinant fab molecules or sera from hcvinfected patients with sjögren's syndrome were incubated with human fc fragment fixed on western blots. after human fc fragments were transferred onto nitrocellulose membranes, the experiments were performed as described above. competitive inhibition assays were carried out to determine the binding affinities of fc-binding fab molecules. in brief, microtitre plates were coated with human fc fragment (0.5 g/well) and blocked with 5% skim milk. after one hour incubation at room temperature, plates were washed with pbst twice. each purified fab was incubated first with various concentrations of soluble fc fragment diluted in pbs (ranging from 2.5 to 80 g/ml) at room temperature for 1 hr. then, the mixtures were added to the wells to react with coated human fc fragment. after incubating at room temperature for 1 hr, plates were washed with pbst six times. thereafter, hrp-conjugated goat anti-human or light chain antibodies (jackson immunoresearch laboratories) were incubated at room temperature for 1 hr to detect bound fab protein. after washing with pbst six times, tmb substrate (sigma) was added to each well for development. reaction was stopped with 1 n hcl and signal intensity was measured at od 450 nm. 2.6. dna sequence analysis. light and heavy chain inserts of fc-binding clones were sequenced and analyzed. primers complementary to constant region of heavy chain (seqgb: 5 -gtc gtt gac cag gca gcc cag-3 (21 mers)) and lambda light chain (seqlb: 5 -gaa gtc act tat gag aca cac-3 (21 mers)) were used. the imgt/v-quest international immunogenetics information system (http://www.imgt.org/)was used to compile and analyze sequence data in parallel with germline gene. modeling. rfl11, an anti-human igg fc fab, was found to possess the highest affinity among several clones screened in this study and modeled as an scfv scaffold using the homology modeling program. to obtain a reliable light chain model for rfl11, sequence alignment of the light chains of rfl11 and rf61 antibodies (pdb id: 2j6e) [13] was used, showing 97.6% or 92.4% of homology, exclusive or inclusive of cdrs, respectively. similarly, to build the heavy chain model of rfl11, the heavy chain sequence showed a high similarity when compared with another antibody (pdb id: 3h0t) [22] , exclusive (98.8%) or inclusive (91.7%) cdrs. homology model of rfl11 was generated by modeler v.9.4 program in discovery studio v.3.5 (accelrys software inc., san diego, ca, usa) using the crystallographic structures of the light chain of rf61 (pdb id: 2j6e) and the heavy chain of the antibody (pdb id: 3h0t) as structural templates. both structures have good resolution and belong to homo sapiens. thus, the interface of the template structure between l chain and h chain of rf61 was employed to build the light chain-heavy chain interface of rfl11 scfv model. owing to the light chains of rfl11 and rf61 antibodies with high homology cdrs, rfl11 and rf61 may bind to the similar epitope of igg fc (see table 1 ). the complex structural model of rfl11 interacting with igg fc antigen was built by aligning rfl11 and the complex structure of rf61-igg fc to obtain an rfl11-igg fc complex instead of using docking methods [23] to avoid uncertain orientation of the interaction between these two proteins. the cdrs of rfl11 interacting with igg fc antigen were energy minimized using dreidinglike force field from discovery studio v.3.5 for a few steps to remove any inappropriate contacts and obtain a reliable complex structure. two fab-displaying phage libraries were constructed from a hepatitis c virus infected patient with sjögren's syndrome. the numbers of heavy chain combined with either or light chain were 2.1 × 10 6 and 1.3 × 10 7 , respectively. to evaluate the ratio of clones lacking correct inserts in the libraries after amplification, fifty clones of each library were selected for restriction analysis to confirm the presence of a 750 bp fragment containing both heavy and light chain genes. the results revealed that over 93% (14/15) of the clones selected from two libraries had correct gene inserts (data not shown). the fc-binding phages were enriched by four rounds of panning. the input phage number of 10 11 was applied for each panning processing. it was generally seen that the eluted phage titer of final round was about one order higher than that of first panning (figure 1(a) ). after each panning, 4 the scientific world journal abbreviations: (s) and (m) denote the side chain and the main chain, respectively. * denotes the residue of somatic mutation. 15 clones were randomly selected to check the presence of dna inserts with correct length. the results showed that more than 90% of the clones contain a fragment of 750 bp in plasmid dna after second around panning (data not shown), indicating that the panning eliminated most of the phage clones not displaying fab fragments on their surfaces, which accounted for less than 10% of phage clones in the original antibody library we constructed. thereafter, the gene iii fragment was removed from phagemid dna purified from e. coli culture after 4th panning. the expression of iptg-induced fab antibodies under the nonreducing condition was clearly detected and shown in figure 1(b) . with the exception of one clone with incomplete nucleotide sequence, the immunoglobulin isotyping anaysis indicated that 13 of 14 rf molecules contained light chain. we did not find any antibody with light chain. human fc. all the clones containing light chain of fab were further tested for binding activity and specificity. gg3 and gg48 clones with high binding specificity to fc [24] and lpsl and bl12 clones with anti-lps and anti-red-blood surface ag were applied as positive and negative controls, respectively. these selected fab fragments showed significant binding activity to fc but not to bsa (figure 1(c) ). moreover, their recognition of human fc fragment immobilized on western blot was tested. the results showed both serum of hcv-infected patient with ss disease (figure 2(b) ) and the recombinant fab molecule as represented by rfl11 (figure 2(c) ) could clearly recognize fc protein. no fcbinding activity was observed when an irreverent fab molecule was applied (figure 2(d) ). in order to test the binding specificity, unrelated antigens including collagen, klh, lps, calf thymus ssdna, and thyroglobulin were used in elisa test. as shown in figure 3 , the representative rfl11 fab antibody as well as the positive gg3 clone showed specific binding activity to fc fragment but minimal binding to the panel of unrelated antigens. the binding specificity and affinity of rfl11 was further determined by competitive inhibition assay. as demonstrated in figure 4 , 89% inhibitory effect was found on the binding reactivity of rfl11 fab against coated human fc fragment in the presence of a concentration of 40 ug/ml of free fc fragment. when performed in parallel, close to 50% of inhibitory effects were seen using positive gg3 fab antibody and none were detected using negative bl12 fab molecule. the value for rfl11 antibody was 6 × 10 −8 m calculated using the klotz plot method [25] . the nucleotide sequences of vh and vl region of fc-binding clones were determined. when compared to potential germline, the light chain and heavy chains of rfl11 were determined and shown the highest homology to vl1-16 and vh6-1 ( table 2) . three nucleotide differences were identified in cdr3 region of the light chain gene. two changes in nucleotide positions 277 and 279 (gat to cag) resulted in amino acid residue that changed from aspartate (d) to glutamine (q). the third change in nucleotide 284 (agc to aac) led to amino acid residue that changed from serine (s) to asparagine (n) (figure 5(a) ). similarly, two nucleotide changes in 158 and 171 of cdr2 of the heavy chain of rfl11 mutated tyrosine (y) to phenylalanine (f) and lysine (k) to asparagine (n), respectively. as compared to jh6b germline gene, four continual residues were somatically mutated making nyyy to ttdf in cdr3 region ( figure 5(b) ). compared to sequences in the cdr domains of rf61 rf molecule, light chain shows the most similarity except cdr3. rfl11 has replacement of aspartate (d) and serine (s) with glutamine (q) and asparagine (n). for heavy chain, significant dissimilarity on cdr was shown on cdr2 and cdr3 with additional residues. besides, the framework of rfl11 to 2j6e was high similarity (figure 6 ). igg fc antigen. to investigate the interaction of rfl11 with igg fc, the six cdrs of rfl11, all of which make contact except cdr-l2 loop and the h2 and h3 loops of rfl11 that dominate the interaction with fc, are shown in figure 8 . from the interaction between cdr-l2 loop and igg fc, we found that phe85 side chain and tyr49 side chain on the h2 loop correspondingly act on asp356 and lys439 on the igg fc, respectively, and the interactions involved are anion-pi interaction and cation-pi interaction, respectively. in addition to the two interactions, some h-bondings are also involved, as exemplified below. the side chain of arg50 acts on the side chains of gln438 and tyr436 via two h-bondings; the side chains of ser51 and asn52 interact with the main chain of gln438 and produce h-bondings; moreover, an h-bonding is also produced between the side chain of asn52 and the side chain of gln438. the cdr-h3 loop forms a finger-like structure extending into the bottom of a deep pocket of the igg fc, which is composed of arg355, ser442, pro352(l), and pro352(h). four different interactions are generated by the h3 loop and fc, namely, ion interaction, cation-pi interactions, hbonding, and hydrophobic interactions. the igg fc-binding surface contains a main charged residue arg355 which is involved in the ion interaction to asp104 and the cationpi interactions to tyr106 and tyr107 on the cdr-h3 loop ( figure 8) . moreover, the side chain of phe105 on the cdr-h3 loop interacts with pro352 in the h/l chain of c 3 domains via two hydrophobic contacts. the thr102 side chain and thr103 side chain on the cdr-h3 loop, respectively, interact with ser444 and ser442 on the igg fc, both resulting in the formation of h-bonding. in this study, we described the isolation and characterization of rfl11 fab directed against human igg fc from a patient with sjögren's syndrome and hcv infection. being different from physiological rf arising from normal antibody responses to aid exogenous antigen clearance, these rfs may be involved in the pathogenesis of the patients with ss/hcv infection. the isolated igg rf was proven to bind specifically to human igg fc fragment. the binding affinity was 6 × 10 −8 m, as determined by competitive inhibition assays ( figure 4) t 107 the sequences of putative germline counterpart were included for comparison. sequence gaps were introduced to maximize alignment as indicated by blank spaces. the boundaries of framework region (fr) and complementarity-determining region (cdr) were indicated above each germline gene sequences. serum sample from a patient with sjögren's syndrome revealed the presence of igg rfs (figure 2 ). although phagederived fabs may represent the artefacts of recombination of h and l chains during library construction, the characteristic of isolated rfl11 could still be a hint to verify the existence of high affinity rf in patients with sjögren's syndrome. our data suggest that igg type rfs may occur in some patients with sjögren's syndrome and that such igg type rfs may have binding affinities higher than those of igm or igg rfs isolated from other autoimmune diseases [12, 13, 26] . to further study and characterize these pathogenic rfs, it is important to comprehend the interaction between rf and fc as well as to realize the influence of somatic mutations on antibody genes. after a comparison of the heavy chain and light chain variable gene sequence with germline counterpart, rfl11 was identified by ten probable somatic mutations, two in frameworks (tyr88phe (vl) and gln16his (vh)) and eight in cdrs (asp93gln and ser95asn (l3); tyr54phe and lys58asn (h2); asn108thr, tyr109thr, tyr110asp, and tyr111phe (h3)) ( figure 6 ). unlike the antibody gene variations isolated in our previous works, where a substantial amount of somatic mutations occurs in cdrs or even in the framework regions in order to fight against exogenous pathogens, these pathogenic rfs caused by immune system disorders, however, possess a sequence similar to the endogenous germline sequence on the genetic level, except that critical mutations occur on the cdrs to provide a binding-specific ability to the igg fc domain. this implies that the generation of rf autoimmune antibodies is attained by critical changes which convert physiological rfs into pathogenic rfs. in general, output phage value after enrichment implies the success of the panning process if greater than 50-fold [27] . at lower enrichment levels, nonspecific and low affinity the scientific world journal 9 l2 h3 l1 h1 l3 h2 cdr color rfl11 scfv-fc complex model figure 8 : the interface structure of the rfl11-fc was focused on the cdr-h2 (colored in blue) and cdr-h3 (colored in magenta) loops in this panel. human igg fc antigen was colored in cyan. the colors of the six cdrs of rfl11 were the same as that in figure 6 . the format of residues of fc antigen and h2/h3 loops is abbreviated as three words and residues codes for distinguishing between antigen and antibody, respectively. the green and red dotted lines in the above panel represent h-bonding and anion-cation interactions of rfl11 with fc, respectively. in the below panel, the green, red, and yellow dotted lines show h-bonding, cation-pi, and hydrophobic interactions, respectively. binders could cause outgrowth in the output phage after amplification. moreover, in the panning process, although eluted phage titer with mild increasing only raised one order of magnitude after final round of panning, we still enriched and isolated specific phage binders from the original library successfully (figure 1(a) ). it is interesting to show that the output phage numbers in our panning process had instant increment after the third cycle, although it dropped slightly in the second round (figure 1(a) ). this result agrees with other studies [28, 29] . after sequencing of isolated clone following the final panning, almost all clones were identified to be similar. it is another verification to convince an efficient panning process resulting in high affinity clone to become dominant in the final library pool. from the results of competitive inhibition assay, a high binding affinity of rfl11 fab having anti-human igg fc capability was confirmed. rfl11 shares high similarity with the l chain amino acid sequence of rf61 autoantibody, which has been reported, but is different from another autoantibody (pdb id: 1adq, lower binding affinity ∼ 6 × 10 −5 m) already published [12] . in addition, cdrs h2 and h3 on the h chain of rfl11 have two and three more amino acids than those of rf61, respectively, but the amino acid sequences on the frameworks are highly similar. despite the fact that rfl11 and rf61 come from different immunoglobulin isotypes (igg and igm types, resp.), their antigens are both igg fc; accordingly, it can be reasonably inferred that the epitopes recognized by rfl11 and rf61 have a high similarity, and the docking simulation of rfl11 antibody and igg fc antigen by using zdock program [23] also revealed an epitope highly similar to that corresponding to rf61. in order to establish a reliable structure model of rfl11 antibody-fc complex, we used structural alignment and cdr loop minimization to replace docking. from this approach, the dominating interactions of h2 and h3 loops between rfl11 and igg fc can be more clearly observed to further investigate why rfl11 demonstrates a superior binding affinity to rf61, as indicated from the experiments. table 1 illustrates the epitope residues on igg fc. gln438 and lys439 on fc individually form h-bonding and cationpi interactions with the cdr-h2 loop, so we can infer that gln438 and lys439 are key residues for both rfl11 and rf61 on the igg fc epitope. however, from the paratope of the cdr-h2 loop, asn52 and phe48 on the rfl11 cdr-h2 loop, generated by somatic mutation, respectively, form h-bonding and anion-pi interactions with respect to gln438 and asp356, indicating the importance of somatic mutation to the cdr-h2 loop. compared with the rf61 cdr-h2 loop, the two additional amino acids ser51 and asn52 on the rfl11 cdr-h2 loop both interact with gln438 on fc with a strong hbonding, showing that the rfl11 cdr-h2 loop has a binding affinity stronger than the rf61 cdr-h2 loop to igg fc. the rfl11 cdr-h3 loop has three more amino acids than the rf61 cdr-h3 loop, and these three amino acids belong to the four amino acids (thr102, thr103, asp104, and phe105) of rfl11 produced by somatic mutation. h-bonding and hydrophobic and ion interactions are formed between igg fc and the four amino acids, among which asp104 forms a critical strong ion interaction with igg fc arg355. moreover, tyr106 and tyr107 also both contribute to the formation of cation-pi interaction with arg355. as shown in table 1 , ion interaction is formed between rf61 and arg355 of igg fc via asp98 and asp100c. thus, arg355 is a key residue to the igg fc epitope. based on the comparison made according to table 1 , it is inferred that the rfl11 cdr-h3 loop has a binding affinity to igg fc similar to that of the rf61 cdr-h3 loop, but there might be some difference in the fc epitope. also, from the structure model, it is observed that the rfl11 cdr-h3 loop forms a finger-like structure extending into the bottom of a deep pocket of igg fc, which is different from the ellipse-like structure bound to the igg fc formed by rf61. due to the finger-like structure, the cdr-h3 loops of rfl11 can interact with each other as a dimer and together form interactions with igg fc (see figure 7) , providing a binding affinity to the cdr-h3 loops of rfl11 higher than the rf61-h3 loops. from the binding structure of rfl11 and rf61 with fc, although a novel epitope was not found, we confirmed that igg type rf is different from igm type rf and discovered a different cdr loop, particularly a substantial difference between h2 and h3 paratopes. our study showed that rfl11 has a stronger binding affinity than rf61, and both the somatic mutation and computational structural biology investigation are in line with the experiments. in this study, we illustrated that high specificity and high binding affinity rfs may possibly exist in ss patience. by analyzing the isolated rf sequences, we managed to explain the reasons why the isolated rfl11 may possess high affinity to igg fc from the structure and simulation perspectives. although a direct relationship between the isolated rfs and autoimmune diseases has not been established, we have demonstrated how these meaningful rfs change the paratopes on cdr through somatic mutation to induce pathogenic high binding affinity and provided insightful information and guidance useful for future rf researches. salivary and oral components of sjogren's syndrome characterization of b cell lymphoma in patients with sjögren's syndrome and hepatitis c virus infection circulating monoclonal immunoglobulins in sjögren syndrome: prevalence and clinical significance in 237 patients hepatitis c virus, sjögren's syndrome and b-cell lymphoma: linking infection, autoimmunity and cancer sjögren syndrome associated with hepatitis c virus: a multicenter analysis of 137 cases rheumatologic manifestations of chronic hepatitis c infection rheumatoid arthritis. pathophysiology and implications for therapy functional analysis of rheumatoid factor-producing b cells from the synovial fluid of rheumatoid arthritis patients analysis of immune complexes in synovial effusions of patients with rheumatoid arthritis rheumatoid factor, complement, and mixed cryoglobulinemia pre-rheumatoid arthritis: predisposition and transition to clinical synovitis structure of human igm rheumatoid factor fab bound to its autoantigen igg fc reveals a novel topology of antibody-antigen interaction crystal structure of a human autoimmune complex between igm rheumatoid factor rf61 and igg1 fc reveals a novel epitope and evidence for affinity maturation phage display of combinatorial antibody libraries therapeutic antibody expression technology blockade of autoantibodyinitiated tissue damage by using recombinant fab antibody fragments against pathogenic autoantigen novel autoantibody markers for early and seronegative rheumatoid arthritis antiidiotypic monobodies for immune response profiling rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface a dominant antigenic epitope on sars-cov spike protein identified by an avian single-chain variable fragment (scfv)-expressing phage generation and characterization of anti--enolase single-chain antibodies in chicken hepcidin revisited, disulfide connectivity, dynamics, and structure zdock: an initial-stage proteindocking algorithm identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage immunoexpression library measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay possible presence of enhancing antibodies in idiopathic thrombocytopenic purpura assembly of combinatorial antibody libraries on phage surfaces: the gene iii site construction and characterization of recombinant single-chain variable fragment antibodies against toxoplasma gondii mic2 protein phage-derived monoclonal anti-lua the authors thank the national science council for financial support (nsc 99-2628-b-038-002-my3). they are also grateful to the national center for high-performance computing in taiwan for computer time, discovery studio software, and facilities. the authors declare that there is no conflict of interests regarding the publication of this article. key: cord-0016924-vvzv1gc4 authors: sakkas, andreas; heil, sebastian; kargus, steffen; rebel, martin; mischkowski, robert a.; thiele, oliver c. title: tocilizumab: another medication related to osteonecrosis of the jaws? a case report and literature review date: 2021-04-14 journal: gms interdiscip plast reconstr surg dgpw doi: 10.3205/iprs000153 sha: acb85570addb3598a93c6672e58401ed83741ba8 doc_id: 16924 cord_uid: vvzv1gc4 introduction: medication-related osteonecrosis of the jaw (mronj) is a serious complication in patients receiving antiresorptive medication, such as bisphosphonates and denosumab, for different oncologic and non-oncologic diseases. here, we report a case of mronj in a patient treated with tocilizumab, a humanized anti-interleukin-6 receptor antibody that effectively treats moderate to severe rheumatoid arthritis in adults. case description: a 45-year-old female patient diagnosed with severe rheumatoid arthritis, who had been undergoing intravenous tocilizumab therapy for three years without history of bisphosphonate use, was referred to our department. four weeks previously, several teeth in the maxilla and mandible were removed under local anesthesia by her dentist. two weeks after the extractions, she felt pain in both jaws. we diagnosed wound dehiscence and delayed healing of the alveolar bone after the tooth extractions. digital volume tomography showed persistent dry alveolar sockets. the patient underwent surgical debridement of necrotic bone, and intravenous antibiotics were administered in hospital. five months later, wound dehiscence reoccurred in the same regions. histopathological analysis of bone biopsies revealed a diagnosis of mronj. four months later, wound dehiscence occurred in the left maxillary alveolar ridge, and local bone resection was performed under antibiotic treatment. twenty-four months after the last surgery, wound dehiscence had healed completely without signs of recurrence. discussion: osteomyelitis of the jaw in patients treated with tocilizumab has not been reported often. this case confirms the potential role of this interleukin-6 receptor inhibitor in the pathogenesis of mronj and shows that patients who receive tocilizumab with mronj-like symptoms should be closely monitored. the pathomechanism of mronj under tocilizumab therapy remains unclear, so dental practitioners, maxillofacial surgeons, and rheumatologists should look for signs of mronj in patients receiving tocilizumab to prevent mronj onset. dry alveolar sockets. the patient underwent surgical debridement of necrotic bone, and intravenous antibiotics were administered in hospital. five months later, wound dehiscence reoccurred in the same regions. histopathological analysis of bone biopsies revealed a diagnosis of mronj. four months later, wound dehiscence occurred in the left maxillary alveolar ridge, and local bone resection was performed under antibiotic treatment. twenty-four months after the last surgery, wound dehiscence had healed completely without signs of recurrence. discussion: osteomyelitis of the jaw in patients treated with tocilizumab has not been reported often. this case confirms the potential role of this interleukin-6 receptor inhibitor in the pathogenesis of mronj and shows that patients who receive tocilizumab with mronj-like symptoms should be closely monitored. the pathomechanism of mronj under tocilizumab therapy remains unclear, so dental practitioners, maxillofacial surgeons, and rheumatologists should look for signs of mronj in patients receiving tocilizumab to prevent mronj onset. keywords: medication-related osteonecrosis of the jaw, tocilizumab, interleukin-6 receptor inhibitors, rheumatoid arthritis, osteomyelitis zusammenfassung einleitung: die medikamenten-assoziierte osteonekrose des kiefers (mronj) ist eine schwerwiegende komplikation bei patienten unter antiresorptiver therapie wie beispielsweise bisphosphonaten oder denosumab, die im rahmen verschiedener onkologischer und auch nichtonkologischer therapien verabreicht werden. hier möchten wir von einer manifesten mronj bei einer patientin berichten, die bei rheumatoider arthritis mittels tocilizumab, einem humanisierten, monoklonalen antikörper gegen den interleukin-6 (il-6)-rezeptor behandelt wurde. in 2014, the american association of oral and maxillofacial surgeons (aaoms) recommended a nomenclature change from bisphosphonate-related osteonecrosis of the jaw (bronj) to medication-related osteonecrosis of the jaw (mronj) because a growing number of osteonecrosis cases were associated with other antiresorptive and antiangiogenic drugs [1] , [2] . in 2014, the aaoms defined mronj as "the presence of exposed necrotic bone or bone that can be probed through an intraoral or extraoral fistula in the maxillofacial region that has persisted for longer than eight weeks, occurring in patients undergoing treatment with antiresorptive or antiangiogenic agents with no history of radiation therapy or obvious metastatic disease to the jaws" [1] . the clinical manifestation of mronj is multifactorial and mainly depends on the duration of antiresorptive therapy, oral or intravenous application, combined treatment with corticosteroids or diverse chemotherapies, and local risk factors such as surgery-related tissue trauma and oral health of the patient [3] , [4] . except classic bisphoshonates, the role of humanized monoclonal antibodies such as denosumab, bevacizumab, and adalimumab, as well as chimeric monoclonal antibodies such as rituximab in mronj pathogenesis has been described in several publications [2] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] . targeted therapies have been developed to control the growth and survival of malignant cells by interfering with specific molecular routes involved in carcinogenesis. however, recent publications investigating the safety of cancer target therapies have indicated that oral complications are still important in contemporary cancer treatment [2] . since the receptor activator of nuclear factor kappa-b ligand (rankl) and tnf-α have similar biological features and rankl inhibition by denosumab is associated with mronj, interleukin-6 receptor inhibition may also be associated with mronj [2] . tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor. interleukin-6 plays a critical role in naïve t cell differentiation into th17 cells in cooperation with transforming growth factor-β. interleukin 6 is involved in the development of immunological and inflam-matory reactions. autoimmune diseases like rheumatoid arthritis are associated with abnormally high interleukin-6 levels. tocilizumab binds soluble and membrane-bound interleukin-6 receptors, preventing interleukin-6 from exerting its pro-inflammatory effects. neutralizing interleukin-6 with inhibitors such as tocilizumab reduces the progression of autoimmune diseases [11] , [12] . tocilizumab can be used to treat moderate to severe rheumatoid arthritis and systemic juvenile idiopathic arthritis, mostly combined with methotrexate. encouraging results of tocilizumab use in the treatment of covid-19 affected patients mainly in china due to the recent coronavirus pandemic have also been reported [21] , [22] . as well as its positive anti-inflammatory activity, a series of negative side effects have been associated with tocilizumab therapy, including upper respiratory tract infections, nasopharyngitis, headache, hypertension, and elevated total cholesterol. the risk of medication-related osteonecrosis of the jaw in patients taking tocilizumab have been well investigated to date. the lack of sufficient clinical reports means the frequency and severity of mronj caused by interleukin-6 receptor inhibitor cannot be reliably assessed [13] , [14] , [15] . three case reports have described the clinical occurrence of mronj in patients treated with tocilizumab [13] , [14] , [15] . here, we present a new case of osteomyelitis in the maxilla and mandible of a patient affected by severe rheumatoid arthritis that was aggravated by tocilizumab and developed into osteonecrosis. mronj has not been assessed in patients treated with interleukin-6 receptor inhibitors so far. the additional case of mronj related to tocilizumab use presented here suggests that immunosuppressive medication may cause mronj in patients who have not previously been exposed to bisphosphonates. a 45-year-old female patient presented to our craniomaxillofacial surgery department (ludwigshafen hospital in germany) in march 2017 with persistent pain in the maxilla and mandible after multiple tooth extractions. the patient's general medical history revealed that she had been diagnosed with severe rheumatoid arthritis, which had been treated with monthly intravenous infusions of 162 mg tocilizumab and weekly subcutaneous infusions of 15 mg methotrexate for the last three years. comorbidities included latent hypothyreosis, insulin-dependent diabetes mellitus type ii, chronic renal insufficiency ii, and arterial hypertension. no history of smoking or alcohol abuse was reported. the patient did not have history of bisphosphonate or corticosteroid therapy. four weeks before being referred to our department, several of the patient's teeth were extracted from the maxilla and mandible under local anesthesia by her dentist. no intraoperative complications were reported during the extractions. two weeks after surgery, the patient felt severe pain in both jaws. the pain persisted despite appropriate analgesic therapy, and the patient came to our department four weeks after surgery. an intraoral clinical examination revealed infected, dry alveolar sockets in the maxilla and mandible (regions #15-14, #11-27, #33-36 and #45-46) without signs of mucosal abscess or purulent discharge ( figure 1 ). moderate marginal periodontitis of the remaining teeth was also diagnosed, with 2 mm attachment loss, 2-3 mm probing depth, and a horizontal bony defect. the patient was able to swallow and open her mouth normally without pain. extraorally, no lymphadenopathy or other alterations were evident. a panoramic radiograph obtained on the day of her visit on march 2017 revealed regions of osteosclerosis, subperiosteal bone deposition at the extraction sites, and persistent unremodeled bone in the alveolar sockets surrounded by periapical hypermineralized lines ( figure 2) . a generalized periodontitis combined with tooth loss and horizontal bone loss was also diagnosed radiologically. a digital volume tomography in sagittal view showed persistent bony trabecular architecture and absence of bony lysis -the classical radiologic signs of "persistent dry alveolar sockets" (figure 3 ). based on these findings, a diagnosis of mronj with exposed necrotic bone was suspected. we considered the bony lesion to be stage 2 mronj based on the aaoms severity staging system; necrotic bone was exposed and infected (as evidenced by pain and erythema at the sites of extraction) without purulent drainage [1] . the patient underwent surgical debridement of the necrotic alveolar bone and local mucosal closure under general anesthesia. the postoperative orthopantomograph showed reduced alveolar bone height without sequesters after surgical revision ( figure 4 ). ampicillin/sulbactam 3 g (unacid ® , pfizer pharma gmbh, germany) were administered intravenously three times a day, and appropriate analgesia was administered for 7 days under hospitalization. a nasogastric tube was used for continuous feeding during this period to avoid soft tissue manipulation at the surgical sites. during hospitalization, the surgical sites were treated with chlorhexidine mouth rinses (0.12%) under continuous antibiotic therapy. after 7 days, the patient was discharged home and followedup daily in our outpatient clinic. the local treatment consisted of irrigating the mouth with chlorhexidine and appropriate mouth hygiene. after hospitalization, no more antibiotics were administered. on review, there was a considerable improvement in her physical condition; her systemic symptoms had objectively subsided and the pain reduced, decreasing the analgesic intake. two weeks post-operation, we noticed a complete remission of the clinical symptoms, without further dehiscence of the alveolar bone. in august 2017, following an uneventful 5-month period, the patient presented in our department with recurring pain. intraoral dehiscence of the alveolar bone and accumulated food debris in the left side of the maxilla and mandibular region were detected. an initial conservative therapeutic approach of oral chlorhexidine rinses (0.12%) and oral antibiotics was carried out for 5 days to try and achieve secondary wound closure without bone debridement. the patient was monitored closely. after this conservative treatment, avital alveolar bone exposure was still present without pus secretion and intraoral wound healing was uneventful. the treatment with tocilizumab was not interrupted during treatment in our clinic. the clinical and radiographic examination revealed mandibular osteonecrosis and the patient underwent surgery under general anesthesia. this involved partial decortication of the infected maxilla and mandible (the residual anterior-posterior alveolar process and the bicortical plate exposed to vital bone) with preservation of the alveolar inferior nerve and the basal cortical margin. after resection, residual infected and necrotic tissues were removed from the remaining avital bone surface by a rotating burr and diamond burr in an attempt to prevent mronj recurrence. the exposed alveolar inferior nerve was protected from all cutting instruments during surgery. the wound was closed after periosteal releasing incisions, to achieve a tension-free adaption of the soft tissues. as described earlier, an adjuvant cycle of antibiotics was administered and a nasogastric tube was fitted under hospitalization for 5 days. a postoperative orthopantomograph was performed in august 2017 after surgical decortication showed vital alveolar bone ( figure 5 ). the sagittal view of the digital volume tomography showed persistent bony trabecular architecture without signs of osteonecrosis ( figure 6 ). no neural alterations were detected on the day after surgery. sutures were removed 2 weeks after surgery and no intraoral wound dehiscence or new bone exposure was detected. the surgical specimen was fixed in neutral-buffered formalin and sent to the pathological anatomy department of our hospital, where it was decalcified in formic acid, embedded in paraffin, sectioned at 4-µm thickness, and stained with hematoxylin-eosin. the histopathological analysis of the decalcified samples showed areas of bone necrosis with inflammatory cell infiltration, several basophilic bacterial colonies, empty haversian canals without residual osteocytes/osteoblasts, and fewer haversian blood vessels, thus confirming the clinical diagnosis of mronj (figure 7) . on december 2017, 4 months after clinical recurrence and surgical treatment, the patient returned to our department with recurring pain in the posterior maxilla. bone exposure and 2× 3 mm wound dehiscence in the left maxillary alveolar ridge (region #26-27) were observed, without signs of abscess. we performed a surgical revision with rotating and diamond burrs to remove necrotic bone tissues and tension-free wound closure under local anesthesia. oral antibiotics were again administered for 5 days. twenty-four months after the last surgical treatment, no further complications or symptoms of recurrence were detected. the 2014 aaoms position paper outlined that mronj can be diagnosed if the following are present [1]: 1. current or previous treatment with antiresorptive or antiangiogenic agents 2. exposed bone or bone that can be probed through an intraoral or extraoral fistula in the maxillofacial region that has persisted for longer than 8 weeks 3. no history of radiation therapy to the jaws or obvious metastatic disease of the jaws mronj caused by intravenous and oral bisphosphonates has been extensively researched over the last years. inhibitors of rankl (denosumab), angiogenesis (bevacizumab and rituximab), tyrosine kinase receptors (sunitinib), and tnf (adalimumab) have been related to mronj in published case reports. this prompted an international nomenclature change from bronj to mronj [1] , [19] , [20] , [23] , [24] , [25] , [26] . histologically, mronj appears similar to osteomyelitis, and it is not clear whether infection is the cause or consequence of bone exposure. interleukin-6 is a cytokine involved in immunological and inflammatory reactions [12] , [27] , [28] . interleukin-6 mediates a wide spectrum of biological activities including activation of t cells, differentiation of b cells, induction of acute phase reactants, proliferation of fibroblasts, and damage to cartilage and joints. the role of interleukin-6 as an anti-inflammatory cytokine is mediated through its inhibitory effects on tnf-α and interleukin-1, and activation of interleukin-1 receptor antagonist and interleukin-10 [27] , [28] . autoimmune diseases like rheumatoid arthritis are associated with abnormally high interleukin-6 levels. tocilizumab, a humanized monoclonal antibody specific for the interleukin-6 receptor, binds soluble and membrane-bound interleukin-6 receptors, thereby preventing interleukin-6 from exerting its pro-inflammatory effects [27] , [28] . tocilizumab was approved for use in the united states in 2010 and current indications include refractory rheumatoid arthritis and polyarticular and systemic forms of juvenile idiopathic arthritis. tocilizumab is considered a disease-modifying antirheumatic drug that improves signs and symptoms of the disease and reduces destruction of cartilage and tissue. tocilizumab is given by intravenous infusion every 4 weeks in doses of 4-12 mg/kg depending on the indication and body weight. it is applied mostly in combination with methotrexate, if other antirheumatic drugs and tnf-α blockers have proven to be ineffective or were not tolerated [11] , [12] . it can also be used as a monotherapy by patients who do not tolerate methotrexate. tocilizumab slows down the progression of arthritis and can improve physical function of patients. the treatment of systemic juvenile idiopathic arthritis with tocilizumab has also been established [29] . tocilizumab is currently available in vials of 20 mg/ml under the brand name roactemra ® (hoffmann-la roche ag, switzerland). recent scientific reports declare the intravenous administration of tocilizumab in the treatment of the covid-19 related acute respiratory insufficiency with encouraging results [30] , [31] , [21] , [22] . due to an overproduction of proinflammatory cytokines and especially il-6, a large number of covid-19 affected patients in critical situation experience a "cytokine storm" [31] . this storm contributes to increased disease severity as well as aggravation of prognosis, and tocilizumab contributes to symptom improvement by reducing the il-6 expression associated inflammation [31] . however, further studies are needed to validate the ability of tocilizumab to restore t cell counts in covid-19 patients. the aim of this article was to describe an original case of tocilizumab-related mronj in a patient affected by severe rheumatoid arthritis. this patient was receiving an intravenous infusion of 162 mg tocilizumab every 4 weeks, combined with 15 mg methotrexate subcutaneously every week. two cases of mronj following the administration of tocilizumab have been reported since 2012. however, in these cases, bisphosphonates or other antiresorptive drugs were administered concurrently. the first case, reported by ebker et al. in 2013, was a 74-year-old female patient with rheumatoid arthritis treated with infliximab and tocilizumab [13] . the patient presented with a leftsided perimandibular abscess after extraction of the left second lower premolar. there were signs of advanced bronj with exposed bone and extraoral fistula formation in the left submandibular region three months after extraction. the panoramic radiograph showed a persistent extraction socket surrounded by periapical hypermineralized lines in the left mandible. the patient was receiving bisphosphonates for osteoporosis as well as methotrexate and prednisolon for rheumatoid arthritis, therefore the role of tocilizumab in the onset of mronj could not be confirmed. however, ebker et al. did point out that tocilizumab may be (at least partly) responsible for the pathomechanism of mronj. another report in 2013 described mandibular osteomyelitis in a 60-year-old japanese woman diagnosed with rheumatoid arthritis following tocilizumab therapy [14] . the authors described mandibular osteomyelitis as a potential complication of tocilizumab treatment, highlighting its use as a risk factor in oral surgery [14] . the clinicians diagnosed a swelling of the right submandibular region extended to the cervical and axillary lymph nodes as well as a right lower gingival ulcer with necrotic bone and intraoral pus discharge. they proceeded with wide bone debridement and resection of the necrotic tissues under antibiotics. however, methotrexate and sodium risedronate hydrate were also administered, so the role of tocilizumab in the pathogenesis of mronj could not be confirmed ( table 1 ). the first case of mronj in a patient treated with tocilizumab for rheumatoid arthritis without treatment with bisphosphonate or other mronj-related drugs was reported by bindajhil et al. in 2018 [15] . they presented the case of a 71-year-old female with intermittent spontaneous bleeding from mandibular left gingiva for 6 months after multiple extractions ( table 1 ). the panoramic radiograph revealed massive bone destruction and large radiolucency at the extraction sites, extending inferiorly up to the superior cortex of the mandibular canal. cone beam tomography showed expanded lucency in the body of the left mandible, with erosion of the buccal cortical bone, significant loss of bone structure, and reduced height of the alveolus. the radiographic and histological features after surgical treatment led to the diagnosis of mronj [15] . based on the expanded therapeutic potential of tocilizumab due to the current covid-19 pandemic and regarding its clinical relevance in oral surgery procedures according to the above mentioned clinical reports, imaculada de queiroz rodrigues et al. evaluated the effect of pretreatment with tocilizumab on bone remodeling in rats after tooth extraction [31] . in this study, the mandible of 48 rats was clinically and histomorphologically evaluated after intravenous administration of tocilizumab and extraction of the first lower left molar. according to its results, reduced osteoclast count, increased histological signs of infection, myeloid suppression and induction of response in neutrophil infiltration of the alveolar mucosa were demonstrated. the suppression of il-6 in rats is likely to contribute significantly to inflammatory alveolar bone destruction [31] . in the present case, maxilla and mandibular osteonecrosis developed after tooth extractions under tocilizumab treatment, without a history of bisphosphonate therapy but with concomitant methotrexate treatment. the persistent uneventful bone and mucosal healing after the extractions led us to diagnose mronj. the bony lesion was defined as stage 2 mronj according to the aaoms staging system because the exposed necrotic bone was infected (as evidenced by pain and erythema at extraction sockets) without purulent drainage [1] . radiological signs included osteosclerosis, cortical disruption, osteolysis, subperiosteal bone deposition, and thickening of lamina dura. osteosclerosis is due to mineralization without balanced bone resorption; extraction sockets persist because bone remodeling is inhibited after tooth removal [17] . delayed healing after tooth extraction and persistence of pain in combination with clinical surface irregularity and persistent sockets pointed to a suspected diagnosis of tocilizumab-related mronj. the patient received intravenous antibiotic cycles during hospitalization, after which she underwent surgery twice, involving debridement of the lysed alveolar bone after her clinical symptoms recurred. histopathological analysis of the surgical specimen confirmed the diagnosis of mronj. to date, the potential role of tocilizumab in the pathomechanism of mronj has not been well investigated [13] , [14] , [15] . however, we have noticed that tocilizumab may facilitate infectious healing complications in patients undergoing oral surgery because of its immu-nosuppressive effects. this case report together with other published cases of tocilizumab-related osteonecrosis of the jaw reinforces the view that tocilizumab contributes to the onset of jaw osteonecrosis. although bisphosphonates and bevacizumab may also contribute to jaw osteonecrosis, it is too early to compare the time of onset, clinical progress, and outcomes of bisphosphonate-and bevacizumab-related jaw osteonecrosis. we recognize several limitations to this case report. tocilizumab was not the only treatment administered to our patient (the chemotherapy drug and immune suppressant methotrexate was also given), so we cannot draw valid conclusions regarding the role of tocilizumab in mronj pathogenesis. further prospective studies with sufficient patient numbers and a standardized evaluation protocol are needed to confirm whether tocilizumab treatment is related to mronj. patient and medication factors, such as treatment duration, concomitant therapies, diabetes mellitus or other autoimmune diseases, presence of marginal or apical periodontitis before treatment, and patient mouth hygiene should also be investigated to fully define the role of tocilizumab in mronj pathogenesis. furthermore, it remains unknown to what extent tocilizumab contributes to mronj after treatment is discontinued. for example, the effect of denosumab on bone metabolism dissipates after 6 months because it is not retained in the bone, unlike bisphosphonates [18] , [19] . the effect of interleukin-6 inhibitors such as tocilizumab on bone metabolism is probably explained by temporal interactions with their receptor. this suggests that tocilizumab only causes mronj when the patient is either taking the drug or has received the drug in the past few months. in summary, considering the nature of case reports like ours and the current lack of prospective studies, we can only assume causality between interleukin-6 inhibitors such as tocilizumab and mronj. with the promising future perspectives of tocilizumab use in immunoinflammatory diseases and in covid-19 treatment, our clinical report as well as the research of imaculada de queiroz rodrigues et al. [31] and previous case reports showed that in the clinical setting, surgeons need to pay attention to side effects of tocilizumab, especially due to increased risk of postsurgical infection. this clinical case recommends a dental examination before prescribing tocilizumab as well as periodic dental checkups during treatment. although imaging is essential to evaluate the extent of the disease and to assess the treatment response, obtaining accurate and detailed medical history is also critical for mronj diagnosis. we reported a case of jaw osteonecrosis in a patient following tocilizumab therapy. future prospective studies that consider other confounding factors, medical comorbidities, and concomitant medication are needed to determine the pathophysiology and occurrence of mronj in patients treated with tocilizumab. a regular dental checkup before and during tocilizumab administration is strongly recommended to prevent mronj recurring or to detect lesions at earlier stages. furthermore, the number of medications related to mronj onset is increasing; therefore, when prescribing drugs such as tocilizumab, attention must be paid to the well-known risk factors for mronj. rheumatologists should also be involved in treatment decisions since autoimmune diseases are common comorbidities. this research was conducted in full accordance with ethical principles, including the world medical association declaration of helsinki. the patients' data were referenced to with the understanding and written consent of the patient, and all data were also anonymized and deidentified prior to analysis. reporting was based on the recommendations of the strengthening the reporting of observational studies in epidemiology (strobe) initiative [32] . american association of oral and maxillofacial surgeons. american association of oral and maxillofacial surgeons position paper on medication-related osteonecrosis of the jaw--2014 update new non-bisphosphonate drugs that produce osteonecrosis of the jaws osteonecrosis of the jaw onset times are based on the route of bisphosphonate therapy oral bisphosphonate-induced osteonecrosis: risk factors, prediction of risk using serum ctx testing, prevention, and treatment. j oral maxillofac surg osteonecrosis of the mandible associated with bevacizumab therapy. oral surg oral med oral pathol oral radiol evaluation of a surgical treatment of denosumab-related osteonecrosis of the jaws. oral surg oral med oral pathol oral radiol osteonecrosis of the jaw in a crohn's disease patient following a course of bisphosphonate and adalimumab therapy: a case report adalimumab: another medication related to osteonecrosis of the jaws? case rep dent sunitinib related osteonecrosis of jaw: a case report. oral surg oral med oral pathol oral radiol clinical study evaluating the effect of bevacizumab on the severity of zoledronic acid-related osteonecrosis of the jaw in cancer patients long-term safety and efficacy of tocilizumab, an anti-il-6 receptor monoclonal antibody, in monotherapy, in patients with rheumatoid arthritis (the stream study): evidence of safety and efficacy in a 5-year extension study comparison of tocilizumab monotherapy versus methotrexate monotherapy in patients with moderate to severe rheumatoid arthritis: the ambition study fulminant course of osteonecrosis of the jaw in a rheumatoid arthritis patient following oral bisphosphonate intake and biologic therapy. rheumatology (oxford) gms interdisciplinary plastic and reconstructive surgery dgpw 2021 tocilizumab: another medication related to osteonecrosis possible negative effect of tocilizumab on bronj in a patient with methotrexate-associated lymphproliferative disorder osteomyelitis of the mandible exhibiting features of medication-related osteonecrosis in a patient with history of tocilizumab treatment treatment with infliximab: implications in oral surgery? a case report osteonecrosis of the jaw in patients with inflammatory bowel disease treated with tumour necrosis factor alpha inhibitors evaluation of a surgical treatment of denosumab-related osteonecrosis of the jaws. oral surg oral med oral pathol oral radiol denosumab and antiangiogenetic drug-related osteonecrosis of the jaw: an uncommon but potentially severe disease osteonecrosis of the mandible due to anti-angiogenic agent, bevacizumab. oral maxillofac surg tocilizumab treatment in covid-19: a single center experience effective treatment of severe covid-19 patients with tocilizumab association of osteonecrosis of the jaws and poems syndrome in a patient assuming rituximab sunitinib related osteonecrosis of jaw: a case report. oral surg oral med oral pathol oral radiol adalimumab: another medication related to osteonecrosis of the jaws? case rep dent antitumor necrosis factor therapy for inflammatory bowel disease: a review of agents, pharmacology, clinical results, and safety arzneistoff-profile. 4 govi pharmazeutischer verlag the role of transsignalling via the agonistic soluble il-6 receptor in human diseases tocilizumab in patients with systemic juvenile idiopathic arthritis; efficacy data from the placebo-controlled 12-week part of the phase iii tender trial new therapeutic opportunities for covid-19 patients with tocilizumab: possible correlation of interleukin-6 receptor inhibitors with osteonecrosis of the jaws tocilizumab, a potent interleukin-6 receptor inhibitor, decreases bone resorption and increases the rate of bacterial infection after tooth extraction in rats strengthening the reporting of observational studies in epidemiology (strobe): explanation and elaboration we would like to thank bacon editing ® for language editing of this manuscript. the authors declare that they have no competing or financial interests, either directly or indirectly, in the products listed in the study. key: cord-0019975-u1n96ucc authors: wei, wenyang; lu, wanpeng; chen, xiaolong; yang, yunfeng; zheng, mengkai title: use of network pharmacology to investigate the mechanism of the compound xuanju capsule in the treatment of rheumatoid arthritis date: 2021-08-10 journal: biomed res int doi: 10.1155/2021/5568791 sha: 7140260f9cc7467ee53ce76941aff69df15f3d39 doc_id: 19975 cord_uid: u1n96ucc objective: to clarify the therapeutic mechanisms of compound xuanju capsule-treated rheumatoid arthritis (ra) based on network pharmacology tactics. method: the tcmsp, tcmid and stitch databases were used to screen the active ingredients and targets in the compound xuanju capsule; the omim, ttd, pharmgkb and genecards databases were applied to screen the ra-related disease targets. then, the obtained targets were imported into cytoscape 3.7.1 software to construct the active ingredient-target network and the ra-related disease-target network. the active ingredient-target ppi network, the ra-related disease-target ppi network and the common target ppi network were built by using the string platform and cytoscape 3.7.1 software. the go and kegg analyses of the common targets were analyzed by using the metascape and bioinformatics online tools. results: a total of 51 active ingredients and 513 corresponding ingredient targets were harvested from the compound xuanju capsule; 641 ra-related disease targets were obtained. after two ppi networks were constructed and merged, 116 ra-related targets of compound xuanju capsules were identified and analyzed. 116 ra-related targets of compound xuanju capsules are mainly involved in the biological processes and molecular functions, such as the cytokine-mediated signaling pathways, the response to lipopolysaccharide and the blood vascular development, the cytokine activity, the cytokine receptor binding and the receptor regulator activity. furthermore, 116 ra-related targets of compound xuanju capsules are concentrated in signaling pathways such as the il-17, tnf, th17 cell differentiation, toll receptor and ra signaling pathway. conclusion: the compound xuanju capsule had the action characteristics of multiple components, multiple targets, and multiple pathways in the treatment of ra, which might primarily reduce the release of proinflammatory factors (such as il-6 and tnf-α) and increase the production of anti-inflammatory factors (such as il-10) by regulating inflammation-related signaling pathways (such as il-17), thereby alleviating the inflammatory damage and improving the bone tissue repair. rheumatoid arthritis (ra), a common autoimmune dysfunction disease characterized by the chronic inflammation of the joint synovium and the systemic vasculitis, can severely impair the physical function and the quality of life [1] . worldwide, approximately 1% of the population is affected by ra, and the ra prevalence in women is about three times higher than that in men [2] . the pathogenesis of ra is relatively complicated and related to immune imbalance, genetic factors, environment factors, and so on, in which the participation of cytokines plays an important role [3, 4] . the com-mon therapeutic drugs for ra include the nonsteroidal antiinflammatory drugs, the disease-modifying antirheumatic drugs, glucocorticoids and biologic agents. while these drugs are taken, there are also the long-term side effects such as increased risk of infection and potential fatal liver damage [5] . traditional chinese medicine (tcm) has been used to treat ra for more than three thousands of years in china. modern pharmacological researches demonstrated that the effective active ingredients in chinese medicine could weaken the activity of ra and protect joints from deformity [6] . in tcm theory, ra belongs to the category of therapeutic concept "bi zheng ", which is related to the deficiency of vital qi as well as the invasion of "wind (feng)", "cold (han)", "damp (shi)", "stasis (yu)" and other evil qi [7, 8] . tcm can regulate the body function as a whole through a multifaceted approach and multiple mechanisms [9] , therefore, it usually adopts the principles of warming the meridian, dispersing the cold and relieving the pain in ra treatment. the compound xuanju capsule, as a chinese patent medicine, is composed of black ants (xj), herba epimedii (epimedium brevicornu maxim., yyh), fructus cnidii (cnidium monnieri (l.) cuss., scz), and fructus lycii (lycium barbarum l., gqz). the black ants, as the primary drug, can nourish the kidney and liver, invigorate the circulation of blood and remove blood stasis, drive wind, and disperse cold [10] . herba epimedii, as the subject drug, can tonify the kidney, strengthen yang, and dispel wind and dampness [11] . fructus cnidii, as the adjuvant drug, can warm the kidney, invigorate yang, and dissipate cold [12] . fructus lycii, as the envoy drug, can tonify the liver and kidney and firm bones [13] . the combined use of four drugs has a good therapeutic effect on ra in clinical practice by warming and tonifying kidney-yang and removing wind and cold [14] . in addition, the compound xuanju capsule could inhibit th17 cells to secrete il-17, while the secretion of th17 and il-17 played an important role in the pathogenesis of ra [15] . furthermore, earlier researches have shown that the compound xuanju capsule had the immunomodulatory and anti-inflammatory effects [16] . it had a significant inhibitory effect on the phagocytic function of the reticuloendothelial system and the delayed allergic reaction in mice and significantly inhibited the increase in the amount of abdominal exudate and the number of white blood cells induced by carboxymethyl cellulose stimulation. at the same time, it could also inhibit the formation of cotton ball granulation tissue. modern pharmacological studies have also found that black ants contained rich formic acid and a variety of trace elements, had obvious anti-inflammatory effect, could reduce the tissue damage, and eliminate free radicals to promote bone formation [17] ; icariin, the core component of epimedii, could inhibit the inflammatory response and the synthesis of inflammatory substances and enhance the nonspecific immune function and cellular immunity [18] ; osthole, a bioactive coumarin compound, was mainly contained in fructus cnidii and could significantly inhibit the release of inflammatory cytokines tnf-α and il-6 and the inflammatory mediator no [19] . however, the pharmacodynamic basis and more molecular mechanism of compound xuanju capsule-treated ra are still unknown. the network pharmacology method of tcm based on the analysis of network big data can effectively uncover the potential compatible action mechanism of drugs, thus providing a powerful tool for accelerating the modernization of tcm research and development [20] . in the present study, the method of tcm network pharmacology was adopted to further analyze the potential active ingredient, related targets and molecular signaling pathways of compound xuanju capsule-related ra, with the aim of providing more theoretical basis for the effectiveness of compound xuanju capsuletreated ra. capsule. the active ingredients of black ants (xj), herba epimedii (yyh), fructus lycii (scz) and fructus cnidii (gqz) in the whole prescription were collected by the traditional chinese medicine systems pharmacology (tcmsp) database and analysis platform (http://lsp.nwu.edu.cn/tcmsp.php), traditional chinese medicine integrated database (tcmid, http://www.http://megabionet.org/tcmid), natural products database of traditional chinese medicine of shanghai institute of organic chemistry (http://www.chemcpd.csdb.cn) and the related literature of the compound xuanju capsule [21, 22] . according to the integrative absorption, distribution, metabolism and excretion (adme) model, bioavailability (ob) and drug-likeness (dl) were regarded as the screening criteria for active ingredients. the active ingredients were remained which were satisfactory with criteria of ob ≥ 30% and dl ≥ 0:18 for further study [23] . to obtain the target of the each active ingredient in the compound xuanju capsule, the potential targets were predicted from the tcmsp and stitch databases (https://string-db.org). and then, the targets corresponding to the active ingredients screened from the two target databases were standardized in the uniprot database (http://www.uniprot.org), with the properties set to "reviewed" and "human" [24] . subsequently, these standardized targets were merged and the duplicated items were removed. finally, an active ingredient-target network was constructed using cytoscape 3.7.1 software, and the network topology analysis was carried out. the degree, a topological parameter, was applied to screen the kernel active ingredients and the corresponding targets of the compound xuanju capsule. targets. with "rheumatoid arthritis" to be the keyword, ra-related targets were retrieved from three public online databases, namely, the therapeutic target database (ttd, http://bidd.nus.edu.sg/group/cjttd), the pharmgkb database (https://www.pharmgkb.org), the online mendelian inheritance in man (omim) database (http://www.omim.org) and the genecards database (https://www.genecards.org) [25, 26] . these targets were also sent to the uniprot database for normalization, and then, the duplicate targets were removed. eventually, the ra-related disease target network was built by using cytoscape 3.7.1 software as well. network and screening of the common targets. the active ingredient targets of the compound xuanju capsule and the ra-related disease targets were analyzed using the string database, and "homo sapiens" was limited. two ppi networks of the active ingredient targets of the compound xuanju capsule and the ra-related disease targets were built and merged through cytoscape 3.7.1 software. afterward, the common network (the intersection part of two ppi networks) was further analyzed by using cytoscape 3.7.1 2 biomed research international software, and the common targets were obtained. additionally, we further screened targets whose degree value ranked in the top 20 as the core targets of compound xuanju capsule-related ra. ontology (go) [27] biology process annotation and the kyoto encyclopedia of genes and genomes (kegg) [28] pathway enrichment analysis of the common targets were carried out through the metascape online database (http:// metascape.org), which got the potential signal pathways, related biological processes, cell components, and molecular functions of the active components in the compound xuanju capsule. the results of go enrichment analysis and the bubble diagram of kegg pathway enrichment were drawn using the bioinformatics online tools (http://www.bioinformatics .com.cn). the cluego plug-in in cytoscape 3.7.1 software was applied for constructing the visual results of the target proteins involved in signal pathways. capsule. in the present study, we searched all the reported chemical components in the compound xuanju capsule through tcmsp, tcmid, and natural products database of traditional chinese medicine of shanghai institute of organic chemistry. a total of 478 ingredients were collected, among which 46 was found in black ants, 130 in epimedium, 114 in fructus cnidii, and 188 in wolfberry. according to the screening criteria of the adme, including the ob and dl, 87 active ingredients of the compound xuanju capsule were biomed research international obtained after duplicated targets were removed. in addition, three antirheumatic active ingredients of xuanju were found from literatures, namely, citral, formic acid, and β-sitosterol [29] . including all the above active ingredients, a total of 90 active ingredients of the compound xuanju capsule were obtained. network. stitch and tcmsp databases were used to predict the targets of the active ingredients. after the "homo sapiens" targets were remained and the duplicated targets were removed, 51 active ingredients and 513 corresponding action targets were screened, as shown in table 1 . the above targets were imported into cytoscape 3.7.1 software to construct the active ingredient-target network diagram ( figure 1 ). the network contained 570 important nodes and 936 edges. among them, the formic acid, quercetin, kaempferol, luteolin, and stigmasterol had higher degree values, ptgs2, ptgs1, prkaca, gabra1 and rxra tar-gets had higher degree values. the top 10 active ingredients and the corresponding targets are shown in table 2 . searching for ra-related targets through ttd, pharmgkb, omim and genecards databases, when "homo sapiens" targets were remained and the duplicated targets were removed, a total of 641 targets were obtained. the above targets were submitted in the cytoscape 3.7.1 software to construct the ra-related disease-target network ( figure 2 ). analysis. the string online database was used for ppi network topology analysis. the analyzed ppi relationship data results (.tsv format file) were imported into the cytoscape 3.7.1 software, and then, the visualized ppi network of the active ingredient targets ( figure 3 ) and the ppi network of ra-related disease targets ( figure 4 ) were obtained. among them, the ppi network of the active ingredient targets involved 500 nodes (interaction figure 3 : the ppi network of the active ingredient targets that consists of 500 nodes and 9488 edges. the yellow rounds represent the protein targets acted by the active ingredient, and the black lines denote the interaction relationship between the protein targets. 6 biomed research international proteins) and 9488 edges (interaction relationships); the ppi network of ra-related disease targets involved a total of 625 nodes (interaction proteins) and 19389 edges (interaction relationships). 3.5. screening of the common targets. in the cytoscape 3.7.1 software, the cytonca plug-in was applied to merge two ppi networks and obtain a visualized intersection target network ( figure 5 ), which consisted of 116 common nodes and 2241 edges. as shown in figure 5 , these key targets were highly correlated in the potential target network relationship of compound xuanju capsule-treated ra, including il-6, vegfa, tnf, akt1, mapk3, cxcl8, il10, il1β, jun and casp3. the 116 common targets were ranked according to the degree value, and the top 20 core targets of compound xuanju capsule-related ra are shown in table 3 . figure 6 . the go-mf analysis indicated that the core targets of compound xuanju capsule-related ra mainly affected the cytokine activity, the receptor regulator activity, the receptor ligand activity, cytokine receptor binding, phosphatase binding, transcription factor binding, and other molecular functions in the treatment of ra. the go-bp analysis showed that the core targets of compound xuanju capsule-related ra could be involved in a majority of biological processes in the treatment of ra, such as the cytokine-mediated signaling pathways, the response to lipopolysaccharide, the response to molecule of bacterial origin, the response to bacterium, the the go-cc analysis indicated that the core targets of compound xuanju capsule-related ra were primarily enriched in the membrane rafts, the membrane microdomain, the cytoplasmic vesicle lumen, the secretory granule lumen, the extracellular matrix and so on. 3.7. kegg pathway analysis. the kegg pathway analysis was carried out to further explore which pathways were related to the screened common targets. among the top 20 pathways (sorted by the degree value), there were mainly the il-17 signaling pathway, the tnf signaling pathway, the th17 cell differentiation pathway, the toll receptor signaling pathway, the ra pathway, the fluid shear stress and atherosclerosis pathway, and so on (figure 7) . using the cluego plug-in in cytoscape 3.7.1 software, the visualization results of the signal pathway of the target proteins involved were constructed. it was found that the efficacy of the compound xuanju capsule was mainly achieved by regulating the il-17 signaling pathway, the tnf signaling pathway, the th17 cell differentiation pathway, the toll receptor signaling pathway, and so on, as shown in figure 8 network pharmacology is a research method to clarify the disease occurrence and development from the perspectives of systems biology and biological networks, through the collection of ingredients and action targets; an interaction network is constructed to further investigate the regulation effect of tcm on different signals, thereby revealing the action mechanism of tcm [30] . based on the network pharmacology tactics, this study deeply explored the potential figure 5 : the common target ppi network that consists of 116 nodes and 2241 edges. the 116 rounds represent the potential protein targets of compound xuanju capsules against ra, and the black lines denote the interaction relationship between the protein targets. the color and size of the round indicate the size of the degree value; the degree value corresponds to the round color from yellow to purple. 8 biomed research international mechanism of the chinese patent medicine compound xuanju capsule in the treatment of ra. 51 active ingredients of compound xuanju capsules, such as formic acid, quercetin, kaempferol, luteolin and stigmasterol, were screened through multiple large databases. it was predicted that there were 116 potential targets of the compound xuanju capsule in the treatment of ra, such as il-6, gapdh, vegfa, tnf and akt1. five potential mechanism pathways of compound xuanju capsule-treated ra were analyzed and summarized, namely, the il-17 signaling pathway, the tnf signaling pathway, the th17 cell differentiation pathway, the toll receptor signaling pathway and the ra pathway. among the 51 active ingredients selected in this work, formic acid, quercetin, kaempferol, luteolin, stigmasterol, anhydroicaritin and so on were the top 10 active ingredients according to the degree value, which might be the main active ingredients of the compound xuanju capsule against ra. researches have demonstrated that formic acid as one of the biologically active substances of black ants was the material basis for its anti-inflammatory, analgesic, antibacterial and antirheumatic effects and immune regulation and prevention of arteriosclerosis [17, 31] . quercetin can improve morning stiffness, arthralgia, and other clinical symptoms of ra patients and reduce disease activity scores [32] . kaempferol can inhibit the migration and invasion of fibroblast-like synovial cells in ra by blocking the activation of the mapk pathway and inhibit the reorganization of the actin cytoskeleton during cell migration, thereby reducing the arthritis symptoms of collagen-induced arthritis rats [33] . luteolin can reduce the proliferation of fibroblast-like synovial cells in ra by inhibiting the nf-κb and jak/stat signal pathways [34] . stigmasterol is a natural plant product with anti-inflammatory activity, which can inhibit the signal pathway of the inflamed joints, improve the antioxidant status, and transfer inflammation [35] . anhydroicaritin has the function of repairing the bone tissue; in addition, it can promote the angiogenesis by increasing the expression of proangiogenic factors such as vegfa and mcp-1, thus providing more nutrients for bone formation [36] . therefore, the above-mentioned active ingredients might be all the medicinal material bases of the compound xuanju capsule in the treatment of ra, which had an important reference value for subsequent clinical and experimental research. each node in the network represents the protein corresponding to the action target; thus, 116 common nodes involved in the intersection network indicated that there were a total of 116 potential action targets of the compound xuanju capsule in the treatment of ra. additionally, the size and color of the nodes represent the degree value of the target protein, and the connection between the nodes represents the potential interaction between the target proteins [37] . the target protein in the network relationship is greatly correlated with the node size, the node color, and the connections, which increase with them [38] . hence, il-6, vegfa, tnf, akt1, mapk3, cxcl8, il10, il1β, jun, casp3 and other targets showed an obvious correlation in the network relationship of compound xuanju capsules in the treatment of ra, as shown in figure 5 . this demonstrated that the 9 biomed research international compound xuanju capsule had higher binding activity with these targets which could be used as the potential action targets. it had shown that tnf-α was increased in the synovium and cartilage of ra patients, which could promote the cartilage destruction by inhibiting the collagen synthesis, stimulating the fibroblasts and chondrocytes to produce the prostaglandins and collagenase, stimulating the chondrocytes to secrete metalloproteinases, inducing the differentiation of peripheral blood mononuclear cells into osteoclasts, etc. [39] . meanwhile, tnf-α can increase the production of the proinflammatory factors (il1β, il-6, etc.) and chemokines (cxcl8, etc.) through the cascade reaction and further aggravate the inflammatory response [40] . il-6 is a major inflammatory mediator and induced osteoclast precursors to differentiate into the real osteoclasts during bone metabolism [41] . thus, il-6 is the main factor for further destruction of bone and cartilage. as the negative regulator of the proinflammatory cytokines, 1l-10 can reduce the release of the proinflammatory cytokines [42] . one of the earliest observed phenomena in synovitis is the formation of new blood vessels. vegf is the key molecule involved in the reg-ulation of angiogenesis which can promote the infiltration of inflammatory cells into the joint and lead to synovial hyperplasia and progressive bone destruction [43, 44] . akt1 is believed to be closely related to the production of ra synovial fibroblasts [45] . the mapk gene participates in signaling pathways that can reduce the oxygen free radicals, inhibit the cell proliferation, promote the apoptosis, reduce the inflammatory response, and inhibit the angiogenesis, thus effectively inhibiting ra progression [46] . jun, closely associated with the proliferation, differentiation, maturation, and function of the osteoblast, plays an important role in maintaining the dynamic balance of bone formation and bone resorption [38] . the results of the current study showed that the known active ingredients of the compound xuanju capsule could primarily act on 116 targets in the treatment of ra. among the 116 targets, il-6, vegfa, tnf, akt1, mapk3, cxcl8, il10, il1β, mapk8, jun and other targets had higher degree values, indicating that these targets might be the core targets of the compound xuanju capsule against ra, and the corresponding action mechanism was characterized by multiple components and multiple targets. the go and kegg pathway enrichment analysis were conducted on 116 common targets. the go results found that the cytokine activity and the cytokine receptor binding were closely related to the occurrence of ra in terms of molecular functions. in addition, the signal pathways and vascular development mediated by the cytokines in biological processes had a certain impact on ra; this was also corroborated with the results that the compound xuanju capsule might act on 166 potential core targets in the treatment of ra. kegg pathway enrichment analysis results demonstrated that the main signaling pathways regulated by the compound xuanju capsule were the il-17 signaling pathway, the tnf signaling pathway, the th17 cell differentiation pathway, the toll receptor signaling pathway, the ra pathway, the liquid shear stress and atherosclerosis pathways, etc. it had shown that the tnf signaling pathway was the key pathway derived from the enrichment analysis and an important pathway in the inflammatory response [47] . the occurrence and development of ra are related to the abnormal expression of il-6, il1β, tnf and ptgs2 in the tnf signaling pathway [48] . il-17 not only causes the inflammation but also stimulates the osteoclast differentiation to cause bone and cartilage damage. at the same time, il-17 can also stimulate the production of many types of chemokines and aggravate the inflammatory response. furthermore, il-17 can induce the prostaglandins to enter the inflammatory sites through the cyclooxygenase-2 and then damage the synovial surface and articular cartilage [49] . therefore, the il-17 signaling pathway plays an important role in the course of ra. the ra pathway includes the t cell receptor signaling pathway, the toll receptor signaling pathway, the vegf signaling pathway, the osteoclast differentiation, and the th17 cell differentiation, all of which are related to the immune regulation and inflammation. among them, the abnormal t cell-mediated immune response is the main pathogenesis of ra. in the diseased synovial tissue, a large number of activated t lymphocytes gather around the blood vessels. the activated t lymphocytes interact directly with synovial macrophages, secrete the cytokines, and stimulate the synovial cells to release collagenase and protease, which further caused the cartilage and bone damage [38] . the toll receptor signaling pathway directly triggers the intracellular bactericidal mechanism or induces the production of immune inflammatory factors on the basis of identifying pathogenic microorganisms to expand nonspecific defenses. research manifested that the toll receptors were significantly expressed in the pathological tissues of ra patients [50] . the vegf signaling pathway inhibits the expression of cytokines in arthritis, reduces the production of collagen, inhibits the proliferation of synovial cells, and weakens the invasion ability [41] . th17 and treg cells are two subtypes of cd4 + t cells. among them, th17 cells can highly secrete inflammatory cytokines such as il-17, il-6 and il-22, mediating inflammatory reactions and autoimmune reactions in the body [51] . abnormal activation of th17 cells will cause the imbalance of th17/treg cells and play a key role in autoimmune diseases such as ra, especially in local inflammatory response and bone destruction, while long-term local inflammation will induce and worsen the destruction of ra articular cartilage [52] . cells in the body can not only sense the stimulation of various chemical factors in the external environment but also live in various mechanical environments, such as the pressure of fluid as well as shear stress caused by the fluid flow. in the interaction between the extracellular matrix and the signal molecules on the cell membrane, these mechanical stresses can be sensed by cells, thereby activating intracellular signaling pathways, transforming the mechanical signals into the intracellular biochemical signals, and thus affecting cell shape, adhesion and migration, gene expression, differentiation, and even cell death [53] . these mechanical factors play a decisive role in the changes of cellular functions such as cardiovascular disease, bone formation, and organ development. the present study results indicated that the main signaling pathways regulated by the compound xuanju capsule included the il-17 signaling pathway, the tnf signaling pathway, the toll receptor signaling pathway, the th17 cell differentiation pathway, the ra pathway and the liquid shear stress and atherosclerosis pathway. furthermore, the inflammatory signal pathways were the main pathway. this suggested that the compound xuanju capsule might mainly regulate the inflammatory pathways to protect the bone tissue and improve the clinical symptoms of ra patients such as joint swelling and pain. moreover, the above research results predicted by the network pharmacology method were mutually confirmed with the observation results of the compound xuanju capsule in the clinical treatment of ra [14, 15] . ra is a chronic inflammatory autoimmune disease with the complex pathogenesis [35] . the results of the present study suggested that ra was related to the abnormality of the il-6-based multitarget and il-17 pathway-based multisignal pathways; thus, the pathogenesis of ra had the characteristics of multitarget and multipathway. to some extent, it explained the phenomenon that there was no good effect in the single target treatment of the disease in clinic. tcm has the characteristics and advantages of multicomponent, multitarget, and multipathway in the treatment of diseases. therefore, it is still a research hotspot to find drugs for ra treatment. in conclusion, based on the network pharmacology methods, the current study found that the compound xuanju capsule, in the treatment of ra, might mainly act on 116 targets based on il-6 through 51 active ingredients such as formic acid, quercetin and kaempferol, and then regulated the il-17 signal pathway, the tnf signal pathway, the th17 cell differentiation pathway, the toll receptor signaling pathway, the ra pathway, etc., to further affect the release of the proinflammatory factors and the anti-inflammatory factors, thereby alleviating inflammatory damage and improving bone tissue repair. the above results reflected the mechanism and advantages of the compound xuanju capsule in treating ra with multiple components, multiple targets, and multiple pathways, and also confirmed that inhibiting the secretion of inflammatory factors was one of its major mechanisms. we have presented all our main data in the form of figures and tables. the datasets supporting the conclusions of this article are included within the article. the authors declare that there are no conflicts of interest. wenyang wei was responsible for data analysis and original diagram preparation. wenyang wei, wanpeng lu, and xiaolong chen wrote the manuscript. yunfeng yang supervised the whole research. wenyang wei and mengkai zheng revised the manuscript. rheumatoid arthritis classifying rheumatoid arthritis by traditional chinese medicine zheng biomed research international multi-center cross-sectional study advances in the treatment of rheumatoid arthritis: costs and challenges one year in review 2020: pathogenesis of rheumatoid arthritis anti-rheumatoid arthritis effects in adjuvant-induced arthritis in rats and molecular docking studies of polygonum orientale l. extracts traditional chinese medicine on treating active rheumatoid arthritis: a protocol for systematic review and meta-analysis the treatment of rheumatoid arthritis using chinese medicinal plants: from pharmacology to potential molecular mechanisms herbal compounds for rheumatoid arthritis: literatures review and cheminformatics prediction nanometer preparation of traditional chinese medicine for rheumatoid arthritis effects of xuanju extract on benign prostatic hyperplasia herba epimedii: an ancient chinese herbal medicine in the prevention and treatment of osteoporosis the effect of the major components of fructus cnidii on osteoblasts in vitro therapeutic potential of chinese herbal medicines in alcoholic liver disease clinical study of compound xuanju capsule combined with methotrexate in the treatment of refractory rheumatoid arthritis effect of compound xuanju capsules on th17 cells/interleukin 17 in patients with rheumatoid arthritis study on immunoregulatory and anti-inflammatory effects of compound xuanju capsule new progress in pharmacological research of ant preparation the effect of icariin on bone metabolism and its potential clinical application osthole alleviates inflammation by down-regulating nf-kappa b signaling pathway in traumatic brain injury network pharmacology and molecular docking analysis on molecular targets and mechanisms of huashi baidu formula in the treatment of covid-19 systematically deciphering the pharmacological mechanism of fructus aurantii via network pharmacology to explore the potential mechanism of shanhaidan granules in the treatment of erectile dysfunction based on network pharmacology a systems pharmacology approach to investigate the mechanism of oryeong-san formula for the treatment of hypertension network pharmacologybased strategy for predicting active ingredients and potential targets of gegen qinlian decoction for rotavirus enteritis homotherapy for heteropathy active components and mechanisms of qiang-huo-sheng-shi decoction for treatment of rheumatoid arthritis and osteoarthritis study on the multitarget mechanism of sanmiao pill on gouty arthritis based on network pharmacology gene ontology: tool for the unification of biology using the kegg database resource prospect of treatment of chronic diseases by polyrhachis vicina roger integrating network pharmacology and experimental models to investigate the mechanism of huanglian jiedu decoction on inflammatory injury induced by cerebral ischemia medicinal use and nutrition of ants quercetin, a plant polyphenol, has potential for the prevention of bone destruction in rheumatoid arthritis kaempferol inhibits the migration and invasion of rheumatoid arthritis fibroblast biomed research international synoviocytes by blocking activation of the mapk pathway chlorogenic acid and luteolin synergistically inhibit the proliferation of interleukin-1β-induced fibroblast-like synoviocytes through regulating the activation of nf-κb and jak/stat-signaling pathways stigmasterol protects rats from collagen induced arthritis by inhibiting proinflammatory cytokines protective effect of anhydroicaritin against peritonitis in mice mechanisms of cortex phellodendri-herba tuberculate speranskia in treatment of rheumatoid arthritis based on network pharmacology effect and mechanism of yao medicine compound containing cissus pteroclada on rheumatoid arthritis in rats and its q-marker prediction role of tumor necrosis factor-α mediated nuclear factor kappa b signaling pathway in antiogenesis in rheumatoid arthritis anti-receptor activator of nuclear factor κb ligand antibody treatment increases osteoclastogenesis-promoting il-8 in patients with rheumatoid arthritis mechanism of caulis sinomenii on rheumatoid arthritis based on network pharmacology interleukin-10 paradox: a potent immunoregulatory cytokine that has been difficult to harness for immunotherapy vascular endothelial growth factor a (vegfa) polymorphisms in chinese patients with rheumatoid arthritis the pathogenic role of angiogenesis in rheumatoid arthritis pathogenesis of rheumatoid arthritis mechanism of shaoyao gancao decoction in treatment of rheumatoid arthritis based on network pharmacology study on the mechanism of millettia speciosa champ. in the treatment of rheumatoid arthritis based on network pharmacology classical and paradoxical effects of tnf-α on bone homeostasis interleukin-17a: a unique pathway in immune-mediated diseases: psoriasis, psoriatic arthritis and rheumatoid arthritis research progress on the correlation between toll-like receptor and rheumatoid arthritis regulatory t cells in rheumatoid arthritis showed increased plasticity toward th17 but retained suppressive function in peripheral blood th17/treg-related transcriptional factor expression and cytokine profile in patients with rheumatoid arthritis molecular basis of rheological modulation of endothelial functions: importance of stress direction the authors thank the members of their laboratory and their collaborators for their research work. key: cord-0012833-5xr3mmvr authors: liao, xiao-ying; deng, qiang-qiang; han, li; wu, zhi-tao; peng, zhao-liang; xie, yuan; wang, guang-ji; aa, ji-ye; pan, guo-yu title: leflunomide increased the renal exposure of acyclovir by inhibiting oat1/3 and mrp2 date: 2019-07-24 journal: acta pharmacol sin doi: 10.1038/s41401-019-0283-z sha: 36b10575b28f5cdc0c341aa9623aec00d9094e0f doc_id: 12833 cord_uid: 5xr3mmvr rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (lef) and the antiviral drug acyclovir to reduce the high risk of infection. acyclovir is a substrate of organic anion transporter (oat) 1/3 and multidrug resistance-associated protein (mrp) 2. considering the extraordinarily long half-life of lef’s active metabolite teriflunomide (ter) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of lef on the disposition of acyclovir. here we used a specific mrp inhibitor mk571 and probenecid (oat1/3 and mrp2 inhibitor) to assess the effects of mrp2 and oat1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. we showed that lef and probenecid, but not mk571 significantly increased the plasma concentration of acyclovir. however, kidney and liver exposures of acyclovir were increased when coadministered with lef, probenecid or mk571. the kidney/plasma ratio of acyclovir was increased to approximately 2-fold by lef or probenecid, whereas it was increased to as much as 14.5-fold by mk571. consistently, these drugs markedly decreased the urinary excretion of acyclovir. ter (0.5−100 μmol/l) dose-dependently increased the accumulation of acyclovir in mrp2-mdck cells with an ic(50) value of 4.91 μmol/l. ter (5 μmol/l) significantly inhibited the uptake of acyclovir in hoat1/3-hek293 cells. these results suggest that lef/ter increased the kidney accumulation of acyclovir by inhibiting the efflux transporter mrp2, which increased its kidney/plasma ratio and renal injury risk. however, the inhibitory effects of lef/ter on oat1/3 reduced the tubular cells’ uptake of acyclovir and increased the plasma concentration. leflunomide (lef) is an immunosuppressive drug that has been widely used in the clinic for the treatment of rheumatoid arthritis (ra) [1] , as well as kidney/lung transplantation [2] . most lef is rapidly metabolized to teriflunomide (ter) in the liver and in the blood ( fig. 1) [2, 3] . the plasma concentration of ter is highly variable among patients, from 3 to 150 mg/l, and the clearance of ter in the plasma is very slow, with a half-life of approximately two weeks [2] . kidney is also responsible for its elimination [2] . we recently reported that lef could downregulate the expression of multidrug resistance-associated protein (mrp) 2 in the liver [4] . however, its effect on the function of mrp2 still needs to be explored. in addition, ter was reported to increase the auc of an oat3 substrate, cefaclor [5] (https://www.accessdata.fda.gov/ drugsatfda_docs/label/2016/202992s002lbl.pdf). ter was suggested to be an inhibitor of oat3. because the structures of oat1 and oat3 are highly homologous [6] , ter may also have an inhibitory effect on oat1. acyclovir is a synthetic nucleoside analog for the first-line treatment of herpetic virus [7] [8] [9] . this drug is eliminated primarily in urine (60%-90%) as the unchanged form [10] . given that its renal clearance greatly exceeds the glomerular filtration rate (three-fold) [10, 11] , tubular secretion plays an important role in its renal clearance. acyclovir was reported to be transported by multiple transporters [12] , including oat1/3 [13, 14] and mrp2 [15] . however, previous studies have focused on the role of the uptake transporters oats in transporting acyclovir [14, 16] ; there is limited information about efflux transporters, such as mrp2, in its renal excretion and renal accumulation. compared with other patients, ra patients are often at a higher risk of developing infections [17, 18] , such as herpes zoster and herpes simplex virus [17, 19] . lef-treated patients may be prescribed acyclovir for the treatment of herpetic infection. acyclovir is a common cause of kidney injury [20, 21] . crystal precipitation is widely believed to be the cause of nephrotoxicity [22] . in addition, nephrotoxicity may occur in the absence of crystal formation [23] . in other words, acyclovir could also cause direct injury to tubular cells [7, [24] [25] [26] . therefore, reduced function of the efflux transporters may result in increased intracellular accumulation of acyclovir and subsequent detrimental nephrotoxic consequences. because of the potential interactions of lef with both uptake/efflux transporters, oat1/3 and mrp2, the effect of lef on the renal disposition of acyclovir may be complicated and deserves further investigation. the objectives of the current work were to explore whether lef could influence the renal accumulation of acyclovir through coinhibiting oat1/3 and mrp2. in this study, the specific mrp inhibitor mk571 and probenecid (inhibitor of both oat1/3 and mrp2) were used to assess the effects of mrp2 and oat1/3 on the pharmacokinetics and tissue distribution of acyclovir. the rats were randomly divided into four groups: (1) control group: acyclovir alone (30 mg/kg); (2) lef group: acyclovir (30 mg/ kg) + lef (30 mg/kg); (3) pro group: acyclovir (30 mg/kg) + probenecid (150 mg/kg); (4) mk571 group: acyclovir (30 mg/kg) + mk571 (10 mg/kg). acyclovir was dissolved in isotonic saline and intravenously administered. lef, probenecid and mk571 were all intraperitoneally injected (ip) 30 min before acyclovir administration. after intravenous administration of acyclovir, blood samples were collected at 5, 10, 30, 60, 120, 240, and 480 min in the four groups (n = 4 per group). for the urinary excretion study, urine samples (n = 4 per group) were collected before dosing and at different time intervals (0-2, 2-4, 4-8, 8-12, and 12-24 h), and the urine volume was recorded. the plasma concentration and the urinary concentration of acyclovir were determined by liquid chromatography-mass spectrometry tandem mass spectrometry (lc-ms/ms) (lcms-8030; shimadzu, kyoto, japan). the plasma concentration of ter was determined by an lc-ms/ms method as previously established in our lab [27] . for tissue distribution, 2 h after acyclovir administration, rats (n = 3 at each time point in each group) were sacrificed via exsanguination from the abdominal aorta under anesthesia. livers and kidneys were rapidly dissected, washed with saline, dried and weighed. every 200 mg of the tissue samples was homogenized with 1 ml of distilled water and stored at −80°c until analysis. blood samples were collected and centrifuged to harvest plasma samples at the same time. cell culture mdckii cells stably expressing the human transporter mrp2 (mrp2-mdck) were kindly supplied by prof xiao-yan chen (shanghai institute of materia medica, shanghai, china). the cells were routinely cultured in dmem medium supplemented with 10% fetal bovine serum and 100 u/ml penicillin and streptomycin. hek293 cells overexpressing human oat1 (nm_153276) or human oat3 (nm_001184732) were transfected with hoat1-pcdna3.1 (+) or hoat3-pcdna3.1 (+). control hek293 cells were obtained in parallel via transduction of an empty pcdna3.1(+) vector. cell lines stably expressing empty vector (mock-hek293) and transporters (hoat1-hek293 and hoat3-hek293) were obtained by g418 selection and then maintained in dmem supplemented with 10% fbs and 0.5 mg/ml g418 at 37°c with 5% co 2 . effects of lef/ter on the accumulation of acyclovir in mrp2-mdck cells to further investigate the role of mrp2, accumulation experiments in mrp2-mdck cells were performed as previously described [28] [29] [30] [31] . briefly, cells were seeded at a density of 2 × 10 5 cells/well on a 24-well plate. after 48 h of seeding, the cells were washed twice with warm (37°c) hbss and then preincubated with 0.3 ml of hbss solution for 15 min at 37°c. then, acyclovir (200 μmol/l) in the presence or absence of test compounds was incubated for 60 min. at the end of the studies, the medium was aspirated, and the cells were washed three times with ice-cold hbss. subsequently, the samples were frozen at −80°c before lc-ms/ms analysis. the ic 50 value for lef/ter on the accumulation of acyclovir in mrp2-mdck cells was determined from the maximum and minimum extremes of the relevant non-linear regression plot by graphpad prism 5 (san diego, ca, usa) (maximal inhibition = 100%, non-inhibited control = 0%). transcellular transport assays mrp2-mdck cells at a density of 3 × 10 5 cells/cm 2 were grown on a polycarbonate membrane filter membrane on transwell inserts (0.4 μm pore size, 6.25 mm diameter; costar, corning, ny, usa) for 5 d, and monolayers with a teer-value above 420 ω/cm 2 were utilized for the studies. before starting the transport studies, the apical (a) and basolateral (b) chambers were washed twice with prewarmed hbss (37°c), and then, the cells were equilibrated for 30 min in the presence or absence of inhibitors. hbss containing acyclovir (10 μmol/l) with or without inhibitors was added to the donor side (either the apical or basolateral side). aliquots (50 μl) were collected from the acceptor compartment at 60 min for lc-ms/ms analysis. inhibitory effect of lef/ter on oat1/3 in hoat1-hek293 and hoat3-hek293 cells for inhibition studies, 6-cf was used as the specific fluorescent substrate of oat1 and oat3 [32] . the 6-cf (5 μmol/l) uptake process with or without lef (1-100 μmol/l) or ter (0.5-50 μmol/l) was conducted as described above, with the exception that hbss containing probenecid (200 μmol/l) was used as a positive inhibitor, and the incubation time was set at 10 min. at the end of the uptake studies, the medium was aspirated, and the cells were washed three times with ice-cold hbss. then, the cells were lysed with 300 μl of 0.1 mol/l sodium hydroxide. the content of 6-cf was measured with excitation and emission wavelengths at 485 and 528 nm with a microplate reader (bio-tek instruments, usa), respectively. the protein content of the solubilized cells was determined using a bca protein assay kit. the fluorescence intensity was normalized to total protein. ic 50 values were calculated using nonlinear regression with an appropriate model by graphpad prism 5. the acyclovir uptake process was conducted as described above, with the exception that hbss containing lef (10 μmol/l), ter (5 μmol/l) and probenecid (200 μmol/l) was used as inhibitors, and the incubation time was set at 30 min. the uptake medium containing 200 μmol/l acyclovir, with or without the inhibitors above, was then added to each well. at the end of the study, the acyclovir content was determined by lc-ms/ms. lef/ter cellular uptake assays to estimate whether ter was a substrate of oat1 or oat3, cellular accumulation for ter was performed in hoat1-hek293 and hoat3-hek293 cells. the uptake process was conducted as described above, with the exception that hbss containing ter (20 μmol/l) was used as the incubation media and the incubation time was set for 20 min. the uptake medium containing ter (20 μmol/l) with or without probenecid (200 μmol/l) was then added to each well. the uptake procedure was also terminated as described above. the the permeability coefficients of acyclovir (p app ) and the efflux ratio (er) were calculated according to the following equation: where c t = the concentration of acyclovir on the receiver side, v = the loading volume on the receiver side, area = membrane surface area (0.33 cm 2 ), c 0 = the initial concentration on the donor side, and t = incubation time. where p a to b and p b to a represent the permeability from the apical to basolateral sides and from the basolateral to apical sides, respectively. pk parameters were calculated in a non-compartmental analysis utilizing winnonlin software (pharsight 6.2, nc, usa). the plasma clearance (cl p ) of acyclovir was calculated using the following equation: the auc 0-∞ is the area under the plasma concentration-time profile after iv administration. the renal clearance (cl r ) of acyclovir was calculated using the following equation: a total is the total cumulative amount of acyclovir excreted in urine over 24 h. the data are presented as the mean ± sd. student's two-tailed unpaired t-test was used for comparisons between two groups using graphpad prism 5. p < 0.05 was considered to be statistically significant. lef increased plasma exposure of acyclovir and decreased its urinary excretion probenecid was used as a conventional inhibitor of oat1/3 and mrp2. the specific mrp inhibitor mk571 was employed to investigate its pure inhibitory effect on mrp2. coadministration of lef increased the auc 0-∞ of acyclovir by 53% (p < 0.001), decreased its total body clearance by 45% (p < 0.05), and prolonged the mean residence time (mrt) by 33% (p < 0.05) compared to single acyclovir administration (fig. 2a , table 1 ). probenecid reduced the total body clearance of acyclovir by 72% and significantly increased its auc 0-∞ by 2.52fold (p < 0.001) ( table 1 ). the mrp2 inhibitor mk571 had no effect on the plasma concentration and plasma clearance of acyclovir. the plasma concentration of ter was detected in the lef group. during 0-8 h after acyclovir administration, the mean plasma concentration of ter varied from 25.9 to 37.8 μg/ml (fig. 2b) . the peak concentration reached at 2 h and did not change obviously until acyclovir was almost completely eliminated (8 h after administration). for administration of acyclovir alone, 78.4% of the dose was recovered from the rat urine through 24-h cumulative urinary excretion of acyclovir (fig. 3 ). the combination with lef reduced the cumulative urinary excretion of acyclovir to 42.2% (p < 0.001) (fig. 3) , and cl r was reduced to 34.6% (p < 0.001) ( table 1) . mk571 and probenecid showed stronger inhibition of urinary excretion and reduced cl r to 32.9% (p < 0.001) and 9.8% (p < 0.001) ( table 1) . the liver and kidney concentrations of acyclovir in the coadministration groups were significantly increased compared to acyclovir administration alone at 2 h (fig. 4) . lef increased the accumulation of acyclovir in the liver and kidney to 3.2-fold (p < 0.05) and 4.2-fold (p < 0.05), respectively. coadministration of mk571 or probenecid had a stronger effect on the accumulation of acyclovir in the liver and kidney at 2 h, but the increased acyclovir concentration in kidney was proportional to its increased plasma concentrations in the lef and pro groups (fig. 4a, b) . the tissue/ plasma concentration ratios in the liver and kidney after lef treatment were increased to 1.71-fold (p < 0.05) and 2.23-fold (p < 0.05), respectively. in the pro group, the values were 1.51-fold and 2.45-fold, respectively. in contrast, mk571 only significantly increased acyclovir concentrations in the kidney and liver but did not significantly change the plasma exposure. therefore, probenecid's effect on the tissue/plasma concentration ratio in the liver and kidney was similar to lef, and mk571 treatment increased the ratios to 5.8-fold (p < 0.001) and 14.5-fold (p < 0.01), respectively (fig. 4c, d) . to understand the mechanism of the interaction between lef and acyclovir, we examined the effect of lef/ter on the cellular accumulation of acyclovir in mrp2-mdck cells. a typical mrp2 inhibitor, mk571 (20 μmol/l), increased acyclovir accumulation in mrp2-mdck by 3-4-fold (p < 0.001) (fig. 5a) . ter, but not lef, increased the accumulation of acyclovir in mrp2-mdck cells in a concentration-dependent manner, with an ic 50 of 4.91 μmol/l (fig. 5b) . consistent with the accumulation assay results, acyclovir in mrp2-mdck cells showed greater permeability in the b to a direction compared with that in the a to b direction (fig. 5c) . the er value of acyclovir was 2.27. ter (10 μmol/l) and mk571 reduced the permeability of acyclovir (the er value was reduced to 1.46). inhibitory activity of lef/ter in hoat1-hek293 and hoat3-hek293 cells we investigated whether lef/ter had a potential inhibitory effect on oat1/3. ter, the main lef metabolite, inhibited the uptake of 6-cf, a fluorescent substrate of oat1/3, in hoat1-/3-hek293 cells with ic 50 values of 3.39 μmol/l and 0.87 μmol/l (fig. 6c, d) . the prodrug lef showed a weaker inhibition on oats. the estimated ic 50 value of lef on oat3 was 4.1 μmol/l, but there was no significant inhibitory effect on oat1 (fig. 6a, b) . furthermore, to investigate whether ter was a substrate of oat1/3, ter uptake analysis was conducted on oat1/3-transfected cells. the transfected cells could not increase the cellular uptake of ter compared to mock cells, and the presence of probenecid did not alter the intracellular uptake of ter in both transfected cell lines (fig. 7a) , indicating that ter was not a substrate of oat1/3. furthermore, acyclovir uptake assays were performed using these transfected cells (fig. 7b) . the uptake of acyclovir was higher in hoat1-hek293 and hoat3-hek293 cells than mock-hek293 cells. similar to the oat inhibitor probenecid, ter (5 μmol/l) inhibited oat-mediated acyclovir transport, while lef (10 μmol/l) only inhibited oat3-mediated acyclovir transport. oat1/3 on the renal basolateral membrane are responsible for the uptake of anionic drugs from the blood, while apical transporters, such as mrp2, ensure their efflux into the tubular lumen [33] [34] [35] . inhibition of uptake or efflux transporters by a coadministered drug can alter their renal distribution. previous studies have focused on the basolateral uptake of acyclovir by oat1/3 in vitro and in vivo [13, 14] . no in vivo studies have been conducted on the contribution of mrp2 to acyclovir disposition. in the present study, the inhibitory effect of lef and its metabolite ter on oat1/3 was evaluated. because lef will be converted to its active metabolite ter in a very short time in vivo [2, 27] , ter was employed to assess the impact of lef on the related transporters in subsequent in vitro studies. the calculated ic 50 values of ter on oat1 and oat3 were 3.39 μmol/l and 0.87 μmol/l, respectively (fig. 6c, d) . the inhibition of ter on oat3 was stronger than that on oat1 (fig. 6) . at the same time, ter inhibited the efflux of acyclovir in mrp2-mdck cells in a concentration-dependent manner with an ic 50 value of 4.91 μmol/l (fig. 5) . to the best of our knowledge, this is the first study to identify ter, not lef, as a potent mrp2 inhibitor. in general, inhibition of renal uptake transporters is believed to reduce the potential nephrotoxicity of their substrates by reducing renal accumulation. for example, probenecid could reduce the nephrotoxicity of aristolochic acid i (aai) and cidofovir in the clinic by inhibiting oat1/3 [36, 37] . however, the success of probenecid with aai and cidofovir came from the fact that neither aai nor cidofovir is a mrp2 substrate [31, 38] . in fact, for drugs that are both substrates of efflux/influx transporters, caution should be taken in interpreting the results. if lef/ter only inhibited the uptake transporters oat1/3, the acyclovir concentration in the kidney should be reduced. however, the kidney concentration of acyclovir (at 2 h) and the kidney/plasma ratio (at 2 h) were significantly increased by coadministration of lef. a similar effect was observed with probenecid, an inhibitor of oat1/3 and mrp2 (fig. 4) . the results implied that the efflux transporters for transporting acyclovir may be inhibited by lef or probenecid, as well as uptake transporters. the ter kidney concentration at that time was determined to be 70 μmol/g tissue (data not shown), which was much higher than its oat1/3 and mrp2 ic 50 values. in addition, lef increased the liver concentration (at 2 h) and liver/plasma concentration ratio (at 2 h) of acyclovir, which might also result from the inhibition of mrp2, located in the canalicular membrane of hepatocytes. to clarify the contribution of mrp2 to acyclovir disposition in the kidney, the specific mrp inhibitor mk571 was employed. notably, lef and probenecid increased the auc 0-∞ of acyclovir and decreased its renal clearance, while the plasma concentration was not changed by mk571 (fig. 2 and table 1 ). unlike lef and probenecid, mk571 increased the kidney concentration of acyclovir without any change to its plasma concentration, which led to an almost 14.5-fold increase in its kidney/plasma ratio (for lef and probenecid, the increase in kidney/plasma ratio was approximately 2-fold) (fig. 4d) . the reason for this phenomenon fig. 4 effects of lef, probenecid (pro) and mk571 on the tissue distribution and the tissue/plasma concentration ratios of acyclovir. a and b open columns on the left represent the acyclovir concentrations in liver and kidney, respectively. the black columns on the right are the plasma acyclovir concentrations. c liver/plasma concentration ratio, (d) kidney/plasma concentration ratio. each bar represents the mean ± sd in triplicate. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control) leflunomide interacts with acyclovir xy liao et al. could be explained by the overall intrinsic clearance concept introduced by sugiyama [39, 40] : for drugs that are influenced by both efflux and uptake transporters, particularly for those whose efflux clearance into bile/urine is greater than the basolateral efflux (efflux into the blood), the reduction of the uptake capacity may affect the plasma concentration-time profile more than the target tissue concentrations. in contrast, if the efflux process is blocked, the target tissue concentration will be dramatically increased, while the plasma concentration is not significantly affected [41] . in this study, mk571 increased acyclovir kidney concentration, but it did not change its plasma exposure, which was consistent with the fact that only mrp transporters were inhibited. this finding implied that the efflux transporters mrp may play a critical role in acyclovir kidney disposition, such as mrp2, which may be the critical factor in determining the effect of lef and probenecid on acyclovir disposition in the kidney. from another perspective, it is clear that the increase of acyclovir plasma exposure in combination with lef and probenecid came from the inhibition of oat1/3. meanwhile, the increased kidney/ plasma ratios of acyclovir after lef or probenecid treatment were much lower than that by mk571, which implied that the reduced function of the uptake transporters oat1/3 partly alleviated the renal accumulation of acyclovir via mrp2 inhibition. the elevated kidney concentration of acyclovir after lfe administration was a combined effect of oats and mrp2 (illustrated in fig. 8 ). lef/ter is also a substrate of bcrp [42] , and the inhibition of bcrp may contribute to the renal accumulation of acyclovir by lef. however, the mk571 inhibition results suggested that mrps dominated the cumulative urinary excretion of acyclovir (fig. 3) . in our study, acyclovir disposition in the kidney was influenced by both efflux and uptake transporters. the potential ddi risk is relatively easy to estimate if only one type of transporter is involved. for example, mrp2 mutation is associated with impaired renal elimination and nephrotoxicity of methotrexate [43] . for drugs that are multiple-transporter perpetrators such as lef, attention should be paid to assess the different contribution of efflux/influx transporters before making conclusions, especially for drugs with narrow therapeutic windows. for example, nonsteroidal anti-inflammatory drugs (nsaids), which have also been reported to be nephrotoxic [44, 45] , are well-known oat1/3 and mrp substrates [46, 47] . the combination of nsaids with methotrexate could result in serious toxicity, including an increased risk of nephrotoxicity [48, 49] . an increased risk of acute kidney injury was also observed with the concomitant use of nsaids with acyclovir or valacyclovir (a prodrug of acyclovir) [20, 50] . in conclusion, our work indicated that lef increased the systemic exposure of acyclovir and increased the tissue/plasma ratios in rat liver and kidney. ter, the major metabolite of lef, is a potent mrp2 and oat1/3 inhibitor. the results suggested that lef increased the renal exposure of acyclovir mainly by inhibiting mrp2, while it inhibited the uptake transporters oat1/3, which resulted in increasing plasma exposure of acyclovir. . the values shown are the mean ± sd of experiments performed in triplicate. ***p < 0.001 compared to mock-hek293. ## p < 0.01; ### p < 0.001 compared to non-inhibitors in the transfected cell lines leflunomide interacts with acyclovir xy liao et al. fig. 8 schematic diagram illustrating the inhibitory mechanisms of lef, probenecid and mk571 on the transport of acyclovir. lef is rapidly converted to ter in vivo, which can inhibit the uptake and efflux of acyclovir mediated by oat1/3 and mrp2. probenecid, at a high dose in this study, exhibited a stronger inhibitory effect on oat1/3 and mrp2. mk571 did not have an obvious effect on oat1/3 and inhibited the efflux of acyclovir into the urine. the inhibition of oat1/3 reduced the uptake of acyclovir into the cells and increased the plasma concentration, while the inhibition of mrp2 increased the accumulation in the tubular cells and reduced its urinary excretion eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update clinical pharmacokinetics of leflunomide in vitro metabolism studies on the isoxazole ring scission in the anti-inflammatory agent lefluonomide to its active alpha-cyanoenol metabolite a771726 leflunomide increases hepatic exposure to methotrexate and its metabolite by differentially regulating multidrug resistance-associated protein mrp2/3/4 transporters via peroxisome proliferator-activated receptor alpha activation human organic anion transporter hoat3 is a potent transporter of cephalosporin antibiotics, in comparison with hoat1 physiology, structure, and regulation of the cloned organic anion transporters oral acyclovir induced hypokalemia and acute tubular necrosis a case report antiviral resistance in herpes simplex virus and varicella-zoster virus infections: diagnosis and management utility of ultradeep sequencing for detection of varicella-zoster virus antiviral resistance mutations metabolic fate of radioactive acyclovir in humans effects of probenecid on the pharmacokinetics and elimination of acyclovir in humans multiple interactions of cimetidine and probenecid with valaciclovir and its metabolite acyclovir. antimicrob agents chemother benzylpenicillin inhibits the renal excretion of acyclovir by oat1 and oat3 inhibitory effect of jbp485 on renal excretion of acyclovir by the inhibition of oat1 and oat3 expression of multidrug resistance associated protein 5 (mrp5) on cornea and its role in drug efflux assessment of the role of renal organic anion transporters in drug-induced nephrotoxicity risk and severity of herpes zoster in patients with rheumatoid arthritis receiving different immunosuppressive medications: a case-control study in asia a systematic review of viral exposures as a risk for rheumatoid arthritis the risk of herpes zoster in patients with rheumatoid arthritis in the united states and the united kingdom the incidence, risk factors, and clinical outcomes of acute kidney injury (staged using the rifle classification) associated with intravenous acyclovir administration intravenous acyclovir and renal dysfunction in children: a matched case control study rapidly progressive acute renal failure due to acyclovir: case report and review of the literature combination of ceftriaxone and acycloviran underestimated nephrotoxic potential? pedia nephrol comparison of the novel hk-2 human renal proximal tubular cell line with the standard llc-pk1 cell line in studying drug-induced nephrotoxicity reversible renal failure in renal transplant patients receiving oral acyclovir prophylaxis proteomic characterization of acyclovirinduced nephrotoxicity in a mouse model inhibition of hepatic cytochrome p450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity efavirenz reduces renal excretion of lamivudine in rats by inhibiting organic cation transporters (oct, oct) and multidrug and toxin extrusion proteins (mate, mate) mdr1 and bcrp transporter-mediated drug-drug interaction between rilpivirine and abacavir and effect on intestinal absorption structure affinity relationship and docking studies of flavonoids as substrates of multidrug-resistant associated protein 2 (mrp2) in mdck/mrp2 cells aristolochic acid i is a substrate of bcrp but not p-glycoprotein or mrp2 analysis of three-dimensional systems for developing and mature kidneys clarifies the role of oat1 and oat3 in antiviral handling renal drug transporters and drug interactions the organic anion transporter (oat) family: a systems biology perspective mechanisms of renal anionic drug transport critical role of organic anion transporters 1 and 3 in kidney accumulation and toxicity of aristolochic acid i pharmacokinetics and renal effects of cidofovir with a reduced dose of probenecid in hiv-infected patients with cytomegalovirus retinitis functional involvement of multidrug resistance-associated protein 4 (mrp4/abcc4) in the renal elimination of the antiviral drugs adefovir and tenofovir in vitro-in vivo extrapolation of transporter-mediated clearance in the liver and kidney impact of genetic polymorphisms of transporters on the pharmacokinetic, pharmacodynamic and toxicological properties of anionic drugs physiologically based pharmacokinetic modeling to predict transporter-mediated clearance and distribution of pravastatin in humans leflunomide and its metabolite a771726 are high affinity substrates of bcrp: implications for drug resistance a mutation in the drug transporter gene abcc2 associated with impaired methotrexate elimination long-term assessment of nsaid prescriptions and potential nephrotoxicity risk in adult kidney transplant recipients need for a judicious use of nonsteroidal anti-inflammatory drugs to avoid communityacquired acute kidney injury stereoselective inhibition of methotrexate excretion by glucuronides of nonsteroidal anti-inflammatory drugs via multidrug resistance proteins 2 and 4 species difference in the inhibitory effect of nonsteroidal anti-inflammatory drugs on the uptake of methotrexate by human kidney slices the effects of nonsteroidal antiinflammatory drugs on methotrexate (mtx) pharmacokinetics: impairment of renal clearance of mtx at weekly maintenance doses but not at 7.5 mg the role of drug transporters in the kidney: lessons from tenofovir association between concomitant use of acyclovir or valacyclovir with nsaids and an increased risk of acute kidney injury: data mining of fda adverse event reporting system gyp and jya designed the paper. xyl, qqd, and lh carried out the experiments. zlp, ztw, yx, and gjw analyzed the data. xyl and gyp prepared the paper. jya, gyp, and gjw critically revised the paper. conflict of interest: the authors declare that they have no conflict of interest. key: cord-0016426-2nsdlm0i authors: zaatout, nawel title: presence of non-oral bacteria in the oral cavity date: 2021-03-31 journal: arch microbiol doi: 10.1007/s00203-021-02300-y sha: 47b79c36d843d2dafe011851cec4891911b4af37 doc_id: 16426 cord_uid: 2nsdlm0i a homeostatic balance exists between the resident microbiota in the oral cavity and the host. perturbations of the oral microbiota under particular conditions can contribute to the growth of non-oral pathogens that are hard to kill because of their higher resistance to antimicrobials, raising the probability of treatment failure and reinfection. the presence of these bacteria in the oral cavity has been proven to be associated with several oral diseases such as periodontitis, caries, and gingivitis, and systemic diseases of importance in clinical medicine such as cystic fibrosis, hiv, and rheumatoid arthritis. however, it is still controversial whether these species are merely transient members or unique to the oral cavity. mutualistic and antagonistic interactions between the oral microbiota and non-oral pathogens can also occur, though the mechanisms used by these bacteria are not clear. therefore, this review presents an overview of the current knowledge about the presence of non-oral bacteria in the oral cavity, their relationship with systemic and oral diseases, and their interactions with oral bacteria. supplementary information: the online version contains supplementary material available at 10.1007/s00203-021-02300-y. the oral cavity is a complex and dynamic environment and is the primary gateway to the human body (zarco et al. 2012; craig et al. 2018) . various studies have identified over 1000 species from the oral cavity that forms the oral microbiota (mahasneh et al. 2017; gao et al. 2018) . however, only a tiny fraction is causing oral infections such as dental caries and periodontitis (kreth and merritt 2009; dewhirst et al. 2010 ). an imbalance of microbial flora contributes to the growth of various clinically important pathogens, that are generally considered ''non-oral'' bacteria, such as gramnegative enteric rods (gnrs), enterococci, and staphylococci (al-ahmad et al. 2009; van winkelhoff et al. 2016 ). non-oral bacteria are non-resident, super-infectious microorganisms that are not generally considered a common part of the oral microbiota. their eradication from the dental biofilms seems to be more challenging due to their higher resistance to antimicrobials, raising the probability of treatment failure and reinfection (souto et al. 2006 ). there has been a great deal of confusion in the literature regarding their natural reservoir and their ability to colonize the oral cavity. previous studies have revealed that they may occur in high numbers and shift from transitory species to colonizers of the oral cavity in immunocompromised individuals (simões-silva et al. 2018; arirachakaran et al. 2019) . however, some studies have shown that they can colonize healthy subjects too (ranganathan et al. 2017; chinnasamy et al. 2019) . moreover, systemic colonization and infections associated with non-oral bacteria isolated from the oral cavity have been revealed (arirachakaran et al. 2019; ghapanchi et al. 2019) , making the oral cavity an extra-hospital reservoir (kearney et al. 2020) . currently, there are a limited understanding and limited information regarding the pathogenesis of non-oral bacteria in the oral cavity. to the best of our knowledge, there are no reviews on the role of non-oral bacteria in the oral cavity and their relationship with the oral microbiota. therefore, this review examines the current knowledge about the most extensively studied non-oral bacteria in the oral cavity and also provides an overview of the interactions between the oral microbiota and non-oral bacteria. non-oral bacteria in the oral cavity: transitory species or colonizers? non-oral bacteria are commonly found in other parts of the human body (nares or gut). they can accidentally be introduced into the mouth by food, water, contact with animals, mouthing and chewing items, etc. nevertheless, nowadays, there is a controversy about whether the oral cavity is an entry point or an important reservoir for this group of bacteria and whether they are merely transient or unique to this niche (zuanazzi et al. 2010; vieira colombo et al. 2016 ). there has been strong evidence that they might colonize the oral ecosystem (souto and colombo 2008a; gonçalves et al. 2007a; da silva-boghossian et al. 2011) . patients positive with certain subgingival non-oral species, most notably acinetobacter baumannii and pseudomonas aeruginosa, are reported to show a higher percentage of periodontal sites with suppuration on probing (silva-boghossian et al. 2013), greater periodontal attachment loss (da silva-boghossian et al. 2013; van winkelhoff et al. 2016) and much more aggressive forms of periodontitis. furthermore, some of these bacteria isolated from the oral cavity, such as enterococci, were found to be genetically different from isolates from other parts of the human body (vidana et al. 2011) , which could potentially lead to another understanding of the ecosystem of the oral cavity. the disturbance of the "equilibrium" (due to medical treatments, biological changes, or inadequate hygiene) between commensal bacteria and the host immune system could be the reason for the shift of non-oral bacteria from transitory species to colonizers (handal et al. 2003; dahlen 2009; tada and hanada 2010) , and could enhance the subsequent morbid microbial communities in the compromised host (botero et al. 2007a; vieira colombo et al. 2016) . however, in normal oral health conditions, one should not expect these microorganisms to overcome in proportions the very well adapted oral species (van winkelhoff et al. 2016) . the most extensively studied species in the oral cavity are species of enterobacteriaceae, staphylococcus aureus, enterococcus faecalis, pseudomonas aeruginosa, and acinetobacter baumannii. the presence of unique and specific virulence factors can help in distinguishing between these different species. enterobacteriaceae is a family of gram-negative rods that have stood out in the healthcare environment due to the variety of severe infections that they can cause and their resistance to antibiotics (leão-vasconcelos et al. 2015) . their presence in the oral ecosystem is perhaps due to the ingestion of contaminated drinking water, food or poor personal hygiene (barbosa et al. 2001; gonçalves et al. 2007b) . the prevalence of gnrs in the oral environment is extremely variable, and it is still not clear whether they are colonizing or merely transient bacteria. this is probably due to the use of single-sample techniques that do not allow the differentiation between transient presence and persistent presence (martinez-pabon et al. 2010) . however, it has been shown that the prolonged transportation time of the samples may encourage the multiplication of gnrs, leading to higher positive results (ali et al. 1996) . moreover, numerous studies on gnrs pathogenesis in the oral ecosystem have shown that (1) they can persist within the subgingival environment after periodontal debridement and surgery , (2) they are implicated as key pathogens in cases of refractory periodontitis (edwardsson et al. 1999) , (3) they are detected at greater frequency and in higher proportions in patients with failing implants (listgarten et al. 1999 ) and (4) they are usually associated with oral mucosal infections in immune-compromised patients. in these patients, oral mucosal infections may spread to the respiratory system and trigger life-threatening infections (scannapecio et al. 2009; tada and hanada 2010) . furthermore, their virulence factors are conferred through several properties that give the ability to adhere and invade the host's tissues (kazemian et al. 2017) . such as the release of enterotoxins and endotoxins, elaboration of extracellular leukotoxins, degradation of immunoglobulins igg and iga, suppression of lymphocyte proliferation and elaboration of collagenolytic and other proteolytic enzymes (barbosa et al. 2001) . nevertheless, the gnrs are rarely identified at the species level, and they are referred to as "enterics" (martinez-pabón et al. 2010) . however, the group is made up of a wide variety of bacterial species, which are incongruent in pathogenicity, virulence and antibiotic susceptibility (arirachakaran et al. 2019) . at the species level, some authors have found that some gram-negative rods can dominate among oral species in some cases, like pereira et al. (2013) who found that k. pneumoniae is the dominant bacterial species in cases wearing removable maxillary prosthesis with and without denture stomatitis lesions. also, according to zhu et al. (2008) , there exists an important correlation between the presence of k. pneumoniae in the oral cavity and the risk of pneumonia by aspiration of these bacteria in people suffering from a stroke. moreover, its ability to degrade elastin (which is perceived to be a marker of p. aeruginosa in the aetiology of lower respiratory tract infections (beatty et al. 2005) ) could contribute to its virulence (goncalve et al. 2007 ). thurnheer and belibasakis (2015) observed that when escherichia coli are given the appropriate nutritional and environmental conditions, they can endure and even dominate among oral species in a polymicrobial biofilm. however, back-brito et al. (2011) have found considerably higher numbers of enteric bacteria in the oral cavities of hiv-positive patients, and enterobacter cloacae were the most frequently isolated species (table 1 , the search strategy is in the supplementary file, table s1 ). interestingly, it was found that the presence of candida albicans in the oral cavity can increase the growth and the swarming activity of proteus mirabilis (kart et al. 2020 ). although the anterior nares are considered the primary ecological niche for staphylococcus (kearney et al. 2020) , their presence in the oral cavity is unquestionable (soni et al. 2017 ) but controversial (smith et al. 2001 ), as it is not clear whether they play a part in the ecology of the healthy oral flora or not (smith et al. 2003a; blomqvist et al. 2015) . however, many authors have indicated that the oral cavity functions as a potential reservoir for s. aureus infections in immunosuppressed patients (agwu et al. 2015; merghni et al. 2015) (table 1 ) and might cause some oral diseases such as periodontitis and dental caries (fritschi et al. 2008; merghni et al. 2014) ; and systemic diseases such as heart disease, chronic kidney disease, orofacial granulomatosis and crohn's disease (gibson et al. 2000; zuanazzi et al. 2010; simões-silva et al. 2018 ). oral s. aureus has also been recognized as an aetiological factor of infective endocarditis (carmona et al. 2002) . persson and renvert (2014) found that s. aureus is present at higher amounts in biofilms obtained from implants with peri-implantitis than peri-implant healthy subjects. other studies have revealed that s. aureus was found at higher levels in the oral cavity and with greater prevalence in periodontitis than non-periodontitis subjects (souto et al. 2006; persson et al. 2008) , while fritschi et al. (2008) found higher levels of s. aureus in aggressive than chronic periodontitis subjects. consequently, s. aureus was pointed out as a contributor to the microbial profiles that could differentiate between aggressive and chronic forms of the disease. moreover, s. aureus was found at higher levels in the oral cavity of patients with rheumatoid arthritis than healthy controls (jackson et al. 1999) and was the most frequently isolated species in the oral cavities of hiv-positive patients (back-brito et al. 2011 ). the ability of s. aureus to cause such a diverse array of problems is due to its arsenal of virulence factors that are coordinately expressed during different stages of infection, such as superantigens, toxins such as β-toxin, matrix-binding surface adhesins, biofilm formation and tissue-degrading enzymes such as proteases, lipases, nucleases, and collagenases (lowy 1998; merghni et al. 2014; lima et al. 2019 ). e. faecalis is not yet considered a normal inhabitant of the oral cavity (kouidhi et al. 2011 ) but has been isolated from various oral conditions, including periodontitis and dental caries (zhu et al. 2010; kouidhi et al. 2011) (table 1) . it is perceived to be the predominant infectious agent associated with primary and secondary endodontic infections (vidana et al. 2011 ) because of its ability to reside within different layers of the oral biofilm, and co-aggregate with different saliva bacteria, which leads to failure of endodontic therapy (al-ahmad et al. 2010) . moreover, it has been found that e. faecalis can preserve viability in root canals ex vivo for at least 12 months (sedgley et al. 2005) ; this is perhaps due to its ability to form biofilms (al-ahmad et al. 2009 or colonize multi-species supragingival biofilms (thurnheer and belibasakis 2015) . furthermore, coexistence between enterococci and c. albicans has been observed in immunocompromised patients (almståhl et al. 2001 (almståhl et al. , 2008 . the origin of these opportunistic bacteria in the oral cavity is not yet clear. wang et al. (2011) demonstrated that the prevalence of e. faecalis in the root canal system had been correlated with its occurrence in saliva. meanwhile, some authors suggested nosocomial transmission from environmental surfaces in dental surgeries due to the robust nature of the microorganisms (vidana et al. 2011; lins et al. 2019) , while others proposed foodborne transmission (zehnder and guggenheim 2009 ). however, vidana et al. (2011) examined the genetic relationship between e. faecalis from root canals and isolates from different host sources and found that isolates from the root canals were not related to those from the typical gastrointestinal microflora, and none of these patients was recorded to have enterococci in their saliva. likewise, cole et al. (1999) did not find any members of this species in the saliva probes from 10 infants. further investigations are needed to minimize the dissemination of virulent and multidrug-resistant clones to the oral cavity. in addition to their role in oral diseases, subsequent systemic colonization and infection associated with an oral source of enterococci have been found; okui et al. (2015) reported a case of infective endocarditis of oral origin caused by e. faecalis, while arirachakaran et al. (2016) isolated oral enterococci from hiv patients. the most studied virulence factors of e. faecalis include biofilms, aggregation substance, gelatinase, lipoteichoic acid, the cytolysin toxin, surface adhesins, extracellular superoxide, sex pheromones, and hyaluronidase. each of these factors might be associated with many phases of endodontic infections, periapical inflammation, and systemic diseases (kayaoglu and ørstavik 2004; anderson et al. 2016; komiyama et al. 2016 ). pseudomonas aeruginosa is a gram-negative bacillus that most often affects the lower respiratory system and is associated with nosocomial infections (watanabe et al. 2009 ). it can be part of the transient oral microbiota but seldom colonize the oral cavity, which is perhaps due to its strong aerobic character (arirachakaran et al. 2019 ). however, studies using molecular biology methods have revealed that its presence in the oral cavity is underestimated and it is much higher in complex biofilms (wade 2013; souza et al. 2018 ). moreover, these species have many virulence properties such as the ability to adhere to and form biofilms on tissues and abiotic surfaces (smith and iglewski 2003b) , along with their ability to produce and secrete extracellular enzymes and toxins (smith and iglewski 2003b; pihl et al. 2010 ) as well as the expression of multiple antimicrobial resistance elements (livermore 2002) . p. aeruginosa has also been identified in the periodontal pockets of immunocompromised subjects (nakou et al. 1997 ) and might be an important pathogen in periodontitis and gingivitis vieira colombo et al. 2016) (table 1) . moreover, they are perceived to be the main pathogen in chronic obstructive pulmonary disease and biofilms on vehicles at intubation (ewan et al. 2015) . their passage into the lungs may occur by passive aspiration of the bacterial microbiota released in saliva or eased by medical devices such as bronchoscopes and endotracheal tubes (scannapieco et al. 2009 ). lately, oral p. aeruginosa has been associated with oral squamous cell carcinoma (al-hebshi et al. 2017) and chronic kidney disease (simões-silva et al. 2018 ). additionally, focal necrotizing lesions have been found in the oral mucosa of hiv-positive patients, which are different from periodontal disease patterns and are related to the presence of oral p. aeruginosa (souza et al. 2018 ). a. baumannii is a gram-negative bacillus often found in the hospital environment. it is among the red list group of eskape pathogens (e. faecium, s. aureus, k. pneumoniae, a. baumannii, p. aeruginosa, and enterobacter species) announced as a critical priority pathogen by world health organization (who) (who 2017). there are not many reports on the incidence of a. baumannii in the oral cavity or its association with oral diseases; though some studies have found that it is significantly associated with suppuration in chronic periodontitis patients, aggressive periodontitis and root canal infections (da silva-boghossian et al. 2013; vidana et al. 2011; souto et al. 2014) , especially in patients with human immunodeficiency virus (gonçalves et al. 2007a) . also, the likelihood of a subject being refractory to periodontal treatment increases when a. baumannii is present (colombo et al. 1998) . furthermore, it is a major pathogen in ventilator-associated pneumonia, which is a massive problem in hospitals, particularly in intensive care units (lee et al. 2012; martinez-lamas et al. 2014) , and was isolated from patients suffering from heart disease (zuanazzi et al. 2010) . major virulence factors that were studied in a. baumannii isolated from the oral cavity are lipocalins production, biofilm formation, siderophore-mediated iron-acquisition system, outer membrane protein a, desiccation resistance and the ability to bypass the glucose metabolism, which can be considered as one of the key factors that help this bacteria survive in a nutrition-deficient environment (richards et al. 2015; priyadharsini et al. 2018) . in the oral cavity, where resources are limited, collaborations between species are needed to survive and endure. some studies have shown the physical and metabolic interactions that exist between members of the oral microbiota and nonoral species; they can be mutualistic interspecies interactions (coaggregation) to form biofilms or antagonistic interactions to prevent the integration of a non indigenous bacterial species (table 2) . however, the biological mechanisms underlying these interactions are not yet clear. coaggregation is defined as cell-to-cell adhesion in which cells of a species adhere more or less specifically to different species (kolenbrande 2000) . this mechanism is involved in the establishment and maintenance of biofilms (kolenbrander et al. 2010) . for instance, in periodontitis patients, an association was found between gnrs and porphyromonas gingivalis with tannerella forsythia; both members of the "red complex" bacterial species are associated with severe forms of periodontitis (socransky et al. 1998) . ardila et al. (2011 ardila et al. ( , 2012 also reported a positive subgingival correlation between gnrs and p. gingivalis, and between gnrs and aggregatibacter actinomycetemcomitans. likewise, e. faecalis strains coaggregated with fusobacterium nucleatum (johnson et al. 2006) , which was able to co-aggregate with helicobacter pylori (andersen et al. 1998 ) and s. aureus (tawara et al. 1996; lima et al. 2019) . fusobacterium is considered a key microorganism in the process of coaggregation among different genera and might work as a bridge between early and late colonizers (andersen et al. 1998; souto and colombo 2008b) . previous studies have demonstrated that f. nucleatum utilizes the surface protein radd to bind and form a dual-species biofilm with other oral species (park et al. 2016; lima et al. 2017) . moreover, da silva-boghossian et al. (2011) demonstrated that p. aeruginosa seemed to have synergism with a. actinomycetemcomitans, raising the risk of periodontal disease. nonetheless, in the same study, the presence of e. faecalis, or s. aureus in association with a. actinomycetemcomitans decreased the risk of periodontal disease. however, other studies have revealed that s. aureus and e. faecalis were detected at higher levels and with greater prevalence in periodontitis than the non-periodontitis subjects (fritschi et al. 2008; persson et al. 2008 ). the differences in methods of detection and ecological variables may account for the data variability amongst these studies. antagonistic relationships are also detected in such intricate microbial communities. nutritional competition between two early colonizers of the oral cavity and e. faecalis was observed. it was shown that the presence of e. faecalis in the oral plaque causes a significant reduction in the numbers of streptococcus oralis and streptococcus mutans (thurnheer and belibasakis 2015) , which is in line with other studies demonstrating that e. faecalis dominates numerically over s. mutans in dual-species biofilms (deng et al. 2009; li et al. 2014) . moreover, okuda et al. (2003) found that streptococcus oralis, actinomyces naeslundii, streptococcus mutans, prevotella intermedia, prevotella nigrescens, and streptococcus sobrinus, produce bacteriocin-like inhibitory proteins against h. pylori. the fact that subjects with good oral hygiene harbor less h. pylori in their mouths could also be due to the inhibitory activity of the early colonizers of dental biofilms, such as oral streptococci, over that species (anderson et al. 1998 ). likewise, watanabe et al. (2009) demonstrated that a substance called the ''new-antipseudomonal substance'' derived from streptococcus sanguinis could have bactericidal activity against a. baumannii and p. aeruginosa. nevertheless, these complex and dynamic interactions remain unknown. more profound studies focusing mainly on quorum sensing are needed to understand how non-oral bacteria regulate their genes and coordinate cooperative behaviors in the presence of oral bacteria. the complex and dynamic interactions in the oral ecosystem between oral and non-oral bacteria are far from being wholly unraveled, and the pathogenetic mechanisms used by these microorganisms are still unclear. nevertheless, it is clear that non-oral bacteria are not passive bystanders and could play an essential role in oral and systemic diseases. some non-oral bacteria, such as those covered by this review, are becoming major microbes in the oral cavity and they are increasingly isolated from healthy subjects. this review highlighted the possible role, versatility, and pathogenic potential of non-oral bacteria in the oral cavity. however, some studies that were used displayed some limitations. most of the studies available on this subject were cross-sectional studies. longitudinal studies are also needed to track the presence of these bacteria over an extended period. assessing quantitatively, the presence of non-oral bacteria is of utmost importance and not just counting on presence/absence. furthermore, molecular biology methods are also needed to see whether non-oral bacteria are genetically different from isolates from other parts of the human body. despite the limitations, the presence of non-oral bacteria in the oral cavity is clearly worrisome. it needs more attention to broaden our understanding of the oral ecosystem and develop novel and more adequate preventive and therapeutic approaches, as well as diagnostic applications so that we can control the spread of non-oral bacteria and render them incapable of damaging the host. presence of helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture baseline burden and antimicrobial susceptibility of pathogenic bacteria recovered from oral lesions of patients with hiv/aids in south-western uganda endodontic and salivary isolates of enterococcus faecalis integrate into biofilm from human salivary bacteria cultivated in vitro antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth inflammatory bacteriome featuring fusobacterium nucleatum and pseudomonas aeruginosa identified in association with oral squamous cell carcinoma prevalence of 6 putative periodontal pathogens in subgingival plaque samples from romanian adult periodontitis patients microflora in oral ecosystems in primary sjögren's syndrome microflora in oral ecosystems in subjects with radiation-induced hyposalivation helicobacter pylori adheres selectively to fusobacterium spp enterococcus faecalis from food, clinical specimens, and oral sites: prevalence of virulence factors in association with biofilm formation positive correlations between presence of gram negative enteric rods and porphyromonas gingivalis in subgingival plaque relationship between gram negative enteric rods, aggregatibacter actinomycetemcomitans, and clinical parameters in periodontal disease highly-active antiretroviral therapy and oral opportunistic microorganisms in hiv-positive individuals of thailand non-oral, aerobic, gram-negative bacilli in the oral cavity of thai hiv-positive patients on highly-active anti-retrovirus therapy medication staphylococcus spp., enterobacteriaceae and pseudomonadaceae oral isolates from brazilian hiv-positive patients. correlation with cd4 cell counts and viral load subgingival occurrence and antimicrobial susceptibility of enteric rods and pseudomonads from brazilian periodontitis patients pseudomonas aeruginosa degrades pulmonary surfactant and increases conversion in vitro phenotype, genotype, and antibiotic susceptibility of swedish and thai oral isolates of staphylococcus aureus occurrence of periodontopathic and superinfecting bacteria in chronic and aggressive periodontitis subjects in a colombian population frequency of detection of periodontopathic and superinfecting bacteria in hiv-positive patients with periodontitis an update on the controversies in bacterial endocarditis of oral origin. oral surg oral med oral pathol oral radiol endod chronic nail biting, orthodontic treatment and enterobacteriaceae in the oral cavity humoral immunity to commensal oral bacteria in human infants : salivary secretory immunoglobulin a antibodies reactive with streptococcus mitis biovar 1, streptococcus oralis, streptococcus mutans, and enterococcus faecalis during the first two years of life clinical and microbiological features of refractory periodontitis subjects child weight gain trajectories linked to oral microbiota composition prevalence of staphylococcus spp and candida spp in the oral cavity and periodontal pockets of periodontal disease patients association of red complex, a. actinomycetemcomitans and non-oral bacteria with periodontal diseases suppuration-associated bacteria in patients with chronic and aggressive periodontitis bacterial infections of the oral mucosa occurrence of enteric rods, staphylococci and candida in subgingival samples influence of streptococcus mutans on enterococcus faecalis biofilm formation the human oral microbiome the microbiota of periodontal pockets with different depths in therapy-resistant periodontitis dental and microbiological risk factors for hospital-acquired pneumonia in non-ventilated older patients staphylococcus aureus and other bacteria in untreated periodontitis oral microbiomes : more and more importance in oral cavity and whole body isolation of enterococcus faecalis in the saliva samples of patient candidates for liver transplantation oral staphylococcal mucositis. oral surg oral med oral pathol oral radiol endod clinical and microbiological profiles of human immunodeficiency virus (hiv)-seropositive brazilians undergoing highly active antiretroviral therapy and hiv-seronegative brazilians with chronic periodontitis periodontal disease as reservoir for multi-resistant and hydrolytic enterobacterial species the incidence of oral gram-negative bacteria in patients with parkinson's disease antibiotic resistance in bacteria isolated from subgingival plaque in a norwegian population with refractory marginal periodontitis oral carriage of staphylococci in patients with rheumatoid arthritis oral staphylococcus in older subjects with rheumatoid arthritis coaggregation interactions between oral and endodontic enterococcus faecalis and bacterial species isolated from persistent apical periodontitis effect of porphyromonas gingivalis vesicles on coaggregation of staphylococcus aureus to oral microorganisms coaggregation ability of weissella cibaria isolates with fusobacterium nucleatum and their adhesiveness to epithelial cells altered metabolomic profile of dual-species biofilm: interactions between proteus mirabilis and candida albicans virulence factors of enterococcus faecalis: relationship to endodontic disease oral colonization by nosocomial pathogens during hospitalization in intensive care unit and prevention strategies the oral cavity revealed as a significant reservoir of staphylococcus aureus in an acute hospital by extensive patient, healthcare worker and environmental sampling oral microbial communities: biofilms, interactions, and genetic systems oral multispecies biofilm development and the key role of cell-cell distance interbacterial adherence between actinomyces viscosus and strains of streptococcus pyogenes, streptococcus agalactiae, and pseudomonas aeruginosa pseudomonas aeruginosa in the oral cavity and sputum of patients with cystic fibrosis interbacterial adhesion between pseudomonas aeruginosa and indigenous oral bacteria isolated from patients with cystic fibrosis enterococcus species in the oral cavity : prevalence, virulence factors and antimicrobial susceptibility antibiotic resistance and adhesion properties of oral enterococci associated to dental caries bacterial and host interactions of oral streptococci enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital outbreak of imipenem-resistant acinetobacter calcoaceticus-acinetobacter baumannii complex harboring different carbapenemase gene-associated genetic structures in an intensive care unit diversity of streptococcus mutans strains in bacterial interspecies interactions identification and characterization of a novel fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with streptococcus gordonii the oral bacterium fusobacterium nucleatum binds staphylococcus aureus and alters expression of the staphylococcal accessory regulator sara comparison of genotypes, antimicrobial resistance and virulence profiles of oral and non oral enterococcus faecalis from brazil, japan and the united kingdom comparative microbiological characteristics of failing implants and periodontally diseased teeth multiple mechanisms of antimicrobial resistance in pseudomonas aeruginosa: our worst nightmare? staphylococcus aureus infections probiotics: a promising role in dental health new clone of st-187 acinetobacter baumannii responsible for an outbreak in an intensive care unit screening for subgingival occurrence of gram-negative enteric rods in periodontally diseased and healthy subjects adhesive properties and extracellular enzymatic activity of staphylococcus aureus strains isolated from oral cavity assessment of adhesion, invasion and cytotoxicity potential of oral staphylococcus aureus strains mitsis f (1997) periodontal microflora of hiv infected patients with periodontitis ecological and immunopathological implications of oral bacteria in helicobacter pylori infected disease detection of identical isolates of enterococcus faecalis from the blood and oral mucosa in a patient with infective endocarditis in vitro antimicrobial activity of ten medicinal plants against clinical isolates of oral cancer cases characterization of fusobacterium nucleatum atcc 23726 adhesins involved in strainspecific attachment to porphyromonas gingivalis opportunistic microorganisms in individuals with lesions of denture stomatitis cluster of bacteria associated with periimplantitis tannerella forsythia and pseudomonas aeruginosa in subgingival bacterial samples from parous women effects of clinical isolates of pseudomonas aeruginosa on staphylococcus epidermidis biofilm formation in silico analysis of virulence genes in an emerging dental pathogen a. baumannii and related species microbiological study of hiv-related periodontitis subgingival prevalence rate of enteric rods in subjects with periodontal health and disease code blue: acinetobacter baumannii, a nosocomial pathogen with a role in the oral cavity pseudomonas aeruginosa and periodontal pathogens in the oral cavity and lungs of cystic fibrosis patients : a casecontrol study a randomized trial of chlorhexidine gluconate on oral bacterial pathogens in mechanically ventilated patients oral streptococci and nitrite-mediated interference of pseudomonas aeruginosa survival of enterococcus faecalis in root canals ex vivo prevalence of enterococcus faecalis at multiple oral sites in endodontic patients using culture and pcr oral colonization of staphylococcus species in a peritoneal dialysis population: a possible reservoir for pd-related infections? in vitro activity of chlorhexidine against enteric rods, pseudomonas and acinetobacter from human periodontitis the ecology of staphylococcus species in the oral cavity staphylococcal species in the oral cavity from patients in a regional burns unit microbial complexes in subgingival plaque opportunistic microorganisms in oral cavity according to treatment status in head and neck cancer patients prevalence of enterococcus faecalis in subgingival biofilm and saliva of subjects with chronic periodontal infection detection of helicobacter pylori by polymerase chain reaction in the subgingival biofilm and saliva of non-dyspeptic periodontal patients prevalence of non-oral pathogenic bacteria in subgingival biofilm of subjects with chronic periodontitis prevalence of pseudomonas aeruginosa and acinetobacter spp in subgingival biofilm and saliva of subjects with chronic periodontal infection oral infection by pseudomonas aeruginosa in patient with chronic kidney diseasea case report opportunistic respiratory pathogens in the oral cavity of the elderly methicillin-resistant staphylococcus aureus and candida albicans on denture surfaces integration of non-oral bacteria into in vitro oral biofilms inhibition of methicillinresistant staphylococcus aureus colonization of oral cavities in newborns by viridans group streptococci non-oral gram-negative facultative rods in chronic periodontitis microbiota enterococcus faecalis infection in root canals-host-derived or exogenous source? martins do soutomaciel da silva-boghossian rc (2016) periodontal-disease-associated biofilm : a reservoir for pathogens of medical importance the oral microbiome in health and disease relationship of biofilm formation and gele gene expression in enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment bactericidal activity in filtrated supernatant of streptococcus sanguinis against multidrug-resistant pseudomonas aeruginosa global priority list of antibioticresistant bacteria to guide research, discovery and development of new antibiotics the oral microbiome in health and disease and the potential impact on personalized dental medicine oral carriage of yeasts and coliforms in stroke sufferers: a prospective longitudinal study prevalence, phenotype, and genotype of enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis prevalence of potential bacterial respiratory pathogens in the oral cavity of hospitalised individuals key: cord-0055042-a4zeifia authors: surabhi, surabhi; cuypers, fabian; hammerschmidt, sven; siemens, nikolai title: the role of nlrp3 inflammasome in pneumococcal infections date: 2020-12-14 journal: front immunol doi: 10.3389/fimmu.2020.614801 sha: d044ff890a385af23dd129d8a6886b5792cf6caa doc_id: 55042 cord_uid: a4zeifia inflammasomes are innate immune sensors that regulate caspase-1 mediated inflammation in response to environmental, hostand pathogen-derived factors. the nlrp3 inflammasome is highly versatile as it is activated by a diverse range of stimuli. however, excessive or chronic inflammasome activation and subsequent interleukin-1β (il-1β) release are implicated in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and diabetes. accordingly, inflammasome inhibitor therapy has a therapeutic benefit in these diseases. in contrast, nlrp3 inflammasome is an important defense mechanism against microbial infections. il-1β antagonizes bacterial invasion and dissemination. unfortunately, patients receiving il-1β or inflammasome inhibitors are reported to be at a disproportionate risk to experience invasive bacterial infections including pneumococcal infections. pneumococci are typical colonizers of immunocompromised individuals and a leading cause of community-acquired pneumonia worldwide. here, we summarize the current limited knowledge of inflammasome activation in pneumococcal infections of the respiratory tract and how inflammasome inhibition may benefit these infections in immunocompromised patients. the human innate immunity axis plays a pivotal role in detection of pathogen-or damageassociated molecular patterns (pamps and damps) and contributes to a crucial inflammatory response. to sense pamps and damps, innate immune cells express pattern recognition receptors (prrs). prrs are classified into five families: toll-like receptors (tlrs), nucleotide-binding and oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene (rig)-i-like receptors, c-type lectin receptors, and absent in melanoma 2 (aim2)-like receptor (alr) (1) . furthermore, other molecules such as cyclic gmp-amp synthase can sense pathogen-derived dna (2) . inflammasomes are one of the most recently discovered classes of nlrs (3) . to date, 22 human nlrs are described. among them, nlr and pyrin domain containing receptor 3 (nlrp3) is by far the best characterized (4) . a wide range of stimuli including bacterial pore forming toxins can activate the nlrp3 inflammasome (5) . the subsequent release of interleukins (il) il-1b and il-18 induces a diverse range of protective host pathways aiming to eradicate the pathogen (6) . however, uncontrolled and excessive hyper-inflammation can be a driver of several inflammatory and autoimmune diseases (7, 8) . implication of the nlrp3 inflammasome in inflammatory diseases has provided new avenues for designing drugs which target the inflammasome and its signaling cascade. however, it is observed that patients who receive nlrp3 or il-1b inhibitors are disproportionately susceptible to bacterial infections (9) . therefore, it is of high importance to understand the role of nlrp3 inflammasome in bacterial pathogenesis. nlrp3 inflammasome is a multi-protein complex comprising of a sensor nlrp3 protein, an adaptor apoptosis-associated specklike protein (asc), and the zymogen procaspase-1 (10) . the cytosolic nlrp3 protein contains an n-terminal pyrin domain (pyd), a central nacht domain, and a c-terminal leucine-rich repeat (lrr) domain. the nacht domain possesses adenosine triphosphatase (atpase) activity and comprises of nucleotidebinding domain (nbd), helical domain 1 (hd1), winged helix domain (whd) and helical domain 2 (hd2) (11) . the asc domain is a bipartite molecule that contains an n-terminal pyd domain and a c-terminal caspase activation and recruitment domain (card). procaspase-1 consists of an n-terminal card, a central large catalytic p20 subunit, and a c-terminal small catalytic p10 subunit (12) . in resting macrophages, the nlrp3 and pro-il-1b concentrations are insufficient to initiate activation of the inflammasome (13) . therefore, the nlrp3 inflammasome is activated in a two-step process. the first, so called priming step, is initiated via the inflammatory stimuli which are detected by tlrs, tumor necrosis factor receptors (tnfr) or il-1r. these actions activate downstream the transcription factor nf-kb. nf-kb, in turn, upregulates the expression of nlrp3 and pro-il-1b. in contrast, the priming step does not affect the expression of asc, procaspase-1 or il-18 (14) (15) (16) . following priming, a second activation step is essential for the assembly of the inflammasome. nlpr3 is highly diverse in nature and a wide range of stimuli can activate it. common activators of the nlrp3 inflammasome are pathogens (17) , extracellular atp (18), pathogen associated rna, proteins and toxins (5, 19, 20) , heme (21) , endogenous factors (amyloid-b, cholesterol crystals, uric acid crystals) (22) (23) (24) , and environmental factors (silica and aluminum salts) (24, 25) . these activators do not directly interact with the inflammasome but rather cause various changes at the cellular level. these include changes in cell volume (26) , ionic fluxes (27), lysosomal damage (28) , ros production, and mitochondrial dysfunction (29) . the second activation step is essential for cells such as macrophages and epithelial cells. in contrast, human monocytes can release mature il-1b already after priming (30, 31) . upon activation, oligomerization of the nlrp3 complex occurs via homotypic pyd-pyd interaction of the sensor and adaptor protein, and card-card interaction of the adaptor and procaspase-1 ( figure 1 ). following assembly, recruited procaspase-1 is converted to bioactive caspase-1 through proximity induced auto-proteolytic cleavage (32) . subsequently, caspase-1 cleaves the cytokine precursors pro-il-1b and pro-il-18 into mature forms. simultaneously, caspase-1 cleaves gasdermin-d (gsdmd). after proteolytic cleavage, the c-terminal gsdmd (gsdmd-c) remains in the cytosol, while gsdmd-n anchors the cell membrane lipid. the lipid binding allows gsdmd-n to enter the lipid bilayer. subsequent gsdmd-n oligomerization within the membrane results in pore formation leading to cell swelling and lysis. the pores serve thereby as protein secretion channels for il-1b and il-18. this form of a programmed inflammatory cell death is called pyroptosis (33) (34) (35) . however, lytic gsdmd-n dependent secretion of il-1b does not apply universally to all cell types. studies on neutrophils have shown that gsdmd-n does not localize at the plasma membrane. instead, it co-localizes with membranes of azurophilic granules and lc3 + autophagosomes resulting in a non-lytic pathway dependent il-1b secretion which depends on autophagy machinery (36) . alongside with il-1b, other pro-inflammatory cytokines, eicosanoids, and alarmins are released into the extracellular space. these actions accentuate the inflammatory state by recruiting additional inflammatory immune cells of different lineages (37) (38) (39) . apart from the canonical, non-canonical nlrp3 inflammasome activation is described (40) . non-canonical inflammasome activation is triggered by caspases-4/5 in humans (41) . however, the noncanonical form can sense only gram-negative bacteria. therefore, it potentially does not play a role in gram-positive bacterial infections (40) . nlrp3 inflammasome signaling is implicated in the onset of a number of diseases, including gout (24), atherosclerosis (23), type іі diabetes (42, 43) , cryopyrin-associated periodic syndrome (caps) (44) , various types of cancer (45) , and inflammatory bowel disease (ibd) (46) . in the following, we give just two examples of the role of nlrp3 in autoinflammatory and auto-immune diseases. caps summarizes three auto-inflammatory diseases caused by mutations in the nlrp3 gene. these include familial cold auto-inflammatory syndrome (fcas), muckle-wells syndrome (mws), and neonatal onset multisystem inflammatory disease (nomid). in most cases, caps manifests during the childhood and is characterized by spontaneous nlrp3 activation and excessive il-1b production resulting in frequent episodes of fever, skin rashes, joint and eye inflammation. in severe cases, children can suffer from periorbital edema, amyloidosis, polyarthralgia, growth retardation, and death (47, 48) . in vivo studies with transgenic mice expressing the human diseaseassociated r258w (mws) or a350v and l351p (fcas) mutations in the nlrp3 gene demonstrated the detrimental role of il-1b in these diseases (49, 50) . genetic deletion of the il-1r efficiently rescued nlrp3 a350v and partially nlrp3 l351p mice from neonatal lethality (49) . rheumatoid arthritis (ra) is a chronic autoimmune disease characterized by persistent synovial inflammation and hyperplasia of the diarthrodial joints and progressive destruction of cartilage and bone (51) . in particular, chondrocytes-and monocytes-derived tnf and il-1b are associated with hyper-inflammatory processes in affected joints (52) . at the local level, even low concentrations of il-1b induce production and secretion of matrix metalloproteinases, which are mainly involved in destructive processes (53) . furthermore, il-1b assists in t helper type 17 (th17) cells differentiation and subsequent il-17a production. both processes further contribute to the hyper-inflammatory state of ra (54) . these data implicate nlrp3 inflammasome as one of the contributing factors to ra progression. in line with this, several studies have shown that nlrp3 and other inflammasome-related genes are highly upregulated in monocytes, macrophages, and dendritic cells of ra patients. furthermore, nlrp3 gene polymorphisms (e.g., rs35829419, rs10754558, rs4612666) were associated with ra manifestation and pathogenesis [reviewed in (55) ]. although the above mentioned diseases affect different organs and are diverse in nature, they are also characterized by a common feature, namely elevated levels of il-1b. it is of significance to mention that il-1b release is not limited to nlrp3 inflammasome activation. a variety of mechanisms, including aim2 inflammasome activation, which also plays a crucial role in bacterial detection, can result in production and release of il-1b [reviewed in (56) ]. therefore, treatments target various components of the signaling cascade and particularly il-1. several strategies that combat il-1 action have undergone substantial clinical trials. some of them are summarized in table 1 . anakinra, rilonacept, and canakinumab are clinically approved il-1 inhibiting drugs and the best studied agents (73) . anakinra is an il-1 receptor antagonist and is used among others for the treatment of ra, acute gouty arthritis, and caps. it blocks the action of both il-1a and il-1b (74) (75) (76) . clinical trials with anakinra reported elevated numbers of infectious episodes in anakinra-treated patients as compared to the placebo-treated group during the first 6 months of treatment. furthermore, the incidence of serious infections was increased. these infections comprised mainly of cellulitis, pneumonia, and bone and joint infections as well (57) . rilonacept is a dimeric fusion protein that contains two il-1 receptors attached to the fc portion of human igg1. similar to anakinra, it blocks the activity of il-1 isoforms but does not interact with the il-1 receptor. rilonacept is used to treat caps and the most reported side-effects include skin reactions and upper respiratory tract infections (58) . canakinumab is a monoclonal igg1 that specifically targets il-1b and is commonly used for treatment of periodic fever syndromes, mws, and acute gouty arthritis (59, 60) . among the various side effects, respiratory tract infections were the most reported side effect in clinical trials (60) . all il-1 inhibiting strategies are well tolerated in the majority of patients. the most common adverse effect is a dose-dependent figure 1 | nlrp3 inflammasome activation by pneumococci. pneumococci secrete two major virulence determinants: pneumolysin (ply) and hydrogen peroxide (h 2 o 2 ). in neutrophils (pmn), ply-mediated nlrp3 activation is a result of k + efflux. k + efflux activates nlrp3 inflammasome resulting in caspase-1 activation and subsequent cleavage of pro-il-1b into mature form. in macrophages, ply-mediated nlrp3 inflammasome activation is among others dependent on atp. the released il-1b stimulates epithelial cells. as a result, they release chemoattractants, including cxcl1 and cxcl2. both chemokines are involved in processes resulting in neutrophil influx. furthermore, il-1b is involved in t helper type 17 cells differentiation and subsequent il-17a release. in contrast to ply, h 2 o 2 suppresses nlrp3 inflammasome in macrophages (mf) resulting in pro-il-1b accumulation in these cells (apc, antigen presenting cell). skin irritation at the injection site. however, a substantially increased incidence of bacterial infections of the respiratory tract caused by gram-positive bacteria, including pneumococci, staphylococcus aureus (sa), and/or group a streptococci (gas) are reported (77) . furthermore, il-1 inhibiting therapies were associated with a higher incidence of fatal infections as compared to the placebo treated group. therefore, treatment with il-1 inhibiting drugs is not recommended for patients with an ongoing infection or with a history of severe infections (59, 77, 78) . pneumococci, sa, and gas are frequent colonizers of the upper respiratory tract (79) . colonization is usually asymptomatic in healthy individuals. however, imbalances in the immune system can lead to severe, invasive and even life-threatening diseases such as pneumonia and sepsis. the occurrence of the more severe forms of infection is commonly found in children younger than 5 years of age, elderly, and immuno-compromised population (80) . due to the immunosuppressive nature of il-1 inhibiting agents, patients undergoing treatment seem to be at higher risk to develop infections caused by these bacteria (77, 81) . in general, inflammation plays a crucial role in infectious diseases. impaired or insufficient inflammatory response can result in prolonged and/or recurrent infections. in contrast, excessive hyper-inflammation is associated with fatal outcome (82, 83) . pneumococci colonize the nasopharyngeal cavity of 20%-50% of children and 8%-30% of adults. they have been implicated as the most common etiologic agent of communityacquired pneumonia (80, 84) . however, only limited number of studies investigated the role of inflammasome in pneumococcal infections and contrary results are reported. for example, one murine model study reported that nlrp3 −/− mice are more susceptible to pneumococcal pneumonia (85) . in contrast, a study on pneumococcal meningitis showed that mice with an active nlrp3 signaling have higher clinical scores, suggesting that nlrp3 activation contributes to brain injury (86) . since the incidence of respiratory tract infections is elevated in patient receiving il-1 inhibiting agent, we will solely focus on the role of nlrp3 in respiratory pneumococcal infections. two of the most important secreted pneumococcal virulence determinants are hydrogen peroxide (h 2 o 2 ) and the cholesteroldependent cytolysin, pneumolysin (ply) (87) . both factors are implicated in inflammasome activating and suppressive processes. based on the pneumococcal serotypes used for the infection, the nlrp3-dependent il-1b secretion by human cells varies. macrophages infected with serotypes that are associated with invasive diseases and express low/non-hemolytic ply (serotypes 1, 7f and 8), release lower amounts of il-1b as compared to macrophages infected with serotypes expressing a fully active ply (serotypes 2, 3, 6b, 9n) (88, 89) . being poor activators of the inflammasome, the invasive serotypes are potentially less efficiently recognized by the innate immune system and therefore, are less susceptible to immuno-mediated clearance. however, the exact mechanism of nlrp3 activation by ply is unknown. this process is most likely of indirect nature (figure 1 ). studies on human neutrophils have shown that plymediated nlrp3 activation is a result of potassium ion (k + ) efflux. experimental inhibition of k + efflux in neutrophils resulted in impaired caspase-1 activation and subsequently in diminished il-1b processing (figure 1 ). furthermore, it was shown that lysosomal destabilization did not play a role in plymediated il-1b processing in neutrophils (90) . in general, il-1b induces the production of chemoattractants, such as cxcl1 and cxcl2 by lung epithelial cells, which enhance neutrophil influx (91) and subsequent bacterial clearance at the site of infection (92, 93) . studies on pneumococcal infections of mouse peritoneal neutrophils indicate that nlrp3 inflammasome is mainly responsible for il-1b secretion, while the aim2 and nlrc4 inflammasomes are dispensable in these type of immune cells (94) . furthermore, neutrophil-derived il-1b is involved in activation of th17 cells. th17-derived il-17a acts as an additional chemoattractant-stimulating agent (95, 96) and indirectly mediates neutrophilia in the infected organs ( figure 1 ) (97) . nonetheless, neutrophil influx alone is not sufficient to clear pneumococci and macrophage influx is essential to ensure bacterial elimination (93, 98) . a study by hoegen and colleagues demonstrated that ply was a key inducer of nrlp3 inflammasome and il-1b expression in human differentiated thp-1 cells (86) . in contrast to neutrophils, nlrp3 inflammasome activity was dependent on lysosomal destabilization, release of atp, and cathepsin b activation (86) . furthermore, nlrp3 inflammasome activating synergistic effects of ply and tlr agonists in dendritic cells and macrophages are reported (85, 88) . however, this is rather a general inflammasome activating/priming effect. apart from nlrp3, ply can also activate aim2 inflammasomes (99) . in contrast to ply, only one study investigated the pneumococci-derived h 2 o 2 and inflammasome interplay. by utilizing mouse bone marrow-derived macrophages (mbmdms), ertmann and gekara have shown that mbmdms infected with pneumococci accumulate large amounts of pro-il-1b and procaspase-1 (100) . detection of the processed forms of il-1b and caspase-1 was highly delayed and remained undetectable until 12 h post infection. however, the ratio of processed il-1b and caspase-1 to their precursors was still very low (100) . in contrast, spxb-mutant strain, which lacked h 2 o 2 production, showed an intrinsically increased capacity to activate the inflammasome. the authors suggested that pneumococci employ h 2 o 2 -mediated inflammasome inhibition as a colonization strategy (100) . although the study provides highly relevant new insights into pneumococci-host interplay, verification of these results in human host system is warranted. however, host factors upstream or downstream of nlrp3 inflammasome also play a crucial role in colonization and infection processes. studies in aged mice have suggested that an increase in endoplasmic reticulum stress and enhanced unfolded protein responses contribute to diminished assembly and activation of the nlrp3 resulting in failed clearance of pneumococci (101) . in support of this, krone and colleagues demonstrated that aged mice shows a delayed clearance of pneumococci in the nasopharynx as compared to young mice (102) . the authors attributed the observed phenotype to the impaired innate mucosal immune responses in aged mice, including nlrp3 and il-1b suppression. furthermore, lemon and colleagues demonstrated a prolonged colonization of il1r -/adult mice as compared to wild-type mice (93) . the prolonged colonization was linked to reduced numbers of neutrophils at early stages of infection and reduced macrophage influx at later time points of carriage in il1r -/mice (93) . apart from the bacterial pore-forming toxins, microbial rna has also been implicated as a direct nlrp3 inflammasome activator (19) . studies showed that even small fragments of staphylococcal or group b streptococcal (gbs) rna are sufficient for inflammasome activation in human thp-1-derived and mouse macrophages (103, 104) . based on detailed analyses of gbs and mouse macrophages interplay, gupta and colleagues proposed that bacteria-mediated activation of nlrp3 inflammasomes requires bacterial uptake, phagolysosomal acidification, and toxin-mediated leakage. subsequently, the free accessible bacterial rna interacts with nlrp3 and activates the inflammasome cascade (104) . whether such mechanism applies to pneumococcal infections, remains to be elucidated. in general, tightly controlled inflammasome activation in pneumococcal pneumonia is one of many important host defense mechanisms contributing to bacterial clearance (56) . however, excessive nlrp3 activation can also lead to uncontrolled pyroptosis. the disproportionate gasdermin-d mediated cell membrane rupture in a variety of lung cells may result in a release of plethora of alarmins, including processed antigens, atp, hmgb1, reactive oxygen species, cytokines, and chemokines (105) . these prompt an immediate reaction from resident and recruited immune cells leading to a pyroptotic chain reaction with subsequent excessive tissue pathology (106) . furthermore, pathogen-associated antigens might disseminate to other organs resulting in a severe systemic hyperinflammatory response (105) . whether such actions apply to pneumococcal pneumonia remains to be shown. besides the crucial role of nlrp3 inflammasome activation in inflammation, many studies implicate nlrp3 inflammasome in the pathology of several autoimmune and auto-inflammatory disorders. currently, the most common therapy for such diseases involves the use of immuno-suppressive, cytokine inhibiting therapies, such as il-1 inhibitors. the immuno-suppression of patients by these agents results in side effects that often include respiratory tract infections caused by pneumococci. however, only a limited number of studies investigated the role of the nlrp3 inflammasome in pneumococcal respiratory infections. future studies, especially those considering the complex interplay of human genetics, immuno-suppressive status, and age with pneumococcal colonization are needed to better understand the role of nlrp3 inflammasome in such infections. ss and ns conceived the concept for this review article. ss, fc, and ns wrote the manuscript. all authors contributed to the article and approved the submitted version. pattern recognition receptors and inflammation the cgas/sting pathway detects streptococcus pneumoniae but appears dispensable for antipneumococcal defense in mice and humans the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-beta nod-like receptors: master regulators of inflammation and cancer bacterial exotoxins and the inflammasome bacterial recognition pathways that lead to inflammasome activation targeting the nlrp3 inflammasome in inflammatory diseases nlrp3 inflammasome and inflammatory diseases il-1b is an innate immune sensor of microbial proteolysis the nlrp3 inflammasome: molecular activation and regulation to therapeutics structural mechanism for nek7-licensed activation of nlrp3 inflammasome caspases: pharmacological manipulation of cell death the nlrp3 inflammasome: an overview of mechanisms of activation and regulation tnfr2 induced priming of the inflammasome leads to a ripk1-dependent cell death in the absence of xiap cutting edge: traf6 mediates tlr/il-1r signaling-induced nontranscriptional priming of the nlrp3 inflammasome cutting edge: nf-kappab activating pattern recognition and cytokine receptors license nlrp3 inflammasome activation by regulating nlrp3 expression modulation of inflammasome pathways by bacterial and viral pathogens extracellular atp activates the nlrp3 inflammasome and is an early danger signal of skin allograft rejection bacterial rna: an underestimated stimulus for innate immune responses zika virus infection induces host inflammatory responses by facilitating nlrp3 inflammasome assembly and interleukin-1b secretion induction of nlrp3 inflammasome activation by heme in human endothelial cells amyloid b directly interacts with nlrp3 to initiate inflammasome activation: identification of an intrinsic nlrp3 ligand in a cell-free system nlrp3 inflammasomes are required for atherogenesis and activated by cholesterol crystals gout-associated uric acid crystals activate the nalp3 inflammasome silica crystals and aluminum salts activate the nalp3 inflammasome through phagosomal destabilization cell volume regulation modulates nlrp3 inflammasome activation orchestration of nlrp3 inflammasome activation by ion fluxes lysosomal destabilization activates the nlrp3 inflammasome in human umbilical vein endothelial cells (huvecs) mitochondrial reactive oxygen species induces nlrp3-dependent lysosomal damage and inflammasome activation priming is dispensable for nlrp3 inflammasome activation in human monocytes differential requirement for the activation of the inflammasome for processing and release of il-1beta in monocytes and macrophages crystal structure of procaspase-1 zymogen domain reveals insight into inflammatory caspase autoactivation cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death inflammasomeactivated gasdermin d causes pyroptosis by forming membrane pores gasdermin d is an executor of pyroptosis and required for interleukin-1beta secretion n-gsdmd trafficking to neutrophil organelles facilitates il-1b release independently of plasma membrane pores and pyroptosis inflammasome-dependent release of the alarmin hmgb1 in endotoxemia hmgb1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with cxcl12 and signaling via cxcr4 rapid induction of inflammatory lipid mediators by the inflammasome in vivo recent advances in the mechanisms of nlrp3 inflammasome activation and its inhibitors nlrp3 inflammasome activation downstream of cytoplasmic lps recognition by both caspase-4 and caspase-5 activation of the nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced il-1b in type 2 diabetes upregulated nlrp3 inflammasome activation in patients with type 2 diabetes inflammasome as a promising therapeutic target for cancer nlrp3 inflammasome and inflammatory bowel disease mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and muckle-wells syndrome familial cold autoinflammatory syndrome: phenotype and genotype of an autosomal dominant periodic fever inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity a mutation in the nlrp3 gene causing inflammasome hyperactivation potentiates th17 cell-dominant immune responses the pathogenesis of rheumatoid arthritis evidence that cytokines play a role in rheumatoid arthritis the pivotal role of interleukin-1 in the clinical manifestations of rheumatoid arthritis myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts role of the nlrp3 inflammasome in autoimmune diseases inflammasomes in pneumococcal infection: innate immune sensing and bacterial evasion strategies us food and drug administration (fda) rilonacept and canakinumab us food and drug administration (fda) use of canakinumab in the cryopyrin-associated periodic syndrome identification of a selective and direct nlrp3 inhibitor to treat inflammatory disorders effects of gevokizumab on glycemia and inflammatory markers in type 2 diabetes gevokizumab in the treatment of autoimmune non-necrotizing anterior scleritis: results of a phase i/ii clinical trial population pharmacokinetic modeling of ly2189102 after multiple intravenous and subcutaneous administrations glyburide inhibits the cryopyrin/nalp3 inflammasome glycemic durability of rosiglitazone, metformin, or glyburide monotherapy a comparison of glyburide and insulin in women with gestational diabetes mellitus an inhibitor of interleukin-1beta converting enzyme, reduces joint damage in two murine models of osteoarthritis s)-1-((s)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethylbutanoyl)-pyrrolidine-2-carboxylic acid ((2r,3s)-2-ethoxy-5-oxotetrahydro-furan-3-yl)-amide (vx-765), an orally available selective interleukin (il)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of il-1beta and il-18 olt1177, a b-sulfonyl nitrile compound, safe in humans, inhibits the nlrp3 inflammasome and reverses the metabolic cost of inflammation a randomized, double-blind study of amg 108 (a fully human monoclonal antibody to il-1r1) in patients with osteoarthritis of the knee interleukin 1 inhibitors efficacy of anakinra in gouty arthritis: a retrospective study of 40 cases anakinra for rheumatoid arthritis: a systematic review interleukin-1-receptor antagonist in type 2 diabetes mellitus anakinra, a recombinant human interleukin-1 receptor antagonist (r-methuil-1ra), in patients with rheumatoid arthritis: a large, international, multicenter, placebo-controlled trial unveiling the efficacy, safety, and tolerability of anti-interleukin-1 treatment in monogenic and multifactorial autoinflammatory diseases port d'entree for respiratory infections -does the influenza a virus pave the way for bacteria? front microbiol insights into the interaction between influenza virus and pneumococcus risk of serious infection in patients with rheumatoid arthritis treated with biologic versus nonbiologic disease-modifying antirheumatic drugs blood-brain barrier pathology and cns outcomes in streptococcus pneumoniae meningitis inflammasome activation mediates inflammation and outcome in humans and mice with pneumococcal meningitis streptococcus pneumoniae: transmission, colonization and invasion pneumolysin activates the nlrp3 inflammasome and promotes proinflammatory cytokines independently of tlr4 the nlrp3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through atp-dependent lysosomal cathepsin b release the role of streptococcus pneumoniae virulence factors in host respiratory colonization and disease the nlrp3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia serotype 1 and 8 pneumococci evade sensing by inflammasomes in human lung tissue neutrophil il-1b processing induced by pneumolysin is mediated by the nlrp3/asc inflammasome and caspase-1 activation and is dependent on k+ efflux the interleukin-1b/cxcl1/2/neutrophil axis mediates host protection against group b streptococcal infection the role of myeloid cells in prevention and control of group a streptococcal infections sensing of interleukin-1 cytokines during streptococcus pneumoniae colonization contributes to macrophage recruitment and bacterial clearance nlrp3/asc/caspase-1 axis and serine protease activity are involved in neutrophil il-1b processing during streptococcus pneumoniae infection the il-23-il-17 immune axis: from mechanisms to therapeutic testing interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease neutrophilic nlrp3 inflammasome-dependent il-1beta secretion regulates the gammadeltat17 cell response in respiratory bacterial infections cellular effectors mediating th17-dependent clearance of pneumococcal colonization in mice critical roles of asc inflammasomes in caspase-1 activation and host innate resistance to streptococcus pneumoniae infection hydrogen peroxide release by bacteria suppresses inflammasome-dependent innate immunity nlrp3 inflammasome activation in aged macrophages is diminished during streptococcus pneumoniae infection impaired innate mucosal immunity in aged mice permits prolonged streptococcus pneumoniae colonization human nlrp3 inflammasome senses multiple types of bacterial rnas rna and b-hemolysin of group b streptococcus induce interleukin-1b (il-1b) by activating nlrp3 inflammasomes in mouse macrophages inflammasomes and pyroptosis as therapeutic targets for covid-19 differential neutrophil responses to bacterial stimuli: streptococcal strains are potent inducers of heparin-binding protein and resistin-release the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 surabhi, cuypers, hammerschmidt and siemens. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0001647-0zkeoa1z authors: ten oever, jaap; kox, matthijs; van de veerdonk, frank l; mothapo, khutso m; slavcovici, adriana; jansen, tim l; tweehuysen, lieke; giamarellos-bourboulis, evangelos j; schneeberger, peter m; wever, peter c; stoffels, monique; simon, anna; van der meer, jos wm; johnson, melissa d; kullberg, bart-jan; pickkers, peter; pachot, alexandre; joosten, leo ab; netea, mihai g title: the discriminative capacity of soluble toll-like receptor (stlr)2 and stlr4 in inflammatory diseases date: 2014-11-19 journal: bmc immunol doi: 10.1186/s12865-014-0055-y sha: 63771fc756683bf14c7f7e169990c7268b35d8ed doc_id: 1647 cord_uid: 0zkeoa1z background: the extracellular domains of cytokine receptors are released during inflammation, but little is known about the shedding of toll-like receptors (tlr) and whether they can be used as diagnostic biomarkers. methods: the release of stlr2 and stlr4 was studied in in-vitro stimulations, as well as in-vivo during experimental human endotoxemia (n = 11, 2 ng/kg lps), and in plasma of 394 patients with infections (infectious mononucleosis, measles, respiratory tract infections, bacterial sepsis and candidemia) or non-infectious inflammation (crohn’s disease, gout, rheumatoid arthritis, autoinflammatory syndromes and pancreatitis). using c-statistics, the value of stlr2 and stlr4 levels for discrimination between infections and non-infectious inflammatory diseases, as well as between viral and bacterial infections was analyzed. results: in-vitro, peripheral blood mononuclear cells released stlr2 and stlr4 by exposure to microbial ligands. during experimental human endotoxemia, plasma concentrations peaked after 2 hours (stlr4) and 4 hours (stlr2). stlr4 did not correlate with cytokines, but stlr2 correlated positively with tnfα (r(s) = 0.80, p < 0.05), il-6 (r(s) = 0.65, p < 0.05), and il-1ra (r(s) = 0.57, p = 0.06), and negatively with il-10 (r(s) = -0.58, p = 0.06), respectively. stlr4 had a similar area under the roc curve [auc] for differentiating infectious and non-infectious inflammation compared to crp: 0.72 (95% ci 0.66-0.79) versus 0.74 (95% ci 0.69-0.80) [p = 0.80], while stlr2 had a lower auc: 0.60 (95% ci 0.54-0.66) [p = 0.0004]. crp differentiated bacterial infections better from viral infections than stlr2 and stlr4: auc 0.94 (95% ci 0.90-0.96) versus 0.58 (95% ci 0.51-0.64) and 0.75 (95% ci 0.70-0.80), respectively [p < 0.0001 for both]. conclusions: stlrs are released into the circulation, and suggest the possibility to use stlrs as diagnostic tool in inflammatory conditions. toll-like receptors (tlrs) are germline-encoded receptors that recognize microbial structures called pathogenassociated molecular patterns (pamps), either alone or in combination with co-receptors. besides regulating innate and adaptive immune responses, tlr signaling plays an important role in the pathogenesis of several inflammatory diseases, and tight regulation is crucial in order to prevent hyperinflammation [1, 2] . immune signaling is regulated at multiple levels, and the release of extracellular domains of immune receptors such as cytokine receptors represents an important regulatory mechanisms [3] . similar negative regulation accounts for modulation of tlr function [1, 2] , and soluble forms of tlr2 and tlr4 have been recently described [4, 5] . the release of these soluble proteins increases upon cell activation and they exert inhibitory activity on tlr signaling [4, 5] . soluble forms of tlrs have been detected in pleural fluid, amniotic fluid, saliva, breast milk and plasma [4, [6] [7] [8] [9] [10] . timely knowledge of the etiology of inflammatory conditions is crucial. not only does it facilitate appropriate treatment, but also unnecessary interventions may be avoided. in light of the critical shortage of new antibiotics, reduction in antibiotic prescription is warranted. the concept of measuring soluble pattern recognition receptors (prrs) for the diagnosis of infections has been previously proposed for the tlr4-coreceptor cd14 [11, 12] . however, analysis of soluble tlrs have up till now only been used in the diagnostic workup of pleural effusion and intra-amniotic infections [8] [9] [10] 13] . the aim of the present study was to gain more insight into the release of stlr2 and stlr4 in-vitro and to investigate the kinetics of monocytic tlr2 and tlr4 expression and plasma levels of their soluble counterparts during experimental endotoxemia (intravenous lps administration in healthy volunteers). furthermore, we hypothesized that stlr2 and stlr4, being soluble forms of receptors that play pivotal roles in pathogen recognition by cells of the innate immune system, are differentially released during various inflammatory diseases, with higher levels in inflammatory conditions of infectious origin. as such, we evaluated the ability of stlr2 and stlr4 levels to discriminate between infectious and noninfectious inflammatory pathologies. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of healthy individuals after informed consent. briefly, pbmcs were isolated by density gradient centrifugation using ficoll-paque plus (ge healthcare, zeist, the netherlands) and collecting the white interphase. cells were washed twice in cold pbs and concentrations were adjusted to 5 × 10 6 cells/ml in rpmi-1640, supplemented 2 mm l-glutamine, 1 mm pyruvate and 50 μg/ml gentamicin (gibco invitrogen, carlsbad, ca). mononuclear cells (5 × 10 5 ) in a 100-μl volume were added to round-bottom 96-well plates (greiner, nurnberg, germany) and incubated with either 100 μl of culture medium (negative control), or lps from e. coli o55:b5 (10 μg/ml; sigma-aldrich, st louis, mo), pam3cys (10 μg/ml) or heat-killed e. coli atcc 35218 (10 7 micro-organisms/ml). after 24 hour incubation at 37°c, the supernatants were stored at -80°c until measurement of stlr2, stlr4 and il-6. this study was part of a larger endotoxin trial registered at the clinicaltrials.gov registry under the number nct00783068 which was approved by the local ethics committee of the radboud university medical center [14] . the 11 healthy male volunteers included in the present study provided written informed consent. briefly, subjects were prehydrated during 1 h before lps administration by infusion of 1.5 l 2.5% glucose/0.45% saline solution, followed by 150 ml/h starting at the time of lps administration until 6 h afterwards and 75 ml/h until the end of the experiment. us reference escherichia coli endotoxin (lps derived from e. coli o:113; clinical center reference endotoxin, national institutes of health, bethesda, md) was administered as an intravenous bolus (2 ng/kg). edta anticoagulated blood was collected from an arterial line. in order to determine expression of tlr2 and tlr4, blood was collected in edta-containing vacutainers. the following directly conjugated mouse anti-human antibodies were used: tlr2: cd282 pe (mouse igg2a, tlr 2.1 clone, ebioscience, san diego, ca), tlr4: cd284 pe-cy7 (mouse igg2a, hta125 clone ebioscience, san diego, ca), and cd14 ecd (mouse igg2a, rmo52 clone immunotech, beckman coulter, marseille, france). isotype and fluorochrome matched controls from beckman coulter were used. cell buffer solution was used containing 0.5% bovine serum albumin in phosphate buffered saline and 0.1% sodium azide. rabbit serum (invitrogen, carlsbad, ca) for blocking was diluted to 20% with cell buffer solution. red blood cell lysis was performed using 0.075 m ammonium chloride (nh4cl, ph7.4), freshly prepared. 1 ml of blood was mixed with 20 ml of nh4cl lysing solution and was left at room temperature for 10 minutes. after centrifuging for 5 minutes at 500 g the supernatant was discarded. the cell pellet was resuspended in 50 ml of pbs and centrifuged again. after this washing step the cell pellet was resuspended in 0.5 ml cell buffer solution. 0.1 ml of this cell suspension was mixed with 0.1 ml 20% rabbit serum and left at room temperature for 10 min. subsequently, cells were incubated with the appropriate antibody concentration mixture for 15 min in the dark at room temperature. after washing, samples were resuspended in 0.5 ml cell buffer solution and analyzed on a beckman coulter fc500 flow cytometer (beckman coulter, miami, fl). monocytes were gated in a side scatter vs. cd14 plot. fluorochrome matched isotype controls, non-stained samples, as well as cells incubated with only a secondary antibody, were used to set the photo multiplier detectors. the tlr2 and tlr4 expression was analyzed within cd14 + monocytes. plasma concentrations of stlr2 and stlr4 were measured in healthy controls, and two groups of patients and compared to that of the most used inflammatory biomarker, c-reactive protein (crp). edta anticoagulated blood from the various groups of patients was prospectively collected during planned laboratory blood assessment for clinical purposes, or was available from previous clinical studies, as indicated. plasma was obtained by centrifugation for 10 minutes at 2000 g. the study has been carried out in the netherlands in accordance with the applicable rules concerning the review of research ethics committees and informed consent. inclusion and exclusion criteria for the inflammatory disorders are shown in table 1 . table 2 shows the demographic characteristics of the healthy controls and the patients included. the first group consisted of patients with infectious diseases in whom plasma was obtained ≤24 hours after presentation: viral lower respiratory tract infections (lrti) (n = 25; table 3 ), measles [15] (n = 43), infectious mononucleosis caused by either epstein-barr virus (ebv) [16] or cytomegalovirus (cmv) infection (n = 16), bacterial and viral respiratory co-infections (n = 20; table 3 ), bacterial sepsis [17] , stratified into sepsis, severe sepsis and septic shock [18] (n = 156), and candidemia [19] (n = 26). the second group comprised of patients with non-infectious inflammation: crohn's disease (n = 15), gout (n = 36), autoinflammatory syndromes (n = 15), rheumatoid arthritis (n = 20), and pancreatitis [20] (n = 22). patients with autoinflammatory syndromes consisted mainly of patients with well-known, genetically confirmed autoinflammatory diseases, like hyperimmunoglobulin-d syndrome, familial mediterranean fever, muckle-wells syndrome and tumor necrosis factor receptor-1 associated syndrome (traps). pancreatitis was of biliary origin in 45%, none developed necrosis and all had negative blood cultures. samples were taken ≤24 hours after presentation, except for crohn's disease and reumatoid arthritis. those were taken during an exacerbation of the disease ( table 1 ). the disease activity of rheumatoid arthritis was measured with disease activity score (das) in 28 joints (das28) [21] . the definition of active rheumatoid arthritis is a das28 > 3.2. the mean das28 was 4.49 (range 3.40-6.40, of whom 7 patients had a score >5.1, indicating high disease activity). cytokine and stlr2 and stlr4 measurement stlr2 and stlr4 concentrations were measured by a commercial elisa kits (uscn life science, inc., wuhan, china) with a lower limit of detection of 0.312 ng/ml and 0.156 ng/ml, respectively. a commercial elisa (sanquin, amsterdam, the netherlands) with a minimal detection level of 1.56 pg/ml was used for the determination of interleukin (il)-6 concentrations in supernatants. il-6, tumor necrosis factor (tnf)α, il-1ra and il-10 concentrations in plasma were determined using a luminex assay (bio-plex cytokine assay, biorad, hercules, ca), with a sensitivity of 6 pg/ml, 20 pg/ml, 72 pg/ml and 6 pg/ml, respectively. crp concentrations were measured with a commercial elisa, with a lower detection limit of 5 mg/l (ibl international, hamburg, germany). samples were diluted when appropriate. cytokine and stlr concentrations in the in-vitro and endotoxemia experiments are expressed as mean ± sem. for the assessment of correlations spearman correlation coefficient was calculated. the mann-whitney u-test was used for the comparison of two groups in the biomarker study. additionally, to correct for the potential influence of age and sex on the biomarker concentrations, we performed multiple linear regression analysis (forced entry method) with the biomarker of interest as dependent variable and age, sex and the assigned group (infection/no infection or bacterial/viral infection) as independent variable. receiver operating characteristics (roc) curve statistics were applied to calculate sensitivity and specificity. in order to determine the diagnostic accuracy of the combination of biomarkers, logistic regression analysis was used to estimate the predicted probabilities, which were subsequently used for the generation of a roc curve. the method described by delong was used for comparing areas under roc curves (auc) [22] . all tests were two-sided, and p < 0.05 was considered statistically significant. data were analyzed using graph pad prism 5 (graphpad software, la jolla, ca) and medcalc version 11.3.1.0 (medcalc software, mariakerke, belgium). in-vitro release of soluble tlrs by human pbmcs stlr2 and stlr4 were below the detection limit (6 and 8 ng/ml, respectively) in the supernatants of unstimulated pbmcs. after stimulation with lps, pam3cys or heat-killed e. coli, significant amounts of il-6, stlr2 and stlr4 were released by pbmcs in the supernatant, although shedding of stlrs was not confined to stimulation of its corresponding cell surface receptor (figure 1 ). stlr2 and stlr4 release during human endotoxemia stlr2 and stlr4 plasma concentrations displayed a distinct pattern after lps infusion ( figure 2 ). before lps administration, stlr2 and stlr4 levels were undetectable or low in all volunteers. stlr4, tnfα, il-6 and il-10 concentrations increased after lps infusion and reached a peak concentration at 2 hours lps infusion; stlr2 and il-1ra peaked after 4 hours. the mean peak values (± sem) were 357 ± 94 ng/ml for stlr2, 10.5 ± 2.3 ng/ml for stlr4, 836 ± 288 pg/ml for tnfα, 926 ± 145 pg/ml for il-6, 90 ± 17 pg/ml for il-10, and 26081 ± 2213 pg/ml for il-1ra, respectively. the auc of stlr4 showed no correlation with the aucs of stlr2 (r s 0.03, p = 0.94), il-6 (r s -0.07, p = 0.83), tnfα (r s -0.07, p = 0.80), il-10 (r s 0.22, p = 0.52), and il-1ra (r s 0.14, p = 0.69). however, stlr2 showed a strong positive correlation with tnfα (r s 0.80, p = 0.003), il-6 (r s 0.65, p = 0.03). stlr2 showed a trend towards a positive correlation with il-1ra (r s 0.57, p = 0.06), and a negative with il-10 (r s -0.58, p = 0.06), respectively. cell-surface expression of tlr2 and tlr4 on monocytes varied extensively among the subjects without a clear pattern and did not correlate with stlr2 and stlr4 plasma levels ( figure 2 ). figure 3 shows the circulating concentrations of crp, stlr2 and stlr4 in various infectious and non-infections inflammatory diseases. 394 patients and 29 healthy volunteers were included. for determination of crp, samples of 351 patients and 11 healthy volunteers were analyzed. crp, stlr2 and stlr4 circulating concentrations were significantly higher in patients with infection compared with patients with non-infectious inflammation (figure 4 , groups a and b). after correction for age and sex, the presence of an infection was still positively associated with crp, stlr2 and stlr4: unstandardized coefficients 85 (95% ci 64-106, p < 0.001), 23 (95% ci 12-34, p < 0.001), and 6.2 (95% ci 4.2-8.2, p < 0.001), respectively. age, but not sex, was also positively associated with the three biomarker concentrations. furthermore, compared with patients suffering from viral infections, patients with bacterial infections displayed higher concentrations of crp and stlr4, but not stlr2 (figure 4 , groups c and d). multivariate analysis with correction for age and sex showed all three biomarkers to be independently associated with the presence of a bacterial infection. unstandardized coefficients for crp, stlr2 and stlr4 were 113 (95% ci 79-147, p < 0.001), 19 (95% ci 0.2-39, p = 0.04) and 6.2 (95% ci 2.2-9.0, p = 0.01), respectively. neither sex, nor age proved to influence the concentrations of crp, sltr2 and stlr4. in the patients with bacterial or fungal sepsis, the presence of a malignancy (n = 27; without malignancy n = 155) was associated with higher concentrations of crp (152 vs 127 mg/l, p = 0.07), stlr2 (62 vs 23 ng/ml, p = 0.001) and stlr4 (7.2 vs 4.3 ng/ml, p = 0.15), although this only reached statistical significance for stlr2. the discriminative value of stlr4 levels to identify infectious versus non-infectious inflammation was similar compared with crp: auc of 0.72 (95% ci 0.66-0.79) and 0.74 (95-% ci 0.69-0.80), p = 0.80 (table 4, figure 5 ). stlr2 performed worse: auc 0.60 (95% ci 0.54-0.66), p = 0.0004 compared to the auc of crp. at a specificity of 95%, circulating concentrations of stlr2 above 47 ng/ml, stlr4 above 18.9 ng/ml, and crp above 150 mg/l had a sensitivity of 32%, 16% and 28%, respectively, to identify an infectious process. combination of biomarkers showed no improvement of the auc (table 4) . crp levels showed good value to discriminate between bacterial and viral infections with an auc of 0.94 (95% ci 0.90-0.96). stlr4 levels displayed a significantly lower auc compared with crp: 0.75 (95% ci 0.70-0.82), p < 0.0001. stlr2 was a poor discriminator between patients with a bacterial or viral infection (table 4, figure 5 ). panel analysis with two biomarkers was comparable to the performance of crp alone. the cut-off values for the figure 1 release of il-6, stlr2 and stlr4 after stimulation for 24 hours of peripheral blood mononuclear cells with either medium (control) several microbial stimuli. data are expressed as means ± sem (n = 6). the concentrations of il-6, stlr2 and stlr4 after incubation with medium are below the detection limit. ten oever et al. bmc immunology 2014, 15:55 discrimination of bacterial infections from viral infections with a specificity of 95% were for crp 67 mg/l (sensitivity 82%), for stlr2 79 ng/ml (sensitivity 23%) and for stlr4 10.6 ng/ml (sensitivity 28%). in the present study, we demonstrate that stlr2 and stlr4 are released in-vitro and in-vivo after challenge with microbial ligands such as lps. significantly elevated plasma concentrations of stlrs are present in the circulation during experimental human endotoxemia, and high circulating concentrations of stlr4 are found in patients with infections compared to patients with non-infectious inflammation, as well as in patients with bacterial infections compared with viral infections. however, the value of stlr2 and stlr4 as additional diagnostic biomarkers is low as both new markers do not surpass crp in accuracy. in addition to the release of stlr2 and stlr4 from stimulated immune cells [4, 5] , constitutive release of stlrs has been demonstrated in various biological fluids such as saliva, breast milk, and amniotic fluid [10, 23] . in plasma, stlr2 represented by several polypeptides, has been found by others [4] , although the concentrations are low. to avoid both harmful or insufficient inflammatory responses, inhibition and activation of the immune system needs to be properly balanced. various negative regulators of tlrs have been described [1] of which stlr2 and stlr4 constitute an important first-line negative regulatory mechanism [4] [5] [6] [23] [24] [25] . stlr2 either interferes with cd14-mediated triggering of membranebound tlr2, dimerizes with tlr2 on the cell surface, or competes with cellular tlr2 for microbial ligands [4] . the complex formed by stlr4 and md-2 probably blocks the interaction between membrane-bound tlr4 and its ligand [25] . the rapid elevation of stlr2 and stlr4 in plasma upon lps administrations, similar to that of pro-inflammatory cytokines, indicates that this feedback mechanism is rapidly activated. consistent with our in-vitro data, the release of stlrs in to the circulation demonstrates that immune modulation mediated by tlrs is not limited to the stimulation of the corresponding receptor on the cell membrane of immune cells. since both stlr2 and stlr4 dampen inflammation by disrupting tlr-mediated pro-inflammatory responses [4] [5] [6] [23] [24] [25] , it might be possible that the counter regulatory mechanisms mediated by stlrs extend to interference with endogenous tlr ligands. although the kinetics of stlr2 and stlr4 concentrations parallel those of anti-inflammatory cytokines, quantitatively they appear to be differentially regulated. plasma stlr4 levels did not show any correlation with both il-10 and il-1ra and for stlr2, a negative correlation with il-10 was found. interestingly, while in-vitro release of stlr2 and stlr4 by immune cells is comparable, their in-vivo concentrations differ strongly, with much higher concentration in the circulation of stlr2: this suggests a much more rapid clearance of stlr4 from circulation. this may imply that these anti-inflammatory mechanisms are regulated at a different level and are potential complementary strategies to reduce inflammation. in recent years, an important role for tlr signaling has been discovered in oncogenesis, particularly in inflammation-driven tumors [26] . although the relationship between cell-bound tlr2 and its release as a soluble form is not clear-cut, the observed higher concentrations of stlr2 in the (small) group of patients with an underlying malignancy may reflect the increased expression of tlr2 as seen in some forms of cancer [26] . alternatively spliced tlr4 mrna encodes the soluble form of tlr4 [5] . as such, we did not expect a correlation between the membrane expression of tlr4 and circulating stlr4. on the contrary, stlr2 results from posttranslational processing: endocytosis of cell surface receptor is followed by conversion into stlr2 intracellularly [4] . in previous monocyte stimulation experiments, membrane-bound tlr2 correlated negatively with supernatant stlr2 [4] . we did not observe the downregulation of cell surface tlr expression on monocytes of individuals during endotoxemia. possible explanations for this lack of correlation are that (1) monocytes are detected only in very low numbers at 2 hours after lps injection [27] and this subpopulation may well have a different tlr expression than more active monocytes that have marginated at this time-point; (2) we only examined the expression of tlrs on monocytes (cd14 + ), however, other circulating cell subsets such as neutrophils or platelets also express tlr2 [28, 29] , all potentially contributing to the plasma concentrations of stlrs; (3) the soluble receptors are derived from an intracellular pool, not directly from the cell surface [4] ; and finally (4) besides being shed, membrane bound tlr is influenced by tlr trafficking between intracellular compartments and the cell membrane [30] . an important aspect of this study is the possibility to use soluble tlrs as diagnostic markers. rapid and reliable differentiation of non-infectious inflammatory disorders from infections, and the classification of infections according to their microbiological etiology is essential for optimal treatment of these conditions. so far, only a small number of studies have been published on stlrs as diagnostic biomarkers. a few studies from the same group reported that intrauterine infections in pregnant women are characterized by elevated levels of stlr1, stlr2, stlr6 and stlr4 in the amniotic fluid [9, 10, 13] , supporting the concept of stlr release during infections. we assessed the value of stlr2 and stlr4 levels to discriminate between several inflammatory conditions. stlr2 and stlr4 were elevated in response to inflammatory insults and particularly stlr4 showed a good specificity to discriminate between an infection and a non-infectious inflammatory conditions such as gout, crohn's disease, rheumatoid arthritis or autoinflammatory syndromes. moreover, stlr4 concentrations show a high specificity for discriminating between bacterial and viral infections using high cut-off values, but sensitivity was low. we have to mention however that the overall discriminative value of stlr levels was not superior to that of crp in the relatively small group of patients assessed in this study future larger validation studies should demonstrate the overall value of stlr2 and stlr4 levels for the diagnosis of infections and autoinflammatory diseases in relation to that of classic inflammatory markers. furthermore, besides stlrs, other soluble pattern recognition receptors such as the soluble mannose receptor that are shed during cell stimulation with β-glucans are also interesting candidates for new and potentially more specific diagnostic biomarkers [31] . our study also has limitations. firstly, it included a relatively limited number of clinical conditions, and it is not possible to extrapolate our results to the entire panel of infectious or non-infectious inflammatory diseases. secondly, we studied groups of inflammatory conditions as a whole, rather than focusing on correlation with other clinical information or outcome. the present study is an important initial proof-ofprinciple report on the role of stlr2 and stlr4 during a broad panel of human infections and autoinflammatory diseases. shedding of stlr2 and stlr4 is not confined to stimulation of its corresponding cell surface receptor, but it is a broader effect upon stimulation of innate immune cells through pattern recognition receptors. we report the significant increase of stlr2 and stlr4 both in experimental models of human endotoxemia, as well as in the circulation of patients with infections. this suggests an important role of soluble tlrs in the modulation of inflammation during infections and the potential to use these tests as diagnostic markers. therefore, larger validation studies in larger patient cohorts are warranted in order to be able to draw definitive conclusions regarding the diagnostic usefulness of stlr2 and stlr4 in human inflammatory diseases. negative regulation of toll-like receptor-mediated immune responses toll-like receptors in the pathogenesis of human disease soluble cytokine receptors soluble forms of toll-like receptor (tlr)2 capable of modulating tlr2 signaling are present in human plasma and breast milk cutting edge: naturally occurring soluble form of mouse toll-like receptor 4 inhibits lipopolysaccharide signaling soluble tlr2 is present in human amniotic fluid and modulates the intraamniotic inflammatory response to infection human parotid saliva contains soluble toll-like receptor (tlr) 2 and modulates tlr2-mediated interleukin-8 production by monocytic cells expression of soluble toll-like receptors in pleural effusions soluble toll-like receptor 1 family members in the amniotic fluid of women with preterm prelabor rupture of the membranes amniotic fluid soluble toll-like receptor 4 in pregnancies complicated by preterm prelabor rupture of the membranes lipopolysaccharide binding protein is a potential marker for invasive bacterial infections in children evaluation of potential biomarkers for the discrimination of bacterial and viral infections amniotic fluid soluble toll-like receptor 2 in pregnancies complicated by preterm prelabor rupture of membranes differential ex vivo and in vivo endotoxin tolerance kinetics following human endotoxemia characterization of the acute inflammatory response in measles infection il-18 serum concentration is markedly elevated in acute ebv infection and can serve as a marker for disease severity procalcitonin as an early indicator of outcome in sepsis: a prospective observational study sccm/esicm/ accp/ats/sis international sepsis definitions conference cytokine gene polymorphisms and the outcome of invasive candidiasis: a prospective cohort study changes in adaptive and innate immunity in patients with acute pancreatitis and systemic inflammatory response syndrome modified disease activity scores that include twenty-eight-joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach soluble forms of toll-like receptor 4 are present in human saliva and modulate tumour necrosis factor-alpha secretion by macrophage-like cells soluble tlr2 reduces inflammation without compromising bacterial clearance by disrupting tlr2 triggering interaction of soluble form of recombinant extracellular tlr4 domain with md-2 enables lipopolysaccharide binding and attenuates tlr4-mediated signaling stat3-driven upregulation of tlr2 promotes gastric tumorigenesis independent of tumor inflammation endotoxaemia modulates toll-like receptors on leucocytes in humans evidence of toll-like receptor molecules on human platelets toll-like receptors stimulate human neutrophil function localisation and trafficking of toll-like receptors: an important mode of regulation martinez-pomares l: fungal recognition enhances mannose receptor shedding through dectin-1 engagement we gratefully acknowledge foekje stelma for the collection of patient samples. the authors declare that they have no competing interests. jto: included patients for the biomarker study, performed the elisas, performed the statistical analysis and drafted the first manuscript. mk and pp: performed the human endotoxemia study. km: performed the in-vitro experiments and the elisas. fvdv, as, as, ms, mj, tlj, lt, ps, pmw, pcw: included patients for the biomarker study. jwm, b-jk, ap: contributed to conception and design of the study. labj and mgn contributed to the conception and design of the study and the analysis and interpretation of the data. all the authors revised the manuscript critically for important intellectual content and approved the final version before submission. key: cord-0020036-06eoyi7n authors: tong, xiang; yu, dongdong; yu, li; chen, weiqian; wen, yanling; gu, pengcheng title: exploring the role of monocyte chemoattractant protein-1 in fibroblast-like synovial cells in rheumatoid arthritis date: 2021-08-11 journal: peerj doi: 10.7717/peerj.11973 sha: 49889c231da4a56a39fd7f00e55932cc0c9544b7 doc_id: 20036 cord_uid: 06eoyi7n background: rheumatoid arthritis (ra) is a chronic systemic inflammatory disease with persistent synovitis. in the present study, the impact of monocyte chemoattractant protein-1 (mcp-1) was explored to determine methods for the diagnosis and treatment of ra. methods: first, fibroblast-like synoviocytes (flss) were obtained from a collagen-induced rat ra model. next, mcp-1-overexpression plasmid and small interfering rna were transfected into human and rat flss. cell counting kit-8 (cck-8), transwell migration and flow cytometry assays were used to analyze cell proliferation, migration and apoptosis of flss following mcp-1 transfections, respectively. furthermore, western blotting was used to analyze the expression levels of p-p38, p-pi3k, pi3k, cd31, vegf, tnf-α and il-β in flss following mcp-1 transfection. in addition, reverse transcription-quantitative pcr and elisas were used to analyze the expression levels of c-reactive protein (crp), estrogen receptor, mcp-1 and pentraxin-3 in patients with clinical ra, followed by correlation analysis of clinical data. finally, expression validation, diagnostic and protein-protein interaction (ppi) network analysis of mcp-1 were performed. results: mcp-1 promoted fls proliferation and migration, and affected the apoptosis of flss. in addition, the expression levels of p-p38, p-pi3k, pi3k, cd31, vegf, tnf-α and il-β were also affected by mcp-1. in patients with clinical ra, the expression level of mcp-1 was increased. moreover, crp expression level was significantly up-regulated in ra. clinically, mcp-1 was strongly correlated with tender joint count, swollen joint count, visual analog scale for general health and disease activity score 28 (das28)-mcp-1, and was moderately correlated with das28 and das28-crp. ppi analysis showed that mcp-1 mainly interacted with other inflammatory cytokines. conclusion: in conclusion, mcp-1 may play a significant regulatory role in ra, and could be used as a measurement index of clinical ra activity. the ra rat model was immunologically induced by bovine type ii collagen. forty-five rats were purchased from zhejiang university, hangzhou, china. male sprague-dawley rats (age: 8 weeks) were feed adaptively for 1 week. next, type ii collagen was solubilized in 0.1 m acetic acid to a final concentration of 2 mg/ml and placed in a refrigerator at 4 • c overnight. the next day, type ii collagen was mixed with an equal volume of incomplete freund's adjuvant and stirred on ice until completely fused as an antigen emulsion. rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg). no animals exhibited signs of peritonitis following the administration of 10% chloral hydrate. rats were then subcutaneously inoculated with 0.3 ml antigen emulsion on days 0 and 7. the rats in the normal control group were injected with an equal volume of normal saline. first, rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (300 mg/kg) and euthanatized by cervical vertebra dislocation before experiments. no animals exhibited signs of peritonitis following the administration of 10% chloral hydrate. furthermore, no pain, suffering, disease or other symptoms occurred during the process of euthanasia. next, the synovial tissue was collected and washed 3-5 times on ice with sterilized pbs. third, the synovial tissue was sliced into one mm 3 thick slices and placed into a petri dish containing 0.2% type ii collagenase and incubated at 37 • c. next, the supernatant was collected every 60 min and centrifuged for 5 min (1000 rpm/min) to collect the cell pellets. subsequently, cells were filtered through the 200-mesh stainless steel filter and inoculated in a culture flask at 37 • c. finally, when the cells had reached 90% fusion, the cell passage was carried out. the cells passaged to the second generation were used for subsequent experiments. vimentin, a marker of flss (zhang et al., 2018) , was used for fls identification. in the culture plate, the slides containing the cells were washed with pbs thrice (3 min/time), fixed with 4% paraformaldehyde for 15 min and washed with pbs. the slides were then permeated with 0.5% triton x-100 at room temperature for 20 min. slides were washed with pbs thrice (3 min/time) and blocked with goat serum at room temperature for 30 min. the slides were incubated with diluted primary antibody (vimentin antibody, bs-0756r, bioss) and incubated overnight at 4 • c in a humid box. the slides were washed with pbs thrice (3 min/time) and incubated with the diluted secondary antibody at 37 • c for 1 h. then, the slides were washed with pbs thrice (3 min/time). finally, 3, 3 -diaminobenzidine (dab) color processing and observation under a microscope were performed to capture the image. the cytoplasm of flss was brownish yellow due to the expression of vimentin. all experimental procedures were approved by the animal ethics committee of the first affiliated hospital of the college of medicine, zhejiang university (approval no.20201172) and carried out accordance with the zhejiang province experimental animal management regulations. the human flss (purchased from shanghai xinyu biotechnology co., ltd) and rat flss were cultured in complete medium in a thermostatic incubator at 37 • c with 5% co 2 . the human mcp-1 and rat mcp-1-overexpression plasmids [constructed by the pcdna3.1 (+) vector] were purchased from beijing bomade genetic technology co., ltd. in addition, chemosynthetic human sirna and rat sirna were obtained from huzhou hippo biotechnology co., ltd. empty vector and scrambled sirna were used as the normal controls for overexpression and sirna transfection, respectively. previous studies have demonstrated that mcp-1 could enhance the growth rate through a transfection experiment (kuroda et al., 2005; zhang et al., 2015) . in the present study, human and rat flss were transfected with mcp-1-overexpression plasmid and sirna for further analysis. one day before transfection, 5.0−8.0 × 10 4 cells were inoculated with 2 ml serum-free medium into a 6-well plate. transfection was performed when cell confluence reached 70-90%. first, 250 µl serum-free medium and 0.8 µg dna or sirna (final concentration, 100 nm) were mixed. next, 250 µl serum-free medium and 2 µl llipofectamine r 3000 (invitrogen; thermo fisher scientific, inc.) were also mixed. these two solutions were incubated at room temperature for 5 min. subsequently, the two mixtures (dna or sirna+ llipofectamine 3000) were mixed and incubated at room temperature for 20 min. the normal medium in the 6-well plate was replaced with 1.5 ml medium. a total of 500 µl of dna/ lipofectamine 3000 or sirna/ lipofectamine 3000 mixtures were added dropwise into the 6-well plate and shaken gently. after 4-6 h, serum-free medium was replaced with complete medium. the cell transfection efficiency was detected after 48 h. in the present study, reverse transcription-quantitative pcr (rt-qpcr) and immunofluorescence analysis were used to analyze the mcp-1 transfection efficiency at the mrna and protein level, respectively. cell samples from normal control, mcp-1overexpression and mcp-1-silencing groups were obtained for rt-qpcr. total rna was extracted using a trizol r kit (cat. no. 10296028; invitrogen; thermo fisher scientific, inc.). nucleic acid concentration analyzer was used to detect the concentration and purity of total rna. superscript iii rt reverse transcription kit (cat. no. 11752050; thermo fisher scientific, inc.) was used to synthesize the cdna. sybr r qpcr mix (cat. no. 4472920; thermo fisher scientific, inc.abi-invitrogen) was used for the rt-qpcr. each experiment was repeated for three times. actin was used as the internal reference. the relative gene expression levels were calculated as fold-changes using the 2 − ct method (livak & schmittgen, 2001) . it is found that mcp-1 is expressed in the cytoplasm in the immunofluorescence analysis (kuroda et al., 2005; lazar et al., 2010; zhang et al., 2015) . therefore, immunofluorescence was used to detect the transfection efficiency in present study. for immunofluorescence analysis, before adding the fluorescent secondary antibody (flow-cyt/if, sheena selvey, diluted 200 times), the slides were soaked three times (3 min/time) with pbs tween-20 (pbst). diluted fluorescent secondary antibody was added to the slides and incubated in a humid box at 37 • c for 1 h. slides were then washed with pbst for three times (3 min/time). next, dapi was added to the slides and incubated in the dark for 5 min for nuclear staining. the extra dapi was washed away with pbst for 4 times (5 min/time). the slides were sealed with a sealing solution containing anti-fluorescence quencher and observed under a fluorescence microscope at a magnification of 400 x. normal human and rat flss were used as the controls. the cell suspension was prepared and inoculated into the 96-well plate (100 µl per well; three replicate wells per condition) and cultured for 24 h to allow cells adhere to the wall. mcp-1-overexpression plasmid or sirna was transfected into cells and incubated in complete medium for 48 h. cck-8 solution (10 µl) was then added to the cells and incubated for 1 h. the absorbance was measured at 450 nm using a microplate reader. the absorbance value was directly proportional to the cell proliferative ability. prior to the experiment, the cells were starved in serum-free medium for 12 h. following digestion with trypsin, the cells were centrifuged for 3 min (1,000 rpm/min) to discard the supernatant. next, the cells were resuspended in pbs and centrifuged to discard the supernatant. the cells were resuspended in serum-free medium containing 0.1% bsa and counted. the serum-free medium containing 0.1% bsa was used to adjust the cell density to 1 × 10 5 cells/ml. complete medium (700 µl) and cell suspension (200 µl) were added to the 24-well plate and inside the transwell chamber, respectively. tweezers were used to carefully transfer the chamber to a 24-well plate, avoiding the formation of air bubbles. the 24-well plate was gently placed into the 37 • c cell incubator containing 5% co 2 for 24-48 h. after gently removing the medium from the upper chamber, the cells of the upper chamber were wiped removed with a pbs-soaked cotton swab. the bottom of the chamber was immersed in a 10% methanol solution to fix the cells for 30 s, and then transferred to pure water to wipe off the methanol solution. the bottom of the chamber was immersed in the crystal violet dye solution for 2 min and washed with pure water until the background was clear. the number of cells passing through the membrane was counted under a microscope at a magnification of 100 x. the cell culture fluid was collected in a suitable centrifuge tube for later use. the adherent cells were washed with pbs. trypsin without ethylenediaminetetraacetic acid was used to digest the cells. the previously collected cell culture fluid was used to disperse gently all the adherent cells. the cells were centrifuged for 5 min (1,000 rpm/min) to collect the cells and discard the supernatant. they were then resuspended with ∼1 ml pbs (pre-cooled at 4 • c) and centrifuged again to discard the supernatant. the mixture (4 ml 4x binding buffer and 12 ml deionized water) was used to resuspend the cells and adjust the cell concentration to 1-5 × 10 6 cells/ml. the cell suspension (100 µl) and annexin v-fitc (5 µl) was mixed in a 5-ml flow tube incubated at room temperature for 5 min in the dark. next, 10 µl of 20 µg/ml propidium iodide (pi) solution and 400 µl of pbs were added for flow cytometry analysis. in our previous study, the cell lysis method was used to detect the expression levels of mcp-1 and vegf by western blotting (tong et al., 2020) . similarly, in a study by du et al. (2016) the cells were lysed for western blotting to determine the expression levels of mcp-1 and vegf. in the present study, western blotting was used to detect the expression levels of mcp-1, phosphorylated (p)-p38 mitogen-activated protein kinase (p38), p-pi3k, pi3k, cd31, vegf, tnf-α and il-β. extracting solution of protein (cat. no. mdl91201; mdl) was used for protein extraction in the logarithmic growth phase cells. bicinchoninic acid protein concentration determination kit (cat. no. md913053; mdl) was used for the detection of protein concentration. sodium dodecyl sulfate polyacrylamide gel electrophoresis prefabricated adhesive kit (cat. no. md911919, mdl) was used for the isolated protein bands. the protein bands on the gel were transferred to a polyvinylidene fluoride membrane (iseq00010, emd, millipore) and incubated with the primary and subsequent secondary antibodies (md912565, mdl, diluted 2000 times). finally, the efficient chemiluminescence kit was used to visualize protein bands. bands from three separate western blotting experiments were analyzed by quantiscan software. actin was used as an internal control for protein detection. result was presented as target protein/internal reference, which was regarded as the relative expression of target protein. unpaired t test was used for statistical significance analysis. in the present study, the diagnostic criteria of ra were in accordance with the american college of rheumatology in 1987. the detailed inclusion criteria for patients with ra were as follows: (i) patients presented with morning stiffness for at ≥1 h (≥6 weeks); (ii) patients presented with swelling in ≥3 joints (≥6 weeks); (iii) patients presented with swelling of the wrist, palm, finger or proximal interphalangeal joint (≥6 weeks); (iv) patients presented with symmetrical joint swelling (≥6 weeks); (v) patients presented with typical radiological changes of ra in the hand; (vi) patients presented with rheumatoid nodules under the skin; and (vii) patients had a positive rheumatoid factor (titer, >1:32). patients presented with more than four of the above symptoms were diagnosed with typical ra. patients complicated by hypertension, coronary atherosclerotic heart disease, diabetes, active infection, connective tissue disease and pregnancy were excluded. according to the above inclusion and exclusion criteria, 13 patients with ra and 13 healthy individuals were enrolled in the study. during the rna extraction process, two inappropriate samples were discarded. the whole blood samples of these individuals were obtained for rt-qpcr. total rna was extracted using a trizol r kit (cat. no. 10296028; invitrogen; thermo fisher scientific, inc.). a total of 0.25 ml liquid sample was added with 0.75 ml lysate rls and then shaken vigorously for 30s, and incubated at 15−30 • c for 10min. 0.2 ml chloroform was added to each 0.75 ml rls, violently shaken for 15 s and placed at room temperature for 5 min. after centrifuged for 10 min (1000 rpm/min) at 4 • c, the upper colorless aqueous phase was transferred to a new ep tube. 1 times volume 70% ethanol was mixed and transferred to adsorption column ra. after centrifuging for 45s (12000 rpm/min), the waste solution was discard. 0.5 ml of protein-removing solution re was added and centrifuged for 45s (12000 rpm/min). the waste solution was discarded. 0.5 ml of rinsing solution rw was added and centrifuged for 45s (12000 rpm/min). the waste solution was discarded (repeat once). in order to remove the rinsing solution as much as possible, mixture was centrifuged again for 2 min (13000 rpm/min). finally, rnase free water was added for elution. nucleic acid concentration analyzer was used to detect the concentration and purity of total rna. superscript iii rt reverse transcription kit (cat. no. 11752050; thermo fisher scientific, inc.) was used to synthesize the cdna. sybr r qpcr mix (cat. no. 4472920; thermo fisher scientific, inc.abi-invitrogen) was used for the rt-qpcr. each experiment was repeated for three times. actin was used as the internal reference. the relative gene expression levels were calculated as fold-changes using the 2 − ct method (livak & schmittgen, 2001) . all primers used in the present study are shown in table 1 . all experimental procedures were approved by the clinical research ethics committee of the first affiliated hospital of the college of medicine of zhejiang university (approval no. 2020861). written informed consent was obtained from all patients. elisa kits were used to detect the content of estrogen receptor (er), pentraxin-3 (ptx3), mcp-1 and c-reactive protein (crp) in normal and ar serum samples, respectively. briefly, samples and enzyme-labeled reagents were added to each well and incubated at 37 • c for 60 min. the washing buffer was added to each well and discarded after being motionless for 30 s (5 thrice). color development reagent and stop solution were used for the dye (15 min) and stop reaction, respectively. finally, the optical density value of each hole was measured at a wave length of 450 nm. in the blood of ra and healthy individuals, tender joint count (tjc), swollen joint count (sjc), visual analog scale for gener al health (gh) and disease activity score 28 (das28) were calculated. the mcp-1 data was added to the das28 calculation formula as follows (liou et al., 2013; prevoo et al., 1995) : .39xln(mcp-1) +0.014x(gh). the r 4.0.2 package was used to analyze the correlation among the expression of tjc, sjc, gh and mcp-1 in the sample. shapiro-wilk test was used to verify whether each group of values was normally distributed (p > 0.05 means normally distributed). pearson correlation coefficient and spearman's correlation coefficient were used for normally and non-normally distributed data, respectively. expression validation, diagnostic and protein-protein interaction (ppi) network analysis of mcp-1 was performed using the gse103578 and gse128813 datasets, which were obtained from the geo database (edgar, domrachev & lash, 2002) . the search tool for the retrieval table 1 primer sequences used for the reverse transcription-quantitative pcr. actin (human)-f(internal reference) 5 -gacaggatgcagaaggagattact-3 actin ( of interacting genes/proteins (string) database (https://string-db.org/) was used to construct the ppi network. subsequently, cytoscape software (http://www.cytoscape.org) was used for the visualization of the ppi network. all statistical analysis was performed using graphpad prism (graphpad software, inc.). p < 0.05 was considered statistically significant. one-way anovas were used to analyze the rt-qpcr results. data are presented as the mean ± sd. vimentin was used to identify isolated rat flss, which was shown in fig. 1 . the mrna expression level of mcp-1 were analyzed following 48 h of transfection. following overexpression and silencing, the expression level of mcp-1 in both human and rat flss were significantly increase and decreased, respectively (figs. s1a and s1b). immunofluorescence was used to further validate the transfection efficiency of mcp-1 at the protein level. under a fluorescence microscope, the cell nucleus was marked with blue fluorescence. the mcp-1 protein was marked with green fluorescence. following the overexpression and silencing of mcp-1, the expression level of mcp-1 in human flss exhibited an increased and decreased trend, respectively, compared with normal human flss (figs. s1c and s1e). however, the trend was not obvious enough, which may be associated with the state of the cells. rat flss also showed a similar trend after mcp-1 transfection (figs. s1f and s1h). this indicated that the transfection was successful and could be used for subsequent experiments. the results of the cck-8 analysis showed that following mcp-1-overexpression, the cell viability of flss in humans and rats was enhanced, which indicate that mcp-1 promoted cell proliferation. in the mcp-1-silencing group, the viability of flss in humans and rats was weakened, which inhibited cell proliferation (fig. 2) . transwell migration assay was used to detect the migratory ability of flss following mcp-1 transfection. mcp-1-overexpression promoted the migratory ability of flss, as compared with the control group. nevertheless, the migratory ability of flss was weakened following mcp-1-silencing (figs. 3a-3f). flow cytometry was used to detect the effect of mcp-1 on the apoptosis of flss. the apoptotic ability of flss was weakened by mcp-1 overexpression. on the contrary, the apoptotic ability of flss was enhanced when mcp-1 was silenced (fig. 4) . quantitative analysis of the apoptotic rate of early apoptotic cells (q3 quadrant) showed that the apoptotic ability of mcp-1 overexpressing cells was weakened in flss (fig. s2 ). p-p38 is an important kinase involved in the regulation of the mapk signaling pathway (liu et al., 2019b; zhou et al., 2019) . pi3k and p-pi3k are important kinases involved in the regulation of the pi3k/akt/mtor signaling pathway (liu et al., 2019a) . tnf-α and il-β are typical pro-inflammatory cytokines (charrad et al., 2016) . in addition, cd31 and vegf are members of the immunoglobulin superfamily and vegf family, respectively (melinte et al., 2012) . western blotting was used to detect the expression levels of mcp-1, p-p38, p-pi3k, pi3k, cd31, vegf, tnf-α and il-β following mcp-1 transfection. in human flss, the overexpression of mcp-1 stimulated the expression of p-p38 and p-pi3k, pi3k, cd31, vegf, tnf-α and il-1β. the opposite phenomenon was observed in the mcp-1-silencing group (figs. 5 and 6 ). in the rat flss, following mcp-1 overexpression and silencing, the p-pi3k and pi3k proteins exhibited increased and decreased trends, respectively, albeit not significant. the expression level of other proteins was consistent with that in human flss (figs. 7 and 8) . to investigate the expression level of mcp-1 in clinical ra, 13 patients with ra and 13 healthy individuals were enrolled in the present study. the clinical information of these individuals is presented in table s1 . blood and serum samples of these individuals were collected for rt-qpcr and elisa analysis, respectively. in rt-qpcr verification, the mrna expression level of mcp-1 in patients with ra was increased, as compared with that in healthy individuals, which was consistent with previous studies (fig. 9a ) (koch et al., 1992; trzybulska et al., 2018) . crp, er, mcp-1 and ptx3 are commonly used indicators of patient infection. in patients with ra, the protein levels of crp and er was increased, as compared with that in healthy individuals (figs. 9b and 9c). however, the protein level of mcp-1 in patients with ra was not obvious change, as compared with that in healthy individuals (fig. 9d) . the protein level of ptx3 in patients with ra was decreased, as compared with that in healthy individuals (fig. 9e) . crp, er, mcp-1 and ptx3 may play a significant regulatory role in improving arthritis. the shapiro-wilk test showed that the data on tjc, sjc, gh and mcp-1, among others, in the ra samples were normally distributed. however, when the total sample was tested, the distribution was not normal. pearson's correlation analysis was performed to evaluate the association between mcp-1 expression and tjc, sjc, gh in patients with ra. the results showed that there was no correlation between the three indicators (tjc, sjc and gh) and mcp-1. the spearman's correlation coefficient was used for the total sample. the results showed that mcp-1 was strongly correlated with each score (fig. 10) . mcp-1 was strongly correlated with tjc, sjc, gh and das28-mcp-1 (figs. 10b, 10d, 10e and 10f) and moderately correlated with das28 and das28-crp (figs. 10a and 10c ). the expression verification results showed that compared with the control group, the expression level of mcp-1 in ra group had an increased tendency without significant difference (fig. s3a ). in the receiver operating characteristic (roc) analysis, the area under curve (auc) of mcp-1 was 0.611 (fig. s3b) . small sample size in dataset may account for the lack of significant differences and diagnostic value in results. in addition, in order to understand the interaction of mcp-1 with other proteins in ra. a ppi network was constructed (fig. s3c) . the results showed that mcp-1 mostly interacted with inflammatory cytokines. tnf-α and il-β. in patients with clinical ra, the expression levels of mcp-1 was higher than that of healthy individuals. ppi analysis showed that mcp-1 mainly interacted with other inflammatory cytokines (il4, il6, il10, il13, cxcl8, tnf). ra is an inflammatory disease, and mcp-1 is a major pro-inflammatory cytokines (he et al., 2006; scott, wolfe & huizinga, 2010) . the experimental results in this article suggested that mcp-1 may play an important role in the formation and development of ra. mcp-1 can promote the proliferation, migration and differentiation of flss (tong et al., 2020) . flss are mesenchymal cells in the synovial joint and play an important role in the pathogenesis of ra (liu et al., 2020) . mcp-1 is a potent chemokine involved in ra immune regulation and inflammatory processes (pavkova goldbergova et al., 2012) . recent -pedersen, 2001; pavkova goldbergova et al., 2012) . in addition, the ability of flss to secrete mcp-1 is increased in joint damage caused by bacterial infection (scian et al., 2011) . in the present study, the expression of mcp-1 was increased in the blood of patients with clinical ra, which is consistent with previous reports. mcp-1 is an important chemokine that regulates the migration and tissue infiltration of monocytes/macrophages (karimian et al., 2017; zhu et al., 2014) . previous studies have shown that mcp-1 can also stimulate the migration, invasion and proliferation of tumor cells (dutta et al., 2018; küper, beck & neuhofer, 2016) . in the present study, the overexpression of mcp-1 promoted the cell proliferation and migration and inhibited the apoptosis of flss. however, mcp-1-silencing reversed this effect. therefore, we hypothesized that mcp-1 could play an important role in the pathogenesis of ra by regulating flss. the expression level of p-p38 is up-regulated in osteoarthritis cartilage or isomycininduced chondrocytes, which suggested that p-p38 may play a role in the progression of osteoarthritis (ji et al., 2019; liu et al., 2019b) . we found that mcp-1 could regulate the expression of p-p38. therefore, it is speculated that mcp-1 may regulate articular cartilage or chondrocytes through p-p38, which could regulate the pathological mechanism of rheumatoid osteoarthritis. the expression levels of pi3k and p-pi3k are reduced when inflammation is inhibited (feng & qiu, 2018) . in addition, it is found that the inhibition of the pi3k/akt/mtor signaling pathway accelerates cell apoptosis and autophagy to reduce the development of ra (feng & qiu, 2018; liang, li & gong, 2020; liu et al., 2019a) . we found that mcp-1 could regulate the expression levels of pi3k and p-pi3k. therefore, it is speculated that mcp-1 might regulate pi3k/akt/mtor signaling pathway through pi3k, and thus played an important role in apoptosis and autophagy of flss. in addition, mcp-1 could regulate the inflammatory progression of ra by regulating pi3k and p-pi3k. however, the specific molecular mechanism remains to be further research. the expression levels of cd31 and vegf were increased in the invasion area of ra, which implies that cd31 and vegf may play an important role in the ra development (ishikaw et al., 2002; melinte et al., 2012) . we found that mcp-1 could regulate the expression levels of cd31 and vegf. therefore, we speculated that mcp-1 could play an important role in the formation and development of ra by regulating cd31 and vegf to affect the invasion of flss cells. certain pro-inflammatory cytokines, such as tnf-α and il-1, stimulate inflammation and degradation of bone and cartilage, which play a significant role in the pathogenesis of ra (alam, jantan & bukhari, 2017; mateen et al., 2016) . at present, anti-cytokines appear to be effective drug molecules for the treatment of ra (alam, jantan & bukhari, 2017; mateen et al., 2016) . we found that mcp-1 could regulate the expression of proinflammatory cytokines tnf-α and il-1. therefore, we speculated that mcp-1 could regulate the degree of inflammation in ra by regulating proinflammatory cytokines. in brief, it is suggested that mcp-1 may regulate the pathological mechanism of ra from multiple aspects by regulating these proteins. the deletion of the erα gene can inhibit the protective effect of e2 on synovitis and joint destruction (engdahl et al., 2014) . in addition, erβ activation also plays a role in the improvement of arthritis (yang et al., 2010) . the expression level of crp is elevated and regarded as a biomarker in ra (atzeni et al., 2017; wasserman, 2018) . in ra, the level of ptx3 has been found to be higher than that in the control group (balbaloglu & ozcan, 2020) . as compared traditional biomarkers, ptx3 has been found to be a sensitive non-invasive biomarker of clinical arthritis activity in ra (sharma et al., 2018) , indicating that er, crp and ptx3 may play a significant role in improving arthritis. in the present study, the expression level of crp was increased in patients with clinical ra, which was consistent with the findings of previous studies. however, er and ptx3 had a deviation, which requires additional larger samples for further study. sjc, tjc, gh and das28 are designated as the measurement indicators of clinical arthritis activity (prevoo et al., 1995) . it has been hypothesized that mcp-1 and das28-mcp-1 in the blood may be conducive to monitoring ra activity (liou et al., 2013) . in the present study, mcp-1 had a strong correlation with each score in the total sample. this suggested that the adapted mcp-1 is a helpful marker of ra clinical disease activity. the present study had certain limitations. first, the clinical sample size is too small, leading to a certain degree of error in elisa analysis. further research on a larger sample size is required. second, exogenous mcp-1 needs to be used for further functional validation experiments. third, the expression levels of migratory proteins, p38, akt, erk and c-jun in the mapk signaling pathway should be further analyzed. in addition, whether the addition of mcp-1 inhibitors would impede the changes observed in the present study needs to be clarified. mcp-1 promoted fls proliferation and migration and inhibited the apoptosis of flss. in addition, overexpression of mcp-1 also promoted the expression of p-p38, p-pi3k, pi3k, cd31, vegf, tnf-α and il-β. however, mcp-1-silencing reversed this effect. this suggests that mcp-1 promotes the progression of ra inflammation. in patients with clinical ra, the expression level of mcp-1 was increased. moreover, crp expression was significantly up-regulated compared with healthy individuals. clinically, mcp-1 was strongly correlated with tender joint count, swollen joint count, visual analog scale for general health and disease activity score 28 (das28)-mcp-1, and was moderately correlated with das28 and das28-crp. this further suggested that mcp-1 might play a significant regulatory role in ra, and could be used as a measurement index of clinical arthritis activity. the authors received no funding for this work. nucleus pulposus cells derived igf-1 and mcp-1 enhance osteoclastogenesis and vertebrae disruption in lumbar disc herniation. international journal of clinical and experimental pathology 7:8520-8531. induction of monocyte chemoattractant protein-1 by nicotine in pancreatic ductal adenocarcinoma cells: role of osteopontin activation of rxr by bexarotene inhibits inflammatory conditions in human rheumatoid arthritis fibroblast-like synoviocytes long non-coding rna thril mediates cell growth and inflammatory response of fibroblast-like synoviocytes by activating pi3k/akt signals in rheumatoid arthritis blood monocyte chemotactic protein-1 (mcp-1) and adapted disease activity score28-mcp-1: favorable indicators for rheumatoid arthritis activity mir-125 regulates pi3k/akt/mtor signaling pathway in rheumatoid arthritis rats via parp2 eosinophils attenuate arthritis by inducing m2 macrophage polarization via inhibiting the iκb/p38 mapk signaling pathway activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting sirna analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method understanding the role of cytokines in the pathogenesis of rheumatoid arthritis vegf and cd31 expression in arthritic synovium and cartilage of human knee joints dominant negative mcp-1 blocks human osteoclast differentiation modified disease activity scores that include twenty-eight-joint counts. development and validation in a prospective longitudinal study of patients with rheumatoid arthritis potential role of fibroblast-like synoviocytes in joint damage induced by brucella abortus infection through production and induction of matrix metalloproteinases acute phase reactant, pentraxin 3, as a novel marker for the diagnosis of rheumatoid arthritis eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update monocyte chemoattractant protein-1 promotes the proliferation, migration and differentiation potential of fibroblast-like synoviocytes via the pi3k/p38 cellular signaling pathway the effect of caveolin-1 knockdown on interleukin-1β-induced chemokine (c-c motif) ligand 2 expression in synovial fluid-derived fibroblast-like synoviocytes from patients with rheumatoid arthritis diagnosis and management of rheumatoid arthritis rheumatoid arthritis: common questions about diagnosis and management light microscopic characterization of the fibroblast-like synovial intimal cell (synoviocyte) endogenous estrogen regulation of inflammatory arthritis and cytokine expression in male mice, predominantly via estrogen receptor alpha lair-1 shedding from human fibroblast-like synoviocytes in rheumatoid arthritis following tnf-α stimulation sequential and timely transfection of hepatocyte growth factor and monocyte chemotactic protein-1 ameliorates hyperkinetic pulmonary artery hypertension in rabbits kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating nf-κb/mapk signaling and protecting chondrocytes the authors declare there are no competing interests. • xiang tong conceived and designed the experiments, performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.• dongdong yu performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.• li yu, weiqian chen and yanling wen analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.• pengcheng gu conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. the following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): all experimental procedures were approved by the clinical research ethics committee of the first affiliated hospital, college of medicine, zhejiang university (no.2020861). the following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):all experimental procedures were approved by the animal ethics committee of the first affiliated hospital, college of medicine, zhejiang university (no.20201172). the following information was supplied regarding data availability:raw data are available as supplemental files. supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.11973#supplemental-information. key: cord-0002486-p7duonkr authors: lin, ze-min; yang, xiao-qian; zhu, feng-hua; he, shi-jun; tang, wei; zuo, jian-ping title: artemisinin analogue sm934 attenuate collagen-induced arthritis by suppressing t follicular helper cells and t helper 17 cells date: 2016-11-29 journal: sci rep doi: 10.1038/srep38115 sha: fbf232152712b5e379797c6b0008bb7926001bb7 doc_id: 2486 cord_uid: p7duonkr sm934 is an artemisinin analogue with immunosuppressive properties and potent therapeutic activity against lupus-like diseases in autoimmune mice. in this report, the therapeutic efficacy and underlying mechanisms of sm934 on rheumatoid arthritis (ra) was investigated using collagen-induced arthritis (cia) in dba/1j mice. we demonstrated that sm934 treatment alleviate the severity of arthritis in cia mice with established manifestations. the therapeutic benefits were associated with ameliorated joint swelling and reduced extent of bone erosion and destruction. further, administration of sm934 diminished the development of t follicular helper (tfh) cells and th17 cells and suppressed the production of pathogenic antibodies, without altering the proportion of germinal center b cells. ex vivo, sm934 treatment inhibited the bovine type ii collagen (cii) induced proliferation and inflammatory cytokines secretion of cii -reactive t cells. in vitro, sm934 impeded the polarization of naïve cd4(+) t cells into tfh cells and the expression of its transcript factor bcl-6. moreover, sm934 decreased the il-21-producing cd4(+) t cells and dampened the il-21 downstream signaling through stat3. these finding offered the convincing evidence that artemisinin derivative might attenuate ra by simultaneously interfering with the generation of tfh cells and th17 cells as well as the subsequent antibody-mediated immune responses. artemisinin (qinghaosu) is a sesquiterpene lactone bearing a peroxy isolated from artemisia annua l 1,2 . artemisinin and its derivatives have been widely used in the world as first-line antimalarial drug for decades. the interaction between artemisinin and fe 2+ leads to the cleavage of peroxide bond in artemisinin, which result in the release of c-centered free radicals, the potent alkylating species then directly impair functions of malarial mitochondria [3] [4] [5] . in addition to the outstanding antimalarial activity, artemisinin and its derivatives possess significant immunosuppressive effects both in vitro and in vivo [6] [7] [8] [9] [10] [11] . we have previously reported that, among synthesized artemisinin derivatives with various structures, sm934, the novel water soluble artemisinin analogue exhibits promising therapeutic effect on multiple autoimmune diseases by suppressing the differentiation and expansion of pathologic t helper cells [12] [13] [14] and accumulation of plasma cells 15 . nevertheless, the impact of artemisinin analogues on t follicular helper (tfh) cells, the critical participator in germinal center (gc) formation, remains largely unclear. germinal centers (gcs) are specialized structures that develop within b cell follicles of secondary lymphoid tissues where antigen-specific b cells undergo somatic hypermutation and affinity maturation, and then differentiate into antibody-producing plasma cells and memory b cells [16] [17] [18] . as a recently identified cd4 + t cell subset, tfh cells, have been found to be present in gcs, provide selection signals and developmental cues for the proliferation, differentiation and class switching of antigen-selected high-affinity gc b cells [19] [20] [21] [22] . the phenotypic and functional features of tfh cells include expression of the chemokine receptor cxcr5, costimulatory molecules such as inducible t cell costimulatory (icos), programmed death 1 (pd-1), as well as the master transcription factor b-cell cll/lymphoma-6 (bcl-6) and the cytokine interleukin-21 (il-21) 19 . t helper 17 (th17) cells, are highly proinflammatory effector t cells that are characterized by the production of high amounts of il-17, il-21, and il-22. recent research indicated that th17 cells have greater capacity to provide cognate b cell help than t helper 1 (th1) cells, and support higher expansion of antigen specific b cells 23 . moreover, il-17 itself could drive b cells to undergo preferential isotype class switch recombination and favor spontaneous gc formation in mouse model of autoimmunity 24, 25 . il-21 signaling is important for gc persistence and function, b cell isotype switching and response to protein antigen. both of tfh and th17 cells are considered as the major source of il-21, conversely, il-21 facilitates tfh and th17 cells lineage commitment in an autocrine fashion 26, 27 . operating in the same way as il-6, il-21 signals through the signal transducer and activator of transcription 3 (stat3), in promoting or sustaining tfh and th17 development 26, 27 . rheumatoid arthritis (ra) is an autoimmune disease characterized by synovial inflammation, cartilage damage and bone destruction. persistent presence of circulating autoantibodies and dysregulated lymphocyte activation have been indicated to be associated with the pathogenesis of ra [28] [29] [30] . increased frequency of circulating tfh cells accompanied by higher levels of serum il-21 have been reported in patients with rheumatoid arthritis and systemic lupus erythematosus (sle) 31, 32 . accumulated reports indicated that tfh cells may account for ra pathogenesis by providing a fostering milieu for self-reactive b cells, and circulating tfh cells may serve as a biomarker of the pathogenesis and for evaluating the therapeutic responses of ra patient 33, 34 . the th17 population, have been shown to play a key role in orchestrating inflammatory response, contribute to the extracellular matrix destruction and bone resorption in ra, by means of inducing il-6 and matrix proteases in synoviocytes from ra patients 35, 36 . here we report the effect of sm934 on the collagen-induced arthritis (cia) in dba/1 mice, the widely used experimental model to investigate the pathogenesis and therapeutic agents of ra. we found that sm934 ameliorated the severity of arthritis in cia mice via inhibiting development of tfh and th17 cells as well as autoantibodies production. additionally, we demonstrated that sm934 could prohibit the il-21-mediated signaling pathway by preventing the activation of stat3. to investigate the effect of sm934 on arthritis, the cia model in dba/1j mice was used. cia mice with established rheumatoid arthritis manifestations such as erythema and edema in joints were orally administrated with saline, mtx or sm934 for 40 days (fig. 1a) . overall arthritis severity in cia mice was assessed by macroscopic clinical scoring. administration of sm934 (10 mg/kg) significantly attenuated the clinical signs of arthritis since 4 days after treatment, indicated by the reduction in the mean arthritis score, the therapeutic effect was comparable with the methotrexate (mtx, 1 mg/kg), a disease-modifying antirheumatic drug (dmard) treatment control used in comparison with arthritic group (fig. 1b) . representative photographs displaying morphological changes of left hind paws of mice from individual group on day 14 after treatment are shown in fig. 1c , macroscopic evidence of arthritis such as erythema or swelling was markedly observed in saline-treated cia mice compared with normal dba/1j mice, while sm934 and mtx treatment significantly ameliorated arthritis severity in cia mice. sm934 reverted bone destruction in cia mice. micro computed tomography (micro-ct) scan was performed to validate the efficiency of sm934 on bone erosion in cia mice. three-dimensional reconstruction of hind right paws by micro-ct demonstrated severe bone erosion within their periarticular bone of paws of the saline-treated cia mice, in marked contrast to normal dba/1j mice, as well as to sm934-treated mice ( fig. 2a) . moreover, as shown in fig. 2b , saline-treated cia mice showed a remarkable decrease of the bone volume/total volume (bone volume fraction, bv/tv), trabecular number (tb. n.), trabecular thickness (tb. th.), and increase of the trabecular spacing (tb. sp.) compared with healthy controls. sm934 treatment significantly prevented these changes in bv/tv, tb. th. and tb. sp. although the trabecular number was higher in sm934-treated group compared to the saline-treated group, there was no statistically significant difference. collagen-specific autoantigen is essential for cia pathogenesis 37, 38 , the effect of sm934 on serum antibodies (abs) production was assessed at the end of treatment. serum ab levels from individual mouse were examined using isotype-specific elisas. the serum levels of anti-cii total igg, igg1 and igg2a antibody decreased significantly after sm934 treatment, compare with saline-treated mice (fig. 3a) . moreover, we used mice immunized with sheep red blood cells (srbcs) and ovalbumin (ova) to confirm the impact of sm934 on the robust antibody response. sm934 treatment significantly lower serum level of srbc-specific and ova-specific antibody respectively, compared with saline-treated control mice ( supplementary fig. s1 ). since the autoreactive t cells plays a pivotal role in the pathogenesis of cia mice, cii-induced t cell proliferation was conducted using b cell-depleted splenocytes from different groups. cii-stimulated b cell-depleted splenocytes obtained from cia mice treated with sm934 exhibited less proliferation in comparison with the cells from saline-treated ones (fig. 3b ). the levels of inflammatory mediators were measured in supernatants of cii or medium cultured b cell-depleted splenocytes. results indicated that the amounts of il-6, il-17, il-21, il-4 and tnf-α markedly elevated in the cell cultures of saline-treated cia mice. sm934 treatment lowered the amount of il-6, il-17 and il-21 in cultured cells responded to cii stimulation, while the concentration of il-1β , il-4 and tnf-α did not changed much (fig. 3c ). taken together, the suppressive effects on cii-specific antibodies production and t cell response, suggesting that sm934 might participate in a t cell-dependent immune response in cia mice. fig. s2 ) changed notably after sm934 treatment. in order to further confirm the effect of sm934 on tfh and th17 cells in vivo, we introduced the srbc-and ova-immunized mice treated with sm934 for 9 day and 7 day respectively. as expected, in both experiments, sm934 treatment significantly diminished tfh population in comparison with saline-treated ones. sm934 treatment also significantly suppressed the th17 expansion in ova-immunized mice, whereas no statistical significance was observed between srbc-immunized mice treated with sm934 and with saline ( supplementary fig. s3 ). to further determine whether sm934 exerted an direct impact on the development of t helper cells, naïve cd4 + t cells from splenocytes of c57bl/6 mice were induced towards to tfh cell differentiation in the presence or absence of sm934 for 4 days, using the naïve cd4 + t cells cultured under neutral conditions as th0 control. as we reported previously, sm934 (10 μ m) could significant impede differentiation of th1 and th17 cells and exerted little influence on treg polarization in vitro 13 , we thus sought to identify the impact of sm934 on the tfh cells differentiation. as shown in fig. 5a , the frequency of tfh cells were significantly decreased when treated with sm934 (10 μ m). we simultaneously examined whether sm934 modulated the expression of bcl-6, the unique transcript factor for tfh programming. as shown in fig. 5b , the cell culture under tfh differentiation condition demonstrated a sharp increase of bcl-6 expression, while sm934 treatment largely abrogated the intracellular expression of bcl-6. apart from costimulatory signals providing cognate help to gc b cells differentiation, il-21, one of the primary cytokines derived from tfh and th17 cells, acts directly on the formation of plasma cells and the maintenance of gcs 39 . in cia mice, we observed elevated level of il-21 on the cd4 + t cells, in line with the up-regulated of il-21 mrna expression. sm934 treatment induced pronounced decrease of the percentage of cd4 + il-21 + cells (fig. 6a) . accordingly, real-time pcr analysis revealed that il-21 expression in splenocytes of sm934-treated cia mice decreased to approximately 23% of saline-treated ones (fig. 6b) . we further investigated what effect of sm934 would have on the downstream signaling of il-21 on t cells. b cell-depleted splenocytes from normal c57bl/6 mice stimulated with il-21 were simultaneously cultured with or without sm934 (10 μ m) for 0 to 60 minutes. we observed the sm934 treatment significantly inhibited the phosphorylation of stat3 at 5, 30 and 60 minutes time points (fig. 6c) . collectively, these results revealed that sm934 could abolish the il-21 secretion and downregulate its downstream signaling through stat3. recent years, artemisinin and its derivatives with immunosuppressive activities have been investigated as promising therapeutic candidates for multiple autoimmune disorders. in present study, we demonstrated that sm934 collagen-induced arthritis is the most widely used animal model for the evaluation of novel therapeutic strategies for rheumatoid arthritis and shares both immunological and pathological features of human ra. there are considerable evidence implied that cii-reactive cd4 + t cells as the primary mediators of disease induction and complement-fixing anti-cii autoantibody production by b cells should be the major immune mechanism instigate the local inflammatory response resulting production of cytokines and inflammatory mediators [40] [41] [42] . our results showed that sm934 treatment could prevent the aggravation of arthritis manifestation and suppress the anti-cii antibody production, especially for the igg2a subclass, which dominated the anti-cii response in cia. further, sm934 inhibited the expansion of tfh cells and th17 cells, the potent supporters for germinal center reaction and generation of antigen specific b cells in autoimmune diseases. intriguingly, sm934 treatment exerted little impact on the frequency of gc b cell, and did not affected the th1 or treg proportion either. it is well documented that the gc b cells are precursors of antigen-specific memory b cell and long-lived plasma cells (pc). as for the role of tfh cells, gc b cells and th cell-b cell interaction in an antigen-specific response, hu and colleagues 43 suggested a reasonable illustration, using a srbc-challenged mice and co-culture experiment, that it was the generation of tfh cells, rather than direct activation b cells by tfh cells, should be the primary mechanism modulating igg antibody production. moreover, there are plenty of molecular and cellular underpinnings in the development of b cell memory and pc formation, including molecular interactions that organize at cellular interfaces of th cells and gc b cells. reports have identified numbers of surface molecules implicated in the checkpoint of gc b cell development: cd40-cd40l and ligation play an essential role in class switch recombination and gc formation 44 ; icos-b7rp-1(icosl) costimulation is critical for production of cytokines lead to b cell activation and differentiation 43 ; cd30-cd153 interactions have an inhibitory effect antibody production in vivo 45 . together, during the formation of antibody-producing cells, tfh cells provide necessary help to gc b cells in terms of costimulatory signals and differentiation factors. in current study, sm934 impeded the propagation of tfh cells and controlled autoantibody production in cia mice without altering the population of gc b cells. we speculated that the suppression on the generation tfh cells and il-21, the critical executor of b cell fate, should be responsible for this phenomenon. nevertheless, what specific action would sm934 have on memory b cells and long-live plasma cells in ra status, could it regulate the expression of surface molecules on tfh cells were unknown and required further investigation. similar as our previous reports 12, 13 , in the present study, sm934 retarded the development of th17 cells both in the autoimmune animals and cellular culture in vitro. accumulating reports have indicated a distinct role of th17 cells in supporting b cell responses. bauquet et al. 46 demonstrated that th17 cells and tfh cells shared several features, including the expression of icos and il-21. patakas et al. 23 stated that th17 cells have a significant role earlier in the initiation/development of the gc responses, by using t cell and b cell receptor transgenic mice specific for model antigens. mitsdoerffer et al. 24 found that even il-17 on its own, could drive class switch recombination in myelin oligodendrocyte glycoprotein (mog)-immunized 2d2 tcr transgenic mice. as one of the major producer of il-17 and il-21, th17 cells might not only act as proinflammatory characters 47 but also incitement of excessive gc reaction and antibody production in ra pathology. in light of these properties, the subsequent effect of the suppression on th17 cells by sm934, could facilitate the general repression of antigen-specific antibody production in cia mice as well as in srbc-and ova-challenged mice. il-21 exerts pleiotropic actions on the immune system. besides engagement in the function of t cells, natural killer (nk) and dendritic cells (dcs), il-21 exerts profound influence on b cell biology. our results showed that sm934 treatment inhibited the production of il-21 from cd4 + t cells and abolished the downstream signaling of il-21 through stat3, coincided with the decreased level of antibodies and reduction of tfh cells and th17 cells. actually, il-21 serves as an autocrine factor secreted by tfh and th17 cells that promotes or sustains tfh and th17 lineage commitment in a stat3-dependent manner 27, 48 . moreover, stat3 not only plays as the transduction element of il-21 signaling, but also binds the il21 promoter directly and modulates il-21-responsive genes 49 . therefore, sm934 treatment could interfere il-21 circuit including il-21 per se and stat3 activation, which thus reinforce the inhibitory effects of sm934 on tfh and th17 cells. furthermore, il-21 can induce various fate on b cells, depending on the interplay with costimulatory signals and on the developmental stage of a b cell: in b cells that encounter antigen and receive t cell help, il-21 induces survival, proliferation, isotype switching, and differentiation to antibody-secreting pcs; in those b cells receiving signals via bcr alone, as can be the case for some autoantigens, or via tlr, il-21 costimulation causes apoptosis 50 . these features of il-21 suggested that there might be a discrepancy between the mechanisms of sm934 on the spontaneous autoimmune diseases and antigen-induced immune responses, relevant to il-21, which is worthy an attentive investigation in further studies. animal ethical statement. the animal experiment was carried out in strict accordance with the institutional ethical guidelines on animal care and were approved by the institute animal care and use committee animals. male dba/1j, female c57bl/6 and balb/c mice were purchased from shanghai laboratory animal center of the chinese academy of sciences. all mice were housed in a pathogen-free facility and rabbits were housed in clean-grade animal cabin with free access to standard laboratory water and food, and kept in a 12 h light/dark cycle with controlled humidity (60-80%) and temperature (22 ± 1 °c). collagen-induced arthritis. the male dba/1j mice were randomly divided into 4 groups (n = 8 per group), 3 groups were immunized at the tail base with 100 μ g bovine type ii collagen (cii, tokyo, japan) in 0.1 m acetic acid emulsified equal volume complete freund's adjuvant (cfa) containing mycobacterium tuberculosis strain h37rv (wako pure chemical industries ltd., osaka, japan). a boost injection of 100 μ g collagen-incomplete freund's adjuvant (ifa) emulsion was given in the same manner 3 weeks later. from day 10 after booster immunization, immunized groups were orally administered with saline, mtx (1 mg/kg/day) or sm934 (10 mg/kg/day) for consecutive 40 days, while normal controls were administered with saline. the arthritis severity of mice was monitored every two days. at the end of treatment, all 4 groups of dba/1j mice were sacrificed, and serum, hind paws and splenocytes were then collected. clinical assessment of arthritis. the clinical severity of arthritis was scored as previously described 51 . briefly, each limb was graded based on a scale of 0 to 4 according to the following scale: 0 = normal; 1 = detectable arthritis with erythema one or several digits; 2 = erythema and moderate swelling extending from the ankle to the midfoot; 3 = severe swelling and redness from joint to digit; and 4 = maximal swelling with ankyloses. the severity was described as the cumulative score of four limbs (the maximum score for each mouse is 16). three-dimensional reconstruction of the hind knee and ankle joints were obtained by micro-ct examination (inveon mm system, siemens preclinical solutions) at the end of treatment. briefly, after the mice in different groups being killed using ether anesthesia, the hind limbs were removed and fixed in 4% paraformaldehyde. the samples were scanned with micro-ct, and images were acquired at an effective pixel size of 8. srbcs. saline or sm934 (10 mg/kg) were orally administered for consecutive 9 days after immunization. five of the unimmunized female balb/c mice were used as normal controls. ova-immunized c57bl/6 mice. naïve female c57bl/6 mice were immunized with ova-cfa emulsion as described previously 52 , then were treated with saline or sm934 (10 mg/kg/day) for consecutive 7 days. five of the unimmunized female c57bl/6 mice were used as normal controls. flow cytometric analysis. single-cell suspensions were prepared from spleens or from cell cultures. antibodies for surface staining, percp-cy5.5-conjugated anti-cd3, pe-conjugated anti-cd4, fitc-conjugated anti-pd-1, apc-conjugated anti-cxcr5, alexa fluor 647-conjugated anti-gl7 and fitc-conjugated anti-pd-1 were purchased from bd bioscience. for intracellular staining, splenocytes were first stained with surface markers followed by fixation and permeabilization using foxp3 staining buffer set purchased from ebioscience 53 . cells were labelled intracellularly pe-conjugated anti-bcl-6 (ebioscience, san diego, ca, usa), pe-conjugated anti-il-17, apc-conjugated anti-ifn-γ or percp-cy5.5-conjugated anti-foxp3 (bd biosciences, san diego, ca, usa). flow cytometry was performed on 4-laser/13-color bd lsrfortessa (bd biosciences) and data were analyzed using flowjo software (tree star, ashland, or). antibody and cytokine assays. at the end of the animal experiments, serum were collected and levels of specific antibodies were measured by enzyme-linked immunosorbent assay (elisa). serum titers of anti-cii or anti-srbc igg were determined as previously described 51, 54 . briefly, 96-well elisa plate were coated with bovine cii (50 μ g/ml) or extracted srbc membranes (20 μ g/ml) (prepared as described previously 55 ) overnight at 4 °c. each diluted serum sample was added and incubated. horse-radish peroxidase (hrp) conjugated goat anti-mouse igg (h + l), igg1and igg2a abs (invitrogen, san diego, ca, usa) were used. the optical density is measured spectrophotometrically at 450 nm. cytokines in sera and culture supernatants were detected using mouse il-6, il-17a (bd biosciences, san diego, ca) and il-21 (r&d systems, minneapolis, mn) elisa kits according to the manufacturer's instructions. splenocytes activation assay. splenocytes isolated from cia mice of indicated groups were incubated with anti-b220 (ra3-6b2) monoclonal antibody (mab) to deplete b cells by immunomagnetic negative selection as describe previously 51 . the resulting cells were cultured (4 × 10 6 cells/ml) with medium alone or cii (10 μ g/ml) respectively for 72 hours. total splenocytes from ova-immunized mice of each group were stimulated with ova (100 μ g/ml) for 48 hours. after incubation, for 96-well flat-bottom plates, [ 3 h] thymidine assay was used to evaluate the cell proliferation. for 24-well plates, the supernatants were collected to determine levels of cytokines by elisa. in vitro tfh cell differentiation. purification of naïve splenic cd4 + t cells (cd4 + cd44 -cd62l + ) from normal c57bl/6 mice was conducted using our previously reported methods 56 . naïve cd4 + t cells were cultured with anti-cd3 mab (5 μ g/ml) and anti-cd28 mab (2 μ g/ml) for 4 days under different skewing conditions: th0: anti-ifn-γ (10 μ g/ml), anti-il-4 (10 μ g/ml, bd bioscience); and tfh: anti-ifn-γ (10 μ g/ml), anti-il-4 (10 μ g/ml), il-21 (50 ng/ml, peprotech). sm934 (10 μ m) was added to the culture simultaneously. gene expression analysis. the total rna was isolated from splenocytes of cia mice treated with saline or sm934 (10 mg/kg) using rnasimple total rna kit (tiangen biotech, beijing, china). and a one-step real-time pcr assay was performed with sybr green pcr reagents (qiagen, valencia, ca, usa). relative quantitation of mrna expression of il-21 and gapdh was calculated using the δ δ ct method. the primers sequence were as follows: for il-21, 5′ -gga ccc ttg tct gtc tgg tag-3′ (forward) and 5′ -tgt gga gct gat aga agt tca gg-3′ (reverse); and for gapdh, 5′ -gcc tca agg tat tgc tgg ac-3′ (forward) and 5′ -acc ttg ttt gcc agg ttc ac-3′ (reverse). western blotting. b cell-depleted splenocytes from normal c57bl/6 mice (4 × 10 6 cells/ml) were stimulated with/without il-21 (10 ng/ml, peprotech, rocky hill, nj) and sm934 (10 μ m) for 0 to 60 minutes. cell cultures or splenocytes isolated from cia mice of individual group were directly lysed in sodium dodecyl sulfate (sds) sample buffer containing protease inhibitor cocktail (roche life science, mannheim, germany) 57 . whole cell lysates were fractionated on 10% sds-polyacrylamide gel electrophoresis (page) and western blotted with antibodies to phosphorylated stat3 (cell signaling technology, beverly, ma) and gapdh (kangchen biotechnology, china). the densities of the bands were quantified with a computerized densitometer (image j launcher, broken symmetry software). statistical analysis. statistical analysis was performed using graphpad prism 6.0 statistical software. comparisons between 2 groups were performed using an unpaired 2-tailed t-test. for experiment involving multiple groups, one-way analysis of variance (anova) followed by turkey's multiple comparison test was used, except for analysis of clinical scores which used two-way anova followed by dunnett's multiple comparison test. p values less than 0.05 were considered significant. qinghaosu (artemisinin): chemistry and pharmacology high-level semi-synthetic production of the potent antimalarial artemisinin artemisinin directly targets malarial mitochondria through its specific mitochondrial activation ferrous ion induced cleavage of the peroxy bond in qinghaosu and its derivatives and the dna damage associated with this process unified mechanistic framework for the fe (ii)-induced cleavage of qinghaosu and derivatives/analogues. the first spin-trapping evidence for the previously postulated secondary c-4 radical a novel artemisinin derivative, 3-(12-beta-artemisininoxy) phenoxyl succinic acid (sm735), mediates immunosuppressive effects in vitro and in vivo investigation of the immunosuppressive activity of artemether on t-cell activation and proliferation anti-inflammatory properties and regulatory mechanism of a novel derivative of artemisinin in experimental autoimmune encephalomyelitis suppressive effect of a novel water-soluble artemisinin derivative sm905 on t cell activation and proliferation in vitro and in vivo the new water-soluble artemisinin derivative sm905 ameliorates collagen-induced arthritis by suppression of inflammatory and th17 responses artemisinin analogue sm934 ameliorates the proteinuria and renal fibrosis in rat experimental membranous nephropathy sm934 treated lupus-prone nzb x nzw f1 mice by enhancing macrophage interleukin-10 production and suppressing pathogenic t cell development oral administration of artemisinin analog sm934 ameliorates lupus syndromes in mrl/lpr mice by inhibiting th1 and th17 cell responses artemisinin analogue sm934 ameliorates murine experimental autoimmune encephalomyelitis through enhancing the expansion and functions of regulatory t cell therapeutic effects of the artemisinin analog sm934 on lupus-prone mrl/lpr mice via inhibition of tlr-triggered b-cell activation and plasma cell formation annual review of immunology intraclonal generation of antibody mutants in germinal centres plasma cells: finding new light at the end of b cell development follicular helper cd4 t cells (tfh) molecular programming of b cell memory t follicular helper (tfh) cells in normal and dysregulated immune responses posttranscriptional t cell gene regulation to limit tfh cells and autoimmunity th17 effector cells support b cell responses outside of germinal centres proinflammatory t helper type 17 cells are effective b-cell helpers interleukin 17-producing t helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune bxd2 mice il-21 induction of cd4+ t cell differentiation into th17 cells contributes to bleomycin-induced fibrosis in mice il-21 is produced by th17 cells and drives il-17 production in a stat3-dependent manner immunologic mechanisms in the pathogenesis of rheumatoid arthritis evolving concepts of rheumatoid arthritis allogeneic mesenchymal stem cells inhibited t follicular helper cell generation in rheumatoid arthritis increased frequency of circulating follicular helper t cells in patients with rheumatoid arthritis expansion of circulating t cells resembling follicular helper t cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus identification of follicular helper t cells as a novel cell population potentially involved in the pathogenesis of rheumatoid arthritis (hum3p. 251) high frequencies of activated b cells and t follicular helper cells are correlated with disease activity in patients with new-onset rheumatoid arthritis interleukin-17a promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing mmp2 and mmp9 expression through nf-κ b/hif-1α pathway the roles of ifn-γ versus il-17 in pathogenic effects of human th17 cells on synovial fibroblasts chronic development of collagen-induced arthritis is associated with arthritogenic antibodies against specific epitopes on type ii collagen serum transfer of collagen-induced arthritis in mice il-21 regulates germinal center b cell differentiation and proliferation through a b cell-intrinsic mechanism collagen-induced arthritis in mice animal models of rheumatoid arthritis and their relevance to human disease the genetic and immunopathological processes underlying collagen-induced arthritis b7rp-1 blockade ameliorates autoimmunity through regulation of follicular helper t cells cd40/cd154 interactions at the interface of tolerance and immunity promotion of b cell immune responses via an alum-induced myeloid cell population the costimulatory molecule icos regulates the expression of c-maf and il-21 in the development of follicular t helper cells and th-17 cells adiponectin exacerbates collagen-induced arthritis via enhancing th17 response and prompting rankl expression functional stat3 deficiency compromises the generation of human t follicular helper cells opposing roles of stat1 and stat3 in il-21 function in cd4+ t cells il-21: an executor of b cell fate 5r)-5-hydroxytriptolide attenuated collagen-induced arthritis in dba/1 mice via suppressing interferon-γ production and its related signaling s-adenosyl-l-homocysteine hydrolase inactivation curtails ovalbumin-induced immune responses a novel pancreatic β -cell targeting bispecific-antibody (bsab) can prevent the development of type 1 diabetes in nod mice insights into the role of bcl6 in follicular th cells using a new conditional mutant mouse model evaluation of primary and secondary responses to a t-cell-dependent antigen, keyhole limpet hemocyanin, in rats therapeutic effects of dz2002, a reversible sahh inhibitor, on lupus-prone nzbxnzw f1 mice via interference with tlr-mediated apc response identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay this study was supported by national natural science foundation of china (nsfc) (81273524 and 81322049). supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord-0022420-8xnhl3zl authors: guagnano, maria teresa; d’angelo, chiara; caniglia, daniela; di giovanni, pamela; celletti, eleonora; sabatini, emanuela; speranza, lorenza; bucci, marco; cipollone, francesco; paganelli, roberto title: improvement of inflammation and pain after three months’ exclusion diet in rheumatoid arthritis patients date: 2021-10-09 journal: nutrients doi: 10.3390/nu13103535 sha: 8c092bfc81eae144ffbbe29215e49480792a513a doc_id: 22420 cord_uid: 8xnhl3zl introduction: rheumatoid arthritis (ra) is a chronic systemic autoimmune disease affecting the synovial joints and causing severe disability. environmental and lifestyle factors, including diet, have been proposed to play a role in the onset and severity of ra. dietary manipulation may help to manage the symptoms of ra by lowering inflammation and potentially decreasing pain. methods: in 40 patients with long-standing ra with stable symptoms and treated with conventional (c-) and biological (b-) disease modifying anti-rheumatic drugs (dmards), the effect of a 3-month diet avoiding meat, gluten, and lactose (and all dairy products; privative diet) was evaluated in comparison with a control balanced diet including those foods. both diets were designed to reduce weight since all patients were overweight or obese. patients were randomly assigned to one of the diets, and ra was clinically assessed at time 0 (t0), through the visual analogue scale (vas), for pain, and the disease activity score of 28 joints (das 28) for ra activity. patients were also administered the short form health survey (sf-36) and the health assessment questionnaire (haq). at t0, a blood sample was collected for laboratory tests and adipokines measurements, and anthropometric measurements were compared. these evaluations were repeated at the end of the 3 months’ dietary regimens. results: a significant decrease in vas and the improvement of the overall state of physical and mental health, assessed through sf-36, was observed in patients assigned to the privative diet. both dietary regimens resulted in the improvement of quality of life compared to baseline values; however, the change was significant only for the privative diet. with either diet, patients showed significant decreases in body weight and body mass index, with a reduction in waist and hips circumference and lower basal glucose and circulating leptin levels. a privative diet was also able to significantly reduce systolic (p = 0.003) and diastolic (p = 0.025) arterial pressure. the number of circulating leukocytes and neutrophils, and the level of hs-c-reactive protein also decreased after 3 months of the meat-, lactose-, and gluten-free diet. conclusions: our results suggest that a privative diet can result in a better control of inflammation in ra patients under stable optimized drug treatment. rheumatoid arthritis (ra) is a chronic systemic inflammatory disease typically affecting the synovial joints, in which autoimmunity drives dysregulated proinflammatory a total of 40 patients with ra, fulfilling the classification criteria of the american college of rheumatology (acr)/european league against rheumatism (eular) [28] , were recruited among those attending the rheumatology clinic of the hospital "ss. annunziata" in chieti. all were females with ages ranging between 31 and 72 years (mean age ± sem: 52.23 ± 1.61), and all had been treated with the same optimized therapy for at least one year, consisting of a combination of biological (b-) and conventional (c-) disease modifying antirheumatic drugs (dmard) (n = 7), or monotherapy with a biological drug (n = 22), or c-dmards only (n = 11), showing stable disease activity, assessed with the scales described below. comorbidities (diabetes, dyslipidemia, celiac disease), treatment with medium to high doses of corticosteroids (i.e., above 7.5 mg/day of prednisone equivalent), and current or previous dietary regimens with avoidance of meat, gluten, or milk represented exclusion criteria. the dietary habits of the patients were assessed by interview, showing a substantial adherence to the mediterranean diet, which is prevalent in the region of the study, and remained unchanged in the previous years. however, individual consumption of meat, gluten, or milk was not quantified since this was not the purpose of the study. moreover, patients were asked about physical activity and important lifestyle changes (significant increase in physical activity, job change, etc.). all patients reported low/moderate physical activity (e.g., casual walk, bike riding, jogging, or swimming in the pool) and no changes in lifestyle occurred in the previous year. low-dose steroid therapy (less than 7.5 mg prednisone equivalent/day) and occasional use of nonsteroidal anti-inflammatory drugs (nsaids) or analgesics were allowed throughout the study. all the participants gave informed consent for study enrollment, as required from the local ethics committee for biomedical research which approved the study (committee project no. 10/20) . the investigations adhered to the declaration of helsinki, revised in 2013. enrolled patients were randomly assigned to one of the two experimental groups and asked to follow a diet excluding meat, gluten and lactose (group a) or a balanced diet (group b). groups were similar in age (mean ± sem group a: 50.60 ± 2.24 years; mean ± sem group b: 54.10 ± 2.09 years; p = 0.269) and main anthropometric characteristics. diets were designed by expert physicians at the obesity center of the "ss. annunziata" hospital in chieti; both diets gave an intake of about 1500 kilocalories (kcal)/day (as part of the treatment, since all patients were overweight/obese), and were optimized according to current guidelines for balanced composition in macronutrients [29] , daily intake of cholesterol (<300 mg/die), saturated fatty acids (<10% of total energy intake) [30] , oligosaccharides (<15% of total energy intake), and dietary fiber (25-30 g/die) [31] . moreover, for both types of diet, the total protein intake was 50% from animal and 50% from vegetable proteins. the macronutrient composition of the meat-, gluten-, and lactose-deprived diet was as follows: 56% of total kcal from carbohydrates, 16% from proteins, and 28% from fat. the main contributors to protein intake were fish (50.1%) for animal protein and flour products (16.1%), legumes (19.3%), and fruit and vegetables (14.5%) for vegetable protein. the macronutrient composition of the balanced diet was as follows: 56% carbohydrates, 17% proteins, and 27% fats. the main contributors to protein intake were poultry meat (28.8%) such as chicken and turkey and milk (products) (7.1%), for animal protein, and flour products (31.1%), legumes (15.7%), fruit and vegetables (17.3%) for vegetable protein. these data and the detailed description of the diets are reported in supplemental tables s1-s3. red meat was absent in both diets, and all dairy products were eliminated in diet a. the diets were to be adhered to strictly every day, and detailed items allowed are listed in the supplemental tables. in addition to the diets, advice for a correct lifestyle was provided. in particular: drinking at least 2 l/day of water/liquids, and walking at least 30 min/day, or 1 h in the gym 1-2 times/week, and general advice: do not smoke, do not drink alcohol, sleep regularly respecting the normal sleep/wake rhythm at the time of recruitment, time 0 (t0), all patients underwent clinical evaluation of ra through an objective physical articular examination, also used to calculate the disease activity score of 28 joints (das 28) (see below). the visual analogue scale (vas) was used for pain assessment, asking the patient to locate on a 100 mm line the point that best identified the intensity of pain in the previous week (where 0 represents no pain and 100 mm represents the maximal pain perceived) [32] . patients were also asked how the disease condition affected their quality of life by administration of the short form health survey (sf-36) [33] and the health assessment questionnaire (haq) [34] . the activity of ra was evaluated by the 4-parameter das 28 using esr [35] . all the above-described measures were repeated after 3 months of diet (t1) in both groups a and b. at the follow-up visit at t1, patients were inquired about adherence to the prescribed diets, and this was total for those who completed the 3 months of the study. patients underwent blood sample collection at t0 by venipuncture, and a complete blood count, and clinical chemistry tests were performed, including oral glucose tolerance test (ogtt), homeostasis model assessment (homa) index, insulin level, serum lipid profile, erythrocyte sedimentation rate (esr), high-sensitivity c-reactive protein (hs-crp), transaminase levels, total proteins, albumin, and transferrin. serum aliquots, obtained after blood clotting and centrifugation, were stored at −80 • c, until assayed for circulating cytokines and adipokines evaluation. anthropometric measurements, patients' height, weight, and calculation of the body mass index (bmi) (kg/m 2 ), arterial blood pressure, and bioimpedance analysis (bia) [36] were also recorded. bia parameters were: muscle mass, fat mass, bone mass, water, and basal metabolism. all the above-described evaluations obtained at t0 were repeated after 3 months (t1) of the deprived (group a) or balanced (group b) diet. selected cytokines and adipokines were measured using the specific quantikine ® elisa kits (human leptin immunoassay, human adiponectin immunoassay, human tnfα immunoassay, human il-10 immunoassay, and human infγ immunoassay) purchased from r&d systems (minneapolis, mn, usa). all elisa assays were performed on serum samples collected at t0 and t1, in the same batch, following the manufacturer's instructions. qualitative variables were reported as frequency and percentage, and quantitative variables as median and interquartile range (iq). the chi-square test was used to compare proportions. nonparametric statistics was used to compare the subjects at t0 and after the dietary intervention (wilcoxon matched-pairs signed-rank test), and to compare the patients randomly assigned to group a or b at t0 and t1 (mann-whitney u-test). spearman's rank correlation coefficient was calculated to assess the relationship between the variables changes at t1 vs. t0. the threshold of statistical significance was set at p = 0.05. data analysis was performed on graphpad prism 6 software, version 6.01, 2012. a total of 12 patients withdrew from the study before t1, and this left 15 cases in group a and 13 in group b who completed the 3 months of the study without departure from the prescribed diet. causes of poor compliance were difficulties to adhere to diet(s), also due to family reasons, in 9 patients (4 in group a and 5 in group b), and nonspecific gastrointestinal complaints (bloating, nausea) in 3 patients (1 in group a and 2 in group b). none of the patients reported worsening of symptoms or a flare of ra as reasons for leaving the trial. to evaluate pain perception and its changes with dietary regimen, the vas score was used ( figure 1 ). in group a, a significant decrease in vas was found after the diet (t0 median: 50, iq: 35-80; t1 median: 40, iq: 10-60; p = 0.003). in group b, with the control diet, the vas score also decreased, but not significantly. the composite clinimetric index das 28 was used to evaluate changes in ra activity after 3 months of diet ( figure 2 ). the das 28 score was not significantly different before and after either diet. figure 3 summarizes the eight scales included in the sf-36 questionnaire, showing the overall state of physical and mental health in patients at the beginning (t0) and after 3 months' diet (t1). note that higher values indicate a better health status. in group a, the t1 score (median: 59, iq: 46-74) was significantly increased (p < 0.001) with respect to t0 values (median: 42, iq: 30-54), whereas in group b no significant change was detected. the differences at t1 tended to favor the outcome of diet a (p = 0.056). the composite clinimetric index das 28 was used to evaluate changes in ra activity after 3 months of diet ( figure 2 ). the das 28 score was not significantly different before and after either diet. the composite clinimetric index das 28 was used to evaluate changes in ra activity after 3 months of diet ( figure 2 ). the das 28 score was not significantly different before and after either diet. figure 3 summarizes the eight scales included in the sf-36 questionnaire, showing the overall state of physical and mental health in patients at the beginning (t0) and after 3 months' diet (t1). note that higher values indicate a better health status. in group a, the t1 score (median: 59, iq: 46-74) was significantly increased (p < 0.001) with respect to t0 values (median: 42, iq: 30-54), whereas in group b no significant change was detected. the differences at t1 tended to favor the outcome of diet a (p = 0.056). figure 3 summarizes the eight scales included in the sf-36 questionnaire, showing the overall state of physical and mental health in patients at the beginning (t0) and after 3 months' diet (t1). note that higher values indicate a better health status. in group a, the t1 score (median: 59, iq: 46-74) was significantly increased (p < 0.001) with respect to t0 values (median: 42, iq: 30-54), whereas in group b no significant change was detected. the differences at t1 tended to favor the outcome of diet a (p = 0.056). when the eight items of the sf-36 questionnaire were considered apart, in group a we observed a significant increase in the state of health (sh), the vitality (v), the social activities (sa), and the role limitations due to the emotional state (re) scores. in group a, the increase in the v score was inversely correlated with pain reduction, assessed by the vas (rho = −0.546, p = 0.027). in group b, the increase in the eight items of sf-36, with respect to t0, were smaller and did not reach statistical significance, with sh, v, and mental health (mh) scores showing no variation with respect to t0 (data not shown). the items on the patient's physical, psychological, and social dimensions in the haq test are organized so that a lower score indicates a better quality of life. both the deprived and the balanced diet were associated with improved quality of life in ra patients compared to baseline; however, the difference reached significance (p < 0.05) only for patients belonging to group a ( figure 4 ). when the eight items of the sf-36 questionnaire were considered apart, in group a we observed a significant increase in the state of health (sh), the vitality (v), the social activities (sa), and the role limitations due to the emotional state (re) scores. in group a, the increase in the v score was inversely correlated with pain reduction, assessed by the vas (rho = −0.546, p = 0.027). in group b, the increase in the eight items of sf-36, with respect to t0, were smaller and did not reach statistical significance, with sh, v, and mental health (mh) scores showing no variation with respect to t0 (data not shown). the items on the patient's physical, psychological, and social dimensions in the haq test are organized so that a lower score indicates a better quality of life. both the deprived and the balanced diet were associated with improved quality of life in ra patients compared to baseline; however, the difference reached significance (p < 0.05) only for patients belonging to group a ( figure 4 ). the anthropometric measures are reported in table 1 . patients randomly assigned to group a or b at t0 were not significantly different for all variables considered (penultimate column of table 1 ). after 3 months of the two dietary regimes, patients showed a significant decrease in body weight and bmi (table 1 ). all patients were overweight/obese at t0. in particular, eight (50%) females in group a and eight (50%) in group b were overweight and five (41.7%) females in group a and seven (58.3%) in group b were obese, but this distribution is not statistically significant (p = 0.662) (data not shown). both dietary regimes were also significantly effective in reducing waist and hips circumference. interestingly, in group a, a significant reduction in systolic (sys) (p = 0.003) and diastolic (dia) (p = 0.025) arterial pressure was achieved, but this did not occur in group b. among the bia parameters, no change in muscle mass and basal metabolic rate was observed after diet in either group, whereas fat mass was significantly reduced in both. the last column of table shows the statistical differences between variables in group a and b at time t1. it is possible to note that there are statistically significant differences between the two diets in muscle mass and water, which have higher values in group a, and in systolic blood pressure, which has a lower median value in group a. in group a, the lower weight, bmi, and fat mass, were significantly correlated with increased v scores in the sf-36 questionnaire (rho = −0.527, p = 0.038; rho = −0.546, p = 0.045; rho = −0.510, p = 0.033, respectively). moreover, statistically significant correlations between weight loss, reduction in hips circumference, and the rp score (used to assess role limitations due to physical health; rho = −0.610, p = 0.009 and rho = −0.576, p = 0.013, respectively) were found. in group b, after 3 months of the control balanced diet, a significant decrease in fat mass and water content have been measured ( table 1) . the improvement in the haq score observed in both groups can be traced back to the moderate calorie restriction to which patients were subjected, such that the produced weight reduction significantly correlated with haq in group b (rho = 0.586, p = 0.044). the metabolic, hematological, and biochemical parameters analyzed are reported in table 2 . patients randomly assigned to groups a or b at t0 did not differ significantly (penultimate column of table 2 ). basal glucose levels in patients after 3 months of either dietary regimen showed a statistically significant decrease ( table 2 ). the deprived diet was also effective in significantly reducing the homa index [37] in group a. no significant changes in fasting insulin levels, blood lipid profile, total proteins, or albumin were recorded ( table 2 ). no differences were observed between the groups at t1 (see last column). the number of circulating leukocytes and neutrophils, and the level of hs-crp were also significantly decreased in group a, together with a significant reduction in transferrin (p = 0.013). the decline in leukocyte numbers was inversely correlated with an increase in the physical pain score (pp) item in the sf-36 test (rho = −0.579, p = 0.026). circulating levels of leptin were significantly decreased after 3 months of diet in both groups a and b, whereas adiponectin was unaffected. the reduction in leptin caused a significant increase in the adiponectin/leptin ratio in both group a ( figure 5 , top third panel) and b (figure 6, top third panel) . the serum levels of the three cytokines measured (tnfα, il-10 and infγ) were not significantly different before or after either diet. our study enrolled ra patients with a long-standing, well-controlled condition under stable pharmacological treatment, and subjected them to a trial diet with the exclusion of three food items (meat, gluten, and lactose) that have been suspected of aggravating the symptoms and worsening the disease course, in comparison with a control balanced diet including those foods. the results demonstrate that both diets, despite not affecting the disease activity, measured by the 4-parameter das28, had a tendency to reduce the arthritic pain perception and to improve the quality of life of the patients. moreover, metabolic and inflammatory parameters showed a trend to normalize, and this, together with a significant lowering of systolic blood pressure might impact the prognosis and the long-term cardiovascular risk of ra [38, 39] . we hypothesized that the two diets may be influencing disease perception and ra burden in several ways, through a range of mechanisms, among them normalizing the adiponectin-to-leptin ratio which is a sensitive index of the inflammatory status [40] , adipose tissue dysfunction, and reducing cardiometabolic risk [41] . the decrease in inflammation was reflected by the reduction in hs-crp, but not in esr and tnf-α. these modifications may explain the lack of effect on ra activity, measured by das-28, and the changes in glucidic homeostasis (homa index), as well as mental and physical health [42, 43] . both diets had significant impact on weight reduction, thus lowering the bmi, and bia revealed a significant decrease in fat mass, with lesser changes for other measurements. this lowering of weight was indeed one of the therapeutic goals of the diets, which were designed to have a low calorie content, considering that all patients selected were overweight/obese, despite following a habitual mediterranean diet. however, only the exclusion diet induced significant changes in adipokines, crp, and glucidic control compared to t0. moreover, it achieved a significant decrease in both sys and dia blood pressure, and the systolic was reduced also when compared with t1 levels of diet b. therefore, we cannot ascribe both diets' results to the simple loss of extra weight and fat. in our ra patients, 3 months of a deprived diet was able to significantly reduce total leukocytes and neutrophils number, and crp and transferrin levels as well. comparing the two diets at time 1, we found statistically significant differences in the median value of sys blood pressure (lower in the deprived diet group) and in median values of muscle mass and water (higher in the deprived diet group). several types of diet or nutritional supplements in ra have received increased attention in recent years, and dietary intervention studies in ra have been published [20, [44] [45] [46] [47] [48] . a 3-month mediterranean-based diet in ra patients ensured clinical benefits, with a significant reduction in das28 and improved quality of life assessed by haq and sf-36 compared to ra control patients on their usual diets [49] . in that study, only recommendations were given, and the diets differed under all aspects. however, there is limited but univocal evidence to suggest that the mediterranean diet is beneficial in the prevention and treatment of pathological conditions, including ra [5, 18, 50] . the protective effect of the mediterranean diet on ra disease activity may be due to changes in the gut microbiota of the patients as shown in the study prevention with mediterranean diet-(predimed): [51] . the mediterranean diet shows many similarities to the dietary schemes adopted in our study, but we failed to observe a reduction in disease activity. we must also point out that all our patients, though not assessed with precision, habitually followed a mediterranean-style diet before entering the study. enrolled patients did not show significant differences at the recruitment time, also considering the different pharmacological treatment received. the low-calorie content was equal for the two diets, and animal protein was provided in both, thus ruling out other possible explanations based on vegetarian diets or fasting. among rheumatic patients who switched from an omnivorous to a vegetarian diet, an attenuation of disease activity has been reported [48] , and fasting, even for short duration, or fast mimicking eating methods have been shown to provide some advantageous outcomes to ra patients [52] . some dietary components may affect chronic inflammatory diseases, although a large prospective study of 56,075 danish middle-aged men and women failed to detect a higher risk of any inflammatory disease, or ra specifically, for low fiber or/and high meat intake [53] . both diets in our study excluded red meat, and only chicken and turkey were allowed in diet b (control diet). obesity in ra has been found to be associated with higher das28, tender joint counts, inflammatory marker levels, patient global evaluation and pain scores, as well as physical function scores [54] [55] [56] . however, it is not associated with increased mortality [57] . many of our patients were obese; their disease activity was kept under control by pharmacological treatment, and it did not change throughout the study, even after weight reduction. altered body composition is very frequent in ra, and the majority may present with loss of lean mass, the so-called rheumatoid cachexia. only a non-significant reduction in muscle mass was observed for both diets in our study; however, this resulted in a significant difference at t1, with lower mass in group b. leptin levels, which are raised in obese patients, are markedly increased in ra and related to general inflammatory status and disease activity [58] . in our study, both diets showed a tendency to lower the leptin levels, resulting in an increase in the adiponectin/leptin ratio, significant only in the exclusion diet. diet has a central role in increasing inflammatory disease risk and progression. several nutrients may have protective activity for ra symptoms, whereas others may have a harmful role. the use of fish-rich diets, among them the mediterranean diet, or fish-oil supplementation in long-standing ra, have proven beneficial in meta-analyses [59] , so the effect of substituting fish for meat as a source of animal protein in our study may be questioned. different types of fish were allowed though, with widely variable content in omega-3 and other components, so the question is purely speculative and difficult to answer. this study has some strengths and several limitations. the small number of patients enrolled calls for the intention to be a pilot study, and the high number of dropouts, mainly due to the problems of maintaining strict diets for a long time (i.e., not allowing for holidays) reduced the final study group to 28 ra patients. the patients were all female and overweight or obese, so they might not be representative of all cases. individual histories might have differed too, as did ongoing and past treatments, as well as their microbiota; future studies should take all these variables into account. no intermediate time point was used to check the study course for its three months' duration. aside from these limitations, our study has some strengths: the prescription of clearly identified diets, and their balanced composition of macronutrients. both groups had to adhere to a strict diet, and the therapeutic goal of the diet was to reduce excess weight. lastly, the diet had to be continued for a reasonable time, 3 months, in order to observe more stable changes. although attenuating effects on ra pathology were observed in our and others' dietary intervention studies, none of them have been successfully implemented in clinics so far. maybe the reasons are due to the poorly characterized mechanisms underlying the improvement in ra patients, or the availability of ever more effective drugs that make dietary alternatives less appealing. moreover, all dietary interventions require changes to the patients' daily lifestyle and eating behaviors, and that requires commitment, endurance, persuasion, and time. the results obtained appear to confirm that exclusion of three common food items, all reported as suspected of worsening of symptoms in ra, with the inclusion of fish, may carry additional health benefits to overweight patients compared with those obtained by a standard weight-reduction program (diet b). the immunological correlates of ra were not sensitive to either diet, but measures of general physical and mental state, pain perception and quality of life showed a tendency to improve with both. the better preservation of muscle mass and the lower systolic blood pressure achieved with the exclusion diet a shows that weight loss in our patients was not the only explanation, since both diets had this result. the aim of preventing complications of ra might be better achieved by the exclusion diet, so a diet prescription should be included in therapeutic plans for ra. supplementary materials: the following are available online at https://www.mdpi.com/article/10 .3390/nu13103535/s1, table s1 : macronutrients (daily intake). table s2 : privative diet (deprived of meat, gluten and lactose) (1500 kcalories). table s3 : balanced diet (1500 kcalories). author contributions: r.p., e.c. and m.t.g. conceptualization; d.c., e.c. and m.t.g. study and diets design; e.c., e.s., d.c. and m.b. collected data and visited the patients; c.d., p.d.g., m.t.g. and r.p. analyzed the results; m.t.g., l.s., f.c. and r.p. supervised all phases of the study; c.d. wrote the first draft of the manuscript; all authors contributed to the interpretation of results. all authors have read and agreed to the published version of the manuscript. funding: no specific funding was available for this project. institutional review board statement: the study was conducted according to the guidelines of the declaration of helsinki, and it has been approved by the ethics committee of the provinces of chieti and pescara and of the "g. d'annunzio" university of chieti-pescara (ethics committee project no.10/20). all participants signed written informed consent for participation in the study. the data that support the findings of this study are available from the corresponding author upon reasonable request. rheumatoid arthritis the pathogenesis of rheumatoid arthritis are we really what we eat? nutrition and its role in the onset of rheumatoid arthritis the nutrition-gut microbiome-physiology axis and allergic diseases how the mediterranean diet and some of its components modulate inflammatory pathways in arthritis update on the epidemiology, risk factors, and disease outcomes of rheumatoid arthritis aging, obesity, and inflammatory age-related diseases interrelation of diet, gut microbiome, and autoantibody production microbiota and probiotics in health and hiv infection the role of diet in triggering human inflammatory disorders in the modern age dysbiosis and the immune system crossing the barriers: revisiting the gut feeling in rheumatoid arthritis the gut-joint axis in rheumatoid arthritis dietary habits and nutrition in rheumatoid arthritis: can diet influence disease development and clinical manifestations? modificatin of gut microbiota in inflammatory arthritis: highlights and future challenges antibodies against dietary antigens in rheumatoid arthritis patients treated with fasting and a one-year vegetarian diet dietary interventions for rheumatoid arthritis mediterranean food pattern in rheumatoid arthritis the mediterranean diet, fish oil supplements and rheumatoid arthritis outcomes: evidence from clinical trials rheumatoid arthritis and dietary interventions: systematic review of clinical trials nutrition and quality of life referring to physical abilities-a comparative analysis of a questionnaire study of patients with rheumatoid arthritis and osteoarthritis diet and rheumatoid arthritis symptoms: survey results from a rheumatoid arthritis registry the role of meat in the expression of rheumatoid arthritis diet and rheumatoid arthritis: red meat and beyond the molecular basis of lactose intolerance milk-the promoter of chronic western diseases gluten-free diet in nonceliac disease rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative pali-schöll, i. the secrets of the mediterranean diet. does [only] olive oil matter? nutrients scientific opinion on dietary reference values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids, and cholesterol scientific opinion on dietary reference values for carbohydrates and dietary fibre pain: a review of three commonly used pain rating scales the mos 36-item short-form health survey (sf-36). i. conceptual framework and item selection measures of functional status and quality of life in rheumatoid arthritis: health assessment questionnaire disability index (haq), modified health assessment questionnaire (mhaq), multidimensional health assessment questionnaire (mdhaq), health assessment questionnaire ii (haq-ii), improved health assessment questionnaire (improved haq), and rheumatoid arthritis quality of life (raqol) the disease activity score (das) and the disease activity score using 28 joint counts (das28) in the management of rheumatoid arthritis new indexes of body fat distribution, visceral adiposity index, body adiposity index, waist-to-height ratio, and metabolic disturbances in the obese homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man increased cardiovascular risk in rheumatoid arthritis: mechanisms and implications cardiovascular events in early ra are a result of inflammatory burden and traditional risk factors: a five year prospective study adiponectin to leptin ratio reflects inflammatory burden and survival in covid-19 adiponectin-leptin ratio: a promising index to estimate adipose tissue dysfunction. relation with obesity-associated cardiometabolic risk the role of leptin/adiponectin ratio in metabolic syndrome and diabetes. horm adipose tissue dysregulation at the onset of psychosis: adipokines and social determinants of health the role of dietary fiber in rheumatoid arthritis patients: a feasibility study dietary fish oil and olive oil supplementation in patients with rheumatoid arthritis. clinical and immunologic effects dietary n-3 fatty acids and therapy for rheumatoid arthritis the effects of the mediterranean diet on rheumatoid arthritis prevention and treatment: a systematic review of human prospective studies controlled trial of fasting and one-year vegetarian diet in rheumatoid arthritis an experimental study of a mediterranean diet intervention for patients with rheumatoid arthritis diet as a modulator of intestinal microbiota in rheumatoid arthritis impact of mediterranean diet on disease activity and gut microbiota composition of rheumatoid arthritis patients. microorganisms 2020 fasting mimicking diets: a literature review of their impact on inflammatory arthritis intake of dietary fibre, red and processed meat and risk of late-onset chronic inflammatory diseases: a prospective danish study on the "diet, cancer and health" cohort do interventions with diet or dietary supplements reduce the disease activity score in rheumatoid arthritis? a systematic review of randomized controlled trials dietary risk factors for the development of inflammatory polyarthritis: evidence for a role of high level of red meat consumption the role of diet in rheumatoid arthritis impact of obesity on remission and disease activity in rheumatoid arthritis: a systematic review and meta-analysis circulating leptin level in rheumatoid arthritis and its correlation with disease activity: a meta-analysis fish oil and rheumatoid arthritis: past, present and future we thank the patients and their supportive families for participating with enthusiasm in the study. the authors declare that they have no competing interests. key: cord-0001164-64p3wpav authors: huang, shang-hui; zhao, li-xiang; hong, chao; duo, cui-cui; guo, bing-nan; zhang, li-juan; gong, zheng; xiong, si-dong; gong, fang-yuan; gao, xiao-ming title: self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin date: 2013-06-10 journal: plos one doi: 10.1371/journal.pone.0064951 sha: 955cb107f58aff48a58de2473c2fe04e42bfa1e3 doc_id: 1164 cord_uid: 64p3wpav we have recently reported that calreticulin (crt), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant crt (rcrt) exhibits extraordinarily strong immunological activities. we herein further demonstrate that rcrt fragments 18–412 (rcrt/18-412), rcrt/39-272, rcrt/120-308 and rcrt/120-250 can self-oligomerize in solution and are 50–100 fold more potent than native crt (ncrt, isolated from mouse livers) in activating macrophages in vitro. we narrowed down the active site of crt to residues 150–230, the activity of which also depends on dimerization. by contrast, rcrt/18-197 is almost completely inactive. when rcrt/18-412 is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. additionally, rcrt/18-412 oligomers are much better than monomers in binding to, and uptake by, macrophages. inhibition of macrophage endocytosis partially blocks the stimulatory effect of rcrt/18-412. we conclude that the immunologically active site of crt maps between residues 198–230 and that soluble crt could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization. calreticulin (crt) is a 46 kda ca 2+ -binding glycoprotein in the endoplasmic reticulum of eukaryotic cells [1] [2] . it folds into 3 domains including a lectin-like globular n domain (amino acid residues , a proline-rich p domain (residues 198-308) and a ca 2+ -binding c domain (residues 309-412) [3] [4] [5] . crt can also appear at the surface of various types of cells exhibiting multiple biological functions [6] [7] [8] [9] [10] [11] [12] . recently it has been shown that soluble crt is present in the sera of patients with rheumatoid arthritis and with sle [13] [14] and that natural crt (ncrt), isolated from human or mouse tissues, can directly activate macrophages in vitro [15] . additionally, rcrt/39-272, a prokaryotically-expressed murine crt fragment covering amino acid residues 39-272 fused with an n-terminal his-tag, was extraordinarily potent in activating b cells and macrophages in vitro and also in eliciting specific ab production in mice [16] . this recombinant polypeptide also exhibited potent adjuvanticity, effectively assisting igg production against conjugated target ags with or without t cell help [16] [17] . however, molecular mechanisms underlying this phenomenon are far from clear. recent x-ray crystographical studies by kozlov [18, 19] . the sequence of rcrt/39-272 encompasses most of the globular n domain (aa residues , and we have previously shown that it possess lectin-like activity (selective binding with polysaccharides including carrageenan, alginic acids, and hyaluronic acids in elisas) [16] , implying that the prokaryotically expressed recombinant polypeptide retained the lectin activity of crt. it is of interest to determine if destroying the carbohydrate binding and/or peptide-binding sites (by deleting first half of the n domain sequence) would also abolish the potent immunological activities observed in rcrt/ 39-272. additionally, the fact that rcrt/39-272 is substantially more potent than ncrt in activating macrophages(see below) raised concerns regarding the possibility of lps contamination in the e. coli.-expressed recombinant product. the ''lps contamination'' hypothesis suggests tight binding between bacterial lps and rcrt and also that, due to a synergistic effect, the lps-crt complex is a more potent immune activator than free lps and rcrt alone. based upon the observation that interaction between the n and c domains of crt influences its structural stability as well as functional activity [20] , a ''c-domain deletion'' hypothesis has also been postulated, suggesting that deletion of the c-domain (as in rcrt/39-272) leads to exposure of an immunologically active site (ias) in the n and/or p domains of crt. the present study compares the biochemical characteristics of ncrt and a series of truncated rcrt polypeptides and investigates the molecular mechanisms underlying the potent immunological activities of soluble rcrt. the results arising from this study have important implications for our understanding of the potential role of soluble crt in immunopathological conditions. native crt was extracted from mouse livers by (nh 4 ) 2 so 4 precipitation followed by ion exchange chromatography on a deae-a50 column using a linear gradient of 280-500 mm nacl for elution. samples of the eluted fractions were assayed by sds-page (fig. 1a) , which showed that the eluent between 360-380 mm nacl contained protein bands of the expected molecular weight for ncrt (55 kda), which showed approximately 90% homogeneity judging by density of the major band in coomassie blue (cbb)-stained gel (fig. 1c ). recombinant murine crt fragment with an n-terminal his-tag and without c-terminal kdel) was expressed in e. coli and affinity purified using a ni 2+ -column. the resultant rcrt/18-412 product contained 3 major protein bands at 60, 46 and 32 kda (figs. 1b & 1c); the two larger bands (namely rcrt-60 kda and rcrt-46 kda), but not the smaller one (cp32), were recognized by polyclonal rabbit-anti-crt antisera (crt-abs) in western blot (wb) (fig. 1d ). purified ncrt, but not bsa or recombinant enhanced green fluorescence protein (regfp, 28 kda with a his-tag), was positively recognized by crt-abs. as evidenced by native page analysis, a substantial amount of rcrt/18-412 formed higher-molecular-weight oligomers, whilst ncrt existed mostly in monomeric form (figs. 1e & 1f). in our previous study, rcrt/39-272 could effectively activate mouse macrophages in vitro [16] . interestingly, rcrt/18-412 was as effective as rcrt/39-272 in inducing no 2 2 production by [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] were analyzed by a sds-page 12% gel followed by cbb staining. fractions 6-10 were combined, dialyzed against pbs, and then adjusted to 1 mg/ml for use as ncrt. (b) a ni-column was employed for purification of rcrt/18-412 from a lysate of iptg-induced e. coli harboring the expression vector for rcrt/18-412. samples of e. coli before and after iptg induction were loaded in lanes, flow through (binding buffer), wash through (20 mm imidazole), resultant rcrt/18-412 (300 mm imidazole), and stripping through were loaded, in the order of 1-6 into a sds-page 12% gel followed by cbb staining. samples of ncrt, rcrt/18-412, bsa and regfp were compared in cbb-stained sds-page 12% gel (c) and native-page (e), followed by wb using rabbit anti-crt polyclonal antibody for detection (d, f). the secondary ab was hrplabeled goat-anti-rabbit igg, with opd as substrate. doi:10.1371/journal.pone.0064951.g001 mouse peritoneal macrophages in vitro, indicating that the presence, or deletion, of the c-domain did not affect the immunological activity of crt ( fig. 2a ). purified ncrt was also able to activate macrophages in vitro, but with a 50-100-fold lower potency than rcrt/18-412 ( fig. 2a) . although rcrt/18-412 and rcrt/39-272 preparations had been repeatedly treated with polymyxin b (an efficient lps inhibitor) prior to functional assays, the possibility that bacterial lps might form a tight complex with rcrt, thereby resisting pmb treatment, was always a concern. figs. 2b & 2c demonstrate that ncrt was not able to bind lps in elisas, implying that formation of tight lps-crt complexes in the expressing e. coli cells is perhaps an unlikely event. moreover, lps and ncrt at sub-optimal concentrations did not show any synergistic effect in activating macrophages (fig. 2d) , providing circumstantial evidence against the ''lps contamination'' hypothesis. contaminating rpl2 does not contribute to rcrt activity when preparing rcrt/18-412, cp32 was a relatively persistent contaminant protein (figs. 1 & 3) . to characterize this protein, the cp32 band was sliced out of sds-page gels and then (i) used to immunize c57/bl6 mice for preparation of specific antisera; and (ii) subjected to q-tof mass spectrometry analysis for sequence identification. the resultant antisera (cp32-abs) were able to recognize the immunizing protein band, but not the rcrt-60 kda and rcrt-46 kda bands, in wb (fig. 3b) . the ms result identified cp32 as bacterial 50s ribosomal protein l2 (rpl2), which was confirmed by positive recognition of recombinant rpl2 (rrpl2, commercially available) by cp32-abs (fig. 3b) . it is noteworthy that rrpl2 was unable to activate macrophages, as evidenced in no 2 2 production assays (fig. 3c ), and also that rrpl2 was not recognized by crt-abs (figs. 4c & 4d). these results exclude the possibility that the contaminant cp32 could make a substantial contribution to the immunological activities of rcrt. 2 in the culture supernatant was then determined using griess reagent and the results are expressed as mean no 2 2 concentration (mm) 6 sd. lps-based elisas were performed for detection of lps binding with crt (b) or lactoferrin (c). lf or ncrt (2 mg/ml) were added to wells in polyvinyl plates pre-coated with lps (10 mg/ml), with bsa as a negative control. combination of polyclonal rabbit abs against crt, or lactoferrin, and hrp-labeled goat-anti-rabbit igg was used for detection with opd as substrate. the results are expressed as absorbance at od492 nm6sd. for sinergy analysis (d), freshly isolated mouse peritoneal macrophages were stimulated with ncrt (0.3-30 mg/ml) in the presence, or absence, of lps (0.1 ng/ml) for 24 h. cells in medium alone (medium) or stimulated with lps (0.1 ng/ml) alone (lps) were included as controls. tnf-a in the culture supernatant was then quantitated using an elisa kit and the results are expressed as mean concentration (pg/ml)6sd. these are representatives of 3 independent experiments. doi:10.1371/journal.pone.0064951.g002 since rcrt and ncrt differ in their ability to form oligomers in solution, we next asked if the potent immunological activities of rcrt polypeptides were the result of self-oligomerization. a sephadex g-75 column was employed for fractionation of rcrt/ 18-412 oligomers and monomers. fig. 4a shows that rcrt/18-412 was successfully separated into 3 peaks, designated sequentially as orcrt (higher-molecular-weight rcrt/18-412 oligomers, major peak), mrcrt-60 kda (monomeric rcrt/18-412, 60 kda) and mrcrt-46 kda (monomeric rcrt-46 kda, aa18-386, see below). guided by the sds-page results ( , revealed their molecular mass as 46.78 kda and 43.57 kda, respectively. it can therefore be calculated that rcrt-46 kda is a degradation product of rcrt-60 kda less the c-terminal 26 amino acid residues. as shown in fig. 5a , orcrts were modestly more effective than unfractionated rcrt/18-412 in eliciting tnf-a production by murine macrophages in vitro, while mrcrt-60, mrcrt-46 and ncrt were 50-100-fold less active than orcrts by comparison. a similar conclusion was also drawn when no 2 2 production was taken as a measure for macrophage activation (fig. 5b) . moreover, s.c. immunization of balb/c mice with orcrts or unfractionated rcrt/18-412 (in the absence of adjuvant) elicited high titer serum igg capable of recognizing both orcrts and mrcrts in elisas (figs. 5c-5f). by contrast, mrcrt-60 kda (monomeric rcrt/18-412) and mrcrt-46 kda (monomeric rcrt/18-386) were almost non-immunogenic in parallel experiments. in order to assess whether the ias is located in the n or p domain, the following fragments were designed: rcrt-n (residues 18-197, full length n domain), rcrt/120-250 (partial n, half p domains), rcrt/150-230 (partial n, one third p domains), rcrt/120-308 (partial n, full length p domain), and rcrt/ 198-308 (full length p domain). all, but rcrt/198-308 (very low yield and poor solubility), were successfully expressed in e. coli and affinity-purified. both rcrt/120-250 and rcrt-n formed homodimers as well as higher-molecular-weight species (fig. 6a) . rcrt/120-250 was as potent as rcrt/39-272 and rcrt/18-412 in terms of eliciting no 2 2 production by murine macrophages, while rcrt-n was almost completely inactive (fig. 6b ). similar to rcrt/120-250, rcrt/120-308 also formed homodimers and higher-molecular-weight oligomers and showed strong macrophage-stimulatory activity in vitro (table 1) . rcrt/150-230, which contains a single cysteine residue (cys163) and could only form homodimers (fig. 6a) , was approximately 10 fold less effective than rcrt/39-272 and rcrt/120-250 but nevertheless substantially more potent than mrcrts and ncrt (fig. 6b) . a mutant form of rcrt/150-230 (namely rcrt/150-230-c163a) was prepared by substituting the cysteine residue at position 163 with alanine. the c163a mutant was neither able to form homodimers/oligomers in solution nor activate macrophages in vitro (figs. 6c-6e). these results map the ias of crt to residues 150-230 and suggest that the n-domain probably does not contribute to the immunological activity of the molecule. moreover, the importance of oligomerization for its immunological activities is further confirmed. in order to investigate the mechanisms for the relationship between rcrt oligomerization and its potent immunological activities, fractionated orcrts and mrcrt-60 kda (monomeric rcrt/18-412) were conjugated with fitc and then compared for ability to stain macrophages. after 30 min incubation at 4uc, fitc-orcrts showed stronger binding to murine macrophages than fitc-mrcrts, as evidenced by flow cytometric analysis and confocal laser scanning microscopy (fig. 7a&b) . substantially more fitc-orcrts than fitc-mrcrts were endocytosed by macrophages after 30 min incubation at 37uc (fig. 7b) . moreover, monodansylcadaverine (mdc), an endocytosis inhibitor [21] , partially suppressed no 2 2 production by macrophages under stimulation with orcrts, while lps-triggered macrophage activation was unaffected by the same treatment (fig. 7c ). in this study, we have examined different hypotheses (i.e. cdomain deletion hypothesis, lps contamination hypothesis, rpl2 hypothesis and oligomerization hypothesis) for explanation of the much stronger immunogenicity and immunostimulatory activities of rcrt than ncrt. our data strongly suggests that selfoligomerization of the rcrt polypeptides is a key factor for their strong immunological activities. as summarized in table 1 , all four rcrt fragments containing residues 120-250 (including rcrt/18-412, rcrt/39-272, rcrt/120-250 and rcrt/120-308) self-oligomerized and exhibited potent macrophage activating ability in vitro and strong immunogenicity in vivo, while ncrt, which existed mainly in monomeric form, showed only modest stimulatory activities towards macrophages and was non-immunogenic in mice (figs. 1 & 3) . moreover, fractionated rcrt/18-412 oligomers were 50-100 folds more active than mrcrts in activating macrophages in vitro (fig. 5) . the n domain polypep-tide (rcrt/18-197) also formed higher-molecular-weight oligomers (fig. 6a ), but it is almost completely inactive in terms of stimulating macrophages in vitro (fig. 6b ) and inducing ab responses in vivo (table 1) . clearly, oligomerization is necessary but not sufficient to arm the rcrt polypeptides with potent immunological activities. in general, oligomerized (aggregated) proteins are more immunogenic than their monomeric counterparts. however, the immunogenicity of orcrts is by far the most impressive and not comparable by other protein aggregates. even in the absence of any adjuvant, minute amount (1 ng/mouse) rcrt/18-412 or rcrt/39-272 can elicit strong igg responses in mice ( fig. 2; ref. 16 ). most, if not all, other protein antigens are unable to induce igg production in t-cell-deficient nude mice, yet rcrt/39-272 and rcrt/18-412 could do so relatively efficiently [16, 22] . moreover, the potent adjuvanticity of the rcrt polypeptides is also quite phenomenal. for instance, rcrt/39-272 (mostly in oligomeric forms) is able to assist the production of igg abs against fused target proteins or conjugated polysaccharides in healthy mice or t-cell-deficient nude mice [17, 22] . dimerization/oligomerization of soluble crt was also observed by previous investigators. for example, jorgensen et al documented that shielding of the free cys163 in the n domain is the main reason that ncrt exists mainly in monomeric form under physiological conditions. under partial unfolding conditions such as high temperature or low ph, however, the free cys could be exposed and subsequently help crt oligomerization [23] . ncrt (isolated from human placenta) formed homodimers and higher-molecular-weight species through disulfide bonding as well as non-covalent association, and that oligomerized ncrt showed higher binding affinity to peptides and denatured proteins [23] . in the case of prokaryotically expressed rcrt polypeptides (the folding of which may differ from ncrt), all cys residues in sequence could contribute to its olimerization, although it might not be absolutely necessary that all 3 cys residues have to be available for inter-molecular cross-linking at the same time. mancino and colleagues illustrated that self-oligomerized rcrt could better assist hla folding in vitro [24] . there are 3 conserved cysteine residues in the amino acid sequence of crt. cys105 and cys137 form intramolecular disulfide bonds, while cys163 is free [25] [26] . it is likely that rcrt polypeptides are unable to form appropriate intra-molecular disulfide bonds like in ncrt, thereby allowing all 3 cysteine residues to participate in self-oligomerization, although formation of higher-molecularweight oligomers could also occur through non-covalent association of crt [23, 27] . crt is considered one of the heat shock proteins (hsps) that share many immunological and biochemical activities [28] [29] . interestingly, self-oligomerization also occurs to other hsps such as grp94 and hsp90, which is likely associated with their chaperone function [30] [31] [32] . koslov et al and chouquet et al have recently solved the crystal structure of the lectin site as well as a peptide-binding site in the crt n domain [18, 19] , which apparently play important roles in the physiological function of crt. however, our data maps the ias of crt to a region of 80 residues between aa150-230 (fig. 6 ). as rcrt-n was almost completely inactive in functional assays (fig. 6, table 1 ), the ias may be narrowed down further to aa198-230 in the p domain, although a series of truncated synthetic peptides covering this region would be needed for a concrete conclusion. interestingly, this sequence of 30 amino acid residues contains the ra shared epitope (se)-binding site (residues 217-224) of crt recently mapped by ling et al. [33] . such coinciding results from 2 independent groups further emphasize the importance of the aa198-230 region of the p domain to the immunological activities of crt. it has been documented that the crt p domain adopts a hairpin-shaped structure, its sequence is consisted of 3 copies of a repeat motif (type 1: ixdpxxxk-pedwd) followed by 3 copies of another repeat motif (type 2) [34] [35] [36] . interestingly, the se-binding site almost completely overlaps the type 1 motif [33] . it is reasonable to suggest that the type 1 motif might also represent the core of ias of crt, responsible for direct biding with activation receptors on the surface of immune cells. functional comparison data showed that rcrt/120-308 and rcrt/120-250 (possessing 3 type 1 repeats, the latter without type 2 repeats) are 10 times more active than rcrt/120-230 (with 2 type 1 repeats, no type 2 repeats) in activating macrophages (table 1, fig. 6b ), implying that (i) type 2 repeats do not contribute to the immunological activity of the molecule; and (ii) the presence of 3 copies of the type 1 motif is of crucial importance for the potent immunological function of orcrts. it can be envisaged that oligomerization of crt multiplies its binding avidity to immune cells with receptors for the type 1 motif, thereby enabling it to deliver stimulatory signals to the cells in a highly efficient manner. we further predict that the high-avidity binding of orcrts to macrophages may easily trigger their uptake process. indeed, orcrts can activate macrophages in an endocytosis-dependent pathway (fig. 7) . perhaps orcrts could use certain intracellular sensors to deliver immunostimulatory activity to the responding immune cells. an example of an intracellular sensor for endocytosed polymeric proteins is the nod-like receptor (nlr) protein nlrp3, a key component of the nlrp3 inflammasome and an important intracellular sensor for microbial ligands and endogenous danger signals [37] . masters and colleagues demonstrated that soluble oligomers of islet amyloid polypeptide (iapp), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, could be endocytosed and trigger the nlrp3 inflammasome and generate mature il-1b [38] . finally, the region of aa150-230 of crt has a 98% homology between mouse and human. it would be of interest to examine if there is a functional relationship between the se binding site and the ias of crt. irrespective of such a relationship, oligomerization might occur to extracellular crt released by tissue cells thereby converting crt into a highly active form, which may play important roles in the development and pathogenesis of autoimmune disorders in humans. purification of ncrt ncrt was purified from mouse livers using a modification of a previously described methods [39, 40] . briefly, fresh mouse liver cells (erythrocytes depleted) were collected and centrifuged at 1200 rpm for 5 min. the cell pellet was lysed in 3 volumes of lysis buffer (1% triton-x 100, 0.2 mm pmsf in pbs) for 30 min on ice, followed by centrifugation at 35,000 g for 60 minutes. the supernatant was then precipitated using (nh 4 ) 2 so 4 and the final precipitate dissolved in binding buffer (150 mm nacl, 20 mm tris, ph7.4) followed by dialysis against this buffer. the sample was applied to a deae sephadex a50 column (1062 cm, ge healthcare, us) which was then sequentially washed with binding buffer and washing buffer (280 mm nacl, 20 mm tris, ph7.4) at 1 ml/min to remove contaminating proteins. the fractions were eluted with a linear salt (280-500 mm nacl) gradient. the preparation of rcrt/39-272 and regfp was as previously described [16] and the other rcrt polypeptides used in this study were prepared using the same prokaryotic system. specific primers were as follows: 59-cccaagcttctaatctgtggggtcatc-gatcttg-39. the rcrt/150-230-c163a mutant was constructed using takara mutan best kit (takara biotechnology co. ltd) following manufacturer's instruction. the mutant primer was as follows: sense: 59-attcacacacctataca-cactgatt-39, antisense: 59-tcatcatccttagcccgga-tatcct-39. all proteins were desalted by passing through pd10 columns (pierce, rockford, il, usa). protein concentration was determined using coomassie protein assay reagent (pierce, rockford, il, usa). all recombinant proteins were used at over 90% purity as judged by cbb-stained sds-page gels. the separated protein bands in sds-page gels were electroblotted onto pvdf membranes, at a constant current of 250 ma in transbuffer (50 mm tris, ph 8.0, containing 0.192 m glycine and 20% methanol), using a bio-rad trans-blot cell. the strips were incubated for 1 hr at room temperature in blocking buffer (tbs containing 5% nonfat milk), followed by a overnight incubation at 4uc with constant agitation in indicated antibody diluted in blocking buffer. after 3 washes with tbs containing 0.05% tween 20, strips were incubated for 1 hr with hrpconjugated secondary antibody (southern biotechnology associates inc., usa) and visualized using the ecl detection system as recommended by the manufacturer (applygen technologies inc., beijing, china). lps-based and crt-based elisas were as previously described [41] . briefly, elisa plates were coated at 4uc overnight with rcrt or lps and subsequently incubated with blocking solution (1% bsa in pbs) for 2 hrs at 37uc. the wells were washed five times with pbs containing 0.05% tween 20 (pbs-t) prior to incubation at 37uc with 100 ml of diluted mouse sera or with indicated protein (ncrt and lactoferrin) followed by corresponding antibody in triplicate. after 5 washes with pbs-t, the plates were further incubated with hrp-labeled goat-antimouse or goat-anti-rabbit igg abs (southern biotechnology associates inc., al., usa) for 1 hr at 37uc. the reaction was developed with 100 ml of o-phenylenediamine (opd, sigma) for 5 min and stopped with 100 ml 2 m h 2 so 4 . optical density (od) was measured at 492 nm in an elisa spectrophotometer (titertek multiscan plus mk ii; icn flow laboratories, irvine, uk). all cells were cultured in complete r10 medium: rpmi-1640 supplemented with 10% (v/v) fetal bovine serum (hyclone, usa), penicillin/streptomycin (100 u/ml), l-glutamine (2 mm), and b-me (5610 25 m). for preparation of mouse peritoneal macrophages, mice were injected i.p. with 3% thioglycollate (1 ml/ mouse) and the macrophages retrieved from the peritoneum 3 days later using a syringe. the resultant cells were.90% positive for f4/80 marker, as determined by facs analysis. relative macrophage stimulation activity (rmsa) of crt is estimated using ncrt as reference, which is able to induce tnf-a production by macrophages in vitro at a concentration of 10 mg/ml or above (see fig. 5a ). the listed results are based on several batches of independent experiments including fig. 5a . b) ability to elicit specific igg responses in healthy balb/c mice after s.c. immunization, in the absence of adjuvant, with 100 mg protein and a booster immunization with 50 mg protein a fortnight later. the mice were monitored for up to 28 days after the second immunization. nd: not detected; -: not immunogenic; +: strong humoral response; 2/+: weak response. freshly prepared c57/bl6 mouse peritoneal macrophages (1.5610 5 cells/well) were stimulated with rcrt fragments, or ncrt, or lps, in r10 medium in 96-well tissue culture plates for 24 hrs in a 5% co 2 incubator at 37uc. the concentration of tnf-a in the culture supernatant was determined using elisa kits (biolegend, san diego, usa) following the manufacturer's instructions. the concentration of no 2 2 in the supernatant was determined by griess reagent. standard curves were established using nano 2. female c57bl/6 mice between the age of 6-8 weeks were purchased from the model animal research center, nanjing, china. all animals were maintained under specified-pathogen-free (spf) conditions and animal usage was conducted according to protocols approved by the soochow university institutional animal care and use committee. for immunization with rcrt, mice were immunized s.c. at the base of the tail with 100 mg protein in total 100 ml pbs. when booster immunization was needed, 50 mg of protein in 200 ml pbs was injected intraperitoneally (i.p.). for immunization with page gel slices containing cp32, the band was cut out with a razorblade and then frozen in liquid nitrogen and emulsioned with cfa, which was then injected s.c. into female c57/bl6 mice. serum samples were collected by tail bleeding, aliquoted and kept at 220uc until use. a sephadex g-75 (ge healthcare, us) column of 8062 cm was employed. 5 ml of rcrt/18-412 at 10 mg/ml was loaded into the column, followed by elution with 0.9% nacl at 20 ml/h and collected every 2 ml. protein was labeled by fitc using fluorotag tm fitc conjugation kit (sigma, us) according to the manufacturer's instructions. in brief, 1 mg of protein was dialyzed against 0.1 m na 2 co 3 , ph 9.5 at 5 mg/ml. fluoresceine isothiocyanate (fitc, sigma) was dissolved in the same buffer with dmso at 1 mg/ml and 50 ml of this solution was added to the protein. the sample was gently mixed for 2 h at rt. separation of labeled protein from unbound fitc was performed on a sephadex g-25 column. flow cytometric analysis 10 6 freshly isolated peritoneal macrophages were stained with apc-anti-f4/80 and then incubated with 15 mg/ml of fitc-orcrt, fitc-mrcrt or fitc-ova for 30 min at 4uc. the binding of crt by macrophages was determined by flow cytometry (bd facs canto ii, us). macrophages (1.5610 5 /well) were plated onto poly-l-lysine coated glass slides and allowed to adhere. the cells were then incubated in a total volume of 200 ml 0.5% bsa in pbs with 15 mg/ml fitc-orcrt, fitc-mrcrt or fitc-ova for 30 min at 4uc or 37uc. after washing to remove unbound protein, macrophages were fixed in 1% paraformaldehyde and stored at 4uc until microscopic analysis. finally, 1% of glycerol was added and slides were counterstained with 1 mg/ml dapi. the cells were imaged with a nikon confocal microscope system a1. all experiments were repeated at least 3 times and the results are expressed as mean6standard deviation of the mean (sd). statistical analysis was performed using the independent-samples t test or two-side paired t test between groups using the spss 14.0 program (spss, chicago, il). differences were considered statistically significant at p,0.05. calreticulin: one protein, one gene, many functions definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver golgi delineation of the lectin site of the molecular chaperone calreticulin oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin calreticulin is expressed on the cell surface of activated human peripheral blood t lymphocytes in association with major histocompatibility complex class i molecules cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin activation of human monocyte cell line u937 via cell surface calreticulin cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of lrp on the phagocyte calreticulin in tlymphocytes. identification of calreticulin in t-lymphocytes and demonstration that activation of t cells correlates with increased levels of calreticulin mrna and protein regulation of peripheral t cell activation by calreticulin candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes calreticulin is released from activated neutrophils and binds to c1q and mannan-binding protein cd91-dependent programming of t-helper cell responses following heat shock protein immunization functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease ajuvanticity of recombinant calreticulin calreticulin as a hydrophilic chimeric molecular adjuvant enhances igg responses to the spike protein of sars coronavirus structural basis of carbohydrate recognition by calreticulin x-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin adjuvanticity of recombinant calreticulin fragment 39-272 in assisting anti-b-glucan igg responses in t celldeficient mice dimerization and oligomerization of the chaperone calreticulin calreticulin recognizes misfolded hla-a2 heavy chains calreticulin, a ca2 + -binding chaperone of the endoplasmic reticulum human placental calreticulin characterization of domain structure and post-translational modifications the metal ion binding properties of calreticulin modulate its conformational flexibility and thermal stability heatshock proteins as activators of the innate immune system heat shock proteins as regulators of the immune response heat-induced chaperone activity of hsp90 endoplasmic reticulum chaperone grp94 subunit assembly is regulated through a defined oligomerization domain substrate-binding characteristics of proteins in the 90 kda heat shock protein family identification of the rheumatoid arthritis shared epitope binding site on calreticulin trosy-nmr reveals interaction between erp57 and the tip of the calreticulin pdomain nmr structure of the calreticulin p-domain nmr structures of 36 and 73-residue fragments of the calreticulin p-domain nlrp3: an immune sensor of cellular stress and infection activation of the nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced il-1beta in type 2 diabetes human placental calreticulin: purification, characterization and association with other proteins a single purification procedure for the major resident proteins of the er lumen: endoplasmin, bip, calreticulin and protein disulfide isomerase heat shock protein 60: specific binding of lipopolysaccharide key: cord-0054340-tlg21uma authors: mong, nikolett; tarjanyi, zoltan; tothfalusi, laszlo; bartykowszki, andrea; nagy, aniko ilona; szekely, anett; becker, david; maurovich-horvat, pal; merkely, bela; nagy, gyorgy title: largely accelerated arterial aging in rheumatoid arthritis is associated with inflammatory activity and smoking in the early stage of the disease date: 2020-11-26 journal: front pharmacol doi: 10.3389/fphar.2020.601344 sha: 498118ea64ae49a54c0dfc5d9e0ac3cbac9fe88a doc_id: 54340 cord_uid: tlg21uma background: rheumatoid arthritis (ra) patients have a shorter life expectancy than the general population primarily due to cardiovascular comorbidities. objectives: to characterize arterial aging in ra. patients and methods: coronary calcium score (ccs) were available from 112 ra patients; out of these patients, follow-up ccs were measured for 54 randomly selected individuals. control ccs were obtained from the mesa database (includes 6,000 < participants); arterial age was calculated from ccs. results: ra patients were significantly older (10.45 ± 18.45 years, p < 0.001) in terms of the arterial age than the age-, gender-, and race-matched controls. the proportion of ra patients who had zero ccs was significantly less (p < 0.01) than that of those in the mesa reference group. each disease year contributed an extra 0.395 years (p < 0.01) on the top of the normal aging process. however, the rate of the accelerated aging is not uniform, in the first years of the disease it is apparently faster. smoking (p < 0.05), previous cardiovascular events (p < 0.05), and high blood pressure (p < 0.05) had additional significant effect on the aging process. in the follow-up study, inflammatory disease activity (crp > 5 mg/l, p < 0.05) especially in smokers and shorter than 10 years of disease duration (p = 0.05) had the largest impact. conclusion: arterial aging is faster in ra patients than in control subjects, particularly in the first 10 years of the disease. inflammation, previous cardiovascular events, and smoking are additional contributing factors to the intensified coronary atherosclerosis progression. these data support that optimal control of inflammation is essential to attenuate the cardiovascular risk in ra. rheumatoid arthritis (ra) is a heterogeneous autoimmune condition; it affects 0.5-1% of the population and is associated with disability and systemic complications (turesson et al., 2006; prete et al., 2011; das and padhan, 2017) . both genetic and environmental factors have a central role in the pathogenesis of the disease, and cigarette smoke is the strongest known environmental factor (mcinnes and schett, 2007; baka et al., 2009; mcinnes and schett, 2011) . in ra, ongoing inflammation leads to cartilage destruction, bone erosions, and subsequent joint deformities. although the current treatment strategy, principally the widespread use of biological therapies, improved the outcome of the disease, the mortality rate is still considerably higher among patients with ra than among healthy persons and systemic complications, especially cardiovascular (cv) risk due to ra, and represent a significant challenge (liu et al., 2017; nagy et al., 2018) . although biologicals have a beneficial effect on the cv risk in ra, tnf and il6 inhibitors often increase the total cholesterol and triglyceride levels (roubille et al., 2015; gabay et al., 2016; giles et al., 2019) . in addition to the traditional cardiovascular risk factors (hypertension, diabetes, smoking, hyperlipidemia, alcohol, and physical inactivity), the effect of chronic inflammation on cardiovascular mortality is a rapidly developing field of interest (ormseth et al., 2015) . elevated crp level is considered as a cardiovascular risk factor (yousuf et al., 2013; fonseca and izar, 2016) . it is noteworthy that the risk of acute myocardial infarction (ami) in ra is similar to the risk of ami in diabetes mellitus (nurmohamed et al., 2015) . coronary artery disease in ra appears more often in multivessel form (karpouzas et al., 2014) . in ra, the inflammation is associated with the presence of high-risk plaques (aubry et al., 2007; karpouzas et al., 2014) . coronary calcium score (ccs) is a well-established diagnostic marker showing calcium deposits in coronary arteries. it is known to be influenced by several factors including age, gender, race (greenland et al., 2018) , smoking (shaw et al., 2006) , high crp levels (defined as higher than 5 mg/ l), cardiovascular disease, high blood pressure, and diabetes, although the connection between diabetes and ccs is controversial (raggi et al., 2004; giles et al., 2009) . the ccs assessment is a noninvasive method that has a great value in cardiovascular risk stratification, showing a significant association with the medium-or long-term occurrence of major cardiovascular events (o'rourke et al., 2000; neves et al., 2017) . the prevalence of coronary artery calcium (cac) increases with age, ranging from 5% in a middle-aged cohort to more than 50% in an elderly cohort . a meta-analysis including asymptomatic individuals indicated that those with coronary artery calcification above the median have an 8.7-fold increased risk of future coronary events (o'malley et al., 2000) . in addition, there are data indicating that progression in ccs is associated with higher risk of myocardial infarction (raggi et al., 2000; raggi et al., 2003) , and coronary artery calcification adds information to the prediction of overall mortality . it has been proposed that cac can be used to estimate the arterial age in adults. although the increased cardiovascular risk is widely accepted in ra, the risk factors associated with the chronic autoimmune disease are less clear. the traditional cardiovascular risk scores underestimate the real cardiovascular risk in ra (crowson et al., 2012; kawai et al., 2015; wahlin et al., 2019) . ccs is better in cv risk stratification in ra than in combinations of the traditional cv risk factors (korley et al., 2017; karpouzas et al., 2020) . here, we investigated the baseline and follow-up ccs of ra patients and studied its progression over time. our present result underscores the impact of inflammation on the cv risk in ra, especially in the first ten years of the disease. all ra patients were recruited in the rheumatology outpatient department of the semmelweis university (polyclinic of hospitaller brothers of st. john of god, budapest, hungary). patients ≥ 18 years of age and diagnosed with ra (n 112) according to the 2010 american college of rheumatology/ european league against rheumatism classification criteria (aletaha et al., 2010) were enrolled. exclusion criteria included concomitant autoimmune disease, except sjögren's syndrome, malignant diseases, chronic infections with or without fever, and known psychiatric disease. the demographic data and the clinical parameters of the patients are summarized in tables 1, 2 . hypertension, diabetes mellitus, and hyperlipidemia were evaluated based on the standard criteria; smoking history was recorded (smoker/nonsmoker). disease activity was evaluated by using the 28-joint counts and erythrocyte sedimentation rate-based (disease activity score/das28) score at each visit. medications were recorded including glucocorticoids, nsaids, conventional and targeted disease-modifying antirheumatic drugs (dmards), and statins. both national and institutional ethics committees approved the study, and informed consent was obtained from each individual [approval number: if 567-4-2016] . this work was carried out in accordance with the helsinki declaration. control population: the multi-ethnic study of atherosclerosis (mesa) database was used as control. mesa is a prospective cohort study with an aim to investigate predictors of cardiovascular risk factors; coronary artery scan was performed in 6,814 participants without apparent cardiovascular problems. we refer to this population as "healthy" population or "mesa" population (blaha et al., 2016) . age-, gender-, and race-matched control data were generated by using the online ccs calculator (https://www. mesa-nhlbi.org/calcium/input.aspx). based on demographic data and the measured ccs, the online calculator provided the estimated probability of having higher than zero calcium, and the 25th, 50th (median), 75th, and 90th ccs percentiles of in a "healthy" (i.e., without apparent cardiovascular disease) population. using these percentiles and inverse quantile transformation, we simulated 100 age-, gender-, and raceadjusted ccs for each patient in our study. in this way, the control population consisted of 100 age-, gender-, and racematched subjects from the mesa database for each ra patient, altogether 11,200 subjects. all ra participants underwent non-contrast-enhanced, prospectively ecg-triggered scan of the heart using a 256-slice multidetector ct (brilliance ict 256; philips healthcare, best, the netherlands) at the heart and vascular center of semmelweis university. images were acquired in cranio-caudal direction during a single breath hold in inspiration, at 78% of the r-r interval, with a slice thickness of 2.0 mm. the following acquisition parameters were used: 128 × 0.625 mm detector collimation, 270 ms gantry rotation time, 120 kv tube voltage, and 30 ma s tube current. the quantification of cac was performed on the axial images on a per-patient and per-vessel basis using a semi-automatic software (heartbeat-cs, philips healthcare, best, the netherlands). ccs were computed by the standard calcium scoring algorithm of agatston (agatston et al., 1990) . follow-up ccs measurement was performed for 54 patients. arterial age is an easy to understand intuitive concept; it shows the apparent age of arteries using healthy population as a reference. therefore, to help the clinical interpretation of the results, ccs were transformed into "arterial age" using the formula of mcclelland et al. (2009): arterial age 39.1 + 7.25 log(ccs + 1). (1) to facilitate the statistical inference, we introduced an additional variable called artage_dif. the variable artage_dif measures difference between the observed and the control arterial ages. for each patient, there were 100 age-, gender-, and race-matched controls, and we obtained artage_dif by subtracting the median of the corresponding controls from each observed value. stata version 15 (statacorp lp, college station, tx) was used for statistical analysis and r (r core team, 2017) with several additional packages such as ggplot2 (wickham, 2016) for additional programming tasks and visualization. the difference between the measured and mesa control population values was tested using the one-sample alternative of the tests; that is, we assumed that simulated control population is not different from the "true" population. percentages of subjects having nonzero ccs in the observed and computer-predicted populations were compared with exact binomial test. the effect of factors that may influence ccs was studied by graphical analysis followed by univariate testing and multivariate linear regression modeling. due to highly nonnormal distribution of the data, we gave preference to nonparametric methods such as rank-based tests or we took advantage of the "robust" option available for many procedures in stata. to illuminate the trends, we fitted locally weighted polynomial regression commonly known as lowess. descriptive summaries such as proportion, means, and standard deviation (sd) are provided for all clinically relevant baseline variables, and statistical significance level was set to p < 0.05, two-tailed. ccs were measured in 112 ra patients; control ccs were obtained from the mesa database. figure 1 upper panel compares the two arterial age distributions. both histograms can be split into two parts. there is one single outstanding bar which represents subjects with zero ccs, and the arterial age of these subjects is by definition exactly 39.1. a characteristic feature of both curves a rightly skewed distorted bell-shape kind of part ranging from 39.1 up to hundred. these parts correspond to subjects having higher than zero ccs. although the general features are similar, there are noteworthy differences between the two histograms. smaller percentage of ra patients have zero ccs, and compared to controls, the center of the histogram is shifted to right, toward older ages. these visual impressions were confirmed by the statistical analysis. the ccs calculator gave the probability for each patient having higher than zero calcium. the average of these predicted probabilities was 0.517, while the found ratio is 0.642. the difference between the groups is highly significant (p 0.008). the assumption that arteries are older in ra compared to the matched controls was further tested with the help of variable artage_dif, which is the difference of the observed value from the median of the controls. statistical theory suggests that if there is no difference between the mesa and ra populations, then the distribution of artage_dif has symmetric distribution around zero. figure 1 lower panel shows that the difference distribution is not symmetric but clearly right skewed with a median of 6.34 years. this difference from the expected zero is highly significant (z 5.51, p < 0.0001). because artage_dif has right skewed distribution (figure 1 lower panel) , the mean difference is higher than the median. as table 2 shows, the mean difference is 10.45 (sd: 18.45) years, which means that the arteries of the ra patients in average are 10.45 years older than those of their mesa counterparts' (p < 0.001). as expected, the inflammatory markers (crp and esr) strongly correlated with each other and with the clinical disease activity (das28), data not shown. the correlation between artage_dif and the disease duration is also significant (r 0.22, p < 0.05). comorbidities, autoantibodies (anti-cyclic citrullinated peptide/rheumatoid factor), smoking, and the arterial age figure 2 displays the dependence of artage_dif on categorical covariates. the differences between nonsmokers/smokers, patients without and with cardiovascular events, and patients with normal and high blood pressure were significant (p 0.016, 0.029, and 0.023, respectively). by contrast, neither the presence of rheumatoid factor (rf), anti-cyclic citrullinated peptide (accp), nor diabetes proved to be predictive. we also checked if the previous statistical conclusions remain valid if we exclude diabetic patients' data from the analysis. seventeen patients (15.1%, table 1 ) had diabetes. excluding these individuals, 64.3% of the remaining patients had ccs above zero. this ratio is still significantly higher than the age-and gendermatched mesa control (49.4%, p 0.004). the same is true for the difference from the matched medians (6.12 years, p < 0.001). existing cardiovascular disease or history of cardiovascular events is associated with higher ccs (lehker and mukherjee, 2020) . subjects with existing cardiovascular condition were excluded from the mesa study, while 17 patients in our ra study group (15.18%, table 1 ) had preexisting cardiovascular conditions. therefore, the question arises to what extent the observed differences are due to the cardiovascular events. to answer this question, we split the ra study group into four subpopulations using the criteria that a patient had a zero ccs or the ccs was above it and that a patient had or had not previous cardiovascular disease. in the left panel of supplementary figure s1 , proportions of patients with zero ccs are compared. both ra subgroups are significantly different from their corresponding matched samples although the difference is much more pronounced in patients with previous cardiovascular disease (supplementary figure s1 ) (p 0.023 and p 0.005 respectively). such difference between the two ra patient subgroups is not seen in the right panel of supplementary figure 1 | arterial age in rheumatoid arthritis. upper panel: arterial age, converted from the observed and age-, gender-, and race-adjusted simulated coronary calcium scores. the figure shows the resulting distributions of the observed and simulated data. the shaded bars are the simulated control subjects, and the transparent bars with red contours represent the observed values. lower panel: arterial age distributions of patients with ra and the control populations are different. artage_dif is the difference between the observed and the median of the control arterial ages. if there is no difference between ra patients and control subjects, then the histogram of artage_dif must be symmetric around zero. using wilcoxon signed test, we showed that this null hypothesis is unlikely (p < 0.001). frontiers in pharmacology | www.frontiersin.org november 2020 | volume 11 | article 601344 figure s1 , and the medians of the matched arterial age differences are practically the same (16.7 and 18.1 years) for patients without and with cardiovascular disease. both of them are significantly different from the expected zero (p < 0.001). these data suggest that the effect of ra on the ccs is mostly due to the accelerated progression rate. cardiovascular disease is an additional risk factor because patients with cardiovascular disease have accelerated conversion rates from being ccs negative to ccs positive. the rate of conversion is significantly higher not only to control (p 0.005) but also compared to ra patients without cardiovascular disease (p 0.012). the analysis presented above suggests that the rate of the calcium build-up process in the control and ra populations is markedly different. indeed, the time from the onset of the disease (disdur) was the only variable which showed significant correlation with the arterial age acceleration (data not shown). however, as the left panel of figure 3 shows, the aging process is not constant. it increases monotonically (r s 0.249, p 0.008), but the fitted nonparametric regression line (span 0.9) suggests that the increase is faster in the first 10 years. nevertheless, to get a numerical estimate of the arterial aging relative to the control, we fitted linear regression between disdur and artage_dif. the fitted regression line is displayed in the right panel of figure 3 . the slope of the regression line is 0.395 (95% ci 0.10-0.68), significantly different from zero (t 2.74, p 0.007). the meaning of this 0.395 is that difference from the control increases by 0.395 years in every year of the disease; thereafter, in average, every year with ra contributes 0.395 extra arterial years. fifty-four patients had a follow-up ccs measurement. supplementary table s1 summarizes the das, crp, and esr values at the time of the two measurements. generally, there is strong correlation between these values (data not shown). the average time between the ccs scans was 1.28 ± 0.35 years. the annual arterial aging rate was calculated by dividing the difference between the second and first arterial age measurements with the length of the time interval between the two measurements. the least-square estimates with the corresponding 95% confidence intervals are shown in the left panel of figure 4 . the estimated aging rate is 1.44 (95% ci 0.77-2.11), which is in good agreement with the previous regression estimate. nevertheless, from the further analysis, we excluded two subjects who converted during the observation period (i.e., their first ccs was zero and the second above zero, with estimated aging of 8.1 and 12.8 years, respectively), because from a statistical viewpoint, they were gross outliers. without these two values, the rate estimate was additional regression analysis showed that the aging process is significantly faster in patients who had elevated crp (>5 mg/ l) level (p 0.024) and in case the time since diagnosis is less than 10 years (p 0.05). the least-square approach allowed us to investigate the combined effect of two or more risk factors. figure 4 demonstrates that in smokers with elevated crp levels, the arterial aging rate is doubled. the additionally computed nonparametric test confirmed the significance of the elevated crp (p 0.0494). the middle and the right panels of figure 4 further demonstrate the effect of crp and disease duration <10 years on the arterial aging. figure 4 | follow-up data: the effect of inflammation, cv diseases, smoking, and ra disease duration on the arterial aging. the arterial aging rate was calculated by dividing the difference of the two consecutive arterial age estimates with the time interval between the two measurements. the left panel shows least-square estimates of five risk factors on the rate: smoking, disease duration ≤10 years (disdur < 10), history of cardiovascular disease (cvevent), crp 5 > 5 mg/l, and concomitant antihypertensive drug taking (bp_high). when the 95% confidence intervals do not cross zero the effects are significant, which is true only for crp5 (p 0.024) and disdur < 10 (p 0.05). in the absence of any risk factors (which includes that the disease started more 10 years ago), the aging rate is close to zero (base). the combined effect of two risk factors such as crp and smoking shown in the figure is an estimated marginal effect. the two boxplots illustrate the effect of crp5 and disdur < 10. frontiers in pharmacology | www.frontiersin.org november 2020 | volume 11 | article 601344 from the fifty-four patients in the follow-up study, thirty-one received biological therapy (twenty-seven patients anti-tnfalpha, four patients non-anti-tnf-alpha bdmards) and twenty-three csdmard therapy only. the yearly arterial aging progression rate in the follow-up group in patients receiving bdmard therapy was numerically lower (1.16 ± 0.35) than that in patients receiving csdmards (1.73 ± 058); the difference (0.57 ± 0.67) was not significant (t −0.85, p 0.389). primarily due to cardiovascular comorbidities, patients with ra die significantly earlier (nurmohamed et al., 2015; nagy et al., 2018) , and the risk of sudden cardiac death is doubled in ra compared to the general population (maradit-kremers et al., 2005; naranjo et al., 2008; karpouzas et al., 2014; jagpal and navarro-millan, 2018) . due to the accelerated cardiovascular risk, the precise risk evaluation is essential. our present data confirm and extend previous observations regarding the increased cardiovascular risk in ra. here, we show for the first time the profound effect of ra on the arterial age, compared to the mesa population. older arterial age was associated with smoking, previous cardiovascular events, and hypertension. the follow-up substudy was self-controlled and highlighted the importance of other additional factors. ongoing inflammation (crp > 5 mg/l), especially in smokers, and shorter disease duration (<10 years) accelerated arterial aging according to our follow-up data. therefore, the increased cardiovascular risk due to ra increases with the disease years, but the augmentation is not linear, and in the first 10 years of the disease, the arterial aging is apparently more pronounced. inflammation plays a central role in the pathogenesis of atherosclerosis; elevated levels of c-reactive protein (crp), interleukin-6, and n-terminal pro-hormone b-type natriuretic peptide (ntprobnp) correlate closely with cardiac events (sabatine et al., 2004) . epidemiological studies suggested that chronic inflammation is associated with higher cardiovascular risk. in addition, inflammation is a recognized risk factor of ami in ra (meissner et al., 2016) . a number of inflammatory mediators have been widely studied, both as surrogate biomarkers and as causal agents, in the pathophysiological network of atherogenesis and plaque vulnerability (libby et al., 2002) . moreover, it was suggested that inflammatory processes and cytokines are similar in ra and in atherosclerotic vascular diseases (plein et al., 2020) . low disease activity is associated with decreased risk of cve in ra . in patients with ra, inflammatory markers, disease severity, and rf positivity were found to be associated with the risk of atherosclerosis (sattar et al., 2003) . acpa and rf are both unfavorable prognostic factors in ra; in accordance with our present data, both autoantibodies are independent of the accelerated arterial aging in ra (berendsen et al., 2017) . the age is an independent risk factor for cardiovascular diseases; nevertheless, often the atherosclerotic disease burden is discordant with a patient's chronological age. calcium is a general component of the atherosclerotic plaque, but not that of the normal vessel wall (o'rourke et al., 2000) . because of this structural difference, calcium is an accurate index of atherosclerotic disease burden and a useful tool to estimate the risk of cardiovascular adverse outcomes (raggi et al., 2000; kondos et al., 2003) . previous studies showed the importance of age-specific ccs percentiles to predict the occurrence of a cardiovascular event in patients with a similar risk profile (wong et al., 2002) . ccs is a widely accepted marker of coronary atherosclerosis. in the mesa population, a doubling of the ccs increased the probability of a coronary event by 25% in a 3.8-year follow-up period. importantly, this predictive value was relatively stable across different ethnic groups (detrano et al., 2008) . similar to our present data, ra severity was associated with the greater prevalence of coronary artery calcification than the mesa population (giles et al., 2009) . the framingham risk score includes age, gender, total and hdl cholesterol, blood pressure, diabetes, and smoking. however, long-standing patients with ra had higher framingham risk scores than patients with early disease or control subjects. furthermore, long-standing inflammation represents additional cardiovascular risk . moreover, the presence of cac has been shown in early ra as well (logstrup et al., 2017) . the lack of diabetes effect in our study was somewhat surprising because it is generally presumed that the ccs is independently associated with incident coronary heart disease in diabetes (malik et al., 2017) . framingham risk based on arterial age is more predictive of short-term incident coronary events than framingham risk based on the observed age (mcclelland et al., 2009) . according to recently published data, coronary artery calcification increases with higher total prednisone dose; by contrast, methotrexate and other csdmards do not influence coronary plaque progression (karpouzas et al., 2020) . furthermore, dmard and tnf-α antagonists are associated with reduced risk of myocardial infarction, stroke, and cardiovascular death (micha et al., 2011; westlake et al., 2011) . we could demonstrate neither positive nor negative effect of the applied drug therapy on the arterial aging process. it was somewhat surprising that anti-inflammatory drug therapy did not have a clear effect on the arterial aging process, in our present study. this could be explained by the relatively low number of patients and the limited period covered in the follow-up study. by contrast, in a recent study, another cardiovascular marker, vascular stiffness, significantly and consistently improved in ra patients, treated with biological or conventional synthetic dmard therapy. ra has profound impact on the vasculature; already at time of the diagnosis, early ra patients have reduced vascular distensibility (plein et al., 2020) . impaired endothelial function, a key event in the progression of atherosclerosis, was observed in ra. in addition to synovial lesions inflammation leads to vessel wall involvement as well. it was reported that endothelial dysfunction can be improved during anti-tnf-alpha therapy (hürlimann et al., 2002) . however, the limited study power prohibits to draw any definite conclusions regarding the frontiers in pharmacology | www.frontiersin.org november 2020 | volume 11 | article 601344 effect of statins and targeted therapies on arterial aging. there are other limitations of our work as well: most of our patients had moderate disease activity, and untreated patients with early disease were not included in this study. ra significantly accelerates arterial aging; additionally to other risk factors, inflammation might be the pathophysiological link between ra and the increased calcification process. further largescale studies are needed to investigate the potential clinical benefit of ccs measurement in ra patients with risk factors for ischemic coronary heart disease. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by both national and institutional ethics committees. if 567-4-2016. the patients/participants provided their written informed consent to participate in this study. nm performed design of the study, data collection, first draft of the manuscript, and data interpretation; zt performed design of the study, data collection, and data interpretation; lt performed statistical analysis; ab, as, and ain performed data collection; db and pmh performed data interpretation; and bm and gn performed design of the study, manuscript preparation, and data analysis. all authors have approved the submitted version. all authors have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. this publication was supported by the national research, development, and innovation office of hungary (nvkp_16-1-2016-0017 national heart program) and by hungarian scientific research fund grant k 131479. the authors are grateful for the investigators, the staff, and the participants of the mesa study. ai nagy was supported by the jános bolyai scholarship of the hungarian academy of sciences.thematic excellence programme (2020-4.1.1.-tkp2020) of the ministry for innovation and technology in hungary, within the framework of the therapeutic development and bioimaging programmes of the semmelweis university. quantification of coronary artery calcium using ultrafast computed tomography low disease activity (das28≤3.2) reduces the risk of first cardiovascular event in rheumatoid arthritis: a time-dependent cox regression analysis in a large cohort study differences in atherosclerotic coronary heart disease between subjects with and without rheumatoid arthritis rheumatoid arthritis and smoking: putting the pieces together anticyclic citrullinated peptide antibodies and rheumatoid factor as risk factors for 10-year cardiovascular morbidity in patients role of coronary artery calcium score of zero and other negative risk markers for cardiovascular disease utility of the framingham risk score to predict the presence of coronary atherosclerosis in patients with rheumatoid arthritis usefulness of risk scores to estimate the risk of cardiovascular disease in patients with rheumatoid arthritis an overview of the extraarticular involvement in rheumatoid arthritis and its management coronary calcium as a predictor of coronary events in four racial or ethnic groups high-sensitivity c-reactive protein and cardiovascular disease across countries and ethnicities comparison of lipid and lipid-associated cardiovascular risk marker changes after treatment with tocilizumab or adalimumab in patients with rheumatoid arthritis coronary arterial calcification in rheumatoid arthritis: comparison with the multi-ethnic study of atherosclerosis exploring the lipid paradox theory in rheumatoid arthritis: associations of low circulating low-density lipoprotein concentration with subclinical coronary atherosclerosis coronary calcium score and cardiovascular risk anti-tumor necrosis factor-alpha treatment improves endothelial function in patients with rheumatoid arthritis cardiovascular co-morbidity in patients with rheumatoid arthritis: a narrative review of risk factors, cardiovascular risk assessment and treatment prevalence, extent and composition of coronary plaque in patients with rheumatoid arthritis without symptoms or prior diagnosis of coronary artery disease impact of cumulative inflammation, cardiac risk factors, and medication exposure on coronary atherosclerosis progression in rheumatoid arthritis brief report: the ability of the 2013 american college of cardiology/american heart association cardiovascular risk score to identify rheumatoid arthritis patients with high coronary artery calcification scores electron-beam tomography coronary artery calcium and cardiac events clinical risk factors alone are inadequate for predicting significant coronary artery disease coronary calcium risk score and cardiovascular risk inflammation and atherosclerosis ten-year progression of coronary artery, carotid artery, and aortic calcification in patients with rheumatoid arthritis left ventricular function at two-year follow-up in treatment-naive rheumatoid arthritis patients is associated with anti-cyclic citrullinated peptide antibody status: a cohort study coronary artery calcium score for long-term risk classification in individuals with type 2 diabetes and metabolic syndrome from the multiethnic study of atherosclerosis cardiovascular death in rheumatoid arthritis: a population-based study distribution of coronary artery calcium by race, gender, and age arterial age as a function of coronary artery calcium (from the multi-ethnic study of atherosclerosis [mesa]) cytokines in the pathogenesis of rheumatoid arthritis the pathogenesis of rheumatoid arthritis impact of disease activity and treatment of comorbidities on the risk of myocardial infarction in rheumatoid arthritis systematic review and meta-analysis of methotrexate use and risk of cardiovascular disease mechanisms of vascular comorbidity in autoimmune diseases cardiovascular disease in patients with rheumatoid arthritis: results from the quest-ra study coronary artery calcium score: current status cardiovascular comorbidity in rheumatic diseases prognostic value of coronary electron-beam computed tomography for coronary heart disease events in asymptomatic populations american college of cardiology/american heart association expert consensus document on electron-beam computed tomography for the diagnosis and prognosis of coronary artery disease cardiovascular effects of biological versus conventional synthetic diseasemodifying antirheumatic drug therapy in treatment-naïve, early rheumatoid arthritis extra-articular manifestations of rheumatoid arthritis: an update identification of patients at increased risk of first unheralded acute myocardial infarction by electron-beam computed tomography progression of coronary calcium on serial electron beam tomographic scanning frontiers in pharmacology | www.frontiersin.org prognostic value of coronary artery calcium screening in subjects with and without diabetes a language and environment for statistical computing the effects of tumour necrosis factor inhibitors, methotrexate, nonsteroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis acute changes in circulating natriuretic peptide levels in relation to myocardial ischemia explaining how "high-grade" systemic inflammation accelerates vascular risk in rheumatoid arthritis prognostic value of coronary artery calcium screening in asymptomatic smokers and non-smokers prognostic value of cardiac risk factors and coronary artery calcium screening for all-cause mortality multiple extra-articular manifestations are associated with poor survival in patients with rheumatoid arthritis performance of the expanded cardiovascular risk prediction score for rheumatoid arthritis is not superior to the acc/ aha risk calculator tumour necrosis factor antagonists and the risk of cardiovascular disease in patients with rheumatoid arthritis: a systematic literature review ggplot2: elegant graphics for data analysis coronary calcium and cardiovascular event risk: evaluation by age-and sexspecific quartiles glossary accp: anti-cyclic citrullinated peptide ami: acute myocardial infarction cac: coronary artery calcium ccs: coronary calcium scores ci: confidence interval crp: c-reactive protein ct dmard: disease-modifying antirheumatic drugs ecg: electrocardiography esr: erythrocyte sedimentation rate hdl: high-density lipoprotein il-6: interleukin-6 lowess: locally weighted scatterplot smoothing mesa: multi-ethnic study of atherosclerosis nsaid: nonsteroidal anti-inflammatory drug ntprobnp: n-terminal pro-hormone b-type natriuretic peptide ra: rheumatoid arthritis rf: rheumatoid factor tnf: tumor necrosis factor frontiers in pharmacology | www we are grateful for the outstanding work of zsófia tarcza, ultrasound technician. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2020.601344/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 mong, tarjanyi, tothfalusi, bartykowszki, nagy, szekely, becker, maurovich-horvat, merkely and nagy. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0025165-bmyfrglp authors: hossain, rajib; quispe, cristina; herrera-bravo, jesús; islam, md. shahazul; sarkar, chandan; islam, muhammad torequl; martorell, miquel; cruz-martins, natália; al-harrasi, ahmed; al-rawahi, ahmed; sharifi-rad, javad; ibrayeva, manshuk; daştan, sevgi durna; alshehri, mohammed m.; calina, daniela; cho, william c. title: lasia spinosa chemical composition and therapeutic potential: a literature-based review date: 2021-12-28 journal: oxid med cell longev doi: 10.1155/2021/1602437 sha: 8ed004092dc113b56e77b3e42d6eb5683a318aa5 doc_id: 25165 cord_uid: bmyfrglp lasia spinosa (l.) is used ethnobotanically for the treatment of various diseases, including rheumatoid arthritis, inflammation of the lungs, bleeding cough, hemorrhoids, intestinal diseases, stomach pain, and uterine cancer. this review is aimed at summarizing phytochemistry and pharmacological data with their molecular mechanisms of action. a search was performed in databases such as pubmed, science direct, and google scholar using the keywords: “lasia spinosa,” then combined with “ethnopharmacological use,” “phytochemistry,” and “pharmacological activity.” this updated review included studies with in vitro, ex vivo, and in vivo experiments with compounds of known concentration and highlighted pharmacological mechanisms. the research results showed that l. spinosa contains many important nutritional and phytochemical components such as alkanes, aldehydes, alkaloids, carotenoids, flavonoids, fatty acids, ketones, lignans, phenolics, terpenoids, steroids, and volatile oil with excellent bioactivity. the importance of this review lies in the fact that scientific pharmacological evidence supports the fact that the plant has antioxidant, anti-inflammatory, antimicrobial, cytotoxic, antidiarrheal, antihelminthic, antidiabetic, antihyperlipidemic, and antinociceptive effects, while protecting the gastrointestinal system and reproductive. regarding future toxicological and safety data, more research is needed, including studies on human subjects. in light of these data, l. spinosa can be considered a medicinal plant with effective bioactives for the adjuvant treatment of various diseases in humans. traditional, herbal, and ayurvedic medicine comprise an important and prestigious form of treatment for various diseases and conditions in different locations all over the world from the beginning of human civilization on earth [1] . several plants and their corresponding preparations have been used for various therapeutic purposes for a long time ago. the history of traditional, herbal, and ayurvedic medicine is the eldest to establish a treatment pattern [1] . lasia spinosa (l.) thwaites, often known as chengmora/ sibru in assames, kata-kachu in bengali, janum-saru in manipuri, kohila/mahakohila/engilikohila in sri lanka, zawangzang in mizoram, and laksmana in sanskrit [2] [3] [4] , belongs to the araceae family [5] . it is found in asia-bangladesh, china, the indian subcontinent, myanmar, thailand, indo-china, indonesia, and papua new guinea [6] . briefly, l. spinosa is an aquatic or terrestrial plant, shortstemmed spiny heirs with underground rhizome that usually occurs in wet forests, open marshes, wetlands, or in permanently standing water [7] . l. spinosa is a large marsh plant with the stem stout 1 m high and the leaves broadly arrowshaped in outlines, 20-30 cm long deeply divided into 4-6 pairs of narrow side lobes. the petiole is 30-40 cm long, veins beneath the petiole and peduncle prickly [8] . morpho-anatomical feature of l. spinosa has been recently reported by lakshmi et al. [9] . the plant is harvested from the wild for its edible leaves and various medicinal uses. sometimes, it is also cultivated as a vegetable crop along ponds margins [10] . recently, it has been reported that fusarium fujikuroi caused leaf spot on l. spinosa in china [11] . with regards to their biological effects, the tender leaves and rhizomes of this plant, used as a vegetable and in indigenous medicine, have been recommended for a variety of conditions [12] [13] [14] [15] . given the multiple potentialities of this plant, this review provides up-to-date data on l. spinosa chemical composition and biological effects based on the scientific reports found in the databases. in this study, the literature on botanical classification of l. spinosa, ethnomedicinal applications, secondary metabolites, biological properties were compiled, reviewed, and summarized. for the compilation of all written papers on this species, scientific search engines such as pubmed, sciencedirect, springerlink, web of science, scopus, wiley online, scifnder, and google scholar have been used. in this study, the literature on botanical classification of lasia spinosa, ethnomedicinal applications, secondary metabolites, and biological properties were compiled, reviewed, and summarized. for searching, the next mesh terms were used: "phytotherapy", "plants", "medicinal", "plant extracts/ administration & dosage", "plant extracts/isolation & purification", "plant/chemistry", "structure-activity relationship", "disease models", "animal", and "plant extracts/ toxicity". using the chemsketch version 12.01 program, chemical structures were drawn. the scientific names of the plants have been verified according to the plantlist [16, 17] . inclusion criteria: works published in english on lasia spinosa that highlighted the following data: chemical compounds isolated from each genus, preclinical pharmacological research highlighting molecular mechanisms, and in vitro/in vivo pharmacological studies that contained the concentration and dose at which the chemical compounds studied were pharmacologically active and toxicological data. the most important results obtained were summarized in the tables. exclusion criteria: abstracts, letters to the editor, papers in languages other than english, studies that did not have dose-effect correlations, and studies that did not have proven molecular mechanisms which underline the pharmacology. l. spinosa is a medicinally important plant, traditionally used by different ethnic communities all over the world. there are various reports on l. spinosa medicinal and economical properties. often used for treating colic, tuberculosis of lymph nodes, swollen lymph nodes, rheumatism/ rheumatoid arthritis, injuries, snake bites, and insect bites, this plant is also recommended as effective for the treatment of sore throat, constipation, to purify the blood, on lung inflammation, bleeding cough, and uterine cancer [14, [18] [19] [20] . rhizomes (roots) are most often used as a remedy for hemorrhoids in sri lanka and malays and to confer protection for some of the above conditions, because of their high fibre content and antioxidant compounds [15] . besides, leaves and stalks have demonstrated profound antihelminthic, anticestode, and antinematode efficacy [12, 15, 18, 19, 21, 22] . the root decoction is also useful in gastrointestinal diseases and stomachache [4] , while also stimulating liver function [22] . paste from tender leaves is externally used in burns [4] . l. spinosa whole plant contains several essential phytochemicals, including alkaloids, flavonoids, tannin, saponins, steroids, terpenoids, and varying amounts of micronutrients, like zinc (zn), magnesium (mg), calcium (ca), iron (fe), copper (cu), manganese (mn), and molybdenum (mo) ( table 1) . nutritional analysis of l. spinosa showed that it contains proteins (17.6 kcal/100 g), fats (1.16 kcal/100 g), and carbohydrates (35.7 kcal/100 g), with a nutritive value of 224 kcal/100 g [2, 23] . in another study, the protein, fats, and carbohydrate content on a dry weight basis were 17.9, 3.8, and 45.5 g/100 g edible portion for protein, fats, and carbohydrate, respectively, for l. spinosa leaf, with a nutritive value of 288.5 kcal/100 g [24] . l. spinosa roots/rhizome contains dietary fibre, ca, and provitamin a carotenoids [18, 25] . l. spinosa leaf contains 15.4 g of fibre, 250 mg of ca, 19.2 mg of fe, and 455 mg of vitamin c for 100 g edible portion on a dry weight basis [24, 26] . in fresh weight, other studies reported content of proteins, fats, and carbohydrates of 3:68 ± 0:28, 0:44 ± 0:03, and 4:78 ± 0:38 g/100 g, respectively, the mineral content of 158:08 ± 3:98, 321:73 ± 7:00, 73:17 ± 2:37, 53:86 ± 3:86, and 0:92 ± 0:08 mg/100 g for ca, k, p, mg, and fe, and 2:99 ± 0:11 and 0:28 ± 0:01 mg/100 g of vitamins c and e, respectively [27] . when specifically addressing the different extracts prepared from l. spinosa, hexane extracts leaves and root contains the alkaloid berberine [28] , lignan (e.g., lyoniresinol, meridional, secoisolariciresinol; 5,5 ′ -dimethoxysecoiso-lariciresinol; 2-(4-hydroxy-3,5-dimethoxybenzyl)-3-(4hydroxy-3-methoxybenzyl)-1,2-butanediol; (7 ′ s,8s,8r)-4,4 ′ -dihydroxy-3,3 ′ 5,5 ′ -tetra methoxy-7 ′ ,9-eproxylignan-9 ′ -ol-7-one; 5,5 ′ -dimethoxy-lariciresinol; 5 ′ -methyoxlariciresinol, dihydrodehydrodiconifery alcohol; syringaresinol) [27] [28] [29] , aldehyde (e.g., p-hydroxy benzaldehyde) [30] , phenolic (e.g., procyanidin a1) [31] and other compounds (e.g., 4-hydroxybenzoic acid, 2-(4 ′ -methoxyphenyl)-ethanol, 4methoxyphenyl alcohol, 1-tetracosane) [30] , from stem carotenoids (e.g., α-carotene, β-carotene, β-carotene-5,6, 5′ , 6′-diepoxide; 5, 6, 5′, 6′-diepoxy-5, 8, 5′,8′-tetrahydro-β, cis-neoxanthin, and unidentified carotenoids i, ii, iii, and iv) are isolated [24, 32, 33] . the aerial parts of l. spinosa contain terpenoids (e.g., limonene, aqualene, caryophyllene), volatile oil (e.g., methyl octadec-6,9-dien-12-ynoate, α-glyceryl-linolenate α-pinene, α-selinene, camphene, δ-3-carene, camphor) [21, 34] , phenolic compounds (e.g., 4-hydroxybenzoic acid, morin, cinnamic acid, syringic acid, gentisic aid) [21, 28, 34] , fatty acids (e.g., methyl ester of oleic acid, palmitic acid, stearic acid, epoxyoleic acid) [34] , steroids (e.g., spinasterone, βsitosterol, γ-sitosterol, stigmasterol, campesterol, crinosterol) [21, 34] , alkane (e.g., hexatriacontane and heptacosane) [35] . the whole plant contains phenolics (e.g., gentisic acid, isovanilic acid, syringic acid, chlorogenic acid, p-hydroxy benzoic acid, (+)-catechin) [28] , flavonoids including flavonoid glycosides and flavonoid aglycones (e.g., vitexin, [28, 32, 35] , and ketone (e.g., hexahydrofarnesyl acetone) (21) . the chemical structures of such compounds are shown in table 2 and figure 1. oxidative stress is the basis of premature ageing of the body, the basis of disease, and is triggered by free radicals [29] more precisely occurs as a result of the imbalance between the amount of reactive oxygen produced in the body and its ability to eliminate it [30, 31] . oxidative stress can be alleviated by approaching a balanced lifestyle that includes a healthy diet and sports [32] . physical exercise reduces cellular oxidation by deep oxygenating tissues, eliminating stress, and relaxing the body [33] . on the other hand, the diet has a very important role, and the best treatment against oxidative stress is antioxidants [34] . they are found in many herbs and can kill free radicals [35, 37] . medicinal plants usually contain a high level of antioxidants that can counteract the oxidative stress process linked to a disease [38, 39] . the free radical scavenging activity of l. spinosa leaves extracts on 1,1-diphenyl-2-picrylhydrazyl (dpph) had been assessed and showed significant antioxidant activities [40] . the ethyl acetate fraction showed the highest free radical scavenging activity (ic 50 = 16:42 μg/ml) when compared to the positive control-butylated hydroxytoluene (bht). at the same time, the aqueous fraction also exhibited moderate antioxidant potential (ic 50 = 73:20 μg/ml) [40] . in dpph and abts assay, ethanol extract (leaves) showed antioxidant activity (sc 50 = 17:25 μg/ml and 16.47 μg/ml, respectively). antioxidant activity is due to the presence of high levels of polyphenolic compounds [38] . in a study performed with different extracts of l. spinosa aerial parts, the highest free radical scavenging activity (dpph) was stated to the methanol extract (ic 50 = 0:48 ± 0:04 μg/ml), whereas in the metal chelating activity of ferrous ions (fe 2+ ) assay, the highest activity was observed for hexane extract (ic 50 = 0:55 ± 0:08 μg/ml) [23] . in another study, the antiradical activity (1/ec 50 ) of l. spinosa leaf determined by the dpph method was 0.1 [24] . the same 3 oxidative medicine and cellular longevity study reported a total phenolic content of 6.4 mg gallic equivalents/g and total flavonoid content of 4.4 retinol equivalent in l. spinosa leaf [24] . in other studies, l. spinosa showed a total phenolic content of 2.1 mg gallic equivalents/g and low antioxidant activity, through ferric reducing antioxidant power (frap) and oxygen radical absorbance capacity (orac) assays, in comparison to indigenous vegetables from southern thailand, such as young cashew leaves (anacardium occidentale l.) and mon-pu (glochidion zeylanicum (gaertn.) a.juss.) [27] . inflammation is part of the complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, or irritants [41] and a protective response involving immune system cells, molecular mediators, among others [42] [43] [44] . in lipopolysaccharide-induced raw 264.7 macrophages, the anti-inflammatory activity of l. spinosa leaf extract was addressed [45] , is stated that it can activate the nuclear factor-(nf-) κappa b, and nuclear factor erythroid 2-related factor 2/heme-oxygenase-1 (nrf2/ho-1) pathways and to suppress mitogen-activated protein kinase (mapk) and phosphoinositide-3-kinase/protein kinase b (pi3k/ akt) pathways. furthermore, l. spinosa leaf extract suppresses the upregulation enzyme inos (nos2), cox2, and proinflammatory cytokines (tnf-α, il-1β, and il-6) and increases cytokines (il-10) which produced antiinflammatory effect [40] . in another study, the anti-inflammatory activity of l. spinosa hydroalcoholic extract in xylene-induced ear oedema model mice was assessed, being stated a significant inhibitory effect on oedema formation 17.1% at 250 mg/kg and 27.9% at 500 mg/kg. an inhibitory potential was also stated in a carrageenan-induced paw oedema model rat, and it was highest at 3 h, with 26.72% inhibition at 250 mg/ kg and 38.70% at 500 mg/kg, when compared to the standard drugs (diclofenac sodium (10 mg/kg): 29.52%, and phenylbutazone (100 mg/kg): 40.47%) (figure 2 ) [46] . antimicrobial. an antimicrobial agent is that able to kill or stop microorganisms' growth. for that, antibacterial and antifungals are used to fight bacterial and fungal infections, respectively [47] [48] [49] . specifically addressing antibacterial, their prolonged use is closely related to a marked decrease root/ rhizome lignan lyoniresinol, meridinol, secoisolariciresinol; 5,5′-dimethoxysecoisolariciresinol; 2-(4-hydroxy-3,5-dimethoxybenzyl)-3-(4-hydroxy-3methoxybenzyl)-1,2-butanediol; (7′s,8s,8r)-4,4′-dihydroxy-3,3′5,5′ -tetramethoxy-7′,9-eproxylignan-9′-ol-7-one; 5,5′-dimethoxy-lariciresinol; 5′-methyoxlariciresinol, dihydrodehydrodiconifery alcohol; syringaresinol alam et al., [36] ; [28, 29, 32] phenolic procyanidin a1 [30] whole plant phenolic gentisic acid, isovanilic acid, syringic acid, chlorogenic acid, p-hydroxy benzoic acid, (+)-catechin [28] flavonoids (glycosides and aglycones) vitexin, vitexin 2"-o-β-d-glucopyranoside; isorhamnetin 3-o-rutinoside, morin, apigenin, 3′-methyl quercetin-3-o-α-l-rhamnopyranosyl-(1/6) β-dglucopyranoside; triglochinin [27, 28, 31] ketone hexahydrofarnesyl acetone [21] 4 oxidative medicine and cellular longevity in the number of enteric bacteria, thus, having a major negative impact on health and wellbeing [50, 51] . in this sense, the consumption of probiotics and a prebiotics-rich diet may help to replace the destroyed gut microbiota [52] . stool transplants may also be proposed for patients with difficulty in recovering from prolonged antibiotic treatment, as for recurrent clostridium difficile infections [53, 54] . the organic extracts (hexane, chloroform, ethyl acetate; 300 μg/disc) and essential oil of l. spinosa aerial parts showed potent antibacterial activity against escherichia coli, methicillin-resistant staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, and enterococcus faecalis in comparison of standard antibiotics (tetracycline 30 μg/ disc, streptomycin 30 μg/disc, and erythromycin 15 μg/disc), 5 oxidative medicine and cellular longevity except for methanol extract [23] . in another study, l. spinosa leaves methanol extract showed moderate antimicrobial properties against bacillus subtilis, e. coli, bacillus cereus, s. aureus, candida albicans, aspergillus niger, and vibrio para hemolyticus at 400 μg/disc by disc diffusion assay [36] . in other studies, the methanol extract of l. spinosa edible parts did not show antimicrobial activity against c. albicans [46] . [55] [56] [57] . this cytotoxicity is done in different ways: some bioactive compounds can affect the genetic material of cells, and others act by blocking the access of malignant cells to the nutrients needed for division and multiplication [58, 59] . the cytotoxic potential of l. spinosa extracts has also been assessed. brine shrimp lethality bioassay technique was applied to determine the cytotoxic potential of crude extracts. the aqueous extract from l. spinosa leaves showed moderate cytotoxicity (lc 50 = 98:66 μg/ml) in brine shrimp lethality bioassay [40] , while the methanol extract from the whole plant led to significant cytotoxic effects (ic 50 = 13:49 μg/ml) on brine shrimp [36] . in the castor oil-induced diarrheal mice model, both standard antimotility drug loperamide and hydroalcoholic extract from l. spinosa root significantly reduced the number of stools in a dose-dependent manner compared to the negative control group. the mean number of stools found was 11.6 for 250 mg/kg and 8.2 for 500 mg/kg b.w., whereas for the standard drug (5 mg/kg) was of 5.6.l. spinosa root extract at 250 mg/kg b.w, causing a little increase in the latent period, but at 500 mg/kg b.w. led to a significant increase [46] . l. spinosa root extract exhibited a potent antidiarrheal activity, supporting their traditional use for diarrhea. 5.6. antihelminthic. helminthic infections continue to be the major people's health hazard, especially in those living in tropical developing countries [60] . l. spinosa leaves methanol extract significantly exhibited paralysis and triggered worms' death, especially at high doses (100 mg/ml) against pheritimaposthuma [61] , in a hymenolepisdiminuta-rat animal model [12] and infected mice with trichinellaspiralis (800 mg/kg; p.o.) [19] . diabetes mellitus is a chronic metabolic disease with numerous complications, like retinopathy, neuropathy, and peripheral vascular insufficiency. several synthetic agents are available for diabetes treatment, but several side effects have been reported [62] . plant-based medicinal products have been used since ancient times to manage diabetes in traditional medicine in many countries all over the world. l. spinosa stem hydroalcoholic extract have revealed antidiabetic activity at 200 and 400 mg/kg (p.o.) in dexamethasone (10 mg/kg s.c.)-induced diabetic albino rats by preventing serum glucose levels rise triggered by dexamethasone [63] and significantly reducing the triglycerides levels [64] . this extract also ameliorated hyperglycemia, and it likely has greater therapeutic potential as they may also exert beneficial effects on the clinical course of noninsulin-dependent diabetes mellitus (niddm), hypertension, and coronary artery disease [63] . a study developed by shafie et al. [65] showed inhibitory effects of different parts (leaves, stems, and roots) of l. spinosa extracts (aqueous hot/cold, ethanol) against pancreatic lipase, α-amylase, and α-glucosidase. 5.8. antihyperlipidemic. l. spinosa leaves have also the potential to prevent hyperlipidemia-induced pancreatitis in rats at concentrations of 400 and 800 mg/kg (p.o.) while exerting cardioprotective effects by significantly increasing serum high-density lipoprotein-cholesterol (hdl-c) at 100 mg/kg, p.o., and triton-x 100 at 480 mg/kg, i.p. in an induced hyperlipidemic animal model [3] . antinociceptive. antinociception is the action or process of blocking the detection of a painful or injurious stimulus by sensory neurons, and antinociceptives are agents that block painful stimulus [66, 67] . the acetic acid-induced writhing test is used for detecting both central and peripheral analgesia, whereas the hot plate is most sensitive to centrally acting analgesics [68] . in acetic acid-induced writhing and hot plate-induced pain in mice the hydroalcoholic extract of l. spinosa roots revealed antinociceptive activity in mice, being stated 37% and 50% writhing inhibition at 250 mg/kg and 500 mg/kg b.w., respectively, while increased pain threshold [46] . on the other hand, the methanol extract from l. spinosa leaves at 400 mg/kg led to a significant decrease in the number of writhes and elongated the reaction time in the acetic acid writhing method and radiant heat tail flicking method, respectively [69] . a study revealed that l. spinosa leaves ethanol extract has gastroprotective effects. in albino rats with indomethacin (5 mg/kg, p.o.) and cold restrain stressinduced ulcers, 3 doses (100, 200, and 400 mg/kg, p.o.) of l. spinosa extract were tested, with gastroprotective effects being mainly conferred by the extractability to create a defensive layer in stomach, through scavenging free radicals and inhibiting lipid peroxidation [70] . in gastric secretion studies, l. spinosa significantly evidenced a tendency to decrease gastric juice, free acidity, and total acidity [70] . thus, after isolation of the individual compounds present in the extract, those responsible for the observed effect can be used both to treat ulcers and to reduce their severity. 5.11. effect on reproductive activity. testosterone plays an important role in sertoli and leydig cell proliferation and hyperplasia that can increase the testis size [71] . testosterone is also involved in spermatogenesis and the growth and development of testis and male accessory reproductive glands [64] . the hydroalcoholic extract of l. spinosa rhizomes was revealed to be able to increase the serum testosterone levels and sperm count in male rats at 40 g/kg b.w. these data were confirmed afterward by an increase in testicular weight and sperm count, with the increase in the absolute weight of testis being attributed to the elevation of androgen biosynthesis leading to an increase in serum testosterone levels [64] . the most important pharmacological properties are summarized in table 3 and figure 3 . 6 oxidative medicine and cellular longevity anthelmintic [61] 7 oxidative medicine and cellular longevity distilled water ↑serum testosterone [64] abbreviations: abts: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; bht: butylatedhydroxytoluene; dpph: 1,1-diphenyl-2-pecrylhydrazyl; ho-1: heme-oxygenase-1; l-name: n-nitro-l-arginine methyl ester; ldl-c: low-density lipoprotein cholesterol; mapk: mitogen-activated protein kinase; nf-κb: kappa b; nrf2: nuclear factor erythroid 2-related factor 2; pi3k/akt: phosphoinositide-3-kinase/ protein kinase b; vldl-c: very-low-density lipoprotein cholesterol. 8 oxidative medicine and cellular longevity oral administration in an acute analysis of 5, 10, 20, and 40 gm/kg of an extract, there were no mortality or physiological changes demonstrated. in the subchronic assay for 28 gm/ kg, administration of 5 or 20 gm/kg of extract for 28 gm/ kg, no animal deaths were announced that day. no differences in haematological parameters were noticed in either case [52] . therapeutic limitation of natural bioactive compounds from lasia spinosa results from the relatively reduced bioavailability of bioactive compounds. in addition, numerous interactions with other prescription drugs may occur. interactions between medicinal plants interfere with the metabolism or elimination of the drug/chemotherapy from the body. drug metabolism/elimination is mediated by enzymes that metabolize drugs in the cytochrome p450 (cyp) family and drug transport proteins. these interactions can change the concentration of drugs in the body [72] . interactions between plants and drugs may occur due to inhibition or activation by plant phytochemicals of cyp enzymes or drug transport proteins that metabolize the drug [73] . some therapeutic pharmacological agents must be activated by cyp to be effective. once cyps are inhibited, such drugs that need to be activated will be ineffective. there may be interactions between plants and drugs that lead to increased elimination of drugs due to cyp activation, which could lead to subtherapeutic exposure to drugs and could lead to therapy failure [74] . some plant-drug interactions due to cyp inhibition may lead to the accumulation of cytotoxic drugs due to delayed clearance and may increase drug toxicity due to high doses of drugs. cancer patients are already taking several medications at the same time due to other conditions associated with cancer and comorbidities, which present a risk of drug interactions [75] . the use of herbs/herbal products may further increase the risk of these potentially harmful interactions that interfere with the impact of the drug. natural plant sources have contributed to many drug developments. in this study, we comply with the traditional uses, pharmacological properties, and chemical constituents of l. spinosa, information that can be useful for further research. many phytochemicals present in l. spinosa may be responsible for its biological effects in various test systems, but more studies are needed to identify and characterize the active compounds responsible for the pharmacological activities of this hopeful medicinal plant. future directions must be oriented to toxicological studies which are scarce, and there are necessary new reports to ensure the safety of this plant. besides, clinical studies are required to confirm the preclinical biological effects in humans. the authors declare that they have no competing interests. the traditional medicine and modern medicine from natural products qualitative estimation of the presence of bioactive and nutritional compound in lasia spinosa: an important vegetable plant used by the bodos of kokrajhar district evaluation of antihyperlipidemic activity of methanolic leaves extract of lasia spinosa and its role in prevention of hyperlipidemia induced pancreatitis in rats a study on ethnomedicinal usage of plants among the folklore herbalists and tripuri medical practitioners: part-ii iucn red list of threatened species acoraceae and araceae anti-oxidant activity and total phenolic contents in cucurbita moschtata, lasia spinosa and limnocharis flava morpho-anatomical feature and phytochemical assessments of lasia spinosa (l)thwaites first report of leaf spot on lasia spinosa caused by fusarium fujikuroi in china anticestodal efficacy of lasia spinosa. extract against experimental hymenolepis diminuta. infections in rats traditional knowledge on wild food plants in andhra pradesh ethnobotanical uses of some plants by tripuri and reang tribes of tripura some nutritional aspects of lasia spinosa (kohila) best practice in research -overcoming common challenges in phytopharmacological research the wealth of india: an encyclopedia of india's raw material resources efficacy of lasia spinosa leaf extract in treating mice infected with trichinella spiralis anticestodal efficacy of folklore medicinal plants of naga tribes in north-east india some ayurvedic important plants from district kamrup (assam) iamm, institute of aryuveda and alternative medicine (iaam) phenolic profile, essential oil composition and bioactivity of lasia spinosa (l.) thwaites relationship between antioxidant properties and chemical composition of some thai plants the effect of maturity on carotenoids of lasia spinosa stem and the effects of cooking on in-vitro bioaccessibility of carotenoids the food ingredients of different extracts of lasia spinosa (l.) thwaites can turn it into a potential medicinal food nutrients value and antioxidant content of indigenous vegetables from southern thailand physicochemical evaluation, preliminary phytochemical investigation, fluorescence and tlc analysis of leaves of the plant lasia spinosa (lour) thwaites the therapeutic potential of anthocyanins: current approaches based on their molecular mechanism of action inflammatory and oxidative stress markers-mirror tools in rheumatoid arthritis cucurbita plants: from farm to industry lifestyle, oxidative stress, and antioxidants: back and forth in the pathophysiology of chronic diseases diet, lifestyle and cardiovascular diseases: linking pathophysiology to cardioprotective effects of natural bioactive compounds dietary supplements, vitamins and minerals as potential interventions against viruses: perspectives for covid-19 avocado-soybean unsaponifiables: a panoply of potentialities to be exploited antimicrobial and cytotoxic activity from lasia spinosa and isolated lignan ficus plants: state of the art from a phytochemical, pharmacological, and 10 oxidative medicine and cellular longevity toxicological perspective pharmacological properties of chalcones: a review of preclinical including molecular mechanisms and clinical evidence the effect of silver nanoparticles on antioxidant/pro-oxidant balance in a murine model antioxidant property, cytotoxicity and antimicrobial activity of lasia spinosa leaves cannabinoids and inflammations of the gut-lung-skin barrier oxidative stress and inflammation interdependence in multiple sclerosis current concepts on the reno-protective effects of phosphodiesterase 5 inhibitors in acute kidney injury: systematic search and review the treatment of cognitive, behavioural and motor impairments from brain injury and neurodegenerative diseases through cannabinoid system modulation-evidence from in vivo studies antiinflammatory effects of lasia spinosa leaf extract in lipopolysaccharide-induced raw 264.7 macrophages antinociceptive, antiinflammatory and anti-diarrheal activities of the hydroalcoholic extract of lasia spinosa linn. (araceae) roots the burden of the serious and difficult-to-treat infections and a new antibiotic available: cefiderocol antimicrobial resistance in bacterial pathogens among hospitalised patients with severe invasive infections cinnamomum species: bridging phytochemistry knowledge, pharmacological properties and toxicological safety for health benefits prevalence and antimicrobial resistance of klebsiella strains isolated from a county hospital in romania bioactive compounds as potential agents for sexually transmitted diseases management: a review to explore molecular mechanisms of action probiotics: versatile bioactive components in promoting human health american journal of gastroenterology lecture: intestinal microbiota and the role of fecal microbiota transplant (fmt) in treatment of c. difficile infection prospects and challenges for intestinal microbiome therapy in pediatric gastrointestinal disorders immunohistochemical expression of tgf beta (tgf-beta), tgf beta receptor 1 (tgfbr1), and ki67 in intestinal variant of gastric adenocarcinomas histochemical and immunohistochemical evidence of tumor heterogeneity in colorectal cancer chitosan nanoparticles as a promising tool in nanomedicine with particular emphasis on oncological treatment therapeutic potential of neoechinulins and their derivatives: an overview of the molecular mechanisms behind pharmacological activities paclitaxel: application in modern oncology and nanomedicine-based cancer therapy phytochemicals as cure of worm infections in traditional medicine systems. emerging trends in zoology in-vitro evaluation of anthelmintic activity of lasia spinosa leaves alternative therapies useful in the management of diabetes: a systematic review evaluation of antidiabetic activity from the stem of lasia spinosa in dexamethasone induced diabetic albino rats acute to subchronic toxicity and reproductive effects of aqueous ethanolic extract of rhizomes of lasia spinosa thw. in male rats. thai nutritional composition, antioxidative and inhibitory effects against pancreatic lipase, α-amylase and α -glucosidase of lasia spinosa epibatidine: a promising natural alkaloid in health genistein: an integrative overview of its mode of action oxidative medicine and cellular longevity properties, and health benefits anti-nociceptive activity of hygrophila auriculata (schum) heine antinociceptive activity of leaves of lasia spinosa gastroprotective potential of lasia spinosa in albino rats management of endocrinopathies in pregnancy: a review of current evidence interactions of clinical relevance associated with concurrent administration of prescription drug and food or medicinal plants: a systematic review protocol association of nutraceutical supplements with longer telomere length a novel nutraceutical formulation can improve motor activity and decrease the stress level in a murine model of middle-age animals cytochrome p450 enzyme mediated herbal drug interactions (part 1) key: cord-0016447-bda4e3fo authors: takeuchi, tsutomu; tanaka, yoshiya; tanaka, sakae; kawakami, atsushi; song, yeong-wook; chen, yi-hsing; rokuda, mitsuhiro; izutsu, hiroyuki; ushijima, satoshi; kaneko, yuichiro title: safety and effectiveness of peficitinib (asp015k) in patients with rheumatoid arthritis: final results (32 months of mean peficitinib treatment) from a long-term, open-label extension study in japan, korea, and taiwan date: 2021-03-03 journal: rheumatol ther doi: 10.1007/s40744-021-00280-5 sha: 5392aa7dadd507e499ef3758ca059629e6b742eb doc_id: 16447 cord_uid: bda4e3fo introduction: this final analysis of a long-term extension (lte) study assessed the safety, tolerability, and effectiveness of peficitinib (asp015k), a pan-janus kinase inhibitor, in asian patients with rheumatoid arthritis (ra). methods: patients had previously completed the 12-week phase 2b (raj1), or 52-week phase 3 (raj3 and raj4) peficitinib studies in japan, korea, and taiwan, and received oral peficitinib 50 or 100 mg/day. dose increase to 150 mg/day or reduction to 50 mg/day was permitted. efficacy endpoints included american college of rheumatology (acr)20/50/70 response rates, 28-joint disease activity score with c-reactive protein (das28-crp), and acr components. safety endpoints included treatment-emergent adverse events (teaes), and incidence rates (irs) of adverse events of special interest per 100 patient-years (py). results: overall, 843 patients received peficitinib for a mean 32.0 months (maximum 85.2 months), and most (64.4%) received peficitinib 100 mg/day as a maximum dose. respective acr20/50/70 response rates were maintained from baseline (week 0 of lte; 71.6, 52.1, and 34.7%) to end of treatment (78.7, 63.3, and 44.1%); continuous improvements in acr components and das28-crp were observed from the baselines of preceding studies and throughout the lte. overall, 796/843 (94.4%) patients experienced teaes; most were severity grade 1/2. most common teaes were nasopharyngitis (47.0%) and herpes zoster (17.3%). drug-related teaes leading to permanent discontinuation occurred in 140 (16.6%) patients, and irs (95% confidence interval) per 100 py of serious infections, herpes zoster-related disease, and malignancies were 2.7 (2.1, 3.4), 7.3 (6.2, 8.6), and 1.2 (0.9, 1.8), respectively. two deaths occurred during the study; one each from diffuse large b cell lymphoma and pneumonia, which were, respectively considered probably and possibly related to study drug. conclusions: improvements in effectiveness variables were maintained during this long-term study of peficitinib in asian patients with ra; peficitinib was generally well tolerated over a mean 32 months’ duration. trial registration: clinicaltrials.gov. nct01638013, retrospectively registered on 11 july 2012 https://clinicaltrials.gov/ct2/show/nct01638013. supplementary information: the online version contains supplementary material available at 10.1007/s40744-021-00280-5. of adverse events of special interest per 100 patient-years (py). results: overall, 843 patients received peficitinib for a mean 32.0 months (maximum 85.2 months), and most (64.4%) received peficitinib 100 mg/day as a maximum dose. respective acr20/50/70 response rates were maintained from baseline (week 0 of lte; 71.6, 52.1, and 34.7%) to end of treatment (78.7, 63.3, and 44.1%); continuous improvements in acr components and das28-crp were observed from the baselines of preceding studies and throughout the lte. overall, 796/843 (94.4%) patients experienced teaes; most were severity grade 1/2. most common teaes were nasopharyngitis (47.0%) and herpes zoster (17.3%). drug-related teaes leading to permanent discontinuation occurred in 140 (16.6%) patients, and irs (95% confidence interval) per 100 py of serious infections, herpes zosterrelated disease, and malignancies were 2.7 (2.1, 3.4), 7.3 (6.2, 8.6), and 1.2 (0.9, 1.8), respectively. two deaths occurred during the study; one each from diffuse large b cell lymphoma and pneumonia, which were, respectively considered probably and possibly related to study drug. conclusions: improvements in effectiveness variables were maintained during this longterm study of peficitinib in asian patients with ra; peficitinib was generally well tolerated over a mean 32 months' duration. rheumatoid arthritis (ra) is a chronic disease requiring long-term treatment; therefore an understanding of the longterm effectiveness, safety, and tolerability of an ra therapy is key. interim results from an open-label longterm extension (lte) study of peficitinib (asp015k), a pan-janus kinase inhibitor, in asian patients with ra, have been published. this final analysis of the lte study assessed the long-term safety, tolerability, and effectiveness of peficitinib in asian patients with ra, over a mean 32 months of treatment. this study showed that improvements in american college of rheumatology response and other effectiveness variables were maintained during long-term peficitinib treatment, and peficitinib was generally well tolerated in asian patients with ra. these final data support peficitinib use for the long-term management of ra in asian patients. this article is published with digital features, including a summary slide, to facilitate understanding of the article. to view digital features for this article go to https://doi.org/10.6084/ m9.figshare.13582829. rheumatoid arthritis (ra) is a chronic condition characterized by systemic inflammation and joint destruction, leading to severe disability and premature mortality [1] . the ra treatment landscape has been strengthened by the development of the janus kinase (jak) inhibitors, a class of targeted synthetic diseasemodifying antirheumatic drugs (dmards) [2] . jak inhibitors target enzymes belonging to the jak family with varying specificity and selectivity, thereby inhibiting a number of proinflammatory processes involved in the pathogenesis of ra [2] . the jak inhibitors tofacitinib (preferential jak 1 and 3 inhibitor), baricitinib (jak 1 and 2 inhibitor), and upadacitinib (jak 1 inhibitor) have undergone phase 3 clinical trials, and are approved for the treatment of ra in the usa, europe, and asia [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . peficitinib (asp015k) is an oral, once-daily, pan-jak inhibitor that has demonstrated efficacy and tolerability in asian patients with ra, as a monotherapy in a 12-week phase 2b study (raj1), and in two 52-week phase 3 randomized controlled trials, in combination with dmards (raj3) or methotrexate specifically (raj4) [13] [14] [15] . peficitinib has been approved in japan, korea, and taiwan, for the treatment of ra [16] [17] [18] . ra is a chronic disease requiring long-term treatment; therefore an understanding of the long-term effectiveness, safety, and tolerability of an ra therapy is key. interim results (over a mean treatment duration of 22.7 months) from an open-label long-term extension (lte) study of peficitinib in ra patients who completed the raj1, raj3, or raj4 clinical trials have been published [19] . here, we report the final safety and effectiveness data after completion of this lte study of peficitinib. this open-label, lte study was conducted at 165 sites in japan, nine sites in korea, and nine sites in taiwan from june 2012 to september 2019. eligible patients had previously completed the raj1, raj3, or raj4 study [13] [14] [15] 19] , and consequently the duration of study treatment varied between patients (supplementary fig. s1 ). in the raj1 phase 2b study, patients were randomized (1:1) to receive peficitinib monotherapy or placebo for 12 weeks with a 4-week follow-up (without peficitinib treatment) [13] . raj3 was a phase 3 study in which patients with an inadequate response to dmards were randomized (1:1:1:2) to receive peficitinib 100 mg/day, peficitinib 150 mg/day, placebo or etanercept. the peficitinib and etanercept treatment duration was 52 weeks, while patients in the placebo arm were switched at week 12 to either dose of peficitinib. patients in the etanercept control reference group of raj3 were not included in the extension study [14] . in the raj4 phase 3 study, patients with an inadequate response to methotrexate were randomized (1:1:1) to receive peficitinib 100 mg/day, peficitinib 150 mg/day or placebo in combination with methotrexate, with treatment duration of 52 weeks, and patients in the placebo arm switched to either dose of peficitinib at week 28 [15] . the lte study design has been previously described [19] . briefly, patients received oral peficitinib 50 mg/day (if transferring from raj1) or 100 mg/day (if transferring from raj3 or raj4) once daily after breakfast as the starting dose. the daily dose of peficitinib could be increased in patients with no safety issues from 50-100 mg/day at the investigator's discretion, or from 100 to 150 mg/day in those with insufficient clinical response [28-joint disease activity score (das28) erythrocyte sedimentation rate c 3.2] after 4 weeks of peficitinib treatment. after the increase, the dose could be reduced from 100 mg/day or 150 mg/day to 50 mg/day at the discretion of the investigator. subsequent to the publication of interim results, and following approval of peficitinib in japan in march 2019, all japanese patients receiving peficitinib 50 mg/day had their dose increased to 100 mg/day, or discontinued if the dose increase was not possible. the efficacy and safety of peficitinib were assessed by the investigator at each patient visit. study drug administration could be suspended, interrupted, or discontinued based on certain criteria, as previously described [19] . inclusion criteria have been described previously [19] . briefly, patients were required to have completed treatment with peficitinib and undertaken assessments at week 16 for the raj1 study, and week 52 for the raj3 and raj4 studies. patients were excluded if they were judged unsuitable to participate in the study for any reason by the investigator, or if they had taken any of the contraindicated therapies detailed in supplementary methods. concomitant medications and therapies were permitted or prohibited as described in supplementary methods. efficacy was evaluated in the overall patient population and in patients grouped by their preceding study. assessment of the long-term efficacy of peficitinib included american college of rheumatology (acr)20, acr50, and acr70 response rates; changes from baselines of the preceding studies in das28 based on c-reactive protein (crp); proportions of patients achieving das28-crp-defined remission (\ 2.6) and das28-crp-defined low disease activity (lda; b 3.2); changes from the baselines of the preceding studies in clinical (cdai) and simplified (sdai) disease activity indices; proportions of patients achieving cdai-and sdai-defined remission (b 2.8 and b 3.3, respectively); acr/european league against rheumatism (eular) boolean-based definition of remission; and changes from the baselines of the preceding studies in the core set of acr components (see supplementary methods) [20] . safety outcomes included treatment-emergent adverse events (teaes), which were defined as any adverse event (ae) that started or worsened in severity after the first dose of study drug in raj2 to the end of the final observation. aes of special interest were assessed per 100 patientyears (py), and included serious infections, malignancies, herpes zoster-related disease (including varicella), and venous thromboembolism (vte; post hoc analysis). mean (standard deviation [sd]) changes from baseline in hematologic, biochemical, and select laboratory parameters were recorded throughout the lte study. vital signs were collected at each study visit, and an electrocardiogram was obtained every 48 weeks and at end of treatment (eot)/ early termination. patient populations and statistical analyses have been defined previously [19] . briefly, efficacy analyses were conducted on the full analysis set (fas; all patients who received c 1 dose of study drug and had measurements for any of the efficacy endpoints), and the safety analysis set (saf) included all patients who received c 1 dose of study drug. the planned sample size was approximately 800 patients treated with peficitinib. this was based on the number of patients from raj1 who participated in the lte study, the number of patients planned to be enrolled in raj3 (excluding the etanercept reference arm), and the number of patients planned to be enrolled in raj4. statistical analyses have been defined previously [19] . briefly, teaes were summarized according to the medical dictionary for regulatory activities (meddra, version 11.1) system organ class (soc) and preferred term (pt). patients reporting c 1 ae for a given meddra pt were counted only once for that pt. patients reporting c 1 ae within a soc were counted only once for the soc total. other safety variables were analyzed descriptively. missing data acr components, das28-crp, and safety variables at eot were summarized by the last observation carried forward (locf) method for each component available, and then calculated. all outliers were included in the analyses. this study was conducted in accordance with good clinical practice, the international council on harmonisation of technical requirements for registration of pharmaceuticals for human use guidelines, and local laws and regulations. the study protocol and amendments were reviewed and approved by an institutional review board or independent ethics committee (supplementary table s1 ) at each study site, and safety data were reviewed by an independent data and safety monitoring board. each patient provided written informed consent prior to treatment initiation. this analysis, and the clinical trials from which data were included, followed the principles of the declaration of helsinki. interim data have been previously reported for the raj2 study [19] , and here we report the final data. of 873 patients screened, 843 were enrolled and included in the saf (201, 225, and 417 patients from raj1, raj3, and raj4 studies, respectively) ( supplementary fig. s2 ). a total of 837 (99.3%) patients were included in the fas. demographics and baseline characteristics have been reported previously [19] . the majority of patients were female (619/843, 73.4%), from japan (806/843, 95.6%), with a mean age of 55.7 years, and a mean (sd) duration of ra of 6.2 (5.6) years (supplementary table s2 ). discontinuations were reported for 330 (39.1%) patients; the most frequently reported reasons were ae (13.4%), other (7.9%), and lack of efficacy (7.6%) (fig. 1 ). mean peficitinib exposure was 32.0 months (max. 85.2 months) ( the acr20, acr50, and acr70 response rates were maintained throughout the lte study from baseline (71.6, 52.1, and 34.7%, respectively) to eot (locf; 78.7, 63.3, and 44.1%, respectively) in the overall population (fig. 2) , and in patients from studies raj3 and raj4 ( supplementary fig. s3) . a sudden decrease in the acr20 response rate was observed at week 192 in raj3, but the number of patients was considerably lower (n = 8) than at other time points (supplementary fig. s3 ). for patients from raj1, response rates initially increased from baseline and were then maintained until eot ( supplementary fig. s3 ). improvements (reductions) in the core set of acr components [tender joint count at 68 joints (tjc68), swollen joint count at 66 joints (sjc66), subject's global assessment of pain (sgap), subject's global assessment of disease activity (sga), physician's global assessment of disease activity [pga], and health assessment questionnaire-disability index (haq-di)] were observed from the baselines of the preceding studies, and continued during the extension study ( supplementary fig. s4 ). the acr20 response rates were sustained during the lte study for maximum peficitinib doses of both 100 mg/day and 150 mg/day. in patients with a maximum peficitinib dose of 50 mg/day, acr20 response rates were maintained during the extension study after an initial increase from baseline of the lte study (week 0) (fig. 3) . mean changes from baseline in das28-crp, cdai, and sdai scores remained consistent over time for the overall study population (fig. 4) , and in patients from raj3 and raj4 ( supplementary fig. s4 ). where raj1 was the preceding study, mean das28-crp, cdai, and sdai scores decreased from baseline, and were then sustained until eot ( supplementary fig. s4 ). in the overall study population, the table 3 ). the proportions of patients with teaes and saes leading to permanent discontinuation of study drug were 16.6 and 8.9%, respectively (table 2) . drug-related teaes and drug-related saes were reported in 656 (77.8%) and 113 (13.4%) patients, respectively (table 2) , and there were 92 (10.9%) patients who presented with drug-related teaes leading to permanent discontinuation ( table 2 ). the proportions of patients for each category of ae were generally higher in the raj1 study compared with raj3 and raj4 (supplementary table s4 ). death was reported for two (0.2%) patients during the study; one patient died from diffuse large b-cell lymphoma and one patient from pneumonia. these events were considered by the investigator to be probably and possibly related to the study drug, respectively (supplementary results). after study completion, there was one death due to uterine sarcoma, and this was considered by the investigator to be possibly related to the study drug (supplementary results). the overall incidence rates [irs; 95% confidence intervals (cis)] per 100 py of serious infections, herpes zoster-related disease, and malignancies, were 2.7 (2.1, 3.4), 7.3 (6.2, 8.6), 1.2 (0.9, 1.8), respectively (fig. 6) . in a post hoc analysis of vte, the ir (95% ci) was 0.1 (0.0, 0.4) per 100 py in the overall period, which related to two cases (one case of pulmonary artery thrombosis and one case of deep vein thrombosis) between 24 (fig. 7) . for herpes zoster-related disease, irs of teaes increased slightly with higher doses [50 mg/day: 3.1 (1.0, 9.5); 100 mg/day: 6.9 (5.6, 8.5); and 150 mg/day: 8.6 (6.7, 11.0)] (fig. 7) . the irs of malignancy-related teaes were higher for peficitinib 100 mg/day [1.6 (1.1, 2.5)] compared with 50 mg/day [1.0 (0.1, 7.0) and 150 mg/day [0.6 (0.3, 1.5)] (fig. 7) . there was a low incidence (95% ci) of vte-related teaes per 100 py for all maximum dose groups [50 mg/day: 0.0; 100 mg/day: 0.1 (0.0, 0.5); and 150 mg/day: 0.1 (0.0, 0.9)] (fig. 7) . there were 16 patients with serious herpes zoster-related disease (supplementary table s5 ). the total exposure to peficitinib among patients who had at least one ae of special interest was 2216.7, 2031.4, 2271.4, and 2277.8 py for serious infections, herpes zoster-related disease, malignancy, and vte, respectively. increases in blood creatine kinase and decreases in lymphocytes were observed from baseline of the extension study (week 0), and were reported as teaes in 97/843 (11.5%) and 43/843 (5.1%) patients, respectively ( this open-label extension study, with mean 32.0 months of peficitinib treatment, and peficitinib exposure of 2277.9 py, supports the results of the interim analysis (1615.3 py of peficitinib exposure) [19] , and provides further evidence of the effectiveness and safety of peficitinib for long-term use in asian patients with ra. longer-term efficacy outcomes were either maintained or improved during this extension study in patients receiving peficitinib after completing the raj1, raj3, or raj4 studies [13] [14] [15] 19] . both interim and final analyses of the extension study showed that acr20, acr50, and acr70 response rates were maintained from baseline (71.6, 52.1, and 34.7%, respectively) to eot (78.9, 61.4, and 42.7%, respectively, for the interim analysis [19] , and 78.7, 63.3, and 44.1%, respectively, for the final analysis). the acr20 response rate was lower at baseline (week 0) for patients transferring from raj1 compared with raj3 and raj4, which was likely due to some patients in raj1 receiving lower doses of peficitinib and/or the shorter treatment period in this study, as discussed previously [19] . unsurprisingly, a similar trend was observed for the acr20 response rate in patients receiving the maximum dose of 50 mg/day peficitinib. in patients who received maximum doses of 100 mg/day or 150 mg/day peficitinib, acr20 response rates were maintained during the lte study. other measures of efficacy, das28-crp, cdai, sdai, tjc68, sjc66, sgap, pga, sga, and haq-di, showed an improvement from the baselines of the preceding studies, and continued to show improvement during the current study. this was consistent with trends seen in the interim analysis of this lte study (cdai and sdai not reported) [19] . an improvement from b fig. 5 the proportion of patients achieving remission/lda in relation to a das28-crp \ 2.6, b das28-crp b 3.2, c cdai b 2.8, d sdai b 3.3, and e acr/eular boolean-based definition of remission (fas). *includes locf. acr american college of rheumatology, cdai clinical disease activity index, das28-crp disease activity score in 28 joints based on c-reactive protein, eot end of treatment, eular european league against rheumatism, fas full analysis set, lda low disease activity, locf last observation carried forward, sdai simplified disease activity index teaes were defined as any ae that started or worsened in severity after initial dose of study drug in the extension study until the end of the final observation ae adverse event, sae serious adverse event, saf safety analysis set, teae treatment-emergent adverse event a possibly or probably related to study drug, as assessed by the investigator or records where relationship was missing b national cancer institute common terminology criteria for adverse events (nci-ctcae): grade 3, severe or medically significant; grade 4, life-threatening; grade 5, death related to ae c one death during the study due to diffuse large b-cell lymphoma was considered probably related to the study drug. one death during the study due to pneumonia and one death after the end of the study due to uterine sarcoma were considered possibly related to the study drug baseline was also observed for measures of remission in the final analysis of this lte study, and by eot more than half of patients (60.4%) had achieved das28-crp-defined remission, and 75.3% had achieved das28-crpdefined lda. the safety profile of peficitinib in the final analysis of the lte study was consistent with observations from the preceding studies and the c fig. 6 incidence of adverse events of special interest per 100 py during the overall period for a serious infections, b herpes zoster-related disease, and c malignancies (saf). py were calculated from initial dose up to first incidence of the event for patients who had c 1 event, and from initial dose through follow-up for patients who had no events; incidence rate was calculated as (100 9 number of patients who had c 1 incidence)/total py. ci confidence interval, ir incidence rate, py patient-years, saf safety analysis set interim lte study analysis, indicating that peficitinib was generally well tolerated over a treatment duration of up to 7 years [13] [14] [15] 19] . of note, the proportion of patients reporting each category of ae was generally higher in the raj1 study compared with raj3 and raj4, perhaps due to the longer mean treatment exposure in patients from the raj1 study (47.1 months) versus raj3 and raj4 (28.1 and 26.9 months, respectively). teaes were similar in frequency for each peficitinib dose, and most events were grade 1 or 2 in severity. the most commonly reported teae was nasopharyngitis, and its incidence did not increase with higher doses of peficitinib. we compared the incidences of aes of special interest observed in our study of japanese, korean, and taiwanese patients with studies of other jak inhibitors in asian populations. there were no notable differences in the incidences of aes of special interest between the phase 3 studies (raj3 and raj4) [14, 15] [19, 21, 22] . studies have reported that jak inhibitors can increase the risk of herpes zoster infection, particularly in asian patients [21] [22] [23] [24] , so it was not surprising that the most frequently occurring serious infection in our study was herpes zoster-related disease, which had an ir (95% ci) of 7.3 (6.2, 8.6) per 100 py. this was consistent with irs (95% ci) per 100 py from the interim analysis of the extension study [6.8 (5.6, 8. 3)], and was similar to results reported for other jak inhibitors in asian populations [5.9 (5.2, 6.6) for tofacitinib and 6.2 (4.9, 7.7) for baricitinib] [19, 22, 25] . in our study, there were no major differences among the incidences of teaes for the different doses of peficitinib; however, irs (95% ci) per 100 py were slightly higher in patients receiving peficitinib 150 mg/day compared with 100 mg/day for serious infections [3.0 (2.0, 4.5) and 2.3 (1.6, 3.3), respectively] and herpes zoster-related disease [8.6 (6.7, 11.0) and 6 herpes zoster-related disease malignancies venous thromboembolism incidence rate (95% ci) per 100 patient-years 50 mg/day 100 mg/day 150 mg/day fig. 7 incidence of treatment-emergent adverse events of special interest per 100 py during the overall period (saf). py were calculated from initial dose up to first incidence of the event for patients who had c 1 event, and from initial dose through follow-up for patients who had no events; incidence rate was calculated as (100 9 number of patients who had c 1 incidence)/total py. ci confidence interval, ir incidence rate, py patient-years, saf safety analysis set general risks, such as advanced age, are identified. there were no major differences in malignancy irs (95% ci) per 100 py in our extension study [1.2 (0.9, 1.8)] and those reported previously for tofacitinib [0.8 (0.6, 1.1)] and baricitinib [1.0 (0.5, 1.7)] in asian populations [21, 22] . it was difficult to interpret the irs of malignancy-related teaes in our study, due to the low numbers of patients with malignancies; however, there appeared to be no evidence of dose-dependency. there have been concerns around the potential for thromboembolic events in ra patients receiving tofacitinib or baricitinib, leading to dose restrictions in this group of patients [26] . the two cases of vte reported for our extension study were considered not related to peficitinib. the strengths and limitations of this study have been discussed in full previously [19] . specifically, our study provides data from a large number of patients for a mean treatment duration of 32.0 months. furthermore, the patients received a variety of peficitinib dosing regimens, which is likely to be more representative of routine clinical practice than the preceding trials. key limitations include the lack of either a placebo or active comparator arm. it was also difficult to compare different dose groups due to the lack of randomization and the ability to adjust the dose, which may have resulted in individual patients experiencing different disease states during the course of treatment. additionally, it should be taken into consideration that almost all patients were from japan, a population known to be particularly susceptible to herpes zoster reactivation when treated with tofacitinib [24] , and also to respond uniquely to biologic dmards [27] . improvements in acr response and other clinical effectiveness variables were maintained over a mean treatment duration of 32 months alt alanine aminotransferase, ast aspartate aminotransferase, eot end of treatment, hdl high-density lipoprotein, ldl low-density lipoprotein, saf safety analysis set, sd standard deviation a eot assessments and tests were to be performed promptly once administration of peficitinib had ended; in cases of early termination of peficitinib administration, these assessments and tests were to be performed within 2 days of the last dose of peficitinib, if possible in asian patients with ra, and peficitinib was generally well tolerated for a period of up to 7 years. these final data are consistent with the interim analysis of the lte study, and support peficitinib use for long-term management of ra in asian patients. yws reports a grant from astellas pharma, inc. yhc reports grants for research and clinical trials from taiwan ministry of science and technology, taiwan department of health, taichung veterans general hospital rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative clinical efficacy of launched jak inhibitors in rheumatoid arthritis upadacitinib: first approval pharmaceuticals and medical devices agency (pmda) japan. new drugs approved in fy 2019 us food and drug administration. fda approves xeljanz committee for medicinal products for human use (chmp) report on the deliberation results: xeljanz tablets 5 mg report us food and drug administration committee for medicinal products for human use (chmp) report on the deliberation results: olumiant tablets 2 mg, 4 mg korea approves olumiant pills for treatment of rheumatoid arthritis abbvie receives european commission approval of rinvoq tm (upadacitinib) for the treatment of adults with moderate to severe active rheumatoid arthritis [media release efficacy and safety of the oral janus kinase inhibitor peficitinib (asp015k) monotherapy in patients with moderate to severe rheumatoid arthritis in japan: a 12-week, randomised, doubleblind, placebo-controlled phase iib study efficacy and safety of peficitinib (asp015k) in patients with rheumatoid arthritis and an inadequate response to conventional dmards: a randomised, double-blind, placebo-controlled phase iii trial (raj3) efficacy and safety of peficitinib (asp015k) in patients with rheumatoid arthritis and an inadequate response to methotrexate: results of a phase iii randomised, double-blind, placebo-controlled trial (raj4) in japan peficitinib for the treatment of rheumatoid arthritis: an overview from clinical trials drug details: 50 mg smyraf (peficitinib hydrobromide drug details: 100 mg smyraf (peficitinib hydrobromide safety and effectiveness of peficitinib (asp015k) in patients with rheumatoid arthritis: interim data (22.7 months mean peficitinib treatment) from a long-term, open-label extension study in the american college of rheumatology preliminary core set of disease activity measures for rheumatoid arthritis clinical trials efficacy and safety of tofacitinib for the treatment of rheumatoid arthritis in patients from the asia-pacific region: post-hoc analyses of pooled clinical study data safety of baricitinib in east asian patients with moderate-to-severe active rheumatoid arthritis: an integrated analysis from clinical trials herpes zoster and tofacitinib: clinical outcomes and the risk of concomitant therapy herpes zoster and tofacitinib therapy in patients with rheumatoid arthritis long-term safety of tofacitinib for the treatment of rheumatoid arthritis up to 8.5 years: integrated analysis of data from the global clinical trials jak inhibitors in covid-19: need for vigilance regarding increased inherent thrombotic risk the japanese experience with biologic therapies for rheumatoid arthritis the authors would like to thank the patients involved in this study, the study investigators, and team staff.disclosures. tt has received grants from astellas pharma, inc., chugai pharma co. ltd, daiichi sankyo co. ltd, takeda pharma co. ltd, abbvie g.k., asahi kasei pharma corp., mitsubishi tanabe pharma co., pfizer japan, inc., eisai co. ltd, ayumi pharma corp., nippon kayaku co. ltd, and novartis pharma k.k.; speaking fees from abbvie g.k., bristol-myers squibb k.k., chugai pharma co. ltd, mitsubishi tanabe pharma co., pfizer japan, inc., astellas pharma, inc., daiichi sankyo co. ltd, eisai co. ltd, sanofi k.k., teijin pharma ltd, takeda pharma co. ltd, and novartis pharma k.k.; consultancy fees from astra zeneca k.k., eli lilly japan k.k., novartis pharma k.k., mitsubishi tanabe pharma co., abbvie g.k., nippon kayaku co. ltd, janssen pharma k.k., astellas pharma, inc., taiho funding. sponsorship for this study and rapid service fee were funded by astellas pharma, inc. medical writing support was funded by astellas pharma, inc.medical writing assistance. medical writing support was provided by anne-marie edwards, mchem, of cello health medergy (europe), and funded by astellas pharma, inc.authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.prior presentation. interim data from this study were published as takeuchi and tanaka et al. safety and effectiveness of peficitinib (asp015k) in patients with rheumatoid arthritis: interim data (22.7 months mean peficitinib treatment) from a long-term, open-label extension study in japan, korea, and taiwan. arthritis research & therapy (2020) 22:47. the final data from this study (32 months mean peficitinib exposure, as included in this manuscript) were presented in part as a virtual poster at the 22nd asia-pacific league of associations for rheumatology, 24-29 october 2020, virtual congress; and were accepted as an abstract to the 2020 jcra annual meeting.compliance with ethics guidelines. this study was conducted in accordance with good clinical practice, the international council on harmonisation (ich) of technical requirements for registration of pharmaceuticals for human use guidelines, and local laws and regulations. the study protocol and amendments were reviewed and approved by an institutional review board or independent ethics committee (supplementary table s1 ) at each study site, and safety data were reviewed by an independent data and safety monitoring board. each patient provided written informed consent prior to treatment initiation. this analysis and the clinical trials from which data were included followed the principles of the declaration of helsinki. key: cord-0031702-07imrw74 authors: anaparti, vidyanand; wiens, dana; o'neil, liam j.; hubbard, erika; robl, robert; smolik, irene; hitchon, carol; lipsky, peter e.; el-gabalawy, hani title: utility of baseline transcriptomic analysis of rheumatoid arthritis synovium as an indicator for long-term clinical outcomes date: 2022-05-03 journal: front med (lausanne) doi: 10.3389/fmed.2022.823244 sha: 9254404c1d437378f769f8cdef821cff8bd405a5 doc_id: 31702 cord_uid: 07imrw74 objective: rheumatoid arthritis is a chronic inflammatory autoimmune disease that can lead to synovial damage, persistent joint pain, and functional disability. our objective was to evaluate baseline synovial transcriptome from early inflammatory arthritis patients (eia) and identify pretreatment biomarkers that could potentially provide insights into long-term functional outcomes of rheumatoid arthritis (ra). methods: synovial biopsies from clinically inflamed knee joints were procured from either 17 eia patients before initiation of disease modifying anti-rheumatic drug (dmard) therapy (dmard-naïve eia) using the minimally invasive closed needle biopsy technique or advanced ra patients undergoing arthroplasty. affymetrix human genome u133 plus 2 microarray platform was used to profile the synovial transcriptome. the cohort was followed clinically for a median of 12.3 years, and patient data was collected at each visit. short-term and long-term clinical outcomes were determined by assessing ra-associated clinical parameters statistical adjustments were made to account for asynchronous clinical visits and duration of follow up. results: based on the transcriptomic analysis, we identified 5 differentially expressed genes (degs), including matrix metalloproteinase (mmp)-1 (fibroblast collagenase) and mmp-3 (stromelysin-1) in dmard-naïve eia patients, relative to advanced ra patients (q < 0.05). dichotomous expression of mmp-1 and mmp-3 mrna and protein was confirmed by qpcr and immunohistochemistry respectively, based on which dmard-naïve eia subjects were classified as mmp-high or mmp-low. hierarchical clustering of transcriptomic data identified 947 degs between mmp-high and mmp-low cohorts. co-expression and ipa analysis of degs in the mmp-high cohort showed an enrichment of genes that participated in metabolic or biochemical functions and intracellular immune signaling were regulated through nf-κb and β-catenin complexes and correlated with markers of systemic inflammation. analysis of short-term clinical outcomes in mmp-high cohort showed a significant reduction in the das-crp scores relative to baseline (p <0.001), whereas area under the curve analyses of modified haq (mhaq) scores correlated negatively with baseline mmp-1 (r = −0.59, p = 0.03). further, longitudinal mhaq scores, number of swollen joints, number of dmards and median follow-up duration appeared to be higher in mmp-low cohort. conclusion: overall, our results indicate that the gene expression profiling of synovial biopsies obtained at the dmard-naive stage in patients with eia categorizes them into subsets with varying degrees of inflammation and can predict the future of long-term clinical outcome. objective: rheumatoid arthritis is a chronic inflammatory autoimmune disease that can lead to synovial damage, persistent joint pain, and functional disability. our objective was to evaluate baseline synovial transcriptome from early inflammatory arthritis patients (eia) and identify pretreatment biomarkers that could potentially provide insights into long-term functional outcomes of rheumatoid arthritis (ra). methods: synovial biopsies from clinically inflamed knee joints were procured from either 17 eia patients before initiation of disease modifying anti-rheumatic drug (dmard) therapy (dmard-naïve eia) using the minimally invasive closed needle biopsy technique or advanced ra patients undergoing arthroplasty. affymetrix human genome u133 plus 2 microarray platform was used to profile the synovial transcriptome. the cohort was followed clinically for a median of 12.3 years, and patient data was collected at each visit. short-term and long-term clinical outcomes were determined by assessing ra-associated clinical parameters statistical adjustments were made to account for asynchronous clinical visits and duration of follow up. results: based on the transcriptomic analysis, we identified 5 differentially expressed genes (degs), including matrix metalloproteinase (mmp)-1 (fibroblast collagenase) and mmp-3 (stromelysin-1) in dmard-naïve eia patients, relative to advanced ra patients (q < 0.05). dichotomous expression of mmp-1 and mmp-3 mrna and protein was confirmed by qpcr and immunohistochemistry respectively, based on which dmardnaïve eia subjects were classified as mmp-high or mmp-low. hierarchical clustering of transcriptomic data identified 947 degs between mmp-high and mmp-low cohorts. coexpression and ipa analysis of degs in the mmp-high cohort showed an enrichment of genes that participated in metabolic or biochemical functions and intracellular immune signaling were regulated through nf-κb and β-catenin complexes and correlated with markers of systemic inflammation. analysis of short-term clinical outcomes in mmphigh cohort showed a significant reduction in the das-crp scores relative to baseline (p<0.001), whereas area under the curve analyses of modified haq (mhaq) scores correlated negatively with baseline mmp-1 (r = −0.59, p = 0.03). further, longitudinal early recognition and treatment initiation in rheumatoid arthritis (ra) patients using appropriately targeted disease modifying anti-rheumatic drugs (dmard) can improve response to therapy, prevent structural damage and facilitate a long-term disease remission (1) (2) (3) (4) (5) . a personalized approach to treatment is only possible when patients can be stratified at the time of their diagnosis and treated based on objective biomarkers that can predict response to specific dmards. ra is a chronic autoimmune condition characterized by synovial cell hyperplasia, joint deformity, bone erosion, and persistent pain, leading to progressive functional disability. the disease evolves as a continuum with early synovitis being the primary clinical manifestation, which in turn is heterogenous in clinical presentation, histopathology, response to therapy and treatment outcomes (6) . in fact, studies undertaken to characterize the cellular composition and molecular mechanisms underlying synovial heterogeneity have identified distinct synovial pathotypes that can refine clinical classification of ra and are associated with clinical variables such as disease duration, systemic inflammation, and radiographic progression. moreover, patient-stratification based on baseline molecular signatures in the synovium of early ra patients was strongly associated with long-term clinical outcomes and predicted future treatment responsiveness to dmards, including biologics (1, 2, 7, 8) . as a means to begin to sub-set patients with early inflammatory arthritis (eia), we applied a microarray-based strategy to evaluate the synovial transcriptome in fine-needle tissue biopsy samples from dmard-naïve eia patients relative to those with established ra. furthermore, we correlated these molecular signatures with clinical outcomes collected from these individuals periodically during a 15-year longitudinal follow-up post-dmard intervention. samples used in this study were obtained from participants recruited into our manitoba early arthritis cohort between 2000 and 2005. the study was approved by the local research ethics boards, and participants provided written informed consent. all patients were enrolled before initiation of a first dmard. after the biopsy, patients were treated according to current guidelines for early ra. clinical data was acquired and captured in a custom database. these individuals were followed longitudinally every 3 months for the first year, and then every 6 months thereafter or as indicated for usual care. visit schedules varied over the course of follow-up and the clinical outcomes were recorded at each visit. no attempt was made to guide their dmard therapy, which was solely based on clinical indications targeting clinical remission. baseline synovial biopsies from dmard-naïve eia patients were obtained using a minimally invasive closed needle biopsy technique (parker-pearson method). all the biopsies were performed on clinically inflamed knee joints. samples were obtained from multiple areas in each biopsied joint, and all samples were adequate for transcriptomic and immunohistopathological analysis. two individuals had bilateral biopsies of their affected knees. as controls, we used synovial tissues from advanced ra (n = 6) patients that were collected from anonymous donors during joint replacement surgery. ra diagnosis was made based on fulfilling the 2010 acr/eular classification criteria, as determined by a rheumatologist (heg/ch). venous blood was collected into sst tm serum separation tubes (bd biosciences) and processed as per the manufacturer's instructions. screening for c-reactive protein (crp), erythrocyte sedimentation rate (esr), anti-cyclic citrullinated protein (anti-ccp) and rheumatoid factor (rf) was performed at a clinical and/or research laboratory at a single tertiary care hospital (health sciences centre, winnipeg, manitoba, canada). analysis of clinical outcomes were undertaken using all the available clinical data for each study participant. because of the asynchronous nature of clinical visits and duration of follow up, area under the curve (auc) of modified health assessment questionnaire (mhaq) scores normalized to duration of follow up was used to estimate the burden of specific disease manifestations such as functional disability. immunohistology h&e (hematoxylin & eosin) staining was carried out on paraffinembedded tissues. total cell counts were determined through light microscopy image analysis. immunohistological analysis and quantification of the same synovial samples was undertaken of oct-embedded tissue blocks for differentially expressed genes (degs) identified in the transcriptomic analysis. tissue homogenization, and total rna isolation (rneasy rna isolation kit, qiagen inc) was carried out on fresh synovial biopsy samples as per manufacturer's instructions. to minimize variability, we collected at least two individual samples from different locations of each joint being used for this purpose. in the case of synovial tissues obtained from patients with late-stage ra (n = 6) at the time of joint arthroplasty, representative samples from each synovial tissue were used to generate total rna, which was then processed in an identical manner to the needle biopsy samples. ten microgram of high-quality rna (28/18s ratio >1) was extracted from these synovial biopsy samples. rna quality was determined on agilent bioanalyzer using the agilent rna 6000 nano kit and quantified on a nanodrop nd-1000 spectrophotometer. total rna with a a260/280 > 2.0 and an rna integrity number (rin) > 8.0 was used for assessing synovial transcriptome. microarray analysis was performed by dr. chao lu (centre for applied genomics, hospital for sick children, toronto). briefly, total rna was converted to complementary rna (crna) and hybridized to affymetrix hu133plus2 chips that had 54,675 probe sets corresponding to the entire human genome. hybridized chips were scanned using an affymetrix genechip scanner 3000. data from the microarray chips was normalized and analyzed using the mas 5.0 algorithm, then imported into arrayassist software (stratagene) and analyzed using significance analysis of microarray (sam) analysis (stanford, california) (9) . mann-whitney u-test, chi-square test, pearson correlation and spearman rank correlation analyses were used as and when required. graphpad prism (v9.1) was used for graphical representation of the results. r packages or ingenuity pathway analysis were used to perform functional network analysis as explained below. weighted gene co-expression network analysis and multi-scale embedded gene co-expression network analysis wgcna algorithm was used to construct co-expressed gene network modules that were assessed further for their functional significance (10) . briefly, raw microarray data files underwent background correction and gcrma normalization resulting in log 2 intensity values compiled into an expression set object (eset). the e-set was then restricted to the top 5,000 probes with the highest variance among the dmard-naïve eia samples. a scale-free topology matrix (tom) was calculated to encode the network strength between probes with a soft thresholding power of 30. tom distances were used to cluster probes into wgcna modules. resulting co-expression networks were trimmed using dynamic tree cutting and the deepsplit function in r. partitioning around medoids (pam) was also utilized to assign outliers to the nearest cluster. the resulting network was formed with a minimum module size of 100, cut height of 1, and merge height of 0.2. modules were given random color assignments and expression profiles summarized by a module eigengene (me). final membership of probes representing the same gene were decided based on strongest within-module correlation to the me value. for each module, me values were correlated by pearson correlation to the clinical data including cohort (mmp-high group = 1, mmp-low group = 0), esr, crp, age, sex, swollen joints, disease duration, tender joints, and total affected joints. significance was determined using an adjusted p-value ≤ 0.2. additionally, megena was applied to the dataset as an independent method of identifying coexpression networks. megena is a multi-scale co-expression gene clustering algorithm, which was used to create additional gene expression networks by applying it on the normalized and filtered e-set as described for wgcna. multi-scale clustering structures were identified using planar filtered networks and resultant gene co-expression modules were also correlated to clinical metadata as previously described for wgcna. the top 40 co-expression modules with significant correlations to the clinical trait of interest, cohort (mmp-high vs. mmp-low) were reported (11). co-expression modules were annotated according to the top overlapping functional category with the most significant p-value and a minimum of four overlapping genes. in the absence of significant overlaps, "unknown" was the assigned annotation. functional enrichment within the gene co-expression modules and relative significance with clinical outcomes was calculated using gene ontology (go), transcriptomic signatures derive from published literature and functional aggregation tools, namely immune/inflammation-scope (i-scope), tissue-scope (t-scope) and biologically informed gene clustering (big-c) (12) (13) (14) (15) (16) . i-scope categorizes gene transcripts into one of a possible 28 hematopoietic cell categories based on matching transcripts known to mark various types of immune/inflammatory cells. t-scope is an additional aggregation tool to characterize cell types found in specific tissues. big-c classifies genes into 53 different groups based on their most probable biological function and/or cellular or subcellular localization. odds ratios and overlap pvalues were calculated using fisher's exact test in r using the fisher test function. statistical significance was obtained using an adjusted p-value ≤ 0.2. in total, 15 dmard-naïve eia patients were enrolled in this longitudinal study and underwent baseline synovial biopsy of an affected knee joint using the parker-pearson technique, prior to initiation of their first dmard. two of these study participants in whom both knees were affected underwent bilateral synovial biopsy. table 1 and supplementary table s1 summarizes the clinical characteristics of eia subjects at baseline and the list of dmards prescribed to them throughout the entire duration of longitudinal follow-up. of this study population, 12/15 table s2) were the most significantly upregulated genes. because mmp-1 and mmp-3 are produced abundantly by the synovial lining layer and are known to play a key role in the progressive joint damage that occurs in ra (17, 18) , we focused on these two molecules as potential biomarkers for classifying the early inflamed synovium (figure 1 ). we noted a dichotomous distribution in the transcript levels of both mmp-1 and mmp-3 in the 17 eia synovial tissue samples (including two individuals who had bilateral synovial biopsy samples). as such, 10/17 synovial tissues (60%) exhibited high transcript levels of both mmp-1 and mmp-3, and 7/17 (40%) exhibited low transcript levels, the latter being comparable to the levels detected in the advanced ra samples (figure 1a) . we confirmed the mmp-1 and mmp-3 expression levels using qpcr ( figure 1b) and showed that there was a very strong correlation between mmp-1 and mmp-3 mrna levels in the dmard-naïve eia patients (r = 0.8897, p = 0.001, supplementary figure s2) . moreover, in analyzing the microarray datasets, we showed that this dichotomous distribution was unique to mmp-1 and mmp-3 as it was not demonstrable with any other mmp or timp transcripts, except for mmp-13, where similar trends were observed (supplementary figure s1; supplementary table s3 ). importantly, in the two individuals who had bilateral synovial samples obtained from both their affected knees joints, there was concordance in the mmp-1 and mmp-3 transcript levels between the two knee joints of the same individual, with one patient exhibiting bilateral high levels, and the other bilateral low levels. this suggests that the mmp-1 and mmp-3 transcript levels were reflective of the individual's pathologic process and not simply related to local factors in each joint. based on these findings, we then categorized each eia patient as being either an mmp-high or mmp-low mrna expressor in their inflamed synovial tissue. we sought to determine whether the grouping of the eia samples based on mmp-1/mmp-3 synovial mrna transcript levels was reflected in the expression of the corresponding proteins, both locally in the synovial tissue using ihc (figure 2a) , and systemically in the circulation using elisa. as shown in figures 2b,c, there were dramatic differences in the expression of both mmp-1 and mmp-3 protein in the synovium between the mmp-high and mmp-low mrna groups. compared to the mmp-low group, the mmp-high group exhibited higher intensity of ihc staining for mmp-1 and mmp-3 in the synovial lining layer and in the sublining areas (figure 2c) . although much of the staining appeared to be extracellular, we were able to demonstrate intense intracellular staining for these proteins in the synovial lining cells (figure 2a) . in contrast to levels observed in the synovial biopsy samples, circulating mmp-1 and mmp-3 levels did not show similar dichotomy (supplementary figure s3) , although the levels of these two proteins were highly concordant in the serum (r = 0.5473, p = 0.0478), and correlated with the degree of inflammation as indicated by crp levels (supplementary figure s3) . we applied unsupervised hierarchical clustering algorithm to identify the spectrum of differentially expressed genes (degs) within the microarray dataset between mmp-high and mmplow groups (figure 3a) . analysis revealed the presence of two distinct clusters based on the gene expression profile. while 622 genes were found to be increased in mmp-high subjects, expression of 325 genes was high in the mmp-low group. wgcna and megena analysis was applied to the transcriptomic data to explore gene co-expression modules and determine biological processes that drive their differential expression in dmard-naïve eia subjects (figures 3b-d) . wgcna analysis yielded 23 co-expressed gene modules (each module was assigned a color), 21 of which correlated with at least one clinical outcome ( figure 3b ). of these, salmon and yellow were the only modules that showed association with the mmp-status of the cohort (r = −0.34 and r = 0.42, respectively). therefore, we selected these modules for further analysis. as shown in figure 3c , subsequent assessment of the yellow module for functional relevance revealed an enrichment for genes involved in metabolic functions (mitochondria, cytoplasm and biochemistry) and was associated with mmp-high group. on the other hand, the salmon module was enriched for fibroblast and stromal signatures (cytoskeleton, secreted and ecm) and associated with the mmp-low group (figure 3c) . we also interrogated gene expression signature in the lightcyan, lightyellow, tan and turquoise modules owing to their correlation with total affected joints, swollen joints, disease duration, esr and crp. these modules were enriched for b cell, t cell, and il-6 gene signatures (figure 3c) . megena was applied to further dissect complex co-regulatory gene networks and explore their interaction with clinical outcomes in dmardnaïve eia patients (figure 3d) . a majority of these modules correlated positively with mmp-status of the patient, followed by esr and crp. most importantly, parent modules were found to be enriched for inflammatory and phagocytic macrophageassociated gene signatures, including those involved in tnf signaling. we also observed an enrichment of the fibroblast transcriptomic signature in module 2.10 and related modules. we then applied ipa bioinformatics tool to identify common transcriptional hubs that were primarily responsible for differential expression of certain genes in mmp-high patients. analysis of curated functional networks revealed direct and indirect relationships with multiple cell-signaling molecules that were centrally connected to nf-κb, β-catenin (ctnnb1) and p38mapk, and converge leading to increased cxcl1 expression (figure 3e) . most of these molecules were acute-phase proteins involved in il-17a signaling in autoimmune diseases such as psoriasis and arthritis (14.3% overlap; p = 0.0000266). given the distinct baseline synovial transcriptomic signatures identified in the cohort of individuals with early, untreated inflammatory arthritis, we next sought to determine whether there were differences in the longitudinal clinical outcomes, when categorized based on mmp dichotomy. patients enrolled in the study were followed for a median of 12.3 years (# clinical visit = 24; iqr 16). clinical assessment (swollen and tender joints), functional scores (mhaq) and medications were recorded at each clinical assessment. baseline clinical features were similar between mmp-high and mmp-low groups (supplementary table s5 ). at the short-term follow-up interval of 31 months, dmard-naïve eia patients displayed a significant reduction in their crp levels, das-crp scores, swollen joint counts and tender joint counts relative to the levels at their baseline visit (figures 4a-d) . interestingly, these changes were highly prominent in the mmp-high group compared to the mmp-low patients (figures 4e-h) . to determine the burden of long-term functional disability, we next assessed mhaq scores across all longitudinal visits, normalized to followup duration (mhaq auc/year). longitudinal mhaq scores showed an increasing trend in mmp low patients compared to mmp high (figure 5a table 2) , based on the mmp status at arthritis onset. importantly, baseline mmp-1, and to a lesser extent mmp3, mrna levels showed an inverse correlation with longterm mhaq auc scores ( figure 5b) . we also observed that the median length of follow up was longer for mmp-low patients (177 months) compared to mmp-high (74 months), though the number of annual visits were similar between the groups (mmp-high vs. mmp-low; 2.03 vs. 1.96 per year). together, these data suggest that the mmp-high group appeared to accrue less long-term disability compared to the mmplow group. we present the results of a broad transcriptomic analysis of baseline synovial tissue samples that were obtained using closed needle biopsy from dmard-naïve eia patients with inflammatory arthritis, most of whom were diagnosed with seropositive early inflammatory arthritis. we defined the transcriptomic signature that was predictive of long-term clinical outcomes in these patients who all received standard care of treatment. the synovium is the primary target organ for the chronic immuno-inflammatory process that characterizes ra, and other chronic arthropathies (19) . it is also well-established that both the systemic and synovial responses to a wide array of available dmard/biologic therapies is heterogeneous, and notoriously difficult to predict based on clinical parameters and circulating biomarkers such as autoantibody profiles and crp. this challenge is further complicated by the unpredictable loss of therapeutic efficacy to currently available ra drugs, necessitating empiric trials of alternative therapies in the hope of recapturing control of the disease. because of this, there has been a longstanding interest in identifying predictive synovial biomarkers early in the disease process that could help classify the inflammatory lesions based on pathotypes which could, in turn, potentially inform difficult clinical decisions (7, 20) . much progress has been made in this area, particularly recently, where large international consortia have provided intriguing new data based on state-of-the-art analyses of the synovial biopsies (3, 7, 21) . yet, despite the availability of sizable cohorts of ra patients who have undergone synovial biopsy in research settings, a key gap is the lack of data regarding the long-term outcomes of these biopsied ra patients in routine clinical settings where individuals typically cycle through several agents, alone or in combination. the data presented in the current study are an attempt to address this gap by providing longitudinal outcome data in a cohort of ra patients who underwent baseline synovial biopsy and were then followed for up to 15 years under routine clinical care. overall, we showed that the transcriptional signature of the synovium of dmard-naïve patients with active ra was heterogenous, and this heterogeneity was primarily defined by dichotomous expression of mmp-1 and mmp-3 genes, both at the gene and protein level. characterization of molecular pathways underlying divergent synovial mmp1/mmp3 expression suggests the presence of distinct types of synovitis, one of which is regulated by nf-κb and β-catenin. importantly, ra patients with high mmp1/mmp3 expression exhibited a significant reduction in their disease activity, and inflammation at a short-term follow-up point and improved physical function when assessed after a prolonged period of dmard therapy. we also observed a difference in the mrna levels of mmp-13 between mmp-high and mmp-low eia subjects. however, these levels did not achieve statistical significance when corrected for false-discovery rate using benjamini-hochberg method. in contrast, short and long-term treatment response in mmp-low cohort was reminiscent of outcomes observed in treatment resistant individuals. taken together, our data suggests a strong association between baseline mmp-status of the synovium and response to dmard treatment, thereby underscoring the diagnostic value of synovial transcriptome at the pre-dmard stage as a predictor of response to ra therapy. additional studies are needed to evaluate whether baseline synovial mmp-status can be used as an indicator to determine short and long-term response to a specific ra drug or is applicable to any dmard therapy (2, 3, 7, 22) . to determine the potential clinical utility of the baseline synovial mmp grouping, we evaluated the clinical outcomes of the cohort over an extended longitudinal timeframe. we defined relatively short-term outcomes after an average of ∼2 years, and long-term outcomes after more than one decade. no attempt was made to guide the subsequent dmard/biologic therapy these individuals received, and they were treated using standard of care. as such, we had serial visits for each member of the cohort, with documentation of joint counts and mhaq scores. our future work will explore the relationship between synovial gene expression and the accrual of radiographic joint damage in both the biopsied joint and non-biopsied joints of hands and feet. since no one visit could be regarded as an endpoint, we chose to perform an area under the curve (auc) analysis as a method to quantify longitudinal outcomes. our study also has other important limitations, the most obvious of which is the small size of the clinical cohort. this may have introduced bias toward specific synovial pathotypes, and indeed may limit the generalizability of the findings. also, we acknowledge the lack of information on the medications prescribed to advanced ra patients, who were anonymous donors undergoing a joint replacement surgery. despite these shortcomings, we were able to clearly delineate two major synovial subsets based on the levels of mmp-1 and mmp-3 mrna and protein expression, both of which paralleled each other. these two mmps are known to play a key role in the pathogenesis and destructiveness of inflammatory arthritis (17, 23, 24) . importantly, at baseline, the two groups were clinically indistinguishable suggesting that there may be potential clinical utility to assessing their synovial expression. as such, it is important to point out that the circulating levels of the mmps did not correlate with their synovial expression levels. using this approach, we unexpectedly demonstrated that the group with the highest baseline synovial expression levels of mmp-1/mmp-3 appeared to accrue less functional disability over time than the group with substantially lower levels, the latter being comparable to the levels we demonstrated in synovial samples obtained from ra patients at the time of joint arthroplasty. this finding seems to be counter intuitive considering the role these molecules play in the destruction of cartilage and connective tissue in the synovial compartment (24) . one potential explanation for this unexpected finding is that the individuals with a high mmp-1/mmp-3 baseline signature are more responsive to dmard therapy. this may be analogous to observations made in the context of malignancies where highly proliferative, activated neoplasms respond better to chemotherapy than do those that are more indolent (25) . however, it needs to be validated in other longitudinally followed early ra cohorts and confirm our observations. evidence suggests synovial phenotypes can range from a myeloid pattern to a lymphoid or a fibroid phenotype defined primarily by the cell-types infiltrating into the synovium [1, 10, 12] . our gene expression analysis or immunohistological staining did not show any evidence of either myeloid lymphoid or pauci-immune phenotypes prior to dmard therapy. we also observed a homogenous infiltration of macrophages (cd68), fibroblasts (cd55) and lymphocytes (cd3 and cd20) in the synovium. this may be due to a low sample size, use of a different methodology for identifying gene expression or a different patient population. the co-expression analysis identified an enrichment of metabolic, and inflammatory genes in the mmphigh subtype, most of which are involved in active intracellular signaling. most of these pathways, including those targeted by dmards regulate the expression of molecules that are involved in the nf-κb pathway (26) . in contrast, mmp-low samples showed a predominance of fibroblast genes that are involved in ecm and cytoskeletal reorganization. ipa curated analysis of genes enriched in mmp-high group identified nf-κb complex, and β-catenin as the two major transcriptional nodes. in patients with early inflammatory arthritis, nf-κb activation in the synovium facilitates proliferation of synovial fibroblasts, modulates tissue-specific immune responses, and perpetuates chronic inflammation by promoting secretion of proinflammatory mediators including mmp1 and mmp-3 in the synovium (26) (27) (28) (29) . further investigations are needed on synovial biopsies collected longitudinally at different time intervals from patients on dmard therapy to validate these hypotheses and shed light on how molecular processes evolve within the synovium in response to treatment in individuals stratified according to their baseline tissue mmp-status. in conclusion, we have demonstrated an mmp-centered synovial heterogeneity in dmard-naïve eia patients, which could reliably predict short-term and long-term response to treatment irrespective of the dmard being administered. if this approach is validated, it may be a valuable metric for clinicians to identify individuals who could have homogenous response to specific treatments and allow them to provide the right treatment to right patient, a step toward precision medicine in ra. the original contributions presented in the study are publicly available. this data can be found here: https://www.ebi.ac.uk/ arrayexpress/experiments/e-mtab-11274. the manitoba early arthritis cohort study was approved by the university of manitoba's research ethics boards, and participants provided written informed consent. he-g and pl conceived research concept. va, dw, lo'n, he-g, rr, and eh analyzed data. va, dw, and eh prepared figures. ch recruited patients, acquired samples, and collected longitudinal clinical data. va, dw, lo'n, eh, pl, and he-g drafted and revised the manuscript, and all co-authors participated in editing the manuscript. all authors contributed to the article and approved the submitted version. manitoba early arthritis cohort study was funded by serial grants from the health sciences research foundation (principal investigator: ch). the study was also supported by individual grants to he-g from the canadian institutes of health research and from astrazeneca uk. the funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. va received postdoctoral fellowships from research manitoba and arthritis society of canada. eh, rr, and pl were supported by a grant from the rilite foundation. synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics synovial tissue signatures enhance clinical classification and prognostic/treatment response algorithms in early inflammatory arthritis and predict requirement for subsequent biological therapy: results from the pathobiology of early arthritis cohort (peac) synovial tissue heterogeneity in rheumatoid arthritis and changes with biologic and targeted synthetic therapies to inform stratified therapy responsiveness to anti-tumour necrosis factor alpha therapy is related to pre-treatment tissue inflammation levels in rheumatoid arthritis patients the clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor alpha expression in the synovium the natural history of rheumatoid arthritis synovial cellular and molecular signatures stratify clinical response to csdmard therapy and predict radiographic progression in early rheumatoid arthritis patients rituximab versus tocilizumab in anti-tnf inadequate responder patients with rheumatoid arthritis (r4ra): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial significance analysis of microarrays applied to the ionizing radiation response wgcna: an r package for weighted correlation network analysis patient ancestry significantly contributes to molecular heterogeneity of systemic lupus erythematosus locally renewing resident synovial macrophages provide a protective barrier for the joint comprehensive transcriptomic analysis of covid-19 blood, lung, and airway altered expression of genes controlling metabolism characterizes the tissue response to immune injury in lupus analysis of trans-ancestral sle risk loci identifies unique biologic networks and drug targets in african and european ancestries early joint erosions and serum levels of matrix metalloproteinase 1, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 in rheumatoid arthritis expression of chemokines and matrix metalloproteinases in early rheumatoid arthritis kelley and firestein's textbook of rheumatology molecular portraits of early rheumatoid arthritis identify clinical and treatment response phenotypes heterogeneity of synovial molecular patterns in patients with arthritis synovial tissue heterogeneity in rheumatoid arthritis in relation to disease activity and biomarkers in peripheral blood matrix metalloproteinases: role in arthritis a tale of two joints: the role of matrix metalloproteases in cartilage biology management of indolent lymphoma: where are we now and where are we going nf-kappa b in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction heterogeneous requirement of ikappab kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy regulation of matrixmetalloproteinase-3 and matrixmetalloproteinase-13 by sumo-2/3 through the transcription factor nf-kappab nfkb2 polymorphisms associate with the risk of developing rheumatoid arthritis and response to tnf inhibitors: results from the repair consortium we acknowledge the invaluable contributions ramandip singh, guoping ma, xiaobo meng, keng wong, and daniela stroescu for their role in data collection, sample storage, sample processing, and data analysis. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-0027848-ectbbz5j authors: wu, tao; zhang, xue-pei; zhang, qian; zou, yao-yao; ma, jian-da; chen, le-feng; zou, yao-wei; xue, ji-meng; ma, ruo-fan; chen, zhong; dai, lie title: gasdermin-e mediated pyroptosis—a novel mechanism regulating migration, invasion and release of inflammatory cytokines in rheumatoid arthritis fibroblast-like synoviocytes date: 2022-02-14 journal: front cell dev biol doi: 10.3389/fcell.2021.810635 sha: 039f0e492266a9b1709fc4baecebd92249730c54 doc_id: 27848 cord_uid: ectbbz5j synovium fibroblast-like synoviocytes (flss) are important participants in the pathogenesis of synovitis and joint destruction in rheumatoid arthritis (ra). pyroptosis is a pro-inflammatory and cell lytic programmed cell death mechanism mediated by gasdermin (gsdm) family proteins. in this study, we demonstrated the increased expression of gsdme and increased levels of gsdme-mediated pyroptosis in ra synovial tissues. in vitro, stimulation with tnf-α plus hypoxia mimicking the inflammatory and hypoxic environment in ra synovium induced gsdme-mediated pyroptosis in ra-flss in combination with the promotion of migration and invasion abilities and the release of inflammatory cytokines (il-6, il-8). moreover, knockdown of gsdme significantly inhibited the proliferation rate, migration/invasion effects and cytokines released through the reduction of gsdme-mediated pyroptosis. the immunohistochemistry results showed that ra patients with high gsdme n-terminal (gsdme-nt) expression, which is the active form of gsdme, showed higher il-6 expression in both lining and sublining layer of synovium than that in patients with low gsdme-nt expression, osteoarthritis and non-inflammatory orthopedic arthropathies. our findings revealed a novel mechanism regulating cell proliferation, migration, invasion and inflammatory cytokines release during the process of gsdme mediated pyroptosis in ra. rheumatoid arthritis (ra) is an autoimmune disease characterized by chronic synovitis accompanied by excessive release of inflammatory cytokines and the destruction of bone and cartilage. although the outcomes of ra patients have achieved considerable improvements due to treatments directed by "treat to target" strategy and other biological reagents, a significant proportion of patients fail to achieve clinical remission and experience radiographic joint damage progression (lillegraven et al., 2012) . it is urgently needed to identify novel therapeutic targets for ra. fibroblast-like synoviocytes (flss) located in the synovium have been identified as important contributors to the pathogenesis of ra due to their involvements in inflammation as well as cartilage and bone destruction (bartok and firestein, 2010) . increasing researches have been concentrated on the mechanisms underlying the abnormal proinflammatory responses and invasive behaviors of ra-flss and, hopefully, possible treatment strategies based on targeting ra-flss. pyroptosis is a form of programmed cell death featuring cell swelling and large bubbles blowing from the plasma membrane mediated by pore-forming gasdermin (gsdm) family proteins . gasdermin is a family of proteins sharing a functionally important gasdermin n domain with pore-forming activity (chadha et al., 2020) . in the absence of activation signals, the gasdermin n domain and c domain are connected to each other and form an autoinhibitory structure (ding et al., 2016) . after cleavage between the n domain and c domain by caspases, the two parts are separated, and the n domain is disinhibited (ding et al., 2016) . the n terminal cleavages then bind to the cell membrane, oligomerize and form pores causing leakage of cell components, which is the pivotal and unique process of pyroptosis distinguished from other manners of cell death (ding et al., 2016) . essentially, pyroptosis is a proinflammatory process because of its cell lytic activity, which mediates excessive release of inflammatory mediators and thus induces and aggravates inflammation . based on this feature, pyroptosis might play a crucial role in the genesis and development of ra. a study has implicated the importance of gsdmd-mediated monocytes pyroptosis in ra pathology (wu et al., 2020) , and ze-qing zhai's latest article revealed a pathogenic role of gsdme in the pyroptosis of monocytes and macrophages in ra (zhai et al., 2021) . however, the occurrence of pyroptosis as well as its mechanism and effect in ra-flss remain unknown. in this study, we illuminated the role of gsdme-mediated flss pyroptosis in ra, providing a novel insight for the mechanisms of inflammatory cytokines secretion and aggressive cell behaviors in ra-flss. lipofectamine rnaimax reagent and opti-mem medium were purchased from thermo fisher scientific (united states). small interfering rnas (sirnas) including gsdme (si-gsdme#1, stb0006132a; si-gsdme#2, stb0006132b; si-gsdme#3, stb0006132c) and control sirnas (si-nc, sin0000001-1-10) were obtained from ribobio (guangzhou, china). the corresponding target sequences for gsdme silencing were si-gsdme#1, cccactgcttctttgtata; si-gsdme#2, caa gcagctgtttatgaca and si-gsdme#3, ggtcctatt tgatgatgaa. primary antibodies including anti-human gsdme monoclonal ab (abcam, ab215191), anti-human gsdme polyclonal ab (proteintech, 13075-1-ap), anti-human-thy1/cd90 ab (abcam, ab181469), anti-human hif-1α ab (abcam, ab51608), anti-human caspase-3 ab (abcam, ab32351), anti-human il-6 ab (abcam ab9324) and anti-gapdh ab (cell signaling technology, #5174) were used in this study. active ra patients fulfilled the 2010 american college of rheumatology (acr) /european league against rheumatism (eular) classification criteria (aletaha et al., 2010) and manifested with knee-joint swollen and pain were recruited from the department of rheumatology at sun yat-sen memorial hospital between november 2020 and june 2021. ra synovium (n = 28) was obtained by parker-pearson needle biopsy. control synovium was obtained from osteoarthritis (oa; n = 4), and non-inflammatory orthopedic arthropathies (orth.a; n = 5) who received arthroplasty, or arthroscopy at the department of orthopedics as we previously described . the clinical characteristics of included participants were listed in supplementary table s1 . the study was performed in accordance with the declaration of helsinki and approved by the medical ethics committee of sun yat-sen memorial hospital (sysec-2009-06 and sysec-ky-ks-012). all participants gave written informed consent. flss were isolated using modified tissue culture method (schumacher and kulka, 1972) and culture as we previously described . flss from passages 3 to 7 were used in this study. for tnf-α treatment, after serum starvation overnight, medium was replaced by serum-free dmem containing recombinant tnf-α (r&d systems, united states) with the indicated concentration. for hypoxia treatment, cells were cultured in a humidified tri-gas incubator (1% o 2 , 5% co 2 , and 94% n 2 ) for the indicated time. knockdown of gsdme transcripts ra-flss (10 (ding et al., 2016) / well) were transfected with sirnas(50 nm, 24 h)using lipofectamine rnaimax reagent and opti-mem medium following the manufacturer's instructions. total rna was prepared from cells using rnaiso reagent (takara, japan). complementary dna (cdna) samples were synthesized with reverse-transcription kit (takara, japan). primers used for amplification of the cdna were as follows (forward, reverse): gsdma, 5′-ctgacaccacttgacagc ct-3′ and 5′-ttcaggaatgggtggtctgc-3'; gsdmb, 5′-tctcagggccatctcagcta-3′ and 5′-agcaccatcctt cttcatcgt-3; gsdmc, 5′-ggaagcaaagacctgaca cc-3′ and 5′-ggaacggtcctgtcacaaca-3; gsdmd, 5′-ctgctccatgagaggcacc-3′ and 5′-aggacgtcc aagtcagagtca-3; gsdme, 5′-tgatggtgacctgat tgcag-3′ and 5′-cgaccactggactcggaaat-3. qpcr was performed using quantitecktm sybr green pcr kit frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 western blot analysis of caspase-3, cleaved caspase-3, gsdme and n-terminal cleavage of ra-flss treated with 40 ng/ml tnf-α under hypoxia conditions for 0-48 h (n = 6). hif-1α serves as a marker of effective hypoxic stimulus, gadph serves as a loading control. (e) video captures of ra-flss under tnf-α + hypoxia treatment (40 ng/ ml, 36 h). blue arrow and yellow arrow point out two bursting cells. yellow asterisks designate the swelling cells. data are shown as mean ± s.e.m and results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way anova. frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 4 (takara, japan). the reactions were initiated with denaturation of cdna templates at 95°c for 30 s, 95°c for 5 s and 60°c for 30 s and amplification for 40 cycles on a roche lightcycler480 sequence detector system (roche, switzerland). the edu staining was conducted using a beyoclick edu cell proliferation assay kit (beyotime, china) according to the manufacturer's protocol. images were captured using an olympus ix73 microscope and the number of cells was manually counted. ihc staining was carried out on the qualified synovium as we previously described . antigens were retrieved by boiling in edta (ph 9.0) for 3 min at 120°c in a pressure cooker. sections were then washed, blocked, and incubated with rabbit anti-gsdme ab (proteintech, 1:100) or mouse anti-il-6 ab (abcam, 1.5 ug/ml) overnight at 4°c. after washing, the sections were incubated with envision mouse or rabbit conjugate (dako corporation, united states) for 30 min at 37°c. the color reaction was completed with the dab-positive substrate and hematoxylin. concentrationmatched normal rabbit igg (cell signaling technology, #2729) and normal mouse igg (santa cruz, sc-2025) were used as a negative control. the mean il-6 expression in the synovium in each field was valuated under a 400× optical microscope using the scoring system (huang et al., 2018) multiplied the intensity of the scoring (0, = absent, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (0 = 0%, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, 4 = 76-100%). the mean score from five different fields was determined as the finial ihc score. for if staining, sections were incubated with rabbit anti-gsdme ab (proteintech, 1:100) and mouse anti-cd90 ab (abcam, 1:100) overnight at 4°c and then incubated with alexa fluor 594 conjugated anti-rabbit igg (cell signaling technology, #8889, 1:800) and alexa fluor 488 conjugated anti-mouse igg (cell signaling technology, #4408, 1:800) for 30 min at 37°c. after staining with dapi slides were observed using a laser confocal microscope (lsm 510; carl zeiss, germany). ra-flss (1.5 × 10 5 / well) treated as indicated were collected and stained with annexin v-fitc/propidium iodide (pi) apoptosis detection kit (elabscience, china). cell death analysis was performed on the bd facs caliber flow cytometer. ra-flss were treated as indicated in 24-well plates and incubated with 1 μg/ml pi (solarbio, china) for 15 min at 37°c. brightfield video and static brightfield / pi channel images of pyroptotic cells were captured using an olympus ix73 microscope. ra-flss (10 (chadha et al., 2020) /well) were seeded into 96-well culture plates and treated as indicated. the supernatant was collected and determined for ldh activity using ldh assay kit (beyotime, china) according to the manufacturer's instruction. ra-flss were collected and the total protein was extracted using column tissue & cell protein extraction kit (epizyme, china) following the manufacturer's protocol. concentrations of extracted protein were determined using the pierce bca assay kit (thermo fisher scientific, united states). expression of proteins were analyzed using western blot as we previously described (ma et al., 2019a) . wound healing assay ra-flss (10 (ding et al., 2016)/well) were seeded in six-well plates and treated as indicated. wound lines were made by scratching the cellular monolayer with sterile 200 µl pipette tips. after 48 h of incubation in serum-free medium, cell migration was measured by calculating that healing area beyond reference line at 0 h using imagej software. migration and invasion assay were performed using transwell chambers as we described previously (ma et al., 2019b) . for migration assay, 2 × 10 4 ra-flss were resuspended by serumfree dmem, placed in the upper chamber, and allowed to migrate for 12 h. for invasion assay, the upper chamber was pre-coated with matrigel (1:5) (bd biosciences, united states) and the same amount of flss were resuspended by serum-free dmem, placed in the upper chamber allowed to invade for 24 h. 10% fbs was placed in the lower chamber as a chemoattractant in both migration and invasion assay. after removal of cells remained on the upper surface of the membrane, the cells on the lower surface were stained with crystal violet staining solution (solarbio, china) manually counted. data were presented as the mean number of cells in five randomly captured 100× fields. supernatants of cells with the indicated treatments were collected and centrifuged (12,000 rpm, 5 min), and secretion of cytokines was analyzed by a protein profiler human cytokine array kit (ary005b, r&d systems, united states) following the manufacturer's protocol. results were quantified using imagej software. secretion levels of il-6, il-8 were also detected using corresponding elisa kits (neobioscience, china) following the manufacturer's protocol. statistical analyses were performed on spss 25.0 statistical software (spss, united states). data were presented as mean ± standard error of mean (s.e.m). comparisons between two groups were analyzed by two-tailed student's t test. multiple groups of samples were analyzed by one-way anova, and pairwise using the data exploration website developed by lewis, m. j., et al. (https://peac.hpc.qmul.ac.uk/) (lewis et al., 2019) , gsdme (dfna5) gene signatures were dissected across different synovial compartments in the pathobiology of early arthritis cohort (peac) and integrated with deep phenotypic profiling. gsdme expression was found in synovial tissue, and this gene was highly expressed in fibroid subtypes (supplementary figure s1a) . furthermore, weighted correlation network analysis (wgcna) showed that gsdme was annotated against thy-1(+) fibroblast cell population (supplementary figure s1b) , which was found to be remarkably expanded in ra synovium compared to control synovium and correlated with severe and figure 3 | suppression of ra-flss pyroptosis by downregulation of gsdme. (a) western blot analysis of casepase-3, cleaved caspase-3, gsdme and hif-1α of ra-flss treated with tnf-α + hypoxia treatment (40 ng/ml, 36 h). gsdme sirna or control sirna was transfected for 24 h before treatment (n = 6). gadph serves as a loading control. (b) flow cytometry analysis (n = 3) and (c) representative phase-contrast images with pi staining of ra-flss treated with or without tnf-α + hypoxia (40 ng/ml, 36 h) after 24 h of transfection with gsdme sirna (si-gsdme) or control sirna (si-nc). data are shown as mean ± s. e.m and results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way anova. frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 6 persistent inflammation in ra (croft et al., 2019; wei et al., 2020) . to confirm the expression of gsdme in ra synovial tissue, 5 active ra patients with distinct knee joint pain were recruited, biopsied, and ihc staining was then conducted on these samples. gsdme full-length protein (gsdme-fl) expression was detected in both the lining and sublining layers ( figure 1a) . as the key step of pyroptosis, gsdme-fl is cut into an n-terminal fragment (gsdme-nt) with pore-forming activity and a c-terminal fragment by the activated cleaved caspase-3 and then cell pyroptosis is induced (ding et al., 2016; wang et al., 2017; jiang et al., 2020) . to explore the existence of gsdmemediated pyroptosis in synovium from ra patients and "less inflamed" disease controls, western blot analysis were performed for the detection of activated gsdme-nt in synovial tissue extracts from ra, oa, and orth.a patients. as shown, gsdme-nt and gsdme-fl expression was significantly increased in synovium extracts from ra patients compared to those from orth.a patients, but statistic difference was not found in either gsdme-fl or gsdme-nt levels between oa synovium and orth.a synovium ( figure 1b, supplementary figure s2 ). immunofluorescence staining of ra and orth.a synovium also revealed colocalization of gsdme with cd55 in the lining area and thy-1 flss in the sublining area, suggesting a link between flss and gsdme-mediated pyroptosis in ra synovium as detected above (figures 1c, supplementary figure s3 ). furthermore, in flss cultured in vitro, gsdme mrna, not gsdma, gsdmb, gsdmc, or gsdmd, was the most abundantly transcribed, but no statistically significance was found between the mrna levels of ra-flss and orth. a-flss ( figure 1d ). protein levels of gsdme-fl and gsdme-nt were also detected by western blot. results showed that gsdme-fl protein expression was increased in ra-flss compared to orth.a-flss, but gsdme-nt expression showed no difference, possibly due to the absence of disease microenvironment in vitro ( figure 1e ). it has been well recognized that the inflammatory and hypoxic microenvironment in ra synovium facilitates the abnormal activation of ra flss. to mimic the microenvironment in ra synovium, ra-flss were treated with tnf-α and exposed to hypoxia (1% o 2 ). the ldh release test (figure 2a) showed that treatment with 40 ng/ml tnf-α plus a 36-h hypoxia exposure represented the optimal trigger for lytic cell death of ra-flss and used in the subsequent experiments. the proportion of lytic cell death determined by flow cytometry analysis and microscopic pi staining was also significantly increased after treatment with tnf-α plus hypoxia ( figures 2b,c) . moreover, characteristic figure 4 | suppression of proliferation, migration and invasion of ra-flss through inhibition of gsdme-mediated pyroptosis. (a) decrease in ra-flss proliferation by knockdown of gsdme measured by edu experiment (n = 6, scale bar = 100 μm). (b,c) inhibition of ra-flss migration and invasion activities by gsdme knockdown. after 24 h of transfection with gsdme sirna (si-gsdme) or control sirna (si-nc), ra-flss migration was determined by wound healing assay (b, n = 6, scale bar = 200 μm) and transwell chamber assay (c, n = 6, scale bar = 100 μm), invasion was determined for by matrigel-coated transwell assay (c, n = 6, scale bar = 100 μm). data are shown as mean ± s.e.m and results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way anova. frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 swelling of cells, formation of large bubbles from plasma membrane, and eventually cell rupture of ra-flss were observed after treatment with tnf-α plus hypoxia, establishing the characteristic morphology of the pyroptosis process ( figure 2e) . consistent with the abovementioned evidences, increased expression of gsdme-nt in ra flss treated with tnf-α plus hypoxia was detected by western blot (figure 2d ), further suggesting that tnf-α plus hypoxia triggers gsdme-mediated pyroptosis in ra-flss. moreover, increases in hif-1α, gsdme-fl and cleaved caspase-3 expression were also observed after tnf-α plus hypoxia treatment ( figure 2d ). the silencing efficiency of gsdme sirna was confirmed by qrt-pcr and western blot (supplementary figures s3a,b) . after tnf-α plus hypoxia treatment, the expression levels of hif-1α, cleaved caspase-3, gsdme-fl and gsdme-nt were significantly increased ( figure 3a) . however, the protein expression levels of gsdme-fl and gsdme-nt were decreased by gsdme sirna transfection in both the control group and treatment group. meanwhile, knockdown of gsdme lowered the rate of lytic cell death in both the control and treatment groups. (figures 3b,c) . the results indicated that downregulation of gsdme mitigated ra flss pyroptosis induced by tnf-α plus hypoxia. excessive proliferation, abnormal migration and invasion are the basic pathologic characteristics of ra flss. results of edu assay showed that both tnf-α+hypoxia treatment and gsdme knockdown restrained the proliferation of ra-flss ( figure 4a ). on the other hand, silencing gsdme suppressed ra-flss migration and invasion abilities stimulated by tnf-α+hypoxia treatment ( figures 4b,c) . taken together, these results suggested a pivotal role of gsdme-mediated pyroptosis in the phenotypic transformation of ra flss. pyroptosis is characterized by cell rupture accompanied by release of a great quantity of cell components, including proinflammatory cytokines and chemokines, which are key regulators of cell behaviors in ra flss. to further investigate the underlying mechanisms, supernatants of ra flss after tnfα plus hypoxia treatment were collected and detected by a proteome profiler array kit to identify the key cytokines and chemokine responding to gsdme knockdown. after tnf-α+hypoxia stimulation, supernatant il-6, il-8 and cxcl1 level were remarkedly increased. reversely, gsdme knockdown attenuated il-6, il-8 and cxcl1 release in response to tnf-α and hypoxia treatment. (figures 5a,b) . the regulation of il-6, il-8 and release by gsdme knockdown was further validated by corresponding elisa assay ( figure 5c ). based on the aforementioned results, suppression of gsdmemediated ra-flss pyroptosis dramatically decreased the levels of il-6 released in vitro, implicating a novel mechanism by which il-6 and other cytokines may be excessively released under inflammative and hypoxic circumstance such as ra. to verify this hypothesis in synovial samples, twenty-one ra, four oa and five orth. a patients with qualified synovium were subjected to ihc analysis of the synovial il-6 expression. il-6 expression was observed in both the lining and sublining layers of the synovium ( figure 5d ). the median ihc scores of both lining and sublining il-6 expression were significantly higher in ra patients than that in oa [lining: 2.6 (1.2, 5.5) vs. 1.0 (0.9, 1.8), p = 0.049; sublining: 2.2 (1.2, 2.6) vs. 0.7 (0.5, 1.0), p = 0.011], or orth. a patients [lining: 2.6 (1.2, 5.5) vs. 1.2 (0.7, 1.6), p = 0.037; sublining: 2.2 (1.2, 2.6) vs. 1.0 (0.4, 1.7), p = 0.047]. ra patients were further classified into a high gsdme-nt expression group (n = 15) and a low gsdme-nt expression group (n = 6) according to the expression level of gsdme-nt in orth.a determined by western blot analysis of synovial tissue extracts. patients with expression levels greater than that noted in orth.a were classified as high gsdme-nt expression; otherwise, patients were considered to have low gsdme-nt expression. ra patients with high gsdme-nt expression possess higher il-6 ihc scores than those with low gsdme-nt expression [lining: 2.8 (2.4, 5.6) vs. 1.1 (0.8, 3.4), p = 0.047; sublining: 2.2 (1.4, 2.8) vs. 1.0 (0.8, 1.8), p = 0.029], suggesting that gsdmemediated pyroptosis can obviously affect the synovial il-6 level ( figure 5e ). in the present study, for the first time, we reported the occurrence of gsdme-mediated pyroptosis in ra-flss under the inflammatory and hypoxic synovial microenvironments. moreover, our results indicated that gsdme-mediated pyroptosis contributes to the proliferation, migration, invasion, and release of inflammatory cytokines in ra-flss, further providing a novel direction for ra pathology research and treatment development. as a basic cell biological process, miscellaneous forms of cell death have been studied in ra-flss, and the most well-known is apoptosis-a non-proinflammatory programmed cell death pattern. ra-flss are resistant to apoptosis and present with excessive proliferation and aggressive cell behaviors, including enhanced secretion of inflammatory and chemotactic cytokines and increased migrative and invasive ability. induction of apoptosis in ra-flss through certain therapies is therapeutically beneficial in ra (pope, 2002; smith et al., frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 8 2010). differ from apoptosis, pyroptosis exhibits a proinflammatory peculiarity. due to the formation of gasdermin pores on the plasma membrane, cells undergo osmolysis, and excessive cellular contents, including inflammatory cytokines and danger-associated molecular patterns (damps), are released into extracellular compartment chadha et al., 2020) , thereby inducing and exaggerating inflammation. pyroptosis has been implicated in cancer biology and infectious disease (wang et al., 2017; sarhan et al., 2018; jiang et al., 2020; karki et al., 2021) . moreover, several researches in osteoarthritis indicated that gsdmd-mediated flss pyroptosis participates in hif-1αinduced synovial fibrosis in knee osteoarthritis (zhao et al., 2018; zhang et al., 2019) . a recent study also showed that gsdmdmediated monocytes pyroptosis was involved in persistent inflammatory cytokine release in ra pathology (wu et al., 2020) . the latest research ultimately studied the pathogenic role of gsdme in monocytes, macrophages and animal models of ra, suggesting that gsdme might be a potential therapeutic target for ra (zhai et al., 2021) . however, involvement of gsdme-mediated pyroptosis in the function of ra-flss has not yet been reported. interestingly, we found that both full-length gsdme and gsdne-n terminal cleavage was detected in ra-flss in synovial tissue in situ and cultured in vitro, constituting the figure 5 | alleviation of il-6 and other inflammatory cytokines release by downregulation of gsdme-mediated ra-flss pyroptosis. (a,c) after sirna transfection and tnf-α + hypoxia treatment, cell culture supernatant was collected and the levels of cytokines and chemokines were measured using proteome profiler human cytokine array kit from r&d systems (a,b). supernatant levels of il-6, il-8 were further verified by elisa (n = 3) (c). (d,e) he and ihc staining for il-6 of synovium tissues from ra (n = 21), oa (n = 4) and orth.a (n = 5) patients (scale bar = 100 μm). ra synovium tissues were futher divide into low gsdme-nt level (n = 6) and high gsdme-nt level (n = 15) groups based on matched gsdme-nt level determined by western blotting. data are shown as mean ± s. e.m and results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using one-way anova. frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 9 prerequisite of pyroptosis. in addition, by mimicking the particular microenvironment in which ra-flss are situated inflaming and hypoxic circumstances due to long-term and severe synovitis (fearon et al., 2016) , we observed that a larger proportion of ra-flss undergo lytic cell death along with activated gsdme-nt expression and typical morphological features, strengthening the occurrence of gsdme-mediated ra flss pyroptosis in inflammatory and hypoxic microenvironments. to study the roles of gsdme-mediated pyroptosis in ra-flss activation, we silenced gsdme expression with sirna transfection and then determined the cell death rate, proliferation rate, migration and invasion ability, and inflammatory cytokines secretion of ra-flss. with gsdme suppression, the level of tnf-α+hypoxia-induced pyroptosis was significantly downregulated, along with restrained cell proliferation rate, migration/invasion ability and secretion of cytokines (especially il-6). these results indicated that gsdme-mediated pyroptosis may contribute to the overactivation and aggressive behavior of ra-flss, inflammatory mediators leakage into the extracellular space and inflammatory cell recruitment from blood to synovium, which are critical mechanisms of cartilage and bone erosion and persistent synovitis ( figure 6) . the pathogenic role of il-6 in ra has been widely demonstrated. il-6 is an important regulator of b-cell maturation, t cell differentiation and bone homeostasis (mcinnes and schett, 2007; garbers et al., 2018) . il-6 increases the expression of receptor activator of nuclear factor-κb ligand (rankl), an important factor for osteoclast differentiation, by osteoblasts, disturbing osteoclast-osteoblast balance and exacerbating bone destruction (mcinnes and schett, 2007; garbers et al., 2018) . fibroblasts are major contributors to synovial inflammation and bone/cartilage destruction and are dominant producers of a variety of cytokines, especially il-6 in ra, flss-derived il-6 also regulates the production of matrix enzymes, such as matrix metalloproteins (mmps), aggrecanases and cathepsins, aggravating articular damage (mcinnes and schett, 2007; nguyen et al., 2017) . as a differentiation factor of th17 cells, il-6 is pivotal in local inflammation. previous studies have shown that the high concentrations of il-6 in synovial fluids positively correlate with disease activity and risk of radiographic progression in arthritis (mcinnes and schett, 2007; larsson et al., 2015; nguyen et al., 2017) . the application of il-6targeted therapy, such as tocilizumab, significantly reduces il-6 expression in ra synovium, demonstrating improvements in ra therapy (kanbe et al., 2011; kang et al., 2019) . nonetheless, understanding the mechanism of il-6 (and probably other cytokines/chemokines) secretion by ra-flss through gsdme-mediated pyroptosis still provides a new perspective for developing more effective treatments in ra. regrettably, most experiments in this study were conducted in ra-flss cultured in vitro. of note, the absolute level of pyroptosis induced by tnf-α+hypoxia remained less than 10%, indicating that only a small fraction of ra-flss underwent pyroptosis. however, a single spark can start a prairie fire. recent studies reported a mechanism of cytokines release in the sublytic phase before the truly membrane-lytic pyroptosis phase (xia et al., 2021; zhou and abbott, 2021) , which might explain the significant effects resulting from pyroptosis of a small number of cells. due to the limitation of direct detection methods of gsdme-nt in situ in synovial tissue, the actual pyroptosis level of ra-flss in vivo still requires further verification. a more reliable and accurate method to detect pyroptosis levels in tissue and larger scale of samples and patient cohort data are required for correlation analysis between gsdme-mediated flss pyroptosis and clinical parameters in the future. another limitation of this research is the lack of animal study. but thanks to zhai's work, we can derive figure 6 | illustration of a novel regulation of inflammatory cytokines secretion and aggressive cell behaviors in ra-flss through gsdme-mediated pyroptosis. frontiers in cell and developmental biology | www.frontiersin.org february 2022 | volume 9 | article 810635 a key information from the cia model of gsdme knockout mice that gsdme −/mice exposed to collagen exhibited a reduced incidence of arthritis and lower clinical arthritis scores than wild-type mice, providing strong support of our results. in conclusion, our study demonstrates a novel mechanism for modulating cell proliferation, migration, invasion and inflammatory cytokines secretion by gsdme mediated pyroptosis in ra-flss. these results provide new insight into the crosstalk between the inflammatory and hypoxic synovial microenvironment and the abnormally activated ra flss, thus suggesting a new direction for flss targeted therapy for ra. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. the studies involving human participants were reviewed and approved by medical ethics committee of sun yat-sen memorial hospital (sysec-2009-06 and sysec-ky-ks-012). the patients/participants provided their written informed consent to participate in this study. all authors contributed to the final manuscript. tw, x-pz, and qz contributed equally to this work as participating in acquisition of synovium samples from needle biopsy, experiments planning and performing, statistical analysis, and drafting and revising this manuscript. ld, y-yz, j-dm helped design the study and critically revised the data and this manuscript. l-fc, y-wz, and j-mx helped with collection of the clinical materials and data analyzing. r-fm and zc helped with collection and acquisition of surgical synovium samples and clinical data. all authors read and approved the final manuscript for publication. rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis mechanistic insights into the role of pyroptosis in rheumatoid arthritis distinct fibroblast subsets drive inflammation and damage in arthritis pore-forming activity and structural autoinhibition of the gasdermin family hypoxia, mitochondrial dysfunction and synovial invasiveness in rheumatoid arthritis interleukin-6: designing specific therapeutics for a complex cytokine 17β-estradiol upregulates il6 expression through the erβ pathway to promote lung adenocarcinoma progression the caspase-3/gsdme signal pathway as a switch between apoptosis and pyroptosis in cancer inhibition of map kinase in synovium by treatment with tocilizumab in rheumatoid arthritis targeting interleukin-6 signaling in clinic synergism of tnf-α and ifn-γ triggers inflammatory cell death, tissue damage, and mortality in sars-cov-2 infection and cytokine shock syndromes interleukin-6 and tumor necrosis factor alpha in synovial fluid are associated with progression of radiographic knee osteoarthritis in subjects with previous meniscectomy molecular portraits of early rheumatoid arthritis identify clinical and treatment response phenotypes remission and radiographic outcome in rheumatoid arthritis: application of the 2011 acr/eular remission criteria in an observational cohort a novel function of artesunate on inhibiting migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients serum matrix metalloproteinase-3 as a noninvasive biomarker of histological synovitis for diagnosis of rheumatoid arthritis activation of the peroxisome proliferator-activated receptor γ coactivator 1β/nfatc1 pathway in circulating osteoclast precursors associated with bone destruction in rheumatoid arthritis cytokines in the pathogenesis of rheumatoid arthritis autocrine loop involving il-6 family member lif, lif receptor, and stat4 drives sustained fibroblast production of inflammatory mediators apoptosis as a therapeutic tool in rheumatoid arthritis caspase-8 induces cleavage of gasdermin d to elicit pyroptosis duringyersiniainfection needle biopsy of the synovial membrane -experience with the parker-pearson technic pyroptosis: gasdermin-mediated programmed necrotic cell death apoptosis in the rheumatoid arthritis synovial membrane: modulation by disease-modifying anti-rheumatic drug treatment chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin notch signalling drives synovial fibroblast identity and arthritis pathology complement c1q synergizes with ptx3 in promoting nlrp3 inflammasome over-activation and pyroptosis in rheumatoid arthritis attenuation of rheumatoid arthritis through the inhibition of caspase3/gsdmemediated pyroptosis induced by tnf-α increased hif-1α in knee osteoarthritis aggravate synovial fibrosis via fibroblast-like synoviocyte pyroptosis nlrp1 and nlrp3 inflammasomes mediate lps/atp-induced p-yroptosis in k-nee o-steoarthritis gasdermin e permits interleukin-1 beta release in distinct sublytic and pyroptotic phases down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappab activation we gratefully thank all the patients and the members of the medical staff who generously collaborated with this research. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.810635/ full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-0024923-4fx2w448 authors: ciaffi, jacopo; mitselman, dmitri; mancarella, luana; brusi, veronica; lisi, lucia; ruscitti, piero; cipriani, paola; meliconi, riccardo; giacomelli, roberto; borghi, claudio; ursini, francesco title: the effect of ketogenic diet on inflammatory arthritis and cardiovascular health in rheumatic conditions: a mini review date: 2021-12-14 journal: front med (lausanne) doi: 10.3389/fmed.2021.792846 sha: b5bfce2609917a5975302b3c68501e45e6320a1e doc_id: 24923 cord_uid: 4fx2w448 the principle of ketogenic diet (kd) is restriction of carbohydrates to a maximum of 5–10% of the total daily caloric intake, aiming at shifting body metabolism toward ketone bodies. different studies suggested promising results of kd to help patients to lose weight, to reduce insulin requirements in diabetes, to supplement cancer protocols, to treat neurological conditions and to optimize control of metabolic and cardiovascular diseases. however, literature about the anti-inflammatory properties of kd in rheumatic diseases is still limited. the beneficial effects of weight loss in patients with inflammatory arthritis can be explained by biomechanical and biochemical factors. obesity is associated with macrophage activation and production of pro-inflammatory cytokines including tnf-α, il-1b, and il-6. the clinical effect of kd may be primarily attributed to improvement of insulin sensitivity. insulin resistance is associated with an increase of tnf-α, il-1α, il-1β, il-6, and leptin. moreover, reduction of body's adipose tissue and weight loss account for part of the anti-inflammatory effects and for the impact of kd on cardiovascular health. in rheumatoid arthritis, fasting was shown to be effective in reducing disease symptoms, possibly through the production of β-hydroxybutyrate (bhb), the main ketone body. bhb may exert inhibitory effects also on il-17 and intermittent fasting improved the clinical manifestations of psoriatic arthritis. in ankylosing spondylitis, current literature doesn't allow to draw conclusion about the effects of kd. future prospective studies will be needed to elucidate the potential beneficial effects of kd on specific domains and clinical outcomes in patients with inflammatory arthritis. ketogenic diet (kd) is characterized by marked carbohydrate restriction, usually to <50 grams a day, and not in a single meal. in a standard kd, carbohydrates should represent about 5-10% of the total daily caloric intake, while the rest of macronutrients consists of proteins (20%) and fats (70-75%) (1) . the concept of kd was proposed for the first time in 1921 as a substitute for fasting (2) . in the early 20th century, before the introduction of anti-epileptic drugs, fasting was the method of choice to manage epilepsy (3) . but fasting, although efficient, cannot be maintained for a long period of time. therefore, in 1921, dr. wilder proposed kd as a suitable method to induce a metabolic state similar to fasting, through the production of ketone bodies, but without caloric restriction. this method was widely used as a treatment for epilepsy during the fourth and fifth decade of 20th century but then dramatically decreased when new anti-epileptic drugs were introduced. kd experienced a reemergence in recent years as a means for weight loss and the physiological concepts behind the dietary regimen gained new scientific interest (3) . in the present mini review, we summarize available literature regarding the potential role, pathophysiology and clinical implications of kd in inflammatory arthritis. literature review was limited to published primary research, including basic science, cohort studies, intervention and observational trials, and review articles indexed in pubmed. the following search terms were used: "ketogenic diet" and "arthritis" or "rheumatoid arthritis" or "psoriatic arthritis" or "ankylosing spondylitis." as the intent of the review was narrative, inclusion was based on relevance, as deemed so by the authors, to one of the three subcategories of interest: (1) ketogenic diet in rheumatoid arthritis (ra); (2) ketogenic diet in psoriatic arthritis (psa); (3) ketogenic diet in ankylosing spondylitis (as). additionally, articles reporting the effects of kd on cardiovascular health in patients with rheumatic diseases were considered relevant. the balance between formation (ketogenesis) and degradation (ketolysis) controls circulating levels of ketone bodies, in a process mainly regulated by the secretion of insulin and glucagon (4) . among the important physiological changes induced by kd there is insulin reduction (5) and the hormonal changes caused by kd, with decreased insulin and increased glucagon levels, favor gluconeogenesis. under conditions of marked carbohydrate restriction, the body primary energy source switches from glucose toward ketones and fatty acids which are obtained from dietary fat and proteins but also from endogenous sources such as glycogen and adipose stores through lipolysis (6) . the accelerated mobilization rate of fatty acids from adipose tissue leads to conversion of acetyl-coa into ketone bodies, in a process known as ketogenesis (7) . ketogenesis takes place primarily in the liver, which can produce ketone bodies in two ways (8) . the first way is the oxidation of fatty acids to acetyl-coa, which are the building blocks of the ketone bodies, or by conversion of amino acids directly into ketone bodies (9) . this results in the synthesis of β-hydroxybutyrate (bhb), acetoacetate and acetone. bhb is the primary and most abundant ketone body found in bloodstream. however, the liver cannot use ketone bodies due to the lack of the enzyme succinyl coa:3-ketoacid coa transferase. therefore, ketone bodies are utilized as a fuel by extra-hepatic tissues, thus sparing glucose metabolism. in these circumstances, ketone bodies replace most of the glucose required by the brain, while liver gluconeogenesis provides the limited amount of energy needed by glucose-dependent tissues such as red blood cells, retina and renal medulla (7) . the mean time for achieving nutritional ketosis is generally 3 days, although longer periods up to 10 days have been documented (10) . during the first 3 days, the ketosis induction phase, the patient may experience adverse effects including headache, nausea, asthenia, fatigue, constipation. these effects tend to disappear at the end of the induction phase (10) . a different type of adverse effects may be seen in long-duration kd, including gastroesophageal reflux disease, uric acid increase, electrolyte imbalance and hyperlipidemia (11) . insulin reduction and improvement of insulin sensitivity contribute to the clinical effects of kd. insulin resistance is an impaired response to insulin stimulation of target tissues, primarily liver, muscle and adipose tissue (12) . insulin signal is conducted through complex intracellular mechanisms regulated by multiple kinase enzymes (13) . one of the most prominent applications of kd is for the purpose of weight loss (14, 15 ) and a meta-analysis of 60 dietary trials of weight loss suggested insulin reduction as a primum movens of weight loss (16) . another clinical manifestation of kd related to glucose metabolism and insulin regulation can be observed in patients diagnosed with glucose transporter 1 (glut1) mutation, which have impaired absorption of glucose from the blood, leading to a reduced supply of nutrients to the brain that manifests as epilepsy. in these patients, kd caused a significant improvement of seizure frequency (17) . furthermore, kd has been evaluated as part of cancer treatment protocols in brain, colon, breast, and lung tumors. these studies resulted in beneficial effects when used alongside chemotherapy, radiotherapy, or both (9) . additionally, kd can be used in the setting of alzheimer's disease, with an increase in cognitive function (18) , and therapeutic effects have been proposed also for neurologic conditions characterized by substantial motor dysfunction (19) . systemic inflammation is regulated by the production of proand anti-inflammatory cytokines. alterations in the balance of these mediators results in reduction or increase in systemic inflammation (20) . the effects of kd on systemic inflammation are related to three main drivers: (1) insulin reduction, (2) bhb synthesis, (3) glucagon increase (21) . additionally, since insulin reduction leads to weight loss, all the anti-inflammatory effects of weight loss should be taken into account as well (figure 1 ). insulin and chronic hyperinsulinemia are associated with an increase of the pro-inflammatory cytokines tnf-α, il-1α, il-1β, il-6, and leptin (22) . leptin is a unique cytokine due to its adipose-derived origin. adipose tissue is considered not only as the body's energy reservoir, but also as an endocrine tissue that can produce proinflammatory cytokines (23) . furthermore, weight loss is a relevant part of the anti-inflammatory effects of kd and reduction of body's adipose tissue has an influence on the secretion of hormones such as leptin (24) . bhb has a double effect on nlrp3 inflammasome complex (nic). nic is a protein complex involved in monocyte-induced inflammation (25) . when activated, nic functions as an inducer of caspase 1, which cleaves pro-il-1β. after being cleaved, pro-il1β becomes functional il-1β (26) . the activation of nic requires two steps: first the stimulus from toll-like receptor 4 (tlr4) that promotes the synthesis of nic proteins, and then a second step which is their assembly. bhb suppresses tlr4 stimulus and the assembly of nic individual proteins (27) . furthermore, bhb functions as a ligand to hydroxycarboxylic acid receptors (hcar). hcar, also known as gpr109a, is a g protein-coupled receptor predominantly expressed in adipose and immune cells (28) . activation of hcar suppresses proinflammatory cytokine production, including tnf-α, il-6, il-12, and il-1 (29) . the increase in glucagon is a direct effect of the decrease in insulin. nevertheless, glucagon is also a potent hormone that affects many systems, including the immune system (30) . glucagon exerts its effect by activating the cyclic adenosine monophosphate (camp) pathway. the effects of camp activation vary between tissues and even between cells of the same tissue. in dendritic cells, the increase in camp suppresses the release of pro-inflammatory mediators including tnf-α, il-17, ifn-γ, and promotes anti-inflammatory cytokine production such as il-10 (31). t cells have shown to reduce proliferation and production of il-2 (31) and to increase production of il-4 and il-5, both interleukins that promote th2 differentiation (32) . in smooth muscle cells, camp inhibits proliferation and suppresses the release of cytokines such as il-1ß and il-8 (30) . the beneficial effects of weight reduction can be explained by biomechanical and biochemical issues. weight loss reduces the load exerted on joints (33) . increased adipose tissue has been associated with local and systemic inflammation (23) . obesity is implicated in macrophage activation and production of proinflammatory cytokines including tnf-α, il-1b, and il-6 (34). as mentioned previously, insulin reduction precedes weight loss (16) . therefore, insulin reduction can be considered a benefit of weight loss. part of insulin pro-inflammatory effect is exerted through inhibition of anti-inflammatory cytokines production (35) . kd, besides the potential anti-inflammatory properties, is also the best non-surgical treatment for weight reduction. a meta-analysis of 53 studies including 68.128 participants suggested that higher-fat, low-carbohydrate diet, was the best intervention to achieve the weight loss and weight maintenance targets (36) . in ra, it is important to distinguish between intentional and unintentional weight loss. intentional weight loss is beneficial in improving the clinical aspects of ra, while unintentional weight loss can worsen the manifestation of ra (37) . a retrospective analysis of electronic medical records of 178 patients diagnosed with ra demonstrated obese and overweight patients who achieved weight reduction exceeding 5 kg had a three-fold higher probability to experience improvement in ra symptoms in comparison with patients who did not succeed in losing weight (38) . similar results were observed in psa, where several disease activity parameters improved after weight loss treatment with very low energy diet, in a dose responder manner (39) . several studies have been conducted to investigate the role of dietary interventions in ra (40) (41) (42) (43) (44) . available evidence suggests the potentially beneficial effects of anti-inflammatory diets on disease activity (45) . products such as red meat, salt or high-fat diet may trigger inflammation, while fruit, vegetables and fish may exert an anti-inflammatory action (46, 47) . nevertheless, there is no specific dietary recommendation in ra. a systematic review of 70 dietary studies revealed that fasting, omega 3 and vitamin d3 significantly reduced ra symptoms (48) . fasting and calorie restriction, in particular, were associated with improvement of ra activity, with stronger effects on subjective symptoms (49) . during ramadan, muslims refrain from eating and drinking from dawn to sunset. it results in a month of intermittent fasting. studying the effects of fasting during ramadan, su et al. (50) observed non-significantly higher das-28 scores before than during ramadan and significant improvement in morning stiffness and functional disability. current literature suggests the benefit of fasting in treatment of ra (49) (50) (51) (52) and, as previously mentioned, the metabolic state induced by fasting can be considered similar to kd. on this basis, it is conceivable a role for kd in ra but the available knowledge outlines little efficacy (53, 54) . fraser et al. (54) found that fasting, but not kd, significantly decreased serum il-6 levels and improved disease activity in ra patients. however, in these studies kd was protracted only for 7 days, in order to reproduce the effects of fasting. it has been demonstrated that longer periods are needed to have a response on pain control (55) and to negatively affect oxidative stress (56) . it is possible that the tested period of treatment was too short to obtain significant results in ra. kd may affect ra in several different ways. first, bhb suppresses macrophages and neutrophils' synthesis of il-1 by inhibiting nic and thus reducing tnf-α (24, 57) . secondly, bhb has been shown to suppress proinflammatory interleukins including il-1, il-12, and il-6 by activation of hcar (29) . furthermore, bhb may inhibit the release of il-1β and il-18 mediated by nlrp3, contributing to the anti-inflammatory role of kd (58). psa is associated with several metabolic abnormalities. insulin resistance, hypertension, diabetes, and hyperuricemia are common comorbidities defining the spectrum of the systemic psoriatic disease (59) . literature about the effects of nutritional interventions in psa is limited (60, 61) , with no specific dietary indication for psa patients. in psoriasis, it has been shown that a ketogenic nutritional regimen led to significant improvement in disease activity indices (62) . through the assessment of nuclear magnetic medicine metabolomic profile, it was also possible to demonstrate a marked amelioration in biochemical parameters indicative of metabolites related to psoriasis (62) . it has been suggested that, in psoriatic patients, kd may facilitate weight loss and modulate systemic inflammation resulting in a quick response to systemic therapy (63) (64) (65) . we could not retrieve studies about kd in psa but some information can be derived from a study conducted on psa patients during the ramadan fasting (66) . adawi et al. demonstrated that intermittent fasting improved the clinical manifestation of psa, including psa disease activity scores, enthesitis and dactylitis. furthermore, the patients' improvement was independent of changes in the patients' weight (66) . psa has been strongly associated with th17 and il-17a increase (67) . in addition to the aforementioned kd effects, bhb induces the production of il-10 via dendritic hcar, resulting in an inhibitory effect on th17 (68, 69) . similar to ra and psa, there is little evidence that specific dietary interventions influence the activity of as. macfarlane et al. (70) conducted a systematic review of 16 publications, including 10 full-text articles, to investigate which dietary regimen induced the best clinical results in as. the authors concluded that reduction in starch intake, exclusion of dairy products, consumption of fish and fish oil or probiotic supplementation did not improve as symptoms. patients affected by as and, in general, by axial spondyloarthritis (axspa), are characterized by an increased cardiovascular risk (71) (72) (73) . mediterranean diet has been shown to exert a protective role on cardiovascular morbidity (74, 75) . when the impact of mediterranean diet was investigated in axspa patients, both the subjective perception of pain, acute phase reactants and disease activity improved after 6 months of nutritional intervention (76) . one of the key pathogenetic mechanisms of as is the impairment of immunomodulatory function of regulatory t cells, resulting in enhanced il-17 and other proinflammatory cytokines production, with proliferation of pro-inflammatory t cell subsets (77) . this process leads to inflammation in the enthesis and ileum of patients with active disease (78) . th17 differentiation is facilitated by tgf-β and il-6, while il-23 is determinant to stabilize and maintain th17 activation and secretion of pro-inflammatory cytokines (79) . interestingly, kd alters the gut microbiome, with ketone bodies directly inhibiting the growth of gut bacteria. data obtained from mice models suggest that, reducing the colonization levels of gut bacteria, kd may mediate the lack of intestinal th17 induction (80) . kd can thus induce changes in host metabolites. the alteration of gut microbiota may have downstream consequences for immune cells, reducing levels of intestinal th17 cells (80) . moreover, bhb was shown to affect microbial-mediated immunomodulation in addition to its ability to inhibit the nlrp3 inflammasome with consequent anti-inflammatory effects (80) . in ra, the risk of cardiovascular diseases has been reported to be higher than in the general population, in particular for stroke, heart failure, myocardial infarction and atrial fibrillation (81) (82) (83) (84) . patients with ra also have increased mortality rate independently from the presence of other cardiovascular risk factors (85) . additionally, high prevalence of cardiovascular comorbidities and increased mortality related to cardiovascular diseases has been demonstrated in patients with psa and as (71, (86) (87) (88) (89) (90) . robust evidence outlines a pivotal role of inflammation and immune system dysregulation in the pathogenesis of atherosclerosis and endothelial damage (91) (92) (93) . biologic and non-biologic anti-rheumatic therapies may exert a protective role on cardiovascular outcomes in patients with rheumatic diseases (94) and optimizing the control of disease activity has been associated with reduction of cardiovascular events in ra, psa and as (95) (96) (97) (98) . assessment and management of cardiovascular risk factors is therefore essential in the follow-up of patients with rheumatic diseases. although no study analyzed the effects of kd on cardiovascular health of individuals with inflammatory arthritis, the potential applications of kd in modulating cardiovascular risk factors and outcomes have been extensively investigated in non-rheumatic patients. a systematic review and meta-analysis of clinical trials carried out to study the efficacy of low-carbohydrate diet on major cardiovascular risk factors demonstrated significant reduction in body weight, bmi, abdominal circumference, blood pressure, plasma triglycerides, fasting plasma glucose, glycated hemoglobin, plasma insulin and plasma c-reactive protein, along with an increase in hdl-cholesterol (99) . an overall beneficial impact of low-carbohydrate diet on cardiovascular health was therefore observed, although a possible duration effect was suggested, with benefits that decrease over time. however, other reviews found controversial results, with no or little difference in changes of cardiovascular risk factors with kd, rising also questions about prolonged adherence to the dietary regimen (14, 100) . in conclusion, current literature suggests that kd might be associated with improvement of cardiovascular risk factors, mainly driven by weight loss possibility but further studies are needed to evaluate the long-term effects of kd on cardiovascular outcomes and also to assess which is the optimal macronutrients composition (101). kd is a well-established treatment option used since the 1920s for drug-resistant epilepsy (102) . emerging evidence suggests an adjuvant role of kd in cancer treatment (9, 103) and a possible applicability also in other conditions such as alzheimer's disease (18) and parkinson's disease (104) . however, the most significant results of kd have been obtained in treating obesity, with robust evidence showing improvement in body weight and reduction in levels of cholesterol, triglycerides and blood glucose (4, 14, 105) . moreover, a role of kd in the reduction of cardiovascular risk has been proposed (99) . obesity has been proposed as an environmental factor promoting onset and evolution of autoimmune diseases through a direct involvement of adipokines in their pathogenesis (106) . common mechanistic pathways are shared by obesity and rheumatic conditions (107) and the assessment of obesity status and of possible therapeutic interventions is of relevance when establishing a treatment plan for patients with rheumatic and musculoskeletal diseases (108) . extensive experimental and clinical research suggests that being overweight or obese impacts not only disease activity but also several aspects of the life of patients living with inflammatory arthritis (109) . weight loss improves outcomes in patients with ra (37, 38, 44, 49) , psa (39, 61, 64) and as (110, 111) . besides inflammatory arthritis, metabolic, and eating disorders were suggested to facilitate the occurrence of early clues of connective tissue disorders (112) and obesity was shown to worsen musculoskeletal symptoms also in patients with fibromyalgia (113) . promoting weight loss strategies may be part of the treatment for overweight and obese patients with fibromyalgia but evidence regarding the efficacy of kd in this setting is lacking. obesity is also a known risk factor for development and progression of osteoarthritis (114) . weight-loss programs were shown to ameliorate osteoarthritis symptoms (115) , with exercise and diet therapy leading to significant improvements (116) . low-carbohydrate diet reduced pain intensity in individuals with knee osteoarthritis (117) but, again, the efficacy of a therapeutic kd on different domains of osteoarthritis such as symptoms, pain relief, physical function and health-related quality of life has not been evaluated. in conclusion, literature about the effects of kd on disease activity and patient reported outcomes in inflammatory arthritis is extremely limited. evidence derived from fasting studies suggests a mild beneficial effect. since fasting and kd induce a similar metabolic state, a potential efficacy of kd could be assumed but the available data do not allow to draw conclusions. future prospective, population-based and adequately powered studies of dietary intervention are required to determine whether kd plays a role in the treatment strategy of patients with rheumatic musculoskeletal diseases. all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. this work was supported by progetto 5x1000 anno 2019 (redditi 2018) and project 5x1000 year 2019 (income 2018). ketogenic diets: boon or bane? ketogenic diet: old treatment, new beginning history of the ketogenic diet recent advances in the application of a ketogenic diet for obesity management effect of the ketogenic diet on glycemic control, insulin resistance, and lipid metabolism in patients with t2dm: a systematic review and meta-analysis starvation in man metabolic effects of the very-low-carbohydrate diets: misunderstood "villains" of human metabolism regulation of hepatic fatty acid oxidation and ketone body production ketogenic diet in the treatment of cancer -where do we stand? the use of nutritional supplements to induce ketosis and reduce symptoms associated with keto-induction: a narrative review long-term monitoring of the ketogenic diet: do's and don'ts insulin resistance insulin signaling in arthritis very-low-carbohydrate ketogenic diet vs. low-fat diet for longterm weight loss: a meta-analysis of randomised controlled trials efficacy and safety of very low calorie ketogenic diet (vlckd) in patients with overweight and obesity: a systematic review and meta-analysis temporal associations among body mass index, fasting insulin, and systemic inflammation: a systematic review and meta-analysis therapeutic strategies for glucose transporter 1 deficiency syndrome feasibility and efficacy data from a ketogenic diet intervention in alzheimer's disease how can a ketogenic diet improve motor function? cytokines in the systemic inflammatory response syndrome: a review ketogenic diet exhibits antiinflammatory properties cytokines and abnormal glucose and lipid metabolism adipose tissue, inflammation, cardiovascular disease inflammation, cytokines and insulin resistance: a clinical perspective the nlrp3 inflammasome: an overview of mechanisms of activation and regulation the role of nlrp3 and il-1β in the pathogenesis of inflammatory bowel disease β-hydroxybutyrate deactivates neutrophil nlrp3 inflammasome to relieve gout flares anti-inflammatory effects of the hydroxycarboxylic acid receptor 2 the role of hca2 (gpr109a) in regulating macrophage function glucagon and glucagon-like peptide-1 as novel anti-inflammatory and immunomodulatory compounds the camp pathway as therapeutic target in autoimmune and inflammatory diseases camp up-regulates il-4 and il-5 production from activated cd4+ t cells while decreasing il-2 release and nf-at induction weight loss reduces knee-joint loads in overweight and obese older adults with knee osteoarthritis obesity-induced inflammation: a metabolic dialogue in the language of inflammation insulin inhibits il-10-mediated regulatory t cell function: implications for obesity effect of lowfat diet interventions versus other diet interventions on long-term weight change in adults: a systematic review and meta-analysis obesity, weight loss, and progression of disability in rheumatoid arthritis association of weight loss with improved disease activity in patients with rheumatoid arthritis: a retrospective analysis using electronic medical record data weight loss improves disease activity in patients with psoriatic arthritis and obesity: an interventional study diet and disease symptoms in rheumatic diseases-results of a questionnaire based survey controlled trial of fasting and one-year vegetarian diet in rheumatoid arthritis a vegan diet free of gluten improves the signs and symptoms of rheumatoid arthritis: the effects on arthritis correlate with a reduction in antibodies to food antigens diet and rheumatoid arthritis symptoms: survey results from a rheumatoid arthritis registry the effects of the mediterranean diet on rheumatoid arthritis prevention and treatment: a systematic review of human prospective studies anti-inflammatory diet in rheumatoid arthritis (adira)-a randomized, controlled crossover trial indicating effects on disease activity role of "western diet" in inflammatory autoimmune diseases how the mediterranean diet and some of its components modulate inflammatory pathways in arthritis rheumatoid arthritis and dietary interventions: systematic review of clinical trials managing rheumatoid arthritis with dietary interventions. front nutr ab0244 retrospective study on effects of ramadhan month fasting on rheumatoid arthritis patients neutrophil functions and clinical performance after total fasting in patients with rheumatoid arthritis effects of fasting on disease activity, neutrophil function, fatty acid composition, and leukotriene biosynthesis in patients with rheumatoid arthritis reduction in serum leptin and igf-1 but preserved t-lymphocyte numbers and activation after a ketogenic diet in rheumatoid arthritis patients serum levels of interleukin-6 and dehydroepiandrosterone sulphate in response to either fasting or a ketogenic diet in rheumatoid arthritis patients ketogenic diets and thermal pain: dissociation of hypoalgesia, elevated ketones, and lowered glucose in rats acute oxidative stress and systemic nrf2 activation by the ketogenic diet ketogenic diet: a new light shining on old but gold biochemistry is there a role for diet in the therapy of rheumatoid arthritis? hyperuricemia in psoriatic arthritis: epidemiology, pathophysiology, clinical implications dietary recommendations for adults with psoriasis or psoriatic arthritis from the medical board of the national psoriasis foundation: a systematic review mediterranean diet and psoriatic arthritis activity: a multicenter cross-sectional study effect of very-low-calorie ketogenic diet on psoriasis patients: a nuclear magnetic resonance-based metabolomic study very lowcalorie ketogenic diet may allow restoring response to systemic therapy in relapsing plaque psoriasis very low-calorie ketogenic diet (vlckd) in patients with psoriasis and obesity: an update for dermatologists and nutritionists aggressive weight-loss program with a ketogenic induction phase for the treatment of chronic plaque psoriasis: a proof-ofconcept, single-arm, open-label clinical trial the impact of intermittent fasting (ramadan fasting) on psoriatic arthritis disease activity, enthesitis, and dactylitis: a multicentre study the pathogenesis of psoriatic arthritis the effects of ketogenic diet on the th17/treg cells imbalance in patients with intractable childhood epilepsy hydroxy-carboxylic acid receptor actions in metabolism relationship between diet and ankylosing spondylitis: a systematic review atherosclerosis and cardiovascular disease in the spondyloarthritides, particularly ankylosing spondylitis and psoriatic arthritis cardiovascular risk profile in patients with spondyloarthritis prevalence of type 2 and type 1 diabetes in psoriatic arthritis: an italian study effect of mediterranean diet in diabetes control and cardiovascular risk modification: a systematic review book review: the prevention of cardiovascular disease through the mediterranean diet mediterranean diet in axial spondyloarthritis: an observational study in an italian monocentric cohort deciphering the role of th17 cells in human disease looking beyond th17 cells: a role for tr1 cells in ankylosing spondylitis? interleukin-23-induced transcription factor blimp-1 promotes pathogenicity of t helper 17 cells ketogenic diets alter the gut microbiome resulting in decreased intestinal th17 cells cardiovascular co-morbidity in patients with rheumatoid arthritis: a narrative review of risk factors, cardiovascular risk assessment and treatment atherosclerotic cardiovascular disease prevention in rheumatoid arthritis body mass index and disease activity in chronic inflammatory rheumatic diseases: results of the cardiovascular in rheumatology (carma) project subclinical impairment of myocardial and endothelial functionality in very early psoriatic and rheumatoid arthritis patients: association with vitamin d and inflammation rheumatoid arthritis and cardiovascular risk: retrospective matchedcohort analysis based on the record study of the italian society for cardiovascular disease and risk factors in patients with rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis cardiovascular comorbidities in patients with psoriatic arthritis: a systematic review patients with ankylosing spondylitis have increased cardiovascular and cerebrovascular mortality: a population-based study cardiac and cardiovascular morbidities in patients with psoriatic arthritis: a population-based case control study cardiovascular risk in psoriatic arthritis, a narrative review microvascular endothelial dysfunction in rheumatoid arthritis impact of risk factors associated with cardiovascular outcomes in patients with rheumatoid arthritis endothelial progenitor cells and rheumatic disease modifying therapy targeting inflammation to prevent cardiovascular disease in chronic rheumatic diseases: myth or reality? the effects of tumour necrosis factor inhibitors, methotrexate, nonsteroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis increased cardiovascular events and subclinical atherosclerosis in rheumatoid arthritis patients: 1 year prospective single centre study anti-tnf-alpha agents and endothelial function in rheumatoid arthritis: a systematic review and meta-analysis. sci rep cardiovascular risk in ankylosing spondylitis and the effect of anti-tnf drugs: a narrative review systematic review and meta-analysis of clinical trials of the effects of low carbohydrate diets on cardiovascular risk factors low carbohydrate versus isoenergetic balanced diets for reducing weight and cardiovascular risk: a systematic review and meta-analysis effects of ketogenic diets on cardiovascular risk factors: evidence from animal and human studies ketogenic diet and epilepsy: what we know so far cancer treatment with the ketogenic diet: a systematic review and meta-analysis of animal studies low-fat versus ketogenic diet in parkinson's disease: a pilot randomized controlled trial long-term effects of a ketogenic diet in obese patients obesity in autoimmune diseases: not a passive bystander inflammation, obesity and rheumatic disease: common mechanistic links. a narrative review can chest imaging be used to draw information about body mass index and obesity status? living with arthritis: a "training camp" for coping with stressful events? a survey on resilience of arthritis patients following the covid-19 pandemic association of obesity with inflammation, disease severity and cardiovascular risk factors among patients with ankylosing spondylitis the relationship between body mass index, disease activity, and exercise in ankylosing spondylitis nailfold capillaroscopy in common non-rheumatic conditions: a systematic review and applications for clinical practice fibromyalgia and obesity: a comprehensive systematic review and meta-analysis obesity and osteoarthritis comparing two low-energy diets for the treatment of knee osteoarthritis symptoms in obese patients: a pragmatic randomized clinical trial osteoarthritis, obesity and weight loss: evidence, hypotheses and horizons -a scoping review the effect of low-carbohydrate and low-fat diets on pain in individuals with knee osteoarthritis the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the handling editor declared a shared affiliation with several of the authors jc, lm, vb, ll, rm, and fu at time of review.publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.copyright © 2021 ciaffi, mitselman, mancarella, brusi, lisi, ruscitti, cipriani, meliconi, giacomelli, borghi and ursini. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0027898-ljxjb2rz authors: schonfeldova, barbora; zec, kristina; udalova, irina a title: synovial single-cell heterogeneity, zonation and interactions: a patchwork of effectors in arthritis date: 2021-09-24 journal: rheumatology (oxford) doi: 10.1093/rheumatology/keab721 sha: 4a46129e6483a7ab6e0c97609fa4b4bb2cef2b9b doc_id: 27898 cord_uid: ljxjb2rz despite extensive research, there is still no treatment that would lead to remission in all patients with rheumatoid arthritis as our understanding of the affected site, the synovium, is still incomplete. recently, single-cell technologies helped to decipher the cellular heterogeneity of the synovium; however, certain synovial cell populations, such as endothelial cells or peripheral neurons, remain to be profiled on a single-cell level. furthermore, associations between certain cellular states and inflammation were found; whether these cells cause the inflammation remains to be answered. similarly, cellular zonation and interactions between individual effectors in the synovium are yet to be fully determined. a deeper understanding of cell signalling and interactions in the synovium is crucial for a better design of therapeutics with the goal of complete remission in all patients. ra is a chronic inflammatory disease that mainly affects the synovium that nourishes and supports the joint during homeostasis (disease pathology was reviewed, for example, in [1, 2] ). a better understanding of synovial biology could help in identifying novel drugable targets for the pursuit of achieving sustained remission or, better yet, finding a cure for ra [3] . ra environment and its biomarkers were originally studied indirectly in peripheral blood or synovial fluid; however, better synovial sampling through arthroscopic or ultrasonography-guided biopsies allowed for immunohistochemical and biochemical profiling of the synovial composition leading to better understanding of the disease site (reviewed in [4] ). still, profiling cellular heterogeneity, localisation, and interactions in the synovium and integrating those data is the way forward in disentangling the underlying mechanisms of inflammation as well as those underlying resolution. single-cell technologies allowed for deep characterisation of the synovial tissue heterogeneity; however, recent reviews have been almost exclusively focussed on fibroblasts, macrophages/monocytes, t cells and b cells and omitted other structural and supporting cells such as endothelial cells or peripheral neurons. in this review, we aim to comprehensively describe synovial cell populations, their activation states and association with synovial pathology, as well as identify knowledge gaps and populations remaining to be profiled. subsequently, we will discuss the positioning (zonation) of individual subpopulations of cells in the synovium and how the single-cell profiling can assist in discovering the localisation of those cells. finally, we will provide a summary of the known intercellular interactions as well as identify which tools can be used to predict and confirm further signalling pathways. rheumatology key messages . single-cell technologies have identified the diverse immune and structural cell composition of the synovium. . many human synovial cell populations appear to have their counterparts in the murine synovium. . the cellular localisation and cell-cell interactions in the synovium are yet to be fully deciphered. the field of single-cell sequencing is rapidly growing, and several new methods have been developed to facilitate the in-depth characterisation of different cell types. these novel technologies, their applications, and their integration into existing research frameworks have been recently reviewed (for example in [5] [6] [7] ). single-cell transcriptomics has been extensively used to profile synovial tissue and infer the cellular function and contribution to inflammation (fig. 1) . several methods such as smart-seq2 [8] or 10x genomics [9] are in routine use and others, such as the low-cost microfluidic instrument, were also reported for profiling the synovium in ra [10] . however, some challenges remain in the use of these technologies (reviewed in [11] ). following the best practices during the analysis is crucial to ensure that results are valid and reproducible [12] . mononuclear phagocytes are important contributors to both homeostasis and inflammation. the role of synovial macrophages in the context of ra has been extensively studied in patients as well as in animals (recent reviews: [13] [14] [15] ). previously, tissue-macrophages were found to restrict excessive neutrophil recruitment [16] ; however, recent single-cell experiments revealed substantial heterogeneity among synovial myeloid cells in both mice and humans [17] [18] [19] [20] . single-cell profiling of synovial macrophages in healthy controls, treatment-naïve/-resistant patients with ra, and patients in sustained remission revealed nine distinct subpopulations (table 1 , fig. 2 ) [17] . the association of these subsets to inflammation vs remission was largely determined by the expression of mer tyrosine kinase (mertk); mertk pos macrophages were more abundant in stable remission and healthy controls, while mertk neg subset was relatively scarce in healthy synovium and over-abundant in active ra [17] . similarly, mertk-expressing macrophages were upregulated with anti-inflammatory medication in ra patients [20] and expression of mertk itself was elevated during resolution of inflammation in a murine model of arthritis [21] . furthermore, the ratio of mertk pos to mertk neg macrophages was also found to be predictive of flares' occurrences [17] showing mertk as a potential predictive tool for clinicians. conversely, mertk neg subpopulations show similarity to previously identified pro-inflammatory macrophages [17, 19] . another pro-inflammatory the sample acquisition (e.g. human or murine joint biopsies) is followed by the processing of the sample into a single-cell suspension. population of interest is then sorted by facs and prepared for sequencing (e.g. barcoding the mrna to identify cell source, reverse transcription, preparation of cdna library). raw sequencing data need to be pre-processed and analysed. the findings from single-cell rna sequencing experiments should be experimentally validated. figure created with biorender.com. barbora schonfeldova et al. population was identified in ra patients, marked by high expression of heparin binding egf-like growth factor (hbegf), that interacted with synovial fibroblasts and was predicted to increase fibroblast invasiveness and neutrophil recruitment [20] . similarly, macrophages in a murine ra model, serum transfer-induced arthritis (stia), were identified as a heterogeneous population displaying dynamic changes in their respective abundances over the first 5 days of the disease [18] . this was largely caused by the macrophage infiltration during disease development. at the initial stage, seven subpopulations of macrophages were identified: c-x3-c motif chemokine receptor 1 (cx 3 cr1) þ lining, resistin-like alpha (relm-a) þ interstitial, aquaporin 1 (aqp1) þ interstitial, c-c chemokine receptor type 2 (ccr2) þ infiltrating, major histocompatibility complex class ii (mhcii) þ interstitial, stathmin 1 (stmn1) þ proliferating macrophages and lymphocyte antigen 6 complex, locus c2 (ly6c2) þ monocytes [18] . cx 3 cr1 þ lining macrophages expressed genes associated with immuneregulation, clearance of apoptotic cells, and barrier formation [18] , suggesting important homeostatic function within the synovium. monocytes are important for the development of stia; their absence is associated with lower leukocyte recruitment and restriction of inflammation [21] . furthermore, compared with oa samples that are not defined by immune cell abundance, ra biopsies have a strong myeloid expression signature [22] . thus, profiling of myeloid cells in inflammatory arthritis contributes to a better understanding of this disease. synovial macrophage subpopulations, their associations with pathology, predicted functions and comparison between humans and mice are summarized in table 1 . due to the high levels of intracellular rnases, granulocytes are difficult to profile by transcriptomic approaches. despite those difficulties, several studies were able to profile heterogeneity of granulocytes, mainly neutrophils, during both homeostasis and inflammation [23, 24] . such transcriptomic advances should be applied to investigate the synovial granulocytes considering the importance of neutrophils at the onset of arthritis in the murine models [25] [26] [27] . diversification of neutrophil phenotypes could be of particular interest as low-density neutrophils have been recently identified to [28] [29] [30] ). interestingly, type 2 responses, including an increased number of eosinophils within the joint, were found protective in murine models of arthritis [31, 32] and were mediated by il-4/il-13induced signal transducer and activator of transcription 6 (stat6) activation [31] . granulocyte-associated gene patterns, such as markers of phagocytosis, pattern recognition receptor and complement activation, were apparent in synovial samples of ra patients [22, 33] . therefore, closer profiling of granulocytes within the synovium would be beneficial to determine their contribution to inflammatory arthritis. autoantibodies against igg antibodies (rf) and citrullinated proteins (acpa) are characteristic of ra in certain patients but their presence is not universal, a phenomenon indicative of heterogeneous b-cell responses. gene patterns associated with classical bcell function have been detected only in synovial tissue of patients with ra, not those with oa [22] . moreover, b-cell activation and differentiation-associated gene signature was predictive of increased bone erosion [33] . single-cell characterisation of b cells in the synovium of patients with oa or ra revealed four distinct subpopulations of b cells: naïve b cells (called sc-b1), ighgþcd27þ memory b cells (sc-b2), autoimmuneassociated b cells (sc-b3) and plasmablasts (sc-b4) [19] . the sc-b3 cluster had an elevated expression of markers associated with b-cell activation, interferon stimulation and autoimmunity, and was overabundant in inflamed synovium compared with oa controls [19] . a subset of receptor activator of nuclear factor kappa-b ligand (rankl)-producing b cells was also identified in the synovial fluid and patient tissue [34] . further macrophage populations characterised as mertk pos folr2 posþ lyve1 pos or mertk pos trem2 high were associated with remission, while mertk neg cd48 pos s100a12 pos or mertk neg cd48 pos spp1 pos cd9 pos macrophages were preferentially found in actively inflamed synovium. other populations had no clear association with either remission or inflammation. interestingly, an increased number of mertk pos trem2 low macrophages was found in early undifferentiated arthritis. validation of the functionality of these cells will be necessary to confirm their proinflammatory and autoimmune role in ra. in-depth b-cell profiling may be valuable to a subset of patients; however, as patients manifest with discrete levels of lymphocyte infiltration [33, 35, 36] , the effectivity of drugs targeting b-cell activation may be limited. t lymphocytes t cells form an important part of the adaptive immune system and transcripts associated with t cell function were detected in synovial tissue isolated from patients with ra but not in oa, suggesting their role in inflammation [22] . conversely, th2 responses were found to be protective in murine models of ra [31, 32] , a discrepancy potentially explained by synovial t-cell heterogeneity [19, 33, 37] . scrnaseq of the synovial tissue isolated from patients with either oa or ra revealed three subpopulations of cd4þ [ccr7þ (called sc-t1), forkhead box p3 (foxp3)þ regulatory (sc-t2), pdcd1þ peripheral helper (t ph ) and follicular helper t cells (t fh ) (sc-t3)] and three subpopulations of cd8þ t cells [gzmkþ (sc-t4), gnlyþgzmbþ cytotoxic (sc-t5), gzmkþgzmbþ t cells (sc-t6)] [19] . sc-t3 t ph and t fh cells were found to produce high levels of a chemokine c-x-c motif chemokine ligand 13 (cxcl13), inhibitory receptors [t cell immunoreceptor with ig and itim domains (tigit), cytotoxic t-lymphocyte-associated protein 4 (ctla4)] and were associated with inflammation in the synovium [19] . the population of t ph cells, characterised as pd1 hi cxcr5 -cd4 þ , was found to be expanded in the joints of seropositive ra patients and supported b-cell differentiation and antibody production [37] . similarly, pd-1 high t ph cells producing pdcd1, tigit and cxcr6 were associated with strong inflammation in treatmentnaïve patients with ra [33] . overall, t ph cells may represent a pathological player in ra. studies in psoriatic arthritis showed that expanded t cells are likely retained in the synovium by an increased expression of cxcr3 or cxcr6 whose ligands cxcl10 and cxcl9 or cxcl16 respectively are present in an increased level in the synovial fluid during the disease [38, 39] . although untested, similar retention could apply to pd-1 high t ph in ra as they also express an increased level of cxcr6 [33] . inhibiting the retention of these proinflammatory t cells may present a potential therapeutic target; however, further research is required to validate t-cell relationships to pathology identified by single-cell experiments. tissue maintenance: fibroblasts stromal cells including fibroblasts are important contributors to synovial homeostasis and have a substantial role in the pathology of ra (recently reviewed in [40] [41] [42] ). recent single-cell studies revealed vast heterogeneity among this population and the differential role of distinct fibroblast clusters in ra pathogenesis [19, [43] [44] [45] . scrnaseq profiling of synovial fibroblasts isolated from patients with ra or oa revealed three major subsets: cd34-thy1-, cd34-thy1þ and cd34þ [43] (fig. 3) . cd34-thy1þ were more abundant in ra than in oa, positively correlated with leukocyte infiltration, histological synovitis and hypertrophy [43] . furthermore, cd34-thy1þ fibroblasts expressed factors that drive osteoclastogenesis and triggered the generation of triiodothyronine receptor auxiliary protein (trap)þ osteoclasts cd55þthy1-lining fibroblast were found to be associated with oa while cd34þ and hla-dra hi cd34-thy1þ sublining fibroblasts were preferentially found in ra. population of sublining fibroblasts defined as dkk3þthy1-was not clearly associated with either. figure created with biorender.com. in vitro [43] . cd34þ fibroblasts were characterized by high expression of interleukin 6 (il6), cxcl12, c-c motif chemokine ligand 2 (ccl2). in vitro experiments confirmed their ability to recruit a high number of peripheral blood monocytes [43] . a similar analysis of synovial tissue from ra and oa patients by single-cell technologies identified four fibroblast subpopulations: cd34þ sublining fibroblasts (termed sc-f1), human leukocyte antigen (hla)-dra hi sublining fibroblasts (sc-f2), dickkopf wnt signalling pathway inhibitor 3 (dkk3þ) sublining fibroblasts (sc-f3) and cd55þ lining fibroblasts (sc-f4) [19] . the hla-dra hi population of fibroblasts (sc-f2) [19] seems to correspond to the previously defined inflammatory cd34-thy1þ fibroblasts [43] . they expressed genes associated with antigen presentation, ifnc signalling, high levels of il6 and cxcl12, and were overabundant in leukocyte-rich ra samples compared with leukocytepoor ra and oa samples [19] . furthermore, ra patients have a higher number of fibroblast activation protein alpha (fapa)þ fibroblasts compared with patients without active inflammation [44] . also, fapaþ fibroblasts were significantly associated with inflammation, leukocyte infiltration, joint damage and pannus formation in stia [44] . in this murine model, scrna-sequencing identified five distinct fibroblast populations [44] . fapaþthy1þ were found to represent sublining fibroblasts which increased the severity and persistence of inflammation, while fapaþthy1-fibroblasts were located to the synovial lining and had a role in bone and cartilage destruction [44] . overall, sublining thy1þ fibroblasts are expanded in both ra and animal models of this disease and associate with increased leukocyte infiltration [19, [43] [44] [45] representing a potential therapeutic target. the comparison of murine and human synovial fibroblasts, their identifying markers and predicted functions are summarized in table 2 . vascularisation: endothelial cells increased vascular permeability is a feature of inflammatory models of arthritis and endothelial dysfunction is a common complication of ra (reviewed, for example, in [27, 46, 47] ). synovial endothelial cells, therefore, seem to have an impact on the propagation of the inflammation. endothelial cells are a heterogeneous population in other tissues with organ-specific characteristics supporting the needs of the local microenvironment [48] [49] [50] [51] [52] . these differences are apparent in ecs' appearance, morphology and transcriptional profile [48] . both human and murine endothelial cells even show sex-specific transcriptome differences across organs such as the aorta, heart and lung [49, 53] . human synovial arterial (podxlþ) and venous [duffy blood group, atypical chemokine receptor (darcþ)] endothelial cells were found to contribute differential cues to the synovial microenvironment [45] . arterial endothelial cells, not venous ones, support the differentiation of synovial sublining fibroblasts [45] . endothelial cells also assist local cell functionality in other organs such as promoting hepatocyte survival and albumin production in the liver [48] . furthermore, capillaries traversing cortical bone from the bone marrow have been recently identified [54] ; however, their contribution to the synovial environment remains to be established. regrettably, endothelial heterogeneity in the joint has not been sufficiently studied. considering the importance of vascular permeability in the immunopathology of ra, it would be interesting to investigate endothelial cell functions of the synovium in steady-state and inflammation. understanding synovial endothelium could lead to the development of novel therapeutics that target vascular permeability and oedema. innervation: peripheral nervous system joint-associated pain is common to both ra and oa [1, 2, 55, 56] . in a murine model of oa, osteoclasts induced axonal growth in the subchondral bone by secreting netrin-1, which was predicted to mediate increased sensitivity of the peripheral nervous system and pain perception [57] . as aberrant innervation of the cartilage is seen in an animal model of inflammatory arthritis [58] , similar effector pathways could mediate joint pain in ra as well. changes in the dorsal horn of the central nervous system in inflammatory arthritis were also detected [58] . furthermore, the loss of sensory innervation in the joint was found to be protective in a patient with psoriatic arthritis [59] . this effect of protective sensory denervation was confirmed in an animal model of inflammatory arthritis [59] . of note, partial denervation of the wrist resulted in self-reported improvement of pain perception in patients with inflammatory arthritis [60] . the peripheral nervous system was found to be formed by a highly heterogeneous population of cells (among many, for example, [61] [62] [63] [64] ). in addition to heterogeneous populations of myelinating and nonmyelinating schwann cells, neural-associated endothelial cells, fibroblasts and leukocytes were also identified [64] . leukocytes of the peripheral nervous system consist of cx3cr1-expressing myeloid cells with cxcl4þ macrophages, also identified in the mouse brain [64, 65] . changes in the populations of cells associated with peripheral nerves were observed in an animal model of spontaneous chronic peripheral neuritis [64] . similarly, sensory and sympathetic neurons innervating the draining lymph nodes were found to be highly heterogeneous, able to communicate with surrounding stromal and immune cells and expand after toll-like receptor 4 stimulation [63] . in conclusion, the heterogeneity of synovial innervation and its contribution to the pathology of ra remain incompletely understood. however, both peripheral and central nervous system seem to be altered in inflammatory arthritis [58] , providing the rationale for the development of pain-modifying drugs that interfere with neural effector pathways to improve the quality of life of patients with both ra and oa [66] . fluorescence imaging and its implication for deciphering organ function have been recently reviewed [67] . advances in confocal microscopy, for example by the addition of airyscan detectors, and in 3d imaging were beneficial for the field of rheumatology as well. however, spatiotemporal localisation of different subsets of immune and structural cells within the synovium has been difficult due to the lack of markers characterizing different subpopulations. as described in the previous section, for most synovial cells, single-cell transcriptomics helped to overcome this issue by providing a characterisation of differentially expressed genes across populations. furthermore, the recent development of spatial transcriptomics such as sequential fluorescence in situ hybridization [68] , slide-seq [69] or commercially available 10x genomics' visium and nanostring is becoming instrumental to further understand the spatiotemporal changes in the synovium. spatial transcriptomics has been used already to study cellular niches and networks in alzheimer's disease [70] or carcinomas [71] and very recently also to uncover signalling and localisation of infiltrating leukocytes in ra [35, 36] . two major interstitial macrophage niches were recently identified in mice by combining transcriptional profiling and fluorescence imaging, namely vasculature and nerve-associated macrophages [72] . lyve1 lo mhcii hi tissue-resident macrophages expressed higher levels of genes associated with antigen presentation and were more closely associated with peripheral nerves [72] . conversely, lyve1 hi mhcii lo macrophages were found to contribute to tissue repair and resided close to the vasculature [72] . these macrophage populations were identified in murine lung, fat, heart and dermis, but also in several human tissues [72] . we compared the expression of markers defining these two interstitial populations to the murine synovial macrophages from culemann et al. [18] and found that synovial relmaþ sublining macrophages resembled lyve1 hi mhcii lo , while synovial mhcii þ interstitial macrophages were more like lyve1 lo mhcii hi . this suggests that relmaþ sublining macrophages are localized closer to the blood vessels and mhcii þ interstitial macrophages near the synovial innervation. this hypothesis is further supported by the resemblance of synovial relmaþ macrophages to human mertk pos folr2 pos lyve1 pos macrophages, which produce high levels of perivascular marker lymphatic vessel endothelial hyaluronan receptor 1 (lyve1) and express markers associated with regulation of vascular endothelial growth factor [17] . however, the exact positioning of synovial interstitial macrophages requires further investigation by imaging technologies. cx 3 cr1 þ lining macrophages were identified as a dense protective barrier which is csf1rand gets replenished by csf1r þ sublining macrophages [18] . based on published confocal microscopy images by culemann et al. [18] and our independent observations, lining macrophages form the uppermost lining layer; however, it is not a dense layer similar to epithelial cells, but it can be rather fragmented. finally, as the peripheral helper t cells were found to have a pathological role in b-cell activation in inflammatory arthritis [19, 33, 37] , these cells need to be localized close to each other. by immunohistochemistry, t ph cells in synovial lymphoid aggregates were found in proximity to b cells [37] . in 40% of patients with ra, leukocyte infiltrates manifest as tertiary lymphoid organ-like structure (reviewed, for example, in [73, 74] ). b cells and t cells seem to have an organized positioning within these structures with ccl21þ cells on the inside and marginal zone b and b1 cell specific protein (mzb1)þ plasma cells on the outside [35] . vickovic et al. [35] hypothesized that tertiary lymphoid organ-like structures retain newly recruited leukocytes; however, the positioning of these structures in respect to the vasculature or other synovial immune and structural cells is unknown. structural cells similar to immune cells, different subsets of fibroblasts localize to different regions of the synovium [19, [43] [44] [45] . cd34-thy1þ fibroblasts were found to reside in the sublining where they surrounded the capillaries [43] . this layer was a few cells thin in oa patients and expanded in the synovium isolated from patients with active inflammation [43] . cd34þ fibroblasts could also be observed in the sublining layer, scattered across both deep and superficial interstitium [43] . finally, cd34-thy1-produced lubricin and formed the synovial lining layer [19, 43, 44] . overall, thy1 expression determines sublining populations of fibroblasts [19, [43] [44] [45] . furthermore, the distance from endothelial cells seems to affect the phenotype of fibroblasts; cells closest to the vasculature express high levels of thy1 and cells furthest express high levels of lubricin [45] . in conclusion, local microenvironment cues are crucial for cell positioning, identity and function. possible methods of how cellular interactions can be studied based on gene expression were nicely summarized in a recent review [75] . in brief, a variety of methods have been developed such as nichenet [76] , soptsc [77, 78] , cellphonedb [79] , cellchat [80] , and many others. each method is using a different algorithm for assessing cell-cell interactions, such as using differential gene expression between and within cell clusters to search for ligand-receptor pairs. these approaches also use different data sources to access ligand-receptor pairs; for example, nichenet is using kegg as its main data source [76] . the assessment of individual strengths and weaknesses of the available methods is not the subject of this review. nevertheless, as emphasized in the recent review [75] , these approaches should be considered mainly for hypothesis generation and require subsequent experimental validation. additionally, spatial transcriptomics can help with understanding cellular interactions by providing site-specific expression profile of the synovium [35, 36] . the ligand-receptor analysis was used to identify the notch signalling axis between fibroblasts and endothelial cells, which shapes fibroblast identity in the synovium [45] . based on this analysis, wei et al. [45] identified that synovial arterial endothelial cells express notch ligands jagged canonical notch ligand 1 (jag1) and delta-like canonical notch ligand 4 (dll4), which interact with notch3 on surrounding mural cells and fibroblasts (fig. 4a) . furthermore, the endothelial cells seemed to direct the differentiation of thy1þ sublining fibroblast, which was confirmed in mixed synovial organoids [45] . fibroblasts were also found to be affected by synovial macrophages in an in vitro co-culture [17] . mertk neg synovial macrophages induced an increased expression of mediators of bone and cartilage destruction, inflammation and leukocyte recruitment in fibroblasts [17] (fig. 4b ). vice versa, in the resolution phase of ra, thy1 pos cxcl14 pos sublining fibroblasts were found to abundantly express an activator of mertk, growth arrest specific 6 (gas6) [17] . mertk was previously found to be significantly associated with the resolution of inflammation [17, 20, 21] . synovial fibroblasts further impact the transcriptome of macrophages by secreting pro-inflammatory factors such as tnf and prostaglandin e2 (pge2) and support their development into inflammatory hbegf þ macrophages [20] (fig. 4c) . medications, such as leflunomide, dexamethasone, naproxen and triple therapy could inhibit the fibroblast induced hbegf þ macrophage polarization [20] . for example, naproxen functionally blocked the prostaglandin generation and in turn abrogated hbegf mrna and granulocyte colony-stimulating factor (gcsf) protein production [20] , showing the importance of both cellular heterogeneity and interactions in understanding the molecular action of antiinflammatory drugs. usage of spatial transcriptomics [81] revealed an upregulated expression of genes such as lymphotoxin beta (ltb), ccl19, cxcl13, cd52, membrane spanning 4-domains a1 (ms4a1) and cd79a, which are associated with t-cell and b-cell function and crosstalk [35, 36] . especially, cxcl13 expression by t ph to activate b cells is of interest as it is confirmed in other sequencing studies [19, 37] . furthermore, signalling lymphocytic activation molecule 5 (slamf5), required for t-cell and b-cell interaction, was upregulated on peripheral and follicular helper t cells and a blockade of slamf5 abrogated pd1 hi cxcr5 -cd4 þ t-cell effect on b cells in an in vitro co-culture [37] suggesting an additional therapeutic target (fig. 4d) . however, this approach would most likely be restricted to seropositive patients. additionally, patients with inflammatory arthritis have a varying degree of b-cell and t-cell infiltration [33] , further complicating this approach. recent advances in single-cell technologies allowed for deeper profiling of synovial cells, identification of their respective markers, activation states and provided predictions of their functionality (fig. 5) . studying cellular organization is virtually impossible without an appreciation of the characteristic cellular markers; therefore, we envisage that the field of rheumatology will soon be enriched with many studies profiling cellular zonation in the synovium. the spatial characterisation will further uncover which cells are likely to interact with each other, leading to a better understanding of signalling pathways in the synovium both in the steady-state and during inflammation. single-cell transcriptomics already led to the development of computational methods allowing for the prediction of ligand-receptor interactomes but, as mentioned before, these tools should be used for hypothesis generation and subsequently experimentally verified. apart from cellular heterogeneity, the extracellular matrix composition, function and localisation creating unique niches was recently reviewed as well [82] . thus, the interactions between synovial cells and the extracellular matrix should also be considered to fully comprehend the maintenance and function of this complex tissue. furthermore, bulk rnaseq was already shown to be able to stratify patients better than histological assessment [83] . however, scrnaseq, once the analysis becomes cheaper and more abundant, could provide even more precise patient classification into treatment groups as it can identify presence/absence of specific pathological cell subsets. examples of interactions identified in human or murine synovium (a) arterial endothelial cells were found to release notch3 ligands dll4 and jag1 that bind notch3 receptor on mural cells and sublining fibroblasts. the notch signalling gradient shapes the identity of fibroblasts where fibroblasts closer to the endothelium will develop into thy1þcd34-fibroblasts and cells further from it will become thy1-lining fibroblasts. (based on [45] ) (b) co-culture of proinflammatory mertk neg cd206 neg macrophages with fibroblasts caused the fibroblasts to adopt a proinflammatory phenotype and secrete bone and cartilage destructing mediators (mmp1/3, rankl) and inflammatory cytokines (il6) or chemokines (cxcl8, ccl2, ccl20) [17] . (c) proinflammatory fibroblasts were found to secrete tnf or pge2, which induce expression of hbegf and gcsf in macrophages, inducing pro-inflammatory phenotype [20] . (d) peripheral helper t cells and b cells communicate in the synovium through multiple pathways, for example via secretion of cxcl13 by t cells [19, 37, 81] or via upregulation of slamf5 [37] . figure created with biorender.com. we identified several limitations in the current literature. firstly, single-cell transcriptomics is uncovering a correlative relationship between cellular states and sustained inflammation or remission. the role and contributions of each subpopulation should be investigated in greater detail to decipher causal effects. nevertheless, single-cell profiling can provide an excellent overview of the cell distribution in tissues, describe markers instrumental for imaging, and aid in hypothesis generation. secondly, the selection of markers used for sorting prior to single-cell rna sequencing can affect the subsequent analysis and some cellular populations can be unknowingly disregarded. furthermore, different markers for identification of cells, different clustering strategies and analyses make the comparison of different datasets rather challenging. potentially, an integrated meta-analysis comparing various datasets could verify current findings. lastly, certain murine models of inflammatory arthritis are dependent only on an innate immune system; thus, profiling of adaptive immune cells in such cases is missing. single-cell transcriptomics is revolutionizing the rheumatology field. we believe that the extensive amount of literature identifying single-cell heterogeneity of the synovium will lead to better spatial characterisation of the synovium and identification of pathological interactions, which can be blocked by medical interventions to the therapeutic benefit of patients. fig. 5 prediction of the zonation of immune and structural cells in the synovium cx 3 cr1þ lining macrophages and lining fibroblast create the 'barrier' layer of the synovium [18, 19] . based on our analysis, mhcii high macrophages could be positioned near the innervation, while relm-aþ macrophages could be located near the vasculature, similarly to the lung interstitium [72] . notch signalling axis is instructing the positioning of fibroblasts with mural cells closest to the vasculature, thy1þcd34-fibroblasts near the vasculature and mural cells with thy1-cd34-lining fibroblast at the opposing end [43, 45] . cd34þ fibroblasts are positioned both in the immediate sublining and deep interstitium [43] . b.s. planned, researched data, and wrote the manuscript. k.z. and i.a.u discussed the content, contributed to the planning, reviewed and edited the manuscript. funding: no specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article. disclosure statement: the authors have declared no conflicts of interest. data are available upon reasonable request by any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and statistical analysis plan (sap) and execution of a data sharing agreement (dsa). all data relevant to the study are included in the article. the immunology of rheumatoid arthritis why remission is not enough: underlying disease mechanisms in ra that prevent cure synovial tissue research: a state-of-the-art review the single-cell sequencing: new developments and medical applications integrative single-cell analysis single-cell sequencing techniques from individual to multiomics analyses smart-seq2 for sensitive full-length transcriptome profiling in single cells massively parallel digital transcriptional profiling of single cells single-cell rna-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation eleven grand challenges in single-cell data science current best practices in single-cell rna-seq analysis: a tutorial synovial tissue macrophages: friend or foe? synovial macrophages in rheumatoid arthritis: the past, present, and future macrophage heterogeneity in the context of rheumatoid arthritis resident macrophages cloak tissue microlesions to prevent neutrophil-driven inflammatory damage distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis locally renewing resident synovial macrophages provide a protective barrier for the joint defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry hbegfþ macrophages in rheumatoid arthritis induce fibroblast invasiveness nonclassical ly6c(-) monocytes drive the development of inflammatory arthritis in mice synovial tissue transcriptomes of long-standing rheumatoid arthritis are dominated by activated macrophages that reflect microbial stimulation single-cell landscape of bronchoalveolar immune cells in patients with covid-19 single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection the multifactorial role of neutrophils in rheumatoid arthritis rheumatoid arthritis synovial fluid neutrophils drive inflammation through production of chemokines, reactive oxygen species, and neutrophil extracellular traps chemokines in rheumatic diseases:pathogenic role and therapeutic implications ros-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies low-density granulocytes activate t cells and demonstrate a nonsuppressive role in systemic lupus erythematosus low-density granulocytes are a novel immunopathological feature in both multiple sclerosis and neuromyelitis optica spectrum disorder synovial single-cell heterogeneity th2 and eosinophil responses suppress inflammatory arthritis in vivo expansion of activated foxp3þ regulatory t cells and establishment of a type 2 immune response upon il-33 treatment protect against experimental arthritis molecular portraits of early rheumatoid arthritis identify clinical and treatment response phenotypes expression of fcrl4 defines a pro-inflammatory, rankl-producing b cell subset in rheumatoid arthritis threedimensional spatial transcriptomics uncovers cell type dynamics in the rheumatoid arthritis synovium exploring inflammatory signatures in arthritic joint biopsies with pathologically expanded peripheral t helper cell subset drives b cells in rheumatoid arthritis polyfunctional, proinflammatory, tissue-resident memory phenotype and function of synovial interleukin-17aþcd8þ t cells in psoriatic arthritis single-cell sequencing reveals clonal expansions of proinflammatory synovial cd8 t cells expressing tissuehoming receptors in psoriatic arthritis restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes fibroblast-like synoviocytes in rheumatoid arthritis: surface markers and phenotypes transformation of fibroblast-like synoviocytes in rheumatoid arthritis; from a friend to foe functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis distinct fibroblast subsets drive inflammation and damage in arthritis notch signalling drives synovial fibroblast identity and arthritis pathology vascular involvement in rheumatic diseases: 'vascular rheumatology' endothelial dysfunction and inflammation: immunity in rheumatoid arthritis single-cell rna sequencing unveils unique transcriptomic signatures of organ-specific endothelial cells the in vivo endothelial cell translatome is highly heterogeneous across vascular beds singlecell rna sequencing maps endothelial metabolic plasticity in pathological angiogenesis single cell analysis of endothelial cells identified organ-specific molecular signatures and heart-specific cell populations and molecular features sex-dependent differences in the secretome of human endothelial cells a network of trans-cortical capillaries as mainstay for blood circulation in long bones cartilage damage in osteoarthritis and rheumatoid arthritis-two unequal siblings subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain peripheral and central nervous system alterations in a rat model of inflammatory arthritis protective effect of sensory denervation in inflammatory arthritis (evidence of regulatory neuroimmune pathways in the arthritic joint) patient satisfaction and outcomes of partial wrist denervation in inflammatory arthritis unbiased classification of sensory neuron types by large-scale single-cell rna sequencing the emergence of transcriptional identity in somatosensory neurons lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential redefining the heterogeneity of peripheral nerve cells in health and autoimmunity singlecell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation update on novel pharmacological therapies for osteoarthritis advances in fluorescence microscopy techniques to study kidney function transcriptome-scale super-resolved imaging in tissues by rna seqfishþ slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution spatial transcriptomics and in situ sequencing to study alzheimer's disease multimodal analysis of composition and spatial architecture in human squamous cell carcinoma two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches tertiary lymphoid organs in rheumatoid arthritis tertiary lymphoid structures: autoimmunity goes local deciphering cell-cell interactions and communication from gene expression nichenet: modeling intercellular communication by linking ligands to target genes single cell transcriptomics of human epidermis identifies basal stem cell transition states cell lineage and communication network inference via optimization for single-cell transcriptomics cellphonedb: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes inference and analysis of cell-cell communication using cellchat visualization and analysis of gene expression in tissue sections by spatial transcriptomics location, location, location: how the tissue microenvironment affects inflammation in ra rituximab versus tocilizumab in anti-tnf inadequate responder patients with rheumatoid arthritis (r4ra): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial synovial single-cell heterogeneity key: cord-0028307-zde2yn62 authors: wu, shan-shan; hao, li-jun; shi, yuan-yuan; lu, zhuo-jian; yu, jia-lin; jiang, si-qi; liu, qing-ling; wang, ting; guo, shi-ying; li, ping; li, fei title: network pharmacology-based analysis on the effects and mechanism of the wang-bi capsule for rheumatoid arthritis and osteoarthritis date: 2022-02-25 journal: acs omega doi: 10.1021/acsomega.1c06729 sha: 1dfc5eb0c53d065e8dfd545925feaf5ba84ac131 doc_id: 28307 cord_uid: zde2yn62 [image: see text] wang-bi capsule (wb) is a traditional chinese medicine (tcm)-based herbal formula, and it has been used in the treatment of rheumatoid arthritis (ra) in china for many years. additionally, wb is also used as a supplement to the treatment of osteoarthritis (oa) in clinical practice. our research aimed to reveal the therapeutic effects and underling mechanism of wb on ra and oa through computational system pharmacology analysis and experimental study. based on network pharmacology analysis, a total of 173 bioactive compounds interacted with 417 common gene targets related to wb, ra, and oa, which mainly involved the pi3k-akt signaling pathway. in addition, the serine–threonine protein kinase 1 (akt1) might be a core gene protein for the action of wb, which was further emphasized by molecular docking. moreover, the anti-inflammatory activity of wb in vitro was confirmed by reducing no production in lipopolysaccharide (lps)-induced raw264.7 cells. the anti-ra and oa effects of wb in vivo were confirmed by ameliorating the disease symptoms of collagen ii-induced ra (cia) and monosodium iodoacetate-induced oa (mia) in rats, respectively. furthermore, the role of the pi3k-akt pathway in the action of wb was preliminarily verified by western blot analysis. in conclusion, our study elucidated that wb is a potentially effective strategy for the treatment of ra and oa, which might be achieved by regulating the pi3k-akt pathway. it provides us with systematic insights into the effects and mechanism of wb on ra and oa. rheumatoid arthritis (ra) is a chronic autoimmune inflammatory disease, mainly characterized by pain, continuum multiarticular synovitis, swollen joints, as well as cartilage and bone damage, which can seriously impair physical function and quality of life. 1, 2 osteoarthritis (oa) is a degenerative disease, resulting in synovial inflammation, progressive destruction of articular cartilage, and formation of osteophyte. 3, 4 ra and oa are the common arthritis seen in many populations. 5 additionally, ra and oa share commonalities of disorder, including inflammation, swelling, osteochondral destruction, and disabling symptoms. 6, 7 moreover, ra and oa belong to the category of "bi syndrome" in the theory of traditional chinese medicine (tcm). the etiology of ra and oa is complex and still unclear, while generally believed to be related to genetic predisposition and environmental factors. so far, there is no cure for ra and oa. anti-inflammatory drugs, such as nonsteroidal antiinflammatory drugs and corticosteroids, are commonly used to treat arthritis, but these drugs are associated with severe side effects and lack robust efficacy. 8, 9 tcms have been shown to be effective and safe in clinical applications for thousands of years. 10 moreover, increasing pieces of evidence have demonstrated that tcms, which include tcm formulas, herbal medicines, and other natural products, are precious resources for curing chronic and complex diseases. 11 wang-bi capsule (wb), as a tcm-based herbal formula, has been used in the treatment of ra in china for many years and has shown positive therapeutic effects. 12, 13 in addition, wb is also used as a supplement to the treatment of oa. wb is composed of several medicinal materials, with the characteristics of multicomponents and multitargets. however, wb still lacks sufficient experimental research, and its mechanism of action remains unclear. network pharmacology, describes the complexity between ingredients, targets, and diseases from the perspective of network, which has been applied to understand and elucidate the potential mechanism of multicomponent and multitarget tcms due to the similarity with the overall philosophy of tcms. 14, 15 in the present study, we aimed to reveal the therapeutic effects and underling mechanism of wb on ra and oa. network pharmacology analysis and molecular docking are performed to explore and clarify the potential mechanism of wb. the anti-inflammatory effect of wb was studied with lipopolysaccharide (lps)-induced raw264.7 cells in vitro. at the same time, the effects of anti-ra and anti-oa for wb were evaluated with collagen-induced arthritis (cia) rats and monosodium iodoacetate-induced osteoarthritis (mia) rats in vivo, respectively. moreover, western blotting assays were carried to confirm the previously predicted mechanism of wb. 2.1. network pharmacology analysis. in total, 173 bioactive compounds of medicinal materials, except for sheep bone in wb, were obtained from tcmsp and the literature (table s1 ). among the 173 compounds, 16 shared compounds were derived from two or more medicinal materials (table 1) , which indicated their vital role in wb to a certain extent. in addition, 1145 compound-related targets were acquired from pharmmapper and swiss target prediction databases. moreover, a total of 5387 ra-related targets and 3199 ra-related targets were acquired from genecards, disgenet, and omim databases, respectively. all targets were standardized with the uniprot database. furthermore, 417 common gene targets were obtained after integrating the targets of compounds, ra and oa ( figure 1a ). these common targets were regarded as drugdisease gene targets. in order to reveal the relationship among medicinal materials of wb, active compounds, and drug-disease targets, a medicinal material−component−target (m−c−t) network was constructed by employing cytoscape software, as shown in figure 1b . the m−c−t network, containing 605 nodes and 5657 edges, visually demonstrated the interaction between the drug, compounds, and targets. as present in figure 1c , a protein−protein interaction (ppi) network was established with 417 common targets to analyze the potential protein interactions. in the ppi network, the nodes and edges, respectively, represented the target proteins and their interactions. the number of connections was reflected in the degree value of the node, where the larger and redder the node meant the higher the degree value and the more important a certain node was in this network. 16 similarly, the redder the node, the more important it was in this network, such as serine− threonine protein kinase 1 (akt1) and tumor necrosis factor (tnf). go analysis revealed 760 terms, including 571 biological processes (bps), 67 cellular components (ccs), and 122 molecular functions (mfs). the top 20 of them that were selected in the descending order of p value were analyzed and shown in figure 1d . the bps mainly involved negative regulation of the apoptotic process, inflammatory response, regulation of phosphatidylinositol 3-kinase signaling, and cellular response to lps. the ccs were mainly associated with the cytosol, cytoplasm, and plasma membrane. the mfs were mainly involved in enzyme binding, protein binding, and protein kinase activity. the kegg pathway enrichment analysis obtained 120 related pathways, and the top 20 kegg pathways are shown in figure 1e , which mainly involved pathways in cancer, hepatitis b, proteoglycans in cancer, and pi3k-akt signaling pathway. combined with previous findings, the pi3k-akt signaling pathway attracted our attention, and then, we designed further experiments to verify whether wb could exert anti-ra and oa effects by regulating this pathway. 2.2. molecular docking analysis. molecular docking analysis was performed to verify the accuracy and reliability of interactions between crucial active compounds and key protein of the pathway. based on previous data, 16 shared compounds and akt1 were, respectively, selected as receptors and ligand to conduct molecular docking analysis. the docking results are listed in table 2 . the 16 compounds all showed good affinity with the akt1 protein and the top five were β-sitosterol, oleanolic acid, luteolin, anhydroicaritin, and (−)-taxifolin, respectively. additionally, the previous network pharmacological analysis found that among the 16 shared compounds, the top 5 compounds in degree ranking were anhydroicaritin, oleanolic acid, kaempferol, luteolin, and quercetin (table 1) , respectively. integrating the analysis of molecular docking and network pharmacology, the results reveal that three core compounds (oleanolic acid, luteolin, and anhydroicaritin) may play an extremely vital role in wb. moreover, the docking results of three compounds with the akt1 protein are visualized and shown vividly in figure 2a . 2.3. anti-inflammatory effect of wb on lps-induced raw264.7 in vitro. in vitro experiments, cell viability was first examined with the cck8 kit to evaluate the cytotoxicity of wb, lps, and bbr. the results showed that wb (50−800 μg/ml), lps (1 μg/ml), and bbr (8 μmol/l) had no significant inhibition effect on cell viability, indicating the dose of which had no significant cytotoxicity ( figure 2b ). free radical, such as no, which played vital role in various inflammatory disorders. 17 the anti-inflammatory effect of wb was evaluated on lps (1 μg/ml)-induced raw264.7 cells by using the griess reagent to detect the level of no in the supernatant. as shown in figure 2c , the no production in the model group (lps, 1 μg/ml) was significantly increased compared with the control group (p < 0.001), indicating the successful establishment of the cellular inflammation model. compared with the model group, the different concentrations of the wb group and positive control bbr group significantly inhibited the no production (p < 0.001), demonstrating the anti-inflammatory effect of wb in vitro. in addition, there was significant difference of wb at the concentrations of 50 and 800 μg/ml (p < 0.05). on day 21 after model establishment (d21), the symptoms of rats, including obvious weight loss (p < 0.001), paw volume severe increase (p < 0.001), and high arthritis index score (greater than 4), indicated the successful establishment of the cia model ( figure 3a −c). next, rats in each group received different treatments for 3 weeks. during the treatment, the above-mentioned symptoms of rats in the df group and wb various dose groups gradually alleviated to a varying degree. after administration (d42), the weight of rats in the df group increased significantly compared with the mod group (p < 0.001), while there was no significant difference in wb groups, as presented in figure 3a . in addition, the paw volume of rats in the df group (p < 0.001), combined with wb-m and wb-h groups (p < 0.01) significantly decreased ( figure 3b ). the ai score of rats also reduced significantly in df, wb-m, and wb-h groups (p < 0.001) compared with the mod group ( figure 3c) . moreover, the levels of cytokines including tnf-α, il-1β, and il-10 in serum were detected and shown in figure 3d . the levels of pro-inflammatory cytokines tnf-α and il-1β were markedly increased (p < 0.001), while the level of antiinflammatory cytokine il-10 was significantly reduced (p < 0.001) in the mod group compared with the con group. meanwhile, the expression levels of tnf-α and il-1β were obviously inhibited, and il-10 was markedly raised in df, wb-m, and wb-h groups compared to those of the mod group. furthermore, the histopathological changes of ankle joints in rats are shown in figure 3e . rats in the con group showed an intact ankle joint with a smooth articular surface and normal synovial tissue. on the contrary, the ankle joint in the mod group showed an uneven articular surface and jagged destruction, accompanied by severe proliferation of the synovial tissue, and a large number of inflammatory cells infiltrated the synovial tissue and articular cartilage. interestingly, admin-istration of df, wb-h, and wb-m could effectively alleviate inflammatory cell infiltration, synovial proliferation, and cartilage destruction to a certain extent. what is more, to detect the imaging changes of ankle joints in rats, the micro-ct analysis was performed, and the results are shown in figure 3f . the ankle joint of rats in the con group exhibited a clear ankle joint structure and a smooth articular bone surface. in contrast, rats in the mod group showed typical imaging features of ra, such as severely damaged and blurred ankle joint structure, rough bone surface with serious bone erosion, the unclear structure of multiple metatarsal joints, and the widened space of digital joints. importantly, df and wb-h could evidently ameliorate the bone destruction of ankle joint and reduce bone damage in rats. 2.5. anti-oa effects of wb on mia rats in vivo. the anti-oa effects of wb in vivo were evaluated in mia rats. for this purpose, the body weight, serum cytokines, and pathological changes of knee joints in rats were examined and analyzed. two weeks after modeling, rats were given medication by gavage for 4 weeks. as shown in figure 4a , the body weights of rats in the mod group were significantly lower than that in the con group (p < 0.001), while the body weights in the gh group and wb groups had no significant difference compared with the con group after 4 weeks of administration. in addition, the results of serum cytokines in rats are presented in figure 4b , and the levels of tnf-α and il-1β markedly increased in the mod group, while il-10 significantly decreased. after treatment, the levels of pro-inflammatory factors tnf-α and il-1β in gh, wb-m, and wb-h groups were markedly downregulated (p < 0.001), while the level of anti-inflammatory factor il-10 was obviously upregulated (p < 0.001). moreover, macroscopic observations of femoral condyles and tibial plateaus in knee joints for rats were carried out, and the results are shown in figure 4c . the articular cartilage surface in the con group was intact and smooth, while that of the model group was severely damaged and the subchondral bone was exposed. gh and wb groups could alleviate the cartilage destruction to varying degrees. especially, the wb-m and wb-h groups only presented slight cartilage damage. furthermore, hematoxylin and eosin (he) pathological results showed serious cartilage extensive destruction and widened joint space in the mod group compared with the con group, as shown in figure 4d . by contrast, cartilage destruction in the gh group and wb groups were relieved to varying degrees. especially in the wb-m and wb-h groups, the knee joints of rats presented slight cartilage changes and the joint spaces almost returned to normal. 2.6. effect of wb regulation on ra and oa through the pi3k-akt signaling pathway. the results of network pharmacology and molecular docking analysis revealed that wb may play a role in the treatment of ra and oa by regulating the pi3k-akt signaling pathway, of which akt was the key protein. therefore, to evaluate the ability of wb in regulating the pi3k-akt pathway, the expression levels of akt and p-akt proteins were determined by employing western blotting. as demonstrated in figure 5a , the expression of p-akt/akt was significantly higher in the mod group in comparison with the con group, while wb could diminish the level of p-akt/akt. the results indicated that wb might exert the therapeutic effects through regulating the pi3k-akt pathway ( figure 5b ). ra and oa are common arthritis diseases clinically, and they share common features, such as inflammation, cartilage destruction, and disability in the late stage of the disease. 18−21 in the theory of tcm, ra and oa belong to the category of "bi syndrome", which is mostly attributed to the deficiency of the liver and kidney. the specific pathogenesis of ra and oa is still unclear, and there is no cure for ra and oa at present. some existing medications, such as nonsteroidal anti-inflammatory drugs, can be used for the treatment of ra and oa; however, they are often accompanied by side effects and poor long-term therapeutic effects. in contrast, tcm has the characteristics of low side effects, strong overall effect, addressing both symptoms and root causes, and good long-term effects. correspondingly, tcm has unique advantages in the treatment of chronic and complex diseases. wb is a tcm-based herbal formula and has been used in clinical practice for many years. in the present study, network pharmacology analysis combined with molecular docking and experimental verification was performed to investigate the therapeutic effects and decipher the mechanism of wb on ra and oa. based on the network pharmacology analysis, a total of 173 bioactive compounds were screened from wb, and 417 common gene targets related to wb, ra, as well as oa were identified. after that, the m−c−t network was established. among the 173 compounds, 16 shared compounds were considered to play a vital role in wb. moreover, the top five in the m−c−t network according to the degree value were anhydroicaritin, oleanolic acid, kaempferol, luteolin, and quercetin, respectively. the higher the degree value, the more important it was in the network. in addition, the pi3k-akt signaling pathway and the akt1 protein were regarded as the potential mechanism and core gene target of wb against ra and oa. based on molecular docking, 16 shared compounds all showed good affinity with the akt1 protein, and the lower the value, the better the affinity. the top five in affinity were anhydroicaritin, oleanolic acid, kaempferol, luteolin, and quercetin, respectively. therefore, three compounds (oleanolic acid, luteolin, and anhydroicaritin) and akt1 protein attracted our attention, which were considered to play a vital role of wb in the treatment of ra and oa. as reported in the literature, oleanolic acid showed antiinflammatory activity by inhibiting the release of the lpsmediated high-mobility group box 1 in human umbilical vein endothelial cells. 22 luteolin could ameliorate the development of arthritis caused by the injection of collagen type ii in mice 23 and attenuate oa progression in the mia rat model. 8 anhydroicaritin suppressed rankl-induced osteoclast differentiation. 24 additionally, accumulated evidence showed that the pi3k-akt pathway is involved in various bps, including the development of ra and oa. 25−28 the akt is a pivotal downstream effector of phosphatidylinositol kinase pi3k. besides, current studies have demonstrated that akt positively regulates chondrocyte proliferation as well as cell growth in skeletal development. 26, 29 for experimental verification, the anti-inflammatory effect of wb in vitro was demonstrated by reducing no production in lps-induced raw264.7 cells. in vivo, the anti-ra effects of wb were confirmed by cia rats. wb could ameliorate cia rats, including reversing weight loss, reducing paw volume and arthritis index, reducing the level of inflammatory factors (tnfα, il-1β) and raising the level of anti-inflammatory factor il-10, and alleviating inflammatory cell infiltration in the synovial tissue and bone destruction of the ankle joint. similarly, the anti-oa effects of wb were confirmed by mia rats. wb could ameliorate mia rats, involving in reversing weight loss, decreasing the level of tnf-α combined with il-1β and increasing the level of il-10, and relieving the cartilage destruction of the knee joint. interestingly, wb effectively inhibited the ratio of p-akt/akt, indicating that wb could suppress the pi3k-akt pathway, which was also consistent with previous prediction. this work revealed that wb might exert anti-ra and anti-oa effects by regulating the pi3k-akt pathway. it is the first time to report the mechanism of wb on treating ra and oa. importantly, it has potential significance for understanding the active substances and pharmacological mechanism of tcmbased herbal formula on complex diseases. in summary, a combination of computational system pharmacology analysis and experimental study was performed to explore the therapeutic effects and mechanism of wb on ra and oa in this study. our research systematically indicated that wb might be a useful strategy for treating ra and oa by regulating the pi3k-akt pathway. it provides not only scientific evidence for the clinical application of wb but also insights into our understanding of the action mechanism for wb on ra and oa. in particular, it is of great significance for understanding the pharmacological mechanism of tcms in the treatment of complex diseases. handling. raw 264.7 murine macrophage cells were supplied by the american type culture collection (atcc, manassas, va, usa) and cultured at 37°c with 5% co 2 in a dmem medium containing 10% fbs and 1% penicillin−streptomycin. grade spf female wistar rats (140 ± 10 g) and male wistar rats (160 ± 10 g) were provided by laboratory animal business department of shanghai institute of family planning (shanghai china). all animals were maintained in a room (temperature 25°c , relative humidity 50−65%) under a 12:12 h light and dark cycle and allowed free to food and water. the animals were accommodated for a week before the start of the experiments. all studies were performed according to the guidelines of china pharmaceutical university pharmacy animal experiment center and approved by the animal ethics committee of this institution. 30 in addition, combined with the literature investigation, other compounds with pharmacological activities but not previously meeting the conditions were also included as bioactive compounds. the related targets of bioactive compounds screened from wb were collected from pharmmapper (http://www.lilabecust.cn/pharmmapper/) and swiss target prediction (http:// www.swisstargetprediction.ch/). the related targets of ra and oa were collected from the databases including genecards (https://www.genecards.org/), disgenet (https://www. disgenet.org/), and omim (https://omim.org/). all targets were standardized with the uniprot database (https://www. uniprot.org/) and selected species were limited to "homo sapiens". 31 intersecting the component-related targets and disease-related targets through the venny 2.1.0 tool (https:// bioinfogp.cnb.csic.es/tools/venny/index.html), the common targets obtained were regarded as the potential targets of wb for the treatment of ra and oa. the relationship of medicinal materials, multiple components, and the common targets, which are associated with wb, ra, and oa, were revealed according to the above results. then, the m−c−t network was constructed and exhibited based on cytoscape software (version 3.8.0). 5.3.3. ppi analysis. ppi analysis for the common targets was carried out using the string tool (https://string-db.org/) and species were "h. sapiens". 32 in addition, the ppi network was visualized by employing cytoscape software. 5.3.4. go and kegg pathway enrichment analysis. the david bioinformatics resources 6.8 database (https://david. ncifcrf.gov/) and omicshare tool (https://www.omicshare. com/tools/) were employed to perform kyoto encyclopedia of genes and genes and genomes (kegg) pathway enrichment analysis and gene ontology enrichment analysis, involving bp, cc, and mfs. 30 5.3.5. molecular docking analysis. molecular docking analysis was used to confirm the interactions between core components and key target of wb for treating ra and oa and to verify the accuracy of predictions in network pharmacology. 10 the crystal structure of the candidate protein was downloaded from rcsb pdb (https://www.rcsb.org/) and was subsequently modified by pymol software, involving in removal of water, protonation, as well as energy minimization. 33, 34 the core components were also converted into the pdb format. additionally, the target protein and compounds were converted into the pdbq format using autodock. furthermore, autodock vina was applied to dock the receptor protein with the molecule ligands of core components. 35 5.4. experimental verification of wb for the application in ra and oa treatment. 5.4.1. sample preparation and assessment of cell viability and no production. in an in vitro study, the preparation of wb samples was as follows: wb with a content of 20.03 g was ultrasonically extracted with 100 ml of water at room temperature for 3 h, and after centrifugation, the supernatant was freeze-dried to obtain 9.16 g of powder. before use, the powder was dissolved in the medium. for the cell viability assay, raw 264.7 cells were seeded in a 96-well plate at a density of 5 × 10 3 cells per well and cultured for 24 h. after that, cells were incubated with the medium or with lps (1 μg/ml) in the presence or absence of various concentrations of wb samples for 24 h. then, cck8 was added and incubated for 1 h. the absorbance was measured at the wavelength of 450 nm using a microplate reader (polarstar). for the nitric oxide (no) production assay, raw 264.7 cells (2 × 10 5 cells/well) were plated in a 96-well plate and incubated for 24 h. then, cells were treated in the same way as mentioned above. after that, the supernatant of cells was obtained, and the level of no production was measured using griess reagents. 36 the absorbance was measured at 540 nm. 5.4.2. establishment of collagen-induced ra and evaluation of the anti-ra effect in vivo. the cia model was established based on previously reported methods with modification. 17,37 in brief, bovine collagen ii was dissolved in 0.05 mol l −1 acetic acid overnight at 4°c to obtain the collagen solution (2 mg ml −1 ). then, on day 0, rats were given a primary immunization, respectively, with an intradermal tail vein injection of emulsion (200 μl), obtained by emulsifying collagen solution and cfa at a ratio of 1:1 in an ice bath. on day 7, rats were intradermally rechallenged with collagen ii emulsified in ifa. wistar female rats were randomly divided into six groups: control group (con), model group (mod), positive control diclofenac sodium group (df), wb low-dose group (wb-l), wb medium-dose group (wb-m), and wb high-dose group (wb-h), and each group consisted of eight rats. except for the control group, other groups replicated the cia model according to the above methods. on day 21, the successful establishment of the cia model in rats was determined by the arthritis index (ai) scores greater than or equal to 4. the criteria of ai scores were as follows: (0) normal; (1) erythema and slight swelling of the ankle joint; (2) erythema and slight swelling of the ankle joint to the plantar joint or palmar joint; (3) erythema and moderate swelling of the ankle joint to the plantar joint or palmar joint; and (4) erythema and severe swelling. the total score of each paw for an individual rat was as an ai, with a maximum total score of 16. after that, wb-l, wb-m, and wb-h groups were intragastrically given corresponding doses of wb (247.5, 742.5, and 2227.5 mg kg d −1 ) once a day for 3 weeks, and the df group was treated with diclofenac sodium (6.75 mg kg d −1 ) on the same schedule, and at the same time, con and mod group rats were orally given an equal volume of cmc-na. after treatment, all rats were sacrificed, and then, the serum, synovium, paws, and ankle joints were obtained for further experimental analysis. the body weights of all rats were monitored using an animal electronic scale (tanita, shanghai, china), and the paw volumes of rats were measured with a pv-200 toe volume measuring instrument (techman, chengdu, china) . the ai of all rats in groups was evaluated with the scoring criteria. the levels of serum cytokines in rats, including tnf-α, il-1β, and il-10, were determined with the elisa kits following corresponding manufacturer's protocols. the right paws and ankle joints of rats were dissected and fixed in formalin for 3 days. micro-computed tomography (micro-ct) analysis of the right paws and ankle joints was performed to acquire the threedimensional (3-d) structure images based on the skyscan 1176 micro-ct system (bruker, shanghai, china) and ctvox software. the scanning parameters were as follows: voltage 70 kv, current 142 μa, isotropic voxel size 18 μm, with a 0.5 mm aluminum filter. in addition, the right ankle joints were decalcified in 10% edta and embedded in paraffin. then, the ankle joint tissue sections were stained with he, which were subsequently analyzed by employing an intelligent digital slice scanning system nanozoomer s60 (hamamatsu, beijing, china) and the corresponding ndp. view 2 software. additionally, the scanning mode of all slices was as follows: bright field, 20 times mirror, and fully automatic scanning mode. 5.4.3. establishment of mia and evaluation of anti-oa effect in vivo. the mia model was performed as previously reported. 38, 39 in brief, rats were given an intra-articular injection of 3 mg of mia diluted in 50 μl of saline. wistar male rats were randomly divided into six groups: control group (con, equal cmc-na), model group (mod, equal cmc-na), positive control glucosamine hydrochloride capsule group (gh, 129.6 mg kg d −1 ), wb low-dose group (wb-l, 247.5 mg kg d −1 ), wb medium-dose group (wb-m, 742.5 mg kg d −1 ), and wb highdose group (wb-h, 2227.5 mg kg d −1 ). after that, con group rats were injected with saline (50 μl) into the articular cavity of unilateral knee joints, while the other group rats were injected with mia solution (3 mg/50 μl). subsequently, after 2 weeks of the injection, 36 rats received the treatment once a day by gavage for 4 weeks. after treatment, rats in all experimental groups were sacrificed, and then, the serum, synovium, and knee joints were acquired for further analysis. the body weights and the levels of serum cytokines (tnf-α, il-1β, and il-10) were measured as previously described. in addition, the knee joints in each group were randomly selected for macroscopic observations, which conducted by separating the femoral condyle and tibial plateau and then taking pictures. moreover, the knee joint tissue sections were stained with he similarly as previously mentioned, and subsequently analyzed with the intelligent digital slice scanning system nanozoomer s60 and the ndp. view 2 software. 5.4.4. western blotting assay. the total proteins of synovium were extracted with ripa lysate freshly supplemented with 1% protease and 2% phosphatase inhibitor (beyotime, shanghai, china) before use. then, the proteins were obtained by centrifugation of lysate at 4°c with 12,000 rpm for 15 min (eppendorf, hamburg, germany). subsequently, the protein concentrations were measured with the bca protein assay kit (keygen bio tech, nanjing, china). additionally, the protein samples were denatured at 99°c for 10 min and were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. the separated proteins were transferred from gel onto nitrocellulose (nc) membranes, which were blocked with 5% non-fat milk or bovine serum albumin for 2 h and then incubated at 4°c with the primary antibodies (1:1000) overnight. 40, 41 after that, the membranes were washed three times with tbst (servicebio, wuhan, china) and incubated with second antibodies (1:2000) for 1 h at room temperature. then, the protein bands were visualized by the ecl kit (tanon, shanghai, china) and tanon-5200 gel imaging system (tanon, shanghai, china). the gray value of the bands was quantified with imagej software. 5.5. statistical analysis. the statistical analysis was performed with graphpad prism 8.0 software. all data were presented as mean ± sd. the one-way analysis of variance was applied for comparisons between groups, and p < 0.05 was considered as statistically significant. complete contact information is available at: https://pubs.acs.org/10.1021/acsomega.1c06729 macrophage m1/m2 polarization and rheumatoid arthritis: a systematic review rheumatoid arthritis articular cartilage regeneration by activated skeletal stem cells quercetin alleviates rat osteoarthritis by inhibiting inflammation and apoptosis of chondrocytes, modulating synovial macrophages polarization to m2 macrophages. free radic implications of microrna 21 and its involvement in the treatment of different type of arthritis cartilage and bone destruction in arthritis: pathogenesis and treatment strategy: a literature review luteolin inhibits il-1β-induced inflammation in rat chondrocytes and attenuates osteoarthritis progression in a rat model research of pathogenesis and novel therapeutics in arthritis 2.0 bioactive constituents and the molecular mechanism of curcumae rhizoma in the treatment of primary dysmenorrhea based on network pharmacology and molecular docking guizhi-shaoyao-zhimu decoction possesses anti-arthritic effects on type ii collagen-induced arthritis in rats via suppression of inflammatory reactions, inhibition of invasion & migration and induction of apoptosis in synovial fibroblasts transcriptomics-based analysis of the mechanism by which wang-bi capsule alleviates joint destruction in rats with collagen-induced arthritis wang-bi capsule alleviates the joint inflammation and bone destruction in mice with collagen-induced arthritis. j. evidence-based complementary altern deciphering the synergistic network regulation of active components from sinisan against irritable bowel syndrome via a comprehensive strategy: combined effects of synephrine, paeoniflorin and naringin wutou decoction ameliorates experimental rheumatoid arthritis via regulating nf-kb and nrf2: integrating efficacy-oriented compatibility of traditional chinese medicine erianin, the main active ingredient of dendrobium chrysotoxum lindl, inhibits precancerous lesions of gastric cancer (plgc) through suppression of the hras-pi3k-akt signaling pathway as revealed by network pharmacology and in vitro experimental verification apoptosis effects of imperatorin on synoviocytes in rheumatoid arthritis through mitochondrial/caspasemediated pathways liquiritin from glycyrrhiza uralensis attenuating rheumatoid arthritis via reducing inflammation, suppressing angiogenesis, and inhibiting mapk signaling pathway salicin from alangium chinense ameliorates rheumatoid arthritis by modulating the nrf2-ho-1-ros pathways oleanolic acid and its derivatives: biological activities and therapeutic potential in chronic diseases anhydroicaritin, a srebps inhibitor, inhibits rankl-induced osteoclastic differentiation and improves diabetic osteoporosis in stz-induced mice maslinic acid prevents il-1β-induced inflammatory response in osteoarthritis via pi3k/akt/nf-κb pathways effects of artesunate on chondrocyte proliferation, apoptosis and autophagy through the pi3k/akt/mtor signaling pathway in rat models with rheumatoid arthritis hydrogen sulfide protects against il-1β-induced inflammation and mitochondrial dysfunction-related apoptosis in chondrocytes and ameliorates osteoarthritis loganin attenuates osteoarthritis in rats by inhibiting il-1β-induced catabolism and apoptosis in chondrocytes via regulation of phosphatidylinositol 3-kinases (pi3k)/akt maltol prevents the progression of osteoarthritis by targeting pi3k/ akt/nf-κb pathway: in vitro and in vivo studies network pharmacology based research into the effect and mechanism of xijiao dihuang decoction against sepsis integrating network pharmacology and experimental evidence to decipher the cardioprotective mechanism of yiqihuoxue decoction in rats after myocardial infarction mechanisms of indigo naturalis on treating ulcerative colitis explored by geo gene chips combined with network pharmacology and molecular docking integrated meta-analysis, network pharmacology, and molecular docking to investigate the efficacy and potential pharmacological mechanism of kai-xin-san on alzheimer's disease network pharmacology and molecular docking analyses on lianhua qingwen capsule indicate akt1 is a potential target to treat and prevent covid-19 tunisian olea europaea l. leaf extract suppresses freund's complete adjuvant-induced rheumatoid arthritis and lipopolysaccharide-induced inflammatory responses regional differences in the gut microbiota and gut-associated immunologic factors in the ileum and cecum of rats with collagen-induced arthritis effect of alpinia oxyphylla extract in vitro and in a monosodium iodoacetate-induced osteoarthritis rat model protective effects of dioscin against cartilage destruction in a monosodium iodoacetate (mia)-indcued osteoarthritis rat model casticin suppresses monoiodoacetic acid-induced knee osteoarthritis through inhibiting hif-1α/nlrp3 inflammasome signaling key: cord-0004029-q8e08pq0 authors: smatti, maria k.; cyprian, farhan s.; nasrallah, gheyath k.; al thani, asmaa a.; almishal, ruba o.; yassine, hadi m. title: viruses and autoimmunity: a review on the potential interaction and molecular mechanisms date: 2019-08-19 journal: viruses doi: 10.3390/v11080762 sha: 00dce8da5fcee849b30c9d7a7e358c5855b14ca9 doc_id: 4029 cord_uid: q8e08pq0 for a long time, viruses have been shown to modify the clinical picture of several autoimmune diseases, including type 1 diabetes (t1d), systemic lupus erythematosus (sle), rheumatoid arthritis (ra), sjögren’s syndrome (ss), herpetic stromal keratitis (hsk), celiac disease (cd), and multiple sclerosis (ms). best examples of viral infections that have been proposed to modulate the induction and development of autoimmune diseases are the infections with enteric viruses such as coxsackie b virus (cvb) and rotavirus, as well as influenza a viruses (iav), and herpesviruses. other viruses that have been studied in this context include, measles, mumps, and rubella. epidemiological studies in humans and experimental studies in animal have shown that viral infections can induce or protect from autoimmunopathologies depending on several factors including genetic background, host-elicited immune responses, type of virus strain, viral load, and the onset time of infection. still, data delineating the clear mechanistic interaction between the virus and the immune system to induce autoreactivity are scarce. available data indicate that viral-induced autoimmunity can be activated through multiple mechanisms including molecular mimicry, epitope spreading, bystander activation, and immortalization of infected b cells. contrarily, the protective effects can be achieved via regulatory immune responses which lead to the suppression of autoimmune phenomena. therefore, a better understanding of the immune-related molecular processes in virus-induced autoimmunity is warranted. here we provide an overview of the current understanding of viral-induced autoimmunity and the mechanisms that are associated with this phenomenon. autoimmune diseases (aid) develop as a result of an aberrant immune response in recognizing self and non-self-antigens. currently, there are more than 80 identified types of autoimmune disorders [1] . although the etiologies of several autoimmune disorders remain completely understood, multiple factors have been linked to autoimmune responses, including genetics, age, environment, as well as viral infections. viruses have been considered as major environmental factors that trigger the autoimmune phenomena in genetically susceptible individuals [2, 3] . multiple mechanisms have been proposed to explain the breakdown of self-tolerance by viral infections (figure 1 ). traditionally, it was believed that viruses carry structurally similar antigens to self-antigens, which activate b and t cells and lead to a cross-reactive response against both self-and non-self-antigens, a mechanism known as "molecular mimicry" [4] . molecular mimicry has been described for herpes simplex enteroviruses are considered the main viral candidates for causing type 1 diabetes in humans [15] . seasonal incidence of type 1 diabetes has been reported subsequent to enterovirus infections since 1969 [16, 17] . although the role of enteroviruses in type 1 diabetes has been investigated for more than 40 years, the etiological link is still enigmatic [18] . a higher frequency of enterovirus infections has been reported among siblings who develop type 1 diabetes as compared to nondiabetic controls [19] . additionally, higher titers of enterovirus antibodies were found in pregnant mothers whose children later developed type 1 diabetes [19] . furthermore, association between enteroviral infections and beta cell autoimmunity has been reported on several occasions. for example, in type 1 diabetes-genetically susceptible children, a seasonal detection of autoantibodies matched the seasonal occurrence of enteroviruses infections, suggesting the contribution of enteroviruses to autoimmune diabetes [20] . in another study, signs of enterovirus infections were detected in parallel with the first appearance of diabetes-associated autoantibodies [21] . correspondingly, elshebani et al. found that certain enterovirus strains that are isolated from type 1 diabetes patients affected the function of beta cells, and induced cell destruction in vitro [22] . similarly, enteroviruses have been found in intestinal biopsies from diabetic patients, suggesting the possibility of an ongoing persistent enterovirus infection in the gut mucosa of type 1 diabetes patients [23] . furthermore, a recent systematic review that analyzed 24 published papers documented a significant association between enteroviruses and autoimmunity/type 1 diabetes [24] . coxsackie b virus (cvb) is the most prevalent enterovirus in pre-diabetic and diabetic patients [15] . several studies have reported the presence of cvb rna in the blood of type 1 diabetes patients [25] [26] [27] [28] . juhela et al. found that t cell response to cvb4 (serotypes 4 in coxsackievirus group b) is enhanced in children with type 1 diabetes, and this might be as a result of enterovirus-specific t cell being trapped in the pancreas [29] . in addition, a post-mortem examination of a deceased diabetic child revealed the death of beta cells and the presence of a lymphocytic infiltrate in the islets of langerhans [30] . in further support of this finding, cvb4 was propagated in mouse pancreatic cells, and when those cells were inoculated into susceptible mice, they developed autoimmune diabetes [30] . dotta et al. detected cvb4 in pancreatic tissue from three out of six patients with type 1 diabetes. in fact, the virus was able to infect beta cells originating from non-diabetic donors, leading to beta cell functional impairment and strong natural killer (nk) cells-mediated inflammation [31] . this observation could be attributed to the high expression of type-i interferon (ifn-1) in beta cells following cvb infection, which leads to rapid beta cell death [32] . evidence of inflammation and direct cytolysis of beta cells has been shown to produce autoantigens that initiate autoimmunity in turn [15] . importantly, age seems to play a significant role in the development of virus-induced diabetes. in this regard, infection with cvb4 has been shown to enhance type 1 diabetes in eight-week-old nod mice but not in a younger age group, suggesting that cvb4 infection can contribute to diabetes progression, only if a threshold level of pre-existing autoreactive t cells were accumulated in the pancreatic islets [33] . virus-induced inflammatory cytokines are also thought to play an important role in the induction of autoimmunity in a time-dependent manner. for example, expression of tumor necrosis factor alpha (tnf-α) in the pancreatic islets has been shown to enhance or abrogate autoimmune diabetes depending on the time of expression [34] . the molecular basis of autoimmunity in cvb4 infection is proposed to be a molecular mimicry [35] , where the 2c non-structural cvb protein has a shared sequence with the glutamic acid decarboxylase 65 enzyme (gad65), which is predominantly expressed in pancreatic beta cells [36] . this result aligns with previous studies that have investigated the immunological cross-reactivity caused by sequence similarity between self-and non-self-antigens. particularly gad65 was found to play an important role in the pathogenesis of type i diabetes as a target autoantigen [37] , where the ensuing immune response to gad65 has been detected before the onset of clinical diabetes [35] . in this regard, it is of note that t cells isolated from type 1 diabetes patients were shown to react with both gad65 and 2c protein, however, other studies have demonstrated that healthy control groups can also have reactive t cells to gad65 [38] . this immunological cross-reactivity may not provide the initiative event for triggering diabetes, but can act as an enhancer for disease development [15] . bystander activation was also a suggested mechanism by which cvb4 induce or accelerate diabetes. mouse studies have indicated that by evoking beta cells damage, cvb4 infection promotes the release of self-antigens, which are then presented by macrophages to autoreactive t cells [32] . on the contrary, some studies have not found convincing evidence supporting the cvb-induced diabetes hypothesis [39] . notably, sarmineto et al. showed that although an enterovirus epidemic occurred in cuban population between 2000 and 2001, diabetes rate in the population was not affected [18] . on the other hand, other studies have demonstrated that infection with cvb during the first year of life can have a protective effect against type 1 diabetes [29] . this protection was attributed to the developmental stage of the adaptive immune system where viral infections trigger specific immune responses, including activation of t regulatory (treg) cells and induction of suppressors which collectively hinder autoimmunity [15] . furthermore, cvb infection was found to be protective through enhancing treg cells in individuals who are genetically susceptible to type 1 diabetes but not insulitis, therefore inhibiting islet-specific t cell autoimmunity [17] . however, it is yet to be determined how often cvb can induce autoimmunity, and what are the factors that contribute to beta cells destruction and diabetes development or protection [15] . taken together, molecular mimics of islet autoantigens have been strongly suggested as a factor for cvb-induced autoimmunity. however, bystander activation of autoreactive t cells and rapid death of cvb-infected beta cells are also potential mechanisms that could contribute to diabetes. several studies investigated the role of rotavirus in the induction of autoimmunity in human and animal models, suggesting bystander induction of autoimmunity. based on serological analysis of rotavirus antibodies and islets autoantibodies, honeyman et al. suggested that rotavirus infection might trigger autoimmunity to pancreatic islets in high-risk children [40] . in contrast, a larger study cohort showed that rotavirus infections are unlikely to trigger beta cell autoimmunity in genetically susceptible children [41] . studies of rhesus monkey rotavirus (rrv) infection in nod mice indicated that rrv can induce diabetes without pancreatic infection, but it depends on the presence of insulitis with involvement of th1 activation and proinflammatory cytokines release [42, 43] . interleukin 1 and 6, the candidate proinflammatory cytokines, are known to suppress treg functionality allowing unchecked adaptive autoimmune response. the time of infection, age, and insulitis status seems to affect the outcomes of infection in terms of delaying, accelerating, or not affecting the onset of diabetes [43] . moreover, rotavirus acceleration of diabetes is strain-specific, as infection of mice with rrv but not the crw-8, which is an australian porcine rotavirus serotype that cross react with human rotavirus serotype 3, has enhanced disease development [44] . pane et al. proposed that diabetes acceleration by rotavirus in nod mice occur via bystander activation: rotavirus degraded dsrna induce toll-like receptor 7 (tlr7) signaling, leading to release of type-i interferon and lymphocyte activation, including autoreactive t cells, which in turn exacerbate diabetes-related autoimmunity [42] . although tlr7 has been implicated in the bystander activation, the melanoma differentiation-associated protein 5 (mda5), might also have a role in rotavirus-induced type-i ifn expression, and thus diabetes progression [32] . if viral-induced diabetes can occur through nonspecific mechanisms without pancreatic viral infection, e.g., proinflammatory cytokine response and lymphocyte activation, this suggests that multiple other viruses can produce similar effect [32] , and hence, further studies in the field are necessary. influenza viruses primarily replicate in the respiratory tract; however, viral replication in pancreatic islets and other internal organs has also been reported and linked to bystander activation of the immune system. in 1990, roman et al. reported that influenza virus induced insulitis and diabetes in transgenic mice expressing hemagglutinin in the pancreatic beta cells [45] . moreover, multiple studies and case reports in the last ten years have demonstrated the possible association between influenza virus infection and diabetes development. using both, avian strains (h7n1 and h7n3) and human strain (h1n1), capua et al. reported that influenza viruses are able to grow in human pancreatic cell lines in vitro, whereas they lead to tissue damage and diabetes in turkeys [46] . influenza viruses can reach the pancreas through viremia [47, 48] and replicate in pancreatic cells from both, exocrine and endocrine origins [46] . the virus can also find its way to the pancreas through reflux from the gut to the pancreatic duct, where it finds a tolerant environment containing appropriate cell receptors and susceptible cells. primarily, human pancreatic cells express both alpha-2,3 and alpha-2,6 sialic acid receptors, which theoretically allow both human and avian influenza strains to replicate [46] . clinically, the onset of diabetes and other pancreatic diseases have been linked to influenza infections. in a case report from china in 2012, diabetic ketoacidosis was documented in a young woman after h1n1 infection [49] . in another case, a patient converted from type 2 to type 1 diabetes after influenza a virus infection during h1n1 pandemic in 2009 in japan. this patient had a human leukocyte antigen (hla) pattern associated with type 2 diabetes, and developed deterioration of glycemic control after the infection [50] . these cases have shed light not only on the role of viral infections, but also on the susceptibility of these patients in development of diabetes. in a study investigating type 1 diabetes onset in children hospitalized with 2009 pandemic influenza a (h1n1) virus, it was found that three children (7%) had ketoacidosis as an indication of type 1 diabetes, suggesting that h1n1 virus can be involved in disease development [51] . in addition, a recent study with a large cohort found that young children presented with respiratory infections such as influenza like illness, had increased risk of developing pancreatic islet autoimmunity [52] . several studies have linked upper respiratory infections to transient increase in type-i ifn expression preceding autoimmunity/diabetes development [53] . similarly, it was found that influenza virus stimulated ifn-α production from plasmacytoid dendritic cells (pdc), which is associated with th1-mediated autoimmunity type 1 diabetes development [54] . further, iav infection induced significantly higher pdc-dependent ifn-α expression in pbmcs obtained from diabetic patients compared to those from healthy controls [54] . this increase in ifn-α expression was associated with increased numbers of pdcs, which in turn could contribute to the bystander activation of autoreactive t cells. more recently, it was reported that a highly functional cd8+ t cell response is elicited by alternative reading frame (arf) epitopes encoded by ns1 mrna of iav, which might have important implications on iav-induced autoimmunity [55] . although studies have linked iavs to diabetes, their exact role in disease prognosis and pathogenies is yet to be explored. viral replication is thought to cause cell damage and cytokines production, similar to conditions linked to diabetes prognosis. more specifically, in situ hybridization experiments of pancreatic tissue after infection with influenza virus have indicated that viral nucleoprotein was detected in beta cells, with reduction in the number of cells staining for insulin [46] . this clearly suggests the potential effect of influenza replication on beta cells damage and insulin levels, leading to diabetes. in the last decade, a number of studies had associated influenza infection or vaccination to type 1 narcolepsy (t1n), a chronic sleep disorder associated with autoimmune destruction of hypocretin (hcrt) neurons in the hypothalamus. this disorder is linked to the hla-dqb1*0602/dqa1*01:02 haplotype, t cell receptors (tcr) and other immune loci, suggesting the involvement of autoimmune response in the disease pathophysiology [56, 57] . however, the associated autoantigens are still not identified. observational studied have reported an increase in t1n after the 2009 h1n1 pandemic [58] . a significantly increased risk of narcolepsy in adults and children was also linked to pandemrix vaccination (influenza inactivated vaccine) [59, 60] . interestingly, the use of focetria vaccination (differently formulated inactivated influenza vaccine) was not associated with increased t1n risk [61] . considering the difference in the adjuvants as well as the antigens of both vaccines, as pandemrix is a split-influenza vaccine while focetria is a subunit vaccine resulting in distinct antigen formulations, this highlights the significant contribution of viral antigen composition in the development of autoimmunity [62] . molecular mimicry of iav antigens has been strongly suggested in t1n, in which a similarity between h1n1 specific peptides and hcrt peptides was reported. in a very recent study, luo et al. reported that sequences of t cell receptor (tcr) α and β/complementarity-determining region (cdr) 3 were found in influenza ha nucleoprotein (np) and hcrt tetramer-positive cd4+ t-cells, and also retrieved in inf-γ-secreting cd4+ t-cells stimulated with pandemrix [57] . accordingly, cross-reactivity between hcrt autoantigen and specific influenza sequences might explain how influenza infection or vaccine trigger t1n autoimmunity. not only that, these findings also suggested the possible involvement of antigen spreading and cd8+ t-cells killing of hcrt neurons leading to this disorder, which is a known mechanism reported in other iav-induced autoimmune disorders as well, including diabetes [63] . nonetheless, to confirm these observations, sequence analysis of more tcrs in autoimmune responses is required. collectively, influenza-induced autoimmunity is explained by several mechanisms. in diabetes, the contribution of t cells bystander activation and direct beta cell damage are strongly supported. nonetheless, in other autoimmune disorders such as t1n, molecular mimicry is believed to be the reason for disease initiation. despite these findings, additional mechanistic and epidemiologic studies are necessary to investigate the occurrence of autoimmune disorders following influenza pandemics there is an increasing evidence linking infection with herpesviruses to the development of multiple autoimmune disorders. large epidemiological studies suggested that susceptibility to multiple sclerosis (ms) is gained in early childhood, with viral infections acting as a trigger. consequently, herpesviruses which are a childhood infections, are considered appropriate candidates contributing to ms development [64] . herpesviruses also persist in the host as a latent infection, and when reactivated contribute to disease pathogenesis as observed in systemic autoimmune diseases (sads) [65, 66] . epstein-barr virus (ebv) infection is suspected to have a central role in the pathogenesis of sads [66] . in fact, high viral loads of ebv dna were detected in the blood of systemic lupus erythematosus (sle) patients [67, 68] . furthermore, increased ebv viral mrnas expression was reported in sle patients [69] . other reports linked sle to ebv based on serological analysis. high titers of anti-early antigen (ea) igg and iga were found in sle patients compared to healthy ebv carriers [65, 70] . increased ebv viral load, high titers of ebv antibodies, and abnormal cell-mediated immunity to ebv have been also observed in rheumatoid arthritis (ra) and sjögren's syndrome (ss) patients [65] . these findings bear immediate relevance for the abnormal cell-mediated regulation of ebv infection and frequent virus reactivation that has been observed in ra, ss, and sle patients [65] . ebv-mediated autoimmunity can also be developed through molecular mimicry, in which antibodies against ebv nuclear antigen 1 (ebna1) cross-react with lupus associated autoantigens in sle patients, followed by epitope spreading mechanism which will include more autoantigens [71] . moreover, through tlr3 signaling, ebv infection induces the activation of innate immunity, and the production of type-i ifn and proinflammatory cytokines [72] . inflammation in turn is known to contribute to autoreactivity enhancement and initiation of bystander activation [4] . additionally, it has been found that some ebv proteins that are important in immune evasion and anti-apoptosis, such as early antigen restricted (ea/r), are homologues to cellular proteins such as bcl2. accordingly, this prevents both infected b cells and epithelial cells from apoptosis, and lead to a loss of tolerance and development of autoimmunity [66] . herpesviruses are neurotropic and neurovirulent, which means they can infect cells of the central nervous system (cns) and produce neurological illness. of note, the eye and the cns are immune privileged sites where the self-antigens of these organs are segregated from the adaptive immune system, in part via the blood brain barrier. inflammation at these sites may lead to the loss of this barrier function, allowing immune cell infiltration. herpesviruses-triggered ms can be mediated through direct lyses of cns cells, or by the immunopathogenic host immune responses, increasing the pool of cns specific self-antigens [64] . several reports have indicated the presence of herpes simplex virus (hsv) in active plaques from postmortem brain samples of ms patients [73] . in addition, it was found that the viral gene products following hsv-1 infection drive apoptosis in neuronal progenitor cells [74] . ebv, another herpesvirus that causes infectious mononucleosis (im) has also been linked to ms [75] . moreover, human herpesvirus 6 (hhv-6) was found to be more prevalent in ms plaques than normal ms white matter, and its reactivation has been observed in ms relapse [64] . infection with hsv have been well-linked to herpetic stromal keratitis (hsk), which is an autoimmune corneal disease [1] . it was reported that molecular mimicry is involved in disease pathology for these viruses as well. cornea-specific t-cell clones are found to recognize the hsv-1-derived protein ul6 in a murine model [5] . contrarily, other studies showed that isolated t cells from hsk patients' corneas do no cross react with ul6, suggesting that t cells may induce pathogenesis through bystander destruction in humans [76] . in addition, earlier studies reported cytomegalovirus (cmv) genome in type 1 diabetes patients [77] . also, persistent cmv infection was found to be associated with the induction of antibodies against islet cells [78] . further, gastrointestinal and herpes viral infections have been linked to the induction of celiac disease (cd), which is a life-long autoimmune disorder [3, 79] . in contrast to the aforementioned studies, interestingly, some other reports have shown a protective association between herpesviruses infections and autoimmunity [3, 80, 81] . serum antibodies to cmv, ebv were found to be lower in cd patients compared to healthy controls [81] . jansen et al., found that anti-cmv, ebv, and or hsv-1 igg levels were inversely correlated with transglutaminase type 2 antibody (tg2a) levels, suggesting a protective impact of these viruses in the pathogenesis of cd [82, 83] . the protective role of infections in autoimmunity supports the "hygiene hypothesis", which proposes that cleaner living environment leads to higher incidence of autoimmune disorders [80] . lerner et al. found that higher cd incidences are correlated with less infectious environment [3] . infections can abrogate autoimmunity disorders through several pathways including: post-translational modification of proteins (ptmp) form non-self to self, and therefore reducing their pathogenicity. for example, some microbial agents modify gluten and thus preventing intestine damage [3, 84] . additionally, some infections are able to shift the immune response from th1 to th2, establishing an immunosuppressive state in th1-derived immune diseases, such as cd [80] . in fact, this protective effect has also been observed in parasitic infections. in mouse model, it was reported that helminths modulate the immune system and generate a th2 environment, which protects from autoimmune diseases or relieve symptoms of established autoimmune inflammation [85] . similarly, it was found that ms patients who are infected with helminth parasites have less severe disease compared to uninfected patients [86] . interestingly, the administration of anti-helminthic drugs increased ms-associated disease activity [87] . currently, the use of live helminths to treat ms and other aids is under clinical evaluation [88] . other than immune system modulation towards th2, viral infections, including infections produced by cmv and ebv, can cause apoptosis of the immune cells, thus, attenuating the immune system response, and minimizing autoimmune disease progression [3] . to summarize, the mechanisms by which herpesviruses trigger autoimmunity are variable. both, molecular mimicry and bystander activation were reported in ebv-and hsv-induced autoimmunity. not only that, ebv also has the ability to immortalize autoreactive infected b cells. in addition, as neurotropic viruses, herpesviruses can infect and kill cns cells directly, leading to several aids. infections with multiple other viruses such as measles and mumps (paramyxoviridae family), and rubella (togaviridae family), have been linked to autoimmune disorders. specifically, mumps and rubella infections were linked to the onset of type 1 diabetes [89] . both viruses have the ability to infect and grow in beta cells [90, 91] . further, both viruses result in cns demyelination disease [64] . viruses from the flaviviridae family, such as zika virus (zikv) and dengue virus (denv) have been also associated to autoimmune disorders, including the recently reported zikv-induced guillain-barré syndrome and denv-induced sle and lupus nephritis [92, 93] . human t-lymphotropic virus type 1 (htlv-1) from the retroviridae family has been associated with cns autoimmunity, causing myelopathy/tropical spastic paraparesis [9] . several recent studies aimed to explain the contribution of viral infections in cns disorders. it was suggested that antiviral immune response cross-react with human nmda receptors (2a subunit) [94] , which are responsible for excitatory glutamatergic transmission and are ion channel proteins found in nerve cells [95] . pentapeptide similarity between the nmda 2a receptor and viral proteins have been previously reported [96] . using the same mechanism, other peptide commonalities with high degree of pentapeptide sharing were found between viral peptides and the human distal-less homeobox (dlx) transcription factors expressed during early fetal neurodevelopment (dlx1, dlx2, dlx5, and dlx6) [94] . analysis of the brain-specific dlx self-antigens pentapeptide revealed matching with several viruses that have been related to neurological disorders including rubella and herpesviruses [94] . collectively, these studies support the assumption that viral infections may relate to cns disorders through autoimmune cross-reactions caused by molecular mimicry. in a recent work by kanduc, peptide sharing analysis of five common viruses (borna disease virus, iav, measles, mumps, and rubella) in comparison to human proteome revealed an unexpected massive viral human peptide cross-reactivity [97] . the author explained this finding in the light of the viral eukaryogenesis hypothesis, which describes that the first eukaryotic cell had evolved from an archaeal ancestor of the eukaryotic cytoplasm, a bacterial ancestor of the mitochondria, and a viral ancestor of the nucleus. importantly, the clinical implication of this high viral/human peptide sequence similarity confirms the significant contribution of molecular mimicry as a mechanism in viral induced autoimmunity. considering the available data from epidemiological and experimental animal studies, there is a wide range of viruses that are suspected to initiate an autoimmune response, despite the lack of a clear mechanistic explanation to this phenomenon in most of the cases. table 1 summarizes studies reporting viral induced autoimmunity in different organisms along with proposed mechanisms. importantly, it is clear that there is no single factor responsible for triggering autoimmunity. it seems that the development of autoimmune diseases following viral infections is a multifactorial process that can be affected by different variables. moreover, determining whether viral infection can lead to autoimmunity or protect from certain immune disorders such as diabetes [98] and cd [81] , depends on multiple factors, including virus strain, genetic predisposition, host immune response, infectious dose, and time of infection [99] . table 1 . examples of viral infections that have been linked to autoimmune diseases in different organisms. viral infections are a major trigger of autoimmunity. virus-induced autoimmunity is a multidirectional process. current data suggests that viruses can initiate autoimmunity via several pathways including molecular mimicry, epitope spreading, bystander activation and/or immortalization of infected b cells. to the contrary, a growing evidence is supporting the protective role of viruses against autoimmunity [3] , where viral infections lead to the activation of regulatory immune responses, consequently suppressing the development of autoimmune reactions. this dual effect of viral infections on autoimmunity is orchestrated by different host, viral and environmental factors. accordingly, further epidemiological and molecular research is needed to gain insights about the interplay between viral infections and host autoimmune responses, and to provide a clear mechanistic description on how a viral infection can trigger autoimmunopathies. the role of infections in autoimmune disease editorial: microbial and environmental factors in autoimmune and inflammatory diseases microbes and viruses are bugging the gut in celiac disease. are they friends or foes? front viruses and autoimmunity molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection viral infections and molecular mimicry in type 1 diabetes molecular mimicry, anti-coxsackievirus b3 neutralizing monoclonal antibodies, and myocarditis epitope spreading and molecular mimicry as triggers of autoimmunity in the theiler's virus-induced demyelinating disease model of multiple sclerosis virus infection, antiviral immunity, and autoimmunity molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease experimental autoimmune encephalomyelitis (eae) as a model for multiple sclerosis (ms) west nile virus infection and myasthenia gravis persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading epstein-barr virus rna confers resistance to interferon-alpha-induced apoptosis in burkitt's lymphoma viral trigger for type 1 diabetes: pros and cons seasonal incidence of diabetes mellitus enterovirus and type 1 diabetes: what is the matter? evidence of association between type 1 diabetes and exposure to enterovirus in cuban children and adolescents a prospective study of the role of coxsackie b and other enterovirus infections in the pathogenesis of iddm the first signs of beta-cell autoimmunity appear in infancy in genetically susceptible children from the general population: the finnish type 1 diabetes prediction and prevention study enterovirus infection as a risk factor for beta-cell autoimmunity in a prospectively observed birth cohort: the finnish diabetes prediction and prevention study effects on isolated human pancreatic islet cells after infection with strains of enterovirus isolated at clinical presentation of type 1 diabetes detection of enteroviruses in the intestine of type 1 diabetic patients enterovirus infection and type 1 diabetes mellitus: systematic review and meta-analysis of observational molecular studies coxsackie b virus infection and onset of childhood diabetes detection of coxsackie b virus rna sequences in whole blood samples from adult patients at the onset of type i diabetes mellitus coxsackie b virus infection and beta cell autoantibodies in newly diagnosed iddm adult patients enterovirus rna in blood is linked to the development of type 1 diabetes t-cell responses to enterovirus antigens in children with type 1 diabetes isolation of a virus from the pancreas of a child with diabetic ketoacidosis coxsackie b4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients lessons from the mouse: potential contribution of bystander lymphocyte activation by viruses to human type 1 diabetes acceleration of type 1 diabetes by a coxsackievirus infection requires a preexisting critical mass of autoreactive t-cells in pancreatic islets a dual role for tnf-alpha in type 1 diabetes: islet-specific expression abrogates the ongoing autoimmune process when induced late but not early during pathogenesis autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus antibodies to glutamic acid decarboxylase and p2-c peptides in sera from coxsackie virus b4-infected mice and iddm patients enteroviruses and the pathogenesis of type 1 diabetes revisited: cross-reactivity of enterovirus capsid protein (vp1) antibodies with human mitochondrial proteins coxsackie b virus serology and type 1 diabetes mellitus: a systematic review of published case-control studies association between rotavirus infection and pancreatic islet autoimmunity in children at risk of developing type 1 diabetes rotavirus infections and development of diabetes-associated autoantibodies during the first 2 years of life rotavirus activates lymphocytes from non-obese diabetic mice by triggering toll-like receptor 7 signaling and interferon production in plasmacytoid dendritic cells rotavirus infection accelerates type 1 diabetes in mice with established insulitis rotavirus acceleration of murine type 1 diabetes is associated with a t helper 1-dependent specific serum antibody response and virus effects in regional lymph nodes the expression of influenza virus hemagglutinin in the pancreatic beta cells of transgenic mice results in autoimmune diabetes influenza a viruses grow in human pancreatic cells and cause pancreatitis and diabetes in an animal model national heart, lung, blood institute retrovirus epidemiology donor study-ii (reds-ii). influenza viremia and the potential for blood-borne transmission evidence of viremia in 2 cases of severe pandemic influenza a h1n1/09 the trigger of diabetic ketoacidosis in a young woman with ketosis-prone diabetes conversion to type 1 diabetes after h1n1 influenza infection: a case report detection of respiratory viruses in the 2009 winter season in rome: 2009 influenza a (h1n1) complications in children and concomitant type 1 diabetes onset respiratory infections are temporally associated with initiation of type 1 diabetes autoimmunity: the teddy study a type i interferon transcriptional signature precedes autoimmunity in children genetically at risk for type 1 diabetes increased ifn-α-producing plasmacytoid dendritic cells (pdcs) in human th1-mediated type 1 diabetes: pdcs augment th1 responses through ifn-α production1 influenza a virus infection induces viral and cellular defective ribosomal products encoded by alternative reading frames antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2 autoimmunity to hypocretin and molecular mimicry to flu in type 1 narcolepsy narcolepsy onset is seasonal and increased following the 2009 h1n1 pandemic in china risk of narcolepsy after as03 adjuvanted pandemic a/h1n1 2009 influenza vaccine in adults: a case-coverage study in england as03 adjuvanted ah1n1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland adjuvanted seasonal and pandemic influenza vaccines update on narcolepsy cases associated with pandemrix vaccination in 2009 in the netherlands narcolepsy and influenza vaccination-induced autoimmunity viruses and multiple sclerosis epstein-barr virus in systemic autoimmune diseases detecting epstein-barr virus dna from peripheral blood mononuclear cells in adult patients with systemic lupus erythematosus in taiwan patients with systemic lupus erythematosus have abnormally elevated epstein-barr virus load in blood a new perspective epstein-barr virus early antigen diffuse (ebv-ea/d)-directed immunoglobulin a antibodies in systemic lupus erythematosus patients epstein-barr virus and molecular mimicry in systemic lupus erythematosus epstein-barr virus (ebv)-encoded small rna is released from ebv-infected cells and activates signaling from toll-like receptor 3 herpes simplex virus in postmortem multiple sclerosis brain tissue microglia-induced il-6 protects against neuronal loss following hsv-1 infection of neural progenitor cells epstein-barr virus in multiple sclerosis: a continuing conundrum herpes simplex virus-specific t cells infiltrate the cornea of patients with herpetic stromal keratitis: no evidence for autoreactive t cells association of cytomegalovirus infection with autoimmune type 1 diabetes influence of the epstein-barr virus nuclear antigen ebna 2 on the growth phenotype of virus-transformed b cells factors that increase risk of celiac disease autoimmunity after a gastrointestinal infection in early life infections and autoimmunity-good or bad? infections may have a protective role in the etiopathogenesis of celiac disease herpesvirus infections and transglutaminase type 2 antibody positivity in childhood: the generation r study ethnic differences in coeliac disease autoimmunity in childhood: the generation r study duodenal bacteria from patients with celiac disease and healthy subjects distinctly affect gluten breakdown and immunogenicity parasitic infection and autoimmunity association between parasite infection and immune responses in multiple sclerosis the impact of parasite infections on the course of multiple sclerosis helminth products protect against autoimmunity via innate type 2 cytokines il-5 and il-33, which promote eosinophilia type 1 diabetes and measles, mumps and rubella childhood infections within the italian insulin-dependent diabetes registry mumps and coxsackie b3 virus infection of human fetal pancreatic islet-like cell clusters infection by human cytomegalovirus and rubella virus of cultured human fetal islets of langerhans autoimmunity in guillain-barré syndrome associated with zika virus infection and beyond dengue fever triggering systemic lupus erythematosus and lupus nephritis: a case report peptide sharing between viruses and dlx proteins: a potential cross-reactivity pathway to neuropsychiatric disorders nmda receptor function, memory, and brain aging understanding neuropsychiatric diseases, analyzing the peptide sharing between infectious agents and the language-associated nmda 2a protein the comparative biochemistry of viruses and humans: an evolutionary path towards autoimmunity enteroviruses, type 1 diabetes and hygiene: a complex relationship do viral infections protect from or enhance type 1 diabetes and how can we tell the difference? cocapture of cognate and bystander antigens can activate autoreactive b cells human immunodeficiency virus proteins mimic human t cell receptors inducing cross-reactive antibodies immunization with a peptide of semliki forest virus promotes remyelination in experimental autoimmune encephalomyelitis herpes simplex virus 1 encephalitis associated with voltage-gated calcium channel autoimmunity autoimmune hepatitis following epstein-barr virus infection coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity critical cytokine pathways to cardiac inflammation hhv-6a in vitro infection of thyrocytes and t cells alters the expression of mirna associated to autoimmune thyroiditis delineation of autoantibody repertoire through differential proteogenomics in hepatitis c virus-induced cryoglobulinemia cerebrospinal fluid cyto-/chemokine profile during acute herpes simplex virus induced anti-n-methyl-d -aspartate receptor encephalitis and in chronic neurological sequelae molecular and clinical relationship between live-attenuated japanese encephalitis vaccination and childhood onset myasthenia gravis latent virus infection upregulates cd40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis epstein-barr virus lytic reactivation activates b cells polyclonally and induces activation-induced cytidine deaminase expression: a mechanism underlying autoimmunity and its contribution to graves' disease zika virus and autoimmunity: from microcephaly to guillain-barré syndrome, and beyond the role of epstein-barr virus infection in the development of autoimmune thyroid diseases extrahepatic immune related manifestations in chronic hepatitis c virus infection cutting edge: cd8 t cell-mediated demyelination is ifn-gamma dependent in mice infected with a neurotropic coronavirus role of hck in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice detection of enteroviruses in stools precedes islet autoimmunity by several months: possible evidence for slowly operating mechanisms in virus-induced autoimmunity lung injury combined with loss of regulatory t cells leads to de novo lung-restricted autoimmunity the role of epstein-barr virus in multiple sclerosis: from molecular pathophysiology to in vivo imaging virus-induced autoimmunity: epitope spreading to myelin autoepitopes in theiler's virus infection of the central nervous system varicella zoster virus and relapsing remitting multiple sclerosis the msmv hypothesis: measles virus and multiple sclerosis, etiology and treatment cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation west nile virus infection with hearing loss htlv-1-associated myelopathy/tropical spastic paraparesis rosner, i. hepatitis c virus-related arthritis: characteristics and response to therapy with interferon alpha a human gain-of-function sting mutation causes immunodeficiency and gammaherpesvirus-induced pulmonary fibrosis in mice herpes viruses in multicase families with rheumatoid arthritis and systemic lupus erythematosus cytomegalovirus as a driver of excess cardiovascular mortality in rheumatoid arthritis: a red herring or a smoking gun? influenza a (h1n1) virus infection triggers severe pulmonary inflammation in lupus-prone mice following viral clearance sjögren syndrome associated with hepatitis c virus: a multicenter analysis of 137 cases herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis herpes simplex epithelial and stromal keratitis: an epidemiologic update a review of chikungunya virus-induced arthralgia: clinical manifestations, therapeutics, and pathogenesis correlation between systemic lupus erythematosus and cytomegalovirus infection detected by different methods autoimmunity and hcv infection in porphyria cutanea tarda: a controlled study lupus and leprosy: beyond the coincidence role of viruses and bacteria-virus interactions in autoimmunity immune-mediated systemic vasculitis as the proposed cause of sudden-onset sensorineural hearing loss following lassa virus exposure in cynomolgus macaques thrombocytopenia in patients with chronic hepatitis c virus infection thyroid involvement in hepatitis c virus-infected patients with/without mixed cryoglobulinemia induction of theiler's murine encephalomyelitis virus (tmev)-induced demyelinating disease in genetically resistant mice coxsackievirus and type 1 diabetes mellitus: the wolf's footprints coxsackievirus b1 is associated with induction of β-cell autoimmunity that portends type 1 diabetes immunology in the clinic review series; focus on type 1 diabetes and viruses: the enterovirus link to type 1 diabetes: critical review of human studies hepatitis c virus-induced vasculitis: therapeutic options this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-0012315-51qadwec authors: cavalli, marco; pan, gang; nord, helena; wallerman, ola; wallén arzt, emelie; berggren, olof; elvers, ingegerd; eloranta, maija-leena; rönnblom, lars; lindblad toh, kerstin; wadelius, claes title: allele-specific transcription factor binding to common and rare variants associated with disease and gene expression date: 2016-03-18 journal: hum genet doi: 10.1007/s00439-016-1654-x sha: c216c8e3e6a4ffb79ec77a225742ab51a624b463 doc_id: 12315 cord_uid: 51qadwec genome-wide association studies (gwas) have identified a large number of disease-associated snps, but in few cases the functional variant and the gene it controls have been identified. to systematically identify candidate regulatory variants, we sequenced encode cell lines and used public chip-seq data to look for transcription factors binding preferentially to one allele. we found 9962 candidate regulatory snps, of which 16 % were rare and showed evidence of larger functional effect than common ones. functionally rare variants may explain divergent gwas results between populations and are candidates for a partial explanation of the missing heritability. the majority of allele-specific variants (96 %) were specific to a cell type. furthermore, by examining gwas loci we found >400 allele-specific candidate snps, 141 of which were highly relevant in our cell types. functionally validated snps support identification of an snp in syngr1 which may expose to the risk of rheumatoid arthritis and primary biliary cirrhosis, as well as an snp in the last intron of cog6 exposing to the risk of psoriasis. we propose that by repeating the chip-seq experiments of 20 selected transcription factors in three to ten people, the most common polymorphisms can be interrogated for allele-specific binding. our strategy may help to remove the current bottleneck in functional annotation of the genome. electronic supplementary material: the online version of this article (doi:10.1007/s00439-016-1654-x) contains supplementary material, which is available to authorized users. to date, more than 15,000 single nucleotide polymorphisms (snp) and insertion/deletions have been associated with diseases and other phenotypes as summarized in the nih gwas catalog (hindorff et al. 2011) . it is often assumed that the genetic variant with the highest association is functional; however, this is usually difficult to prove due to linkage disequilibrium (ld) between snps (the 1000 genomes project. 2012). only 15 % of the anticipated functional variants are located in coding regions, and many are believed to act through the regulation of gene expression, which is in agreement with the notion that gene activity is largely genetically controlled (schadt et al. 2008; lappalainen et al. 2013) . identifying the snp with the strongest association to gene expression (esnp) on a haplotype was proposed as a means to finding the variant driving the association to disease, and consequently the nih started the genotype tissue expression project (gtex) to correlate a person's genotype with gene expression in many tissues. however, esnps are also generally in ld with other snps leaving the question of direct abstract genome-wide association studies (gwas) have identified a large number of disease-associated snps, but in few cases the functional variant and the gene it controls have been identified. to systematically identify candidate regulatory variants, we sequenced encode cell lines and used public chip-seq data to look for transcription factors binding preferentially to one allele. we found 9962 candidate regulatory snps, of which 16 % were rare and showed evidence of larger functional effect than common ones. functionally rare variants may explain divergent gwas results between populations and are candidates for a partial explanation of the missing heritability. the majority of allele-specific variants (96 %) were specific to a cell type. furthermore, by examining gwas loci we found 1 3 functionality often unanswered. the encyclopedia of dna elements (encode) project (the encode project consortium 2012) was initiated with the aim of finding all functional elements in the genome. data on chromatin and transcribed genes were generated in cell lines and tissues and, based on these, candidate regulatory elements were suggested. these regulatory elements have been shown to harbor an enrichment of gwas-snps (the encode project consortium 2012), but despite all efforts, to our knowledge, the functionality of around 20 snps have been described, e.g., the ones regulating sort1 (musunuru et al. 2010) , rfx6 (huang et al. 2014 ) and tox3 (cowper-sal et al. 2012) . in these cases, a common feature is that the functional snp is located in a motif for a transcription factor where the alleles differ in their ability to bind the transcription factor (tf) and thus their capacity to regulate one or more genes. after our initial discovery that signals in dna enriched by chromatin immunoprecipitation (chip) may differ between alleles (ameur et al. 2009 ), the strategy has been applied genome wide using next-generation sequencing (chip-seq) data generated by us wallerman et al. 2009 ) and others (kasowski et al. 2010; rozowsky et al. 2011; reddy et al. 2012) . snps with allele-specific (as) tf binding are likely to be functional and in this project we systematically searched for them. we have characterized as-snps in four major encode cell lines and made functional validations. this has resulted in a collection of 9962 candidate functional snps. the genomes of k562 and sk-n-sh were sequenced with paired-end reads to 30× coverage. illumina sequenced reads were aligned to human reference genome hg19 and duplicates were marked with picard. the reads then went through gatk preprocessing including indel realignment (depristo et al. 2011) . variants were called following gatk best practices (https://www.broadinstitute.org/gatk/ guide/best-practices) using haplotypecaller, followed by gvcf file combination and recalibration of variants. raw chip-seq reads (.fastq) were obtained from the encode project database (ftp://hgdownload.cse.ucsc.edu/ goldenpath/hg19/encodedcc/) selecting all the tf chipseq data available at the time of download for four different cell lines: gm12878 (nov. 2013), h1-hesc (jan 2014), k562 (mar 2014) and sk-n-sh (mar 2014). as-snps collections were intersected and filtered with several publicly available databases: nhgri gwas catalog (jan 2014), collection of signal artifact blacklisted encode regions (the encode project consortium 2012), 1000 genomes snps collection (1000 genomes project, phase1_release_v3.20101123) and lymphoblastoid cell lines eqtls collections (lappalainen et al. 2013) . the reference genome (g1) used was the ucsc hg19 assembly based on the genome reference consortium human genome build 37 (grch37), but excluding random and unplaced contigs. the alternative genomes (g2), when not available, were built for the different cell lines using the fastaalternatereferencemaker gatk utility that generates an alternative reference sequence replacing the reference bases at variation sites with the bases supplied by a cell-specific snps collection. the genomes of k562 and sk-n-sh were sequenced at the broad institute as part of this project and the sources for cell-specific snps collections and alternative genomes were: 1. gm12878: snps from 1000 genomes pilot project official release and hiseq from broad and diploid sequence of na12878 genome from m.gerstein laboratory. 2. h1-hesc: snps calls were obtained from b.ren laboratory (january 2014) and g2 built via gatk. 3. k562: wgs and snp calls from broad institute (june 2014) and g2 built via gatk. 4. sk-n-sh: wgs and snp calls from broad institute (june 2014) and g2 built via gatk. a graphical overview of the as-snps selection process is presented in the flowchart in fig. s1 in supplementary material 1. the pipeline follows these steps: (1) each sets of chip-seq reads is aligned to the reference (g1) and alternative (g2) genome using asap (http://www.bioinformatics.babraham.ac.uk/projects/asap/). asap uses bowtie with the following flags: best-l 35-n 1, returning hits with a maximum number of one mismatch permitted in the seed (in this case 35 bp). (2) reads mapped specifically to g1 or g2 are counted at the heterozygous snps. snps with "0" reads mapped on g1 or g2 are discarded. (3) to determine whether the g1/g2 read counts difference is statistically significant, a binomial test is applied against the null hypothesis of an equal g1:g2 coverage. after correcting for multiple testing (benjamini & hochberg or fdr), as-snps with p < 0.05 are selected. (4) as-snps are then intersected with the whole 1000 genomes snps collection to retrieve afs. as-snps with af ≥1 % are considered common; as-snps with af <1 % are considered rare as well as as-snps not present in the 1000 genomes snps collection. (5) extensive filtering of the selected as-snps is performed to remove as-snps that are in centromeric or telomeric regions (ucsc gap in the cases where haplotype-resolved cnvs were available, the null hypothesis in the binomial tests to determine a statistically significant difference in reads count at different alleles (h 0 : p 1 = p 2 = 1/2) was adjusted to take into account the extra chromosomal copies, e.g., if a region was defined as a three-copy cnv and no haplotype info was available, the as-snps overlapping this region were discarded. if haplotype info was available for the same region (namely, which allele was present in a 2:1 ratio), the as-snps overlapping the region were tested against an adjusted null hypothesis (h 0 : p 1 = 1/3|p 2 = 2/3), so as to exclude the copy number bias from the significance of the allele-specific binding difference. regions with four or more copies were excluded from analysis. (6) pruned as-snps selections are finally intersected with collections of gwas or esnps and snps in ld (r 2 > 0.8) with gwas or esnps (proxy snps calculated via the snap tool (johnson et al. 2008 ) which uses genotype data from the international hapmap project) to select candidate functional as-snps for experimental validation. we compared the ratio between the allele with higher read number count over the total read count at hz snps [g1 if g1 > g2|g2 if g2 > g1/(g1 + g2)] to estimate the allele-specific effect of an snp. p values comparing as effects between common and rare as-snps were calculated using twotailed t test. peripheral blood mononuclear cells (pbmcs) were purified from healthy donor buffy coats (department of transfusion medicine, uppsala university hospital, sweden) using ficoll density-gradient centrifugation. the b cells were isolated from pbmcs by positive selection using cd19+ b-cell isolation kit (miltenyi biotec) according to manufacturer's instructions. the cells were cultured in 1 ml volumes in 24-well plates (nunc) in macrophage serumfree medium (life technologies) at the concentration of 3 × 10 6 b cells/ml. the cells were stimulated with a phosphorothioate-modified cpg a oligonucleotide odn2216 (cybergene) at the concentration of 3 μg/ml and incubated for 5 h at 37 °c with 5 % co 2 . the harvested cells were stored in rlt buffer (qiagen) at −80 °c. total rna was extracted from mock and stimulated b cells using rneasy ® mini kit (qiagen). first-strand cdna was synthesized from 1 ng of total rna with oligo (dt) primer using the maxima first strand cdna synthesis kit (thermo scientific). the reaction was performed at 50 °c for 30 min and terminated at 85 °c for 5 min. qpcr gene expression quantifications were performed in 96-well plates using the steponeplus™ real-time pcr system (applied biosystems). reactions were performed in a total 20 µl volume comprising 10 µl jumpstart™ taq readymix™ (sigma-aldrich) + evagreen ® dye 20× (biotium) and 0.25 µg of each primer. all qpcr reactions were performed in triplicate and the ct values were averaged. normalization was performed using the housekeeping gene β-actin to evaluate the relative expression. we set out to discover putative functional genetic variants by detecting snps with a difference in tf binding between alleles at a large scale. we sequenced the genomes of cell lines from myeloid cells (k562) and neuroblastoma (sk-n-sh) (see "materials and methods") and downloaded public data on the genome sequence of b cell (gm12878) and embryonic stem cell (h1-hesc). all public chipseq data from the encode project from these cell lines were downloaded, giving data from on average 66 tfs per cell line (range 27-100). we used the allele-specific alignment pipeline (asap) (http://www.bioinformatics. babraham.ac.uk/projects/asap/) to align the reads to the reference (g1) and alternative (g2) alleles, respectively. we required that at least one chip-seq read mapped to each allele to further support the snp calling. the number of reads mapping to the g1 and g2 allele was counted at all heterozygous positions, and those with a statistically significant difference in the number of reads were identified after correcting for multiple testing and copy number variation (cnv) (see "materials and methods"). data from the 1000 genomes project (the 1000 genomes project 2012) was used to determine if the variants were common with allele frequency (af) ≥1 % or rare <1 %. we used extensive filtering to remove potential false positives in repeated and encode "blacklisted" sequences. the number of reads mapping to the two alternative genomes is presented in table s1 in supplementary material 1. there were only small differences in the number of the aligned reads between the genomes, as in previous studies (rozowsky et al. 2011) , indicating that aligning bias toward the reference genome was well controlled for. in the four cell lines, we found evidence of allelespecific binding of transcription factors to 9962 snps (as-snps). the highest number of as-snps, 4299, was detected in k562 and the lowest number, 1014, in h1-hesc (table 1) , and as expected cells with the highest number of data sets of chip-seq of tfs had the highest number of as-snps. annotation in chromhmm was available for gm12878, k562 and h1-hesc and 17 % of the as-snps were located in active promoters, 17 % in insulators and the rest in distal regulatory elements. since we only consider heterozygous positions in this analysis, we compared the number of heterozygous snps and the number of as-snps that were shared between two or more cell lines. out of the 5,523,883 heterozygous snps, we found 3,296,857 that were unique to one of the cells, whereas 1,580,106, 551,933 and 94,987 were present in 2, 3 and 4 cell lines (table s2 in supplementary material 1). the majority of the as-snps, 9215, were detected in only one cell line, while 324, 29 and 3 were shared between 2, 3 and 4 cell lines, respectively. there was a highly significant difference in the distribution of heterozygous and as-snps in these cells (p < 2 × 10 −16 ), suggesting that most functional gene-regulatory elements are unique to a cell type and that only a small fraction is shared between two or more cell types. out of our as-snps, 1191 were also detected as dhs variants (maurano et al. 2015) . the results from both strategies depend on the alleles that are present in the studied cell types, so it is expected that the overlap will only be partial. most as-snps are common in the population, but notably 16 % (13-19 %) of all as-snps (1563 as-snps) had an af <1 %. out of all heterozygous snps in a cell, 14 % have af <1 % so an equal fraction shows allele-specific tf binding. to estimate the as effect, we calculated the ratio between the allele with higher read number count over the total read count observed at common or rare heterozygous snps (see "materials and methods"). we found a strikingly higher ratio with rare as-snps in all cells except h1-hesc ( fig. 1) , indicating that rare variants may have a larger effect on regulatory elements than common variants. with the exception of h1-hesc, there was no difference in the ratio for af 1-10 % or 1-5 % compared to common alleles (fig s5 in supplementary material 1). our data suggest that rare variants frequently affect the function of regulatory sequences and their effect may be larger than common alleles (lappalainen et al. 2013 ) and that they therefore may contribute to common diseases to a higher degree than rare variants in coding sequences. we wanted to determine how many tfs were needed to detect most as-snps in a cell and therefore investigated the fraction identified by the 20 tfs that showed the highest number of allele-specific binding sites in each cell line. we found that between 92 % and 99 % of all as-snps in each cell line were detected by the top 20 tfs, polymerases or coactivators, and even if chip-sequencing of 100 different dna-binding proteins had been performed, the results from the top 20 are enough to detect most as effects in a (84) 1563 (16) cell. in a project to search b cells, or the other cells studied here, from additional people for as-snps, these tfs can be selected to optimize the chances of finding candidate regulatory variants. we investigated the overlap of the top 20 tfs, polymerases or coactivators between the four different cell lines ( fig. 2a ; table s4 in supplementary material 1). three tfs (ctcf, rad21, yy1) and pol2 detected many as-snps in all cell lines, three lines shared 4 tfs (nrsf, max, usf1 and tbp) and two lines shared 11 tfs (cebpb, znf143, gabp, jund, rfx5, egr1, elf1, nfyb, creb1, tead4 and e2f6) and p300. the number of highly informative tfs that are unique to one cell line varied between five (h1-hesc) and nine (sk-n-sh). some of these unique tfs are previously known to be of central importance for the cell, i.e., pioneer tfs like nanog in h1-hesc and factors important for maturation of b cells such as bcl3, ebf1 and pu1 in gm12878. our data suggest that tfs shared by many cells, pioneer tfs and those important for cell development should be selected when designing a project to identify as-snps in a cell or tissue previously not studied by chip-seq. we then investigated the number of individuals that need to be analyzed to detect most common as-snps in a certain cell type. assuming hardy-weinberg equilibrium (hwe), 33 % (1/3) of all common polymorphisms are heterozygous in one person. there is a huge number of rare variants in the human population and only a small fraction of them are heterozygous in one person. we determined the percentage of common polymorphisms expected to be heterozygous in two to ten unrelated individuals assuming hwe. according to our prediction ( fig. 2b and table s5 ), 66 % (2/3) of all common polymorphisms are expected to be heterozygous in at least one of three unrelated people. for five people, the corresponding figure is 79 % and for ten people 90 %. the likelihood to find at least one heterozygous person depends on the af and is the highest at af = 0.5. at this af, you have 88 % chance of discovering a heterozygote if you study three people, 97 % if you study five and over 99 % in ten people. however, even at af = 0.1, the chance to find one or more heterozygotes is 45 % among three people, 63 % among five and 86 % in ten people. for rare variants (af <1 %), the chances decrease substantially. it is thus possible to search for as-snps among most common polymorphisms in the population by investigating cell lines or tissues from three to ten people, ideally from diverse ethnic backgrounds. the gwas catalog has entries for a wide spectrum of traits that are expressed in different cell types, all of which were not studied in this experiment. molecular events that could be explained by the cells investigated are for example immune-mediated diseases for gm12878 and k562, and neurological diseases for sk-n-sh (table s3 in supplementary material 1). consequently, we searched the catalog for these cell-specific traits. h1-hesc was not investigated. the snp with the strongest association (gwas top hit) was collected from the catalog and snps in high ld (r 2 > 0.8) were identified and intersected with the collection of as-snps. for 36 traits, we found 141 as-snps. we investigated the number of candidate regulatory snps found when searching the gwas catalog using matched random sets of non-as-snps. we found significantly more as-snps than expected by chance with a clear enrichment (5-to 13-fold) in the overlap of as-snps as compared to random sets of non-as-snps (fig s3 in supplementary material 1) . out of the 141 unique as-snps that were candidates to explain gwas signals, only 15 were the particular snp reported in the gwas catalog and the other 126 were in the high-ld interval and 10 of these were rare as-snps (table 2 ; fig. 3a , b and tables s8, s11, s13 in supplementary material 3). we grouped the snps in 1 mb loci, and in immune cells, gm12878 and k562, we found candidate functional snps at 71 loci and in sk-n-sh at 14 loci, giving a total of functional candidates at 85 loci. we compared our candidates with the total number of mapped regions for different diseases and detected tentative functional variants for 11 % of loci mapped for psoriasis, 9 % for sle and self-reported allergy, 8 % for inflammatory fig. 1 allelic effects of rare variants. allele-specific effect (see "materials and methods") at heterozygous snps with a significant difference between the chip-seq reads of alleles g1 and g2 in four different cell lines bowel disease/crohn's disease/ulcerative colitis and 6 % for type 1 diabetes, suggesting new functional regulatory elements for many common diseases. furthermore, we took the top hits from the whole gwas catalog including snps in high ld (r 2 > 0.8) and compared to the list of as-snps. this resulted in 398 (tables s9, s12 , s14 and s15 in supplementary material 3). the relevance of these as-snps is not ascertained, since they were not necessarily detected in the appropriate cell to explain the phenotype, but it is likely that several of them may be functional due to pleiotropic effects. we have found novel c four as-snps, located in three different regulatory elements, interact with several gwas-snps associated with autoimmune diseases putative functional variants that may explain gwas-snps using lymphoblastoid cell lines. some examples are as-snps detected in gm12878 and k562 on a haplotype at the ikzf3/ormdl3 locus, which is associated with both a range of autoimmune diseases described below and cervical cancer. infection with papilloma virus is a strong risk factor for cervical cancer and variation in the immune response to this agent may contribute to disease risk. this reflects that cellular mechanisms may be shared between cells types, and a gene-regulatory element detected in one cell type could be relevant to other types. we have also found novel putative functional variants that may explain eqtl snps (esnps). genetic control of gene activity has been investigated in lymphoblastoid cell lines and esnps have been detected (lappalainen et al. 2013) . we examined whether as-snps in b cells (gm12878) are better candidates to drive the allelic difference in expression. we took snps in ld (r 2 > 0.8) with 16,078 esnps for b cells and investigated how many are as-snps in gm12878. we found 84 esnps that are as-snps, and an additional 362 as-snps that are in ld with an esnp (tables s10 in supplementary material 3). some of the esnps in gm12878 are associated with the expression of repeats and therefore difficult to interpret from a disease perspective. we consider the as-snps as good candidates to drive the allele-specific expression variation and note that only a fraction of esnps shows tentative functional effect, but that many more snps in high ld do. there is a clear enrichment (5-to 13-fold) in the overlap of as-snps and cell-specific gwas and eqtls compared to random sets of non-as-snps (fig s3 in supplementary material 1) . a haplotype at the ikzf3/ormdl3 locus is associated with primary biliary cirrhosis, systemic lupus erythematosus, systemic sclerosis, type 1 diabetes autoantibodies, type 1 diabetes, ulcerative colitis, crohn's disease, inflammatory bowel disease and rheumatoid arthritis (hindorff et al. 2011) (fig. 3c and fig. s2 in supplementary material 1). seven snps detect the associations, but none are as-snps. however, they are in ld with four as-snps: as-snps rs4065275 and rs8076131 separated by 47 bp at one regulatory element, as-snp rs2305479 residing 18 kb away and to as-snp rs12936231 (table s8 in supplementary material 1) located a further 33 kb away at another element (verlaan et al. 2009 ). our data suggest that the as-snps, all detected in the b cell gm12878, are good candidates to drive the association and that they may influence the expression of ikzf3 which is important for b lymphocyte proliferation and differentiation or ormdl3 involved in sphingolipid synthesis. the pattern that several gwas-snps are associated with the same as-snp and that several as-snps are detected at one locus is found in many other instances (fig. 3b) . this suggests that snps at distinct regulatory elements could regulate the activity of the same gene, which is supported by recent data (corradin et al. 2014) . to test if as-snps are enriched in super-enhancers we obtained a collection of ~60,000 superenhancers (dbsuper, http://bioinfo.au.tsinghua.edu.cn/ dbsuper/index.php) defined per cell line and intersected them with our collections of as-snps or random sets of selected non-as-snps (fig. s4 in supplementary material 1). we found a higher fraction of super-enhancers to contain as-snps than what would be expected by chance (p = 2 × 10 −5 ). recently, rs909685 was associated with rheumatoid (okada et al. 2014 ) and primary biliary cirrhosis (liu et al. 2012) . the esnp rs2069235 is in high ld with the gwas/ as-snp rs909685 and associated with the expression of syngr1, coding for a membrane protein associated with presynaptic vesicles in neuronal cells and also expressed in b-and other immune cells. to validate the results from gm12878, we purified b cells from blood donors who had been genotyped using a 200 k immunochip. the cells were either treated with medium only (mock) or stimulated with the oligonucleotide odn2216 (see "materials and methods"), which activated the tf ebf1 (fig. 4a ). chipseq has shown that ebf1 binds to the regulatory element that harbors the gwas/as-snp rs909685. b cells from donors who are homozygous, aa or tt for the alleles, respectively, at this locus showed significant difference in activity of syngr1 (fig. 4b, lower panel) . stimulation with odn2216, which increases the levels of ebf1, significantly decreased the expression of syngr1 for both genotypes (fig. 4b, lower panel) . rs909685 disrupts the motif for pitx3 which is known to interact with nurr1, a factor involved in the regulation of cortisol which suggests a link to inflammatory processes (mages et al. 1994 ). this indicates that syngr1 is controlled by the regulatory element with the as-snp rs909685 giving an allelic difference in expression at basal conditions and that ebf1 acts as a repressor to decrease the expression from both alleles. furthermore, it suggests that differential expression of syngr1 could mediate the genetic predisposition to rheumatoid arthritis and primary biliary cirrhosis at this locus. a gwas has shown that rs7993214 is associated with psoriasis (liu et al. 2008 ) and rs9603616 to rheumatoid 1 3 arthritis (okada et al. 2014 ). the gwas-snps rs7993214 and rs9603616 are in high ld with each other and with the as-snp rs9603612 which is located at a regulatory element bound by ebf1 in gm12878. purified b cells from blood donors, homozygous, cc or gg, for the alleles at rs9603612 were stimulated with odn2216 and we determined the expression of the psoriasis candidate gene cog6. a donor homozygous for the g allele had significantly higher expression than the one homozygous for c. in the cells stimulated with odn2216, the expression of top rs909685 is a gwas-snp with as behavior detected by ebf1 chip-seq reads. rs909685 is in ld with the eqtl snp rs2069235 which is associated with the expression of the syngr1 gene. bottom syngr1 expression in an individual homozygous for the a-or t-allele, respectively, at rs909685, unstimulated (blue) or stimulated with odn2216 (yellow). middle motif for pitx3 at rs909685 which alters the tf-binding motif. c top as-snp rs9603612 is located in an intron of cog6 and is in ld with the gwas-snp rs7993214. rs9603612 showed as behavior with ebf1 chip-seq reads covering the snp with significant difference. bottom expression of cog6 in an individual homozygous for the c-and g-alleles, respectively, of rs9603612, unstimulated (blue) or stimulated with odn2216 (yellow). middle motif for creb1 at rs9603612 which alters the tf-binding motif cog6 increased significantly for both genotypes (fig. 4c, lower panel) . this indicates that cog6 is differentially expressed depending on the allele at rs9603612 and that ebf1 regulates the activity of both alleles. homozygous mutations in cog6 cause shaheen syndrome with hyperkeratosis of the palms and soles, hypohidrosis, intellectual disability and dental enamel hypoplasia. patients with psoriasis also have hyperkeratosis resulting in scaly patches over the body, in the palms of the hands and soles of the feet. the as-snp rs9603612 is located in a motif for creb1, which is activated in psoriatic lesions (funding et al. 2007) . the data thus suggest that the risk of developing psoriasis is mediated by the regulatory element with the functional snp rs9603612 leading to differential activity of cog6. most variants associated with common diseases are in non-coding dna and enriched in regulatory dna (maurano et al. 2012) . many as variants have been detected using reads from dhs analysis (maurano et al. 2015) and were predicted to be caused by as tf binding. as a complementary approach in this study, we present candidate functional snp variants detected using the biased tf allele binding in chip-seq data. chip-seq reads generated for tfs in four different cell lines from the encode project were analyzed to identify cell-specific collections of as-snps. this makes a significant addition to the collection of snps with as chromatin signals. one incentive for the study was to define candidate variant drivers of gwas signals and we identified 141 as-snps meeting the criteria. based on the detailed validations of two snps associated with rheumatoid arthritis/primary biliary cirrhosis and psoriasis, we believe that many others are worth further study. these efforts will make it possible, not only to validate the regulated gene, but also to connect the disease process to upstream regulatory pathways. this will provide a more holistic approach to the understanding of disease processes and help to remove the current bottleneck in the process of translating gwas signals to functional disease mechanisms. most of the detected as-snps are located in distal regulatory elements and are candidates to regulate a nearby gene, while 17 % are located in promoters. recent studies have indicated the presence of chromatin domains with associated signals between snps (waszak et al. 2015; grubert et al. 2015) , but across the genome, waszak et al. (2015) detected 14,559 domains, of which only 25 % were larger than 70 kb. correlation of allelic bias in adjacent snps was also found (maurano et al. 2015) with the same conclusion that most correlations are present in regions with sizes of regulatory elements and only a small fraction extending over 10 kb. this indicates that at a (small) fraction of regions, the as-snps we detect may be correlated to other functional variants which are the drivers of chromatin signals. in b cells, we found 446 as-snps that are eqtl snps or are in high ld with such snps and we regard them as candidates to drive the eqtl. this does not exclude the possibility that many of the other as-snps may be associated with gene expression variation. a recent study in mice (crowley et al. 2015) suggests that more than 80 % of mouse genes have cis-regulatory variation. studies in man have been performed on small sample sets and therefore underpowered to detect the weak effects, so more associations remain to be found as studies increase, e.g., in the gtex consortium. recent data have shown a high number of rare variants in people and the idea is that some of them are functional, and that evolution has not had time to remove them from the population (keinan and clark 2012) . exome sequencing has been performed in large disease cohorts and controls and only a low number of rare coding variants have been associated with disease, indicating that they do not have a large impact on disease risk in the population (fu et al. 2013) . we found a high number of candidate functional as-snps that are rare in the population, which can be compared to 53-80 predicted candidate functional variants per person, most of them rare, in the coding sequence (li et al. 2015; fu et al. 2013) . we have just studied four cell types from one person each, so if all different cells in the human organism would be analyzed, the number of rare candidate-regulatory variants would increase and even more outnumber the rare candidate functional coding variants. we observed a significantly higher difference in g1/ g2 read counts at rare as-snps as compared to common ones, which suggests that rare as-snps may have a large functional effect. this is consistent with findings from eqtls in b cells (lappalainen et al. 2013) , showing that low-frequency alleles have a large effect on expression. it is therefore possible that rare variants in regulatory regions frequently contribute to common disease risk. this possibility has been difficult to study since the correct functional regulatory element needs to be investigated; however, the data we now present points to a collection of candidate regulatory sequences. if rare variants act on regulatory elements in the frequencies we detect, it would add heterogeneity and noise to association studies. rare variants are often specific to an ethnic group, and in one population a set of rare variants may be associated with one common variant on a haplotype, whereas in another population there may be one or more rare variants associated with another common snp. consequently, different gwas and eqtl studies may find the strongest signals to different common snps on the same haplotype that has one or more common functional variant(s). this is consistent with the fact that gwas studies often find the strongest association to alternative snps and with our finding that several gwas-snps at a locus often show association to one or a few as-snps (fig. 3) . the missing heritability has been much debated over the years. rare variants are generally not found in gwas studies and often even filtered out in quality control steps. if rare variants contribute to common diseases in the numbers that we detect them, they may change the proportion of the explained heritability. not only are they frequent, but their allele-specific effect may be larger than for common ones; so combined, this may explain a part of the missing heritability. we are approaching an era when gwas studies will be based on whole genome sequencing, thus making it possible to evaluate the contribution of rare regulatory variants to common disease. in the same way, the rare variants may also obscure associations of gene expression and may be the reason why we detect many more as-snps in ld with an esnp, than as-snps that are esnps. in an attempt to minimize the proportion of false positives during the selection of as-snps, we applied strong filtering and cutoffs to eliminate candidate snps in genomic regions with high repetitive content such as centromeres, telomeres and cnvs. in fact, large regions were inaccessible for analysis due to cnvs and large chromosomal abnormalities, limiting the number of as-snps that could be detected. in future projects to find candidate regulatory snps, care should be exercised to use cell lines without gross structural rearrangements. the number of detected as-snps depends on the number of reads at each allele and in this data set it was limited by the type of data generated in encode. we propose that a project optimized to search for as-snps should improve power by using long paired-end reads to sequence the whole chipenriched dna fragment and by generating a high number of reads. our results indicate that it is possible to predict which tfs will detect most as-snps in a cell or tissue, which has not been analyzed before. starting with the tfs that are expressed in the cell or tissue, one can select those that are shared between two or more of the cells in this study and add pioneer factors and others with verified central importance to the cell. as described, chip-seq of 20 selected tfs from the cells studied here or 20-30 tfs for new cells or tissues is a powerful way to find a large collection of common as-snps. by repeating the experiment in samples from three to ten people, the most common polymorphisms in the human population can be interrogated for allele-specific binding. this strategy can be used to find more candidate functional variants. rare variants are, as always, harder to study. they will be detected in whole genome sequencing, but their functional effect may need to be studied by large-scale functional tests (kheradpour et al. 2013) or by computer modeling. drivers of gwas signals in non-coding regions are located in gene-regulatory elements and result in difference in tf binding between alleles. we have screened for such events in four cell types and found a large number of candidate functional variants. those in ld r 2 > 0.8 in relation to reported snps for traits and expression were identified resulting in hundreds of potential genetic drivers. experimental validation supports snps in b cells that explain psoriasis, rheumatoid arthritis and primary biliary cirrhosis. as many as 16 % of functional candidates are rare with af <1 %. if we assume that they contribute to common disease to the same degree as common variants, they may explain why gwas signals differ between populations and may contribute to the missing heritability. identification of candidate regulatory snps by combination of transcription-factor-binding site prediction, snp genotyping and haplochip combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits breast cancer risk-associated snps modulate the affinity of chromatin for foxa1 and alter gene expression analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance a framework for variation discovery and genotyping using next-generation dna sequencing data analysis of 6,515 exomes reveals the recent origin of most human protein-coding variants mitogen-and stress-activated protein kinase 2 and cyclic amp response element binding protein are activated in lesional psoriatic epidermis genetic control of chromatin states in humans involves local and distal chromosomal interactions a catalog of published genome-wide association studies. www.genome.gov/gwastudies a prostate cancer susceptibility allele at 6q22 increases rfx6 expression by modulating hoxb13 chromatin binding snap: a web-based tool for identification and annotation of proxy snps using hapmap variation in transcription factor binding among humans recent explosive human population growth has resulted in an excess of rare genetic variants systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay transcriptome and genome sequencing uncovers functional variation in humans analysis of loss-of-function variants and 20 risk factor phenotypes in 8,554 individuals identifies loci influencing chronic disease a genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci dense fine-mapping study identifies new susceptibility loci for primary biliary cirrhosis not, a human immediate-early response gene closely related to the steroid/thyroid hormone receptor nak1/tr3 systematic localization of common diseaseassociated variation in regulatory dna large-scale identification of sequence variants influencing human transcription factor occupancy in vivo patchwork: allele-specific copy number analysis of whole-genome sequenced tumor tissue differential binding and co-binding pattern of foxa1 and foxa3 and their relation to h3k4me3 in hepg2 cells revealed by chip-seq from noncoding variant to phenotype via sort1 at the 1p13 cholesterol locus high-resolution dna analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity genetics of rheumatoid arthritis contributes to biology and drug discovery effects of sequence variation on differential allelic transcription factor occupancy and gene expression alleleseq: analysis of allele-specific expression and binding in a network framework :e107 the 1000 genomes project (2012) an integrated map of genetic variation from 1,092 human genomes an integrated encyclopedia of dna elements in the human genome allele-specific chromatin remodeling in the zpbp2/gsdmb/ormdl3 locus associated with the risk of asthma and autoimmune disease molecular interactions between hnf4a, foxa2 and gabp identified at regulatory dna elements through chip-sequencing population variation and genetic control of modular chromatin architecture in humans the authors thank the encode project for generating chip-seq data and bing ren for providing the sequence of h1-hesc. the computations were performed on resources provided by snic through uppsala multidisciplinary center for advanced computational science (uppmax) under project b2010003 and b2011107. sequencing was performed with support of the science for life genomics platform (ngi uppsala). this work was supported by the swedish research council ( conflict of interest the authors declare that there is no conflict of interest associated with this manuscript.open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord-0002445-x4na1o4p authors: fan, zhi-dan; zhang, ya-yuan; guo, yi-hong; huang, na; ma, hui-hui; huang, hui; yu, hai-guo title: involvement of p2x7 receptor signaling on regulating the differentiation of th17 cells and type ii collagen-induced arthritis in mice date: 2016-10-24 journal: sci rep doi: 10.1038/srep35804 sha: 0fc01f8a6bb755f52b6407df7cb8c9accd15450e doc_id: 2445 cord_uid: x4na1o4p interleukin (il)-17 producing t helper (th17) cells are major effector cells in the pathogenesis of rheumatoid arthritis (ra). the p2x7 receptor (p2x7r) has emerged as a potential site in the regulation of inflammation in ra but little is known of its functional role on the differentiation of th17 cells. this study investigates the in vitro and in vivo effects of p2x7r on th17 cell differentiation during type ii collagen (cii) induced experimental arthritis model. in cii-treated dendritic cells (dcs) and dc/cd4(+) t coculture system, pretreatment with pharmacological antagonists of p2x7r (suramin and a-438079) caused strong inhibition of production of th17-promoting cytokines (il-1β, tgf-β1, il-23p19 and il-6). exposure to cii induced the elevation of mrnas encoding retinoic acid receptor-related orphan receptor α and γt, which were abolished by pretreatment with p2x7r antagonists. furthermore, blocking p2x7r signaling abolished the cii-mediated increase in il-17a. blockade of p2x7r remarkably inhibited hind paw swelling and ameliorated pathological changes in ankle joint of the collagen-induced arthritis mice. thus, we demonstrated a novel function for p2x7r signaling in regulating cii-induced differentiation of th17 cells. p2x7r signaling facilitates the development of the sophisticated network of dc-derived cytokines that favors a th17 phenotype. our preliminary study. collectively, th17 cells are involved in multiple pathological processes of ra, and regulation on th17 cell differentiation may be a good therapeutic strategy in the treatment of ra. it is now widely accepted that adenosine triphosphate (atp) is an important extracellular signaling molecule, which has been implicated in a wide variety of biological processes via p2 receptors 19, 20 . the p2x7 receptor (p2x7r) is an atp-gated ion channel belonging to p2 receptors, and has emerged as a potential site in the regulation of inflammation in ra 21 . particularly, the p2x7r allows cation passage through the cell to activate the inflammasomes and production of il-1β , which is obligatory for the th17 development 22, 23 . these findings provide the functional basis supporting the hypothesis that p2x7r may regulate the in vitro and in vivo effects of type ii collagen (cii) on the differentiation of th17 cells. to clarify the role of p2x7r in regulating cii-induced differentiation of th17 cells, we assessed the contribution of p2x7r signaling on cytokine production during initial t cell priming in cii-treated dendritic cells (dcs). in addition, the effect of p2x7r signaling on the th17 cell differentiation was investigated on the cd4 + t cells in coculture with dcs. the animal study was also performed to assess the effectiveness of blocking p2x7r signaling to suppress th17 cell differentiation. human subjects. a total of 19 patients with juvenile ra (8 males, 11 females; mean age 8.71 ± 3.26 years), and 22 healthy age-and sex-matched volunteers served as controls (10 males, 12 females; mean age 8.92 ± 3.45 years) were enrolled into the study at the nanjing children's hospital affiliated to nanjing medical university. all patients and volunteers were han chinese. all patients were clinically assessed according to the detailed diagnostic information obtained from the medical records and physical examinations. the study was approved by the ethics committee of nanjing medical university (nanjing, china) and all methods were performed in accordance with the relevant guidelines and regulations. written informed consent on the use of clinical specimens for medical research was obtained from the parents or legal guardians of the participants. the peripheral blood mononuclear cells (pbmcs) from all study participants were isolated and cultured for the measurement of the p2x7r expression. experimental animals. male dba/1j mice were purchased from the shanghai slac laboratory animal co., ltd. (shanghai, china). all mice were kept under a light-, temperature-and humidity-controlled environment with a constant 12-h light/dark cycle, and access to food and water ad libitum. all animal procedures and study protocols were approved by the experimental animal care and use committee of nanjing medical university and complied with the guide for the care and use of laboratory animals (nih publication no. 85-23, revised 1996). bone marrow-derived dcs were prepared as described previously 24 . briefly, bone marrow precursor cells were obtained from femur and tibia of mice and cultured in fresh dc culture medium consisting of rpmi 1640 medium plus granulocyte-macrophage colony-stimulating factor (10 ng/ml; immunex, seattle, wa, usa) and il-4 (1 ng/ml; peprotech, rocky hill, nj, usa). dcs were induced to mature by lipopolysaccharide (100 ng/ml; sigma-aldrich, st louis, mo, usa). measurement of extracellular atp. the concentration of extracellular atp was detected using enliten ® rluciferase/luciferin reagent (promega, madison, wi, usa). dcs (2.5 × 10 5 cells/well) were incubated in rpmi 1640 medium supplemented with 1% (v/v) fetal bovine serum for 12 h in a 12-well culture plate. an aliquot of 40 μ l conditioned medium was collected as a control sample for baseline atp release. the cells were exposed to bovine cii (chondrex, redmond, wa, usa) at room temperature, and then conditioned medium was collected at the indicated time points. each sample was centrifuged at 600 g for 5 min at 4 °c and 10 μ l of the supernatant was used for atp determination. the concentration of atp was determined by measuring the chemiluminescense with a spectramax luminometer (molecular devices, downingtown, pa, usa) according to manufacturer's instructions. quantitative real-time pcr. rna extraction and quantitative real-time pcr were performed for gene expression analysis as previously reported 25 . briefly, total rna was extracted from cell samples with trizol reagent (invitrogen, carlsbad, ca, usa) in accordance with the manufacturer's protocols. the concentration of rna samples was determined in duplicate by measuring the optical density (od) at 260 nm. purity of rna was ensured by an od260/od280 ratio in the range of 1.8-2.1. rna was reversely transcribed to synthesize cdna using the cdna synthesis kit (fermentas, st. leon-rot, germany). one microliter of cdna was then amplified in a 20-μ l reaction mixture. the reaction was performed according to a modified protocol on the 7500 fast real-time pcr system (applied biosystems, foster city, ca, usa). the β -actin was adopted as an endogenous reference, and the 2 −δδct method was used to calculate the relative expression level of interested genes in each group. primer sequences were shown in supplementary table 1 . proteins were extracted and subjected to western blot analysis as previously described [26] [27] [28] . equal amounts of proteins (30 μ g) were separated by a 10% sds-page and transferred to nitrocellulose membrane (pall, pensacola, fl, usa) by semi-dry blotting. membranes were probed with the primary antibody against p2x7r (santa cruz biotechnology, ca, usa) and visualized using an enhanced chemiluminescence procedure (pierce, rockford, illinois, usa) with biomax films (kodak). the od of each interested band was normalized to that of β -actin band by the image-pro ® plus 4.5 software. purification of cd4 + t cells. single-cell suspensions from fresh spleen were harvested and prepared. magnetic bead separation was used to negatively isolate cd4 + t cells. briefly, single-cell suspensions were depleted by biotin-conjugated specific monoclonal antibodies against cd8 + , b220 + , cd16 + , gr-1 + and ly76 + cells (bd biosciences pharmingen, san diego, ca, usa), anti-biotin magnetic beads, and an ld magnetic bead column (miltenyi biotec, auburn, ca, usa) 24 . the purity of cd4 + t cells was consistently greater than 95% as assessed by flow cytometry. co-culturing cd4 + t cell with dcs. dcs (2 × 10 4 cells per well) incubated with and without two p2x7r antagonists (suramin and a-438079) were cultured with cd4 + t cells (1 × 10 5 cells per well) in the presence of cii (20 μ g/ml) for 72 h. the supernatant was collected for cytokine measurement. induction of cia. cia was induced in 6-to 8-week-old male dba/1j mice as previously described 29, 30 . briefly, the mice were immunized intradermally at the tail base with bovine cii (100 μ l of 1 mg/ml) emulsified in complete freund's adjuvant containing killed mycobacterium tuberculosis (difco, usa) (day 0). on day 21, animals were boosted with 200 μ g of cii emulsified with incomplete freund's adjuvant at the base of the tail. from the primary immunization day, mice were treated with p2x7r antagonists (suramin [30 mg/kg] and a-438079 [5 mg/kg]) or saline i.p every week for five weeks. quantification of cytokines. cell-free culture supernatant from coculture system was collected and the concentrations of il-1β , transforming growth factor (tgf)-β 1, il-23p19, il-12p40, il-12p35 and il-6 were measured by using specific elisas according to the protocol recommended by the manufacturer. a standard curve was generated for each plate over the linear range and used to calculate the absolute concentrations of the indicated cytokines. tissue preparation and histological examination. at the peak of cia (about 35 days after first immunization), mice were sacrificed and their joints were processed for histopathological analysis as previously described 32 . the joints were histologically analyzed and scored, including (i) synovial hyperplasia, (ii) bone or cartilage destruction, (iii) inflammatory cells infiltrate and (iv) vascular proliferation 33 . the pathological changes were graded from 0 to 3 based on the severity of each index as follows: grade 0, no detectable changes; grade 1, mild changes; grade 2, moderate changes; grade 3, severe changes. the total histological score was the sum of the four items and ranged from 0 to 12. statistical analysis. the spss 18.0 for windows was employed in the data analysis. excel (version 2007, microsoft) was also adopted for preliminary data analysis. all data were expressed as mean ± se of individual values. comparisons between two observations in the same experimental protocol were assessed by student paired t test. anova followed by post hoc bonferroni test was applied when multiple comparisons between different groups were made. a value of p < 0.05 was considered statistically significant. increased expression of p2x7r in pbmcs from patients with ra. we tested the expression of p2x7r on pbmcs in 19 patients with ra and 22 age-matched normal individuals using quantitative real-time pcr. as demonstrated in fig. 1 , p2x7r had higher expression levels in the ra group than in the control group, indicating that the p2x7r expression specifically unregulated in ra patients. to investigate whether cii can stimulate the atp release from dcs, the extracellular atp levels of cells exposed to cii were investigated. in response to 5-minute treatment of cii, the extracellular atp concentrations were increased in a dose-dependent manner as compared with unstimulated controls ( fig. 2a) . extracellular atp levels were also measured in dcs cultured with 20 μ g/ml cii for 6, 12 or 24 h. in control wells, atp levels remained constant, whereas continuous treatment increased atp levels up to 6 folds (fig. 2b) . furthermore, cii treatment groups exhibited significantly higher p2x7r levels than the control group (fig. 2c ). in dc cultures treated with apyrase (an atp diphosphohydrolase, 0.5 u/ml), cii failed to elevate the level of p2x7r, ruling out the possible involvement of endogenous atp (fig. 2d ). pretreatment with p2x7r antagonists (suramin and a-438079) abolished the promoting effect of cii on atp release from dcs (fig. 2e ). in the early stages of culture, cell viability was unaffected by cii treatment (fig. 2f) . so, the results reveal that cii-induced atp release from dcs require the presence of p2x7r function. involvement of p2x7r in cii-induced atp release from dc/cd4 + t cell coculture system. we preliminarily studied the effects of three doses (10, 30 and 100 μ m) of suramin and three doses (100, 200 and 300 nm) of a-438079 on the extracellular atp levels in coculture system. suramin completely blocked the cii-stimulated atp secretion at the doses of 30 and 100 μ m (fig. 3a) . we chose a lower dose of 30 μ m of suramin in this study to avoid potential toxicity. only a-438079 at 300 nm could completely block the cii-stimulated p2x7r activation (fig. 3b) . thus, 30 μ m suramin and 300 nm a-438079 were administered in the following experiments to ensure a better competitiveness. these results show that p2x7r is involved in the cii-induced atp release from dc/cd4 + t cell coculture system. to assess the role of cii and p2x7r signaling on cytokine production during th17 cell differentiation, the marrow-derived dcs were cultured in the presence of cii with or without p2x7r antagonists. in response to cii treatment, we found substantial upregulation of the mrnas encoding il-1β , tgf-β 1, il-23p19, il-12p40, il-12p35 and il-6 by approximately 2.5-6-fold above the unstimulated controls (fig. 4a ). we then blocked p2x7r signaling in dcs using p2x7r antagonists (suramin and a-438079). pretreatment with p2x7r antagonists caused strong inhibition of production of th17-promoting cytokines (il-1β , tgf-β 1, il-23p19 and il-6), but not of th1-promoting cytokines (il-12p40 and il-12p35) in cii-treated dcs, confirming the requirement of p2x7r for cii-induced development of a th17-promoting rather than a th1-promoting microenvironment. in addition, we determined the ability of cii to induce cytokine production during th17 cell differentiation in a dc/cd4 + t cell coculture system. cytokine profile in the cii-stimulated coculture supernatant displayed a marked alteration compared with that of controls. we found strong production of the il-1β , tgf-β 1, il-23p19, il-12p40, il-12p35 and il-6, completely consistent with the changing trend of their mrnas (fig. 4b) . preconditioning with p2x7r antagonists only eliminated the release-promoting effects of cii on the above th17-promoting cytokines (il-1β , tgf-β 1, il-23p19 and il-6), but had no effect on the production of il-12p40 and il-12p35. thus, these data provide further evidence supporting the requirement of p2x7r for the induction of a th17-promoting microenvironment by cii. th17 cell differentiation induced by cii required p2x7r signaling. as the addition of cii could stimulate the production of th17-promoting cytokines, we then evaluated the influence of cii on the induction of transcription factors essential for th17 cell differentiation such as retinoic acid receptor-related orphan receptors α and γ (rorα and rorγ t). exposure to cii induced the elevation of mrnas encoding rorγ t and rorα whereas pretreatment with p2x7r antagonists abolished these increases (fig. 5a) . further, cii treatment resulted in a substantial up-regulation of mrna (fig. 5a ) and protein expression (fig. 5b ) of il-17a, which was abrogated by antagonists of p2x7r. moreover, in dc/cd4 + t coculture system, we found that cd4 + il-17a + cells were significantly increased in response to cii, whereas prior exposure to p2x7r antagonists had no effect on the cd4 + il-17a + cell populations in cii-stimulated subjects as compared with unstimulated controls (fig. 5c) . similarly, the mrna (fig. 6a ) and protein expression (fig. 6b ) of il-17a were increased remarkably in splenocytes derived from cii-treated mice. consistently, the expression of th17-transcriptional factors rorα and rorγ t were increased in cii-treated mice (fig. 6a) . furthermore, cii treatment resulted in a significantly higher percentage of cd4 + il-17a + cells (fig. 6c) . these data signified that cii promoted the th17 cell differentiation in mice. however, pretreatment with p2x7r antagonists could abolish cii-induced th17 cell differentiation in mice. to investigate the possibility that the il-17 elevation is due to the direct effect of atp on t cells, we induced th17 polarization of cd4+ t cell in vitro and measured the mrna and protein levels of il-17a in response to 3 mm atp. besides, we measured the expression of p2x7r on t cells cultured with 20 μ g/ml cii for 6, 12 or 24 h by quantitative real-time pcr. it was found that cii caused no change of p2x7r mrna in t cells as compared with unstimulated controls (fig. 7a) . the extracellular atp failed to induce il-17a expression both at the transcriptional and translational levels (fig. 7b,c) , thus excluding the possibility that the il-17 elevation is due to the direct effect of atp on t cells. combined with these findings, we believe that il-17a elevation after cii treatment might be a consequence of the enhanced expression of th17 related cytokines released by dcs. blocking the p2x7r signaling attenuated cii-mediated joint damage of collagen-induced arthritis (cia) mice. compared with the control subjects, there was a higher level of il-17a in synovial tissues in cii-treated mice (fig. 8a ). as shown in fig. 8b , significant increases in paw oedema were recorded in cia mice rather than controls. histological score calculated from microphotographs was depicted in fig. 8c . in addition, histological images suggested that cii leaded to severe synovial hyperplasia, bone or cartilage destruction, vascular proliferation and inflammatory cells infiltration (fig. 8d) . treatment with p2x7r antagonists was effective in suppressing the severity of arthritis and immune responses to cii. these results suggested that blocking the p2x7r signaling attenuated joint damage of cia mice in vivo. the primary novel findings of the present study were that p2x7r was involved in cii-induced atp release from dcs, and required for cii-induced development of a th17-promoting microenvironment. furthermore, we found that cii regulated the th17 cell differentiation by mediating p2x7r signaling both in vivo and in vitro. blocking the p2x7r signaling could attenuate cii-mediated joint damage of cia mice. to our knowledge, this is the first study supporting the requirement of p2x7r for the regulation of th17 cell differentiation by cii. as an important extracellular signaling molecule, atp is a potent danger-associated molecular pattern molecule that may contribute to tissue damage and inflammation 19, 20, 34 . extracellular atp signaling acting through the p2x7r played an important regulatory/modulatory role in the pathogenesis of ra 35 . in ra patients, a high concentration of extracellular atp is present in the synovial fluid 36 . expression of p2x7r was higher in monocytes from patients with ra compared with controls 37 . similarly, we also observed significantly increased p2x7r expression in the ra patients as compared to their normal counterparts. in addition, p2x7r deficiency leads to a lower incidence and lower severity of joint inflammation in animal models of arthritis 38 . recently, it has become evident that p2x7r is involved in atp release in many types of cells (astrocytes, osteoclasts, osteoblasts, etc.) 39, 40 , and the release of atp via the p2x7r is supposed to be an important source of extracellular adenosine 41 . in osteoclasts under normal conditions, efflux via the p2x7r is the primary mechanism for atp releases 40 . although exactly how up regulation of the p2x7r mediates atp release from cells is unclear, a likely mechanism for atp release is directly through the formation of membrane pores caused by transient activation of the p2x7r. nowadays, significant roles for p2x7r signaling in the therapies of ra are emerging. cia, induced by immunization with an emulsion of complete freund's adjuvant and cii, is the most commonly studied autoimmune model of ra to identify potential pathogenic mechanisms of autoimmunity 42 . cia resembles many pathological features of ra, including synovial hyperplasia, mononuclear cell infiltration and cartilage degradation. of the antigen-defined models that are based on cartilage proteins, cia has the shortest duration between immunization and disease manifestation 42 . cii is a major protein in cartilage, the target tissue of ra 42 . the presence of t-cell and b-cell immunity to cii has been well established in the immunopathogenesis of ra [43] [44] [45] . here, we firstly investigated the role of cii on the atp release from dcs. cii dose-dependently increased the concentrations of extracellular atp and prolonged culture with cii resulted in a further elevation of extracellular atp. inflammasome/ il-1β dependent and independent pathways maybe two downstream cellular signaling mechanisms proposed for p2x7r stimulation 46, 47 . interestingly, we also observed that the strong promoting effect of cii on the release of atp was accompanied by a large increase in protein levels of p2x7r. knowing that involvement of the p2x7r in atp release has recently been reported in many cell types 48 , we tested the hypothesis that p2x7r function was required for the cii-induced atp release from dcs. our data showing that the p2x7r antagonists completely abolished cii-stimulated atp release suggest that p2x7r mediates atp release from dcs in response to cii. the effect of apyrase treatment on p2x7r expression strongly suggests a reciprocal relationship between the release of atp and p2x7r function in response of dcs to cii. it has been reported that sustained stimulation of the p2x7r caused phenotypic changes, membrane permeabilization, and eventually cell death 49 , but we did not observe any significant cytotoxicity up to 24 h of exposure to cii. in particular, p2x7r activation also contributes to the th17 cell differentiation. based on these findings, we postulate that p2x7r is involved in the cii-mediated th17 polarization. the cytokines il-1β , tgf-β 1, il-23 and il-6 are sufficient to differentiate th17 cells from naive cd4 + t cells 50,51 , while il-12 is a key player in polarizing cd4 + t cells toward the th1 subset 51 . p2x7r activation enhances the release of th17-biasing cytokines [50] [51] [52] . our data add to this observation by showing that stimulation of p2x7r by cii strongly potentiated the production of il-1β , tgf-β 1, il-23 and il-6 mrna. in the absence of p2x7r signaling, stimulation with cii failed to induce the upregulation of th17-promoting cytokines in dcs. a previous study showed that stimulation of p2x7r by bzatp caused no influence on the release of l-12p70 53 , and miller et al. 54 found that absence of p2x7r did not affect il-12 production. in agreement with these data, we found that cii-stimulated dcs lacking functional p2x7r still possessed the ability to promote the production of il-12p40 and il-12p35. in contrast, absence of the p2x7r did not affect il-1β production 54 , different from our findings in the current study. we speculated that different mediums measured in two studies may explain the discrepancy. notably, the above results indicated that p2x7r was required for cii-induced development of a th17-promoting rather than a th1-promoting microenvironment. interestingly, a study by vergani et al. 50 found that in vitro genetic upregulation of the p2x7r pathway was shown to stimulate th1/th17 cell generation. these findings suggest that there may be different roles of p2x7r for the differentiation of th1 and th17 cells under certain circumstances. co-culturing system of cd4 + t cell and dcs is widely used to study the mechanisms underlying the polarization of naive cd4 + t cells in vitro 55 . we thus investigated the potential roles of p2x7r on the th17 cell differentiation in this co-culturing system. consistent with the mrna findings, blocking the p2x7r signaling had a similar influence on the cytokine profile in the cii-stimulated coculture supernatant. the elisa results enhanced our finding that p2x7r was necessary in the cii-mediated th17 cell differentiation. extracellular atp signaling acting through the p2x7r is a complex and dynamic scenario 56 . a previous study showed that atp stimulation strongly induced p2x7r expression both at the transcriptional and translational levels 57 . subsequently, pores formed in response to p2x7r activation induced additional atp release, initiating a positive feedback loop 58 . from the results of in vitro experiments, we have found a reciprocal relationship between atp release and p2x7r function in response to cii, by which cii-induced p2x7r activation promote the atp release via the formation of membrane pores, meanwhile intensified stimulation of atp enhance the expression of p2x7r. the dynamic association between atp release and p2x7r expression strengthens their functions against challenge with cii. as previously reported, atp activated the p2x7r to induce cytokine responses in inflammatory conditions 59 . in this work, we have explored the release of six inflammatory cytokines (il-1β , tgf-β 1, il-23, il-12p40, il-12p35 and il-6) from dcs in response to cii. considerable research effort has focused on the possible mechanism downstream of atp-p2x7r for the induction of inflammatory cytokines, including nlrp3 inflammasome-dependent il-1β secretion 60 , pkc/mapk signalling pathway for tgf-β 1 61 and p38 mapk pathway for il-6 62 . collectively, we believe the activation of downstream signals of atp-p2x7r axis is likely responsible for the release of inflammatory cytokines induced by cii. further studies are needed to elucidate signalling pathways for the release of inflammatory cytokines induced by cii. given that p2x7r signaling facilitated the development of cytokine milieu important for th17 cell differentiation, we then investigated its influence on the expressions of rorγ t and rorα , essential for th17 cell differentiation 55 . we firstly reported that the mrnas levels of rorγ t and rorα were profoundly elevated following the stimulation of p2x7r by cii. furthermore, we failed to observe the upregulation of the mrnas encoding rorγ t and rorα in dc/cd4 + t coculture system after the blockade of p2x7r, hinting that p2x7r could positively regulate the expressions of rorγ t and rorα . blocking p2x7r signaling abolished the cii-mediated increase in il-17a. importantly, no change was observed in response to cii in the proportion of cd4 + il-17a + cells by treatment with p2x7r antagonists compared with unstimulated controls, proving the sufficient and direct evidence that the p2x7r is a necessary component for the cii-induced differentiation of th17 cells. further analysis in spleen t cells from cia mice showed the same change trends of the above indices for th17 cell differentiation. based on these findings, we propose that cii promotes the differentiation of th17 cells via p2x7r signaling both in vivo and in vitro. interestingly, recent studies suggested the possible involvement of b cells in il-17 production. schlegel et al. 63 demonstrated that in addition to th17 cells, a substantial number of other innate and adaptive immune cells, in particular also b cells participate in the il-17 production during established ra. another study also identified b cells as a major source for rapid, innate-like il-17 production in vivo in response to trypanosoma cruzi infection 64 . p2x7r is expressed on cells of the immune system, including cd4 t cells and b cells 65 . however, it is still unknown whether p2x7r antagonist effect is only specific to cd4 t cells rather than b cells. we speculate that it is possible that p2x7r antagonists induced both inhibition of cd4 t cells and b cells may work simultaneously to abolish the cii-mediated increase in il-17a in vivo. considering the positive effect of p2x7r signaling in the differentiation of th17 cells, we next examined whether p2x7r inhibition attenuated cii-mediated joint damage of cia mice. our results indicated that blockade of p2x7r remarkably inhibited hind paw swelling and ameliorated the pathological changes in ankle joint of the cia mice. these results clearly suggest that p2x7r might be an interesting target for the clinical treatment of ra. since the blockade of p2x7r can attenuate the differentiation of th17 cells promoted by cii in vivo and in vitro, further application of therapeutic regimen targeting p2x7r should be investigated. recently, it has been recognized that gm-csf has the potential in the amelioration of autoimmune diseases by inducing natural tregs. as previously reviewed, although blocking gm-csf or its receptor has been regarded as available potent anti-inflammatory therapies, gm-csf can act as an anti-inflammatory/regulatory cytokine in many situations 66, 67 . it can increase regulatory t-cell numbers and function by modulating differentiation of dcs 68 . interestingly, gathungu et al. 69 demonstrated that elevated gm-csf autoantibody was a risk marker for aggressive crohn's disease behavior and complications including surgery, indicating that deficiency of gm-csf can contribute to a relative immunodeficiency. a case report by rowin et al. 70 revealed that gm-csf treatment leaded to a sustained increase in the foxp3 expression and an enhance capability of foxp3+ cd4+ cd25+ tregs to limit the proliferative capacities of cd4+ t effector cells. our study showed that p2x7r presented positive effect on the differentiation of th17 cells through its pro-inflammatory role and p2x7r inhibition attenuated joint damage of cia mice. given the opposite effects of gm-csf and p2x7r on the regulation of tregs/th17 balance, we postulate gm-csf may possibly affect the expression of p2x7r and hence attenuate cia. further experiments are needed to test the hypothesis. in addition to the induction of cia, cii is proved to be capable of generating eye-mediated immune tolerance 71 and acaid-mediated immune tolerance 72 . the p2x7r is emerging as a novel anti-inflammatory therapeutic target, and various selective p2x7r antagonists are under clinical trials. in view of the potential role of p2x7r in the regulation of inflammation reported in our study and other previous studies 35, 38, 41, 46, 54 , we believe that p2x7r may have a central role in the above immune tolerance. this could have therapeutic implications in other routes of immune tolerance in which cii is involved. in summary, we have demonstrated a novel function for p2x7r signaling in regulating cii-induced differentiation of th17 cells. p2x7r signaling facilitates the development of the sophisticated network of dc-derived cytokines that favors a th17 phenotype. as dc is of critical importance in immunotherapy, p2x7r may become an interesting target for dc-based therapeutic approaches. fortunately, several potent p2x7r antagonists are available that could be helpful for drug screening and in managing patients with ra. immunologic mechanisms in the pathogenesis of rheumatoid arthritis the epidemiology of rheumatoid arthritis rheumatoid arthritis of the cervical spine histopathologic analysis of 16 subcutaneous rheumatoid nodules development of a multimorbidity index: impact on quality of life using a rheumatoid arthritis cohort quality of life and drug compliance: their interrelationship in rheumatic patients cost-effectiveness of tofacitinib in the treatment of moderate to severe rheumatoid arthritis in south korea interleukins and interleukin receptors in rheumatoid arthritis: research, diagnostics and clinical implications eular recommendations for the management of rheumatoid arthritis with synthetic and biological diseasemodifying antirheumatic drugs: 2013 update where do t cells stand in rheumatoid arthritis? altered immunoregulation in rheumatoid arthritis: the role of regulatory t cells and proinflammatory th17 cells and therapeutic implications the role and modulation of ccr6+ th17 cell populations in rheumatoid arthritis role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis rheumatoid arthritis pathophysiology: update on emerging cytokine and cytokine-associated cell targets the pathogenesis of rheumatoid arthritis the potential role of th17 in mediating the transition from acute to chronic autoimmune inflammation: rheumatoid arthritis as a model molecular mechanisms of cytokine and chemokine release from eosinophils activated by il-17a, il-17f, and il-23: implication for th17 lymphocytes-mediated allergic inflammation il-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of nfkappa b ligand/osteoprotegerin balance extracellular adenosine triphosphate and adenosine in cancer p2x(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases modulating p2x7 receptor signaling during rheumatoid arthritis: new therapeutic approaches for bisphosphonates human th17 subsets interleukins 1beta and 6 but not transforming growth factorbeta are essential for the differentiation of interleukin 17-producing human t helper cells up-regulation of th17 cells may underlie inhibition of treg development caused by immunization with activated syngeneic t cells immunization with autologous t cells enhances in vivo anti-tumor immune responses accompanied by up-regulation of gadd45beta artificial microrna interference targeting at(1a) receptors in paraventricular nucleus attenuates hypertension in rats human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay anti-inflammatory activity of sappanchalcone isolated from caesalpinia sappan l. in a collagen-induced arthritismouse model the sympathetic nervous system stimulates anti-inflammatory b cells in collagen-type iiinduced arthritis u87mg glioma cells overexpressing il-17 acclerate early-stage growth in vivo and cause a higher level of cd31 mrna expression in tumor tissues identification of a novel toll-like receptor 7 endogenous ligand in rheumatoid arthritis synovial fluid that can provoke arthritic joint inflammation therapeutic effect of 7,3′-dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting jak2-stat3 signal pathway extracellular atp triggers and maintains asthmatic airway inflammation by activating dendritic cells the human p2x7 receptor and its role in innate immunity synovial fluid atp: a potential substrate for the production of inorganic pyrophosphate expression and function of the p2x(7) purinergic receptor in patients with systemic lupus erythematosus andrheumatoid arthritis absence of the p2x7 receptor alters leukocyte function and attenuates an inflammatory response p2x7 receptors mediate atp release and amplification of astrocytic intercellular ca2+ signaling the p2x7 receptor is an important regulator of extracellular atp levels p2x7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration collagen-induced arthritis enhanced t cell proliferative response to type ii collagen and synthetic peptide cii (255-274) in patients with rheumatoid arthritis persistence of collagen type ii-specific t-cell clones in the synovial membrane of a patient with rheumatoid arthritis specificity of antibodies to type ii collagen in rheumatoid arthritis p2x7 receptor-stimulation causes fever via pge2 and il-1β release a potential therapeutic role for p2x7 receptor (p2x7r) antagonists in the treatment of inflammatory diseases the p2x7 receptor is an important regulator of extracellular atp levels extracellular atp causes apoptosis and necrosis of cultured mesangial cells via p2z/p2x7 receptors long-term heart transplant survival by targeting the ionotropic purinergic receptor p2x7 differential requirement for p2x7r function in il-17 dependent vs. il-17 independent cellular immune responses signaling through purinergic receptors for atp induces human cutaneous innate and adaptive th17 responses: implications in the pathogenesis of psoriasis p2x7-dependent, but differentially regulated release of il-6, ccl2, and tnf-α in cultured mouse microglia dysregulation of the inflammatory response to the parasite, toxoplasma gondii, in p2x7 receptor-deficient mice ox62+ ox6+ ox35+ rat dendritic cells are unable to prime cd4+ t cells for an effective immune response following acute burn injury modulating p2x7 receptor signaling during rheumatoid arthritis: new therapeutic approaches for bisphosphonates paeoniflorin down-regulates atp-induced inflammatory cytokine production and p2x7r expression on peripheral blood mononuclear cells from patients with primary sjögren's syndrome a novel recombinant plasma membrane-targeted luciferase reveals a new pathway for atp secretion role of atp in trauma-associated cytokine release and apoptosis by p2x7 ion channel stimulation neutrophil p2x7 receptors mediate nlrp3 inflammasomedependent il-1β secretion in response to atp activation of p2x7 purinoceptor-stimulated tgf-beta 1 mrna expression involves pkc/ mapk signallingpathway in a rat brain-derived type-2 astrocyte cell line, rba-2 high fatty acids modulate p2x(7) expression and il-6 release via the p38 mapk pathway in pc12 cells b cells contribute to heterogeneity of il-17 producing cells in rheumatoid arthritis and healthy controls trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors rorγ t and ahr that leads to il-17 production by activated b cells inhibition of antigen receptor-dependent ca(2+ ) signals and nf-at activation by p2x7 receptors in human b lymphocytes dual role of gm-csf as a pro-inflammatory and a regulatory cytokine: implications for immune therapy gm-csf: an immune modulatory cytokine that can suppress autoimmunity il-10-producing cd4+ cd25+ regulatory t cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis granulocyte-macrophage colony-stimulating factor autoantibodies: a marker of aggressive crohn's disease granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory t cells eye-mediated immune tolerance to type ii collagen in arthritis-prone strains of mice type ii collagen induces peripheral tolerance in balb/c mice via the generation of cd8+ t regulatory cells this work was supported by national natural science foundation of china (nsfc) (no. 81202345 and 81102256). we are grateful to lei zhang from nanjing medical university for his support on this project. supplementary information accompanies this paper at http://www.nature.com/srep competing financial interests: the authors declare no competing financial interests. key: cord-0032148-k5818jgj authors: walha, roua; dagenais, pierre; gaudreault, nathaly; beaudoin-côté, gabriel; boissy, patrick title: the effects of custom-made foot orthoses on foot pain, foot function, gait function, and free-living walking activities in people with psoriatic arthritis (psa): a pre-experimental trial date: 2022-05-25 journal: arthritis res ther doi: 10.1186/s13075-022-02808-8 sha: c91d66bdd044451255275c6a4c23b4828be0e9c6 doc_id: 32148 cord_uid: k5818jgj introduction: foot involvement is a significant concern in psoriatic arthritis (psa) as it can lead to severe levels of foot pain and disability and reduced mobility and quality of life. previous studies have shown moderate efficacy for custom-made foot orthoses (cfo) in reducing foot pain and disability in people with rheumatoid arthritis. however, evidence on the efficacy of cfo in people with psa is lacking. objectives: to explore the effects of cfo on foot function, foot and lower limb pain, gait function, and free-living walking activities (fwa) in people with psa. methods: a pre-experimental study including twenty participants with psa (mean age: 54.10 ± 9.06 years and disease duration: 11.53 ± 10.22 years) was carried out. all the participants received and wore cfo for 7 weeks. foot and lower limb pain and foot function were measured before and after the intervention using the numerical rating scale (nrs) and the foot function index (ffi). gait function was assessed by recording spatiotemporal parameters (stps) during a 10-m walk test using an instrumented gait analysis system (mobility lab). free-living walking activities (step count, free-living cadence, time spent in different ambulatory physical activities (apa)) were recorded over 7 days using an accelerometer-instrumented sock. results: the ffi reported scores demonstrated severe baseline levels of foot pain (54.46 ± 14.58 %) and disability (46.65 ± 16.14%). statistically and clinically significant improvements in foot pain and foot function and large effect sizes (cohen’s effect size > 1, p < 0.005) were observed after the intervention period. a strong correlation (r = −0.64, p < 0.01) between the cfo wearing time and foot function was demonstrated. however, no significant changes were found for gait stp or free-living walking activities after 7 weeks of cfo use. conclusion: results support the clinical and biomechanical plausibility of using cfo in people with psa to reduce pain and improve foot function. large-scale and controlled studies are needed to confirm these findings. moreover, a multidisciplinary approach including the prescription of exercise therapy and physiotherapy combined with cfo could be required to improve stp and promote apa in people with psa. trial registration: clinicaltrials.gov, nct05075343. retrospectively registered on september 29, 2021 supplementary information: the online version contains supplementary material available at 10.1186/s13075-022-02808-8. psoriatic arthritis (psa) is a chronic inflammatory arthropathy and a complex disease that frequently associates skin psoriasis, musculoskeletal manifestations including axial and/or peripheral arthritis, and several comorbidities such as cardiovascular disease, diabetes, and obesity [1] . foot and ankle problems are prevalent in people with psa and are sometimes among the disease's first and most important musculoskeletal manifestations [2, 3] . several inflammatory features can be observed in the foot and ankle. these include toe dactylitis referred to as sausage-like toes [4] , achille's tendon and plantar fascia enthesitis [4, 5] , metatarsophalangeal and distal interphalangeal joint synovitis [6, 7] , and tenosynovitis of the tibialis posterior, the common peroneal sheath, the long flexor tendons of flexor digitorum longus, and the flexor hallucis longus [8, 9] . all foot compartments can consequently be affected by psa, causing pain, swelling, stiffness, tenderness, and in later stages, deformities leading to severe foot disability, reduced mobility, and quality of life [9] [10] [11] . quantitative gait analysis, including spatiotemporal, kinematic, and kinetic parameter assessment, has been proven useful in evaluating gait function, disease progression, and/or a given intervention's effect on patients' mobility [12, 13] . gait spatiotemporal parameters (stps), in particular, are key metrics in gait function evaluation [14] and are indicators of gait adaptations in people with inflammatory arthritis [15] . instrumented gait analysis studies in people with psa demonstrated impairments in gait stp, including reduced cadence, gait speed, stride length, and increased double support time, indicating impaired gait function and ultimately implying difficulties in walking activities [16] [17] [18] . walking activities, on the other hand, involve moving in diverse situations and environments (e.g., the patient's usual environment). a significant part of daily living activities, including transport/commuting, recreation, and domestic/occupational activities, depend mostly on walking. walking is also the most practical, accessible, prevalent form of physical activity (pa) and the most frequently prescribed activity to meet the recommendations of the international guidelines for pa [19] [20] [21] . inherently, gait and walking are closely related. for instance, a reduced gait speed is a significant predictor of decreased daily walking activity, and it could therefore have significant impacts on daily life [22, 23] . the effects of pa and walking on health outcomes are well-established [24, 25] . it has also been reported that pa may benefit disease activity, muscle strength, fatigue, pain, and quality of life in people with psa [26] . however, a recent systematic review demonstrated low pa levels in people with psa which could be detrimental to psa-associated comorbidities (i.e., cardiovascular health) and the disease per se [26] . importantly a close relationship between foot pain and gait function deficiencies has been demonstrated [27, 28] . this suggests that interventions aiming at improving foot pain and function could eventually improve gait function and promote walking activity and overall pa in people with psa. psa management is mainly based on pharmacological treatments including disease-modifying antirheumatic drugs (dmards), janus kinase inhibitors (jaki), and biological therapies which have been proven efficient on symptom control and disease activity reduction [29] [30] [31] . remarkably, inflammation and foot pain, as well as the related disability, can still be observed in a large proportion of patients on pharmacological therapy [16, 18, [32] [33] [34] . from a physiopathological perspective, mechanical stress and trauma have been suggested as joint and soft tissue inflammation triggers in psa [35] [36] [37] . this could partly explain the high prevalence and persistence of foot and ankle problems, considering the weight-bearing function and the continuous mechanical stress applied to the foot during daily living activities [38] . in this context, experts point out the need for targeted therapies addressing biomechanical abnormalities in the foot to reduce mechanically triggered inflammation and pain in people with psa with persistent foot problems. among such therapies, custom foot orthoses (cfo) are medical devices designed to alter the magnitude and temporal patterns of the reaction forces acting on the plantar foot in order to improve foot and lower limb function and offload painful structures during weight-bearing activities [39] . cfo are common practice in patients with foot problems associated with rheumatic diseases. in people with rheumatoid arthritis (ra), a disease characterized by the high prevalence and severity of foot problems similar to those reported in psa, the use of cfo showed positive effects on foot pain and disability [40] [41] [42] [43] [44] [45] . however, to our knowledge, there are no records in the literature on the efficacy of cfo in people with psa. in addition, despite the similarities in foot problems, ra and psa are different entities with different clinical trial registration: clini caltr ials. gov, nct05 075343. retrospectively registered on september 29, 2021 keywords: psoriatic arthritis, custom foot orthoses, foot pain, foot function, spatiotemporal parameters, free-living walking activities presentations making the direct extension of results from ra to psa inadequate [46] . therefore, the primary objective of this study is to explore the effects of using cfo for a 7-week period on foot function in people with psa. the secondary objectives are to explore the effects of cfo on foot pain, the relationship between the cfo wearing time and foot pain and function, and the effects of cfo on gait function (stp) and free-living walking activities (fwa) in people with psa. a pre-experimental study with pre-test/post-test design was conducted. potential participants were referred to the research team from the rheumatology out-patient clinics at the université de sherbrooke hotel dieu university hospital (chus). the first author (rw) screened the potential participants for eligibility via phone interviews. twenty-two participants with psa met the inclusion criteria and were then recruited. inclusion criteria were being between 20 and 70 years of age, having a confirmed diagnosis of psa by a trained rheumatologist, and having moderate to severe and recurrent foot pain and stable medication over 3 months preceding the recruitment. patients with diabetes, neurological disease, or any musculoskeletal disease that could impact the normal gait pattern who received an intra-articular corticosteroid injection or any conservative foot treatment such as foot orthoses/footwear intervention within the past 3 months were excluded. the study was approved by the ciusss de l'estrie-chus ethics board, and all the participants gave their informed consent before data collection. on a first data collection session (t1) at sherbrooke's research center on aging, demographic and baseline characteristics were assessed. then, foot and lower limb pain, global pain, and foot function were measured using self-reported questionnaires. in this same session, all the participants performed three 10-m walking trials at their self-selected speed. participants were then examined by a trained podiatrist who performed foot casts and designed the cfo. at the cfo delivery visit (t2), the participants' foot and lower limb pain and foot function were evaluated for a second time. this second pre-intervention measurement was planned considering the t1-t2 delay and the fluctuating nature of pain in psa. cfo were then dispensed and adjusted if necessary to improve fit and comfort. the participants were directed to wear the cfo for 7 weeks and they were provided with a diary to record daily cfo wearing time, weekly foot pain intensity, and any changes in medication that occurred during the intervention period. at a final follow-up visit scheduled at the end of the 7-week period (t7), foot and lower limb pain and foot function were re-assessed and the instrumented 10-m walking trials were repeated. free walking activities (fwa) were recorded over seven consecutive days before the cfo first use and after the 7-week intervention period using an instrumented sock (including an imu at the ankle) that the participants wore during waking hours. functional foot orthoses were custom-made for each participant. the cfo were designed by the same experienced podiatrist (gbc) based on a detailed clinical and biomechanical assessment. foot scans were obtained with the occipital structure sensor 3d scanner (occipital inc), the patient in a prone position while the subtalar joint was held in a neutral position. the obtained scan was edited and smoothed using the msoft software (techmed3d inc, levis, qc, canada). three-quarter length cfo were 3d printed in a rigid material (nylon) using a multijet fusion 3d printer (hp, palo alto, ca, usa). the thickness of the cfo ranged between 2 and 3.4 mm depending on the participants' medial arch weight and height. most of the orthoses included a medial arch support, a heel stabilizer, and a metatarsal pad. the degrees and types of corrections added to the cfo were determined specifically for each participant based on the clinical examination and were adjusted at the cfo delivery according to each patient's comfort and tolerance. the participants were taught to wear the cfo progressively during the first 2 weeks to allow for the lower limbs' muscles and structures to adjust to the cfo, and to wear the orthoses for the next 5 weeks, 7 days a week as often as they could. to document adherence to the cfo, all the participants completed a diary to report the daily wearing time in hours. in addition, the participants were advised to wear the cfo with adapted shoes after the general characteristics (e.g., heel height, malleability of the sole, etc.) of such shoes were explained. sex, age, body mass index, professional occupation, and previous history of foot injuries were assessed using a self-reported questionnaire. disease duration and c-reactive protein (crp) levels at baseline were obtained from the patients' medical records. foot type, foot deformities, and foot pain sites were obtained from the podiatrist's clinical examination records. changes in medication during the cfo intervention period were recorded by the participants in the diaries provided to them. foot function was measured using the foot function index (ffi). the ffi is a valid, reliable, and responsive self-reported questionnaire widely used in previous studies to evaluate cfo effects on foot function [47, 48] . the ffi is composed of 23 items divided into three subscales measuring foot pain (ffi-p), foot disability (ffi-d), and foot-related activity limitation (ffi-al). each item of the ffi is recorded on a zero-to-ten numeric rating scale (nrs) allowing for subscale and total score calculation. each subscale score is calculated as the sum of all its items divided by its maximum possible total [47] . the total score is obtained as the sum of all subscales' final percentages divided by the total number of subscales. the values reported are presented as percentages ranging between 0 and 100%, with higher values indicating greater pain, disability, and activity limitation. the minimal clinically important difference (mcid) for the ffi total score was found to be 7 points in patients with plantar fasciitis [49] . foot pain intensity was measured using a zero-to-ten nrs which is a valid, reliable, and responsive tool for pain intensity assessment [50] . the mcid reported for the nrs in people with chronic pain is equal to 2 points [51] . participants were asked to circle the number between 0 and 10 that matched better their average foot pain intensity in the 7 days preceding the data collection. foot pain was assessed more in detail (e.g., pain walking with foot orthoses, pain walking with shoes, pain at the end of the day, etc.) with the ffi pain subscale. moreover, to monitor the evolution of foot pain intensity during the intervention period, the patients were asked to report at the end of each week their perceived foot pain in a diary using zero-to-ten nrs. foot pain and foot function were measured at three time points namely, at first data collection (t1), at cfo delivery (t2), and after the 7-week intervention period (t7). as there were no statistical differences in the ffi and nrs scores between t1 and t2, the average of these two time points was used as a baseline measure while the last time point (t7) represented the final measure. the daily cfo wearing time was recorded in the participants' diaries and it is reported as the average reported time per week. global pain and pain at the knee, hip, and lower back pain were measured at t1, t2, and t7 using the nrs (0, no pain, to 10, worst imaginable pain). similarly to foot pain and foot function, data obtained during the first two time points were used as baseline measures. gait function was assessed using an instrumented gait analysis system. gait spatiotemporal parameters (stps) including cadence, gait cycle duration, gait speed, stride length, double support, swing time, foot strike angle, and stride time variability were recorded using the mobility lab system (apdm wearable technologies) during the 10-m walk test (10mwt). mobility lab is a researchgrade system proven to accurately and reliably estimate stp [52] [53] [54] . mobility lab uses a set of six opal inertial measurement units (imus) and a software that allows for automated and easy extraction of stp. all the participants performed three trials of the 10mwt. to do so, they walked over a 14-m straight walkway with the mobility lab's imus fixed with elastics straps on the chest, the lower back, both wrists, and feet. the 10mwt trials were performed at the participants' comfortable speed and the average of the three trials was calculated. fwa including step count, free-living cadence, and time spent in ambulatory physical activity (apa) intensity-based categories were measured using an instrumented sock (sensoria inc, redmond, wa, usa) including a 9-axis imu positioned at the ankle. the instrumented sock connects automatically, without any manipulation needed from the participants, via bluetooth to a smartwatch (apple watch, series 3) where the raw inertial measures of motion (3d accelerometer) are stored. the data are then transferred to a computer where they are processed to extract walking activities' specific outcomes (see additional file 1 for signal processing and step identification description). step count was assessed as the total number of steps per day. furthermore, to describe the step accumulation pattern throughout the day, the number of steps was assessed for each of the following active event categories based on the number of consecutive steps within each category: 0 to 20 steps, 20 to 60 steps, 60 to 120 steps, 120 to 300 steps, 300 to 600 steps, and more than 600 steps. free-living cadence included cadence averaged for total wearing time (mean cadence/day). moreover, as cadence may vary depending on the number of consecutive steps performed, it was also assessed for each of the above active event categories. apa intensity-based categories were determined using cadence data. they included stepping activities (0 to 59 steps/min), slow walking (60 to 79 step/min), medium walking (80 to 99 step/min), moderate intensity apa (mapa) (100 to 119 step/min), and vigorous apa (vapa) ( > 120 step/ min), as previously defined [55, 56] . the time spent in each of the last three categories was calculated only for purposeful walking (i.e., events composed of more than 60 steps). free-living walking activities were assessed over a period of 7 consecutive days before and after the intervention. to do so, the participants were instructed to wear the instrumented sock during waking hours while they perform daily activities. only valid days defined as days with at least 8 h of recordings and only valid participants with a minimum of one valid day were included in the analyses. adherence to the instrumented sock use was tested by the system itself and by asking the participants to record the daily wearing time in a diary. to avoid influencing the participant's physical activity, they were only told that the study would assess the effects of cfo on foot pain, foot function, and gait stp. given the exploratory nature of this study, the sample size was calculated assuming a large effect size (0.8) for the pre-post differences in the ffi total score. using a two-tailed paired t-test, a significance level α of 5%, a power of 90%, and assuming a 15% dropout rate, a total sample size of 22 participants was required. the shapiro-wilk test was used to examine data distribution and parametric and nonparametric statistical tests were used depending on data distribution. paired t-tests (or the wilcoxon signed-rank test) were used to assess the differences in foot pain, foot function, and global and lower limb pain between baseline and t7. cohen's effect size was calculated to quantify the magnitude of these differences. a one-way repeated measures anova was conducted to determine whether there was a statistically significant difference in weekly recorded foot pain intensity and cfo wearing time over the 7-week intervention period. spearman correlation coefficients were calculated to assess the relationships between the cfo wearing time, foot pain, and foot function at t7. as a 2-week adaptation period was required, correlation analyses were performed using the average cfo wearing time per week calculated for the last 5 weeks of the intervention period. correlation coefficients were considered weak, moderate, and strong for values between 0.1 and 0.3, 0.3 and 0.5, and > 0.5, respectively [57] . the pre-post differences in gait function and free-living walking activities were assessed using the wilcoxon signed-rank test. the significance level was set at 0.05. the statistical analyses were performed using spss version 26 (ibm statistics corporation, armonk, ny). the participant flow throughout the study is presented in additional file 2. twenty-seven participants were initially screened, five were excluded as two did not meet inclusion criteria and three declined to participate. twenty-two participants met the inclusion criteria and participated in the study. of these, one participant was excluded after the podiatry examination because the cfo was considered not clinically appropriate. another patient was excluded because of an acute arthritis flare during follow-up. from september 2019 to august 2021, twenty participants completed the study and were included in the analyses. demographic and baseline clinical characteristics are presented in table 1 . the study sample was composed of 5 males and 15 females with a mean age of 54.10 ± 9.06 years, a mean bmi of 29.3 ± 4.5 kg/m 2 , and a mean disease duration of 11.53 ± 10.22 years. the crp baseline levels were within the normative ranges for 75% of the patients. two participants recorded changes in medication during the intervention period ( table 1 ). the first participant started taking nsaids at the end of the fourth week for generalized body pain, and the second received a new biological therapy starting at week 4. figure 1 presents the ffi (fig. 1a) and nrs (fig. 1b) scores at baseline and after 7 weeks of using the cfo. all the ffi sub-scores and total score decreased at t7 indicating a significant improvement in foot pain (54.46 ± 14.58% at baseline and 34.01 ± 18.94 at t7), foot disability (46.26 ± 19.91% at baseline and 24.13 ± 18.84 at t7), and foot-related activity limitation (41.54 ± 31.35% at baseline and 12.26 ± 17.57 at t7). the mean pre-post difference in the ffi total score was 22.30 ± 24.76% (p = 0.004) and a large effect size (d = 1.25) was reported. a significant improvement in foot pain was also demonstrated through the nrs score which decreased by 2.30 ± 2.98 points and a large effect size was reported (d = 1.19, p = 0.004). global pain was also significantly decreased with a mean difference of 1.95 ± 2.77 and a large effect size (d = 0.90, p = 0.006). regarding lower limb pain, while there were no significant changes in knee and lower back pain, hip pain significantly improved with a mean difference of 1.88 ± 3.38 points and a moderate effect size (d = 0.67, 0.031) ( table 2) . seventeen participants recorded the weekly foot pain intensity and cfo wearing time using the provided diaries. the cfo weekly wearing time and the evolution of foot pain are presented in fig. 2a and b . as it can be observed, there was a tendency for foot pain to decrease during the first 4 weeks and a plateau was observed after this period. the same tendency was also observed for wear time which increased progressively during the first 4 weeks and remained almost stable afterward. the oneway repeated measures anova showed statistically significant changes in weekly foot pain intensity over time f (2.13, 32.02) = 20.28, p = 0.001, and cfo wearing time f (2.27, 34.17) = 5.92, p = 0.005. post hoc tests (with bonferroni adjustment) showed no significant differences in foot pain between the different time points. however, the differences between week 1 and week 7 (mean difference = 1.81, p = 0.09) and weeks 2 and 7 (mean difference = 1.43, p = 0.06) were almost significant. regarding cfo wearing time, post hoc analysis showed a significant decrease between the first week and all the other time points (p < 0.05). correlation coefficients showed significant correlations between the cfo wearing time (hours/ week) and foot pain (r = −0.57, p = 0.023), disability (r = −0.68, p = 0.005), activity limitation (r = −0.49, p = 0.047), and the ffi total score (r = −0.64, p = 0.01) at t7 (fig. 3) . spatiotemporal gait parameters measured before and after the intervention period are presented in table 2 . results show a slight but significant increase and decrease in double support time and swing time, respectively, at t7. however, for all the remaining stp including cadence, gait cycle duration, gait speed, stride length, and foot strike angle, there were no significant differences at t7 compared to baseline. similarly, no significant changes were reported for the effects of cfo on stride time variability. free-living walking activities are presented in figs. 4, 5, and 6. out of the 20 participants, four were excluded from the fwa analyses because they did not accumulate at least one valid day of recordings (i.e., recording time > 8 h) at baseline and t7. consequently, the results presented in this section include data from 16 participants. on average, the patients accumulated a total of 10.79 ± 1.18 h and 9.99 ± 1.02 h of recorded time at baseline and t7, respectively, with no significant differences between baseline and t7 (p = 0.09). the average active time was 3.9 ± 1.7 h at baseline and 3.5 ± 1.6 h at t7 (p = 0.08). the average number of steps per day was 6460 ± 2818 steps/day at baseline and 5836 ± 3079 steps/day at the end of the intervention period with no significant differences between the two time points (p = 0.18) (fig. 4a) . the pattern of accumulating these steps is presented in fig. 4b . as can be observed, the total number of steps was accumulated in short active events composed of 20 to 300 consecutive steps (fig. 4b) . the daily average free-living cadence was 96.68 ± 5.85 steps/min and 96.03 ± 5.58 steps/min before and after the intervention, respectively (p = 0.79) (fig. 5a) . results show that cadence increases considerably for active events of more than 20 steps compared to those of less than 20 steps (fig. 5b) . for active events composed of 20 to 600 consecutive steps, the mean cadence was 96.65 ± 1.10 steps/min at baseline and 96.38 ± 1.02 steps/min at t7. results also show that cadence reaches and crosses 100 step/min only for active events composed of more than 600 consecutive steps, for an average of 100.94 ± 11.06 steps/min at baseline and 101.75 ± 8.73 steps/min at t7 with no significant differences between the two time points (p = 0.44). figure 6 presents minutes spent in apa intensitybased categories including stepping activities (fig. 6a) and slow, medium, brisk walking, and vigorous apa (fig. 6b) the primary purpose of this study was to explore the effects of wearing cfo for a 7-week period on foot function in people with psa. as a secondary objective, we sought to assess the effects of cfo on foot pain, gait fig. 3 correlation between weekly cfo wearing time averaged for the last 5 weeks of the intervention period and the ffi subscales and the total score recorded at t7 fig. 4 step count. a daily averaged steps and b number of steps of the different step-based active episode categories function (instrumented gait stp analysis), and fwa and to assess the relationship between the cfo wearing time and foot pain and function in people with psa with foot involvement. the main findings showed a significant improvement in foot pain (nrs and ffi) and foot function (ffi) after using the cfo for a 7-week intervention period. the ffi total score decreased by 22.30%, and a large effect size (d = 1.25) was reported. interestingly, after only 7 weeks of intervention, this improvement was clinically significant for 70% of the participants since the pre-post differences in their ffi total scores were higher than the mcid previously reported for the ffi total score [49] . research in people with ra has shown some efficacy of cfo on foot pain which is consistent with our findings [41] [42] [43] . however, conflicting results regarding the effects of cfo on foot function have been reported. indeed, while some studies endorsed the effectiveness of cfo on foot function [44, 46] , others reported no significant effects for this outcome [58] [59] [60] . several factors could explain such discrepancies. first, the orthoses type can differ between the studies according to the targeted therapeutical aim (e.g., palliative vs functional). moreover, foot casting and manufacturing materials and techniques can vary from one study to another depending on the latest advances in technologies and on the national practice guidelines making between-studies comparison difficult. in our study, we used functional cfo in all the participants. the materials and techniques of foot casting and manufacturing were standardized for all the participants. however, the degrees and types of corrections were specific for each participant's needs and tolerance. the cfo were also adjusted at delivery and during the following 2 weeks to increase comfort and acceptance if needed. this could explain the positive effects on pain and foot function observed in this study. furthermore, there was no consensus regarding the previous studies' intervention durations and control conditions. some trials used placebo orthoses while others used shoes only as a control condition. as the former are usually made from thick and cushioned materials, they cannot be considered as not having therapeutic effects, thereby attenuating the cfo effects. in contrast, using shoes-only as a control could overestimate the effect of cfo [61] . importantly, the cfo wearing time was not documented in all the previous studies which can compromise their conclusions as a lack of effectiveness could partially be related to insufficient use of the orthoses. in our study, a sufficient wearing time (8h/day) was reported and a strong correlation between the cfo wearing time and the ffi total score was demonstrated. these findings are similar to those reported in a few studies in people with ra that did report wearing time [44, 46, 60] . this observation highlights the relevance of monitoring this variable in future studies and the importance of continuous and regular wearing of the cfo. apart from the intervention characteristics, sampling groups and individual characteristics such as age, sex distribution, disease duration, and baseline levels of foot pain and disability are also important considerations as they may influence the effectiveness of cfo. for instance, data from a previous study [62] showed that shorter disease duration, younger age, and higher baseline values of foot pain and disability predicted improved outcomes after cfo use. in the present study, the participants had a mean age of 54.10 ± 9.06, a median disease duration of 6 years, a high prevalence of foot pain, and severe levels of disability at baseline. therefore, it is not unexpected that the cfo helped relieve pain and improve perceived foot function. in addition to foot pain and disability, the findings showed a significant decrease in global and hip pain. however, the differences were not clinically significant (mean differences < 2 points). knee and lower back pain also improved but the differences did not reach the significance level. although there is evidence that cfo could have beneficial effects on lower limb pain [63] [64] [65] [66] , further studies using specific tools to assess knee, hip, and lower back pain in detail are needed to confirm the beneficial effects of cfo on these outcomes. gait stps are indicators of functional capacity and overall health and autonomy, making them the most relevant and most often measured biomechanical parameters for gait analysis in healthy and pathological populations [13, 67] . we noticed a lack of scientific evidence regarding the effects of cfo on gait stps in people with arthritic foot diseases. for instance, studies in people with ra showed only a minimal or no effect of cfo for increasing gait speed, cadence, and stride length [42, 68, 69] and some studies showed that wearing the cfo allowed for an improvement of gait stability [45, 70] . our findings showed that gait stps were altered compared to the normative values reported for healthy adults [71, 72] . however, there were no significant changes in stp and gait stability after the intervention except for a negligible effect on double support and swing time. these results could be potentially attributed to the short-term nature of our intervention period and the long disease duration reported in this study. gait impairments demonstrated in participants with psa may be acquired over many years. consequently, the patients can develop antalgic walking strategies that may require more extended intervention periods to improve. in addition, the patients could have developed fear-avoidance beliefs which have been reported as significant predictors of poorer response to cfo in other patient populations [73] . another possible explanation for the lack of stp response could be that psa may be more responsive to cfo treatment in earlier stages of the disease. therefore, long-term studies including patients with early disease would be more relevant to assess the effects of cfo on gait stp properly. furthermore, although the 10mwt is reliable and responsive in patients with major gait impairments secondary to neurological diseases such as stroke and parkinson [74] , it might not have been responsive in this study because of the relatively milder gait impairment related to psa and the short intervention's duration and follow-up period [75] . longer time/distance tests should also be considered when assessing gait in people with psa as gait impairments reported in this population could be attributed to common features of inflammatory arthritis such as muscle weakness, reduced range of motion, and deformities [76] [77] [78] and they may be more significant during sustained walking activities. the benefits of physical activity on health are undeniable. results from a recent systematic review [26] suggest that physical activity may positively affect disease activity, muscle strength, fatigue, pain, quality of life, and cardiovascular risk in people with psa. therefore, promoting physical activity in this population should be a health care priority. walking is a practical, accessible, and low-skill mode of physical activity that plays a major role in occupational and social activities of everyday living [79] . the findings of this study reported decreased step counts with large standard deviations (6460 steps/day at baseline and 5836 steps/day at t7) which are below the recommended 10,000 steps/day [80] . similarly, the daily averaged free-living cadence was 96.67 ± 5.85 steps/ min at baseline and 96.03 ± 5.85 steps/min at t7 which is below the naturally selected free-living walking speed (100 steps/min) reported for healthy individuals [56, 81] . besides, the reported time spent in brisk walking did not achieve the minimum of daily 30 min of moderate-intensity physical activity recommended by the international guidelines for pa activity [82] . these results indicate decreased pa levels in participants with psa with foot problems which is consistent with what is reported in a previous systematic review [26] . future studies comparing pa levels between participants with psa and matched healthy controls are needed to confirm these results. we postulated that by alleviating pain, the use of cfo would positively impact walking activities in people with psa. nevertheless, neither the number of steps/day, free-living cadence, nor the time spent in the different intensity-based apa changed after using the cfo. again, the short-term nature of this study could have not fully reflected the effects of cfo. besides, free-living cadence is a measure of ambulatory behavior [56] that is likely influenced by psychoaffective factors that have not been assessed in this study. in a previous study in people with psa, beliefs about pa (e.g., fear of exacerbating pain) have been shown to be associated with activity level [83] . this suggests that future studies should address the patient's behavior which is complex and may require using a multidisciplinary approach including behavioral change strategies, advice, education, prescription of exercise therapy, and cfo to promote physical activity in people with psa. in addition, as this study was carried out over nearly 2 years, natural environmental factors such as seasons/weather, and the occurrence of the covid-19 pandemic, might have influenced the patients' pa [84, 85] . lastly, it should be mentioned that only 16 participants were included in the fwa analyses and there might be not enough power to detect significant changes. also, the average recorded time was nearly 1 h lower at t7 compared to baseline (10.79 h vs 9.99). this could have contributed to the obtained results. to our knowledge, only one study investigated the effects of cfo on physical activity [86] in a population of people with ra. using a self-reported questionnaire, their results showed that a 6-month cfo use improved light-intensity physical activity but not moderate-and vigorous-intensity activities [86] . however, these findings cannot be compared to those demonstrated in the present study because of the different approaches used for pa measurement since using self-reported measures could overestimate pa compared to objective measurements [87] . this study has limitations that should be mentioned. first, there was no control group to control for the placebo effect and the natural evolution of the disease. future studies are encouraged to use prefabricated, sham orthoses and/or a shoe-only condition as comparators to overcome this limitation. the choice of the control condition should be made according to the study aims (e.g., using prefabricated orthoses as a control condition if the aim is to assess the intervention's cost-effectiveness). moreover, if sham orthoses are to be used, researchers are encouraged to document the credibility and the biomechanical effects of these devices. second, the follow-up duration may have been too short to show changes in outcomes such as gait parameters and fwa that may necessitate a longer time to improve. moreover, the small sample size and the high proportion of female participants which is not representative of the gender distribution usually seen in psa could compromise the generalizability of our findings. comorbidities associated with psoriatic arthritis: review and update involvement of the foot in patients with psoriatic arthritis. a review of 26 cases subjective health complaints in individuals with psoriasis and psoriatic arthritis: associations with the severity of the skin condition and illness perceptions -a cross-sectional study dactylitis in psoriatic arthritis: prevalence and response to therapy in the biologic era clinical enthesitis in a prospective longitudinal psoriatic arthritis cohort: incidence, prevalence, characteristics, and outcome enthesitis and dactylitis in psoriatic disease: a guide for dermatologists is there subclinical synovitis in early psoriatic arthritis? a clinical comparison with gray-scale and power doppler ultrasound power doppler ultrasonographic assessment of the ankle in patients with inflammatory rheumatic diseases focussing on the foot in psoriatic arthritis: pathology and management options foot problems in psoriatic arthritis: high burden and low care provision perspectives of patients and health professionals on the experience of living with psoriatic arthritis-related foot problems: a qualitative investigation quantification of human motion: gait analysis-benefits and limitations to its application to clinical problems summary measures for clinical gait analysis: a literature review biomechanical parameters for gait analysis: a systematic review of healthy human gait gait characteristics associated with the foot and ankle in inflammatory arthritis: a systematic review and meta-analysis achilles tendon biomechanics in psoriatic arthritis patients with ultrasound proven enthesitis biomechanics of enthesitis of the foot in psoriatic arthritis uk/ order detai ls. do; jsess ionid= b5d18 50df9 c83e5 73465 7db70 a6205 4c? uin= uk plantar forefoot pressures in psoriatic arthritis-related dactylitis: an exploratory study effectiveness of three different walking prescription durations on total physical activity in normal-and overweight women global participation in sport and leisure-time physical activities: a systematic review and meta-analysis coming of age: considerations in the prescription of exercise for older adults ambulatory physical activity, disease severity, and employment status in adult women with osteoarthritis of the hip factors associated with walking activity in adults with cerebral palsy walking and primary prevention: a meta-analysis of prospective cohort studies physical activity and all-cause mortality: what is the dose-response relation? psoriatic arthritis and physical activity: a systematic review associations of regionspecific foot pain and foot biomechanics: the framingham foot study gait speed as a screening tool for foot pain and the risk of falls in community-dwelling older women: a crosssectional study minimal disease activity and remission in psoriatic arthritis patients treated with anti-tnf-α drugs remission and low disease activity in psoriatic arthritis publications: a systematic literature review with meta-analysis eular recommendations for the management of psoriatic arthritis with pharmacological therapies: 2019 update ultrasound imaging for the rheumatologist. xxxii. sonographic assessment of the foot in patients with psoriatic arthritis mri changes in psoriatic dactylitis--extent of pathology, relationship to tenderness and correlation with clinical indices sonographic analysis of the ankle in patients with psoriatic arthritis association between environmental factors and onset of psoriatic arthritis in patients with psoriasis mechanical strain determines the site-specific localization of inflammation and tissue damage in arthritis the biomechanical link between skin and joint disease in psoriasis and psoriatic arthritis: what every dermatologist needs to know mechanical loading and off-loading of the plantar surface of the diabetic foot athletic footwear and orthoses in sports medicine effectiveness of foot orthoses in patients with rheumatoid arthritis related to disability and pain: a systematic review and meta-analysis systematic review and meta-analysis of effects of foot orthoses on pain and disability in rheumatoid arthritis patients custom foot orthoses for rheumatoid arthritis: a systematic review clinical effectiveness and cost-effectiveness of foot orthoses for people with established rheumatoid arthritis: an exploratory clinical trial a randomized controlled trial of foot orthoses in rheumatoid arthritis the effect of foot orthoses on balance, foot function, and mobility in rheumatoid arthritis: a randomized controlled clinical trial distinguishing rheumatoid arthritis from psoriatic arthritis the foot function index: a measure of foot pain and disability a review of the foot function index and the foot function index -revised minimal important difference: values for the foot health status questionnaire, foot function index and visual analogue scale validity of four pain intensity rating scales clinical importance of changes in chronic pain intensity measured on an 11-point numerical pain rating scale validity of mobility lab (version 2) for gait assessment in young adults, older adults and parkinson's disease accuracy and repeatability of two methods of gait analysis -gaitrite tm und mobility lab tm -in subjects with cerebellar ataxia validity and repeatability of inertial measurement units for measuring gait parameters patterns of adult stepping cadence in the 2005-2006 nhanes using cadence to study free-living ambulatory behaviour statistical power analysis for the behavioral sciences randomized controlled trial for clinical effects of varying types of insoles combined with specialized shoes in patients with rheumatoid arthritis of the foot impacts of foot orthoses on pain and disability in rheumatoid arthritics effectiveness of insole use in rheumatoid feet: a randomized controlled trial selecting control interventions for use in orthotic trials: the methodological benefits of sham orthoses factors predicting the outcome of customised foot orthoses in patients with rheumatoid arthritis: a prospective cohort study the effect of orthotic devices on knee adduction moment, pain and function in medial compartment knee osteoarthritis: a literature review the short-term effect of custom-made foot orthoses in subjects with excessive foot pronation and lower back pain: a randomized, double-blinded, clinical trial is there an evidence-based efficacy for the use of foot orthotics in knee and hip osteoarthritis? elaboration of french clinical practice guidelines convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? the effects of shoes and insoles for low back pain: a systematic review and meta-analysis of randomized controlled trials biomechanics and physiological parameters during gait in lower-limb amputees: a systematic review a comparison of two podiatric protocols for metatarsalgia in patients with rheumatoid arthritis and osteoarthritis the effect of custom moulded ethyl vinyl acetate foot orthoses on the gait of patients with rheumatoid arthritis the effects of foot orthoses on walking stability in patients with early rheumatoid arthritis normative spatiotemporal gait parameters in older adults revealing the quality of movement: a meta-analysis review to quantify the thresholds to pathological variability during standing and walking predictors of response to foot orthoses and corticosteroid injection for plantar heel pain a systematic review of the responsiveness of lower limb physical performance measures in inpatient care after stroke methods for assessing responsiveness: a critical review and recommendations assessment of foot and ankle muscle strength using hand held dynamometry in patients with established rheumatoid arthritis gait analysis of the lower limb in patients with rheumatoid arthritis: a systematic review sarcopenia in autoimmune and rheumatic diseases: a comprehensive review walking as a social practice: dispersed walking and the organisation of everyday practices accumulating 10,000 steps: does this meet current physical activity guidelines? outdoor walking speeds of apparently healthy adults: a systematic review and meta-analysis world health organization 2020 guidelines on physical activity and sedentary behaviour relationship between selfefficacy, beliefs, and physical activity in inflammatory arthritis the impact of covid-19 on physical activity behavior and well-being of canadians the effect of season and weather on physical activity: a systematic review foot orthosis treatment improves physical activity but not muscle quantity in patients with concurrent rheumatoid arthritis and sarcopenia comparison of self-reported versus accelerometer-measured physical activity publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the rheumatologists of the chu hôtel dieu of sherbrooke for their contribution to the participants' recruitment. the use of cfo for a 7-week period improved foot pain and related disability in people with psa. this suggests that cfo may be used in conjunction with pharmacological therapy to relieve pain and improve perceived foot function. however, the results should be confirmed in larger and controlled studies. the effects of cfo on gait function and free-living walking activities were not evident in this study due to the small sample size, short follow-up period, and personal and environmental factors that have not been measured. future rcts with larger sample sizes and longer intervention durations are needed for any conclusions to be drawn. the online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13075-022-02808-8.additional file 1. signal processing and steps identification. authors' contributions rw and pb conceived the study. rw collected and analyzed the data and wrote the first full draft of the manuscript. pb assisted with the analysis of data. pb, gbc, pd, and ng all reviewed the manuscript drafts. all authors read and approved the final manuscript. this research was funded by the canadian msk rehab research network and supported through a donation from janssen pharmaceutical companies to fondation pour la recherche et l' enseignement en orthopédie de sherbrooke. the dataset used and analyzed during the current study is available from walha.roua@usherbrooke.ca on reasonable request. the study was approved by the institutional review board of the ciusss de l'estrie-chus (2019-3182), and all the participants gave their informed consent to participate in the study. the authors declare no competing interests. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-0028500-w35igre9 authors: jung, seung min; kim, wan-uk title: targeted immunotherapy for autoimmune disease date: 2022-02-17 journal: immune netw doi: 10.4110/in.2022.22.e9 sha: 5ed3534adc16770a86c8912bc5ab33ed1daf1a09 doc_id: 28500 cord_uid: w35igre9 in the past few decades, biological drugs and small molecule inhibitors targeting inflammatory cytokines, immune cells, and intracellular kinases have become the standard-of-care to treat autoimmune diseases. inhibition of tnf, il-6, il-17, and il-23 has revolutionized the treatment of autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, and psoriasis. b cell depletion therapy using anti-cd20 mabs has shown promising results in patients with neuroinflammatory diseases, and inhibition of b cell survival factors is approved for treatment of systemic lupus erythematosus. targeting co-stimulatory molecules expressed on ag-presenting cells and t cells is also expected to have therapeutic potential in autoimmune diseases by modulating t cell function. recently, small molecule kinase inhibitors targeting the jak family, which is responsible for signal transduction from multiple receptors, have garnered great interest in the field of autoimmune and hematologic diseases. however, there are still unmet medical needs in terms of therapeutic efficacy and safety profiles. emerging therapies aim to induce immune tolerance without compromising immune function, using advanced molecular engineering techniques. autoimmune diseases are pathologic conditions characterized by dysregulated inflammation against autoantigens and affect 3%-10% of the general population (1) . conventional treatments for autoimmune diseases have suppressed general immune function to modulate uncontrolled inflammation. however, those therapeutic approaches have not been completely successful in heterogeneous patient populations, and their efficacy comes at the expense of side effects, particularly increased risk of infection, usually from non-selective immune suppression. to overcome the limitations of conventional therapies, current treatments aim to more selectively inhibit inflammatory signals while causing minimal disruption to homeostatic immune functions. recent advances in understanding disease pathogenesis and new drug manufacturing techniques have led to the widespread use of targeted immunotherapy to treat autoimmune as knowledge about the pathogenesis of disease is rapidly increasing, numerous biological drugs targeting inflammatory signaling pathways are being developed to treat intractable inflammatory diseases. following successful introduction of biologic therapies to treat autoimmune diseases, the molecular targets have expanded to intracellular kinases. blockade of convergent signals by small molecule kinase inhibitors is of great interest in terms of therapeutic efficacy and long-term safety (4, 5) . this review summarizes current therapeutic approaches that target signaling pathways involved in the pathogenesis of autoimmune diseases and presents emerging immunotherapies intended to induce immune tolerance. because the market for targeted immunotherapy is growing rapidly, we focus on drugs that have received clinical approval to treat autoimmune diseases. inflammation is a natural process by which living organisms repair tissue damage and protect against foreign substances. however, dysregulated immune reactions against self-ags lead to loss of immune tolerance and development of autoimmune disease. autoimmunity arises from central and peripheral defects in tolerance checkpoints and activation of nontolerant immune cells. autoantigens can be induced by release of self-ags from immune-privileged sites, generation of neo-self ag, and molecular mimicry of self-proteins with foreign substances (6) . clinical manifestations of autoimmunity can be diverse, ranging from asymptomatic conditions in the presence of autoantibodies to fulminant autoimmune diseases that cause life-threatening organ damage. development of autoimmune disease can be triggered by environmental factors in genetically susceptible individuals. environmental triggers, including stress, smoking, and infection, induce the pro-inflammatory functions of innate immunity, and promotes the pathologic response of adaptive immunity (7) . although the conventional concept of autoimmunity was dysregulation of the adaptive immune system, growing evidence indicates that the innate immune system is also critical to initiation and progression of autoimmune diseases. as the key players in innate immunity, macrophages and dendritic cells (dcs) are essential to ag presentation and production of pro-inflammatory cytokines such as tnf, il-1β, il-6, il-23, b cell-activating factor (baff, also known as blys or tnfsf13b), and a proliferation-inducing ligand (april, also known as tnfsf13a) (8, 9) . type 1 ifn, critically implicated in the pathogenesis of systemic lupus erythematosus (sle) and its related diseases, is primarily produced by plasmacytoid dcs (pdcs), a specialized subset of dcs (8, 10) . the interaction between macrophages/dcs and t cells/b cells further promotes autoimmune inflammation. https://doi.org/10.4110/in.2022. 22 .e9 targeted immunotherapy for autoimmune disease 3/23 https://immunenetwork.org naïve cd4 + th cells differentiate into distinct t cell subsets depending on the cytokine milieu (11) . t cells play a key role in the pathogenesis of autoimmune diseases through autoantigen recognition, cytokine production, and enhanced cytotoxicity (6) . in recent decades, th17 cells producing il-17 and foxp3 + tregs have been highlighted as therapeutic targets for autoimmune diseases. autoreactive b cells, another major component of adaptive immunity, produce pathologic autoantibodies and activate t cells through ag presentation and cytokine production (6, 7) . autoantibody production is a hallmark of various autoimmune diseases, including ra and sle. anti-citrullinated peptide ab in ra and anti-dsdna ab in sle are representative pathogenic autoantibodies responsible for clinical presentation and disease activity. due to the important role of b cells in autoimmunity, b cell surface molecules are therapeutic targets for various autoimmune diseases. soluble mediators from activated immune cells transduce inflammatory signals by binding to their cognate receptors. once engaged by inflammatory cytokines, the receptors activate the jak family to induce phosphorylation, dimerization, and nuclear translocation of stats (4). gene transcription by stats promotes cell proliferation and differentiation and production of a variety of inflammatory mediators, further exacerbating autoimmune inflammation. although the pathophysiologic mechanisms differ for each autoimmune disease, several common inflammatory pathways could be therapeutic targets for immunotherapy. the key therapeutic targets for the treatment of autoimmune diseases based on our current understanding of the pathogenesis of autoimmune inflammation are depicted in fig. 1 . tables 1-3 list targeted immunotherapies approved for autoimmune diseases or under clinical development based on the promising results, detailed below. this review focuses on the clinical application of targeted immunotherapies in the area of immune-mediated inflammatory diseases. tnf is a proinflammatory cytokine mainly produced by myeloid cells and activated t cells (39) . the pathogenic role of tnf in chronic inflammation was suggested by increased expression of tnf in ra synovium and development of arthritis in tnf-transgenic mice (40, 41) . the success of anti-tnf therapy has revolutionized the treatment strategy for ra patients. since the first approval of infliximab and etanercept in 1998, 4 mabs (infliximab, adalimumab, golimumab, and certolizumab) and one receptor-fc fusion protein (etanercept) have been approved and are currently available for treatment of chronic immune-mediated diseases, including ra, juvenile idiopathic arthritis (jia), ankylosing spondylitis (as), psoriasis, and psoriatic arthritis (psa). such mabs are also indicated for treatment of inflammatory bowel disease and noninfectious uveitis. although tnf is well known to have a pro-inflammatory role, it is a pleiotropic cytokine that depends on the binding of tnf receptor (tnfr) (42,43). tnfr1, constitutively expressed on almost all nucleated cells, is mostly responsible for the inflammatory function of tnf, whereas tnfr2, which is expressed only on specific cell types, such as myeloid-derived suppressor cells, treg cells, and monocytes, is associated with the regulatory function of figure 1 . key therapeutic targets in the pathogenesis of autoimmune diseases. autoimmune inflammation involves the dysregulated activation of immune cells, inflammatory cytokines, and intracellular signaling molecules. key molecules in the inflammatory pathway are therapeutic targets for the treatment of autoimmune diseases. pjia, polyarticular juvenile idiopathic arthritis; uc, ulcerative colitis; cd, crohn's disease; bd, behcet's disease; sjia, systemic juvenile idiopathic arthritis; dira, deficiency of il-1 receptor antagonist; traps, tnf receptor associated periodic syndrome; hids/mkd, hyperimmunoglobulin d syndrome/mevalonate kinase deficiency; fmf, familial mediterranean fever; crs, cytokine release syndrome; car-t, chimeric ag receptor-t cell; ssc-associated ild, systemic sclerosisassociated interstitial lung disease, pmr, polymyalgia rheumatica, nmosd, neuromyelitis optica spectrum disorder; axial spa, axial spondyloarthritis. * despite positive results in phase iii clinical trials, fda did not recommend sirukumab for treatment of ra due to safety issues. il-1α and il-1β, members of the il-1 family, are pro-inflammatory cytokines that are closely associated with innate immune responses. although il-1α and il-1β share biological functions by binding with il-1 receptor 1 (il-1r1), several characteristics distinguish il-1α from il-1β (47) . pro-il-1α, constitutively expressed in mesenchymal cells, is biologically active, whereas pro-il-1β produced by macrophages requires cleavage by caspase-1 to become active il-1β. caspase-1-dependent cleavage of pro-il-1β is mediated by activation of an inflammasome containing either a member of nucleotide binding domain and leucine-rich-repeat-containing targeted immunotherapy for autoimmune disease [nlr] protein family (e.g. nlr family pyrin domain-containing [nlrp] 1, nlrp3, nlr family card domain-containing 4) or a member of pyrin-hin-200 domain-containing protein family (e.g. absent in melanoma 2) (48) . in addition, il-1α is not detected in systemic circulation, in contrast to il-1β, which suggests that the pathogenic role of il-1α in autoimmune diseases is local rather than systemic. at this time, il-1β is considered to be more closely associated with diverse rheumatic diseases than il-1α, including systemic jia, adult onset-still's disease (aosd), and gout, and with hereditary autoinflammatory diseases, such as cryopyrinassociated periodic syndromes (caps) and familial mediterranean fever (49) . upon occupation by il-1α or il-1β, il-1r1 forms a heterotrimeric complex with il-1r3 to recruit myeloid differentiation primary response gene 88 (myd88), which triggers a subsequent kinase cascade (il-1r-associated kinases [iraks], iκb kinase, iκb, and nf-κb) that promotes a pro-inflammatory state (47, 50) . this inflammatory activity of il-1 is regulated by the naturally occurring il-1r antagonist (il-1ra) from the same il-1 family. occupation of il-1r1 by il-1ra leads to a conformational change that hinders formation of the heterotrimeric complex with il-1r3, thereby hampering the il-1-mediated inflammatory process. currently, 3 protein therapeutics have been approved as anti-il-1 therapies: canakinumab, an anti-il-1β mab; anakinra, a recombinant il-1 receptor antagonist; and rilonacept, an il-1r1-fc fusion protein. based on their molecular structures, all 3 drugs block il-1β, and anakinra and rilonacept also inhibit il-1α (47) . as a new therapeutic approach to inhibit il-1 signaling, oral nlrp3 inhibitors, including dapansutrile (olt177), are under investigation (51) . in early clinical development, anakinra was tested as a treatment for ra. despite its proven therapeutic efficacy in patients with ra, anakinra is not recommended as the first-line biologic therapy due to its low cost-effectiveness. anti-il-1 therapy is more widely used in autoinflammatory diseases such as systemic jia, aosd, and caps (52) . other conditions with high inflammatory burdens, such as gout and recurrent pericarditis, are also managed by il-1-targeted therapy. due to the broad role of il-1 in inflammatory diseases, il-1 blockades are expected to offer clinical benefit in treating intractable autoimmune diseases, including sle and systemic sclerosis, and in controlling excessive pro-inflammatory responses, such as cytokine releasing syndrome and macrophage activation syndrome (47, 53) . il-6 is a pleiotropic cytokine produced by various cell types in the context of infection, inflammation, and malignancy. il-6 was initially identified as a 'b cell differentiation factor' or 'b cell stimulation factor' secreted by t cells (54) . despite its crucial role in b cell maturation, anti-il-6 therapy failed to show therapeutic efficacy in patients with multiple myeloma (55) . however, il-6-targeted therapy with tocilizumab, the first anti-il-6rblocking mab, was shown to have significant clinical benefits in ra and was even superior to adalimumab in efficacy (56) . tocilizumab is currently approved for the treatment of ra, jia, aosd, giant cell arteritis, and cytokine releasing syndrome based on its ability to regulate systemic hyperinflammation (54) . recently, therapeutic application of anti-il-6 therapy has been investigated in sle, neuromyelitis optica, and systemic sclerosis (17, 57, 58) . il-6 has multiple biological functions in physiological and pathological conditions. in physiological states, il-6 is responsible for macrophage differentiation to the m2 state, osteoclastogenesis through the increased expression of the rank ligand on osteoblasts, and the acute phase response via the jak-stat pathway in the liver. in pathological targeted immunotherapy for autoimmune disease 8/23 https://immunenetwork.org (inflammatory) conditions, il-6 is critically involved in th17 differentiation by regulating foxp3, rorc, and il-23r expression (59) . il-6 is also important for development of follicular helper t cells and maturation of b cells (60) . il-6 signaling is transduced through a complex of il-6r and glycoprotein 130 (gp130). dimerization of gp130 sequentially activates multiple signaling pathways including jak-stat, mapk, pi3k, and yes-associated protein 1, which translocates to the nucleus and controls the transcription of genes associated with cell growth, proliferation, and inflammation (61, 62) . to interfere with the il-6 signaling pathway, anti-il6 therapy can target il-6, il-6r, gp130, jak, and stat3. however, concerns about side effects limit the potential of gp130 and stat3 as therapeutic targets (63, 64) . thus, anti-il6 mabs, anti-il-6r mabs, and jak inhibitors have been used to block il-6 signaling, and the potential for using soluble gp130 to inhibit the il-6/il-6r complex is under investigation (60). il-17a, commonly known as il-17, is the signature cytokine of th17 subsets of cd4+ t cells, but it is also produced by cd8+ t cells (tc17), γδ t cells, natural killer t cells, group 3 innate lymphoid cells, and neutrophils (65) . in the physiological state, th17 cells contribute to innate immunity by recruiting neutrophils, host defense against bacteria and fungi, and tissue repair at skin and mucosal sites (66, 67) . in pathological conditions, il-17a promotes autoimmunity, synergistically with tnf and other inflammatory chemokines (68) . animal models of inflammatory arthritis and neuroinflammation suggest the predominant role of th17 cells rather than th1 cells in the development of these diseases (69) . th17 cells are derived from naïve cd4 + naïve t cells through repression of foxp3 and activation of stat3 and rorc in the presence of il-1β, il-6 and tgf-β (59). subsequently, il-23 stabilizes pathogenic th17 cells for proliferation and survival (70) . upon binding with il-17a, a unique intracellular cytoplasmic domain termed sef/il-17r of il-17r subunit a (il-17ra) and subunit c (il-17rc) recruit the adaptor protein act1, which triggers ubiquitination of tnf-receptor associated factor (traf) 6 and subsequent activation of the nf-κb, mapk, and activator protein 1 (ap1) pathways and c/ebp transcription factors (71, 72) . a growing body of evidence from animal and human studies suggests that il-23/il-17 axis is an excellent therapeutic target for several autoimmune diseases, such as as, psa, and ra (68, 73) . current anti-il-17 therapies include mabs to il-17a (secukinumab and ixekizumab) and il-17ra (brodalumab). as expected, anti-il-17 therapeutics have produced a dramatic response in psoriasis, psa, and as (73, 74) . unexpectedly, this approach failed to show remarkable clinical efficacy in patients with ra, a th17-dependent disease (75) . this unsatisfactory result could be explained by heterogeneity in the disease itself or differential pathogenic role of il-17a depending on ra stage (e.g. early versus advanced). il-23 is a proinflammatory cytokine secreted by dcs and activated macrophages (76) . as a member of the il-12 family, il-23 is a heterodimer composed of the p40 subunit, which is shared by il-12 (il-12/il-23p40), and the p19 subunit unique to il-23 (il-23p19). although il-12 and il-23 share a structural subunit, il-23 is more critically and widely involved in the pathogenesis of autoimmune diseases. most importantly, il-23 stabilizes the pathogenic features of th17 cells through maintenance of th17 signature genes, suppression of repressive factors, upregulation of il-23r expression, and induction of effector genes (77, 78) . it also promotes the pro-inflammatory functions of dcs and macrophages as an autocrine factor (79) . il-23 interacts with a receptor complex composed of il-12rβ1 (binding with il-12/il-23p40) and il-23r (binding with il-23p19), which are linked with tyrosine kinase (tyk) 2 and jak2, respectively (78) . activation of tyk2 and jak2 leads to the phosphorylation and nuclear translocation of stats, predominantly stat3. blockade of il-23 signaling can be achieved by the targeted inhibition of il-12/il-23p40 or il-23p19. as an anti-il-12/il-23p40 mab, ustekinumab blocks both il-12 and il-23, whereas the anti-il-23p19 mabs (guselkumab, tildrakizumab, risankizumab, and mirikizumab) inhibit only il-23 (80) . similar to anti-il-17 therapy, il-23 inhibition is effective in the treatment of psoriasis and psa (80) . however, both mabs targeting the p40 and p19 subunits failed to improve the clinical course of as, in contrast to anti-il17 therapy. despite the clear pathogenic role of il-23 in preclinical models of as, such unexpected result suggests that il-23 plays a different role in a tissue-and time-dependent context in human autoimmune diseases (73) . in crohn's disease, ustekinumab successfully improved clinical outcomes, and clinical trials of anti-il-23p19 mabs are ongoing ( table 1 ). type 1 ifns, including ifn-α, ifn-β, ifn-ε, ifn-κ, and ifn-ω, were initially identified as soluble antiviral factors (10) . subsequent studies revealed that ifn-α has an essential role in the pathogenesis of autoimmune diseases, especially sle. clinical data from patients with sle showed significantly increased expression of type 1 ifn signatures (81) . the development of sle-like syndrome after recombinant ifn-α therapy also suggests a pathologic role of type 1 ifn in autoimmune disease (82) . although most nucleated cells can produce type 1 ifns, the main cellular source is pdcs (8) . nucleic acid-containing extracellular stimuli, such as rnas and dnas complexed with anti-rna and dna autoantibodies, respectively, interact with toll-like receptor 7 (tlr7) and tlr9 in pdcs to recruit myd88 and activate iraks and trafs, which lead to translocation of ifn regulatory family (irf) 7 and resultant transcription of ifn-α (10,83). type 1 ifns bind with the ifn-α/β receptor (ifnar) which consists of ifnar1 and ifnar2. upon engagement by type 1 ifns, ifnar1 and ifnar2 activate jak1 and tyk2, respectively, which leads to phosphorylation and nuclear translocation of stat1 and stat2. in the nucleus, along with irf9, stat1/stat2 induce the expression of ifn-stimulated genes (10) . in sle, ifn-α promotes the pro-inflammatory response by stimulating myeloid dcs, th1 cells, and b cells and by suppressing tregs (83) . the pathophysiologic significance of ifn-α in sle has led to the development of type 1 ifn-targeted immunotherapy. rontalizumab and sifalimumab, mabs that neutralize ifn-α, were tested in sle but failed to control disease activity (84, 85) . although the role of ifns other than ifn-α is less clear, suppressing only ifn-α while leaving ifn-β and ifn-κ active might explain the inadequate therapeutic effect in sle. recently, anifrolumab, a human mab targeting ifnar1 to block the biological function of all type 1 ifns, has been approved for moderate to severe sle (86) . anifrolumab showed a consistent clinical benefit in terms of reduced disease activity, oral corticosteroid use, and annualized flares. however, patients with lupus nephritis and neuropsychiatric lupus were excluded from the clinical trials. https://doi.org/10.4110/in.2022.22.e9 several new therapeutic approaches to block type 1 ifns are under investigation. to reduce the production of type 1 ifns, drugs are targeting pdcs and signaling molecules, such as tlr, myd88, and irak4 (83) . kinase inhibitors also are under clinical development for various autoimmune diseases based on their ability to inhibit the ifnar downstream signaling pathways. as our understanding of b cell function has expanded, b cells are considered an active participant in autoimmune diseases. interestingly, the pathologic significance of b cells in autoimmunity was highlighted by the success of b cell depletion therapies in ra and multiple sclerosis (ms). beyond their role in ab production, b cells actively participate in autoimmunity through ag-presentation, formation of tertiary lymphoid tissues, and production of cytokines, such as il-6, tnf, ifn-γ, and gm-csf (87) . ab-independent functions of b cells explain the treatment efficacy of b cell depletion therapies in ra and ms despite the absence of autoantibody reduction (88, 89) . b cells are generated in the bone marrow and sequentially differentiate into ag-specific b cells in peripheral tissues. b cell differentiation proceeds toward increasing affinity for an ag through somatic hypermutation and the class-switch recombination of immunoglobulins, accompanied by changes in b cell surface markers (90) . cd20 is expressed exclusively in the b cell lineage from pre-b cells to memory b cells, but not in plasma cells (fig. 1) . during the past several decades, anti-cd20 therapy has been the main tool for b cell depletion. rituximab, a chimeric anti-cd20 mab, redistributes cd20 into lipid rafts, and then activates complement-dependent cytotoxicity and ab-dependent cellular cytotoxicity (adcc) (90) . effective elimination of cd20 + b cells by rituximab exhausts the cellular source of ab-secreting plasmablasts and prevents the ab-independent b cell function, resulting in attenuation of autoimmune inflammation (91). following the clinical success of rituximab in ra and ms, diverse anti-cd20 therapeutics with distinct binding epitopes, different route of administration, and advanced therapeutic efficacy have been developed. ocrelizumab and ofatumumab, humanized anti-cd20 mabs, have been approved for relapsing ms, and clinical trials of other anti-cd20 mabs, obinutuzumab and ublituximab, are ongoing ( table 2) . unfortunately, rituximab was not fully successful in the treatment of sle, one of the stereotypical b-cell-mediated diseases. aside from heterogeneity in patients with sle, treatment failure with rituximab could arise from the presence of autoreactive cd20 − (but cd19 + ) long-lived plasma cells, incomplete depletion of b cells in peripheral blood and tissues, or disease flares during b-cell reconstitution (91) . for broad inhibition of b cells resistant to anti-cd20 therapy, b cell depletion therapy has targeted cd19, which is expressed on cd20 + b cells as well as cd20plasmablasts and some plasma cells. a humanized anti-cd19 mab, inebilizumab, was approved for neuromyelitis optica spectrum disorder (92) and is undergoing clinical trials to treat other inflammatory neurological disorders ( table 2) . another approach to b cell inhibition is to block b cell survival factors, specifically baff and april. baff is critically involved in b-cell maturation and survival after ligation with its 3 distinct receptors: 1) baff receptor (baff-r, known as tnfrsf13c), which is expressed on https://doi.org/10.4110/in.2022.22.e9 targeted immunotherapy for autoimmune disease 11/23 https://immunenetwork.org most b-cell subsets except plasma cells; 2) transmembrane activator, calcium modulator and cyclophilin ligand interactor (taci; known as tnfrsf13b), which is expressed on marginal zone b cells, memory b cells, and plasma cells; 3) b cell maturation ag (bcma; known as tnfrsf17) expressed on plasmablasts and plasma cells (87) . unlike baff-r, taci and bcma also bind with april, which is important for ab class switching and plasma cell survival. given the essential role of b cell maturation, proliferation, and survival, the baff/april system has been considered a promising therapeutic target in sle and its related autoimmune diseases. belimumab, a humanized anti-baff mab, is the first targeted immunotherapy approved for sle by fda.; however, belimumab does not always have universal therapeutic benefits in all patients with sle (93) . ianalumab, a mab against baff-r, has shown promising results in a phase ii trial in patients with primary sjögren syndrome (pss) (29) . as a b cell depletion therapy, ianalumab has 2 modes of action: b-cell lysis by enhanced adcc and blockade of baff signaling. telitacicept, a taci-ig fusion protein, is also under clinical development based on its inhibitory effect on the baff/april system (94). as key regulators in adaptive immunity, t cells play a critical role in the development and progression of autoimmunity. t cell effector functions require ag recognition by tcrs and additional engagement of co-stimulatory receptors. thus, modulation of those costimulatory molecules has been investigated for t cell-targeted therapy in autoimmune diseases and malignancies (95, 96) . cd28 signaling is a well-known co-stimulatory pathway for t cell activation and differentiation through activation of the mapk, akt, and nf-κb pathways (97) . cd28 expressed on cd4+ t cells binds to cd80 or cd86 on ag-presenting cells, which are shared by ctla-4 as an inhibitory counterpart of cd28 (98) . based on counter-regulation of cd28 and ctla-4, a fusion protein composed of extracellular domain of ctla4 and an igg fc region (ctla4-ig) was developed to inhibit cd28 signaling by occupying cd80 and cd86. the first ctla4-ig, abatacept, was approved to treat ra and jia with proven efficacy in the control of inflammatory arthritis (99) . subsequently, another ctla4-igs with improved binding to cd80 and cd86, including belatacept and medi5256, have been investigated for clinical applications (100, 101) . although belatacept was approved for kidney transplant recipients in 2011, the additional clinical benefit of belatacept beyond conventional calcineurin inhibitor-based regimen is controversial (102) . the cd40 pathway is the primary activation signal for t cell effector function. cd80 and cd86 expression is upregulated after ligation of cd40l to cd40 (103) . when cd40l on activated t cells binds to cd40 on target cells, multiple kinase cascades activate transcription factors, such as nf-κb and ap1, to induce cell survival, proliferation and differentiation (104) . cd40 signaling is particularly important for the b cell-t cell interactions involved in t cell-dependent ab responses, germinal center formation, and memory b cell differentiation (105) . due to its critical role in b cell function, the therapeutic potential of targeting cd40 signaling has been investigated in preclinical and clinical studies. a recent phase ii trial of iscalimab, a humanized anti-cd40 mab, showed clinical improvement in patients with pss (106) , and a subsequent phase iii trial is ongoing ( table 2 ). immunotherapies that target other co-stimulatory signaling, such as the inducible t cell costimulator (icos) and ox40 pathways, are also being developed to inhibit the t cell effector https://doi.org/10.4110/in.2022.22.e9 targeted immunotherapy for autoimmune disease 12/23 https://immunenetwork.org function in autoimmune diseases (96). as co-stimulatory molecules expressed on activated cd4+ t cells, icos and ox40 are involved in t cell survival, differentiation, and activation. as previously stated, binding soluble inflammatory mediators to their cognate receptors transduce signals through activation of intracellular kinases, typically the jak family. the jak family, which consists of jak1, jak2, jak3, and tyk2, is associated with cytokine receptors (including il-2r, il-4r, il-5r, il-6r, il-13r, and type 1 ifns), growth hormones and erythropoietin (4) . activation of the jak family leads to phosphorylation, dimerization, and nuclear transformation of stats. in the nucleus, stats contribute to the gene transcription involved in cell differentiation, proliferation, and survival, cytokine production, and angiogenesis (107, 108) . due to the pivotal role of jak in hematopoiesis, jak inhibitors were first evaluated for treatment of hematologic diseases, especially myeloproliferative neoplasm (108) . since ruxolitinib (a jak1 and jak2 inhibitor) was first approved for myelofibrosis in 2011, numerous kinase inhibitors have been approved or investigated for treatment of hematologic malignancies and inflammatory diseases (5) . several jak inhibitors have been approved for ra, psa, and ulcerative colitis based on their proven clinical efficacy (108) . selectivity for jak differs by each jak inhibitor, although differences in therapeutic efficacy have not been demonstrated between jak inhibitors. tofacitinib and baricitinib mainly target jak1/3 and jak1/2, respectively. upadacitinib and filgotinib are second-generation jak inhibitors that selectively inhibit jak1. in contrast to the aforementioned immunotherapies, which regulate cellular activation signals in the extracellular space, jak inhibitors target signaling pathways in the intracellular space. jak-targeted therapy inhibits the convergent signals from multiple cytokine receptors and disrupts the feedback loop (4). as a result, even a partial blockade of select kinases can efficiently downregulate multiple inflammatory signaling pathways, attenuating autoimmune inflammation. it should be noted that in a head-to-head, randomized controlled trial, baricitinib showed a better clinical response than the anti-tnf mab adalimumab (109) . in addition to jak inhibitors, a selective tyk2 inhibitor, deucravacitinib, is awaiting clinical approval as the first oral targeted therapy in psoriasis (110) . tyk2 mediates the intracellular inflammatory signals originating from il-12, il-23, and type 1 ifns. the therapeutic potential of deucravacitinib is currently being investigated in patients with psa, inflammatory bowel diseases, and sle based on mechanistic insights into the pathogenesis of those diseases ( table 3) . bruton tyrosine kinase (btk) inhibitors are also expected to ameliorate autoimmune inflammation by modulating b cell function. btk inhibitors are currently being investigated in various autoimmune disease, including ra, ms, pss, and sle (4). some recent phase ii trials with btk inhibitors showed promising results in patients with relapsing ms (111, 112) . the future direction of kinase inhibition is development of more selective inhibitors with minimal off-target effects and improved organ specificity based on advanced molecular techniques. furthermore, on-target effects caused by unregulated cellular behaviors other than https://doi.org/10.4110/in.2022.22.e9 targeted immunotherapy for autoimmune disease https://immunenetwork.org amelioration of inflammation should be investigated extensively. recent evidence suggests that patients treated with tofacitinib face an increased risk of cardiac events, malignancies, thrombosis, and death, and fda has decided to revise the boxed warnings of jak inhibitors (113) . the chronic course of autoimmune diseases raises safety concerns for long-term drug use, which can substantially influence drug selection by patients and physicians. with better understandings of the immuno-pathogenesis of autoimmune diseases and continuing advances in biotechnology, numerous drugs with novel therapeutic targets and advanced efficacy continue to be developed, showing promisingly efficacious results. notwithstanding all of this, there are still unmet medical needs, such as heterogeneous therapeutic effects and adverse events caused by immunosuppression. to overcome the limitations of current treatments, therapeutic approaches for inflammatory diseases have been diversified and individualized. here, we briefly introduce a new perspective of targeted immunotherapy enabled by advanced molecular engineering techniques. the basic concept of car t cell therapy is administration of autologous t cells containing genetically engineered tcrs to capture tumor-specific ags and eliminate tumor cells by increasing cytotoxicity. following therapeutic success in a patient with diffuse large b cell lymphoma, cd19 car t cell therapy was approved for treatment of b-cell lymphoma and acute lymphoblastic leukemia (114) . cars are engineered receptors composed of an extracellular ag recognition domain, a transmembrane domain, and a intracellular t cell activation domain (115) . the extracellular domain, also known as single-chain fv, is designed as the variable heavy and light chains of a monoclonal ab. ag recognition by extracellular domains activates the immunoreceptor tyrosine-based activation motifs on intracellular domains, leading to cytokine production and lysis of target cells. to improve therapeutic efficacy, the intracellular domain consists of an activation domain, cd3ζ, and a co-stimulatory domain, such as cd28. although car t cells were first introduced in the field of oncology, their therapeutic potential to eliminate pathologic cells extends to the treatment of autoimmune inflammatory diseases. recently, mougiakakos et al. (116) reported a rapid clinical remission with autologous cd19 car t cell therapy in a patients with sle refractory to conventional treatment. in that patient, infused cd19 car t cells were detectable in the peripheral blood for 7 weeks after a single dose with a rapid and sustained decrease in anti-dsdna ab. that is a promising result in that current b cell depletion with anti-cd20 mabs requires regular injections and attention to immunogenicity to maintain the initial therapeutic effect. with the development of engineered t cell therapy, chimeric autoantigen receptor (caar) t cell and car treg therapies have drawn attention as novel therapeutic strategies for autoimmune diseases (117) . the extracellular domain of caar t cells expresses an autoantigen that can interact with ag-specific autoreactive b cells and t cells. caar t cell therapy could be effective in autoimmune diseases with known autoantigens. car treg cells are also being designed with the expectation of an ag-specific immune-regulatory response that cannot be adequately achieved by infusion of polyclonal treg cells. however, https://doi.org/10.4110/in.2022.22.e9 targeted immunotherapy for autoimmune disease such engineered t cell therapies for autoimmune diseases have not yet been widely studied. moreover, despite the hypothetical therapeutic effect of engineered t cell therapies, cytokine releasing syndrome and neurotoxicity could limit enthusiasm for the use of cell therapies in autoimmune diseases, particularly in high inflammation microenvironments (118) . il-2 is a key cytokine for differentiation, activation, and survival of treg cells and is mostly produced by activated cd4 + t cells (119) . tregs, defined by cd4 + foxp3 + cd25 + cd127 low expression, modulate autoreactive immune cells through secretion of anti-inflammatory cytokines (il-10, tumor growth factor-β, and il-35), induction of immunosuppressive enzyme (indoleamine 2,3-dioxygenase) by dcs, inactivation of effector t cells and nk cells, and direct cytotoxicity against cd8 + t cells and nk cells (119, 120) . treg cell deficiency and/or dysfunction are frequently reported in various autoimmune diseases, such as ra, sle, pss, and as (120) . in addition to activating treg cells, il-2 induces survival and proliferation of effector t cells and inhibits th17 cell differentiation (119) . based on its stimulatory effect on conventional t cells, high-dose il-2 therapy was first approved for metastatic renal cell cancer and metastatic melanoma. however, later studies have shown that the main role of il-2 is mediated through the high-affinity il-2r expressed on treg cells. il-2r is expressed in monomeric, dimeric, and trimeric variants, and trimeric il-2r has higher affinity than the monomeric and dimeric forms (121) . because treg cells constitutively express trimeric il-2r at high level, treg cells are highly sensitive to il-2 than effector memory cd4+ t cells. thus, low-dose il-2 therapy (0.3 to 3 million international unit/day) has been evaluated in autoimmune diseases in anticipation that it will induce immune tolerance by means of treg stimulation while having minimal effects on effector t cells (120) . in patients with autoimmune diseases, including sle, psa, and hepatitis c virus-induced cryoglobulinemic vasculitis, low dose il-2 therapy improved clinical outcomes, which was consistent with an increase in treg cells (120) . in clinical trials, low dose il-2 had a tolerable safety profile and was not associated with an increased risk of infection. moreover, in mice, immune responses to vaccination, infection, and cancer were not impaired by long-term treatment with low dose il-2 (122). the favorable clinical trial results have led to the development of engineered il-2 proteins with altered amino acid sequences (il-2 muteins) to improve selectivity and reduce adverse effects (120) . combinations of il-2 and biologic therapies such as tnf-and il-6 inhibition are also under investigation. inducing immune tolerance to autoantigens without disrupting the rest of the immune system is an ideal approach to autoimmune diseases. in this regard, oral tolerance that can induce ag-specific immune tolerance has been investigated for more than a century (123) . gut-associated lymphoid tissue (galt) is the largest immune organ that maintains tolerance to large amounts of food and commensal microorganisms (124) . given the role of galt in intestinal homeostasis and systemic regulation, the tolerogenic effects of fed ags on systemic autoimmune diseases have been studied extensively in preclinical and clinical trials. in animal models, oral tolerance induction was found to effectively prevent and even treat various inflammatory diseases, including inflammatory arthritis, experimental autoimmune https://doi.org/10.4110/in.2022.22.e9 targeted immunotherapy for autoimmune disease encephalitis, diabetes, and uveitis (123) . depending on the dose of ag, low doses of ag promote the generation of treg cells secreting il-4, il-10, and tgf-β, whereas high doses of ag promote the clonal deletion or anergy of specific t cells (125) . however, such mechanisms of immune tolerance are not exclusive and can overlap. early pilot trials and phase ii trials of oral tolerance induction also showed promising results in patients with ra, ms, and uveitis without treatment-related toxicity. however, phase iii trials of ag feeding in patients with ra and ms showed a suboptimal therapeutic effects (125) . despite the experimental efficacy and acceptable safety of ag feeding, unsolved issues need to be addressed to enable successful induction of immune tolerance: 1) protocol for ag feeding (dose and type of ag, use of mucosal adjuvant, and frequency of administration); 2) combined treatment with conventional immunosuppressive drugs; 3) biological and immunological markers that reflect the effectiveness of oral tolerance development; and 4) selection of suitable patients amenable to induced oral tolerance (age, disease onset, and history of desensitization). another approach to ag-specific tolerance is tolerogenic vaccines that deliver autoantigens. tolerogenic vaccines have successfully induced ag-specific treg cells, and promoted autoreactive t cell anergy and apoptosis in animal models of autoimmune diseases (126, 127) . several platforms of tolerogenic vaccines, including protein/peptide-, nanoparticle-, cell-, and dna/rna-based vaccines, have been investigated to enhance immunogenicity in preclinical and clinical research of ms (128) . although a phase iii trial using a peptide vaccine failed to show clinical benefit (129) , the tolerogenic approach is still being studies using several vaccine platforms in patients with ms. recently, krienke et al. (130) reported successful induction of immune tolerance with a noninflammatory mrna-based vaccine in mice model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (mog35-55). although double-stranded rna molecules are inherently proinflammatory in the extracellular environment, their proinflammatory nature was reduced by replacing uridine with 1-methylpseudouridine (m1ψ). the mog35-55-encoding m1ψ-modified single-stranded mrna vaccine prevented disease development and progression in mice. the tolerogenic vaccine induced mog35-55-specific foxp3 + treg cells and suppressed mog33-35-specific th1 and th17 cells. expression of inhibitory molecules such as pd1 and ctla4 was upregulated on ag-specific cells, and the protective effect of the vaccine was abrogated by pd1-and ctla-4-targeted checkpoint inhibitors. nonetheless, development of tolerogenic vaccines is challenging because it is inconclusive and controversial which autoantigens are specific for autoimmune diseases, and multiple autoantigens could be involved in most autoimmune diseases. advances in targeted immunotherapy during the past few decades have revolutionized the treatment and clinical outcomes of patients with autoimmune diseases. current targeted immunotherapies lead to suppression of major pro-inflammatory signaling pathways by blocking inflammatory cytokines, cell surface molecules, and intracellular kinases. despite the tremendous success of targeted therapies in autoimmune disease, unmet medical needs https://doi.org/10.4110/in.2022.22.e9 targeted immunotherapy for autoimmune disease remain in terms of drug efficacy and long-term safety. beyond blocking inflammatory signaling pathways, future therapies aim to induce long-standing immune tolerance while maintaining protective immune functions. hopefully, advances in biotechnology and knowledge of diseases will provide opportunities to develop new drugs with improved therapeutic efficacy and minimal adverse effects. additionally, considering the multifaceted roles of inflammatory mediators and immune cells, the development of more selective and specific drugs will be required together with a more precise understanding of disease pathogenesis. recent insights in the epidemiology of autoimmune diseases: improved prevalence estimates and understanding of clustering of diseases recent advances in (therapeutic protein) drug development kinase inhibition in autoimmunity and inflammation trends in kinase drug discovery: targets, indications and inhibitor design the multiple pathways to autoimmunity immunopathogenic mechanisms of systemic autoimmune disease the multifaceted biology of plasmacytoid dendritic cells the role of monocytes and macrophages in autoimmune diseases: a comprehensive review type i interferons in the pathogenesis and treatment of autoimmune diseases peripheral cd4+ t-cell differentiation regulated by networks of cytokines and transcription factors effect of canakinumab vs placebo on survival without invasive mechanical ventilation in patients hospitalized with severe covid-19: a randomized clinical trial efficacy and safety of sirukumab in patients with active rheumatoid arthritis refractory to anti-tnf therapy (sirround-t): a randomised, double-blind, placebo-controlled, parallel-group, multinational, phase 3 study sirukumab for rheumatoid arthritis: the phase iii sirround-d study olokizumab, a monoclonal antibody against interleukin 6, in combination with methotrexate in patients with rheumatoid arthritis inadequately controlled by methotrexate: efficacy and safety results of a randomised controlled phase iii study the efficacy and safety of clazakizumab, an anti-interleukin-6 monoclonal antibody, in a phase iib study of adults with active psoriatic arthritis safety and efficacy of tocilizumab versus azathioprine in highly relapsing neuromyelitis optica spectrum disorder (tango): an open-label, multicentre, randomised, phase 2 trial tocilizumab in patients hospitalized with covid-19 pneumonia tocilizumab in hospitalized patients with severe covid-19 pneumonia sarilumab in patients admitted to hospital with severe or critical covid-19: a randomised, double-blind, placebo-controlled, phase 3 trial improvement of signs and symptoms of nonradiographic axial spondyloarthritis in patients treated with secukinumab: primary results of a randomized, placebocontrolled phase iii study efficacy and safety of brodalumab, an anti-il17ra monoclonal antibody, in patients with axial spondyloarthritis: 16-week results from a randomised brodalumab in psoriatic arthritis: results from the randomised phase iii amvision-1 and amvision-2 trials bimekizumab efficacy and safety in moderate to severe plaque psoriasis (be ready): a multicentre, double-blind, placebo-controlled, randomised withdrawal phase 3 trial safety and efficacy of subcutaneous ianalumab (vay736) in patients with primary sjögren's syndrome: a randomised, double-blind, placebo-controlled, phase 2b dose-finding trial efficacy and safety of abatacept, a t-cell modulator, in a randomised, double-blind, placebo-controlled, phase iii study in psoriatic arthritis tofacitinib in juvenile idiopathic arthritis: a double-blind, placebocontrolled, withdrawal phase 3 randomised trial tofacitinib for the treatment of ankylosing spondylitis: a phase iii, randomised, doubleblind, placebo-controlled study baricitinib in patients with moderate-to-severe atopic dermatitis: results from a randomized monotherapy phase 3 trial in the united states and canada (breeze-ad5) efficacy and safety of baricitinib combined with topical corticosteroids for treatment of moderate to severe atopic dermatitis: a randomized clinical trial efficacy of upadacitinib in a randomized trial of patients with active ulcerative colitis once-daily upadacitinib versus placebo in adolescents and adults with moderateto-severe atopic dermatitis (measure up 1 and measure up 2): results from two replicate double-blind, randomised controlled phase 3 trials safety and efficacy of upadacitinib in combination with topical corticosteroids in adolescents and adults with moderate-to-severe atopic dermatitis (ad up): results from a randomised, double-blind, placebo-controlled, phase 3 trial filgotinib as induction and maintenance therapy for ulcerative colitis (selection): a phase 2b/3 double-blind, randomised, placebo-controlled trial pathogenic and protective functions of tnf in neuroinflammation are defined by its expression in t lymphocytes and myeloid cells translating molecular insights in autoimmunity into effective therapy tnf blockade induces a dysregulated type i interferon response without autoimmunity in paradoxical psoriasis paradoxical expansion of th1 and th17 lymphocytes in rheumatoid arthritis following infliximab treatment: a possible explanation for a lack of clinical response insights into the biology and therapeutic implications of tnf and regulatory t cells the il-1 family of cytokines and receptors in rheumatic diseases recognition of bacteria by inflammasomes autoinflammation: lessons from the study of familial mediterranean fever interleukin-1 and related cytokines in the regulation of inflammation and immunity pharmacological inhibitors of the nlrp3 inflammasome autoinflammation and autoimmunity across rheumatic and musculoskeletal diseases macrophage activation syndrome in the era of biologic therapy translating il-6 biology into effective treatments biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind, controlled phase 4 trial tocilizumab in systemic sclerosis: a randomised, double-blind, placebo-controlled, phase 3 trial efficacy and safety of an interleukin 6 monoclonal antibody for the treatment of systemic lupus erythematosus: a phase ii dose-ranging randomised controlled trial reciprocal developmental pathways for the generation of pathogenic effector th17 and regulatory t cells interleukin-6: designing specific therapeutics for a complex cytokine interleukin-6-type cytokine signalling through the gp130/jak/stat pathway a gp130-src-yap module links inflammation to epithelial regeneration targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders cytokine signaling in 2002: new surprises in the jak/stat pathway innate il-17-producing cells: the sentinels of the immune system the il-17 family of cytokines in health and disease interleukin 17 is a chief orchestrator of immunity distinct roles of interleukin-17 and t helper 17 cells among autoimmune diseases mechanisms underlying helper t-cell plasticity: implications for immune-mediated disease il-23 drives a pathogenic t cell population that induces autoimmune inflammation distinct functional motifs within the il-17 receptor regulate signal transduction and target gene expression the adaptor act1 is required for interleukin 17-dependent signaling associated with autoimmune and inflammatory disease the il-23-il-17 pathway as a therapeutic target in axial spondyloarthritis discovery of the il-23/il-17 signaling pathway and the treatment of psoriasis a phase ii randomized study of subcutaneous ixekizumab, an anti-interleukin-17 monoclonal antibody, in rheumatoid arthritis patients who were naive to biologic agents or had an inadequate response to tumor necrosis factor inhibitors novel p19 protein engages il-12p40 to form a cytokine, il-23, with biological activities similar as well as distinct from il-12 the il-23-il-17 immune axis: from mechanisms to therapeutic testing insights into il-23 biology: from structure to function effects of il-12 and il-23 on antigen-presenting cells at the interface between innate and adaptive immunity critical role of interleukin (il)-17 in inflammatory and immune disorders: an updated review of the evidence focusing in controversies an update on the role of type i interferons in systemic lupus erythematosus and sjögren's syndrome side effects of high-dose interferon therapy for chronic hepatitis c targeting interferons and their pathways in systemic lupus erythematosus a phase ii study of the efficacy and safety of rontalizumab (rhumab interferon-α) in patients with systemic lupus erythematosus (rose) greth w; cd1067 study investigators. sifalimumab, an anti-interferon-α monoclonal antibody, in moderate to severe systemic lupus erythematosus: a randomised, double-blind, placebo-controlled study trial of anifrolumab in active systemic lupus erythematosus b cell depletion therapies in autoimmune disease: advances and mechanistic insights changes in b-and t-lymphocyte and chemokine levels with rituximab treatment in multiple sclerosis efficacy of b-cell-targeted therapy with rituximab in patients with rheumatoid arthritis monoclonal antibodies directed to cd20 and hla-dr can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells anti-cd20-mediated b-cell depletion in autoimmune diseases: successes, failures and future perspectives inebilizumab for the treatment of neuromyelitis optica spectrum disorder (n-momentum): a double-blind, randomised placebo-controlled phase 2/3 trial smoking and pre-existing organ damage reduce the efficacy of belimumab in systemic lupus erythematosus telitacicept as a blys/april dual inhibitor for autoimmune disease co-stimulatory receptors in cancers and their implications for cancer immunotherapy targeting co-stimulatory molecules in autoimmune disease cd28 costimulation: from mechanism to therapy ctla-4: a moving target in immunotherapy a review on applications of abatacept in systemic rheumatic diseases rational development of lea29y (belatacept), a high-affinity variant of ctla4-ig with potent immunosuppressive properties a cd80-biased ctla4-ig fusion protein with superior in vivo efficacy by simultaneous engineering of affinity, selectivity, stability, and fcrn binding should belatacept be the centrepiece of renal transplantation? activation of human dendritic cells through cd40 cross-linking cd40-cd40 ligand gp39-cd40 interactions are essential for germinal center formation and the development of b cell memory assessment of the anti-cd40 antibody iscalimab in patients with primary sjögren's syndrome: a multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study mechanisms and consequences of jak-stat signaling in the immune system current and future status of jak inhibitors baricitinib versus placebo or adalimumab in rheumatoid arthritis phase 2 trial of selective tyrosine kinase 2 inhibition in psoriasis evobrutinib phase 2 study group. placebo-controlled trial of an oral btk inhibitor in multiple sclerosis safety and efficacy of tolebrutinib, an oral brain-penetrant btk inhibitor, in relapsing multiple sclerosis: a phase 2b, randomised, double-blind, placebo-controlled trial fda requires warnings about increased risk of serious heart-related events, cancer, blood clots, and death for jak inhibitors that treat certain chronic inflammatory conditions car t cell immunotherapy for human cancer engineering strategies to overcome the current roadblocks in car t cell therapy cd19-targeted car t cells in refractory systemic lupus erythematosus is there a place for chimeric antigen receptor-t cells in the treatment of chronic autoimmune rheumatic diseases? recent advances in car t-cell toxicity: mechanisms, manifestations and management biology and regulation of il-2: from molecular mechanisms to human therapy interleukin-2 and regulatory t cells in rheumatic diseases interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy sustained stimulation and expansion of tregs by il2 control autoimmunity without impairing immune responses to infection, vaccination and cancer oral tolerance as antigen-specific immunotherapy anatomical basis of tolerance and immunity to intestinal antigens oral tolerance: therapeutic implications for autoimmune diseases targeting of autoantigens to dec205 + dendritic cells in vivo suppresses experimental allergic encephalomyelitis in mice microparticles bearing encephalitogenic peptides induce t-cell tolerance and ameliorate experimental autoimmune encephalomyelitis emerging therapeutics for immune tolerance: tolerogenic vaccines, t cell therapy, and il-2 therapy a phase iii study evaluating the efficacy and safety of mbp8298 in secondary progressive ms a noninflammatory mrna vaccine for treatment of experimental autoimmune encephalomyelitis key: cord-0029401-317zhrki authors: zeng, liuting; yu, ganpeng; yang, kailin; xiang, wang; li, jun; chen, hua title: efficacy and safety of mesenchymal stem cell transplantation in the treatment of autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and ankylosing spondylitis): a systematic review and meta-analysis of randomized controlled trial date: 2022-03-24 journal: stem cells int doi: 10.1155/2022/9463314 sha: 8c1309c5c8553d9981095c9baa13f1de4fd5bf0f doc_id: 29401 cord_uid: 317zhrki objective: to evaluate the efficacy and safety of mesenchymal stem cell (msc) transplantation in the treatment of autoimmune diseases. methods: the chinese and english databases were searched for clinical research on the treatment of autoimmune diseases with mesenchymal stem cells. the search time range is from a self-built database to october 1, 2021. two reviewers independently screened the literature according to the inclusion and exclusion criteria, extracted data, and evaluated the bias of the included studies. revman 5.3 analysis software was used for meta-analysis. results: a total of 18 rcts involving 5 autoimmune diseases were included. the 5 autoimmune disease were rheumatoid arthritis (ra), systemic lupus erythematosus (sle), inflammatory bowel disease, ankylosing spondylitis, and multiple sclerosis. for ra, the current randomized controlled trials (rcts) still believe that stem cell transplantation may reduce disease activity, improve the clinical symptoms (such as das28), and the percentage of cd4+cd 25+foxp3+tregs in the response group increased and the percentage of cd4+il-17a+th17 cells decreased. the total clinical effective rate of ra is 54%. for sle, the results showed that mesenchymal stem cell transplantation may improve sledai [-2.18 (-3.62, -0.75), p = 0.003], urine protein [-0.93 (-1.04, -0.81), p < 0.00001], and complement c3 [0.31 (0.19, 0.42), p < 0.00001]. for inflammatory bowel disease, the results showed that mesenchymal stem cell transplantation may improve clinical efficacy [2.50 (1.07, 5.84), p = 0.03]. for ankylosing spondylitis, msc treatment for 6 months may increase the total effective rate; reduce erythrocyte sedimentation rate, intercellular adhesion molecules, and serum tnf-α; and improve pain and activity. for multiple sclerosis, the current research results are still controversial, so more rcts are needed to amend or confirm the conclusions. no obvious adverse events of mesenchymal stem cell transplantation were found in all rcts. conclusion: mscs have a certain effect on different autoimmune diseases, but more rcts are needed to further modify or confirm the conclusion. autoimmune diseases are a series of diseases caused by the immune system's response to self-antigens, resulting in self-tissue damage or dysfunction [1] . it mainly includes systemic lupus erythematosus (sle), rheumatoid arthritis (ra), sjogren's syndrome, polymyositis and dermatomyositis [1, 2] . many autoimmune diseases are characterized by the production of autoantibodies, which bind to the host's own proteins or form immune complexes and deposit in tissues. any organ of the body may become a target organ for autoimmunity, including skin, joints, kidneys, and blood vessels. the inflammatory effect caused by autoantibodies is mediated by binding to fc receptors on leukocytes, which is an important cause of downstream tissue damage [3, 4] . meanwhile, autoantibodies can also directly mediate tissue damage in diseases through complement activation [5] . in the development stage of the disease, genetic factors and environmental factors may interact in turn to promote the development of autoimmunity and ultimately lead to tissue inflammation and damage, becoming a chronic disease with multiple organs and multiple system damage [6, 7] . clinically, once diagnosed, patients should be treated with medication in time to avoid further development of the disease causing damage to organs or systems such as the liver and kidney [8] . clinically, commonly used glucocorticoids and traditional disease-improving antirheumatic drugs (dmards) have good anti-inflammatory, pain-relieving, and improving or delaying disease progression effects and are still used as the first-line choice for clinical treatment of rheumatic immune diseases [9, 10] . however, for first-line treatments with single or combined regimens that do not respond well or cannot tolerate them, other treatment options with potential curative effects need to be considered [11] [12] [13] . for example, stem cell transplantation, biological preparations, or new botanical preparations, as well as some antirheumatic drug candidates that may modulate or suppress immunity, in order to alleviate the condition of patients with refractory rheumatism and improve their quality of life. among them, mesenchymal stem cells are one of the most promising therapeutic strategies [14] . since the discovery of mesenchymal stem cells (msc), the understanding of them has continued to deepen. because of its proliferation and differentiation ability, the medical community expects it to be used in the treatment of clinical diseases [14] . in the past few decades, the initial research on msc focused on its differentiation ability, but with the discovery of its immunomodulatory function, the direction of msc-based therapeutic research has changed from the initial regenerative medicine to autoimmune diseases [15] . so far, there have been many reports in the literature on the treatment of autoimmune diseases with msc [16, 17] , and there are more and more researches on the application of msc in clinical trials. one-third of the clinical studies focused on the treatment of autoimmune diseases by msc [18] . studies have found that mscs are weakly immunogenic and did not express major histocompatibility complex (mhc) class ii molecules, apoptotic gene ligands, and t cell costimulatory molecules (b7-1, b7-2, cd40, and cd40l). it did not express or express mhc class i molecules at very low levels, did not induce an immune response in vitro, and also had an immunosuppressive effect. these studies have laid the theoretical foundation for the transplantation of allogeneic bone marrow mscs to treat autoimmune diseases [19] . for some autoimmune diseases (such as rheumatoid arthritis (ra) and sle, crohn's disease, primary sjogren's syndrome, systemic sclerosis, dermatomyositis, ankylosing spondylitis, psoriasis, multiple sclerosis), animal experiments and clinical controlled trials have also shown that msc transplantation can improve the clinical symptoms of the above diseases [18] [19] [20] . due to the relative uncontrollability of cell culture used in these clinical randomized controlled trials, the complexity of clinical trial design, and the implementation factors of effective evaluation measures, there is an urgent need for a comprehensive systematic review and meta-analysis of the clinical controlled trials of mesenchymal stem cells for the treatment of autoimmune diseases. therefore, this study registered the protocol on prosepro in order to provide a complete and comprehensive evaluation and provide new evidence for clinical practice. 2.1. protocol. this systematic review and meta-analysis were conducted strictly in accordance with the protocol registered in prospero (crd42021277144) and prisma guidelines (see supplementary materials) [21] . chinese databases (including cnki, vip database, wanfang database, and sinomed) and english databases (including embase, medline, pubmed, and web of science) were searched from the establishment of the database to oct. 1st, 2021. cochrane library and clinicaltrials.gov were also searched. the research retrieval strategy of embase and pubmed were shown in table s1. 2.3.1. participants. patients who have been diagnosed with any kind of autoimmune disease according to authoritatively recognized standards were included. patients had no restrictions on gender, age, region, etc. methods. the intervention of the experimental group was mesenchymal stem cell (msc), which can be used alone or in combination with other therapies. the intervention measures of the control group were non-msc therapy, which could be traditional therapy, placebo, etc. [22] . the risk of bias was independently assessed by two researchers in accordance with the cochrane risk bias assessment form [23] provided by the cochrane collaboration. two researchers independently screened the literature, extracted data, and assessed the quality of rcts. if there is a disagreement, they will negotiate with the third researcher. 2.5. statistical analysis. revnan5.3 was used for metaanalysis [22] . relative risk (rr) and mean difference (md) are used as the combined effect size of dichotomous variables (such as adverse events and effective rate) and continuous variables (such as sledai score), respectively. the heterogeneity between rcts was tested by chi-square test, and the test standard was p < 0:1. the degree of heterogeneity was judged based on i 2 . when i 2 > 50%, it indicated that there is high heterogeneity, and a random effect model was established. on the contrary, when i 2 < 50%, a fixed effect model was established. a total of 1109 records were retrieved initially, and 23 records were left for further screening after preliminary screening. finally, 18 rcts were included for they meet the search criteria, while 5 records were excluded [24] [25] [26] [27] [28] . the literature screening process is shown in figure 1 . concealment. nine rcts [30, 34, 38, 39, 41-43, 45, 46] described the random sequence generation methods and were rated as low risk of bias. other rcts did not describe the random sequence generation method and were assessed as unclear risk of bias. four rcts [30, 36, 42, 45] describe allocation concealment methods and were assessed as low risk of bias. panés et al. [38] did not perform allocation concealment and was assessed as high risk of bias. other rcts did not describe the allocation concealment methods and were assessed as unclear risk of bias. [31] and tang et al. [32] claimed to use the blind method but did not describe the implementation process; shadmanfar et al. [30] did not mention whether to use blinding; therefore, they were rated as unclear risk of bias. other rcts did not use blinding and their outcomes were subjective indicators; hence, they were rated as high risk of bias. six rcts [29-31, 33, 36, 44] have missing data and did not use appropriate statistical treatment method; hence, they were rated as unclear risk of bias. 3.4. other potential bias. other sources of bias were not observed, and they were rated as low risk of bias. 3.5. rheumatoid arthritis. ra often manifests as joint swelling, joint stiffness, and tenderness in the morning. it is mainly due to the invasion and damage of the cartilage and bone due to synovial hyperplasia, which involves a variety of immune cells and mediated inflammation. three rcts reported msc treatment of ra. however, due to their different data presentation methods, a systematic review was conducted. among all rcts, the use of bone marrow mesenchymal stem cells is generally safe and tolerable. yang et al. [29] showed that after msc treatment, the disease activity was weakened and the clinical symptoms (including das28) were improved. the improvement of most patients' condition lasts for 12 months, and the total clinical effective rate is 54%. two patients in the response group had pain and swelling at 24 weeks, and their esr and crp levels increased. it is also found that the dosage of prednisone acetate in 23 patients in the experimental group gradually decreased after the intervention. for the immune response, it found that the percentage of cd4+cd 25+foxp3+tregs in the response group increased and the percentage of cd4 +il-17a+th17 cells decreased; and the levels of il-6 and tnf-α decreased significantly. shadmanfar et al. [30] shows that msc may improve the patient's standing time and womac total score and reduce the use of methotrexate and prednisolone. it also showed that patients with knee involvement found that knee pain was reduced by more than 50%. álvaro-gracia et al. [31] showed that a moderate proportion of patients meets the comprehensive measure of acr 20/50/70 response, but fewer patients achieve an improvement of 50% or 70%. 3.6. systemic lupus erythematosus. sle mainly manifests as specific skin lesions, fatigue, weakness, fever, and weight loss and other inflammatory symptoms (such as decreased serum c3). the symptoms of multiple organs are related to the involvement of organs. it was mainly evaluated by sledai. if the kidney is involved, urine protein would be used to assess the kidney involvement. 3.6.1. sledai. four rcts reported sledai [32] [33] [34] [35] . the heterogeneity test showed that i 2 = 52%, p = 0:10, considering the moderate heterogeneity among rcts. therefore, the random effects model is used for data analysis. the results show that the sledai in the experimental group was lower than that in the control group (-2.18 (-3.62, -0.75), p = 0:003) (figure 4 ). 3.6.2. urine protein. four rcts reported urine protein [32] [33] [34] [35] . the heterogeneity test showed that i 2 = 0%, p = 0:72, considering the low heterogeneity among rcts. therefore, the fixed effects model is used for data analysis. the results show that the urine protein in the experimental group was lower than that in the control group (-0.93 (-1.04, -0.81), p < 0:00001) ( figure 5 ). 3.6.3. serum c3. three rcts reported serum c3 [33] [34] [35] . the heterogeneity test showed that i 2 = 22%, p = 0:28, considering the low heterogeneity among rcts. therefore, the fixed effects model is used for data analysis. the results show that the serum c3 in the experimental group was higher than that in the control group (0.31 (0.19, 0.42), p < 0:00001) ( figure 6 ). 3.6.4. adverse events. three rcts reported serum adverse events [32, 34, 35] . the heterogeneity test showed that i 2 = 0%, p = 0:74, considering the low heterogeneity among rcts. therefore, the fixed effects model is used for data analysis. the results show that the incidence of adverse events between two groups was of no statistical significance (0.87 (0.33, 2.29), p = 0:79) (figure 7 ). 3.7. inflammatory bowel disease. inflammatory bowel disease is a chronic nonspecific gastrointestinal disease, which is disabling, can seriously affect all aspects of patients' lives, and also causes a heavy burden on the health care system and society. it mainly includes crohn's disease and ulcerative colitis. crohn's disease is an inflammatory bowel disease characterized by chronic inflammation of any part of the gastrointestinal tract, with a progressive and destructive course. the clinical symptoms are mainly diarrhea, abdominal pain, blood in the stool, fever, and fatigue. ulcerative colitis mainly manifests as abdominal pain, rectal pain, bleeding, difficulty in defecation, fever, and fatigue. a total of 4 rcts were included [36] [37] [38] [39] . the heterogeneity test showed that i 2 = 74%, p = 0:009, considering the high heterogeneity among rcts. stem cells international therefore, the random effects model is used for data analysis. the results show that the clinical efficacy of the experimental group is better than that of the control group (2.50 (1.07, 5.84), p = 0:03) ( figure 8 ). a total of 4 rcts were included [36] [37] [38] [39] . the heterogeneity test showed that i 2 = 0%, p = 0:52, considering the low heterogeneity among rcts. therefore, the fixed effects model is used for data analysis. the results show that the incidence of adverse events between two groups were of no statistical significance (0.99 (0.81, 1.22), p = 0:96) ( figure 9 ). ankylosing spondylitis mainly manifests as chronic back pain and stiffness. it may be due to erosion, bone growth and vertebral fusion, and inflammatory damage involving th1/17 and related cytokines. only one rct reported the treatment of ankylosing spondylitis with msc. su et al. [40] found that compared with the fliximab group (control group), msc treatment for 6 months may increase the total effective rate; reduce erythrocyte sedimentation rate, intercellular adhesion molecules, and serum tnf-α; and improve pain and activity. 3.9. multiple sclerosis. multiple sclerosis is an immune disease characterized by chronic demyelination of the central nervous system. in multiple sclerosis patients, monocytes infiltrate into the perivascular space between the arteries and veins and pia mater, axon myelin sheath is lost and destroyed, and glial cell immunoreactivity changes lead to the formation of plaques in multiple parts of the central nervous system. su et al. [40] found that the progression-free survival (pfs) rate, total number of episodes, and average number of episodes each year in the experimental group were lower than the glucocorticoid group (control group), while the quality of life in the experimental group was higher. li et al. [44] also showed that compared with the control group, the overall symptoms of msc-treated patients improved, and the edss and recurrence rate were reduced. however, the summary of other outcomes showed different results. 3.9.1. number of lesions and volume of lesions. three rcts reported number and volume of lesions [41, 42, 46] . for a number of lesions, the heterogeneity test showed that i 2 = 0%, p = 0:62, considering the low heterogeneity among rcts. therefore, the fixed effects model is used for data analysis. the results show that the number of lesions between two groups were of no statistical significance (-1.13 (-3.80, 1.55), p = 0:41) ( figure 10 ). for the volume of lesions, the heterogeneity test showed that i 2 = 75%, p = 0:007, considering the high heterogeneity among rcts. therefore, the random effects model is used for data analysis. the results show that the volume of lesions between two groups were of no statistical significance (-5.08 (-11.33, 1.17), p = 0:11) (figure 11 ). 3.9.2. expanded disability status scale. three rcts reported comparable data of edss [42, 43, 46] . the heterogeneity test showed that i 2 = 85%, p < 0:0001, considering the high heterogeneity among rcts. therefore, the random effects model is used for data analysis. the results show that the edss between two groups were of no statistical significance (0.12 (-1.18, 1.43), p = 0:85) ( figure 12 ). 3.9.3. adverse events. two rcts reported adverse events [42, 45] . the heterogeneity test showed that i 2 = 0%, p = 0:56, considering the low heterogeneity among rcts. therefore, the fixed effects model is used for data analysis. the results show that the adverse events between two groups were of no statistical significance (1.12 (0.81, 1.53), p = 0:50) ( figure 13 ). mscs are a kind of adult stem cells that mainly exist in the bone marrow and have multidifferentiation potential, low immunogenicity, and immunomodulatory properties. in addition to the bone marrow, it can also be isolated and cultured from almost all adult tissues such as the placenta, umbilical cord, cord blood, and adipose tissue. mscs have powerful immune regulation functions, can induce immune tolerance, and promote hematopoiesis and tissue repair. studies showed that mscs have the following characteristics: (1) inhibiting the proliferation of a variety of immune cells including t and b lymphocytes [47] , (2) influencing the secretion of cytokines of immune cells to induce their antiinflammatory effects [48] , and (3) it may also release soluble factors and participate in the regulation of rabbit disease [49] . in addition, mscs do not express major histocompatibility complex (mhc) class i molecules, but mainly express mhc class i molecules, which makes them have low immunogenicity [50] . due to its multidirectional differentiation potential, immune regulation, hematopoietic support, low immunogenicity, and no immune rejection,, mscs have been used in the treatment of refractory and severe autoimmune diseases in recent years, providing patients with safe and effective new treatment options. [51, 52] . studies have found that the mscs of patients with autoimmune diseases has many problems such as changes in the number, abnormal cytoskeleton, decreased migration ability, abnormal multidirectional differentiation potential, and abnormal secretion of basic cytokines [53, 54] . it is currently believed that mscs can inhibit the proliferation of multiple types of allogeneic immune cells [47] and exert immunoregulatory functions on t lymphocytes, b lymphocytes, macrophages, dcs, and nks [17] . in addition, mscs may exert immunomodulatory effects by secreting a variety of regulatory cytokines, such as interleukin-(il-) 4, il-7, il-10, γinterferon (ifn-γ), and prostaglandin e2 (pge2) [55] . t cells under the induction of thymus hormone, and then play a series of immune functions. it has the characteristics of participating in delayed-type allergic reactions, regulating transplantation immunity, promoting the formation of precursor cells to produce antibodies, and regulating cellular immunity by secreting a variety of cytokines. it also has different subtypes such as helper, inhibitory, effector, and cytotoxic t cells [56, 57] . current research has shown that various types of t cells are disordered in patients with autoimmune diseases, and intervention strategies for autoimmune diseases mediated by t cells have become the main direction of new drug development. msc may secrete a variety of soluble cytokines through paracrine pathways, such as nitric oxide (no), pge2, and lumbromine 2,3 dioxygenase (ido) and nutritional factors such as transforming growth factor (tgf)-β3 and tumor necrosis factor (tnf)-α [58, 59] , to inhibit the proliferation of t lymphocytes [60] [61] [62] [63] . this thereby affects the expression of cell surface markers, specific proliferation, the formation of cytotoxic t lymphocytes, th1 type cell production of inf-γ, and th2 type cell production of il-4 [64, 65] . glennie et al. found that msc may suppress t cells in the g0/g1 phase of the proliferation cycle, downregulate cyclin 22 (an important conversion protein in the g1/s phase), and inhibit the p27kipl protein, thereby causing a series of changes in the secretion of soluble cytokines, and ultimately inhibiting the activity of t lymphocytes [66] . in addition to cytokines, msc may also exert an inhibitory effect through direct contact with t lymphocytes. msc expresses pd-1 ligand 1 (pd-l1) and pd-l2 molecules that bind to programmed death protein 1 (pd-1) on the surface of t lymphocytes, causing the activity 8 stem cells international of t lymphocytes to be inhibited and hindering their proliferation. these effects may only be exerted when the msc is in direct contact with t lymphocytes [67] . it can be seen that msc can affect the immune function of t cells through a variety of mechanisms, and the occurrence and development of a variety of diseases involve abnormal immune regulation of t cells. therefore, clarifying the immune regulation of msc to t cells can not only provide a theoretical basis for analyzing its specific mechanism of action in diseases but also provide new ideas for the treatment of immune-related diseases. [68] . current research shows that b cells in autoimmune diseases are the main link in the production of autoantibodies, which is the main direction of drug research and development [69] . the negative regulatory effect of msc on b stem cells international lymphocytes may be caused by direct contact with b cells to produce a series of cytokines and directly secrete some soluble cytokines to act on b cells. this in turn inhibits the proliferation of b cells and reduces the production of plasma cells and memory b cells, resulting in the reduction of b cells secreting antibodies, cytokines, and chemokines [70] . msc can also promote the production of granulocyte-macrophage colony-stimulating factor (gm-csf) through the participation of stem cell antigen 1/lymphocyte antigen 6aie protein and inhibit the maturation of b lymphocytes. 11 stem cells international of c-x-c motfreceptor 4 (cxcr4) and cxcl13 to inhibit b cell differentiation [70, 72] . hermankova et al. found that in the presence of ifn-γ, msc may inhibit the proliferation of b lymphocytes by expressing ido [73] . therefore, similar to t cells, msc may regulate the immune function of b cells through a variety of mechanisms and play different roles in a variety of autoimmune diseases. dendritic cells (dc). dc can efficiently ingest, process, and present antigens and is the body's strongest antigenpresenting cell. mature dc can activate initial t cells and then initiate, regulate, and maintain immune response, while immature dc has strong migration ability, can quickly migrate to the lesion site, and participates in the immune response [74] . the change of dc may damage the immune regulation mechanism, break the balance of natural immune tolerance, and cause autoimmune diseases. in addition, the activation of t cells and b cells by dc is also closely related to the occurrence of autoimmune diseases. therefore, it is believed that dc is the hub of the pathological pathway of autoimmune diseases. the related research of dc on the treatment of autoimmune diseases illustrates the close relationship between dc and autoimmune diseases from another angle [75, 76] . when msc and dc are cocultured, it can inhibit the differentiation of monocytes into dc by downregulating the expression of cd1a, cd86, and hla-dr of mhc class ii molecules. it also inhibits the expression of cd83, inhibits the secretion of tnf-4 from dc1 cells, and enhances the secretion of il-10 from dc2 [77] , thereby changing the dc phenotype from mature to immature stage, leading to immune silence [78] . msc can significantly inhibit the transformation of gm-csf and l-4 leads from cd14+ monocytes to dc. djouad et al. found that msc can secrete il-6 and downregulate the expression of mhc i molecules, cd40, and cd86 on the surface of mature dc, or by secreting tgf-β, pge2 and other cytokines, inhibit the activity of dc, and cause dc to differentiate into immature phenotype [79] . msc affects the maturation of dc through a variety of ways, including the expression of antigen and costimulatory molecules, changes in antigen presentation and migration ability, maintaining the expression of cadherin, and inhibiting the expression of cc motfireceptor 7 (ccr7) and cc motiligand 19 (ccl19), thereby inhibiting the migration of dc and so on [72] . therefore, msc may inhibit the generation, proliferation, antigen presentation, migration, and deformation ability of dc and participate in the differentiation and maturation of dc. in summary, it is currently believed that mscs exert their immune regulation function mainly by inhibiting the proliferation of t lymphocytes, inhibiting the proliferation and differentiation of b lymphocytes, regulating the activity of nks, and preventing the maturation of dcs. in the future, more mscs' immune regulation mechanisms would be revealed. [80] . a variety of msc transplantation treatments, such as bone marrow source, fat source, and cord blood source, can effectively alleviate the symptoms of ra model mice [81, 82] . previous studies have suggested that due to lack of immunogenicity and significant local immunosuppressive ability, mscs from umbilical cord matrix tissue can be used more safely in allogeneic transplantation and can exert their immunomodulatory effects in the body without prior induction and activation and has gradually replaced bone marrow-derived mscs [83] . the specificity of umbilical cord mscs may be due to differences in gene and protein expression profiles, that is, increased expression of immunomodulatory surface proteins, such as cd200, cd273, and cd274, and cytokines such as il-1β, il-8, leukemia inhibitory factor, and tgf-β2 [84] . mscs inhibit the proliferation of t lymphocytes and reduce the expression levels of inf-γ and tnf-α, thereby improving the clinical symptoms of autoimmune encephalomyelitis model mice. in addition, mscs can accumulate in peripheral immune organs, causing immune tolerance to peripheral t stem cells international lymphocytes [85, 86] . studies have found that tgf-β and il-4 are also involved in the immune regulation of ms by mscs [87, 88] . the 3 rcts included in this systematic review showed the therapeutic effect of msc transplantation on ra. showed that after msc treatment, the disease activity was weakened and the clinical symptoms (including das28) were improved. it also found that the dosage of prednisone acetate in 23 patients in the experimental group gradually decreased after the intervention. for the immune response, it found that the percentage of cd4+cd 25 +foxp3+tregs in the response group increased and the percentage of cd4+il-17a+th17 cells decreased; and the levels of il-6 and tnf-α decreased significantly. shadmanfar et al. (2018) shows that msc may improve the patient's standing time and womac total score and reduce the use of methotrexate and prednisolone. álvaro-gracia et al. (2017) showed that a moderate proportion of patients meets the comprehensive measure of acr 20/50/70 response, but fewer patients achieve an improvement of 50% or 70%. in addition, the combination therapy of mesenchymal stem cells and other cytokines will become a new mesenchymal stem cell combination strategy in the future. he et al. through intravenous injection of ifn-γ to patients, "emerging" mesenchymal stem cells, forming an immune microenvironment that is conducive to mesenchymal stem cells to exert their anti-inflammatory and immune regulation functions, to treat autoimmune inflammatory diseases such as ra [89] . compared with the treatment of mesenchymal stem cell transplantation alone, during the three-month clinical observation period, the effective rate of "empowering" mesenchymal stem cell transplantation in the treatment of rheumatoid arthritis has been significantly improved, from 53.3% to 93.3%. this research had become an important advancement in the field of mesenchymal stem cell treatment of rheumatoid arthritis in recent years [89] . at present, the team is conducting a multicenter clinical randomized trial to prove the effect of the therapy in the treatment of diseases such as ra and sle. 2018) used the intra-articular injection method but did not describe the specific dosage. all three have curative effects, but because the same indicators are not reported, they cannot be combined for meta-analysis. and because the rcts with the same dose and infusion methods were few, subgroup analysis was hard to perform. therefore, it is not yet known which dose and which intervention method works best. we may only speculate based on current evidence that 1 to 3 * 10 7 cells (or 1 * 10 6 cells/kg dose) may achieve therapeutic effects through intravenous infusion or intra-articular injection. sle is an autoimmune disease that mainly manifests itself in the formation of autoantibodies and involves multiple organs and multiple systems. sle is common in women of childbearing age, and its clinical manifestations are complex and diverse, and the exact pathogenesis has not been confirmed. at present, the main treatment options for sle are glucocorticoids and immunosuppressive agents. this program has poor curative effect on some patients with refractory lupus and has many adverse reactions, which has a greater impact on the quality of life of patients. animal studies have shown that the mrl/lpr effect of msc alone or combined with cyclophosphamide in the treatment of sle model mice is better than cyclophosphamide alone, which is shown in reversing multiple organ dysfunction in lupus mice and improving proteinuria and renal pathological damage [90, 91] . in addition, studies have confirmed that mscs from different sources can control disease progression and improve disease performance in lupus model mice. cord blood-derived mscs can also effectively relieve the condition of lupus model mice [92] , and fat-derived mscs can improve the immune system damage caused by lupus to a certain extent and can reduce kidney damage [93] . this meta-analysis showed that the sledai and urine protein in the experimental group was lower than that in the control group. the serum c3 in the msc group was also higher than that in control group. in terms of safety, there was no statistical difference in the incidence of adverse events between the msc group and the control group. it can be considered that the safety of msc and the control group (placebo or traditional therapy) is equivalent. other clinical trials also showed that msc transplantation has significant clinical therapeutic effects, which can stabilize the patient's condition and reduce the recurrence of the patient's condition. the patients received msc transplantation without rejection, and mscs have good safety [90, 94] . through a multicenter clinical study on msc transplantation for the treatment of sle, a total of 40 patients from 4 centers were enrolled. the results of the study showed that the overall survival rate after transplantation was 92.5%, and no serious transplant-related adverse reactions occurred [95] . longterm follow-up of 9 patients with refractory sle for up to 6 years showed that there was no increase in serum tumor markers before and 6 years after msc infusion [95] . it shows that in these patients, allogeneic umbilical cord-derived msc transplantation has good safety. in summary, combined with single-arm clinical trials and rcts, for refractory sle, msc transplantation treatment has good safety. for dosage and infusion method, except for the renal artery method used by zeng et al. (2016) , the intravenous infusion method is used for other rcts. and these rcts use different doses (from 1 * 10 6 cells to 2 * 10 8 cells). therefore, it is difficult to evaluate which dose and method of administration are better. we may only speculate based on current evidence that 1 * 10 6 cells to 2 * 10 8 cells msc transplantation may achieve therapeutic effects through intravenous infusion or renal artery. immune dysfunction is believed to play a key role in the occurrence and development of ulcerative colitis. research suggests that mesenchymal stem cells may help tissue regeneration by suppressing inappropriate immune responses and providing various cytokines instead of directly restoring damaged cells [96] . the pathogenesis of ulcerative colitis is unclear. studies have found that in the intestinal mucosa of patients with active 13 stem cells international ulcerative colitis, there is a cytokine storm, especially il 17 levels are significantly increased [97] . the imbalance in the ratio of regulatory t cells (tregs)/helper t cells 17 (th17) may be related to the occurrence and development of ulcerative colitis. only cd4+cd25+regulatory t cells expressed by foxp3 have immunomodulatory effects. the combination of foxp3 and nuclear receptors can significantly inhibit the transcription of interleukin 17, thereby affecting the differentiation of th17 cells [98] . studies have found that rab27a and rab27b are gtpases related to exosomes, which are related to the secretion of exosomes and their docking in the plasma membrane of various cells. compared with the healthy control group, a significant increase in the number of rab27a+ or rab27b+ intestinal immune cells can be observed in the colonic mucosa of the active ulcerative colitis group. this indicates that the immune response mediated by exosomes plays an important role in the pathogenesis of ulcerative colitis [99] . msc can induce the apoptosis of t lymphocytes by secreting exosomes, stimulate monocytes to secrete il10 and tgf β, promote the upregulation of cd4+ cd25+ foxp3+ regulatory t cells, reduce the level of inflammatory factor il 4, and increase the level of antiinflammatory factor il 10 to regulate the immune response. anti-inflammatory factors such as tgf β and il 10 can stimulate mesenchymal stem cells in vitro to secrete exosomes more effectively, which in turn promotes the upregulation of regulatory t cells, reduces intestinal inflammation, and promotes the repair and regeneration of damaged tissues [100, 101] . in addition, animal experiments have shown that mesenchymal stem cells can migrate to the colon and differentiate into vascular endothelial cells to promote the formation of new blood vessels in damaged parts [102] [103] [104] , promote the reconstruction of microcirculation, and thus facilitate the repair of colonic mucosal inflammation. the number of directional migration of stem cells is related to the degree of tissue damage. with the aggravation of the damage, the migration rate of mesenchymal stem cells increases, and the number in the recovery period decreases significantly [105, 106] . when inflammation occurs in the intestine, mesenchymal stem cells can migrate in the body and settle on the surface of the intestinal mucosa and proliferate and differentiate into new colonic mucosal epithelial cells to repair the injured site [107] . brittan et al. found that msc transplantation can colonize and differentiate into intestinal subepithelial myofibroblasts after transplantation and promote intestinal mucosal repair and neovascularization by improving the intestinal microenvironment [108] . in the human body, whether mesenchymal stem cells differentiate directly into intestinal mucosal epithelial cells or myofibroblasts and promote intestinal epithelial cell repair and angiogenesis by improving the intestinal microenvironment still needs further research to confirm. this metaanalysis found that it can improve clinical efficacy. the incidence of adverse events between two groups was of no statistical significance. for the dosage and infusion method, garcia-olmo et al. methods are reported less, it is not known which route of administration is better. and since the doses administered are also diverse, it is not known which method of administration is better. we may only speculate based on current evidence that 1 to 5 * 10 7 cells for msc transplantation may achieve therapeutic effects through intravenous infusion or local injection. ji et al. (2013) found that the progression-free survival (pfs) rate, total number of episodes, and average number of episodes each year in the experimental group were lower than that in the glucocorticoid group (control group), while the quality of life in the experimental group was higher. however, the summary of other outcomes showed that the number and volume of lesions and edss between the experimental group and control group was of no statistical significance. this controversial result is interesting, so more relevant research is needed in the future to amend or confirm the conclusion. however, basic research has found that msc may have the effect of treating multiple sclerosis. multiple sclerosis is a chronic inflammatory demyelinating disease that mainly affects the central nervous system. its pathological characteristics are mainly manifested by cell infiltration of myelin-specific autoreactive t cells and subsequent neuroinflammatory response, demyelination response, and neuronal cell damage. the destruction of axon integrity and the accumulation of irreversible sclerosis are the main causes of irreversible neurological damage [109, 110] . the pathogenesis of multiple sclerosis involves a variety of cells in innate immunity, such as th17 helper t cells 1, treg, microglia, dendritic cells, and macrophages. the destruction of the balance between helper t cells 1 and helper t cells 17 is considered to be an important mechanism leading to the pathogenesis of multiple sclerosis, and regulatory t cells are considered to be a key regulator of the adaptive immune response of multiple sclerosis [111, 112] . although there are many kinds of drugs that can be used for the treatment of multiple sclerosis, most of them can only control the progression of the disease and improve the clinical symptoms of patients, but they cannot completely cure the disease. once the patient's clinical manifestations develop into progressive disability, there is no effective way to protect, repair, and regenerate nerve tissue to restore the patient's nerve function. therefore, myelin and nerve cell regeneration are still the main obstacles to the treatment of multiple sclerosis [113, 114] . in the past 20 years, stem cell transplantation has been considered a potentially effective treatment for invasive multiple sclerosis [115] , and different types of stem cells, even stem cells of the same type but from different sources, have their unique characteristics. mesenchymal stem cells exert their therapeutic effects on multiple sclerosis mainly by regulating the immune response and promoting nerve repair. the regulation effect of rabbit disease is manifested by inhibiting innate and adaptive immune response, inhibiting the proliferation of pathogenic effect cd4+ t cells and b cells, regulating cd8+ t cell 14 stem cells international subsets, inducing the generation of regulatory t cells, and affecting the functions of dendritic cells and natural killer cells. the nerve repair function is produced by secreting a variety of neurotrophic factors, affecting the differentiation of neural stem cells, and promoting remyelination and axon survival [115, 116] . barati et al. found that promoting the production of m2 type microglia and inhibiting the expression of proinflammatory cytokines may be the mechanism for mesenchymal stem cells to treat demyelinating diseases [117] . bone marrow mesenchymal stem cells can improve the symptoms of patients with multiple sclerosis by inhibiting the inflammatory response in the central nervous system, regulating the expression of interleukin 6, stimulating the production of nerve growth factor, and protecting axons [118] . wang et al. showed that the supernatant of bone marrow mesenchymal stem cells can affect the function of cd4+ t cells [64] . it thereby inhibits the secretion of inflammatory factors in the peripheral blood of experimental autoimmune encephalomyelitis and reduces the degree of demyelination in the central nervous system of mice with experimental autoimmune encephalomyelitis. compared with human bone marrow mesenchymal stem cells, mesenchymal stem cells derived from human embryonic stem cells can significantly reduce the clinical symptoms of experimental autoimmune encephalomyelitis and more effectively prevent demyelination. this difference may be related to the high permeability of mesenchymal stem cells derived from human embryonic stem cells [64] . in addition to bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells are also commonly used to treat multiple sclerosis and experimental autoimmune encephalomyelitis. adipose-derived mesenchymal stem cells can pass through the blood-brain barrier and reduce the infiltration of brain b cells, t cells, and macrophages. in experimental autoimmune encephalomyelitis mice treated with adipose-derived mesenchymal stem cells, human leukocyte antigen g is one of the main factors to reduce the severity of the disease [119] . in addition, li et al. [120] found that adipose-derived mesenchymal stem cells can also reduce the th17/treg ratio by releasing leukemia inhibitory factors and reduce the degree of disability in experimental autoimmune encephalomyelitis. kurte et al. [121] observed that transplantation of mesenchymal stem cells before the onset of the disease in experimental autoimmune encephalomyelitis mice or at the peak of the disease has the best therapeutic effect. the findings of strong et al. [122] emphasize the importance of choosing a donor. they injected adipose-derived mesenchymal stem cells from obese and wasting donors into mice with experimental autoimmune encephalomyelitis by intraperitoneal injection. the results showed that adipose-derived mesenchymal stem cells from obese donors failed to inhibit inflammation and clinical symptoms, and adipose-derived mesenchymal stem cells from obese donors increased the secretion of proinflammatory cytokines. cell transplantation through intravenous injection is usually the preferred injection method in experiments, but intranasal administration can bypass the bloodbrain barrier and directly enter the brain through the olfactory and trigeminal nerve pathways, which also provides researchers with another option [123] . for the dosage and infusion method, rcts use different doses (from 5 * 10 7 cells to 6 * 10 8 cells). therefore, it is difficult to evaluate which dose and method of administration are better. we may only speculate based on current evidence that 5 * 10 7 cells to 6 * 10 8 cells msc transplantation may achieve therapeutic effects through intrathecal injection or intravenous infusion. ankylosing spondylitis is an autoimmune disease mediated by immune complexes. the main symptom is a chronic progressive inflammatory disease that invades the spine and affects the patient's sacroiliac joints and surrounding joint tissues. it has a high clinical morbidity and disability rate. the study found that, compared with healthy donors, although bone mscs (bmscs) obtained from patients showed normal proliferation, cell viability, surface markers, and multiple differentiation characteristics, and their immunomodulatory ability was significantly reduced [124] . xie et al. [125] found that because bmscs secreted more bone morphogenetic protein 2 (bmp2) and less noggin (nog), bmscs of as patients had stronger osteogenic differentiation ability than bmscs of normal donors. this state may contribute to the underlying pathological osteogenesis in as. animal studies have shown that after mscs are injected into mice, th17 cells are inhibited and the percentage of cd4+-cd25+-foxp3 +-treg cells increases [126] . in addition, as patients have a low number of treg cells, a low b cell level, and abnormal function [127] . studies have shown that mscs can differentiate t cells into th2 phenotype and inhibit the differentiation of th17 cells, thereby reducing the cytokine levels of th17 cells and promoting the regeneration process of subsequent tissue damage [128] . clinical trials have shown that mscs may help relieve the symptoms of as patients [127] [128] [129] . wang et al. [130] found that the bath ankylosing spondylitis disease activity index (basdai), night pain score (vas) and bath ankylosing spondylitis functional index (basfi) improved. wang et al. [130] found that the bath ankylosing spondylitis disease activity index (bas-dai), night pain score (vas), and bath ankylosing spondylitis functional index (basfi) improved. patients' esr and immunoglobulin g decreased significantly at 3, 6, and 12 months after stem cell transplantation. this systematic review only found one rct related to the treatment of ankylosing spondylitis with msc. it is found that compared with the fliximab group, msc treatment for 6 months may increase the total effective rate; reduce erythrocyte sedimentation rate, intercellular adhesion molecules, and serum tnf-α; and improve pain and activity. for the dosage and infusion method, one rct is injected with 1 * 10 6 cells/kg through intravenous infusion, and it has a certain effect. for multiple sclerosis, the administration methods and dosages of each rcts are varied, and the summary results have no significant curative effect compared with the control group. therefore, the optimal dosage and route of administration are not yet known. we may only speculate based on current evidence that 1 * 10 6 cells/kg dose of msc transplanted by the intravenous infusion method has not been able to observe the therapeutic effect. nevertheless, more rcts are still needed to further determine the key points of mscs in the treatment of ankylosing spondylitis, such as cell source, dosage, route of drug administration, and especially intervention in the most ideal disease stage (early or late). this systematic review and meta-analysis summarized the safety and effectiveness of msc in the treatment of autoimmune diseases (ra, sle, inflammatory bowel disease, multiple sclerosis, and ankylosing spondylitis) and provides relevant evidence for the future clinical research design (such as dose and disease severity) of clinical trials for msc treatment of autoimmune diseases (such as rheumatoid arthritis, sle, inflammatory bowel disease, multiple sclerosis, and ankylosing spondylitis). all data generated or analyzed during this study are included in this published article. our study did not require an ethical board approval because it is a systematic review and meta-analysis. stem cells international autoimmune addison's disease -an update on pathogenesis comparative united states autoimmune disease rates for 2010-2016 by sex, geographic region, and race prediction and prevention of autoimmune disease in the 21st century: a review and preview human autoimmune diseases: a comprehensive update type i interferons in autoimmune disease the role of epigenetics in autoimmune/inflammatory disease systemic autoinflammatory diseases b cell depletion therapies in autoimmune disease: advances and mechanistic insights regulatory t cells: the future of autoimmune disease treatment challenges of autoimmune rheumatic disease treatment during the covid-19 pandemic: a review adverse effects of gluten ingestion and advantages of gluten withdrawal in nonceliac autoimmune disease jak inhibitors for the treatment of autoimmune and inflammatory diseases treg enhancing therapies to treat autoimmune diseases stem cell treatment for patients with autoimmune disease by systemic infusion of culture-expanded autologous adipose tissue derived mesenchymal stem cells mesenchymal stem cell therapy for autoimmune disease: risks and rewards mesenchymal stem cell-derived exosomes and other extracellular vesicles as new remedies in the therapy of inflammatory diseases current research and use of mesenchymal stem cells in the therapy of autoimmune diseases mesenchymal stem cell-based therapy for autoimmune diseases: emerging roles of extracellular vesicles adipose-derived mesenchymal stem cells in autoimmune disorders: state of the art and perspectives for systemic sclerosis mesenchymal stem/stromal cells for rheumatoid arthritis treatment: an update on clinical applications the prisma 2020 statement: an updated guideline for reporting systematic reviews chapter 16: special topics in statistics chapter 8: assessing risk of bias in included studies clinical trial of human umbilical cord blood-derived stem cells for the treatment of moderate-to-severe atopic dermatitis: phase i/iia studies observation on the effect of different doses of human umbilical cord mesenchymal stem cell transplantation for ms efficacy of treatment with umbilical cord mesenchymal stem cells combined with rituximab in patients with systemic lupus erythematosus clinical efficacy of umbilical cord mesenchymal stem cell transplantation in the treatment of lupus nephritis human umbilical cord mesenchymal stem cell therapy for patients with active rheumatoid arthritis: safety and efficacy serum ifn-γ levels predict the therapeutic effect of mesenchymal stem cell transplantation in active rheumatoid arthritis intraarticular knee implantation of autologous bone marrowderived mesenchymal stromal cells in rheumatoid arthritis patients with knee involvement: results of a randomized, triple-blind, placebo-controlled phase 1/2 clinical trial intravenous administration of expanded allogeneic adipose-derived mesenchymal stem cells in refractory rheumatoid arthritis (cx611): results of a multicentre, dose escalation, randomised, single-blind, placebo-controlled phase ib/ iia clinical trial efficacy and mechanism of umbilical cord mesenchymal stem cell transplantation in the treatment of lupus nephritis clinical effects of mesenchymal stem cells combined with mortemacrolide in the treatment of lupus nephritis a randomised double-blind, placebo-controlled trial of allogeneic umbilical cord-derived mesenchymal stem cell for lupus nephritis efficacy of umbilical cord mesenchymal stem cell transplantation in the adjuvant treatment of systemic lupus erythematosus safety and therapeutic effect of mesenchymal stem cell infusion on moderate to severe ulcerative colitis expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase ii clinical trial expanded allogeneic adipose-derived mesenchymal stem cells (cx601) for complex perianal fistulas in crohn's disease: a phase 3 randomised, double-blind controlled trial allogeneic bone marrow-derived mesenchymal stromal cells promote healing of refractory perianal fistulas in patients with crohn's disease analysis of the effect of umbilical cord mesenchymal stem cell transplantation in the treatment of patients with ankylosing spondylitis clinical observation of umbilical cord mesenchymal stem cells combined with rituximab in the treatment of multiple sclerosis in 30 cases adiposederived mesenchymal stem cells (admsc) for the treatment of secondary-progressive multiple sclerosis: a triple blinded, placebo controlled, randomized phase i/ii safety and feasibility study beneficial effects of autologous mesenchymal stem cell transplantation in active progressive multiple sclerosis the potential of human umbilical cord-derived mesenchymal stem cells as a novel cellular therapy for multiple sclerosis human placenta-derived cells (pda-001) for the treatment of adults with multiple sclerosis: a randomized, placebo-controlled, multiple-dose study randomized placebo-controlled phase ii trial of autologous mesenchymal stem cells in multiple sclerosis interactions between human mesenchymal stem cells and natural killer cells human mesenchymal stem cells modulate allogeneic immune cell responses mesenchymal stem cll for the treatment ofsystemiclupus erythematosusis the cure for connective fssue diseases witin connectve tissue? immune-related antigens, surface molecules and regulatory factors in humanderived mesenchymal stromal cells: the expression and impact of inflammatory priming concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing mesenchymal stem cells as an immunomodulatory therapeutic strategy for autoimmune diseases abnormality of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus enhanced apoptosis and senescence of bone-marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus mesenchymal stromal cells and rheumatic diseases: new tools from pathogenesis to regenerative therapies immunomodulatory characteristics of mesenchymal stem cells and their role in the treatment of multiple sclerosis advances in mesenchymal stem cell therapy for immune and inflammatory diseases: use of cell-free products and human pluripotent stem cell-derived mesenchymal stem cells mesenchymal stem cell therapy for cirrhosis: present and future perspectives mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices treatment of perianal fistulas with human embryonic stem cell-derived mesenchymal stem cells: a canine model of human fistulizing crohn's disease impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells mesenchymal stem cells: identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial microengineering of their niche culture and properties of adipose-derived mesenchymal stem cells: characteristics in vitro and immunosuppression in vivo human esc-derived mscs outperform bone marrow mscs in the treatment of an eae model of multiple sclerosis mesenchymal stem cell effects on t-cell effector pathways mesenchymal stem cellbased treatment in autoimmune liver diseases: underlying roles, advantages and challenges immunomodulatory effects of adipose-derived mesenchymal stem cells on the gene expression of major transcription factors of t cell subsets b-cell targeted therapies in human autoimmune diseases: an updated perspective immunomodulatory receptors are differentially expressed in b and t cell subsets relevant to autoimmune disease a stromal cell niche for human and mouse type 3 innate lymphoid cells allogeneic mesenchymal stem cells inhibited t follicular helper cell generation in rheumatoid arthritis mesenchymal stem cells, autoimmunity and rheumatoid arthritis suppression of il-10 production by activated b cells via a cell contactdependent cyclooxygenase-2 pathway upregulated in ifnγ-treated mesenchymal stem cells monitoring dendritic cell activation and maturation plasmacytoid dendritic cells: development, regulation, and function tolerogenic dendritic cells for reprogramming of lymphocyte responses in autoimmune diseases comparative efficacies of long-term serial transplantation of syngeneic, allogeneic, xenogeneic, or ctla4ig overproducing xenogeneic adipose 18 stem cells international tissue-derived mesenchymal stem cells on murine systemic lupus erythematosus immunomodulatory effects of human umbilical cord wharton's jelly-derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells mesenchymal stem cells inhibit the differentiation of dendritic cells through an interleukin-6-dependent mechanism mesenchymal stem cells alleviate experimental rheumatoid arthritis through micro rnaregulated iκb expression cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collageninduced arthritis treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells mesenchymal stem cells in arthritic diseases what makes umbilical cord tissue-derived mesenchymal stromal cells superior immunomodulators when compared to bone marrow derived mesenchymal stromal cells? neuroprotection and immunomodulation with mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis mesenchymal stromal cells ameliorate experimental autoimmune encephalomyelitis by inhibiting cd4 th17 t cells in a cc chemokine ligand 2-dependent manner mesenchymal stem cells enhance the engraftment and myelinating ability of allogeneic oligodendrocyte progenitors in dysmyelinated mice early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination combination of human umbilical cord mesenchymal stem (stromal) cell transplantation with ifn-γ treatment synergistically improves the clinical outcomes of patients with rheumatoid arthritis mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans transplantation of human bone marrow mesenchymal stem cell ameliorates the autoimmune pathogenesis in mrl/lpr mice therapeutic effects of umbilical cord blood-derived mesenchymal stem cell transplantation in experimental lupus nephritis adipose tissue-derived mesenchymal stem cells induce expansion of interleukin-10-producing regulatory b cells and ameliorate autoimmunity in a murine model of systemic lupus erythematosus umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus long-term safety of umbilical cord mesenchymal stem cells transplantation for systemic lupus erythematosus: a 6-year follow-up study mesenchymal stromal cells with enhanced therapeutic properties immune markers and differential signaling networks in ulcerative colitis and crohn's disease interactions among the transcription factors runx1, rorgammat and foxp3 regulate the differentiation of interleukin 17-producing t cells exosome in intestinal mucosal immunity mesenchymal stem cells and acellular products attenuate murine induced colitis genemicrobiota interactions contribute to the pathogenesis of inflammatory bowel disease new cell therapy using bone marrow-derived stem cells/endothelial progenitor cells to accelerate neovascularization in healing of experimental ulcerative colitis topical implantation of mesenchymal stem cells has beneficial effects on healing of experimental colitis in rats bone marrow mesenchymal stem cells repair colonic vascular endothelium in rats with ulcerative colitis stem cell transplantation for inflammatory bowel disease: application of hematopoietic stem cells 19 stem cells international and mesenchymal stem cells migration of mesenchymal stem cells after transplantation in rats with ulcerative colitis efficacy of human umbilical cord mesenchymal stem cells on experimental ulcerative colitis in mice bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon remyelination in multiple sclerosis: from basic science to clinical translation a comprehensive review on the treatment approaches of multiple sclerosis: currently and in the future dendritic cells as therapeutic targets in neuroinflammation neural stem cell-based regenerative approaches for the treatment of multiple sclerosis progressive multiple sclerosis: prospects for disease therapy, repair, and restoration of function treatment of multiple sclerosis by transplantation of neural stem cells derived from induced pluripotent stem cells stem cell therapy for multiple sclerosis cell therapy for multiple sclerosis mesenchymal stem cell mediated effects on microglial phenotype in cuprizone-induced demyelination model cns disease diminishes the therapeutic functionality of bone marrow mesenchymal stem cells adipose-derived mesenchymal stem cells modulate the immune response in chronic experimental autoimmune encephalomyelitis model therapeutic effects of human adipose tissue-derived stem cell (hadsc) transplantation on experimental autoimmune encephalomyelitis (eae) mice intravenous administration of bone marrow-derived mesenchymal stem cells induces a switch from classical to atypical symptoms in experimental autoimmune encephalomyelitis human adipose stromal/stem cells from obese donors show reduced efficacy in halting disease progression in the experimental autoimmune encephalomyelitis model of multiple sclerosis intranasal delivery of biologics to the central nervous system reduced immunomodulation potential of bone marrow-derived mesenchymal stem cells induced ccr4+ccr6+ th/treg cell subset imbalance in ankylosing spondylitis mcp1 triggers monocyte dysfunctions during abnormal osteogenic differentiation of mesenchymal stem cells in ankylosing spondylitis mesenchymal stem cells generate acd4 c025+foxp3t regulatovy t cell population during the differentition process of th1 and th17 cell infusion of umbilical cord mesenchymal stem cells alleviates symptoms of ankylosing spondylitis ankylosing spondylitis and mesenchymal stromal/stem cell therapy: a new therapeutic approach role of stem cells in pathophysiology and therapy of spondyloarthropathies-new therapeutic possibilities? human umbilical cord mesenchymal stem cell transplantation for the treatment of ankylosing spondylitis the authors declare no competing interests. ganpeng yu, liuting zeng and jun li should be co-first authors. prisma 2020 checklist: checklist. key: cord-0003075-ki94q4qv authors: louis, cynthia; burns, chris; wicks, ian title: tank-binding kinase 1-dependent responses in health and autoimmunity date: 2018-03-06 journal: front immunol doi: 10.3389/fimmu.2018.00434 sha: 61be2db10d752ec61e879d76e6328b6d676a0f55 doc_id: 3075 cord_uid: ki94q4qv the pathogenesis of autoimmune diseases, such as rheumatoid arthritis (ra) and systemic lupus erythematosus (sle) is driven by genetic predisposition and environmental triggers that lead to dysregulated immune responses. these include the generation of pathogenic autoantibodies and aberrant production of inflammatory cytokines. current therapies for ra and other autoimmune diseases reduce inflammation by targeting inflammatory mediators, most of which are innate response cytokines, resulting in generalized immunosuppression. overall, this strategy has been very successful, but not all patients respond, responses can diminish over time and numerous side effects can occur. therapies that target the germinal center (gc) reaction and/or antibody-secreting plasma cells (pc) potentially provide a novel approach. tank-binding kinase 1 (tbk1) is an ikk-related serine/threonine kinase best characterized for its involvement in innate antiviral responses through the induction of type i interferons. tbk1 is also gaining attention for its roles in humoral immune responses. in this review, we discuss the role of tbk1 in immunological pathways involved in the development and maintenance of antibody responses, with particular emphasis on its potential relevance in the pathogenesis of humoral autoimmunity. first, we review the role of tbk1 in the induction of type i ifns. second, we highlight how tbk1 mediates inducible t cell co-stimulator signaling to the gc t follicular b helper population. third, we discuss emerging evidence on the contribution of tbk1 to autophagic pathways and the potential implications for immune cell function. finally, we discuss the therapeutic potential of tbk1 inhibition in autoimmunity. the pathogenesis of autoimmune diseases, such as rheumatoid arthritis (ra) and systemic lupus erythematosus (sle) is driven by genetic predisposition and environmental triggers that lead to dysregulated immune responses. these include the generation of pathogenic autoantibodies and aberrant production of inflammatory cytokines. current therapies for ra and other autoimmune diseases reduce inflammation by targeting inflammatory mediators, most of which are innate response cytokines, resulting in generalized immunosuppression. overall, this strategy has been very successful, but not all patients respond, responses can diminish over time and numerous side effects can occur. therapies that target the germinal center (gc) reaction and/or antibody-secreting plasma cells (pc) potentially provide a novel approach. tank-binding kinase 1 (tbk1) is an ikk-related serine/threonine kinase best characterized for its involvement in innate antiviral responses through the induction of type i interferons. tbk1 is also gaining attention for its roles in humoral immune responses. in this review, we discuss the role of tbk1 in immunological pathways involved in the development and maintenance of antibody responses, with particular emphasis on its potential relevance in the pathogenesis of humoral autoimmunity. first, we review the role of tbk1 in the induction of type i ifns. second, we highlight how tbk1 mediates inducible t cell co-stimulator signaling to the gc t follicular b helper population. third, we discuss emerging evidence on the contribution of tbk1 to autophagic pathways and the potential implications for immune cell function. finally, we discuss the therapeutic potential of tbk1 inhibition in autoimmunity. keywords: tank-binding kinase 1, type 1 interferons, germinal center, autophagy, humoral immunity, autoimmunity introduction tank-binding kinase 1 (tbk1) is an ikk-related serine/threonine kinase best known for the induction of antiviral type i interferons (ifn-is) in innate immunity. however, a growing body of evidence highlights the relevance of tbk1 for other responses. in this review, we discuss our present understanding of the role of tbk1 in nucleic acid sensing pathways, antibody responses, and autophagy. we conclude by speculating how these diverse tbk1-regulated responses could potentially culminate in the induction, promotion, and maintenance of autoimmunity, as well as how pharmacological modulation of tbk1 could represent an alternative treatment strategy, particularly in the context of humorally mediated autoimmunity. tank-binding kinase 1 is an ikk-related serine/threonine kinase, best known for the induction of innate antiviral type i ifns. however, tbk1 potentially has much broader functions, which we discuss in this review (figure 1) . tbk1 is ubiquitously expressed in both hematopoietic and non-hematopoietic compartments. germline deletion of tbk1 is embryonically lethal in mice (1), highlighting its homeostatic functions during development. through biochemical studies, tbk1 was shown to be activated by double stranded (ds)-rna (via tlr3-trif), lps (via tlr4-trif), viral rna (via rig-i-mavs), and dsdna (via cgas-sting) in innate immune signaling pathways (2, 3) . trif (tir-domain-containing adapter-inducing ifn β), mavs (mitochondrial antiviral-signaling), and sting (stimulator of ifn genes) are innate immune adaptor proteins that transduce signal downstream of their corresponding sensors to the activation of interferon regulatory factor 3 (irf3). mechanistically, tbk1 activation is thought to occur via trans-autoactivation, in response to adaptor proteins that shuttle tbk1 to specific signaling complexes and direct subcellular localizations, such as to the er-golgi compartments (4) (5) (6) (7) . activated tbk1 then phosphorylates irf3 and induces the production of type i ifn-is (8) (9) (10) (11) (12) . other tbk1 substrates include akt (13, 14) and plk1, which are involved in tlr activation or oncogenicity of cancer cells (15) . closely related to tbk1, ikkε shares 60% homology and is initially thought to participate also in ifn-is induction (8, 9) . subsequent studies show that ikkε is dispensable for ifn-i responses (16) . ikkε is abundantly expressed in t cells and have been shown to regulate a number of t cell responses (17) (18) (19) . interferons are a family of cytokines with potent antiviral priming effects, but are also associated with humorally mediated autoimmune diseases, most notably systemic lupus erythematosus (sle) (20) (21) (22) . recently, tbk1 was also shown to associate with the inducible t cell co-stimulator (icos) in cd4 + t follicular b helper (tfh) cells that support efficient antibody responses (23) . however, the downstream target(s) of tbk1 in icos signaling have not yet been identified. finally, tbk1 is also implicated in promoting autophagy by phosphorylating autophagy receptor proteins, including optineurin (optn), sqstm1/p62, and ndp52 (24, 25). tbk1-mediated regulation of autophagy is currently under evaluation because tbk1 haploinsufficiency is a major risk factor in neurodegenerative diseases (26, 27). autophagy is thought to protect senescent neuronal cells from the accumulation of defective or redundant organelles. tbk1 may likewise physiologically protect long-lived immune cells through autophagy (28). although no particular tbk1 genetic variants have to date been directly linked to the development of autoimmune diseases, the diverse functions of tbk1 may contribute to one or more aspects of autoimmunity, which is the focus of this review. tank-binding kinase 1 has a well described role in activating the transcription factor irf3 to induce type i ifns production (8) (9) (10) (11) (12) . ifn-is are a family of cytokines with pleiotropic functions that have potent antiviral and antimicrobial effects against some intracellular bacteria, but are also implicated in pathogenesis of sle (sle, discussed below). tbk1 is ubiquitously expressed in both hematopoietic and non-hematopoietic compartments and it is activated by sensor-adaptor pairs, including tlr3/4-trif, rig-i-mavs, or cgas-sting, in response to lps, dsrna, virus infection, and cytoplasmic dna, respectively (2, 3). consequently, tbk1 −/− mouse embryonic fibroblasts (mefs) have impaired production of ifn-is (ifn-α and ifn-β) and ifn-inducible chemokines (ccl5 and cxcl10), among other genes, following activation with synthetic dsrna (poly i:c) or viruses, or lps (8, 10) . molecular characterization of the sequence of events leading to tbk1 induction, irf3 activation, and ifn-is production have mainly been performed in cell lines and mefs, in response to lps stimulation or in the context of antiviral responses (2, 3, 8, 10) . elucidating roles for tbk1 in more complex biological settings in vivo has been challenging due to the embryonic lethality of germline tbk1-deficiency in mice. this is thought to be due to tnf-α-induced hepatocyte apoptosis and can be rescued by combined loss of tnf (i.e., tbk1 −/− tnf −/− mice are viable) (1). subsequently, tbk1 has been suggested to control cell survival through pai-2/serpinb2 and transglutaminase 2 in the tnfactivated anti-apoptotic response (29). high levels ifn-α or induction of ifn-stimulated genes (i.e., the "ifn signature") is a remarkably consistent feature of sle and is associated with high titers of affinity-matured autoantibodies and worse disease outcome (20, 21, 22) . a similar ifn signature and correlation with high levels of autoantibodies and disease activity is also found in some patients with ra and primary sjogren's syndrome (30, 31) consistent with a pathogenic role for ifn-α in autoimmunity. consequently, the possibility of targeting tbk1-dependent ifn-is induction has received attention as a treatment strategy (32). among members of the ifn-i family in humans and mice, ifn-α and ifn-β are the best characterized and most broadly expressed. they signal through a shared, ubiquitously expressed heterodimeric receptor (ifnar), and prime a rapid antiviral response that acts directly or indirectly on many cell types, including nk cells, t cells, b cells, dcs, and macrophages (33-35). ifnar signaling mediates early attrition of existing memory cd8 + t cells in response to viral infections, which is thought to permit a more vigorous, diverse, and efficient t cell response emanating from the naïve t cell pool (36). in later stages, ifnar signaling in activated cytotoxic cd8 + t cells (ctls) (37) and nk cells (38) is important for long-term survival against perforin-mediated cytotoxicity, thereby preventing rapid elimination in vivo and sustaining antiviral immunity. ifnar signaling is also required for optimal nk cell effector function through upregulation of granzyme b (38). ifn-is is gaining attention in anti-cancer therapy, where it is generally considered pro-cytotoxic for ctls and presumably nk cells. this is exemplified by the observations that ifnar downregulation in ctls endows colorectal cancers with an immune-privileged niche that promotes aggressive tumorigenesis, associated with poor prognosis, and lessens the response to immunotherapy. conversely, ifnar expression suppresses tumor growth and improves the efficacy of combined anti-cancer chimeric antigen receptor t cell transfer and pd-1 inhibition (39). targeted intratumoral delivery of ifn-i-inducing (i.e., interferogenic) cyclic dinucleotide gmp-adenosine monophosphate (amp), which activates the sting-tbk1 pathway and ifn-is production in endothelial cells, has been shown to control tumor growth by boosting antitumor cd8 + t responses in murine models of melanoma and colon cancer (40). emerging evidence also implicates dysregulated nk cells and cd8 + t cells in sle and potentially ra (41). despite an overall reduction in circulating nk cell number in lupus patients and lupus mouse models, presumably owing to activation-induced death of these cells, nk cells with an activated phenotype infiltrate the kidneys of pre-disease lupus mice and may contribute to tissue injury by releasing cytotoxic granules (42). another study showed that sle patients have an expanded population of ctls, which may contribute to tissue damage (43). further studies are needed to determine whether ifn-is contribute to the activation of these human effector cells. persistent ifn-is exposure, particularly ifn-α, has long been implicated in immune dysfunction and autoimmune diseases, through a number of mechanisms. some patients treated with ifn-α therapy develop autoimmunity, including ra and lupus-like autoimmune syndrome (44, 45). chronic ifn-α overexpression in vivo induces rapid and lethal lupus, with immune complex glomerulonephritis in nzb/w lupus-prone mice (46). such excess ifn-α can also induce sustained b cell proliferation in vivo, accompanied by uncontrolled production of proliferating, short-lived, autoantibody-secreting plasmablasts in secondary lymphoid organs of nzb/w mice (47). pdcderived ifn-is have been shown to increase the translocation of marginal zone b cells to the follicular region of the spleen, which disrupt the ability of marginal zone macrophages to clear apoptotic cells and promote the loss of immune tolerance to apoptotic cell-derived antigens in sle (48). ifn-is also promotes affinity maturation of antibodies by activating dcs to produce il-6 (49). the severity of lupus-related pathology is attenuated with ifnar-deficiency or ifnar-blocking antibody in several murine lupus models (50-52). while the involvement of ifn-is-ifnar signaling is a consistent feature of murine lupus models, there is less consensus in ra. in contrast to the association of ifn-α with humoral autoimmunity, ifn-β has homeostatic and anti-inflammatory functions. in ra synovium, ifn-β reduced the secretion of ra-associated proinflammatory mediators, including il-6, tnf-α, matrix metalloproteinases, and prostaglandin e2 (53). ifn-β also primes an anti-inflammatory phenotype of endothelial cells by upregulating the expression of cd73, an ecto-5′-nucleotidase that produces anti-inflammatory adenosine from amp, at least in neuroinflammation (54). other studies have shown that exogenous ifn-β can inhibit autoimmune collagen-induced arthritis (cia) (55, 56). in contrast, ifn-β deficient mice develop prolonged cia, with a higher incidence relative to control mice (57). ifn-β delivery has been used therapeutically in multiple sclerosis (58) and has been considered for ra (59). monoclonal antibody therapies inhibiting ifn-is signaling or depleting of ifn-overproducing plasmacytoid dcs (pdcs) are under evaluation for the treatment of sle (60, 61). the opposing roles of ifn-α and ifn-β clearly require careful consideration in relation to these potential ifn-is-targeted therapies in autoimmunity. the prevailing concept in sle and murine lupus models is that immune complexes containing autoantibodies bound to self-dna and rna can act as interferogenic stimuli, following fc receptormediated internalization and activation of endosomal tlr7 and tlr9 in pdcs (62) . tlr7-or tlr9-mediated induction of ifn-is, however, does not require tbk1. for instance, tbk1 is not required for ifn-i production in the tlr7-dependent pristaneinduced lupus model (51). tlr9 ligand (cpg-b) induces ifn-is production by b cells and dcs through irf3, but independently of tbk1. autocrine ifnar signaling in b cells is required for enhanced igm and igg2a autoantibody production and these are dominant autoantibody isotypes in murine lupus (63) . using viable tbk1 −/− tnf −/− mice, ishii and colleagues demonstrated functional distinctions between tbk1 signaling in hematopoietic and non-hematopoietic cells for the induction of ag-specific responses in a plasmid-dna immunization model (64) . tbk1 −/− tnf −/− mice had no difference in total serum igg1 and igg2a, suggesting normal b cell function. however, tbk1 −/− tnf −/− mice had completely abrogated primary and secondary antigen-specific igg responses upon vaccination with plasmid-dna, relative to wild type, tnf −/− tbk1 +/+ , myd88 −/− , or trif −/− mice. mechanistically, the dna component of the plasmid-dna vaccine was shown to activate dcs in a tbk1-and ifn-i-dependent manner, but this occurred independently of the cpg dna sensor, tlr9 (64) . along the same lines, alum and hydroxypropyl-β-cyclodextrin adjuvants have been shown to induce cell death and dna release as part of their immunogenic properties and tbk1 −/− tnf −/− mice immunized with these adjuvants had reduced levels of antigen-specific igg1 responses (65, 66) . ishikawa and colleagues subsequently demonstrated that intracellular dna induced dc activation and ifn-is production through the cgas-sting-tbk1 pathway (67). as mentioned, tbk1-dependent ifn-is responses are activated by cytoplasmic nucleic acids. in the autoimmune context, pathogenic tbk1-mediated ifn-is responses can be caused by aberrant self-dna that leads to chronic irf3 activation, such as is the case in trex1 deficiency (68) . trex1 is an endoplasmic reticulum (er)-associated 3′-5′ exonuclease, which degrades cytoplasmic viral dna before sensing occurs. trex1 is also required to clear endogenous retroelements and genomic dna. trex1 deficiency in patients and murine models causes lupuslike autoimmune manifestations. trex1-deficient mice develop aberrant interferogenic responses and features of lupus owing to the cytoplasmic accumulation of endogenous nucleic acids and chronic activation of the tbk1-dependent dna-sensing pathway (68) (69) (70) . mutations in trex1 are associated with human autoimmune disorders, including aicardi-goutières syndrome (71) , familial chilblain lupus (72) , and sle (73) . an inhibitor of tbk1 was effective in treating trex1 −/− mice (74) . in summary, tbk1 is an important signaling kinase for the induction of ifn-is in response to a number of ligands that activate tlr3, tlr4, and the sting pathways. tbk1 may be less relevant in other ifn-is induction pathways, including tlr7 and tlr9. mutations leading to aberrant activation of tbk1 and ifn-is overproduction can contribute to lupus. limiting pathogenic ifn-is production through tbk1 inhibition may alleviate lupus. however, tbk1-driven responses other than ifn-is induction may also contribute to humoral autoimmunity and are discussed in the next section. antibody-mediated autoimmune diseases share underlying immune mechanism(s). high-affinity autoantibodies arise from a gc reaction occurring in the b cell follicles (75, 76) . the gc is a specialized structure in secondary lymphoid tissues, where b cells undergo iterative rounds of somatic hypermutation in ig variable (v) gene segments, class switching and affinity selection, as well as post-translational modifications. normally, the gc reaction is transient (self-terminating) (77) and only b cells expressing affinity-matured, class-switched antibodies specific for the antigen exit gcs, and survive as long-lived memory b cells and/or antibody-secreting plasma cells (78) (79) (80) . however, the gc reaction can persist and give rise to antibody-mediated autoimmunity (81) . intrinsic b cell defects can directly contribute to the development of spontaneous gcs, breakdown of b cell tolerance and humoral autoimmunity, such as tlr7 gene duplication (80) or was (wiskott-aldrich syndrome) protein deficiency (82, 83) in lupus. however, cd4 + tfh cells provide another essential cellular component regulating gc b cells. tfh cells are required for the generation of high-affinity antibodies by promoting the gc reaction, including b cell clonal proliferation, affinity selection and the development of high-affinity antibody-producing cells (75, 84, 85) . tfh cells are characterized by the expression of chemokine receptor cxcr5, which facilitates migration and proximity to follicular b cells. here, they provide cognate help to b cells via stable interactions such as sap (slam-associated protein), costimulatory molecules such as cd40l, and cytokines such as il-21, il-4, and ifn-γ (86) (87) (88) (89) . while tfh cells are critical for an optimal gc reaction and subsequent generation of protective antibodies following immunization, abnormal development and/or function of tfh have also been implicated in loss of tolerance and the development of humoral autoimmunity. an increased tfh population in the gc as well as gc numbers may contribute to aberrant positive selection and autoantibody formation in sle (90) . this is exemplified in sanroque mice, in which exaggerated tfh generation occurs in a cell-intrinsic manner and leads to spontaneous gc formation, and lupus-like pathology (91, 92) . furthermore, adoptively transferred roquin san/ san tfh cells are able to induce spontaneous gc b cell expansion and gc formation in naïve recipient mice (92) . similarly, an enlarged tfh population accompanies increased gc size and more productive humoral responses in immunization models (77) . conversely, mice with conditional bcl6 deficiency in t cells (bcl6 is a transcriptional repressor that regulates both tfh and gc b cell differentiation) have impairment of tfh development, gc reactions, and antibody responses (93, 94) . cd4 transgenic autoreactive t cells deficient in sap (slam-associated protein, which mediates stable t-b interactions critical for gc formation), failed to mount gc reactions, develop igg1 autoantibodies, and autoantibody-mediated arthritis (95, 96) . thus, the size of the tfh population is directly coupled with gc function and ensuing humoral responses. abnormal tfh accumulation may also contribute to the production of pathogenic autoantibodies through enhanced positive selection of self-reactive b cells. in clinical settings, the frequency of tfh-like cells is increased in the peripheral blood of ra patients and correlates with higher elevated levels of anti-ccp (cyclic citrullinated peptide) autoantibodies, as well as disease activity (97, 98) . conversely, treatment responsive, new onset ra patients have a reduced frequency of circulating tfh, which is accompanied by a decrease in anti-ccp antibody (98) . sle patients also demonstrate a similar expansion of tfh-like cells, which correlates with disease activity, frequency of circulating plasmablasts, and anti-double-stranded dna antibody positivity (99) . tfh expansion and its association with autoantibody responses have also been noted in other humoral autoimmune syndromes, including type 1 diabetes (100) and primary sjogren's syndrome (101, 102) . given the robust correlation between tfh numbers and high-affinity autoantibody levels, manipulation of the differentiation program and plasticity of tfh cells may provide new therapeutic options in autoimmune diseases, such as sle and ra. among many determinants of optimal humoral immunity, icos has been consistently associated with gc reactions and the induction of gc-dependent thymus-dependent (td) antibody responses. icos is a critical coreceptor, distinct from cd28, on activated or antigen-experienced t cells (103, 104) and is highly expressed on tfh (105) . through interaction with icos ligand (icosl) on antigen-presenting cells (dcs and b cells), icos delivers robust costimulatory signals that promote tfh positioning and thus supports gc function (106) . icosl −/− mice mount comparable antigen-specific igm and igg3 responses, but have reduced igg1 and igg2a production upon immunization with thymus-independent antigens (107) . icos −/− or icosl −/− mice have defective production of class-switched antibodies against td antigens (particularly igg1, igg2a, and igg2b isotypes, but not igm), along with reduced number and size of gcs and a lack of b cell memory (107) (108) (109) (110) . additionally, mice with a tyrosine-to-phenylalanine point mutation at residue 181 in the cytoplasmic tail of icos have abrogated tfh generation, gc reactions, antibody class switching, and antibody affinity maturation (111) . icos deficiency or antibody-mediated depletion of icos-expressing cd4 + t cells in sle1 lupus mice results in diminished pathogenic tfh expansion, inhibited plasma cell generation, and a reduction in class-switched igg autoantibodies (112) . icosl −/− or b cell-specific icosl −/− mice have markedly inhibited development of proteoglycan-induced arthritis, with notable reductions in tfh and gc b cells, il-21 production, and proteoglycan-specific igg antibody responses (113) . blockade of the icosl pathway ameliorates autoimmune cia, the k/bxn spontaneous arthritis model, and the sle (nzb/ nzw) f1 mouse model, with marked reductions in disease manifestations, numbers of tfh and gc b cells, and pathogenic, class-switched, high-affinity autoantibodies (113) (114) (115) . interestingly, inhibition of cia was observed even when icosl blocking antibody was given after the onset of disease (114) . sanroque mice have excessive icos activation due to genetic mutation of a ring-type ligase that represses icos (91) . these mice develop spontaneous gc in the absence of foreign antigen, increased numbers of tfh cells, spontaneous autoantibodies, including antinuclear antibodies, and lupus-like manifestations, such as glomerulonephritis. intriguingly, although icos or icosl deficiency in sanroque mice substantially reduced autoantibody production, it did not result in complete inhibition of autoantibody production (91) . this observation suggests a contribution from residual gc-independent extra-follicular pathway (85, 113) . thus, icos/icosl signaling drives optimal gc-dependent td antibody responses and inhibition of this pathway abrogates the gc reaction, autoantibody responses, and disease features in humorally mediated autoimmune disease models. indeed, therapies targeting icos/icosl are under evaluation in early phase clinical trials of sle (116) . similar to other cd4 + t cell subsets (th1, th2, th17, and treg cells), tfh development is a multi-step process which involves initial priming of naïve cd4 + t cells by dendritic cells in the t cell zone, followed by expansion and differentiation that are regulated through signaling pathways activated downstream of cytokines and cell surface molecules. subsequent activation of lineage-defining transcription factors (t-bet for th1, gata3 for th2, rorγt for th17, foxp3 for treg cells, and bcl6 for tfh) promotes t cell differentiation (93, 94, 117) . t follicular b helper development can be separated into two stages-(i) naive to bcl6 + pre-tfh and (ii) pre-tfh to mature gc tfh. pre-tfh development follows dc priming in vivo, through an icos costimulation signal and the phosphoinositide-3 kinase (pi3k) pathway. the icos-pi3k pathway instructs tfh differentiation via induction of bcl6 and the subsequent bcl6-dependent expression of cxcr5 on pre-tfh (23, 105) . the activation icos-pi3k signaling alone is, however, insufficient to drive full gc tfh maturation and the gc reaction (23, 111, 118) . for final differentiation of nascent tfh into gc tfh, pre-tfh cells require a second costimulatory signal through icos (119, 120) . this has been demonstrated by the inability of t cell-selective deletion of pi3k components to fully recapitulate the phenotype of cd4 + t cells from icos −/− mice (111, 121) . a recent report identified tbk1 as a unique signaling kinase in the icos pathway (23) . in this study, pedros and colleagues identified a conserved traf-like motif in the cytoplasmic tail of icos (iprox motif), which mediated tbk1 recruitment and activation following a combination of strong tcr and icos signals. these authors showed that by deleting the iprox motif on icos specifically in cd4 + t cells, tbk1 failed to associate with icos. t cells modified in this way displayed severely impaired differentiation into gc tfh and td antibody responses, despite generating pre-tfh cells. a similar effect was obtained by cd4 + t cell-specific tbk1 depletion. in a series of reconstitution experiments, transducing icos and tbk1 constructs into icos −/− tcr transgenic cd4 + t cells, it was shown that intact icos is required for the generation of both nascent and final gc tfh populations, while tbk1 controls progression from the pre-tfh to mature gc tfh phenotype (23) . although the downstream mediators of icos-tbk1 signaling in tfh have not been identified, foxo1 is a potential candidate because icos signaling instructs the tfh program via aktmediated foxo1 phosphorylation (119) . akt has been shown to be a tbk1 substrate in some settings (13, 14, 122) . foxo1 is a transcription factor that, in its active unphosphorylated state, represses tfh programming. foxo1 phosphorylation results in its transient inactivation and cytoplasmic translocation from the nucleus (119) . foxo1 inactivation also reduces foxo1-dependent klf2 expression, together with expression of klf2-dependent chemokine receptors, necessary for optimal repositioning of tfh in the gc (120). foxo1 inactivation, specifically in cd4 + t cells (foxo1 fl/fl :cd4-cre mice) caused defective tregs and systemic autoimmunity, characterized by accumulation of the tfh population, with exaggerated bcl6 induction and gc formation, and production of anti-dna antibodies (123). while foxo1sufficient cd4 + t cells give rise to effector t cells, pre-tfh, and gc tfh upon immunization, foxo1 −/− cd4 + t cells generate pre-tfh cells with higher expression of tfh-defining markers (bcl6, cxcr5, and pd-1) and lowered t cell zone chemokine receptors (cd62l, psgl1) (119). icos-driven foxo1 inactivation thus alters the chemokine receptor profile of pre-tfh, facilitating migration from the t cell zone toward the b cell follicles (119) . conversely, icos/icosl blockade results in the relocation of fully developed tfh back to the t cell zone. this relocation reverses their phenotype toward non-tfh effector t cells, with a consequent reduction in antigen-specific gc b cells, as well as serum antigen-specific igg1 and igg2a responses, indicating collapse of the gc response (120) . this study also concluded that icos is not required for tfh survival or expression of tfh-related transcription factors, but rather, it regulates the expression of tfh homing markers. changes in tfh transcription factors are thus likely to be a secondary effect upon failure to maintain the positioning of pre-tfh and impaired costimulatory signals from follicular b cells (120) . although tbk1 has been identified as an icos-specific signaling kinase required for full maturation of gc tfh, the role of tbk1-mediated foxo1 regulation in this process has not been elucidated. one study using conditional tbk1 −/− in cd4 + t cells (tbk1 fl/fl :cd4-cre mice) and stimulation with tcr and cd28, suggested that basal tbk1 is required for constitutive akt turnover to prevent hyperactivation of akt upon t cell activation (124) . these authors also reported a marked increase in ifn-γ production and activation markers in cd4 + t cells derived from tbk1 fl/fl :cd4-cre mice, indicating the propensity of these cells to become a th1-like population in the absence of basal tbk1 (124) . interestingly, icos −/− or icosl −/− mice also exhibit enhanced th1 responses in secondary lymphoid tissues with marked elevation of ifn-γ in the context of infection (125) (126) (127) . given the role of tbk1 in icos signaling in gc tfh and downstream gc-driven antibody responses, tbk1 inhibition may curtail humoral autoimmunity through an icos-driven gc pathway. importantly, targeting icos/icosl and/or tbk1 may not result in generalized immunosuppression, but rather reverse cell fate decisions in tfh. understanding how tbk1 signals in tfh, how it affects cell fate decisions in t helper cell polarization, positioning and migratory pathways may provide new therapeutic strategies, especially for antibody-mediated autoimmunity. the functional effects of tbk1 extend beyond innate immune signaling. autophagy is a conserved homeostatic process in eukaryotic cells involving sequestration and lysosomal degradation of cytoplasmic contents, including damaged or surplus organelles (mitophagy for mitochondria, pexophagy for peroxisomes, ribophagy for ribosomes, reticulophagy for endoplasmic reticulum), cytotoxic macromolecular aggregates (aggrephagy), and intracellular microorganisms (xenophagy) (128-131). autophagy will not be discussed in detail as it has been extensively reviewed elsewhere (128) (129) (130) (131) . instead, we discuss reports which have implicated tbk1 in autophagic processes and how these translate to immune responses. tank-binding kinase 1-mediated regulation of autophagy has been described in the context of antimicrobial defense (xenophagy), in which intracytoplasmic pathogens are sequestered into autophagosomes and targeted for lytic, lysosomal degradation. tbk1 and its homolog ikkε have been identified as binding partners of the autophagy receptor protein ndp52 that recognizes polyubiquitylated salmonella enterica in human cells. however, only tbk1 is required for xenophagy of s. enterica (132, 133) and mycobacteria (134) . canonical ikks initiate autophagy, while tbk1 knockdown suppresses the maturation of autophagosomes into autolysosomes. mechanistically, tbk1 phosphorylates autophagy receptor proteins, including ndp52, optn on ser177, and p62 (also known as sqstm1) at ser403 (located at the ubiquitin-associated/ uba domain) (132) (133) (134) . phosphorylation increases the affinity of lc3-binding autophagy adaptors for k48-and k63ubiquitinated cytoplasmic bacteria, as well as polyubiquitinated protein aggregates (135) , and it promotes autophagic clearance (132) (133) (134) . knockdown or pharmacological inhibition of tbk1 impairs autophagic killing of s. enterica or m. tuberculosis (133, 134) . mice deficient in autophagic proteins (atg3, atg5, atg7, atg9, and atg16l1) have embryonic lethality (129) . viable conditional autophagy knockout mice often have impaired pathogen clearance, reduced survival, and severe tissue injury due to enhanced inflammasome and cytokine responses, and in some cases, enhanced th17 responses (136) (137) (138) . in the same study, tbk1 was shown to be important for delivery of the lysosomal hydrolase cathepsin d to the autophagolysosomal compartment (134) . tbk1, therefore, appears to play an essential role in late autophagic flux. autophagy is increasingly appreciated for its role in maintaining cell homeostasis through clearance or normal turnover of cytoplasmic contents or defective cellular organelles, including mitochondria (mitophagy). mice deficient for atg5 specifically in neural cells develop progressive decline in motor function in the absence of any disease-associated mutant proteins, accompanied by the accumulation of cytoplasmic inclusion bodies in neurons (139) . damaged mitochondria are detrimental to cellular homeostasis and efficient removal through autophagy is crucial for cell survival, particularly for senescent cells, such as neurons, which cannot dilute cytotoxic contents through cell division (140) . mitochondrial damage induces concomitant pink1-parkin-mediated poly-ubiquitylation of damaged mitochondria and also activates tbk1. in turn, tbk1 can phosphorylate autophagy receptors (optn, sqstm1/p62, and ndp52), thereby enhancing the ability of these receptors to associate with ubiquitinated cargo (e.g., ubiquitin-tagged, depolarized mitochondria) and autophagic membranes (24, 25, 28, 141) . this post-translational modification creates a signal amplification loop that recruits and retains autophagy receptor/tbk1/ubiquitinated cargo complexes, thereby promoting mitophagy. separate mutations that disrupt tbk1's association with optn, or of optn with ubiquitin, abolish the translocation and activation of tbk1, and, therefore, impair mitophagy (25). exome sequencing identifies tbk1 as a neurodegenerative disease gene in amyotropic lateral sclerosis (als) and frontotemporal dementia (26, 27). further, it was shown that mutations of tbk1 at the c-terminal tbk1 coiled-coil domain, resulted in tbk1's dissociation from optn, while preserving its kinase activity (located at the n-terminal ubiquitin-like domain) (27). these studies provide a potential mechanistic basis for tbk's involvement in als. mutations linking optn to impaired autophagy and neurodegenerative diseases have also been characterized (142) . thus, although it has not been directly demonstrated, tbk1-regulated autophagy appears to maintain cellular homeostasis of long-lived neuronal cells through mitophagy. tank-binding kinase 1-regulated autophagy may also be important for regulation of immune cells. as mentioned above, mice deficient in autophagic proteins (atg3, atg5, atg7, atg9, and atg16l1) have neonatal lethality, as do tbk1 −/− mice (1, 129) . in contrast, sting −/− mice are viable, but have impaired tbk1dependent ifn-i responses to cytoplasmic dna (67) . autophagy allows dynamic changes necessary for proper mammalian development through the recycling and provision of macromolecules and clearance of apoptotic bodies. conditional atg7 fl/fl :vav-cre mice (i.e., hematopoietic cell-specific deletion of atg7) overcomes embryonic lethality, but these mice are anemic and lymphopenic, linking autophagy to erythropoiesis and lymphopoiesis (129) . similarly, t cell-specific deletion of atg5 or atg7 (atg5 fl/fl :lck-cre or atg7 fl/fl :lck-cre mice) or innate lymphoid cell (ilc)-specific deletion of atg5 (atg5 fl/fl :nkp46-cre) reduces peripheral t cells and ilc subpopulations, respectively (143, 144) . maturation of naïve t cells depends on autophagy to reduce mitochondrial and er contents through mitophagy and reticulophagy, respectively (143, 145, 146) . defective autophagy in t cells results in accumulated mitochondrial biomass, disturbed ca 2+ homeostasis, higher levels of reactive oxygen species (superoxide), and enhanced susceptibility to apoptosis (143, 145, 146) . in contrast to lymphopenia, the myeloid compartment in atg7 fl/fl :vav-cre mice is expanded (147) , implicating autophagy in the balance between lymphopoiesis and myelopoiesis. it was also recently shown that autophagy is required for full granulopoiesis (148) . autophagy regulates cellular differentiation and activation by accommodating metabolic adaptation, which can occur in parallel and independently of transcriptional regulators. neutrophils from atg7 fl/fl :vav-cre or atg7 fl/fl :cebpa-cre (granulocyte-macrophage progenitor-specific deletion of atg7) mice are numerically expanded, but are unable to complete maturation and, therefore, are functionally defective. in this case, autophagy-mediated lipolysis (lipophagy) provides free fatty acids to support a mitochondrial respiration pathway essential for neutrophil differentiation (148) . autophagy regulates cytosolic processing of antigen for presentation on mhcii in dcs. the absence of atg5 in dcs results in failure to mount full th1 cell immunity to viral infection (149) . tbk1 has also been associated with metabolic adaptation of dc after tlr stimulation, whereby tbk1 phosphorylates akt for the glycolysis which is necessary for dc activation. shrna-mediated tbk1 knockdown in dcs results in a blunted glycolytic shift and reduced ability of these dcs to prime antigen-specific t cells in vitro (14) . in summary, separate lines of evidence have linked autophagy to cell metabolism, tbk1 to autophagy, and tbk1 to metabolism in immune cell development and activation. further studies of tbk1's role in cellular autophagy and metabolism in various immune contexts could allow manipulation of immune function-either for protection against pathogens or rewiring toward tolerance in autoimmunity. in contrast to the maturation defect of neutrophils in the absence of autophagy, macrophages derived from atg5 fl/fl :lysm-cre mice display a heightened proinflammatory phenotype. these mice develop greater hepatitis on a high fat diet and low dose lps and also spontaneous uveitis (150, 151) . autophagy-deficient macrophages activate the nlrp3 inflammasome and develop il-1β-mediated inflammation (151) (152) (153) . similar to macrophages, atg16l1-deficient dcs have heightened activation in graftversus-host disease (154) . loss of function polymorphisms of atg16l1 have been associated with age-dependent development of inflammatory bowel disease (crohn's disease) owing to impaired clearance of ileal pathogens or endogenous protein aggregates, and chronic elevation of inflammatory cytokine responses (155, 156) . interestingly, dc-specific deletion of tbk1 (tbk1 fl/fl :cd11c-cre mice) also display age-related cellular hyperactivation, with marked upregulation of costimulatory molecules on dcs, t cell activation, and autoimmune features (splenomegaly, lymphadenopathy, and tissue infiltration with lymphocytes) (157) . these tbk1 fl/fl :cd11c-cre mice have an increased frequency of activated ifn-γ-producing cd4 + and cd8 + t cells, while tregs remain comparable to tbk1-sufficient mice. consequently, these mice are more sensitive to eae and mount more robust antitumor immunity against poorly immunogenic b16f10 melanoma cells (157) . enhanced macrophage or dc activation in the absence of tbk1 may be due to impaired autophagy, which normally limits age-related inflammasome activation. of note, the age-dependent hyperinflammatory status of autophagy-or tbk1-deficient macrophages and dcs in atg conditional knockout mouse models resembles aging macrophages. these cells shift from an anti-inflammatory to a proinflammatory phenotype, with an age-related reduction in autophagic activity and sensing of endogenous damage-associated molecular patterns (damps) (158, 159) . thus, it is tempting to speculate that tbk1 maintains cellular autophagy and sustains immune cell longevity and homeostasis. hallmarks of accelerated immune cell aging with chronic tbk1 deficiency are also notable in the tbk1 fl/fl :cd19-cre mice (b cell-specific ablation of tbk1) (160). these mice have normal b cell populations in the spleen and bone marrow, but develop age-related dysregulation of the non-canonical nf-κb pathway, uncontrolled production of iga, increased levels of autoantibody antinuclear antigen and anti-dsdna, with nephropathy-like disease (160) . in this study, it was concluded that steady state tbk1 negatively regulates iga class switching in b cells by attenuating noncanonical nf-κb signaling. this effect was thought to be due to tbk1-mediated phosphorylation and degradation of nf-κbinducing kinase, downstream of baff or april signaling (160). intriguingly, it is possible that tbk1 fl/fl :cd19-cre mice phenocopy the aging b cell repertoire because the b1 population (responsible for iga responses) and autoantibody production are enhanced by age (161) . while chronic deficiency of autophagy or tbk1 results in amplified endogenous inflammation to damps in myeloid cells and abnormalities in the b cell repertoire, inhibition of autophagy may be exploited to target long-lived autoimmune populations (discussed below) through acceleration of immune cell aging. autophagy supports long-lived memory immune cells-implication of tbk1 as discussed above, autophagy is cytoprotective in senescent cells, such as neurons. this cytoprotective function also appears to apply in long-lived immune cells. mice with b cell-specific deletion of atg5 or atg7 (atg5 fl/fl :cd19-cre or atg7 fl/fl :cd19-cre mice) have mostly normal b cell development, but are unable to maintain longlived humoral antibody responses owing to the failure to maintain long-lived plasma cells and memory b cells, respectively (162, 163) . in both studies, plasma cells and memory b cells arising from immunization and gc were shown to upregulate components of the autophagic machinery. in plasma cells, autophagy is thought to maintain longevity by reticulophagy (autophagic clearance of redundant endoplasmic reticulum) to limit excessive antibody synthesis and conserve energy balance (162) . further studies are needed to investigate whether tbk1 is involved in reticulophagy and physiological adaptation of plasma cells. autophagy also supports the lifespan of quiescent, antigenexperienced, long-lived, gc-derived memory b cells through mitophagy. atg7 fl/fl :cd19-cre mice mount normal primary antibody responses and have normal differentiation of post-gc memory b cells, but fail to generate secondary antibody responses to influenza virus due to spontaneous death of memory b cells (163) . memory b cells from atg7 fl/fl :cd19-cre mice are unable to efficiently remove damaged mitochondria, resulting in accumulation of reactive oxygen species, lipid peroxidation, and oxidative stress-induced death. interestingly, these mice also develop enhanced th17 responses to viral infection, possibly as a compensatory mechanism (163) . whether tbk1 also regulates mitophagy and, therefore, the survival of memory b cells requires further investigation. mitophagy was shown to support the generation of lcmv-induced memory cd8 + t cells (164, 165) and mcmvinduced memory nk cells (166) . these studies highlight that autophagy is not apparently required for germline t cell development, nor for t cell activation and proliferation, but is important for established effector t cells to generate a pool of memory cells (165, 166) . autophagy is dynamically induced at various stages of immune cell development, activation, and differentiation, but plays a particular role in the formation and maintenance of longlived immune populations, including memory b cells and senescent plasma cells. because long-lived plasma cells or plasma cells deriving from memory b cells can drive persistent autoimmune disease (167) , abrogation of autophagy through tbk1 inhibition might reduce resistance to autonomous cell aging and death, and diminish pathogenic autoantibody responses. in summary, a number of studies demonstrate significant overlap between tbk1 and autophagy, most notably in antimicrobial xenophagy and maintenance of neuronal cell health. similar to autophagy, tbk1 has a complex role in immune cells. it is known that autophagy is dynamically regulated to accommodate rapid metabolic adaptation and organelle turnover associated with cell development, differentiation, activation, and longevity. tbk1 regulates autophagy through post-translational modifications of autophagy adaptor/receptor proteins required for the maturation of autophagosomes. tbk1's involvement in other types of organelle autophagy and metabolic signaling pathways in immune lineage cells and immune responses is of great interest. autophagy supports the extended lifespan of cells, such as neurons, immune memory populations, and long-lived plasma cells. targeting autophagy through the inhibition of tbk1 may provide a novel approach for treating humoral autoimmune diseases. inhibition of autophagy or tbk1 may favor the generation of short-lived effector cells, rather than long-lived memory populations. both autophagy and tbk1 have been shown to regulate the delicate balance between cellular adaptation for efficient immune response and aging-associated autoinflammation. studies exploring how, when, and where tbk1 facilitates autophagy in distinct immune lineages will inform potential modulation of protective or pathogenic immune responses. in this review, we discuss the remarkable functional diversity of tbk1 in the context of humoral autoimmunity. these pathways are summarized in the figure 1 . tbk1 is required for ifn-is production in the context of sensing viral or aberrant cytoplasmic nucleic acids. overactive tbk1 can precipitate ifn-is and lupus, such as is the case for trex1 deficiency. recent literature reports that tbk1 is associated with humoral antibody responses via its recruitment to and activation of icos in the cd4 + tfh population in gcs of lymph nodes. icos is required for full maturation of the gc tfh population, the gc reaction, gc-mediated generation of affinity-matured long-lived plasma cell and memory b cells, and productive gc-derived antibody responses. thus, tbk1 inhibition may be useful in pathogenic autoantibody responses mediated by gc. finally, we highlight the similarity of tbk1 deficiency to that of autophagy-deficiency. therapeutic tbk1 inhibition may therefore lead to premature aging and/or death of pathogenic immune cells, such as long-lived plasma cells, and memory b cells in autoimmune diseases. all authors contributed to the assembly and revision of this review manuscript. (4) deficiency of t2k leads to apoptotic liver degeneration and impaired nf-kappab-dependent gene transcription phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation the tyrosine kinase src promotes phosphorylation of the kinase tbk1 to facilitate type i interferon production after viral infection molecular basis of tank-binding kinase 1 activation by transautophosphorylation crystal structure and mechanism of activation of tank-binding kinase 1 sting activation by translocation from the er is associated with infection and autoinflammatory disease the golgi apparatus acts as a platform for tbk1 activation after viral rna sensing ikkepsilon and tbk1 are essential components of the irf3 signaling pathway triggering the interferon antiviral response through an ikk-related pathway the roles of two ikappab kinase-related kinases in lipopolysaccharide and double stranded rna signaling and viral infection ifn-regulatory factor 3-dependent gene expression is defective in tbk1-deficient mouse embryonic fibroblasts sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway ikappab kinase epsilon and tank-binding kinase 1 activate akt by direct phosphorylation tlrdriven early glycolytic reprogramming via the kinases tbk1-ikkvarepsilon supports the anabolic demands of dendritic cell activation dissection of tbk1 signaling via phosphoproteomics in lung cancer cells multiple functions of the ikk-related kinase ikkepsilon in interferonmediated antiviral immunity the inducible kinase ikki is required for il-17-dependent signaling associated with neutrophilia and pulmonary inflammation inactivation of the enzyme gsk3alpha by the kinase ikki promotes akt-mtor signaling pathway that mediates interleukin-1-induced th17 cell maintenance ikappab kinase epsilon is an nfatc1 kinase that inhibits t cell immune response interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus interferon and granulopoiesis signatures in systemic lupus erythematosus blood systemic lupus erythematosus and the type i interferon system a traf-like motif of the inducible costimulator icos controls development targeted therapeutics in sle: emerging strategies to modulate the interferon pathway type i interferon and lupus irf3-dependent type i interferon response in b cells regulates cpg-mediated antibody production tankbinding kinase-1 delineates innate and adaptive immune responses to dna vaccines dna released from dying host cells mediates aluminum adjuvant activity hydroxypropyl-beta-cyclodextrin spikes local inflammation that induces th2 cell and t follicular helper cell responses to the coadministered antigen sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity trex1 prevents cell-intrinsic initiation of autoimmunity trex1 exonuclease degrades ssdna to prevent chronic checkpoint activation and autoimmune disease autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease mutations in the gene encoding the 3'-5' dna exonuclease trex1 cause aicardi-goutieres syndrome at the ags1 locus familial chilblain lupus due to a novel mutation in the exonuclease iii domain of 3' repair exonuclease 1 (trex1) mutations in the gene encoding the 3'-5' dna exonuclease trex1 are associated with systemic lupus erythematosus cutting edge: inhibiting tbk1 by compound ii ameliorates autoimmune disease in mice germinal centers dynamics of b cells in germinal centres persistent antigen and germinal center b cells sustain t follicular helper cell responses and phenotype high affinity germinal center b cells are actively selected into the plasma cell compartment plasma cell and memory b cell differentiation from the germinal center germinal-center development of memory b cells driven by il-9 from follicular helper t cells dysregulation of germinal centres in autoimmune disease wasp-deficient b cells play a critical, cell-intrinsic role in triggering autoimmunity altered b cell signalling in autoimmunity t follicular helper cell differentiation, function, and roles in disease pathophysiology of t follicular helper cells in humans and mice mice deficient for the cd40 ligand il-4-producing cd4+ t cells in reactive lymph nodes during helminth infection are t follicular helper cells cytokine-secreting follicular t cells shape the antibody repertoire il-21 regulates germinal center b cell differentiation and proliferation through a b cell-intrinsic mechanism defects in germinal center selection in sle a ring-type ubiquitin ligase family member required to repress follicular helper t cells and autoimmunity follicular helper t cells are required for systemic autoimmunity bcl6 and blimp-1 are reciprocal and antagonistic regulators of t follicular helper cell differentiation bcl6 mediates the development of t follicular helper cells optimal germinal center responses require a multistage t cell:b cell adhesion process involving integrins, slam-associated protein, and cd84 the role of follicular helper t cell molecules and environmental influences in autoantibody production and progression to inflammatory arthritis in mice increased frequency of circulating follicular helper t cells in patients with rheumatoid arthritis high frequencies of activated b cells and t follicular helper cells are correlated with disease activity in patients with new-onset rheumatoid arthritis circulating follicular helper-like t cells in systemic lupus erythematosus: association with disease activity follicular helper t cell signature in type 1 diabetes follicular helper t cells may play an important role in the severity of primary sjogren's syndrome b cell depletion therapy normalizes circulating follicular th cells in primary sjogren syndrome icos is an inducible t-cell co-stimulator structurally and functionally related to cd28 expression of icos in vivo defines cd4+ effector t cells with high inflammatory potential and a strong bias for secretion of interleukin 10 icos receptor instructs t follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor bcl6 tfh cells progressively differentiate to regulate the germinal center response impaired germinal center formation and recall t-cell-dependent immune responses in mice lacking the costimulatory ligand b7-h2 icos co-stimulatory receptor is essential for t-cell activation and function icos is critical for cd40-mediated antibody class switching icos is essential for effective t-helper-cell responses inducible costimulator promotes helper t-cell differentiation through phosphoinositide 3-kinase loss of immune tolerance is controlled by icos in sle1 mice b cell-specific expression of inducible costimulator ligand is necessary for the induction of arthritis in mice amelioration of collagen-induced arthritis by blockade of inducible costimulator-b7 homologous protein costimulation b7rp-1 blockade ameliorates autoimmunity through regulation of follicular helper t cells inducible t-cell co-stimulator ligand (icosl) blockade leads to selective inhibition of anti-klh igg responses in subjects with systemic lupus erythematosus the transcriptional repressor bcl-6 directs t follicular helper cell lineage commitment targeted knock-in mice expressing mutations of cd28 reveal an essential pathway for costimulation icos coreceptor signaling inactivates the transcription factor foxo1 to promote tfh cell differentiation icos maintains the t follicular helper cell phenotype by down-regulating kruppel-like factor 2 phosphoinositide 3-kinase activity in t cells regulates the magnitude of the germinal center reaction tbk1 directly engages akt/pkb survival signaling to support oncogenic transformation regulation of t-cell activation and migration by the kinase tbk1 during neuroinflammation th1 cytokine responses fail to effectively control chlamydia lung infection in icos ligand knockout mice impact of inducible co-stimulatory molecule (icos) on t-cell responses and protection against mycobacterium tuberculosis infection the costimulatory molecule icos regulates host th1 and follicular th cell differentiation in response to plasmodium chabaudi as infection autophagy in immunity and inflammation the role of atg proteins in autophagosome formation autophagy: an emerging immunological paradigm autophagy in antimicrobial immunity the tbk1 adaptor and autophagy receptor ndp52 restricts the proliferation of ubiquitincoated bacteria phosphorylation of the autophagy receptor optineurin restricts salmonella growth tbk-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation serine 403 phosphorylation of p62/sqstm1 regulates selective autophagic clearance of ubiquitinated proteins autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation autophagy in infection, inflammation and immunity atg7 deficiency intensifies inflammasome activation and pyroptosis in pseudomonas sepsis suppression of basal autophagy in neural cells causes neurodegenerative disease in mice compromised autophagy and neurodegenerative diseases the ubiquitin kinase pink1 recruits autophagy receptors to induce mitophagy mutations of optineurin in amyotrophic lateral sclerosis autophagy is essential for mitochondrial clearance in mature t lymphocytes atg5 is essential for the development and survival of innate lymphocytes temporal regulation of intracellular organelle homeostasis in t lymphocytes by autophagy autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in t lymphocytes the autophagy protein atg7 is essential for hematopoietic stem cell maintenance autophagy-dependent generation of free fatty acids is critical for normal neutrophil differentiation in vivo requirement for atg5 in antigen presentation by dendritic cells impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization impaired autophagy in macrophages promotes inflammatory eye disease autophagy-based unconventional secretory pathway for extracellular delivery of il-1beta autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial dna mediated by the nalp3 inflammasome autophagy gene atg16l1 prevents lethal t cell alloreactivity mediated by dendritic cells a crohn's disease variant in atg16l1 enhances its degradation by caspase 3 defective atg16l1-mediated removal of ire1alpha drives crohn's disease-like ileitis the kinase tbk1 functions in dendritic cells to regulate t cell homeostasis, autoimmunity, and antitumor immunity canonical nlrp3 inflammasome links systemic low-grade inflammation to functional decline in aging agedependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system the kinase tbk1 controls iga class switching by negatively regulating noncanonical nf-kappab signaling aging-dependent exclusion of antigen-inexperienced cells from the peripheral b cell repertoire plasma cells require autophagy for sustainable immunoglobulin production essential role for autophagy in the maintenance of immunological memory against influenza infection autophagy is a critical regulator of memory cd8(+) t cell formation autophagy is essential for effector cd8(+) t cell survival and memory formation bnip3-and bnip3l-mediated mitophagy promotes the generation of natural killer cell memory long-lived autoreactive plasma cells drive persistent autoimmune inflammation the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0018506-unh840wg authors: bustami, mona; matalka, khalid z; mallah, eyad; abu-qatouseh, luay; abu dayyih, wael; hussein, nour; abu safieh, nayef; elyyan, yousef; hussein, nagham; arafat, tawfiq title: the prevalence of overweight and obesity among women in jordan: a risk factor for developing chronic diseases date: 2021-06-22 journal: j multidiscip healthc doi: 10.2147/jmdh.s313172 sha: 71aa24aea34445e25e8d6cb1ed09e4f97a371727 doc_id: 18506 cord_uid: unh840wg objective: the study aimed to investigate the prevalence of obesity among jordanian women and its association with a wide range of chronic diseases. methods: subjects were enrolled in the present cross-sectional study based on a random drop-off technique at the obstetrics and gynecology clinics at jordan university hospital. initially, any female 18 years of age and older was asked to enroll in the study. relevant data were gathered using a questionnaire composed of 30 questions, and body mass index (bmi) was determined from each participant’s weight and height. the following variables were collected: socio-demographic, chronic diseases, and health status. each variable’s frequencies were reported, and the 95% confidence interval (95% ci) for each variable was calculated. for association analysis, chi-square analysis was performed with an odds ratio (or) and 95% ci. multinomial logistic regression analysis was applied to a combination of independent variables and a dependent condition with covariate factors. results: the age-standardized prevalence of overweight/obese jordanian women was 70.6% (95% ci 66.0–74.8%). on the other hand, the age-standardized prevalence of only obese women was 36.4 (95% cl 31.9–41.2%). furthermore, the association between age and overweight/obesity was significant (p<0.0001). the percentage of overweight and obesity started to be significant in the 30–39 year age group. moreover, the or for obesity ranged from 2.7 to 7.0 (p<0.05–0.01) for those women with only elementary education. besides, high parity was significantly associated with obesity and elementary education. for chronic conditions, the percentages of hypertension, diabetes, hypertriglyceridemia, osteoporosis, and rheumatoid arthritis were significantly correlated with increased bmi in jordanian women. with age adjustment, however, only hypertension was associated with obese level 3 with or of 7.2 and 95% ci of 2.1–25.1 (p<0.01). conclusion: there is a high prevalence of overweight/obesity among women in jordan, which was related to high parity and low education level. this high prevalence of obesity increased the incidence of chronic diseases, such as hypertension. therefore, community-based multiple strategies are required to combat obesity in jordanian women. obesity and overweight pose a significant public health threat as a rising epidemic in many countries. 1 in recent years, an increase in body mass index (bmi) was linked to the risk burden of non-communicable chronic diseases globally. [2] [3] [4] several epidemiologic studies have shown that the rates of obesity in all ages and among both males and females irrespective of geographical locality, ethnicity, or socioeconomic status are increasing. 5, 6 for instance, the united states national health and nutrition examination survey has revealed data showing the overall prevalence of obesity among adults in 2013-2014 was 37.7%. however, the prevalence of obesity is generally greater in women. 7 the prevalence among women was 40.4% compared with 35.0% among men. 8 furthermore, women with high bmi are marked at higher risk of breast cancer, 9 atherosclerotic cardiovascular disease, 10 type 2 diabetes, 11 hypertension, 12 dyslipidemia, 13 musculoskeletal disorders, 14 endocrine disorders, 15 risk of infertility, 16 and impaired pulmonary function. 17 the overcharging aim to prevent overweight and obesity among women has been highly addressed to governments by the world health organization (who) to prevent premature death due to chronic diseases. 18 aging along with obesity are considered to be the most popular universal contributors to health decline. during 2016, 78% of the total 36,000 jordanian deaths occurred because of chronic diseases. 19 in addition, it was estimated that the prevalence of chronic diseases from 2005 to 2050 in jordan is expected to accelerate rapidly. 20 in a 2008 study, the age-standardized prevalence of obesity in the northern area of jordan was 28.1% in men and 53.1% in women. 21 besides, the same study reported increased obesity percentages in women aged ≥40 years in 2008 compared to the surveys performed in 1994 and 2004. 21 as in other areas, obesity in jordan coincided with an increased odds ratio of diabetes, hypertension, and dyslipidemia. [21] [22] [23] though, weight reduction and control should be the center of secondary prevention strategies of hypertension and cardiovascular disease when it comes to conveying clinical information into practice. recently, we have studied the factors associated with natural menopause age, mainly related to premature and early menopause among jordanian women. 24 although the bmi increase was not a factor in premature or early menopause in the latter study, bmi data in women was alarming. therefore, in the present study, we investigated the prevalence of obesity in relation to age, education, and parity, and its correlation to chronic diseases and its emerging risk factors in jordanian women. this study was designed for cross-sectional research. a study protocol was submitted to the research ethics committee at the jordan university hospital and received approval (approval #38/2015) in early 2016. this study was carried out at jordan university hospital per the declaration of helsinki, whereby every subject has agreed to enroll by willingly signing informed consent. for the selection process, we followed a random drop-off technique to the obstetrics and gynecology clinics atjordan university hospital. any female 18 years of age and older was asked to enroll in the study. investigators discussed the importance of the study to each participant. following the acceptance to enroll, each participant signed informed consent. furthermore, the sample size was calculated to be ≥380 subjects, based on 50% probability of obesity in jordan, 0.05 error and 95% confidence interval (95% ci). 21 four hundred and sixty-eight accepted to enroll in the study. out of the 468, 40 subjects were excluded from the analysis because they were non-jordanian and were residing in jordan as students, ie, they were residing for a short period of time. with the help of the health-trained investigators, each participant answered a questionnaire containing 30 questions. the questions have information about each participant's socioeconomic factors, including marital status, having kids, type of work, household salary, and education status. furthermore, the questionnaire included data on each subject's smoking habits, chronic conditions, and diseases. each subject's weight and height were measured and recorded (seca 700 with stadiometer, hamburg, germany). bmi was calculated and classified (c-bmi) using the international classification recommended by who. c-bmi was as follows: 1 for underweight (<18.5 kg/m 2 ), 2 for standard weight (18.5-24.9 kg/m 2 ), 3 for overweight (25.0-29.9 kg/m 2 ), 4 for obese 1 (30-34.9 kg/m 2 ), 5 for obese 2 (35-39.9 kg/m 2 ), and 6 for obese 3 (>40 kg/m 2 ). means and standard deviations were used to describe the continuous variables and percentages to describe categorical variables. each categorical variable's frequencies were reported, and the 95% confidence interval (95% ci) for each variable was calculated. for association analysis, chisquare analysis was used with the estimation of odds ratio (or) and 95% ci. furthermore, multinomial logistic regression analysis was applied to a combination of independent https://doi.org/10.2147/jmdh.s313172 journal of multidisciplinary healthcare 2021:14 variables and a dependent condition with covariate factors, such as age and socioeconomic variables. a p-value of <0.05 was considered to be statistically significant. all analyses were performed using spss 25 statistical package. the study population consisted of 428 jordanian women with a mean age of 48.6 years (±14.5 sd) ( table 1 ). the majority were married (75.5%), having kids (68.5%), housewives (64%), never smoked (79.2%), and their household income was low (75.2%). besides, half of the population (50.9%) had a college degree, and slightly higher than half (57.7%) considered having good health (table 1) . more than half (59.8%) of the study population had arthritis pain. however, the most common chronic condition that affected the study group was hypertension (29.5%), and to a lesser extent, diabetes and hypertriglyceridemia (table 2) . furthermore, women with ovarian cysts, uterine fibrosis, and osteoporosis were 12-14%. on the other hand, the percentage of autoimmune-related diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and thyroid diseases was 6.1%. the percentage frequency of the study population revealed that the majority of women were overweight ( figure 1a ). this represented an overall c-bmi average of 3.25 (± 1.19) with a 95% ci of 3.14-3.37. according to c-bmi ≥3, the age-standardized prevalence of overweight/obese jordanian women was 70.6% (95% ci 66.0-74.8%). on the other hand, the age-standardized prevalence of only obese women was 36.4 (95% cl 31.9-41.2%). furthermore, data showed that as females aged, the c-bmi significantly increased ( figure 1b ). according to the age groups, the percentage of overweight/obese females increased from ~26% in 20-29-year age groups to a range between 73dovepress 84% in 30 years of age and older (p<0.0001) ( figure 1c) . however, the percentage of women with the standard weight (c-bmi of 2) was kept constant (~20%) from 30 years of age and above. on the other hand, the percentage of obese women kept increasing with age ( figure 1c ). to evaluate if the education level, having kids, or smoking status were an associated factor with prevalence of obesity in jordanian women, we used a multinomial regression analysis adjusted with age as a covariate factor. the results showed that obesity, but not overweight, was related to low education levels ( table 3 ). the adjusted or (aor) values ranged from 2.7 to 7.0 for elementary education and obesity, and these aor values became insignificant with high school and college educations. on the other hand, no significant correlation between high bmi and having kids or smoking status was observed. furthermore, when parity was classified to low (1-2 kids), medium (3-4 kids), grand parity (5-6 kids) and high grand parity (>7 kids), obesity was significantly associated with medium, grand, and high grand parity in comparison to low parity (p<0.05, 0.05, 0.01, respectively) ( table 4) . when age was introduced as a cofactor, only high grand parity was associated with obesity (table 4 ). however, when education was introduced, no significance was noted. besides, low education was significantly correlated with an increase in parity (table 5 ). since overweight/obesity was a major observational factor in the jordanian population, further analysis related to chronic/disease conditions with high bmi was performed ( table 6 ). the percentages of hypertension, diabetes, hypertriglyceridemia, osteoporosis, and rheumatoid arthritis were significantly correlated with increased c-bmi in jordanian women. furthermore, the or ranged between 2.53 and 2.76 for metabolic syndrome-related conditions and was extremely high (9.01) for rheumatoid arthritis. after adjusting for age and socioeconomic variables in the multinomial regression analysis, only hypertension was associated with an obese 3 level and an aor of 7.2 (table 7) . furthermore, rheumatoid arthritis showed a trend towards correlation with overweight and obese 3 (aor 7.6 and 10.3, p=0.058 and 0.056, respectively). on the other hand, diabetes, osteoporosis, and hypertriglyceridemia became insignificantly correlated with overweight/obesity (table 7) . the present study showed that age-adjusted prevalence of overweight and obesity is high (70.6%) in jordanian women. this is similar to females living in the eastern mediterranean regions, including jordan. 25, 26 however, the age-adjusted prevalence for obesity is moderately high (36.4%). the latter is less than the reported study in northern jordan, 21 or in kuwait, saudi arabia, or qatar, but closer to women from lebanon, iran, or tunisia. 25 furthermore, our findings stressed on the association of obesity to low education level and high parity. therefore, a good reason for the differences in the prevalence of obesity in the literature between different populations and studies is the percentage of women with low education levels and the number of parity each woman had in each study. furthermore, we showed that bmi increased steadily from the age group of 30-39 to over 60 years. hence, making it evident that increasing bmi is highly associated with age. this finding is expected since previous studies showed that overweight/obesity is related to age. 27 there were no coinciding studies in jordan that correlate educational level to obesity among women. however, previous research has shown that the prevalence of overweight/ obesity among low socioeconomic levels. 21, 26 herein, we showed that the prevalence of obesity was inversely related to educational levels. women with only elementary education showed to be the highest in obesity. this is in agreement with previous studies that have produced ample evidence that obesity is related to low educational levels, [28] [29] [30] and consequently such low education would disrupt the dynamic factor of the socioeconomic ladder. 31 since an increasing number of pregnancies may contribute to postpartum weight retention, 32, 33 this study showed that increasing parity is significantly related to obesity. 34, 35 furthermore, as a proxy for socioeconomic status, low educational level is inversely associated with parity, and when cofounded with obesity, lower education was highly related to increased odds of obesity due to increase in parity. 34, 36 aging, with its related consequences on health, is considered a significant contributor to health decline. when the accumulation of unhealthy lifestyle behaviors such as low physical activity, high intake of sugar-sweetened beverages, and low intake of fruits and vegetables during aging would increase multiple inflammatory processes and therefore, linkage to chronic inflammatory diseases occurs. [37] [38] [39] [40] in the present study, hypertension, diabetes, hypertriglyceridemia, osteoporosis, and rheumatoid arthritis were all significantly correlated with increased bmi and age. however, after adjusting for age and socioeconomic variables, only hypertension was associated with obesity. the latter points to other risk factors accumulated with aging that may enhance the prevalence of diabetes, hypertriglyceridemia, osteoporosis, and rheumatoid arthritis. furthermore, this reveals the limitations of the present study, which did not include variables such as lifestyle behavior for each subject, obesity family history, and obesity in early adolescence. 41 besides, this study is a cross-sectional observational study, and it is difficult to make inferences regarding causal relationships and obesity. furthermore, although the sample size of this study is adequate, the number of subjects enrolled is limited. finally, some of the questionnaire questions were relatively subjective and depended on each participant's opinion. obesity is an alarming risk factor for hypertension, leading to increased morbidity and mortality among jordanian women. hypertension is a modifiable risk factor for cardiovascular and renal diseases. 42 besides, multiple pathophysiological abnormalities were found to be highly related to hypertension and obesity. the associated abnormalities are inflammation, insulin resistance, increased oxidative stress, abnormal adipokines production, the sympathetic nervous system, and the reninangiotensin-aldosterone systems. 43 thus, early diagnosis and treatment of obesity impact the prevention of hypertension and its consequences. furthermore, obesity is preventable by improving our understanding of the factors that promote obesity from an early age. thus, implementing preventing obesity into clinical practice should increase awareness against high-fat food consumption, reduced physical exercise, and improved sedentary lifestyle. 44, 45 medical education in the clinic regarding obesity and its correlation to metabolic and cardiovascular diseases, adopting new lifestyle habits for weight management should be of high importance. furthermore, this and other previous studies should help health policymakers implement awareness of the background of obesity in jordan. in conclusion, there is a high prevalence of overweight/ obesity among women in jordan, which was related to high parity and low education level. besides, this high prevalence increased the incidence of chronic conditions among obese women. therefore, community-based multiple strategies are required to combat obesity in jordanian women. furthermore, research should be dedicated to supporting women's well-being in jordan's clinics by elevating the association of overweight and obesity with increased morbidity and mortality rates. finally, it is vital to improve our understanding of obesity in women to initiate interventional strategies to control chronic diseases. 95% ci, 95% confidence interval; aor, adjusted odds ratio; bmi, body mass index; c-bmi, classified bmi; or, odds ratio; who, world health organization. obesity collaborators. health effects of overweight and obesity in 195 countries over 25 years who; 2020 tackling obesities: future choices-obesogenic environments-evidence review. london: government office for science overweight and obesity: prevalence, consequences, and causes of a growing public health problem obesity epidemiology worldwide obesity as a disease: the obesity society 2018 position statement the epidemiology of obesity trends in obesity among adults in the united states obesity and adverse breast cancer risk and outcome: mechanistic insights and strategies for intervention cardiovascular disease in women: clinical perspectives type ii diabetes, obesity, and breast cancer risk: the multiethnic cohort obesity and hypertension trends in lipids, obesity, metabolic syndrome, and diabetes mellitus in the united states: an nhanes analysis the impact of obesity on the musculoskeletal system polycystic ovary syndrome, obesity, and pregnancy obesity as disruptor of the female fertility obesity and its implications for covid-19 mortality world health organization. global action plan for the prevention and control of ncds noncommunicable diseases country profiles population division of the department of economic and social affairs of the united nations secretariat obesity in jordan: prevalence, associated factors, comorbidities, and change in prevalence over ten years the prevalence of dyslipidemia among jordanians hypertension in jordan: prevalence, awareness, control, and its associated factors age of natural menopause among jordanian women and factors related to premature and early menopause the relationship between obesity, overweight, and the human development index in world health organization eastern mediterranean region countries an alarmingly high and increasing prevalence of obesity in jordan obesity and related consequences to ageing prevalence of obesity among adults, by household income and education -united states association of overweight, obesity and abdominal obesity with socioeconomic status and educational level in colombia a new scale for measuring socioeconomic status in educational research: development and validation of the australian socioeconomic index 2006 (ausei06) prevalence and characteristics associated with gestational weight gain adequacy associations between pre-pregnancy bmi, gestational weight gain, and prenatal diet quality in a national sample impact of parity on anthropometric measures of obesity controlling by multiple confounders: a cross-sectional study in chilean women parity and overweight/obesity in peruvian women association between parity and obesity: a cross sectional study on 6447 iranian females screen time, adiposity and cardiometabolic markers: mediation by physical activity, not snacking, among 11-year-old children sugar-sweetened beverages and risk of metabolic syndrome and type 2 diabetes: a meta-analysis chronic inflammation in the etiology of disease across the life span smoking, drinking, diet and physical activity-modifiable lifestyle risk factors and their associations with age to first chronic disease obesity in women: insights for the clinician elevated systolic blood pressure and risk of cardiovascular and renal disease: overview of evidence from observational epidemiologic studies and randomized controlled trials hypertension in obesity and the impact of weight loss prevalence of selected chronic, noncommunicable disease risk factors in jordan: results of the 2007 jordan behavioral risk factor surveillance survey assessing risk factors for chronic disease-jordan the authors acknowledge the financial support from the deanship of research and graduate studies at the university of petra (grant numbers 5-4-2015), amman, jordan, and jordan university hospital to facilitate meeting the participants and collecting samples. a special gratitude acknowledgment dedicated to ms. suzan alwawi, university of petra, for technical assistance and finally to all study participants. khalid z matalka reports their current address is matalka's scientific writing, lexington, ma, 02420, usa. all authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval for the version to be published; and agreed to be accountable for all aspects of the work. the authors declare no conflicts of interest for this work. the journal of multidisciplinary healthcare is an international, peerreviewed open-access journal that aims to represent and publish research in healthcare areas delivered by practitioners of different disciplines. this includes studies and reviews conducted by multidisciplinary teams as well as research which evaluates the results or conduct of such teams or healthcare processes in general. the journal covers a very wide range of areas and welcomes submissions from practitioners at all levels, from all over the world. the manuscript management system is completely online and includes a very quick and fair peer-review system. visit http://www.dovepress.com/testimonials. php to read real quotes from published authors. key: cord-0061257-so4j6cms authors: hosseinikhah, seyedeh maryam; barani, mahmood; rahdar, abbas; madry, henning; arshad, rabia; mohammadzadeh, vahideh; cucchiarini, magali title: nanomaterials for the diagnosis and treatment of inflammatory arthritis date: 2021-03-18 journal: int j mol sci doi: 10.3390/ijms22063092 sha: 89143b97e3e8bc8771137e34f8eebd91a3710679 doc_id: 61257 cord_uid: so4j6cms nanomaterials have received increasing attention due to their unique chemical and physical properties for the treatment of rheumatoid arthritis (ra), the most common complex multifactorial joint-associated autoimmune inflammatory disorder. ra is characterized by an inflammation of the synovium with increased production of proinflammatory cytokines (il-1, il-6, il-8, and il-10) and by the destruction of the articular cartilage and bone, and it is associated with the development of cardiovascular disorders such as heart attack and stroke. while a number of imaging tools allow for the monitoring and diagnosis of inflammatory arthritis, and despite ongoing work to enhance their sensitivity and precision, the proper assessment of ra remains difficult particularly in the early stages of the disease. our goal here is to describe the benefits of applying various nanomaterials as next-generation ra imaging and detection tools using contrast agents and nanosensors and as improved drug delivery systems for the effective treatment of the disease. inflammatory arthritis is an autoimmune heterogenous group of joint disorders associated with pain, stiffness, bone erosion, synovial hyperplasia, and progressive joint destruction [1, 2] that aggravates prevailing inflammation to the associated connective tissues, lungs, heart, and skin [3] . the most alarming situation in inflammatory arthritis is the irreversible inflammatory damage [4, 5] . inflammation in arthritis is linked to either any infection or any foreign organism like bacteria, virus, and fungi [6] . inflammation is the prime protective feature of the immune system in preventing from underlying pathogenic infections or injury [7] . normal inflammatory processes of host immune cells in maintaining homeostasis accounts for the mucosal, connective, and epithelial barriers and proinflammatory signaling [8] . all these three natural mechanistic processes help in limiting the overburden of notorious pathogenic bacteria and overactivated immune response [9] . however, any physiological change in the body due to the various etiological factors like stress, genetics, and age leads to an imbalance in homeostasis and the penetration of pathogenic bacteria in the connective tissues and synovial joints, contributing to exaggerated inflammation and pathogenesis of the disease [10] . other reasons for inflammatory arthritis may include a reduction in the amount of cartilage, the tissue that withstands shock and stress in the joint [10] . moreover, inflammatory arthritis can be described in various types, i.e., ankylosing spondylitis where inflammation occurs via fusion of small bones of the spinal vertebrae, making spine hard and less flexible [11] . in some cases, ribs can also be affected and can cause difficult breathing [12] . similarly, gout can be developed due to increased levels of uric acid and defined as inflammation in the joints of feet and toes, resulting in intense pain and a burning sensation [13] . moreover, lyme disease is characterized by the swelling of joints caused by infectious bacterial agents [14] . ra is the most common type of chronic inflammatory arthritis that can affect joints and damages variety of body systems and blood vessels [15, 16] . the prevention of inflammatory arthritis is more effective compared with its treatment as lifestyle modifications, weight loss, and antioxidant rich food are highly effective in mitigating the onset of inflammatory arthritis [17] . as far as the treatment of inflammatory arthritis is concerned, emphasis is placed on the management of pain using heating and ice packs for soothing [18] . however, other practical sorts of treatment for inflammatory arthritis include medications like analgesics and non-steroidal anti-inflammatory drugs (nsaids) such as aspirin, celecoxib, diflunisal, ibuprofen, and naproxen sodium [19] . menthol-based analgesic creams and immunosuppressants are also common modalities used against inflammatory arthritis [20, 21] . nsaids function by restricting prostaglandin synthesis by inhibiting the enzyme cyclooxygenase (cox), thus reducing swelling and pain [22] . in severe conditions where medications remain ineffective, surgery can be performed to replace the joint with an artificial one, especially for hips and knees [23] . physical therapy for the treatment of inflammatory arthritis is also common in some group of patients and involves muscle strengthening around the affected joint [24] . however, administration of medications through the oral and parenteral routes is limited due to low bioavailability, rapid metabolism, poor absorption, first-pass effect, and serious adverse effects [25] . several diagnosis techniques for inflammatory arthritis includes blood tests like erythrocyte sedimentation rate (esr) to assess the levels of inflammation in the body, c-reactive protein (crp), and complete blood count (cbc), as well as joint scans and x-rays [15] . the diagnosis of inflammatory arthritis is a challenging issue due to high time gaps between the incidence of the disease and the possibility of detecting specific markers [26] . nanomedicine has revolutionized the field of medicine and significantly proved to ameliorate the pharmacological and pharmacokinetic patterns of the conventional medications in the treatment of inflammatory arthritis [27] [28] [29] . nanomaterials are sophisticated and smart systems to enhance drug delivery [30] . therefore, nanoparticles (nps) are attractive compounds to stabilize and encapsulate anti-inflammatory drugs, increasing their solubility, half-life, therapeutic index, safety, and efficacy [31, 32] . at a higher level, nps derived from smart formulations may also prompt targetability potential with specific receptors [33, 34] . several nanoformulations enriched with enhanced drug delivery in treating inflammatory arthritis include nsaid-based metallic and polymeric np conjugates (chitosan-dexamethasone nps, i.e., cs-dex nps), (hyaluronic-gold-tocilizumab nps), human serum albumin nps (arginine-glycine aspartic acid attached with gold nano half-shells conjugated with methotrexate (mtx)), solid lipid nps (curcumin cur-loaded slns), surface-engineered liposomes, nanoemulsions (quercetin-loaded nanoemulsionbased gel and intra-articular mtx-associated nanoemulsion) and nanomicelles, and herbal cur-based nps [35] . these nps can be more effective in the size range of 100 to 400 nm. nps can also be decorated with certain antibodies to specifically target the macrophages, playing a major role in inflammatory processes in arthritis [36] [37] [38] . nanomedicine has also been largely involved in the detection of inflammatory arthritis with higher sensitivity, cost-effectiveness, and minimized overdiagnosis owing to its high specificity. nanomaterial drug delivery is advantageous compared with conventional therapy because of its enhanced targeting specificity via controlled drug release, its increased ability to solubilize hydrophobic drugs, its synergistic combinatorial chemistry, and superior ability for drug delivery [39, 40] . advanced in vivo molecular imaging approaches like magnetic resonance imaging (mri) can take images of the internal joint and cartilage [41, 42] . contrast agents used in mri help in the differentiation between normal as well as abnormal tissues via magnetic signals [42] . moreover, superparamagnetic iron oxide nanoparticles (spions), gold, and cs-glyco-nps (spions) are captured by monocytes as well as macrophages and are transported to the inflamed tissues. these nps allow for very high resolution, sensitive detection of discrete forms of inflamed joints and quantification via treating with dex [43] . spions have the remarkable ability to exhibit favorable non-toxicological profiles [42] . with this in mind, as a continuous effort related to the synthesis of nanomaterials and investigation of their potential in biological applications , we currently review here different nanomaterials applied to diagnosis and treatment of inflammatory arthritis. the disease is difficult to diagnose as there are more than 100 various kinds of arthritis and among the various disorders affecting the joints, several symptoms are identical [83] . the following classes will usually be defined as arthritis: inflammatory arthritis, degenerative arthritis, infectious arthritis, and metabolic arthritis. [84] . the most common form of arthritis is the inflammatory arthritis [85] . historically, for the diagnosis of inflammatory arthritis, some common laboratory assays and imaging methods have been carried out. popular laboratory tests include the detection of blood antinuclear antibodies, arthrocentesis (collection and testing of synovial fluid), complement tests, and blood cell counts (white and red blood cells, and platelets) [86, 87] . for example, the sedimentation rate of erythrocytes or esr determines how easily red blood cells deposit at the bottom of a test tube. when there is inflammation in the body, the amount of esr increases [88] . the amount of red blood cells present in a sample of blood is determined by hematocrit or packed cell volume (pcv). low red blood cell levels (anemia) are frequent in patients with arthritis [89] . the rheumatoid factor (rf) monitors the presence of an antibody in most patients with ra [90] . interestingly, for inflammatory arthritis, uric acid and crp increase in gout [91] . on the other hand, biomarkers may allow for the diagnosis of inflammatory arthritis at early stages of the disease. the acr/eular 2010 criteria for the diagnosis of ra focus on the detection of rf and of antibodies against cyclic citrullinated proteins (anti-ccp), while early diagnosis may also include antibodies against carbamylated proteins (anti-carp), mutated citrullinated vimentin antibodies (anti-mcv), cartilage oligomeric matrix protein (anti-comp), serum calprotectin, and 14-3-3 eta protein. imaging methods, on the other hand, may provide a better understanding of the processes that occur in the joint(s) during inflammatory arthritis. x-rays, ultrasound (us), mri, and arthroscopy are imaging techniques that can be employed for inflammatory arthritis [92, 93] . x-rays reveal changes in the joints and damage to the bone seen in inflammatory arthritis. in order to see the condition of synovial tissue, ligaments, tendons, and joints, us is based on sound waves without radiation. mri photographs are even more accurate than x-rays, showing joint damage, including in the muscles and tendons [94] . arthroscopy consists of a thin tube (arthroscope) carrying a flashlight and a camera to peek through the joint. it is used to diagnose any joint debilitating and/or arthritic alterations, to classify bone disorders and tumors, and to evidence the severity of bone inflammation and pain [95, 96] . all methods have many drawbacks such as low precision, poor image resolution, and high cost, despite the extensive use of conventional methods in the diagnosis of arthritis. however, important progress in nanomedicine has been made recently and new platforms for high precision diagnosis of inflammatory arthritis can be introduced by nanotechnology [97, 98] . a selection of imaging methods contribute in the diagnosis and evaluation of inflammatory arthritis, although the proper assessment of arthritis, especially in the early stages of the disease, can be problematic [99] . various studies are therefore ongoing to improve the sensitivity and accuracy of imaging methods to facilitate early stage diagno-sis of inflammation arthritis [100] . in the biomedical area, the engineering of nanoscale materials is increasingly used to fulfill this goal [101] . in the following paragraphs, the use of spio, gold, polymeric nps, and multimodal nanomaterials (cerium and silica nps) will be discussed that could have promising biomedical applications in the imaging of inflammatory arthritis (figure 1 ). for both preclinical animal models and human clinical trials, spions have been used as contrast agents in a wide variety of diseases such as coronary artery disease, malformation, muscular dystrophy, cancer, inflammation, transplant failure, and arthritis [102] . spions are able to picture different types of infection at the cellular level like ra [103] . for example, chih-lung et al. [104] prepared targeted spions for in vivo mri of t cells in ra via attachment of a monoclonal anti-cd3 antibody to carboxylated-polyethylene glycol (peg)-spion (iopc-cd3). serial mri analysis showed a selective reduction of the s/n ratio of iopc-cd33-infused femoral growth plates in the collagen-induced arthritis (cia) model of ra in rats, in agreement with immunohistochemical findings showing that the aggregation of t cells and spions will be in the target area. gold nanostructures including injectable auranofin for ra have been used for medicinal purposes for decades [105] . gold nanostructures have been widely evaluated in a number of imaging techniques, including ct, mri, raman spectroscopy, fluorescence, and photoacoustic imaging owing to its unique light scattering abilities and configurable surface plasmon resonance [106] . the desirable imaging features and simplicity of gold nanomaterial preparation make it a good substrate for selective inflammation and arthritis imaging [107] . marc et al. [108] prepared enlarged gold nanorods with a sharp absorption at 1064 nm and modified them using infliximab and certolizumab antibodies to target tumor necrosis factor alpha (tnf-α). this nanoprobe was used to define inflammation through optoacoustic molecular imaging in arthritic mouse knees. using a fast-scanning optoacoustic imaging device based on a pulsed nd:yag laser and a single centered us transducer, they demonstrated a longitudinal improvement of optoacoustic signal magnitudes after injection of infliximab-but not certolizumab-modified and pegylated control particles on arthritic and healthy control mice. in light of their good light trapping nature, bioactivity, and configurable adsorption capacities, the use of conjugated polymer nanomaterials solved the limitations of traditional contrast agents, being superior imaging substances [109] . for example, a new near-infrared (nir)-ii conjugated polymer nanoprobe (pnp) has been synthesized by chen et al. [110] for use in a cia animal model. tocilizumab-loaded nanopolymers demonstrated highresolution images of swollen and cartilage tissue using nir-ii pmi of the ra joint tissue, indicating its monitoring ability for noninvasively tracking the development of ra disease. in related work, hieu et al. [111] employed a combination of nir imaging and 19f mri with labeled np injection in vitro and in cia mice. the nps were made from poly-(lacticco-glycolic acid) (plga)-peg-folate (folate-np) or from peg-block-plga, loaded with indocyanine green (icg) and perfluorooctyl bromide (pfob). in the inflamed synovial membrane/fluid and the pannus of inflammatory vascular tissue in ra-affected joints, an excess of macrophages was identified. at the early point of time (2 h), the existence of folate as a targeting ligand greatly enhanced the nir signal from inflamed tissue. in another study, shuyi et al. [112] designed a cartilage-targeted cationic nanoprobe to enhance photoacoustic imaging (pai) based on poly-l-lysine-melanin (pll-m)-nps to track the development of arthritis. the in vitro analysis displayed the capability of pll-m-nps to identify various high-sensitivity anionic glycosaminoglycan (gag) concentrations. responsive and consistent visualization of arthritis development was supported by the use of nanoprobe-enhanced pai to recognize changes in gag material. multimodal imaging modalities in which the strengths of more than one imaging technique are combined can also be used for nps. for example, as a multimodal imaging agent, silica-based nps are attractive because of their biocompatibility, photostability, multivalent binding capability, and biodistribution [113] . nanoceria, cerium oxide-based nps, are another form of biocompatible nps applied in arthritis multimodal imaging [114] . the prevalence of arthritis since the start of the post-industrial period has alarmingly risen [115] . historically, the traditional medical diagnosis of arthritis is focused on symptoms of pain and decreased function and computed tomography (joint disturbances) that frequently appear late in the course of the disease [116] . the analysis of biological parameters may be an enticing and realistic alternative. for example, rf and anti-ccp are used for inflammatory arthritis diagnosis based on eular 2010 guidelines [115] . the most promising alternative for the future diagnosis and treatment of inflammatory arthritis are actually nps [97] . nanomaterials such as quantum dots, carbon nanoallotropes, micelles, and liposomes will be discussed in following paragraphs ( figure 1 ). ra is characterized by the development of auto-antibodies, synovial inflammation, and degradation of bones and rf auto-antibodies are the most recognized biomarkers for arthritis as described above. to address this problem, veigas et al. [117] developed a cost-effective and simple approach to detect and quantitatively measure the rf marker. this colorimetric nanosensor was based on crosslinking of the au nanoprobe, resulting in extensive accumulation in the vicinity of the pentameric igm rf. nanoconjugate accumulation causes a change of color from red to purple that can be easily detected by the unaided eye. a limit of detection (lod) of 4.15 ua/ml igm rf was obtained by the nanoplatform. in biological applications, organic-inorganic hybrid nanomaterials have been investigated because of their structural and compositional variations in each section. due to its interparticle plasmonic pairing for photonics-based biosensor applications, a mixture of these hybrid nps with metallic nanomaterial would be desirable [118] . hwang et al. [118] prepared silver/gold (ag@au) core-shell nps)-poly(aniline) hybrid nanostructures (cbcphns) for the early diagnosis of ra. nanohybrids were used in surface-enhanced raman scattering (sers)-based multiplexed detection of auto-antibodies (rf igm and anti-ccp). the lod for rf igm and anti-ccp was 0.93 iu/ml and 0.68 iu/ml, respectively. in animal studies, the relationship between mycoplasma pneumonia (mp) and ra has been confirmed for decades, with an increased risk of ra in patients with mycoplasma pneumonia [119] . jia et al. [120] prepared raman-based immunoassay strip for the accurate detection of mp contagion in blood samples. in this immunoassay, au@ag nps were loaded with two layers of raman dye 5,5 -dithiobis-(2-nitrobenzoic acid) (dtnb) as sers tags. the lod was of 0.1 ng/ml for rf, i.e., 100-fold more accurate than the colorimetric assay. cardiovascular diseases (cvds) and ra are generally associated with human immunodeficiency virus (hiv) infection, a global public health issue [121] . islam et al. [122] designed a unique biosensor based on graphene-based field-effect transistors to detect hiv and its associated diseases (cvds and ra). in this study, amine-functionalized graphene (afg) was integrated with antibodies (anti-ccp for ra, anti-ctn1 for cvd, and anti-p24 for hiv) to sense several biomarkers. via carbodiimide activation, the antibodies were conjugated covalently to afg. the nanosensor was highly susceptible and displayed a linearity for the p24, ctn1, and ccp biomarkers. the lod was of 10 fg/ml for ccp and ctn1 and of 100 fg/ml for p24. ra is one of the most common autoimmune and inflammatory progressive disorders diagnosed with several principal symptoms such as synovial joint damage and cartilage and bone tissue dramatic malformation [123] . approximately 1.5 million people worldwide suffer from ra. the prevalence of this inflammatory disorder in women is almost three times higher than in men [124] . this inflammation could also develop in other tissues like the heart, lungs, kidneys, and pleura. although there are some methods for the treatment of ra, none are reliably effective [123] . there are presently several groups of antiarthritis therapeutic agents use for ra therapy, such as nsaids and disease-modifying antirheumatic drugs (dmards) which includes biologics like an anti-interleukin 6 (anti-il-6) receptor and anti-tnf-α antibodies [125] . although considered strong and effective drugs, their long-term consumption may cause serious side effects. one of the main problems associated with the use of these drugs is that they are widely distributed throughout the body, except at the site of disease and inflamed joints. for this reason, the treatment of chronic arthritis is often associated with the destruction to several important organs like the liver, kidneys, and lungs. therefore, it is necessary to effectively deliver such drugs to the inflamed ra sites in order to increase their efficacy in managing arthritis [125] . nanomaterials can provide adapted tools to address this issue. nanomaterials have been extensively employed to increase the bioavailability, bioactivity, pharmacokinetics, and pharmacodynamics of drugs against ra. it is therefore necessary to expand and investigate new and suitable therapeutic agents for the treatment of ra that exactly and correctly target abnormal joints without damaging other, healthy tissues. a variety of studies employed nps for the therapy of ra, including liposomes, polymeric nps, niosomes, metal nps, quantum dots, slns, and polymeric micelles. the findings of these investigations revealed the performance of these systems due to their specific physicochemical properties such as biocompatibility, lack of toxicity, ability to promote sustained drug release, and selective drug delivery to damaged and inflamed tissues in animal ra models [123, 126] . liposomes are considered successful nanovehicles formed by lipid bilayers surrounding an aqueous core for drug delivery strategy. they are well-known nanoscale systems with reduced toxicity and minor side effects for the administration of therapeutic agents. liposomes as drug delivery nanocarriers have numerous benefits including a low immunogenicity and high biocompatibility, the ability of loading and conjugating both hydrophilic and lipophilic agents, and suitable sizes with physico-chemical features that can improve the stability and prolong the biological half-life of drugs while potentially delivering medicines specifically to damaged and inflamed joints ( figure 2 ) [127] . although liposomes have certain advantages, there are two important challenges remain for a therapeutic application of conventional liposomes. first, the blood circulation time of liposomes is restricted as they can be rapidly removed by phagocytic cells in the liver. second, liposomes typically do not have long-term stability under physiological conditions. the presence of these challenges may lead to the release of encapsulated drugs at off-target sites. several factors may have effects on the instability of liposomes including the osmotic pressure, the hydrolysis of lipid, and a surfactant-induced decomposition. the polymerization process of lipids in bilayers has been evidenced as a powerful method to get the structural integrity of liposomes [5] . mtx is a drug commonly prescribed to treat ra. due to the intrinsic adverse effects of this drug, loading of mtx in liposomes may be a suitable delivery method to diminish its toxicity while retaining its properties and efficacy. the encapsulation efficiency (ee) of mtx (or of any other type of drug) in liposomes is regulated by the features of the liposomes, including their aqueous volume and membrane rigidity. encapsulation may also be affected by the hydrophilic/hydrophobic parts of a drug that may impact its capacity to interact with the liposome membrane bilayer. loading of mtx in liposomes occurs via a passive procedure controlled by the ability of the liposome to trap the aqueous phase containing the drug. this procedure results in lower ee as drug retention is restricted to the size of the aqueous part in the liposome and to the solubility of the drug. in a recent work, guimarães et al. [128] improved the loading of mtx in liposomes by reducing the initial formed solution (20% at a 1:1-v/v-organic:aqueous phase ratio) using a procedure based on ethanol injection, so that the aqueous volume outside the liposomes dissolved in ethanol was diminished, promoting the interaction between mtx and lipids to obtain a suitable size distribution and greater drug ee. they found a small size and suitable polydispersity index (pdi) with higher loaded mtx, the efficiency was more than 30% compared with conventional ethanol injection method. results obtained by nuclear magnetic resonance (nmr) revealed bilateral connections between the drug and the main phospholipid through hydrogen bonding, increasing ee. the authors were thus capable of developing a new pre-concentration ethanol injection technique to achieve higher mtx encapsulation in liposomes, an important advance for the therapy of ra. the presence of liposomes in the blood circulation for several hours is considered the main role in passive targeting, depending on several factors like hydrophobicity, surface charge, and particle size. the diameter of the liposomes is the basic factor that has an effect on blood circulation time and biological release after intravenous injection. moreover, the diameter of liposomes influences their permeation via gaps in leaky synovial vessels and retention in damaged joints. their targeting capability is basically ascribed to the leaky vasculature of the synovial fluid caused by proinflammatory cytokines such as il-1β, il-6, and tnf-α, resembling the boosted the effect of permeability and retention (epr) in tumors. the size of the endothelial gap between cells is a specified range due to the adsorption of plasma proteins and the phagocytosis by the reticuloendothelial system (res), but can differ between patients [127] . in recent study, ren et al. [129] investigated the mechanisms of passive targeting of liposomes and indicated the effect of their biophysical and biochemical features on their retention time in the blood stream. the authors prepared liposomes with various sizes and surface charges and also used different peg chain lengths (1, 2, and 5 kda) and concentrations (5%, 10%, and 20% w/w of total fat by lipid film dispersion and extrusion) and assessed their targeting ability using an nir fluorescence imaging arrangement. they next employed optimal liposome systems (charge, size, etc.) to deliver dex in cia rats. pharmacodynamics studies revealed that dex liposomes significantly increased the anti-arthritic effects of dex in this ra model in vivo. in ra, when the wall of blood vessels becomes inflamed, the vessels may become weakened and enhance in size, or they become leaky in the inflamed joints. in passive targeting, the secretion of nanosize drug delivery carriers via the leaky vasculature and following inflammatory cell-mediated sequestration (elvis) can lead to their accumulation, especially in sites of inflamed joints, and to an increased anti-inflammatory efficacy [127] . in another study, wang et al. [127] prepared polymerized stealth liposomes consisting of 1,2-bis (10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (dc 8,9 pc) and 1,2-distearoylsn-glycero-3-phosphoethanolamine peg (dspe-peg 2000) using the thin film hydration procedure. to increase the integrity of the liposomes and enhance their blood circulation time, the authors used dc 8, 9 pc molecules crosslinked in a bilayer of liposome by ultraviolet (uv) radiation and peg chains in order to make a stealth layer, respectively. the biocompatible liposomes were then administered to arthritic rats, with effective mobilization in the damaged joints. administration of dex via encapsulation in such polymerized stealth liposomes suppressed the concentration of proinflammatory cytokines such as tnf-α and il-1β in joint textures and reduced the swelling of inflamed and damaged joints, overall preventing further progression of ra. in addition, shen et al. [130] prepared new thermosensitive liposomes based on dipalmitoyl phosphatidylcholine (dppc), hydrogenated soyabean phosphatidylcholine (spc), and cholesterol to load the aquatic-soluble drug sinomenine hydrochloride (sin). the liposomal delivery systems with suitable particle size had great compatibility and storage stability, allowing to successfully prevent the release of sin in the blood circulation before reaching target sites in ra rats upon full release via microwave hyperthermia. the thermosensitive liposome delivery systems enhanced the concentration of the drug at the inflamed site of ra by improved controlled release and reduced ra signs without side effects, especially when combining the sin treatment with microwave hyperthermia as an optimized, combined therapy to possibly manage the clinical symptoms of ra. polymeric nps are being prepared from colloidal particles and the diameter ranges considered (1-1000 nm). in fact, polymeric nps have a great potential in the medical field due to their advantageous properties such as biodegradability, biocompatibility, great synthetic flexibility, ability to be precisely tailored, and appropriate mechanical properties [131] . to prevent the macrophage uptake, the surface of nps may be sheathed with stealth polymers like peg, and as the peg covering density and thickness enhance, the polymeric np circulation time increases in the blood. modification of nps via pegylation, a process of covalent conjugation that prevents removal from the reticuloendothelial system, or via conjugation with other small molecules (peptides, vitamins, and antibodies) can greatly prolong the circulation time of the systems in the blood and improve the efficacy of the anti-ra drug being delivered, such as nsaids, corticosteroids, dmards, small interfering rnas (sirnas), and therapeutic peptides [132] . synthetic cationic polymers such as polyethylenemine (pei), poly-l-lysine (pll), and dendrimers are usually utilized to deliver nucleic acids such as dna and interfering rnas (rnai) [133] . among them, pei is the most frequently employed because of numerous protonated amino functional groups, allowing for a higher cationic charge density at physiological ph that facilitates the attachment of nucleic acids via electrostatic adsorption [133] . espinosa-cano et al. [134] demonstrated the benefits of using polymeric nps conjugated with naproxen and dex to decrease inflammation and prevent il-12 expression in macrophages. note that il-12 and il-23 recently appeared as therapeutic targets in the therapy of long-lasting inflammatory disorders in which t cells are the primary dysfunctional immune cells, via either cox-dependent or cox-independent regulation mechanisms. the authors prepared anti-inflammatory polymeric nps by mixing dex and ketoprofen (ket) with suitable chemical and physical properties and that properly accumulated and delivered both drugs in damaged joints. as a consequence, these structures had significant anti-inflammatory effects by reducing the concentrations of joint nitric oxide (no) and the expression of m1 macrophage markers, while enhancing that of m2 macrophage markers, following rapid uptake by the macrophages. this may favor their retention at inflamed locations by the extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration effect (elvise). tofacitinib (tfc) is another candidate for ra therapy as a novel, oral non-traditional janus kinase (jak) inhibitor with similar efficacy and safety to that of other dmards [135] . however, its clinical application has been hindered thus far by its low plasma half-life. bashir et al. [136] designed innovative plga-based nps to promote the sustained release and target-specific delivery of tfc. plga is one of the most commonly employed biocompatible and biodegradable polymers that is applied in various drug delivery systems. hydrolization of plga produces two main monomers in water, e.g., poly-lactic acid (pla) and poly-glycolic acid (pga). the authors encapsulated tfc in plga nps by nanoprecipitation as a new nanodelivery structure to target the inflamed synovium and demonstrated that such tfc-plga-nps supported an adapted tfc pharmacokinetic profile [136] . a study by howard et al. [137] investigated the impact of an cs-sirna np so suppress inflammatory tnf-α expression in macrophages in cia mice. the histological examination of the joints indicated slight cartilage damage and inflammatory cell penetration in anti-tnf-α-treated mice, demonstrating the benefits of such a tnf-α knockdown approach via cs-sirna np on local and systemic inflammation (figure 3) . lee et al. [123] adopted a similar approach using nanostructure polymerized sirna (poly-sirna) targeting tnf-α with thiolated glycol cs (tgc) polymers to control the progression of ra in mice. tnf-α expression was downregulated in vitro via the application of these nps and at arthritic sites in ra mice joints. ha et al. [138] investigated the potential of photothermally organized drug delivery using multifunctional nps (mnps) with a nir irradiated location, with improved therapeutic efficacy for patients suffering from ra and decreased side effects. in this approach, the layer of au film was applied to mtx-encapsulated peg-plga nps, allowing for correct encapsulation of mtx in the mnps. the synergistic interactions of mtx-encapsulated mnps accompanied by nir rays were tested in fibroblast-like ra synoviocytes (flss) and cia mice [138] . the advantage of using nir resonance of the au shell is to accelerate the local release of mtx from the nps. nir images of mtx-loaded mnps showed suitable transfer of the mnps to the inflamed joints. furthermore, repeated administration of mnps encapsulating mtx at a 1/1400 solution had stronger anti-ra effects in cia mice than the mtx solution itself. further-more, combining mtx-encapsulated mnps with nir radiation was more efficient than chemotherapy alone. overall, mtx-loaded mnp therapy with nir irradiation represents a suitable treatment for ra based on a single, low dose of mtx [138] . lee et al. [123] adopted a similar approach using nanostructure polymerized sirna 423 (poly-sirna) targeting tnf-α with thiolated glycol cs (tgc) polymers to control the 424 progression of ra in mice. tnf-α expression was downregulated in vitro via the appli-425 cation of these nps and at arthritic sites in ra mice joints. ha et al. [138] investigated the 426 potential of photothermally organized drug delivery using multifunctional nps (mnps) 427 with a nir irradiated location, with improved therapeutic efficacy for patients suffering 428 from ra and decreased side effects. in this approach, the layer of au film was applied to 429 mtx-encapsulated peg-plga nps, allowing for correct encapsulation of mtx in the 430 mnps. the synergistic interactions of mtx-encapsulated mnps accompanied by nir 431 rays were tested in fibroblast-like ra synoviocytes (flss) and cia mice [138] . the ad-432 vantage of using nir resonance of the au shell is to accelerate the local release of mtx 433 from the nps. nir images of mtx-loaded mnps showed suitable transfer of the mnps 434 to the inflamed joints. furthermore, repeated administration of mnps encapsulating mtx 435 at a 1/1400 solution had stronger anti-ra effects in cia mice than the mtx solution itself. 436 furthermore, combining mtx-encapsulated mnps with nir radiation was more efficient 437 than chemotherapy alone. overall, mtx-loaded mnp therapy with nir irradiation rep-438 resents a suitable treatment for ra based on a single, low dose of mtx [138] . 439 niosomes are vesicles with lamellar morphology that are microscopic in size and are 441 mostly composed of nonionic surfactants and cholesterol (figure 4 ). they display signifi-442 cant advantages as drug delivery systems due to their ability to support a controlled re-443 lease of drugs at a targeted site, to their nonimmunogenic properties and biocompatibility, 444 and to their delayed clearance from the environment [139] . 445 figure 3 . cs-sirna np delivery in macrophages to suppress tnf-α expression. macrophage elimination by cs-sirna nps was established to inhibit a local production of il-1β, il-6, tnf-α, and matrix metalloproteinases (mmps) and thus reduce the pathogenesis of inflammatory arthritis. niosomes are vesicles with lamellar morphology that are microscopic in size and are mostly composed of nonionic surfactants and cholesterol (figure 4) . they display significant advantages as drug delivery systems due to their ability to support a controlled release of drugs at a targeted site, to their nonimmunogenic properties and biocompatibility, and to their delayed clearance from the environment [139] . niosomes can be prepared when nonionic surfactant vesicles self-associate together. 448 several factors affecting the formation of niosomes include the type of non-ionic surfac-449 tant used, the hydration temperature, and the preparation method [140] . primary surfac-450 tant vesicles are generally made of ionic surfactants. work showed that the highest tox-451 icity is associated with cationic surfactants followed by anionic surfactants, with nonionic 452 surfactants displaying the lowest toxicity. for these reasons, niosomes made from 453 nonionic surfactants are ideal choices for transdermal delivery. hydrophilic-lipophilic 454 balance (hlb) values play a significant role in the preparation of niosomes and a range of 455 hlb 4-8 is usually used. common surfactants used to make niosomes include span, brij, 456 and tween [141, 142] . 457 in recent study, rajaram et al. [143] designed a promising system to deliver piroxi-458 cam (pc) encapsulated in a vesicular carrier of nonionic surfactant as transdermal patches, 459 a suitable drug delivery system to increase the solubility of drugs with poor solubility and 460 to increase the retention time of the drug at the site of absorption. this approach may be 461 niosomes can be prepared when nonionic surfactant vesicles self-associate together. several factors affecting the formation of niosomes include the type of non-ionic surfactant used, the hydration temperature, and the preparation method [140] . primary surfactant vesicles are generally made of ionic surfactants. work showed that the highest toxicity is associated with cationic surfactants followed by anionic surfactants, with nonionic surfactants displaying the lowest toxicity. for these reasons, niosomes made from nonionic surfactants are ideal choices for transdermal delivery. hydrophilic-lipophilic balance (hlb) values play a significant role in the preparation of niosomes and a range of hlb 4-8 is usually used. common surfactants used to make niosomes include span, brij, and tween [141, 142] . in recent study, rajaram et al. [143] designed a promising system to deliver piroxicam (pc) encapsulated in a vesicular carrier of nonionic surfactant as transdermal patches, a suitable drug delivery system to increase the solubility of drugs with poor solubility and to increase the retention time of the drug at the site of absorption. this approach may be a useful structure for treatment of ra. another report from paradkar et al. [124] showed the possibility to prepare a niosomal thiocolchicoside topical gel using the thin film hydration procedure, consisting of a molar ratio of span 60:cholesterol. this niosomal gel increased the topical retention time and controlled the pain caused by ra and its side effects at reduced dosing frequency. mujib et al. [144] designed a study to treat ra with the aim of increasing the duration of drug action at lesser side effects. topical gel formulations containing ibuprofen loaded in niosomes were prepared by changing the ratios of various nonionic surfactants (span20, span60, span80, and cholesterol) by the thin film hydration and ether injection methods. the results indicated that the niosomal nanocarrier of ibuprofen gel containing carbopol acted as the basis of an appropriate topical drug delivery system for lagging the duration of the drug. the unique biological, physical, mechanical, chemical, and thermal features of metal nps made them increasingly used systems in the medical and pharmaceutical fields [76] . metal nps are very small mineral particles with at least one dimension in a size range below 100 nm [145] . silver nps (ag nps) have been reported for their anti-inflammatory activities by limiting the production of proinflammatory cytokines such as tnf-α and il-6 and have been beneficial in ra patients [146] . ag nps can also reduce the amount of vascular endothelial growth factor (vegf), a factor produced by epithelial cells that increases antigen sensitivity, exhibits a major function in physiological abnormalities, causes plasma proteins to leak into the extracellular space, leads to a thickening of the airway wall, and increases the t helper type-2 (th2) cell-mediated inflammation (il-9, il-4, il-5, and il-13) [147] . ag nps inhibit the src kinase pathway and block y419 phosphorylation in a dose-dependent manner, reducing the vascular endothelial permeability induced by vegf and il-1β. ag nps also can block vegf and il-1β-induced solute flux and decrease vegf-induced cell production [147] . ag nps can also decrease the expression of the hypoxia-induced factor 1 alpha (hif-1α) that regulates the expression of proinflammatory genes and kill bacteria [147] . finally, ag nps can restrict the secretion of proinflammatory mediators such as tnf-α, il-12, and cox-2 at higher concentrations [25] . m1 macrophages play key roles during the pathogenesis of ra. infiltration of these inflammatory cells leads to the secretion of different inflammatory cytokines. to improve synovial inflammation, m1 macrophages may progress to an m2 anti-inflammatory macrophage phenotype [148] . yang et al. [148] used folic acid modified with silver nanoparticles (fa-agnps) which could be effectively transported into m1 macrophages to stimulate m1 macrophages reduction and then m2 macrophages polarization in order to successful ra therapy. in this model, m1 macrophages were targeted through the folate receptor overexpressed at their cell surface. after entering the cell, these nps dissolve in response to intracellular glutathione and release silver ions. in fact, this process is a key factor in starting a series of anti-inflammatory actions like the removal of reactive oxygen species (ros) to accelerate the polarization of m2 macrophages. these nps accumulate passively in inflamed joints, having strong anti-inflammatory activities and therapeutic effects in mice model of ra with high safety. an et al. [149] prepared au nps from aqueous leaf extracts of m. alliacea with a facecentered cubic form as reported by x-ray diffraction (xrd) and a diameter of 20-30 nm as noted by transmission electron microscopy (tem). the effects of gold were determined using tail-flip examination, a sensitive drug model that operates in the central nervous system (cns). a maximum heat threshold of almost 3.5 times that of the treated vehicle was observed 2 h after treatment. such au nps were capable of reversing complete freund's adjuvant (cfa)-induced thermal hyperalgesia that is detected in physiological conditions. in another study, investigators showed that the reaction between synoviocytes and macrophages was effectively involved in causing inflammatory responses in the synovium. the results indicated that triamcinolone-gold nps (triam-au nps) increased the anti-inflammatory reactions of flss and macrophages through the repolarization of macrophages from an m1 to an m2 phenotype and reduced proinflammatory responses. triam alone reduced the proinflammatory reactions of flss and macrophages. different experiments in various test conditions in vitro/ex vivo (human samples) and in vivo (mice) revealed that triam-au nps effectively repolarized macrophage activity in damaged synovium and improved the appearance of cartilage tissue while triam alone did not prompt fls anti-inflammatory actions nor macrophage repolarization [150] . some researchers assessed the intra-articular adsorption rate of super-paramagnetic iron oxide nanoparticles (spions) covered with poly-vinyl-alcohol (pva-spions) by the synovial membrane in an animal model in vivo. the nps remained in the synovium for almost a week, demonstrating that such systems may provide an effective platform for intra-articular drug delivery especially for the treatment of acute or chronic joint diseases [123] . spions are appropriate nanocarriers with an extensive ability for drug delivery and can be conjugated with several therapeutic agents for controlled release to a target site by an external magnetic field. carneiro et al. [151] prepared a system of magnetic targeting with using gold-covered superparamagnetic iron oxide nanoparticles (au-spions) to target the joints of cia rats. such a system improved ra signs without undesirable side (toxic) effects. similar to this approach, a study published in 2019 showed that magnetic targeting of au-spions remained longer in joints than colloidal au-nps and had greater antioxidant effects than mtx. in this method, au-spions were intra-peritoneally administered in collagen-induced arthritis (cia) rats three times daily. after that, a neodymium magnet was coupled to the right ankle joint for 1 h targeting. the results showed that the au-spions significantly reduced tissue edema, leukocyte infiltration, and tnf-α and il-1β expression in the synovial fluid of cia [39] . in another study, mtx and spions were co-encapsulated in to the slns in order to be utilized as therapeutics and imaging factors, respectively. transmission electron microscopy (tem) images showed that the spions were loaded in to the sln matrix, and mtx encapsulation efficiency amounts were more than 98%. in vitro investigations demonstrated that the formulations exhibited minor toxicity at concentrations lower than 500 µg/ml, by using thp-1 cells. moreover, these slns have been functionalized with anti-cd64 which is a kind of antibody that specially attached to a surface of cell receptor over expressing in ra infected macrophages. the results indicated that the np structures offered suitable and promising outcomes for future theranostics of ra. moreover, the proposed nanoformulations are very hopeful for the future of ra therapy and diagnostic, because of a nontoxic and targeted delivery of drugs and simultaneously permitting in vivo imaging [152] . quantum dots (qds) are inorganic nanomaterials or semiconductor nanocrystals with a size of 1-10 nm, consisting of a central semiconductor core with a shell consisting of inorganic salts (cds, zns) [62] . qds have been employed in several nanotechnological procedures including for drug delivery, bioimaging of abnormal cells, and ra diagnosis and treatment. kalangi et al. [153] tested mercaptopropionic acid (mpa) nanoconjugates surrounded with cd-te qds and celecoxib (a strong cox-2 inhibitor) for bioimaging in carrageenan-induced mice with paw inflammation. the authors demonstrated the ability of qd-celecoxib conjugates as substitutes of radioisotopes to reveal the localization of the drug in sites of inflammation in this animal model and may be of further help to evidence off-targets. slns are novel and interesting pharmaceutical drug delivery platforms with a diameter of 120-200 nm that are extensively used for managed drug delivery, combining the advantages of polymeric nps and oil-in-water emulsions [146] . among various drugs, anti-inflammatory cur has been reported for its benefits in a wide diversity of inflammatory diseases, but its use may be limited by its weak absorption, fast metabolism, and rapid systemic elimination. to overcome such issues, arora et al. [154] evaluated the potential of a cur-sln system to manage joint hyperalgesia and stiffness with enhanced leukocyte count, tnf-α, crp, and oxidative stress in cfa-induced inflammation in rats relative to free cur treatment. they observed a significant, dose-dependent reduction of ra signs in the animals upon cur-sln administration compared with free cur application via decreased oxido-inflammatory and immunomodulatory signals. in another study, bhalekar et al. [155] encapsulated piperine (pi) in slns (pi-slns) using the melt emulsification technique to treat ra. pi is an alkaloid substance originates from the fruits and also roots of piper nigrum types of piperacea group. the authors reported an average particle size of 128.80 nm with a zeta potential of −23.34 mv and an encapsulation efficacy of 78.71%. the prepared sln was administered to cfa-induced inflammatory mice orally and topically. in the study, the franz diffusion cell indicates that pi from the sln gel formulation assembles in the skin. the outcomes of the pharmacodynamic investigation show that both topical and oral pi have a suitable response compared to for example oral chloroquine suspension. alarifi et al. [156] designed formulations of ciprofloxacin (cip) into slns as a means to improve its biopharmaceutical and biochemical properties and its anti-inflammatory properties. the results of the evaluation showed that encapsulation of cip in slns increased its antimicrobial and anti-inflammatory activities in vitro which could significantly improve a treatment by reducing dose-dependent side effects and increasing the range of protective activities. biocompatible and biodegradable polymers are widely used in pharmaceutical processes as useful components for pharmaceutical formulations and also utilized as nanocarriers in drug delivery systems [157] . currently, micelles are based on the use of amphiphilic block copolymers in aqueous solution. they can be employed as nanocarriers for less soluble drugs that may be covalently attached to polymer chains or non-covalently placed in micelles [157] . generally, they are suitable nanovehicles to improve several biochemical and biopharmaceutical properties such as solubility, bioavailability, and targeting of these hydrophobic drugs. they are adapted for use in combination with other therapeutics agents for ra remedy. twenty years ago, an outstanding novel micelle-based system has been developed for the treatment of ra [158] . glucocorticoids (glcs) are among the most critical agents used in ra, but they display serious side effects when applied at high doses. in this regard, wang et al. [159] applied glcs in a rat model of arthritis using a micellar nanosystem, allowing for a targeted, low-dose drug delivery in the damaged joints capable of safely ameliorating their therapeutic efficacy. micelles loaded with dex self-assembled from the amphipathic pegblock-poly(caprolactone) (peg-pcl) polymer through film dispersion were intravenously injected in adjuvant-induced arthritis rats using only a dose of 0.8 mg/kg. the micelles remained in the circulation for a significant period of time and then accumulated in the inflamed joints. micelle-delivered dex decreased joint pain and swelling as well as proinflammatory cytokine expression in both the joint tissues and the blood. peg-pcl micelles led to not only reduced adverse effects on body weight, but also decreased the lymphocyte counts and the blood glucose levels. these findings show that loading dex in peg-pcl micelles may allow for a suitable and effective low-dose gc therapy targeting inflammatory diseases. abdollahi et al. [160] prepared novel dextran stearate polymeric micelles by the dialysis method as a means to transport indomethacin, a nsaid that can successfully decrease the ra's pain and inflammation, in order to decrease its otherwise serious side effects. such micelles delivered a more impact and useful drug that reduced the inflammation in a rat model of arthritis compared with the treatment with indomethacin alone. ra is an inflammatory and chronic disorder related to abnormal function of immune system. in ra, the immune system mainly attacks the joints, causing their inflammation like in the knee, wrist, and hands. this tissue damage can cause long-term pain and improper deformation of the body. in several patients, ra can damage a wide part of our body, including the skin, eyes, hearts, lungs, and blood vessels. the primary goals of treating ra are to control the inflammation and pain and to slow or stop the progression of the disease. early treatment approaches include mtx and sulphasalazine which suppress the immune system. although these drugs are appropriate, suppressing the immune system increases the risk of infection. it is also critical to keep in mind the potential side effects of the drugs, like nausea, abdominal pain, and severe damage to the liver and the lungs. as the effects of these drugs usually last 6-12 weeks, rheumatologists may also administrate them with nsaids to treat pain and inflammation. despite these issues, dmards are still used as first-line therapies. in this article, our goal was to describe several types of nanomaterials for applications in the treatment of ra. the results of this review show that all nanocarriers have unique properties to encapsulate anti-ra drugs and reduce drug side effects. the evaluations also showed that these systems can increase the bioavailability of hydrophobic drugs and enhance their effectiveness for ra. briefly, the present review discusses several useful and successful nanocarriers in order to exactly delivery several therapeutic agents to target sites in ra therapy. table 1 shows various nanostructures as a novel platform in improving function of anti-ra drugs. table 1 . various nanostructures as a novel platform in improving function of anti-ra drugs. liposomes mtx, dex, sin improved controlled release, reduced ra signs, decreased side effects [128] polymeric nps ket, dex, tfc, mtx decreased inflammation [134] niosomes pc, thiocolchicoside, ibuprofen increased drug retention [143] silver nps fa-agnps enhanced anti-inflammatory activities [148] gold nps triam enhanced anti-inflammatory activities [150] iron nps mtx suppression of joint edema and inflammation [152] quantum dots celecoxib localized activity in sites of inflammation [153] solid lipid nps cur, pi, cip enhanced anti-inflammatory activities [154] polymeric micelles dex, indomethacin decreased side effects [159] abbreviations: nps, nanoparticles; mtx, methotrexate; dex, dexamethasone; sin, sinomenine hydrochloride; ket, ketoprofen; tfc, tofacitinib; pc, piroxicam; fa-agnps, folic acid modified with silver nanoparticles; triam, triamcinolone; cur, curcumin; pi, piperine; cip, ciprofloxacin; ra, rheumatoid arthritis; cox-2, cyclooxygenase 2. the expanded use of nanomaterials in a wide variety of biomedical applications, on the other hand, also raises questions about their toxicity. the morphological and physicochemical properties of nanomaterials play an important role in determining their toxicity in various body organs such as the liver, kidney, skin, brain, heart, etc. while nanomaterials have significant toxicological effects in various experimental models, their toxicity can be minimized or eliminated by engineering their surface with various types of natural or synthetic polymers and other compounds. while some work explored the toxicity of nanomaterials for biomedical application, there is thus far no in-depth information available on these aspects. the authors declare no conflict of interest. immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial cluster analysis of autoimmune rheumatic diseases based on autoantibodies. new insights for polyautoimmunity rheumatoid arthritis (ra) and cardiovascular disease macrophage immunomodulation in chronic osteolytic diseases-the case of periodontitis emergent high fatality lung disease in systemic juvenile arthritis bacterial amyloids: the link between bacterial infections and autoimmunity mechanisms underlying the anti-inflammatory and immunosuppressive activity of ruxolitinib γδ t cells in homeostasis and host defence of epithelial barrier tissues low-dose methotrexate for the prevention of atherosclerotic events tendon and ligament mechanical loading in the pathogenesis of inflammatory arthritis international genetics of ankylosing spondylitis consortium. epistatic interactions between killer immunoglobulin-like receptors and human leukocyte antigen ligands are associated with ankylosing spondylitis metagenomic profiling of the pro-inflammatory gut microbiota in ankylosing spondylitis global epidemiology of gout: prevalence, incidence, treatment patterns and risk factors protective immunity and new vaccines for lyme disease the importance of ultrasound in identifying and differentiating patients with early inflammatory arthritis: a narrative review nk cell-derived gm-csf potentiates inflammatory arthritis and is negatively regulated by cis preclinical rheumatoid arthritis and rheumatoid arthritis prevention comparative analysis of the influence of selected physical factors on the level of pain in the course of temporomandibular joint disorders exploration of nanoethosomal transgel of naproxen sodium for the treatment of arthritis impact of cumulative inflammation, cardiac risk factors, and medication exposure on coronary atherosclerosis progression in rheumatoid arthritis analgesics, non-steroidal anti-inflammatory drugs (nsaids), muscle relaxants, and antigout medications effects of ibuprofen intake in muscle damage, body temperature and muscle power in paralympic powerlifting athletes new materials for hip and knee joint replacement: what's hip and what's in kneed? one year in review 2020: fibromyalgia treatment of immune checkpoint inhibitor-induced inflammatory arthritis inflammatory arthritis due to immune checkpoint inhibitors: challenges in diagnosis and treatment delivering the power of nanomedicine to patients today biodegradable nanopolymers in cardiac tissue engineering: from concept towards nanomedicine surface-engineered cancer nanomedicine: rational design and recent progress recent advances of two-dimensional materials in smart drug delivery nano-systems mesoporous silica nanoparticles for drug delivery eco-friendly and sustainable synthesis of biocompatible nanomaterials for diagnostic imaging: current challenges and future perspectives biodegradable nanoparticles decorated with different carbohydrates for efficient macrophage-targeted gene therapy nanomedicine therapies modulating macrophage dysfunction: a potential strategy to attenuate cytokine storms in severe infections a new method for accurate in vivo mapping of human brain connections using microstructural and anatomical information nanoparticle-sirna: a potential strategy for rheumatoid arthritis therapy? cationic block copolymer nanoparticles with tunable dna affinity for treating rheumatoid arthritis metabolic functions of gut microbes associate with efficacy of tumor necrosis factor antagonists in patients with inflammatory bowel diseases magnetic nanoparticles: a new diagnostic and treatment platform for rheumatoid arthritis nanomaterials in biomedical diagnosis challenges in the development of nanoparticlebased imaging agents: characterization and biology radical dendrimers based on biocompatible oligoethylene glycol dendrimers as contrast agents for mri a negative association between urinary iodine concentration and the prevalence of hyperuricemia and gout: a cross-sectional and population-based study in mainland china evaluation of carum-loaded niosomes on breast cancer cells: physicochemical properties, in vitro cytotoxicity, flow cytometric, dna fragmentation and cell migration assay a new formulation of hydrophobin-coated niosome as a drug carrier to cancer cells lawsone-loaded niosome and its antitumor activity in mcf-7 breast cancer cell line: a nano-herbal treatment for cancer in silico and in vitro study of magnetic niosomes for gene delivery: the effect of ergosterol and cholesterol nanotreatment and nanodiagnosis of prostate cancer: recent updates comprehensive evaluation of gene expression in negative and positive trigger-based targeting niosomes in hek-293 cell line nanomaterials for the treatment and diagnosis of alzheimer's disease: an overview stimuli-responsive polymeric nanocarriers for drug delivery, imaging, and theragnosis magnetic delivery of antitumor carboplatin by using pegylated-niosomes fabrication of a new superparamagnetic metal-organic framework with core-shell nanocomposite structures: characterization, biocompatibility, and drug release study in vitro cytotoxicity assay of d-limonene niosomes: an efficient nano-carrier for enhancing solubility of plant-extracted agents diosgeninloaded niosome as an effective phytochemical nanocarrier: physicochemical characterization, loading efficiency, and cytotoxicity assay the synthesis of methotrexate-loaded f127 microemulsions and their in vivo toxicity in a rat model effect of tocopherol on the properties of pluronic f127 microemulsions: physico-chemical characterization and in vivo toxicity a combined theoretical and experimental study to improve the thermal stability of recombinant d-lactate dehydrogenase immobilized on a novel superparamagnetic fe 3 o 4 nps@ metal-organic framework synthesis and characterization of highly efficacious fe-doped ceria nanoparticles for cytotoxic and antifungal activity synthesis, characterization, and intraperitoneal biochemical studies of zinc oxide nanoparticles in rattus norvegicus the synthesis and characterization of a magnetite nanoparticle with potent antibacterial activity and low mammalian toxicity revisiting the cytotoxicity of quantum dots: an in-depth overview green synthesis and characterization of zinc oxide nanoparticles with antibacterial and antifungal activity behavioral effects of zinc oxide nanoparticles on the brain of rats copolymer/graphene oxide nanocomposites as potential anticancer agents newly crocin-coated magnetite nanoparticles induce apoptosis and decrease vegf expression in breast carcinoma cells cancer theranostic applications of mxene nanomaterials: recent updates on facing the sars-cov-2 (covid-19) with combination of nanomaterials and medicine: possible strategies and first challenges atorvastatin-loaded sba-16 nanostructures: synthesis, physical characterization, and biochemical alterations in hyperlipidemic rats gum-based cerium oxide nanoparticles for antimicrobial assay −x) bafe 12 o 19 /xcofe 2 o 4 hard/soft magnetic nanocomposites: synthesis, physical characterization, and antibacterial activities study deferasirox-loaded pluronic nanomicelles: synthesis, characterization, in vitro and in vivo studies anticancer effect of green tea extract (gte)-loaded ph-responsive niosome coated with peg against different cell lines photo-and magnetothermally responsive nanomaterials for therapy, controlled drug delivery and imaging applications scrutinizing the therapeutic and diagnostic potential of nanotechnology in thyroid cancer: edifying drug targeting by nano-oncotherapeutics nanomaterials for parkinson disease: recent progress nanotechnology in ovarian cancer: diagnosis and treatment progress in natural polymer engineered biomaterials for transdermal drug delivery systems nanomaterials for diagnosis and treatment of brain cancer: recent updates advances of non-ionic surfactant vesicles (niosomes) and their application in drug delivery progress in the application of nanoparticles and graphene as drug carriers and on the diagnosis of brain infections how to face skin cancer with nanomaterials: a review the role of structural genes in the pathogenesis of osteoarthritic disorders osteoarthritis and rheumatoid arthritis pannus have similar qualitative metabolic characteristics and pro-inflammatory cytokine response inflammatory arthritis and sicca syndrome induced by nivolumab and ipilimumab antinuclear antibody testing-misunderstood or misbegotten? extractable synovial fluid in inflammatory and non-inflammatory arthritis of the knee erythrocyte sedimentation rate and c-reactive protein measurements and their relevance in clinical medicine comparative analysis of blood parameters of the erythrocyte lineage between patients with chronic periodontitis and healthy patients: results obtained from a meta-analysis anti-citrullinated protein antibodies and high levels of rheumatoid factor are associated with systemic bone loss in patients with early untreated rheumatoid arthritis correlation of serum uric acid with disease activity and c-reactive protein in patients suffering from rheumatoid arthritis diagnosis and management of rheumatoid arthritis; what is the current role of established and new imaging techniques in clinical practice? imaging techniques: options for the diagnosis and monitoring of treatment of enthesitis in psoriatic arthritis clinical applications of advanced magnetic resonance imaging techniques for arthritis evaluation arthroscopic management of septic arthritis of the native shoulder: a systematic review high-resolution mri (3t-mri) in diagnosis of wrist pain: is diagnostic arthroscopy still necessary? nanotechnology application in drug delivery to osteoarthritis (oa), rheumatoid arthritis (ra), and osteoporosis (osp) are nanotechnological approaches the future of treating inflammatory diseases? nanomedicine photoacoustic tomography for human musculoskeletal imaging and inflammatory arthritis detection pet/mri in infectious and inflammatory diseases: will it be a useful improvement nanotechnology in medical imaging: probe design and applications mr imaging of murine arthritis using ultrasmall superparamagnetic iron oxide particles detection of synovial macrophages in an experimental rabbit model of antigen-induced arthritis: ultrasmall superparamagnetic iron oxide-enhanced mr imaging targeted superparamagnetic iron oxide nanoparticles for in vivo magnetic resonance imaging of t-cells in rheumatoid arthritis new oral gold compound for treatment of rheumatoid arthritis bimetallic core-shell nanostars with tunable surface plasmon resonance for surface-enhanced raman scattering label-free ecl immunosensor for the early diagnosis of rheumatoid arthritis based on asymmetric heterogeneous polyaniline-gold nanomaterial antitumor necrosis factor-α antibody-coupled gold nanorods as nanoprobes for molecular optoacoustic imaging in arthritis conjugated polymer nano-systems for hyperthermia, imaging and drug delivery tocilizumab-conjugated polymer nanoparticles for nir-ii photoacoustic-imaging-guided therapy of rheumatoid arthritis imaging rheumatoid arthritis in mice using combined near infrared and 19 f magnetic resonance modalities tracking osteoarthritis progress through cationic nanoprobe-enhanced photoacoustic imaging of cartilage hybrid silica nanoparticles for multimodal imaging pharmacological potential of cerium oxide nanoparticles biomarkers in rheumatoid arthritis, what is new? can synovial pathobiology integrate with current clinical and imaging prediction models to achieve personalized health care in rheumatoid arthritis? front antibody modified gold nanoparticles for fast colorimetric screening of rheumatoid arthritis compartmentalized bimetal cluster-poly (aniline) hybrid nanostructures for multiplexed detection of autoantibodies in early diagnosis of rheumatoid arthritis increased risk of rheumatoid arthritis among patients with mycoplasma pneumonia: a nationwide population-based cohort study in taiwan dual dye-loaded au@ ag coupled to a lateral flow immunoassay for the accurate and sensitive detection of mycoplasma pneumoniae infection rheumatoid arthritis in patients with hiv: management challenges. open access rheumatol a smart nanosensor for the detection of human immunodeficiency virus and associated cardiovascular and arthritis diseases using functionalized graphene-based transistors engineering nanoparticles for targeting rheumatoid arthritis: past, present, and future trends thiocolchicoside niosomal gel formulation for the pain management of rheumatoid arthritis through topical drug delivery peptide-targeted liposomal delivery of dexamethasone for arthritis therapy rheumatoid arthritis and the challenge of using nanoparticles for its treatment improving the anti-inflammatory efficacy of dexamethasone in the treatment of rheumatoid arthritis with polymerized stealth liposomes as a delivery vehicle increased encapsulation efficiency of methotrexate in liposomes for rheumatoid arthritis therapy role of liposome size, surface charge, and pegylation on rheumatoid arthritis targeting therapy sinomenine hydrochloride loaded thermosensitive liposomes combined with microwave hyperthermia for the treatment of rheumatoid arthritis stimuli-responsive polymeric nanomaterials for rheumatoid arthritis therapy nanomedicine for the treatment of rheumatoid arthritis nanomedicines for the delivery of glucocorticoids and nucleic acids as potential alternatives in the treatment of rheumatoid arthritis anti-inflammatory polymeric nanoparticles based on ketoprofen and dexamethasone the first janus kinase (jak) inhibitor for the treatment of rheumatoid arthritis fabrication, characterization and in vitro release kinetics of tofacitinib-encapsulated polymeric nanoparticles: a promising implication in the treatment of rheumatoid arthritis chitosan/sirna nanoparticle-mediated tnf-α knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model formulation design, statistical optimization, and in vitro evaluation of a naringenin nanoemulsion to enhance apoptotic activity in a549 lung cancer cells development and characterization of niosomal gel system using lallementia royaleana benth. mucilage for the treatment of rheumatoid arthritis niosomes: a review on niosomal research in the last decade niosomal nanocarriers for enhanced skin delivery of quercetin with functions of anti-tyrosinase and antioxidant niosomes as nanoparticular drug carriers: fundamentals and recent applications fabrication of non-ionic surfactant vesicular gel for effective treatment of rheumatoid arthritis formulation of ibuprofen loaded niosomal gel by different techniques for treating rheumatoid arthritis impact of response surface methodology-optimized synthesis parameters on in vitro anti-inflammatory activity of iron nanoparticles synthesized using ocimum tenuiflorum linn novel nano therapeutic materials for the effective treatment of rheumatoid arthritis-recent insights anti-inflammatory mechanism of various metal and metal oxide nanoparticles synthesized using plant extracts: a review targeted silver nanoparticles for rheumatoid arthritis therapy via macrophage apoptosis and re-polarization development of biofabricated gold nanoparticles for the treatment of alleviated arthritis pain triamcinolone-gold nanoparticles repolarize synoviocytes and macrophages in an inflamed synovium gold-coated superparamagnetic iron oxide nanoparticles attenuate collagen-induced arthritis after magnetic targeting solid lipid nanoparticles: a potential multifunctional approach towards rheumatoid arthritis theranostics synthesis, characterization, and biodistribution of quantum dot-celecoxib conjugate in mouse paw edema model curcumin loaded solid lipid nanoparticles ameliorate adjuvant-induced arthritis in rats formulation of piperine solid lipid nanoparticles (sln) for treatment of rheumatoid arthritis enhancement of ciprofloxacin activity by incorporating it in solid lipid nanoparticles polymeric micelles for the delivery of poorly soluble drugs: from nanoformulation to clinical approval polymeric micelles for the treatment of rheumatoid arthritis targeted delivery of low-dose dexamethasone using pcl-peg micelles for effective treatment of rheumatoid arthritis indomethacin loaded dextran stearate polymeric micelles improve adjuvant-induced arthritis in rats: design and in vivo evaluation key: cord-0019815-7wb3h1iu authors: ishii, nobuyasu; shimizu, toshiki; ishiura, yoshihisa; amuro, hideki; nishizawa, tohru; tamaki, takeshi; nomura, shosaku title: a single-center retrospective observational study evaluating the favorable predictive factors for the disease control time of treatment with tocilizumab in patients of rheumatoid arthritis date: 2021-08-05 journal: j inflamm res doi: 10.2147/jir.s323577 sha: 81836e247b6f2f46c3cf4382ab1d760d09759fd4 doc_id: 19815 cord_uid: 7wb3h1iu background: tocilizumab (tcz) is humanized monoclonal antibody against the interleukin-6 (il-6) and receptor that has prominent efficacy for the treatment of rheumatoid arthritis (ra). we conducted a retrospective observational study to determine how long tcz controls ra. patients and methods: we retrospectively reviewed the medical records of ra patients treated with tcz. the aim of this study was to evaluate the contribution of clinical parameters to disease control time (dct) in ra patients. results: overall, 144 patients were enrolled in the study. the median age of patients was 66 years (range: 34–85 years). in univariate analysis, dct was significantly increased in patients who had never received previous biologic disease-modifying anti-rheumatic drugs treatment (p = 0.0064). we also analyzed the contribution of the base line value of c-reactive protein (crp) to dct. we divided the patients with ra into two groups according to a cutoff value of 1.000 mg/dl. the median control times were 77.5 months (95% confidence interval [ci]: 44.8–not reached to median) and 34.5 months (95% ci: 17.0–79.3) for patients with high and low crp value, respectively. in univariate analysis, dct was significantly increased in patients with a high crp value (p = 0.0283). multivariate analysis clearly revealed that a high baseline crp value was an independent favorable predictive factor for longer dct (hazard ratio, 0.608, 95% ci: 0.378–0.981, p = 0.0416). conclusion: these data clearly demonstrate that the baseline value of crp was closely associated with long time dct in patients of ra treated with tcz. rheumatoid arthritis (ra) is a systemic inflammatory disease characterized by the destruction of the structural architecture of joints. 1 the pathogenic process of ra is modulated by a series of proinflammatory cytokines including interleukin-1 (il-1), interleukin-6 (il-6) and tumor necrosis factor alpha (tnf-α). recently, these cytokines have become accepted targets of biologic disease-modifying antirheumatic drugs (bdmards). of note, il-6 has a crucial role in the pathogenic process of ra, and inhibiting its signal transduction cascade is a promising strategy for the treatment for ra. tocilizumab (tcz), a humanized monoclonal antibody against the human il-6 receptor, and strongly blocks il-6 signal transduction by inhibiting the binding il-6 to its receptor. 2 numerous clinical reports have demonstrated a significant clinical efficacy of tcz in the treatment of ra. [3] [4] [5] [6] however, precise favorable predictive factors for the treatment of ra with tcz have not been identified. in terms of disease control time (dct) other than disease remission rate, no obvious predictive factors have been reported. therefore, we conducted a retrospective observational study to determine how long tcz controls ra using a statistical technique with a time-to-event analysis. in this study, we revealed that a high value of baseline c-reactive protein (crp) was an independent favorable predictive factor for the longtime disease control of ra treated with tcz. the medical records of all patients with ra who received treatment with tcz from april 2008 to august 2018 at kansai medical university medical center (moriguchi-city, japan) were retrospectively reviewed. the clinical diagnosis of ra was made according to the 2010 american college of rheumatology (acr)/european league against rheumatism (eular) criteria or the 1987 acr criteria. 7,8 data on sex, age and other covariates in the patients of ra, observed before the administration of tcz were obtained retrospectively from the patients' medical records. informed consent was obtained in the form of opt-out on the website (http://www.kmu.ac. jp/takii/hospital/scq14r0000000yu1.html). those who rejected consent were excluded from the study. this retrospective study was performed in accordance with the declaration of helsinki and was approved by the institutional ethics review board for clinical research of kansai medical university medical center (institutional id: 2019156, umin-ctr: umin000040027). complete blood cell count (cbc) and various platelet volume indices were measured using ethylenediaminetetraacetic acid (edta)-treated blood. an automated blood cell counter was used for these analyses (sysmex xe-2100, kobe, japan). the crp concentration was measured using an automatic analyzer (beckman coulter au5400, miami, fl, usa). we created a new outcome measure for this study. the dct was defined as the time from the date patients were initially administrated tcz to the time the treating physician decided to discontinue the tcz treatment due to an inadequate efficacy. the event was classified into three categories: (1) tcz treatment was discontinued due to inadequate efficacy; (2) death of a patient from any reason; (3) tcz treatment was discontinued due to an unacceptable adverse effect. censoring was classified into the four categories: (1) tcz treatment was continued at the time of analysis; (2) tcz treatment was discontinued at the time of analysis due to disease remission; (3) the last observation of patients lost to follow up; (4) patient refusal to continue tcz treatment without obvious disease progression. statistically significant differences between the groups were compared using the chi-square, student's t or mann-whitney u-test. receiver operating characteristics (roc) curve analysis was used to estimate an optimal cutoff value for the various hematological factors. univariate and multivariate analyses of dct were performed using the kaplan-meier product-limit method with the log rank test and the cox proportional hazards model, respectively. the 95% confidence interval (ci) for the disease control rate was calculated using greenwood's method. to calculate the 95% ci of the median control time (mct), the brookmeyer and crowley method was used. all statistical analyses were conducted using the jmp (version 9.0.2) software program for windows (sas institute inc, cary, nc, usa). all statistical tests were two-sided, and p < 0.05 was considered to be statistically significant. overall, 144 patients with ra who were treated with tcz were enrolled in this study. the characteristics of these 144 patients are summarized in table 1 . all the patients were asian (japanese, korean, or chinese), their median age was 66 years (range: 34-85 years), and they included 114 women and 30 men. their median duration of disease of ra was hundred patients had concomitantly received glucocorticoid, whereas remaining 44 patients had not. ninety-five patients had received methotrexate (mtx) in addition to tcz, whereas the remaining 49 patients had received tcz treatment alone. a history of previous bdmards treatment was reported in 84 patients, whereas the remaining 60 patients had been never treated with bdmards. the details of the previous bdmards treatment included 28 regimens of infliximab, 25 regimens of adalimumab, 12 regimens of golimumab, 7 regimens of certolizumab pegol, 27 regimens of etanercept, 14 regimens of abatacept and 1 regimen of tofacitinib. median value of base line crp was 1.329 mg/dl (iqr: 0.3513-3.2138). eighty-four patients had base line crp value higher than or equal to 1.000 mg/dl, whereas the base line crp value of the remaining 60 patients were lower than 1.000 mg/dl. we conducted a series of survival analyses on december 31, 2020. at that time, 34 patients had discontinued tcz treatment due to insufficient efficacy, 1 patient discontinued tcz treatment due to disease remission, 4 patients had died from any cause, 32 patients discontinued tcz treatment due to an unacceptable adverse event, 4 patients refused to continue tcz treatment from any cause, 27 patients were lost to follow-up, and the remaining 42 patients continued tcz treatment with adequate disease control. consequently, the censoring rate was 51.4%. in the 34 patients with insufficient efficacy, 9 patients were primary failure (≤6 months) and 25 patients were secondary failure (≥6 months). the causes of death of four patients were two acute myocardial infarctions, one infection of clostridium difficile, and one primary lung cancer. the kaplan-meier curve for dct is shown in figure 1a . the mct was 59.4 months (95% ci: 36.4-79.3). the 1-, 2-, and 5-year disease control rates were 79.3% (95% ci: 72.5-86.0), 67.8% (95% ci: 59.9-75.7), and 49.9% (95% ci: 40.6-59.1), respectively. in univariate analysis, dct was significantly increased in patients who had never received previous bdmards treatment (p = 0.0064) (table 2, figure 1b ). however, age (p = 0.1017), sex (p = 0.7950), disease duration (p = 0.2225), concomitant use of glucocorticoid (p = 0.5865) and concomitant use of mtx (p = 0.3983) were not statistically significant (table 2) . we also analyzed the contribution of the base line value of crp to dct. because a previous report showed that a cut off value of crp predicted disease remission, we divided the patients with ra into two groups according to a cutoff value of 1.000 mg/dl for crp. 9 the mct was 77.5 months (95% ci: 44.8-nr) and 34.5 months (95% ci: 17.0-79.3) for patients with high and low crp value, respectively (table 2, figure 1c ). in univariate analysis, the dct was significantly increased in the patients with a high crp value (p = 0.0283). next, we compared the various characteristics between the two groups: ra patients with a high and those low crp values. the characteristics of the two groups are summarized in table 1 . there were no significant differences in age, sex, bmi, disease duration, concomitant use of glucocorticoid and concomitant use of mtx between the two groups. although a history of bdmards treatment was a significant predictive factor for a shorter dct, there were no significant differences between the two groups ( table 1) . we also analyzed various hematological indices in two groups ( table 3 ). the erythrocyte sedimentation rate (esr), white blood cell (wbc) count, and platelet count (pc) were significantly increased in ra patients with a high baseline crp value compared with those with a low baseline crp value (p < 0.0001, p = 0.0011 and p < 0.0001 respectively). in contrast, hematocrit (ht), hemoglobin (hb), red blood cell distribution width (rdw), mean platelet volume (mpv), platelet distribution width (pdw) and platelet-large cell ratio (p-lcr) were significantly decreased in ra patients with a high baseline crp value compared with those with a low baseline crp value (p = 0.0139, p = 0.0217, p = 0.0353, p = 0.0009, p = 0.0017 and p = 0.0010 respectively). all other all variates were not significantly different between the two groups. these covariates that correlated with baseline crp value were analyzed using logistic regression analysis followed by roc curve analysis, and the optimal cut off values were determined. using these cut off value, we conducted a series of univariate analysis for dct (table 4 ). in table 4 , figure 1d ). we also conducted a multivariate analysis for dct (table 5 ). multivariate analysis clearly revealed that a high baseline crp value was an independent favorable predictive factor for longer dct (hazard ratio [hr], 0.608, 95% ci: 0.378-0.981, p = 0.0416). in addition, no history of bdmards was also an independent favorable predictive factor for longer disease control (hr, 0.470, 95% ci: 0.275-0.780, p = 0.0031). in contrast, were not significant factors. these results clearly showed that concomitant use of glucocorticoid or mtx in addition to tcz treatment had no additional benefit for disease control of ra compared with tcz monotherapy. crp was firstly reported by tillett and francis at 1930. 10 it was induced in vivo in response to a streptococcal capsular polysaccharide. 11 most crp is produced by hepatocytes, and it forms a pentamer in circulating blood. currently, it is widely accepted as an important acutephase protein and is used as a universal surrogate marker of inflammatory condition in various clinical situations. castel et al clearly showed that il-6 was a major regulator of crp synthesis in adult human hepatocytes. 12 thus, crp is thought to be a candidate predictive factor for the clinical efficacy of tcz treatment, because the target of tcz is the il-6 receptor. pers et al reported that the baseline crp value ≥1.0 mg/dl was a favorable predictive factor for better disease remission in patients with ra treated with tcz. 9 on the basis of their results, we assessed the contribution of baseline value of crp in patients with ra treated with tcz. we clearly showed that a baseline crp value ≥1.0 mg/dl was an independent favorable predictive factor for longtime disease control. these findings strongly suggested that tcz treatment is suitable for patients in whom inflammatory condition have accelerated. unfortunately, we could not obtain adequate data to evaluate the disease remission (for example, disease activity score-28 for rheumatoid arthritis with esr (das28-esr) or clinical disease activity index (cdai) because of the retrospective nature of this study. 13 therefore, we could not assess whether the predictive factors for dct and for the disease remission rate were identical. therefore, a prospective study is warranted. previous experimental data clearly showed that at least distinct two mechanisms are involved in the pathogenic process of ra. il-6 blockade did not affect the pathogenesis of the spontaneous arthritis in tnf-α transgenic mice although it completely inhibited the pathogenic process of collagen-induced arthritis in dba/1j mice. 14 moreover, fujimoto et al showed that anti-mouse il-6 receptor monoclonal antibody inhibited th17 mobilization and pathogenic process of collagen-induced arthritis in dba/ 1j mice. 15 these findings suggested that patients suitable for tcz or tnf-α inhibitors might be quite different. in the past decade, the time-to-event analysis technique has been applied in the clinical research field of ra. numerical studies employed "drug retention" for time-toevent analysis. [16] [17] [18] however, there are some issues regarding the definition of "event" and "censored for drug retention". regarding the definition of drug retention, treatment discontinuation due to disease remission was also treated as an event. therefore, time-to-event analysis has become complicated and unintuitive, and additional subset analyses related to the reason for treatment discontinuation must be performed. therefore, we defined new outcome measures, dct and disease control rate, in this study. using our new outcome measures, discontinuation due to disease remission was excluded from events; therefore, a simple time-to-event analysis was possible. we also assessed the correlation between various hematological parameters and baseline crp value. however, only two factors, hb and p-lcr, were significant variates in the log rank test for dct. previous reports showed that hb and p-lcr were closely correlated with the serum concentration of il-6. 19, 20 however, il-6 induces the production of crp. therefore, we concluded that hb and p-lcr were confounding factors for baseline crp and they were excluded from our multivariate analysis for dct. nevertheless, they are ubiquitous blood biomarkers that predict the longtime disease control of tcz treatment because hb and p-lcr data are obtained from a common complete blood count test. we also revealed that no treatment history with bdmards was an independent favorable predictive factor for longtime disease control for tcz treatment. however, all patients included in this study had no history of treatment with anti-il-6 receptor antibody. there is an enigma that the previous history of treatment failure with bdmards other than anti-il-6 receptor antibody contributed to the clinical efficacy of anti-il-6 receptor antibody treatment whereas all patients had never received anti-il-6 receptor antibody. one possible explanation to this issue is that previous failure of any bdmards treatment might be a selection bias in favor of refractory ra. 21 the patients of refractory ra demonstrated an insufficiency to multiple bdmards targeting various cytokines. in such a situation, a selection of patients who had no treatment history with bdmards could be a "cherry picking", resulting in reduce the likelihood of refractory ra and leading to better efficacy of tcz treatment. despite the retrospective nature and small size of the present study, we revealed that a high baseline crp value might indicate a dominant inflammatory pathogenic process of ra patients for whom tcz treatment might achieve longer disease control. further investigation should lead to a better understanding of therapeutic selection of appropriate patients in the treatment of tcz. c-reactive protein and implications in rheumatoid arthritis and associated comorbidities anti-interleukin 6 receptor antibody treatment in rheumatic disease il-6 receptor inhibition with tocilizumab improves treatment outcomes in patients with rheumatoid arthritis refractory to anti-tumour necrosis factor biologicals: results from a 24-week multicentre randomised placebo-controlled trial interleukin-6 receptor inhibition with tocilizumab and attainment of disease remission in rheumatoid arthritis: the role of acute-phase reactants a randomised, double-blind, parallel-group study of the safety and efficacy of subcutaneous tocilizumab versus intravenous tocilizumab in combination with traditional disease-modifying antirheumatic drugs in patients with moderate to severe rheumatoid arthritis (summacta study) treatment of rheumatoid arthritis with anti-tumor necrosis factor or tocilizumab therapy as first biologic agent in a global comparative observational study the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis acr/eular 2010 rheumatoid arthritis classification criteria predictors of response and remission in a large cohort of rheumatoid arthritis patients treated with tocilizumab in clinical practice serological reactions in pneumonia with a non-protein somatic fraction of pneumococcus du clos tw. c-reactive protein is protective against streptococcus pneumoniae infection in mice interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes disease activity early in the course of treatment predicts response to therapy after one year in rheumatoid arthritis patients interleukin 6 is required for the development of collagen-induced arthritis interleukin-6 blockade suppresses autoimmune arthritis in mice by the inhibition of inflammatory th17 responses drug tolerability and reasons for discontinuation of seven biologics in 4466 treatment courses of rheumatoid arthritis-the answer cohort study differential long-term retention of biological disease-modifying antirheumatic drugs in patients with rheumatoid arthritis by age group from the first registry favorable retention rates and safety of conventional anti-rheumatic drugs in older patients with rheumatoid arthritis anemia in early rheumatoid arthritis is associated with interleukin 6-mediated bone marrow suppression, but has no effect on disease course or mortality correlation between cytokines and coagulation-related parameters in patients with coronavirus disease 2019 admitted to icu defining refractory rheumatoid arthritis we thank j. ludovic croxford, phd, from edanz group for editing a draft of this manuscript. the authors have declared no conflicts of interest. the journal of inflammation research is an international, peerreviewed open-access journal that welcomes laboratory and clinical findings on the molecular basis, cell biology and pharmacology of inflammation including original research, reviews, symposium reports, hypothesis formation and commentaries on: acute/chronic inflammation; mediators of inflammation; cellular processes; molecular mechanisms; pharmacology and novel anti-inflammatory drugs; clinical conditions involving inflammation. the manuscript management system is completely online and includes a very quick and fair peerreview system. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-0005807-bi1tqa7e authors: saklatvala, j. title: tumour necrosis factor α stimulates resorption and inhibits synthesis of proteoglycan in cartilage date: 1986 journal: nature doi: 10.1038/322547a0 sha: 95803b520dd8a2f7d963e89828f3fb56160736a5 doc_id: 5807 cord_uid: bi1tqa7e during inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). of these substances, which include the interleukins, interferons and tumour necrosis factors (tnfs), interleukin-1 (il-1) has been considered potentially a most important inflammatory mediator because of its wide range of effects (reviewed in refs 1, 2). in vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes(3) and stimulates resorption of cartilage(4) and bone(5,6). cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and il-1 is the only cytokine hitherto known to promote it. tnfs are characterized by their effects on tumours and cytotoxicity to transformed cells(7–9), but share some actions with il-1.1 report here that recombinant human tnfα stimulates resorption and inhibits synthesis of proteoglycan in explants of cartilage. its action is similar to and additive with il-1, and it is a second macrophage-derived cytokine whose production in rheumatoid arthritis, or inflammation generally, could contribute to tissue destruction. during inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). of these substances, which include the interleukins, interferons and tumour necrosis factors (tnfs), interleukin-l (il-l) has been considered potentially a most important inflammatory mediator because of its wide range of effects (reviewed in refs i, 2). in vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes 3 and stimulates resorption of cartilage 4 and bone m • cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and il-l is the only cytokine hitherto known to promote it. tnfs are characterized by their effects on tumours and cytotoxicity to transformed cells 7 -9 , but share some actions with il-1. i report here that recombinant human tnf a stimulates resorption and inbibits synthesis of proteoglycan in explants of cartilage. its action is similar to and additive with il-l, and it is a second macropbage-derived cytokine wbose production ' in rheumatoid artbritis, or inflammation generally, could contribute to tissue destruction. two human tnfs have been isolated: tnfa (refs 8, 9) is a product of activated mononuclear phagocytes, tnff3 (ref. 7) of activated lymphocytes. the proteins show about 50% homology in nucleotide sequence and compete for a common class of receptors on the cervical carcinoma line me-180 (ref. 10) . tnfa is probably identical with cachectin ll , a factor that suppresses production of lipoprotein lipase in cultured adipocytes, and may playa part in the development of cachexia during infection l2 . il-i shows some biological similarity to tnf: it is cytotoxic to certain transfonned cells l3 and it suppresses production oflipoprotein lipase in adipocytes l4 . furthermore, cachectin has recently been shown to stimulate production of prostaglandins and latent collagenase by human synovial and dennal fibroblasts in a manner similar to il-lls. in view of these findings, i have investigated the effect of tnfa on both the resorption and synthesis of proteoglycan by explants of cartilage, chondroitin-sulphate-rich proteoglycan is an essential component of the matrix of cartilage since it enables the tissue to resist compression during load-bearing. loss of proteoglycan, such as occurs in rheumatoid arthritis, osteoarthritis and other joint diseases, results in severe impairment of the function of cartilage. il-l is the only purified cytokine known to cause cartilage to degrade its proteoglycan 4 ,16, and to inhibit resynthesis 17 • figure la shows the amount of proteoglycan (measured as percentage of total chondroitin sulphate) released from porcine articular cartilage during 6 days of culture in the presence of human recombinant tnfa or pure porcine il-l. the tnfa caused up to 75% of the proteoglycan to be released, although it was less potent than the il-l, which was significantly active at a 20-fold lower dose (0.5 pm). figure 1 b shows a similar experiment carried out on cartilage from bovine nasal septum which was cultured for a shorter period (48 h): again, the il-l was more potent. the time dependence of the release of proteoglycan from bovine cartilage caused by sub-maximal concentrations of the two agents revealed that their effects were additive. figure ie shows that 50 pm il-l or 290 pm tnfa caused a similar rate of release, and that this was approximately doubled when the agents were combined. maximal stimulation of cartilage by il-i caused more rapid release of proteoglycan than did tnfa (fig.ld) : results for two concentrations of each cytokine demonstrate that responses were maximal. supramaximal doses of the two agents in combination caused a rate of release that was considerably faster than that due to tnfa alone, but was not significantly greater than that seen with ll-1 alone. the failure of tnfa to augment the maximal response to il-i may be because the limit of the chondrocytes' ability to degrade their matrix in vitro was being approached. the enzymatic mechanism by which the proteoglycan is degraded in cartilage is not understood. normally, cartilage proteoglycans aggregate in a specific manner with hyaluronic acid, and it is thought that the large size of these aggregates causes them to be trapped in the matrix. cartilage stimulated by il-i releases fragments of proteoglycan which, as judged by gel filtration, are smaller than nonnal proteoglycan monomers and are unable to aggregate with hyaluronic acid ls . there is no evidence of degradation of their chondroitin sulphate chains. these changes suggest that degradation is by limited proteolysis of the protein core. the fragments of proteoglycan that were released by cartilage stimulated with tnf behaved similarly on gel filtration to those generated by stimulation with il-i (fig. 2) . the bulk of the fragments generated by stimulation with either agent emerged from a sepharose 2b column at a region between the elution positions of intact proteoglycan and proteoglycan digested with papain (which consists largely of single-chain chondroitin sulphate peptides). addition of hyaluronic acid to the prot eo glycan fragments before chromatography caused little or no fonnation of aggregates. this suggested that the hyaluronate binding region was blocked or had been lost. when the proteolgycan fragments were chromatographed under dissociative conditions (4 m guanidine-hcl in the chromatographic buffer) the position of the main peak was unchanged. these experiments showed that chondrocytes activated by tnf or il-i caused a similar limited proteolysis of the proteoglycans. in order to study the effect of tn fa on the synthesis of proteoglycan, cartilage was stimulated for 48 h, and 3is04 was added to the culture medium for the last 6 h. in this procedure the isotope becomes incorporated into newly synthesized sulphated glycosaminoglycan (mainly chondroitin sulphate). at the end of the experiment the medium and · cartilage were digested with papain, and glycosaminoglycan was precipitated from the digests with cetylpyridinium chloride. the amount of radioactivity present in the precipitates was a measure of chondroitin sulphate (and, by inference, proteoglycan) synthesis. in experiments made with porcine articular (fig.3a) or bovine nasal septal (fig.3b) cartilages, tnfa caused a marked suphours for 30 min). each was then transferred to a well of a 96-well multititre plate and incubated under the same conditions in 0.2 ml of culture medium, either with no addition, or with human tnfa or porcine il-l of p15. the medium was changed at 3 days and the culture was terminated after 6 days. human tnfa was a recombinant protein expressed in escherichia coli and purified as described previously7.22. porcine il-l was a natural leukocyte protein purified to homogeneity as described elsewhere!6. the pi 5 form rather than the pi 8 form was used for these experiments. after culture the medium and cartilage were separated. the cartilage was digested completely with papain (see legend to methods. a, pieces of pig arti~ular cartilage were dissected, precultured and then stimulated either with tnfa or porcine il-i for 48 h exactly as described for fig. i , except that the culture medium (dmem) contained 1 % normal bovine serum (heat inactivated) during the stimulation period. for the last 6 h the medium was replaced with s04-free culture medium (still containing tnfa or il-i) to which was added 2.5 !lci ml-i of 35s04 (25-40 ci mg-i ; amersham). after culture, the cartilage pieces were separated from the medium, briefly blotted to remove excess medium, and then weighted. each piece was digested at 65°c for 2 h in 0.2 ml of 0.05 m sodium phosphate buffer ph 6.5 containing 1 mm edta, 2 mm n-acetylcysteine, 28!lg ml-i papain (sigma type iii). samples of medium (0.2 ml) were also digested with 0.1 ml of the papain solution under the same conditions. chondroitin sulphate (0.1 ml of 2 mg ml-i ) was added to all the samples followed by 0.1 ml of cetylpyridinium chloride (10% w/v). samples were centrifuged, the precipitates were washed twice in 3% cetylpyridinium chloride, then dissolved in 0.5 ml of formic acid and added to 5 ml of a scintillation mixture (pico-fluor-30, packard) and counted in a liquid scintillation counter. the radioactivity of the digests of tissue and medium were added together and the results expressed as d.p.m. per mg wet weight of cartilage. b, as for a except that disks of bovine nasal septal cartilage were used (see fig. i ), and the culture medium contained 5% normal bovine serum throughout the experiment. pression of proteoglycan production as judged the incorporation of 35s04 into glycosaminoglycan, but was less potent than il-l. the porcine il-1 inhibited incorporation in porcine articular cartilage in the range 0.1-10 pm; tn fa was 20-fold less active. a similar differential was observed on the bovine cartilage. the lower potency of the human tnfa compared with porcine il-1 may be due to species differences. taken together, the experiments demonstrate that tnfa has a similar action to il-1 on chondrocytes. it causes them to degrade proteoglycan by limited proteolysis and inhibits their synthesis of new proteoglycan. exposure of cartilage to either of these leukocyte products during inflammation could lead to loss of proteoglycan and impairment of function; furthermore, their effects may be additive. pigs l6 , like humans l9 , have two different il-1 proteins: for these experiments the pi 5 il-1 was used rather than the pi 8 form. the il-1s are equipotent on cartilage l6 and compete for the same receptors on porcine synovial fibroblasts: tnfa at 400 times excess over il-l) did not compete for these receptors (t. a. bird and j. s., in preparation). the augmentation of the effect of maximal doses of tnfa by il-1 reported here is consistent with there being different receptors on chondrocytes for the two cytokines. since they are apparently not homologous 8 ,16, il-1 and tnfa would be expected to combine with different receptors. these considerations suggest that chondrocytes (and probably other connective tissue cells) could have two distinct types of receptor (one for il-1 and one for tnf) whose interaction with ligand promotes resorption of matrix polymers while inhibiting their synthesis. such a possibility could have important implications for the pharmacological control of inflammatory tissue destruction. following submission of this manuscript, bertolini et al. 20 have reported that human tnfs, like il-1, stimulated ca2+ release from fetal rat bones. i thank dr m. palladino (genentech) for supplying tnf, dr c. a. dinarello (tufts university) for suggesting the experiments, dr j. tyler (strangeways laboratory) for doing the gel chromatography, mrs v. curry and mrs j. mason for technical help, and the arthritis and rheumatism council for financial support. biochemistry and biology of corona viruses key: cord-0016741-ehvc43de authors: deng, zhengyu; liu, shiyong title: inflammation-responsive delivery systems for the treatment of chronic inflammatory diseases date: 2021-04-15 journal: drug deliv transl res doi: 10.1007/s13346-021-00977-8 sha: 5136829bcfc760ecb8fc2ddc9b0e138230cc3ae6 doc_id: 16741 cord_uid: ehvc43de inflammation is the biological response of immune system to protect living organisms from injurious factors. however, excessive and uncontrolled inflammation is implicated in a variety of devastating chronic diseases including atherosclerosis, inflammatory bowel disease (ibd), and rheumatoid arthritis (ra). improved understanding of inflammatory response has unveiled a rich assortment of anti-inflammatory therapeutics for the treatment and management of relevant chronic diseases. notwithstanding these successes, clinical outcomes are variable among patients and serious adverse effects are often observed. moreover, there exist some limitations for clinical anti-inflammatory therapeutics such as aqueous insolubility, low bioavailability, off-target effects, and poor accessibility to subcellular compartments. to address these challenges, the rational design of inflammation-specific drug delivery systems (ddss) holds significant promise. moreover, as compared to normal tissues, inflamed tissue-associated pathological milieu (e.g., oxidative stress, acidic ph, and overexpressed enzymes) provides vital biochemical stimuli for triggered delivery of anti-inflammatory agents in a spatiotemporally controlled manner. in this review, we summarize recent advances in the development of anti-inflammatory ddss with built-in pathological inflammation-specific responsiveness for the treatment of chronic inflammatory diseases. [image: see text] inflammation is a dynamic immune process mounted by the innate and adaptive immune system to protect living organisms from injurious factors, including viruses (e.g., sars-cov-2), bacteria, fungi, or dead cells [1] [2] [3] . in its controlled form (i.e., acute inflammation), it serves to eliminate pathogens, cellular debris, and inflammatory mediators from organisms, and stimulate tissue repair, resulting in resolution of inflammation and restoration of tissue homeostasis [1] . however, when allowed to proceed unchecked, inflammation may lead to autoimmune or autoinflammatory disorders, such as atherosclerosis, type 2 diabetes, inflammatory bowel disease (ibd), cystic fibrosis (cf), rheumatoid arthritis (ra), alzheimer's disease (ad), and so forth [1] [2] [3] . in such chronic inflammatory diseases, the exact identification of the inflammatory stimulus is often unrevealed and, if revealed, is demanding to eradicate [1] [2] [3] . therefore, it is of great significance to suppress excessive inflammation and enhance resolution via therapeutically targeting chronic inflammatory responses [4] . increasing understanding of inflammatory response has unveiled a rich assortment of anti-inflammatory agents, spanning from aspirin, nonsteroidal anti-inflammatory drugs (nsaids), glucocorticoids, anti-cytokine biologicals to therapeutic nucleic acids for the treatment and management of chronic diseases [5] . notwithstanding these successes, clinical outcomes are variable especially when inflammatory diseases have been established and serious adverse effects are common as a result of compromised host defense. for instance, the risks of additional infections increase for those patients taking antibodies against tumor necrosis factor-α (tnf-α) as anti-inflammatory therapy [1] . moreover, there exist some limitations such as aqueous insolubility (e.g., nsaids and glucocorticoids), low bioavailability, off-target effects, and poor accessibility to subcellular compartments [6] [7] [8] . to resolve these issues of anti-inflammatory agents, the rational development of inflammation-specific and inflammation-responsive drug delivery systems (ddss) has been proposed [6] [7] [8] . clinical inflammation is well known and recognized by classical signs of heat, redness, swelling, pain, and loss of function, and these "macroscopic" features are governed by complex immune-regulatory pathways [7] . for example, the recognition of danger signals by host cells triggers the release of proinflammatory cytokines and chemokines, resulting in increased vascular permeability and angiogenesis (heat, redness, and swelling) [7] , which is reminiscent of well-known enhanced permeability and retention (epr) effect observed in solid tumors [9, 10] . in fact, extravasation through leaky vasculature and ensuing inflammatory cell-mediated sequestration (elvis) effect [7, 11, 12] for macromolecules or nano-objects in inflamed tissues was firstly corroborated in patients with ra [13] . nevertheless, there is overwhelming evidence that numerous cell surface receptors, such as cell adhesion molecules (cams), integrins, folate receptor-β (fr-β), and mannose receptor, are upregulated within inflamed areas upon inflammatory stimulation [7] . taken together, these ubiquitous hallmarks render it possible the design of inflamed tissue-selective antiinflammatory therapy by leveraging passive and/or active targeting approaches [7, 8] . on the other hand, as compared to healthy tissues, pathologically inflamed tissue-associated biochemical signals comprise high oxidative stress (e.g., reactive oxygen species, ros, and reactive nitrogen species, rns), acidic ph (~ 6.4), and overexpressed enzymes (e.g., cyclooxygenases (coxs) and matrix metalloproteinase (mmps)), enabling the development of inflammationtriggered drug delivery platforms [14, 15] . so far, quite a few reviews have been presented to summarize inflammation-specific nanotherapeutics, bioinspired nanocarriers, and oxidation-responsive nanomaterials for the treatment of a variety of chronic inflammatory diseases [16] [17] [18] [19] [20] [21] [22] [23] . however, systematic overviews of antiinflammatory ddss with built-in responsiveness to pathological inflammation-related biochemical signals remain limited [14, 15, 24] . in this contribution, we summarize recent progress on inflammation-responsive ddss as promising anti-inflammatory therapy for chronic inflammatory diseases. the main body of this review is organized according to the responsive properties of systems to specific types of inflammation-associated stimuli, comprising of oxidationresponsive, ph-responsive, enzyme-responsive, and multistimuli-responsive systems (scheme 1). besides, several cytoplasmic reductive milieu-responsive anti-inflammatory ddss are also covered (scheme 1). it is worth mentioning that we provide a summary of the most relevant and pioneering reports by highlighting selected and representative examples throughout the main text. finally, a brief conclusion section is presented to outline the future challenges in this promising field. oxidation-responsive and oxidative stress-eliminating systems as mentioned above, oxidative stress, as a result of the imbalance between oxidants (e.g., ros and rns) and antioxidants (e.g., glutathione (gsh) and vitamin e), is involved in both acute and chronic inflammatory responses [21, 25, 26] . specifically, highly reactive ros including superoxide anion (o 2 •ˉ) , hydrogen peroxide (h 2 o 2 ), hydroxy radical (ho • ), and hypochlorous acid (hocl), are closely connected to rns, such as nitric oxide (no) and peroxynitrite (onooˉ) [21, 26] . concerning higher equilibrium levels of ros and rns, a growing body of literature has been reported to sense, leverage, and modulate oxidative stress for the imaging and treatment of chronic inflammatory diseases [15, 23, 25, 27] . in this part, we will present an overview of oxidation-responsive and oxidative stress-eliminating systems according to the types of reactive moieties. boronic ester motif is well known to be degraded by oxidation with elevated levels of ros or rns at inflammation and tumor sites, and this triggering chemistry has been extensively exploited for the development of oxidation-sensitive nanocarriers for biomedical applications [28] [29] [30] [31] [32] . in this respect, zhang and coworkers systematically explored the therapeutic potentials of nanoplatforms consisting of phenylboronic ester-conjugated β-cyclodextrin (β-cd) for the management of chronic inflammatory diseases [33] [34] [35] [36] [37] [38] . for instance, oxidation-responsive nanoparticles encapsulating antioxidant tempol (a typical free radical scavenger) were fabricated using phenylboronic ester-modified β-cd, lecithin, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-poly(ethylene glycol) (dspe-peg; number-average molecular weight (mw) of peg segment is 2000 da) through nanoprecipitation method, collectively serving as superoxide dismutase (sod)/catalase (cat) mimics [34, 39] . accordingly, hydrophobic and h 2 o 2 -reactive β-cd derivatives within drug-loaded nanovehicles could transform into water-soluble products upon contact with oxidative environment, leading to consumption of ros (e.g., h 2 o 2 ), dissociation of nanovehicles, and release of tem-pol. significantly, it was revealed that such sod/cat mimetic nanosystem was efficiently accumulated in the inflamed colon in vivo with reduced nonselective distribution in healthy organs after oral administration [40] . in three mice colitis models, these oxidative stress-eliminating nanomedicine notably mitigated manifestations and suppressed expressions of proinflammatory mediators, with superior therapeutic efficacy over free tempol and poly(lactide-coglycolide) (plga)-based nonresponsive nanosystem. in their follow-up work, tempol and phenylboronic ester were covalently attached onto β-cd, followed by a coassembly process in combination with lecithin and dspe-peg, affording broad-spectrum ros-scavenging nanoparticles ( fig. 1) [37, 38] . by eliminating multiple ros, including h 2 o 2 , o 2 •ˉ, ho • , and hocl (fig. 1b) , these combinatorial nanoparticles could effectively attenuate oxidative stress-induced inflammation and cell apoptosis as well as inhibit oxidized low-density lipoprotein (oxldl)-induced foam cell formation, which was closely associated with the pathogenesis of atherosclerosis. it was demonstrated that such anti-inflammatory nanoparticles accumulated in atherosclerotic lesions in apolipoprotein e-deficient (apoe − / − ) mice through the elvis effect and significantly inhibited the development of atherosclerosis following intravenous administration [37] . in another separate study, ros-responsive and hydrophobic phenylboronic ester-conjugated α-cyclodextrin (α-cd) derivatives were synthesized and then used to physically encapsulate anti-rheumatic drug dexamethasone (dex), in combination with folic acid-modified dspe-peg (dspe-peg-fa) to fabricate fa-decorated nanoparticles for the treatment of ra [41] . preliminary in vivo evaluations suggested that such targeted nanoparticles were effectively delivered to and became accumulated at inflamed joints in collagen-induced arthritis (cia) mice, enabling the alleviation of joint swelling and cartilage destruction without increased side effects. apart from cd derivative-based oxidation-sensitive nanosystems, phenylboronic ester-tethered macromolecules have also been developed for the diagnosis and treatment of chronic inflammatory diseases [42, 43] . for example, hu and coauthors reported the construction of targeted theranostic nanoplatform encompassing magnetic resonance (mr) imaging-active superparamagnetic iron oxide nanoparticle (spion) and phenylboronic acid-modified dspe-peg [42] . specifically, congo red-linked dspe-peg (congo red is capable of binding to amyloid plaques as targeting agent) and phenylboronic acid-modified dspe-peg were simultaneously utilized to stabilize oleic acid-coated iron oxide nanoparticles through hydrophobic interactions, followed by drug loading of vicinal diol-containing rutin (a powerful phenolic antioxidant with anti-inflammatory activity) through a dynamic covalent boronate ester linkage [42] . as a result, congo red/rutin-magnetic nanoparticles coinjected with mannitol could cross the blood-brain barrier (bbb) into the brain of appswe/ps1de9 transgenic mice and recognize amyloid plaques, allowing for diagnosis of amyloid plaques by mr imaging and selective delivery of therapeutics. furthermore, β-amyloid (aβ)-induced overproduction of oxidative species could actuate the transformation of phenylboronic ester into phenol and ensuing release of rutin, resulting in the alleviation of oxidative stress, and reduction of amyloid plaques and neuronal loss. alternatively, jiang et al. presented ros-responsive polymeric micelles with biomimetic targeting ability to normalize the oxidative and inflammatory microenvironment, and reeducate microglia from an early phase of ad, based on aberrant hyperreactive state of microglia and related microenvironment changes of ad progression [43] . to elaborate, oxidation-triggerable amphiphilic block copolymers (bcps) were firstly synthesized via post-polymerization modification between hydrophobic phenylboronic ester-containing motifs and amine groups on peg-b-polylysine (peg-b-plys), followed by conjugation with a small targeting peptide (klvffaed; ab peptide), which is derived from the binding domain of aβ protein with receptor for advanced glycation end-products (rage) (fig. 2) . through an aβ transportation-mimicked pathway, self-assembled, targeted, and curcumin (an immune-modulatory agent with antioxidant and anti-inflammatory capabilities)-loaded micelles could become accumulated into the diseased regions and exert synergistic effects of polymer-based ros elimination and curcumin-mediated aβ inhibition. it should be noted that such multi-target strategy exhibited gradual normalization of brain microenvironment, efficient neuroprotection, and microglia modulation, resulting in the decrease of aβ plaque burdens and enhancement of cognitive functions in appswe/ps1de9 mice. oxidation-responsive polymeric micelles with biomimetic targeting capability. a proposed mechanism of oxidation-actuated selfimmolative side chain degradation of ros-responsive bcp micelles. b illustration of microglia induced ad microenvironment and modulation mechanisms of brain-targeting and ros-responsive micellar delivery system: (i) ab peptide modified micelles mimic aβ transpor-tation from peripheral into brain parenchyma, (ii) ros-actuated degradation of micelles and triggered release of aβ inhibitive curcumin, and (iii) aβ-mimicked micelle targeting to activated microglia and damaged neurons. reproduced with permission from ref. [43] . copyright 2018, the authors. published by wiley-vch on the other hand, recent studies indicate that elevated levels of trace metal ions, especially iron, copper, and zinc ions, are closely interacted with aβ aggregate deposition and neurotoxicity in ad [44] . hence, metal dysregulation has been considered as a promising therapeutic target for ad therapy [45] . however, orthodox metal chelators are not suitable as potential therapeutic agents against ad due to their inability to efficiently cross bbb and specifically recognize metal ions in various forms [46] . to resolve these problems, qu et al. developed a mesoporous silica nanoparticle (msn)-based ros-responsive system to realize selective delivery of metal chelator [47, 48] . as a proof of concept, 3-carboxyphenylboronic acid was initially anchored onto msn surface, followed by conjugation with adjacent diols of saccharides within human igg through the dynamic boronate ester linkage to trap clioquinol (cq) inside msn pores. such boronate-based and igg-gated msn platform was demonstrated as promising dds for oxidation-triggered delivery of therapeutic metal chelators, showing potential for ad treatment. very recently, lee, wang, and coauthors reported a biomimetic dds derived from macrophage membrane-coated chitosan-based nanoparticles with ros responsiveness for the treatment of atherosclerosis, considering the accumulation of macrophages and increased oxidative stress during the development of atherosclerosis [49] . notably, surface coating with macrophage membrane not only diminished the clearance of nanoparticles from mononuclear phagocytes system (mps) but also guided nanoparticles to inflammatory tissues, where the ros-responsive polymeric scaffold enabled triggered release of encapsulated atorvastatin (at, a lipid-lowering drug). moreover, the macrophage membrane was capable of sequestering proinflammatory cytokines and chemokines to achieve local inflammation suppression. the synergetic effects of pharmacotherapy and sequestration of inflammatory cytokines and chemokines from such a biomimetic dds led to enhanced therapeutic efficiency against atherosclerosis. due to its excellent stability against acid, base, or enzymemediated degradation, thioketal motif that could be efficiently cleaved by diverse oxidants has been increasingly employed for biomedical applications, particularly drug delivery. in 2010, merlin, murthy, and coworkers innovatively reported ros-degradable and thioketal-based polymeric nanoparticles as oral delivery vehicles for small interfering rna (sirna), that could direct encapsulated sirna against tnf-α (tnf-α sirna) to sites of intestinal inflammation and thus inhibit gene expression in inflamed tissue ( fig. 3 ) [50] . when delivered orally, such thioketal nanoparticles (tkns) remained stable in the gastrointestinal tract, protecting tnf-α sirna against harsh conditions. more importantly, at sites of intestinal inflammation with high levels of ros, these tkns selectively underwent ros-triggered degradation and controlled release of tnf-α sirna. in a murine model of ulcerative colitis, it was demonstrated that orally administered tnf-α sirna-loaded nanocomplexes effectively reduced tnf-α messenger rna (mrna) levels in the colon and protected mice from ulcerative colitis. while above-discussed ros-responsive moieties (i.e., boronate and thioketal) are subjected to cleavage and degradation in response to oxidative milieu, hydrophobic thioether motif undergo oxidation-triggered transformation into hydrophilic sulfoxide or sulfone group [26] . by leveraging this intriguing transformation, hubbell, tirelli, and coauthors have elegantly developed a number of oxidationresponsive polymeric nanocarriers (e.g., vesicles and micelles) containing poly(propylene sulfide) (pps) moieties and these nanocarriers exhibited ros-actuated solubility switching and destabilization, activating triggered release of a range of encapsulated payloads [26, [51] [52] [53] . likewise, duvall et al. developed oxidation-sensitive micelles selfassembled from amphiphilic bcps containing hydrophilic poly(n,n-dimethylacrylamide) (pdma) and hydrophobic pps blocks for triggered drug release [54] . it was shown that such destructible fluorophore-loaded micelles efficiently released model drug (e.g., nile red) upon contact with exogenous h 2 o 2 , onooˉ, or endogenous oxidative species generated inside lipopolysaccharide (lps)-stimulated raw264.7 macrophages. along the same line, shuai and colleagues fabricated andrographolide (andro, a labdane diterpenoid with superior anti-inflammatory activity)-encapsulated oxidationdestructive micellar nanocarriers of peg-b-pps for the treatment of atherosclerosis via concomitant antioxidative and anti-inflammatory effects [55] . subsequent detailed evaluations in the mice model of apoe − / − revealed excellent therapeutic effects and low side effects of andro-loaded micelles. very recently, they further explored antirheumatic activity of celastrol (a bioactive component of tripterygium wilfordii with anti-inflammatory activity)-loaded oxidation-responsive micelles of peg-b-pps ( fig. 4) [56] . it was demonstrated that such nanomedicine could effectively accumulate in the inflamed joints and subsequently alleviate major ra-related symptoms including synovial inflammation, bone erosion, and cartilage degradation. analogously, scott group explored anti-inflammatory potentials of ppsbased nanovehicles with distinct morphologies (i.e., spherical micelles; filamentous worm-like micelles, filomicelles; polymeric vesicles, polymersomes) against atherosclerosis [57] [58] [59] [60] . notably, morphology-dependent organ and cellular level biodistributions of nanocarriers assembled from peg-b-pps bcps with identical surface chemistries were disclosed [57] . while spherical micelles and filomicelles were found to associate with liver macrophages and bloodresident phagocytes, respectively, polymersomes were significantly efficient at targeting splenic dendritic cells (dcs; critical mediators of atherosclerotic inflammation) and exhibited remarkably lower uptake by other cells of the mps. furthermore, polymersomes demonstrated superior selectivity for dcs in atherosclerotic lesions in vivo, highlighting the importance of nanostructure morphology in tailoring targeting specificity of nanoscale dds [57] . like sulfur-based derivatives, selenium-containing compounds have been widely used in pharmacochemistry as antioxidants, among which diselenide motif constitutes a promising candidate owing to excellent responsiveness towards both oxidative and reductive milieu [61, 62] . in this respect, liu group reported dually targeting selenium nanoparticles (senps) with antioxidative and neuroprotective activity to inhibit aβ fiber formation in ad [63] . lpffd and tgn (sequence: tgnykalhphng) peptide cofunctionalized targeted senps efficiently crossed bbb and possessed a strong affinity towards aβ species, efficiently suppressing aβ fibrillation by disrupting hydrophobic and electrostatic interactions that are involved in the nucleation process of aβ40. in addition, it was found that targeted senps could mitigate the aβ40 fiber-mediated generation of ros and neurotoxicity in pc12 cells. in another contribution, zhang et al. described positively charged polyprodrug amphiphiles to simultaneously encapsulate hydrophobic superparamagnetic iron oxide nanocubes and negatively charged lethal-7b antisense oligonucleotides via hydrophobic and electrostatic interactions, respectively [64] . to elaborate, lipid-lowering drug simvastatin (sim) was covalently connected to carboxyl-modified zwitterionic poly(carboxybetaine) (pcb) through a diselenide linkage, affording positively charged pcb-se-se-sim with high drug loading content. the formulated mr imaging-traceable nanoparticles could achieve oxidation-actuated corelease of sim and antisense oligonucleotides to decrease differentiation of exogenous neural stem cells and induce secretion of brain-derived neurotrophic factors concurrently, enabling the proliferation of endogenous neural stem cells for ad treatment. inspired by the underlying chemiluminescence chemistry of glow sticks, in which oxalate ester selectively decomposes upon contact with h 2 o 2 to form high-energy and unstable 1,2-dioxetanedione intermediate known to excite nearby fluorophores to release photons, a variety of functional systems containing oxalate moieties and fluorophores have been developed to achieve in vitro and in vivo sensing of ros fig. 3 orally delivered tkns loaded with tnf-α sirna target inflammation and inhibit gene expression in the intestines. a tnf-α-tkns were fabricated by first complexing tnf-α sirna with cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (dotap), followed by loading into nanoparticles composed of ppadt. typical scanning electron microscopy (sem) image showed tnf-α-tkns (scale bar represents 1.5 µm). b orally administrated tnf-α-tkns remained stable in the harsh conditions of gastrointestinal tract, protecting tnf-α sirna and preventing its undesirable release to noninflamed mucosal tissues. however, at sites of intestinal inflammation, high levels of ros triggered degradation of tkns, thus specifically releasing tnf-α sirna. reproduced with permission from ref. [50] . copyright 2010, springer nature (e.g., h 2 o 2 ) [65] [66] [67] . for instance, murthy et al. reported the construction of chemiluminescent nanoparticles formulated from oxalate ester-containing polymer and fluorescent dyes and such oxalate-based nanoparticles were capable of imaging h 2 o 2 with high specificity and sensitivity in the peritoneal cavity of mice during lps-induced inflammatory response [65] . subsequently, lee further explored the antiinflammatory effect of biodegradable polyoxalate nanoparticles featured with ros-triggered release of p-hydroxybenzyl alcohol (hba) [68, 69] . it was revealed that hba-backboned copolyoxalate (hpox) nanoparticles could enable the inhibition of expression of both inducible nitric oxide synthase (inos) and production of tnf-α in lps-activated raw264.7 macrophage cells. in follow-up works, in vivo anti-inflammatory activities of hpox nanoparticles with excellent ros-scavenging capability were investigated as novel therapeutic agents against airway inflammatory diseases [69, 70] . in a murine model of asthma, intratracheal administration of hpox nanoparticles substantially reduced the recruitment of inflammatory cells and expression of pro-inflammatory mediators including interleukin-4 (il-4) and inos (fig. 5 ) [70] . to further deepen our understanding and improve the clinical translation of nanomedicines, visualization of nanomedicines' fate in vivo, including the circulation, accumulation, and distribution at targeted inflammatory site, is also highly desirable. towards this end, ma et al. described a ros-responsive theranostic nanoplatform for two-photon aggregation-induced emission (aie) [71] imaging and antiinflammatory therapy of atherosclerosis [72] . specifically, prednisolone (pred), a synthetic glucocorticoid agent, was conjugated with a two-photon aie luminogen (tp) via ros-cleavable oxalate linkage, forming a theranostic compound tpp (fig. 6 ) [72] . afterwards, tpp was encapsulated into micellar nanoparticles that self-assembled from rosdisintegrable bcp consisting of hydrophobic thioetherincorporated poly(2-methylthioethanol methacrylate) (pmema) and hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (pmpc) [72] . upon oxidative triggering, such theranostic nanomedicine with serial ros fig. 4 schematic representation of in vivo anti-inflammatory mechanism using rosresponsive micelles selfassembled from peg-b-pps for suppressing synovial inflammation and alleviating joint destruction in ra. reproduced with permission from ref. [56] . copyright 2020, american chemical society responsiveness firstly underwent ros-actuated hydrophobic-to-hydrophilic transition and thereby released tpp molecules, followed by ros-triggered cleavage of oxalate linkage and activation of anti-inflammatory pred. moreover, tpp-loaded nanoparticles exhibited strong two-photon fluorescent emission, allowing for a distinct dimensional bioimaging of inflammation site with minimum autofluorescence interference and deep imaging penetration. importantly, impressive anti-inflammatory activity against ra and atherosclerosis in mice model was discovered through fig. 5 schematics of hbabackboned copolyoxalate (hpox) nanoparticles exhibiting oxidation-actuated degradation and triggered release of antioxidant hba for the treatment of airway inflammatory diseases. reproduced with permission from ref. [70] . copyright 2013, elsevier fig. 6 ros-responsive theranostic nanoplatform with two-photon imaging capability and anti-inflammatory activity (top). models of acute lung injury, arthritis, and atherosclerosis were used to evaluate the broad-spectrum inflammation theranostics (bottom). reproduced with permission from ref. [72] . copyright 2020, american chemical society systematic in vivo evaluations, exhibiting reduced inflammatory response and suppressed expression of pro-inflammatory cytokines. sung and coauthors developed an ultrasensitive rosresponsive hollow plga microcapsule that encompassed an anti-inflammatory dexamethasone sodium phosphate (dex-p), an acid-generating precursor consisting of ethanol and ferrous chloride (fecl 2 ), and sodium bicarbonate as a gas-generating agent [73] . in cases of inflamed osteoarthritis (oa), low level of h 2 o 2 could diffuse through the microcapsules to oxidize encapsulated ethanol in the presence of fe 2+ by fenton reaction, creating an acidic microenvironment. subsequently, sodium bicarbonate reacted with acid and thus decomposed to form co 2 gas bubbles, disrupting the plga shells of microcapsules and releasing high dose dex-p to the inflamed site, eventually fighting against joint destruction [73] . bilirubin, as the final product of the metabolism pathway of heme, is a potent endogenous antioxidant capable of scavenging various ros and thereby protecting cells from oxidative stress-mediated damage. jon group originally explored anti-inflammatory activity of nanoparticles self-assembled from pegylated bilirubin for the treatment of colitis, asthma, and psoriasis, by effectively diminishing ros and modulating the immune system [74] [75] [76] . typically, in a mouse model of ulcerative colitis, intravenous injection of nanoparticles of pegylated bilirubin exhibited preferred accumulation at the sites of inflammation and remarkably inhibited the progression of inflammation process in the colon [74] . very recently, xie, pu, and coworkers reported engineered exosomes (exo) with inflammation-tropism and antiinflammatory effects for concomitant imaging and therapy of atherosclerosis (fig. 7) [77] . such exo were derived from anti-inflammatory m2 phenotype macrophages and further electroporated with fda-approved hexyl 5-aminolevulinate hydrochloride (hal), a precursor for biosynthesis of heme. following systemic administration and localization into inflammatory cells, encapsulated hal would undergo intrinsic biosynthesis and metabolism of heme to endogenously generate anti-inflammatory carbon monoxide (co) and bilirubin, which collectively enhanced anti-inflammatory activities and finally alleviated inflammation in atherosclerosis lesions. at the same time, the intermediate product protoporphyrin ix of the heme biosynthesis process allowed for fluorescent imaging and tracking of atherosclerosis. concerning the importance of neutrophils and macrophages in inflammatory diseases, regulating inflammatory responses of neutrophils and macrophages with nanomedicine represented a promising approach for disease fig. 7 engineered macrophage-derived exo with inflammationtropism and intrinsic heme biosynthesis for atherosclerosis imaging and treatment. a typical construction route of hal-encapsulated exo, hal@m2 exo. b the inflammation-tropism and anti-inflammation activity of hal@m2 exo. c the intrinsic biosynthesis and metabolism of heme starting from hal. reproduced with permission from ref. [77] . copyright 2019, wiley-vch management. in this regard, guo et al. fabricated nanoparticles from luminol-conjugated β-cd, which efficiently attenuated neutrophil/macrophage-mediated inflammation and demonstrated desirable efficacies and biosafety in treating both acute and chronic inflammatory diseases (e.g., acute lung injury and atherosclerosis) (fig. 8) [78] . it was found that such bioactive nanoparticles significantly suppressed the production of pro-inflammatory cytokines (e.g., tnfα) and ros (e.g., h 2 o 2 ) by neutrophil/macrophage, and effectively inhibited their migration in vitro. specifically, in apoe − / − mice model of atherosclerosis, these nanoparticles efficiently delayed the progression of atherosclerosis as well as notably stabilized atheromatous lesions. local acidification has been recognized at inflammation sites of diverse acute and chronic inflammatory diseases, such as ischemic sites, asthmatic airways, atherosclerotic plaques, and rheumatic joints [7] . compared to healthy tissues, inflamed tissues generally exhibit lower ph values [7] . for instance, extracellular ph in the site of myocardial ischemia was about 6.5-6.0 and ph as low as 4.2 was confirmed in the fracture microenvironment [79, 80] . this mildly acidic characteristic is attributable to enhanced consumption of energy and oxygen by infiltrated inflammationassociated cells, which transform from aerobic metabolism to anaerobic manner inside inflamed tissues, resulting in oxygen-independent glycolysis and increased production of lactic acid [1, 7] . it should be also mentioned that, in addition to such pathological circumstances, varying degrees of acidic conditions exist in specific cells, tissues, and organs of the human body under normal physiological circumstances. for example, the ph value along the gastrointestinal tract has a well-tuned gradient, spanning from 1.0 to 3.5 in the stomach to 5.5-7.5 in the intestine [81] . besides, subcellular compartments display distinct ph microenvironments, with ph 5.0-6.5 in early and late endosome, ph 4.5-5.0 in lysosome, ph 4.0-6.5 in phagosome, and ph ~ 7.8 within mitochondrial matrix [82] . collectively, this physiological fig. 8 development of anti-inflammatory nanoparticles from luminol-modified β-cd by simultaneously regulating neutrophils and macrophages. reproduced with permission from ref. [78] . copyright 2019, wiley-vch or pathological acidic feature provides a vital biochemical trigger for developing novel ph-responsive ddss for the management of chronic inflammatory diseases [7, 15] . in 2005, murthy group reported acid-sensitive polyketal nanoparticles that were designed to transport therapeutic agents (e.g., dex) to physiologically relevant acidic microenvironment [83] . such polyketal nanoparticles were fabricated from a hydrophobic polymer, poly(1,4-phenyleneacetone dimethylene ketal) (ppadk). importantly, these biodegradable polyketal nanoparticles underwent acid-accelerated degradation into low mw and nonacidic products, in contrast to degradation of polyester-based biomaterials. to further address toxicity concern associated with degradation product (i.e., 1,4-benzenedimethanol) of ppadk, they developed novel polyketal, poly(cyclohexane-1,4-diyl acetone dimethylene ketal) (pcadk), with well-documented toxicity profiles [84] . it was found that sod-encapsulated pcadkbased microparticles enabled efficient scavenging of ros produced by macrophages in cell culture experiments. collectively, polyketal-based biomaterials held great potential for treating inflammatory diseases including myocardial infarction, given their neutral, biocompatible degradation products and their capability to deliver a rich assortment of therapeutic agents [85, 86] . in this aspect, davis et al. demonstrated that a direct cardiac injection of p38 inhibitor (sb239063)-encapsulated microparticles, fabricated from pcadk, improved cardiac function following myocardial infarction (mi) (fig. 9 ) [85] . significantly, pcadk microparticles induced slight inflammatory responses both in vitro and in vivo and could therefore reside in inflamed site and served as a sustained-release reservoir for sb239063, with a release half-life of 7 days at ph 7.4. in vivo measurements showed that sb239063-loaded pcadk microparticles exhibited prolonged reduction of inflammatory signaling and eventually increased functional outcome following a single injection, in contrast to nonresponsive plga-based system. besides ketal-containing systems, acetal-based acidresponsive biomaterials have been extensively explored for numerous biomedical applications [87] [88] [89] [90] . for instance, fréchet et al. elegantly presented an acid-responsive biodegradable nanoparticle from acetal-derivatized dextran with a solubility switching mechanism for controlled delivery of pharmaceutical agents [89] . using this interesting chemistry, zhang and coworkers synthesized acetal-modified cyclodextrin and poly(cyclodextrin) with ph-actuated degradability as biocompatible nanomaterials [91] . water-soluble fig. 9 noninflammatory acidic ph-sensitive microparticlebased delivery system for improved cardiac function following mi. a pcadk microparticles transformed into neutral, nontoxic products through acid-catalyzed hydrolysis of the ketal linkage. b a singleemulsion/solvent-evaporation technique was used to produce large (∼ 20 µm) sb239063encapsulated microparticles. particles were imaged using sem. c microparticles were injected intramyocardially, where they released the encapsulated inhibitor within the infarct zone. reproduced with permission from ref. [85] . α-cd and β-cd as well as their corresponding condensed polymers were transformed into water-insoluble products through acetalation, respectively. with respect to the formulation process, standard emulsion technique was exploited to prepare nanoparticles with size of about 150 nm. alternatively, a self-assembling approach was employed to control the sizes and morphologies of assemblies by taking advantage of well-known host-guest interactions (e.g., β-cd and adamantyl moiety). as expected, nanoparticles from acetalated-cd exhibited acid-triggered hydrolysis, resulting in hydrophobic-to-hydrophilic switching and dissociation of nanoparticles, and thus controlled release of the encapsulated model drug, rapamycin (rap, an immunosuppressant). more importantly, biocompatibility was further demonstrated for cd derivative-based assemblies via comprehensive measurements both in vitro and in vivo. in their subsequent work, superior targeting ability to atherosclerosis lesions was discovered for acid-responsive nanoparticles of acetalated β-cd following intraperitoneal injection in apoe − / − mice [35] . moreover, in vivo therapeutic outcomes revealed that rap-loaded nanomedicine from acetalated β-cd could more efficiently suppress the development of atherosclerosis and effectively improve the stability of atheromatous plaques in comparison with the nonresponsive plga-based delivery system. wang group developed a series of polymer-drug conjugates (pdcs) with acidic ph-labile acylhydrazone linkage for treatment of diverse chronic inflammatory diseases [92] [93] [94] [95] . typically, ph-sensitive prodrug monomer of dex was copolymerized with n(2-hydroxypropyl) methacrylamide (hpma) via reversible additionfragmentation chain transfer (raft) polymerization [92] . as expected, the resultant conjugate released dex upon triggering by acidic milieu (ph 5.0), while remained stable and intact under neutral condition (ph 7.4). notably, in vivo evaluation suggested that such pdcs offered superior, longer-lasting anti-inflammatory activities and joint protection effects when compared to free dex for the management of ra. later on, a comprehensive head-to-head comparison study of four established formulations of dex, including dex-loaded liposomes (l-dex), core-crosslinked micelles (m-dex) with nonselectively cleavable sulfone estercontaining linkage [96] , slow releasing pdcs (p-dex-slow) with acylhydrazone bond, and fast releasing pdcs (p-dexfast) with acylhydrazone benzyl ester linker, were evaluated in a clinically relevant inflammatory arthritis rat model (fig. 10) [94] . it was shown that following a bolus intravenous injection, the formulations with slower drug release rates (i.e., m-dex and p-dex-slow) provided longer duration of therapeutic activity, i.e., better joint protection, than those with relatively faster drug release kinetics. this finding was instructional and inspiring for the future design, development, and optimization of ddss for the clinical treatment of ra and other chronic inflammatory diseases. along this line, sun et al. reported self-assembling pdcs comprising of peg-based derivative and hydrophobic anti-inflammatory pred through cleavable acylhydrazone linker [97] . it was demonstrated that self-assembled polymeric micelles remained intact in circulation for long duration and specifically accumulated in arthritis joints, where the acidic milieu fig. 10 head-to-head comparison study of four established formulations of dex. a schematic representation and chemical structures of four dexamethasone-containing nanomedicine formulations. p-dex-slow and p-dex-fast encompassed acidic phsensitive linkages for drug conjugation. b representative microcomputed tomography (micro-ct) images of the ankle joints of dex-containing nanomedicine-treated adjuvantinduced arthritis rats. p-dexslow and m-dex exhibited an impressive reduction of ankle joint bone erosions, where bone was well-preserved. reproduced with permission from ref. [94] . copyright 2014, american chemical society triggered efficient release of pred, contributing to superior therapeutic efficacy than free pred against cia. as mentioned above, pegylation has been extensively exploited in dds to reduce opsonization by proteins and uptake by macrophages of mps, and prolong plasma residence time following systemic administration [98] . however, after localizing at pathological site, pegylation might hinder cellular uptake and subsequent endosomal escape, forming an obstacle in the realization of therapeutic activity [99] . to resolve this "peg dilemma," sheddable pegylation has been proposed to achieve on-demand depegylation at target sites [99] . in this regard, cui group presented acidic ph-sheddable pegylated plga nanoparticles to improve delivery of therapeutic payloads into chronic inflammation sites [100, 101] . it was demonstrated that hydrazone-based pegylation of nanoparticles significantly increased the distribution and retention of nanoparticles at pathological lesion when intravenously administered in a mouse model of lps-induced chronic inflammation [100] . to further augment selective accumulation and/or retention, binding ligand mannose was additionally decorated onto sheddable plga nanoparticles to achieve inflammation-associated acidic phtriggered conditional targeting [101] . taken together, acidsensitive sheddable ddss hold great promise against chronic inflammatory diseases and comprehensive evaluations need to be conducted in vivo in the future. dated back to 1993, brown et al. reported an intra-articular polymeric microsphere-based controlled release system consisting of natural polyelectrolytes, gelatin and chondroitin 6-sulfate [102] . the microsphere was fabricated by complex coacervation to encapsulate therapeutic protein, radiolabeled catalase ( 14 c-catalase). it was shown that kinetics of degradation of microspheres and concomitant release of 14 c-catalase related to gelatinase activities in the joint fluids. subsequently, they further optimized the formulation process of microsphere from gelatin and chondroitin 6-sulfate with respect to appropriate loading content and degradation kinetics [103] . in addition, biocompatibility of microsphere-based system was corroborated in human synovial fibroblast culture and by intra-articular administration into the knees of mice. together, such novel enzyme-degradable, biocompatible, and noninflammatory microsphere delivery system held great potential for the delivery of bioactive proteins to diseased joints (e.g., oa and ra). annamalai et al. described bioresponsive gelatin microspheres which could be degraded by proteolytic enzymes overexpressed in arthritic flares, leading to spatiotemporally controlled release of anti-inflammatory cytokines for cartilage preservation and repair (fig. 11 ) [104] . to elaborate, gelatin microspheres were fabricated by emulsification of solubilized anionic gelatin, followed by covalent crosslinking with amine-reactive genipin. such negatively charged microspheres were further utilized to sequester cationic cytokines with anti-inflammatory effects (e.g., il-4 and il-13) through electrostatic interactions, acting as minimally invasive delivery formulation for oa. collagenase-triggered degradation of gelatin matrix would result in controlled release of antiinflammatory cytokines, allowing to attenuate the stimulation of chondrocytes and the ensuing secretion of no. due to excellent biocompatibility, tunable composition, and tailor-made physiochemical features, hydrogels have found widespread applications in biomedical engineering, such as drug delivery, cell encapsulation, and regenerative medicine [105] . in the context of drug delivery, biodegradable fig. 11 enzyme-responsive gelatin microspheres were sensitive to proteolytic enzymes (e.g., collagenase) that typically overexpressed in arthritic flares, resulting in spatiotemporally controlled release of anti-inflammatory cytokines for cartilage preservation and repair in oa. reproduced with permission from ref. [104] . copyright 2019, wiley-vch hydrogels with cleavable backbones, crosslinkers, or sidechains enable release of therapeutic agents in a spatiotemporally controlled manner [105] . in this aspect, anseth team reported the construction of a proof-of-concept peg hydrogel with immobilized side-chain, which encompassed human neutrophil elastase (hne)-cleavable substrate [106] . the kinetic rate of enzyme-mediated cleavage reaction within the hydrogel could be tailored by manipulating the type and sequence of amino acid in substrate, holding promise for cellular responsive drug delivery. recently, mejías et al. designed a self-regulated nanoparticle-in-microgel delivery platform, in which microgel decrosslinking was actuated by extracellular hne, and nanoparticle was loaded with nexin-hib20, a potent inhibitor for neutrophil degranulation [107] . successful in vivo delivery of nexinhib20 to the airways and into neutrophils were observed, promoting the resolution of inflammatory response by suppressing recruitment and degranulation of neutrophils, production of pro-inflammatory cytokines in both airway and systemic compartments, as well as neutrophil-derived pathological extracellular vesicles (evs) in lung fluid. on the other hand, supramolecular hydrogels formed by low mw gelators that self-assembled in water through multiple noncovalent interactions have garnered considerable attention, representing a promising alternative to polymeric hydrogels [108] . for instance, xu et al. presented a local delivery system through conjugating tripeptide derivatives with olsalazine, a clinically validated anti-inflammatory prodrug [109] . the resultant amphiphiles self-assembled into nanofibers and then formed supramolecular hydrogels, exhibiting a gel-to-sol transition and controlled generation of bioactive 5-aminosalicylic acid upon azoreductasecatalyzed reduction. in another attempt, karp and coworkers reported an enzyme-responsive hydrogel platform [110, 111] as inflammation-targeting system for local drug delivery to treat chronic inflammatory diseases [112, 113] . in an embodiment, hydrogel microfibers capable of encapsulating anti-inflammatory dex, were generated from amphipathic ascorbyl palmitate containing esterase-cleavable ester linkage [112] . it was demonstrated that these inflammationresponsive hydrogels could exclusively release drug upon enzymatic cleavage and preferentially adhere to inflamed epithelial surfaces both in vitro and in two murine colitis models. in contrast to free dex enemas, dex-loaded hydrogel enemas, administered every other day to mice, contributed to a significant attenuation of inflammation and exhibited lower dex peak serum levels and, thereby, reduced systemic drug exposure. analogously, the same group fig. 12 arthritis flare-responsive hydrogel platform. a schematics of self-assembling process of tg-18 to form supramolecular hydrogel and encapsulation of ta. b high-resolution sem images of taloaded tg-18 hydrogels (scale bar is 2 μm). tg-18 lamellar structures extended to generate highly ordered fibrous structures. the entangled fibers formed bulk injectable hydrogel that could be administered via intra-articular injection. c triggered dissociation of tg-18 hydrogel in response to flare-associated overexpressed enzymes (e.g., mmps) in the inflamed joint. reproduced with permission from ref. [113] . copyright 2018, the authors developed arthritis flare-responsive hydrogel platform by self-assembling a small-molecule amphiphile, triglycerol monostearate (tg-18), to encapsulate corticosteroid triamcinolone acetonide (ta) (fig. 12 ) [113] . it was revealed that ta-loaded hydrogel could achieve on-demand drug release upon contact with enzymes (i.e., esterase or mmps) or synovial fluid from patients with ra. notably, a single locally injected dose of ta-encapsulated supramolecular hydrogel suppressed arthritis activity in the injected paw when compared to equivalent dose of free ta. by combining the efficacy of two clinically used agents, homma team reported the construction of hyaluronic acidmethotrexate (mtx) conjugates through varied cathepsinsensitive linkages for the treatment of oa [114, 115] . through the optimization of linker structure, mw, and stoichiometry of mtx within conjugate, a promising candidate conjugate was identified, capable of inhibiting the proliferation of human fibroblast-like synoviocytes in vitro and knee swelling in rat antigen-induced monoarthritis in vivo [115] . likewise, several nsaids were conjugated onto synthetic dendrimers or linear polymers through enzyme-cleavable linkers in order to alleviate side effects of long-term use of nsaids meanwhile retaining their therapeutic activities [116, 117] . in contrast to the mildly oxidizing extracellular environment, the high concentration of glutathione (1-10 mm) and other small redox molecules with free thiol groups (5-10 mm) provide a promising stimulus to devise ddss for selective intracellular activation [118] . for instance, xiao et al. presented a strategy that combined the unique property of bioreducible, cationic polymers with superior targeting capability of macrophage-specific mannose residues towards efficient rna interference (rnai) for ibd therapy (fig. 13 ) [119] . the mannosylated reducible polymer was synthesized via michael addition reaction between cystamine bisacrylamide (cba) and branched polyethylenimine (bpei), and then modified with d-( +)-mannose through hydrophilic peg spacer. tnf-α sirna-loaded nanosized polyplexes were fabricated through electrostatic interactions, followed by additional stabilization by ionic crosslinker, sodium triphosphate pentabasic (tpp). these polyplexes could efficiently enter macrophages, release encapsulated sirna upon triggering by reductive milieu, and actuate high-level rnai, suppressing tnf-α expression and conferring antiinflammatory efficacy in vitro and ex vivo. in another contribution, zhong, yin, and coworkers reported a targeted drug/ sirna codelivery platform based on reversibly disulfidecrosslinked polymersomes for efficient treatment of ulcerative colitis [120] . apart from reduction-responsive systems for rnaimediated anti-inflammatory therapy, reductive milieusensitive systems have also been explored to transport conventional low mw anti-inflammatory agents and antiinflammatory peptides [121] [122] [123] . for instance, feng and coauthors fabricated reduction-responsive and mtxencapsulated polypeptide-based disulfide-crosslinked nanogels for selective drug delivery in relieving cia [122] . it was found that such nanogels could selectively accelerate the release of mtx upon responding to gsh to cleave fig. 13 electrostatic interactiondriven self-assembling process into mannose-functionalized, sirna-encapsulated nanoparticles for macrophage-targeting delivery and cytosolic reductive milieu-triggered release of sirna. reproduced with permission from ref. [119] . copyright 2013, elsevier ltd disulfide-based crosslinkers. moreover, drug-loaded nanogels exhibited selective accumulation in the inflammatory joints of cia mouse model, effectively inhibiting secretion of proinflammatory cytokines and relieving the progress of cia. because of the large variations in physiological conditions between normal tissues and diseased sites, smart materials with single responsiveness may not achieve the desired goals in a complex pathophysiological microenvironment [124] [125] [126] . instead, materials responsive to multiple physical, chemical, and biological stimuli are highly promising for advanced biomedical applications, including precision drug delivery [124] [125] [126] . as discussed above, oxidative stress, acidic ph, and overexpressed enzymes are important pathological stimuli for drug delivery to inflamed sites. towards developing multi-stimuli-responsive systems, almutairi group presented an innovative approach to devise inflammation-responsive logic gate nanoparticles for triggered delivery of proteins [127] . specifically, they constructed an oxidation and acidic ph dually responsive polythioether ketal by integrating a thioether moiety in ph-responsive polyketal backbone; oxidation-reactive thioether acted as a solubility switch that turned the polymer more hydrophilic when it was subjected to oxidation [51, 52] . higher release kinetics of encapsulated ovalbumin was observed in the presence of both h 2 o 2 and acidic ph. in another report, zhu and coworkers constructed polydatin (pd; a bioactive agent with hepatoprotective and antifibrotic activity)-loaded micelles assembled from ros and acidic ph dually sensitive bcps to ameliorate liver fibrosis [128] . the pd-encapsulated micelle exerted great promise in improving the biocompatibility of pd and exhibited highly effective liver-targeted drug delivery in response to the fibrotic microenvironment. both in vitro and in vivo evaluations demonstrated that pd-loaded micelles could significantly suppress inflammatory response and oxidative stress, reduce hepatocyte apoptosis, and avert activation of macrophages and hepatic stellate cells, endowing them with remarkable efficacy and minimal side effects in the management of liver fibrosis. in an alternative manner, lee et al. developed an oxidation and acid dually responsive antioxidant polymeric prodrug of vanillin, poly(vanillin oxalate) (pvo), in which anti-inflammatory activity of vanillin was blocked via h 2 o 2 -reactive peroxalate ester and acidresponsive acetal linkages [129] . it was revealed that pvo nanoparticles exhibited highly potent antioxidant effects by scavenging ros and inhibiting the production of ros, as well as suppressed the expression of pro-inflammatory chemokines and cytokines in activated macrophages in vitro and in vivo. analogously, daniel and coauthors reported a proof-of-concept example of dual-responsive nanoparticles capable of activating bioactive mmp inhibitor (designated as py-2) upon contact with mmp and ros [130] . exposure of such dually responsive micellar nanoparticles to mmp-12 induced a morphological transformation into micron-scale aggregates, which was beneficial for enhanced retention of nanomedicine in targeted lesions [131, 132] . this change together with the presence of h 2 o 2 , resulted in controlled release of py-2, potentially relieving inflammation by inhibiting enzymatic activities of mmps. sung et al. described a blended nanoparticle system with dual responses to oxidative stress and reduced ph through coassembling ph-responsive cy3-conjugated n-palmitoyl chitosan and ros-degradable poly-(1,4-phenyleneactone dimethylene thioketal) (ppadt) for delivery of curcumin (fig. 14) [133] . notably, owing to the presence of förster resonance energy transfer (fret) between curcumin and conjugated fluorophore cy3, such system enabled in situ monitoring of intracellular release behavior. furthermore, in vivo anti-inflammatory effects of curcumin-loaded nanoparticles were validated in a mouse model with ankle inflammation induced by lps, revealing significant mitigation of oxidative stress. along this line, reactive oxygen species (ros)-sensitive dextran-naproxen conjugate was blended with acid-responsive acetalated dextran polymer, forming a dually responsive nanoparticle with high specificity towards inflammatory microenvironment [134] . in another comprehensive work, a facile and effective approach was proposed to tailor ph/oxidation dual-responsive nanocarriers by combination of ph-sensitive acetalated β-cd and oxidation-responsive phenylboronate conjugated β-cd [135] . by tuning the weight ratio of acidic ph-sensitive material and ros-responsive material, coassembled nanoparticles with desirable ph/oxidation-responsive capability could be easily produced. in response to reduced ph or oxidative stress, encapsulated rap molecules could be released from drug-loaded dually responsive nanoparticles. furthermore, targeting capacity of such dual-responsive nanoparticles was enhanced by surface decoration with a type iv collagen targeting peptide (sequence: klwvlp-kgggc), resulting in an actively targeted nanoplatform. accordingly, rap-loaded targeted nanoparticles displayed more effective in vivo efficiency than passively targeted nanoparticles in the inhibition of neointimal formation in rats. taken together, these developed ph/oxidation dualresponsive nanocarriers held great promise for the management of restenosis and other cardiovascular diseases related to vascular inflammation. on the other hand, santos group presented ph and oxidation sequentially responsive nano-in-micro composites for targeted therapy of ibd [136] . specifically, oxidation-sensitive nanoparticles formulated from a phenylboronic esters-modified dextran were coated with mucoadhesive chitosan, followed by encapsulation in ph-responsive hydroxypropyl methylcellulose acetate succinate (hpmcas) through microfluidics technique, affording nano-in-micro composite particles. the final composites were devised to protect the rifaximin (rif; an intestine-specific antibiotic employed in inducing remission of ibd)-loaded oxidation-responsive nanoparticles from the harsh conditions in the upper gastrointestinal tract (e.g., acidic ph of the stomach) and to unmask them in the intestine, closer to the targeted lesions. upon activating by elevated ros levels, oxidation-responsive nanoparticles would selectively release rif into the inflamed sites. unfortunately, comprehensive in vivo anti-inflammatory evaluations of such promising nano-in-micro composites have not been systematically investigated. in this review, recent progress on the development of inflammation-responsive delivery systems as promising anti-inflammatory therapy in the treatment of chronic diseases is summarized. the rational design of intelligent ddss enables selective, on-demand release and activation of bioactive agents at target disease sites by leveraging inflammation-associated biochemical signals, including oxidative stress, reduced ph, overexpressed enzymes, and reductive microenvironment. in vivo antiinflammatory potentials of inflammation-sensitive ddss have been preliminarily demonstrated via local, oral, or reproduced with permission from ref. [133] . copyright 2014, american chemical society systemic administration in diverse animal models, exhibiting enhanced efficiency as compared to nonresponsive systems. notwithstanding these advances, only a very few inflammation-responsive ddss have been comprehensively explored in various animal models of chronic inflammatory diseases. even worse, excellent anti-inflammatory effects of inflammation-sensitive delivery systems in animal models of chronic inflammatory diseases have not progressed in a clinical trial phase. to our knowledge, no translational studies have been carried out in inflammationresponsive therapies towards the treatment and management of chronic diseases [8, 15] . in contrast, several traditional and nonresponsive anti-inflammatory ddss have succeeded to reach clinical trials [8] . this status is reminiscent of the development process of smart ddss in cancer treatment [137] , challenging the therapeutic benefits of bioresponsive delivery systems over traditional systems in clinical applications. numerous challenges in this burgeoning field remain to be addressed in the future before clinical translation studies. first, for anti-inflammatory therapy, diverse biological barriers impede the selective accumulation of ddss and therapeutic agents at the site of inflammation, compromising in vivo therapeutic efficiency and benefit-to-risk window [1] . second, new and standard preclinical animal models that could thoroughly mimic the pathophysiological characteristics of human chronic inflammation need to be established. thus far, most in vivo therapeutic studies of inflammation-responsive ddss were conducted in murine models of chronic inflammatory diseases, and the collected results could not be directly correlated to humans. third, for rational design and development of inflammation-responsive ddss, the optimal biochemical stimuli should be determined according to disease types and diseased sites. for instance, the extent of oxidative stress and the major type of oxidative species might be dynamically changed in distinct chronic diseases, even at different stages of one disease. while multi-stimuliresponsive systems theoretically could provide better sensitivity and selectivity, their formulations generally involve complicated synthesis and/or fabrication procedures. last but not least, further work is needed to surmount pharmaceutical barriers ranging from biocompatibility and safety of various ddss, cost of goods and manufacturing, to batch-to-batch reproducibility, facilitating potential clinical translations of inflammation-sensitive delivery systems. anti-inflammatory therapy in chronic disease: challenges and opportunities a guiding map for inflammation inflammationnature's way to efficiently respond to all types of challenges: implications for understanding and managing "the epidemic" of chronic diseases resolution of chronic inflammatory disease: universal and tissue-specific concepts anti-inflammatory agents: present and future drug targeting systems for inflammatory disease: one for all, all for one fishing for fire: strategies for biological targeting and criteria for material design in anti-inflammatory therapies advanced nanomedicines for the treatment of inflammatory diseases a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs macromolecular therapeutics in cancer treatment: the epr effect and beyond the arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study novel dexamethasone-hpma copolymer conjugate and its potential application in treatment of rheumatoid arthritis permeability of human synovial membrane to plasma proteins. relationship to molecular size and inflammation inflammation-responsive polymers bioresponsive drug delivery systems for the treatment of inflammatory diseases oxidation-responsiveness of nanomaterials for targeting inflammatory reactions perspectives and opportunities for nanomedicine in the management of atherosclerosis atherosclerosis treatment with stimuli-responsive nanoagents: recent advances and future perspectives nanoparticulate drug delivery systems targeting inflammation for treatment of inflammatory bowel disease biomimetic nanoparticle technology for cardiovascular disease detection and treatment reactive oxygen, nitrogen, and sulfur species (ronss)-responsive polymersomes for triggered drug release reactive oxygen species-responsive drug delivery systems for the treatment of neurodegenerative diseases dancing with reactive oxygen species generation and elimination in nanotheranostics for disease treatment chapter 1-polymers in drug delivery: an update therapeutic strategies by modulating oxygen stress in cancer and inflammation oxidation-responsive materials: biological rationale, state of the art, multiple responsiveness, and open issues fluorescent imaging of reactive oxygen and nitrogen species associated with pathophysiological processes a biocompatible oxidation-triggered carrier polymer with potential in therapeutics biocompatible polymeric nanoparticles degrade and release cargo in response to biologically relevant levels of hydrogen peroxide biocompatible reactive oxygen species (ros)-responsive nanoparticles as superior drug delivery vehicles engineering intracellular delivery nanocarriers and nanoreactors from oxidation-responsive polymersomes via synchronized bilayer cross-linking and permeabilizing inside live cells anti-inflammatory polymersomes of redox-responsive polyprodrug amphiphiles with inflammation-triggered indomethacin release characteristics nanoparticles responsive to the inflammatory microenvironment for targeted treatment of arterial restenosis a superoxide dismutase/catalase mimetic nanomedicine for targeted therapy of inflammatory bowel disease non-proinflammatory and responsive nanoplatforms for targeted treatment of atherosclerosis a targeting nanotherapy for abdominal aortic aneurysms targeted therapy of atherosclerosis by a broad-spectrum reactive oxygen species scavenging nanoparticle with intrinsic anti-inflammatory activity a broad-spectrum ros-eliminating material for prevention of inflammation and drug-induced organ toxicity scavenging ros: superoxide dismutase/catalase mimetics by the use of an oxidation-sensitive nanocarrier/ enzyme conjugate the delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury reactive oxygen species-responsive dexamethasoneloaded nanoparticles for targeted treatment of rheumatoid arthritis via suppressing the irhom2/tnf-α/baff signaling pathway nanotheranostics: congo red/rutin-mnps with enhanced magnetic resonance imaging and h2o2-responsive therapy of alzheimer's disease in appswe/ps1de9 transgenic mice microenvironment remodeling micelles for alzheimer's disease therapy by early modulation of activated microglia the metallobiology of alzheimer's disease therapeutic approaches to alzheimer's disease nanoparticle and other metal chelation therapeutics in alzheimer disease mesoporous silica nanoparticle-based h2o2 responsive controlled-release system used for alzheimer's disease treatment cerium oxide caged metal chelator: anti-aggregation and anti-oxidation integrated h2o2-responsive controlled drug release for potential alzheimer's disease treatment treatment of atherosclerosis by macrophagebiomimetic nanoparticles via targeted pharmacotherapy and sequestration of proinflammatory cytokines orally delivered thioketal nanoparticles loaded with tnf-α-sirna target inflammation and inhibit gene expression in the intestines oxidationresponsive polymeric vesicles oxidation-sensitive polymeric nanoparticles lyotropic behavior in water of amphiphilic aba triblock copolymers based on poly(propylene sulfide) and poly(ethylene glycol) poly(ps-b-dma) micelles for reactive oxygen species triggered drug release aortic plaque-targeted andrographolide delivery with oxidation-sensitive micelle effectively treats atherosclerosis via simultaneous ros capture and anti-inflammation inflammation-targeted celastrol nanodrug attenuates collagen-induced arthritis through nf-kappab and notch1 pathways tailoring nanostructure morphology for enhanced targeting of dendritic cells in atherosclerosis celastrol-loaded peg-b-pps nanocarriers as an anti-inflammatory treatment for atherosclerosis surface engineered polymersomes for enhanced modulation of dendritic cells during cardiovascular immunotherapy an injectable hydrogel platform for sustained delivery of anti-inflammatory nanocarriers and induction of regulatory t cells in atherosclerosis dual redox responsive assemblies formed from diselenide block copolymers from selenite to diselenide-containing drug delivery systems dual-functional selenium nanoparticles bind to and inhibit amyloid beta fiber formation in alzheimer's disease positively charged polyprodrug amphiphiles with enhanced drug loading and reactive oxygen species-responsive release ability for traceable synergistic therapy in vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles real-time imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing oxalate-curcumin-based probe for micro-and macroimaging of reactive oxygen species in alzheimer's disease antioxidant and anti-inflammatory activities of hydroxybenzyl alcohol releasing biodegradable polyoxalate nanoparticles reduction of oxidative stress by p-hydroxybenzyl alcohol-containing biodegradable polyoxalate nanoparticulate antioxidant antioxidant polymeric nanoparticles as novel therapeutics for airway inflammatory diseases aggregationinduced emission: together we shine, united we soar reactive oxygen species responsive theranostic nanoplatform for two-photon aggregation-induced emission imaging and therapy of acute and chronic inflammation controlled release of an anti-inflammatory drug using an ultrasensitive ros-responsive gas-generating carrier for localized inflammation inhibition bilirubin nanoparticles as a nanomedicine for anti-inflammation therapy bilirubin nanoparticles ameliorate allergic lung inflammation in a mouse model of asthma bilirubin nanomedicine alleviates psoriatic skin inflammation by reducing oxidative stress and suppressing pathogenic signaling molecularly engineered macrophage-derived exosomes with inflammation tropism and intrinsic heme biosynthesis for atherosclerosis treatment cyclodextrin-derived intrinsically bioactive nanoparticles for treatment of acute and chronic inflammatory diseases studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance the cytokines and microenvironment of fracture haematoma: current evidence ph-responsive nanoparticles for drug delivery sensors and regulators of intracellular ph polyketal nanoparticles: a new phsensitive biodegradable drug delivery vehicle polyketal microparticles: a new delivery vehicle for superoxide dismutase sustained release of a p38 inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction delivery of nox2-nadph oxidase sirna with polyketal nanoparticles for improving cardiac function following myocardial infarction a new approach towards acid sensitive copolymer micelles for drug delivery stimuli-responsive supramolecular assemblies of linear-dendritic copolymers acetal-derivatized dextran: an acid-responsive biodegradable material for therapeutic applications expansile nanoparticles: synthesis, characterization, and in vivo efficacy of an acid-responsive polymeric drug delivery system facile engineering of biocompatible materials with ph-modulated degradability synthesis and evaluation of a well-defined hpma copolymer-dexamethasone conjugate for effective treatment of rheumatoid arthritis syntheses of click peg−dexamethasone conjugates for the treatment of rheumatoid arthritis nanomedicines for inflammatory arthritis: headto-head comparison of glucocorticoid-containing polymers, micelles, and liposomes macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis glucocorticoid-loaded core-cross-linked polymeric micelles with tailorable release kinetics for targeted therapy of rheumatoid arthritis ph-sensitive polymeric micelles for targeted delivery to inflamed joints pegylation of biopharmaceuticals: a review of chemistry and nonclinical safety information of approved drugs depegylation strategies to increase cancer nanomedicine efficacy acid-sensitive sheddable pegylated plga nanoparticles increase the delivery of tnf-α sirna in chronic inflammation sites acid-sensitive sheddable pegylated, mannosemodified nanoparticles increase the delivery of betamethasone to chronic inflammation sites in a mouse model controlled drug delivery to the joints by enzymatically degradable microspheres gelatin/chondroitin 6-sulfate microspheres for the delivery of therapeutic proteins to the joint bioresponsive microspheres for on-demand delivery of anti-inflammatory cytokines for articular cartilage repair injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications human neutrophil elastase responsive delivery from poly(ethylene glycol) hydrogels neutrophil-targeted, protease-activated pulmonary drug delivery blocks airway and systemic inflammation supramolecular hydrogelators and hydrogels: from soft matter to molecular biomaterials molecular nanofibers of olsalazine form supramolecular hydrogels for reductive release of an anti-inflammatory agent on-demand drug delivery from self-assembled nanofibrous gels: a new approach for treatment of proteolytic disease a single localized dose of enzyme-responsive hydrogel improves long-term survival of a vascularized composite allograft an inflammation-targeting hydrogel for local drug delivery in inflammatory bowel disease towards an arthritis flare-responsive drug delivery system novel hyaluronic acid-methotrexate conjugates for osteoarthritis treatment synthesis and optimization of hyaluronic acidmethotrexate conjugates to maximize benefit in the treatment of osteoarthritis design, synthesis and biological evaluation of enzymatically cleavable nsaids prodrugs derived from selfimmolative dendritic scaffolds for the treatment of inflammatory diseases an enzyme-responsive prodrug with inflammationtriggered therapeutic drug release characteristics disulfide-based self-immolative linkers and functional bioconjugates for biological applications mannosylated bioreducible nanoparticle-mediated macrophage-specific tnf-α rna interference for ibd therapy efficient and targeted drug/sirna co-delivery mediated by reversibly crosslinked polymersomes toward anti-inflammatory treatment of ulcerative colitis (uc) release of anti-inflammatory peptides from thermosensitive nanoparticles with degradable cross-links suppresses pro-inflammatory cytokine production reductionresponsive polypeptide nanogel for intracellular drug delivery in relieving collagen-induced arthritis intra-articular injection of indomethacin-methotrexate in situ hydrogel for the synergistic treatment of rheumatoid arthritis multistimuli responsive macromolecules and their assemblies multi-stimuli responsive polymers-the all-in-one talents multi-stimuliresponsive polymer particles, films, and hydrogels for drug delivery inflammation responsive logic gate nanoparticles for the delivery of proteins nanodrug with ros and ph dual-sensitivity ameliorates liver fibrosis via multicellular regulation inflammationresponsive antioxidant nanoparticles based on a polymeric prodrug of vanillin dualresponsive nanoparticles release cargo upon exposure to matrix metalloproteinase and reactive oxygen species therapeutic enzyme-responsive nanoparticles for targeted delivery and accumulation in tumors enzyme-responsive nanoparticles for targeted accumulation and prolonged retention in heart tissue after myocardial infarction nanoparticles with dual responses to oxidative stress and reduced ph for drug release and anti-inflammatory applications inflammation-responsive drug-conjugated dextran nanoparticles enhance anti-inflammatory drug efficacy a ph/ros dual-responsive and targeting nanotherapy for vascular inflammatory diseases ph and reactive oxygen species-sequential responsive nano-in-micro composite for targeted therapy of inflammatory bowel disease nanoparticles in the clinic: an update the authors would like to acknowledge the support from university of science and technology of china, hefei, anhui province, china. author contribution writing-original draft preparation: zhengyu deng. writing-review and editing: shiyong liu.funding this work was supported by national key research and development program of china (2020yfa0710700) and national natural science foundation of china (nnsfc) project (51690150, 51690154, 52021002, 21925107, and u19a2094).data availability data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. research involving human and animals this article does not contain any studies with human participants or animal models performed by any of the authors. the authors declare that we participated in the development of this manuscript and have approved the final version, and give our consent for this submitted manuscript to be considered in drug delivery and translational research. the authors declare that they have no conflict of interest. key: cord-0036416-87cccwap authors: katsanos, aristeidis h.; kyriakidi, kalliroi; karassa, fotini b.; politis, dimitrios; skamnelos, alexandros; christodoulou, dimitrios k.; katsanos, konstantinos h. title: biomarker development in chronic inflammatory diseases date: 2017-09-23 journal: biomarkers for endometriosis doi: 10.1007/978-3-319-59856-7_3 sha: 8370a2e19f1031426d97aeee8ab1941494726241 doc_id: 36416 cord_uid: 87cccwap chronic inflammatory diseases, such as inflammatory bowel disease—namely, crohn’s disease and ulcerative colitis—psoriasis, multiple sclerosis, rheumatoid arthritis, and many others affect millions of people worldwide, causing a high burden of disease, socioeconomic impact, and healthcare cost. these diseases have common features including autoimmune pathogenesis and frequent co morbidity. the treatment of these chronic inflammatory diseases usually requires long-term immunosuppressive therapies with undesirable side effects. the future of chronic inflammatory disease prevention, detection, and treatment will be greatly influenced by the use of more effective biomarkers with enhanced performance. given the practical issues of collecting tissue samples in inflammatory diseases, biomarkers derived from body fluids have great potential for optimized patient management through the circumvention of the abovementioned limitations. in this chapter, peripheral blood, urine, and cerebrospinal fluid biomarkers used in chronic inflammatory conditions are reviewed. in detail, this chapter reviews biomarkers to fore used or emerging to be used in patients with chronic inflammatory conditions. those include inflammatory bowel diseases, chronic inflammatory conditions of the liver, biliary tract, pancreas, psoriasis, atopic disease, inflammatory skin diseases, rheumatic diseases, demyelination, and also the chronic inflammatory component of various other diseases in general medicine—including diabetes, cardiovascular disease, renal disease, and chronic obstructive pulmonary disease. development of personalized medicine is closely linked to biomarkers, which may serve as the basis for diagnosis, drug discovery, and monitoring of diseases. as personalized medicine is becoming an integral component of modern healthcare, the development of biomarker-based clinical tests emerges as a key challenge. in the conventional setting, chronic inflammatory diseases are assessed by clinical activity indices that measure clinical symptoms or by radiologic or endoscopic indices that measure tissue inflammation. disease subtypes are often defined on the basis of medical history, physical findings, imaging parameters, serum markers, as well as endoscopic and histopathological characteristics of the disease. noninvasive or minimally invasive diagnostic procedures present less burden and risk to patients than, for example, invasive biopsy sampling. they may thus support clinical decision-making by providing information on disease status and prognosis. the requirements of these applications cannot always be met by a single biomarker class. a comprehensive biomarker approach is needed, given its ultimate significance for clinical application. this strategy could lead to an optimized management of chronic inflammatory diseases with existing drugs and may facilitate the development of novel, more effective therapies. chronic inflammation is a component of many disorders that are discussed as per the system involved. major disorders with chronic inflammation involve many diseases including those of the digestive tract, liver, and cardiovascular and nervous systems. diseases such as diabetes and rheumatoid arthritis are also characterized by inflammation. in addition, rejection of allografts also involves chronic inflammatory immune mechanisms, and there is an interest in finding predictive biomarkers of organ rejection after transplantation. this chapter provides an overview of the field of biomarkers in the peripheral blood, urine, or cerebrospinal fluid (csf) that are currently used or can be easily used in combination with other clinical facilities to diagnose, treat, and tailor therapy in patients with chronic inflammatory diseases. for patients diagnosed with inflammatory bowel diseases (ibd) including crohn's disease (cd) or ulcerative colitis (uc), several laboratory markers have been investigated for diagnosis and differential diagnosis of ibd as well as for assessment of disease activity and risk of complications, prediction of relapse, and monitoring the effect of therapy [1, 2] . we can differentiate the biomarkers in three major categories: serological, fecal, and other biomarkers. each category includes other subcategories, for instance, serological biomarkers are differentiated in acute-phase reactants, cytokines, and others [ table 1 ]. serological biomarkers are measurable substances in body fluids (blood), whose application is cheaper, less laborious, less invasive, and more objective compared to the endoscopy-/biopsy-based approach [3] . serological acute-phase proteins are those whose plasma concentration increases (positive acute-phase proteins) or decreases (negative acute-phase proteins) by at least 25% during inflammatory disorders [1] . it is widely accepted that the concentrations of multiple components of the acutephase response do not increase uniformly in all patients with the same illness, although the serological acute-phase proteins commonly increase together. as serum markers can be elevated in a variety of conditions, fecal markers of inflammation would be more specific for ibd, in absence of enteric infection [4] . fecal biomarkers are valuable in their specificity to the gastrointestinal tract. they include a heterogeneous group of substances that either leak from or are generated by the inflamed intestinal mucosa [1] . in the setting of mucosal inflammation in patients with ibd, inflammatory proteins (protein cytokines and markers c of neutrophil activation), leukocyte products, and leukocytes themselves leak from a permeable mucosa. fecal microbiota appear to be attractive biomarkers in ibd, since they provide a noninvasive way to directly monitor changes in the intestinal environment associated with mucosal inflammation. however, they do not necessarily play a causative role in disease. the ability of the organism to compete in the altered intestinal environment is associated with the appearance or disappearance of a microbial group in disease [5] . c-reactive protein (crp), an acute-phase protein, is produced as an acute-phase reactant predominantly in the liver, in response to a variety of acute and chronic inflammatory conditions, and is an important component of the innate immune system [1, 4, 6] . crp is produced mainly by hepatocytes in response to stimulation by interleukin (il)-6, and to a lesser extent in response to tnf-a and il-1b, which are produced at the site of inflammation [1, 4] . crp is an easy and reliably measured biomarker across diagnostic laboratories and has a short plasma half-life of 19 h [6] . crp production is rapidly upregulated, in the presence of an acute-phase stimulus [4] . within 24-48 h the increase may be 500 to 1000-fold higher than under basal circumstances. inversely, to its increase, the reduction of crp may be similarly rapid, as the acute-phase response subsides, with a fall from peak with a half-time of 48 h [1] . once the stimulus disappears, crp concentrations quickly decrease due to crp's short half-life. hence, this makes crp a valuable marker to detect the activity of ibd [4] . crp is an objective marker of inflammation correlating well with disease activity in cd. increased crp levels are associated with better response rates, whereas normal crp levels predict high placebo response rates in clinical trials [2] . the same increasing trend can be observed in uc, although crp levels are generally lower than in cd [4] . several studies have identified increased levels of crp in nearly 100% of patients with cd and approximately 50% of those with uc [7] . crp is the most sensitive biomarker compared to other biomarkers of inflammation in adult population for the detection of ibd and is also used in screening patients with gastrointestinal symptoms. the sensitivity of crp in discriminating cd from irritable bowel syndrome ranges between 70 and 100% and the sensitivity in uc between 50 and 60% [1] . hence, crp is a valuable biomarker in sorting out active cd from functional bowel disorders [6] . moreover, since crp levels tend to be higher in cd than in uc, crp might be used to differentiate both types of ibd [1] . however, the relatively low sensitivity of the crp test in detection of uc prohibits the use of this marker alone to identify patients with symptoms compatible with ibd, without further evaluation [7] . crp distinguishes between active and quiescent ibd and trends with mucosal healing [6] . various studies have been conducted, regarding crp levels and their correlation to different clinical courses [4] . a retrospective analysis of mayo clinic data has associated moderate to greater clinical disease severity, active disease on colonoscopy, and histological severe inflammation with an elevated crp, while 51% of uc patients with active disease also had an elevated crp. in a follow-up investigation, among patients with symptoms of active cd and an elevated crp, 86% exhibited evidence of inflammation at colonoscopy [4, 6] . this suggests that crp has the ability to predict active mucosal inflammation. across studies, however, it has been showed that crp (in both uc and cd patients), when compared to calprotectin and lactoferrin, is less sensitive and has a lower correlation with mucosal inflammation as shown by endoscopy [6] . studies in patients with cd, with clinically inactive disease and elevated crp, have demonstrated that the latter had higher chance of relapse in the following 2 years than those with normal crp. in uc patients, crp elevation was significantly correlated with severe clinical activity and active disease by means of colonoscopy but not with histological inflammation [4] . yet, some patients have had persistent, normal levels of crp despite active disease [7] . these patients often exhibit exclusive ileal disease and low body mass index [4] . for these patients, crp will not be a useful marker for differentiation of quiescent from active disease [7] . generally, the value of crp as a predictor of relapse is controversial, with some studies considering it an accurate predictor and others not. a combination of crp and erythrocyte sedimentation rate (esr) has also been used to predict relapse in patients with cd [4] . esr is the rate at which red blood cells (rbc) migrate through the plasma over the period of 1 h [6] . the esr determination is a commonly performed laboratory test and reflects the changes in the various acute-phase proteins [1] . the test measures the distance that erythrocytes have fallen after 1 h in a vertical column of anticoagulated blood under the influence of gravity [1] . esr is not rapidly responsive to changes in clinical status, as the concentrations of many serum proteins vary and some have long half-lives [4] . esr determinations have been shown to be satisfactory monitors of acute-phase response to disease after the first 24 h [1] . therefore, esr is less suited to detect changes in disease activity, and it fails to exhibit the real clinical course, as it may take several days to decrease even when rapid clinical improvement occurs. hence, esr is an indirect, crude, but fairly rapid assessment of the general acute-phase response [1, 4, 6] . except from erythrocytes morphology and plasma constituents, such as immunoglobulins, esr is influenced by several factors including age, gender, anemia, blood dyscrasias, and pregnancy [4, 6] . although it is still widely used, especially as a biomarker of ibd activity, its usefulness has decreased as new methods of evaluating disease have developed [1, 6] . in uc there is a good correlation between esr and disease activity. in cd the esr appears to be a less accurate measure of disease activity [4] . the increase of esr in increasing disease activity correlates more with colonic disease and does not reflect the disease activity in the small bowel [4] . compared with crp, during the first 24 h of inflammation, esr will peak less rapidly, and may take days to decrease, even if the inflammation has subsided. however, crp tests are costly, frequently unavailable, and more timeconsuming to perform than the esr [1] . besides their function, which is to stop bleeding, platelets also have a recognized role in inflammatory processes [6] . therefore, platelet count correlates with disease activity in ibd [4] . the high platelet number correlates well with disease severity and may persist even after bowel resection in ibd patients [1] . increased platelet concentration in the blood circulation of ibd patients is not yet understood, but is associated with inflammation, as a nonspecific response [1] . thus, their relationship to ibd pathophysiology demands further investigation [6] . platelet count is a potential method for the distinction of ibd from infectious diarrhea [1] . in active ibd, the platelet count may be elevated, while the mean platelet volume is low [6] . causes of this occurrence are unknown, but there might be an association with the thrombopoiesis disturbance often observed in the early stages of systemic inflammatory processes, as occurs in cd and uc [1] . according to studies, in more than 30% of ibd patients, spontaneous platelet aggregation takes place independently of disease severity [1] . while the normal level varies considerably, in practice, the platelet count is routinely available in patients and, if elevated, may alert the clinician about ongoing inflammation [6] . orosomucoid or alpha-1-acid glycoprotein is an acute-phase plasma glycoprotein, which is synthesized primarily in hepatocytes [6] . the levels of circulating orosomucoid have been shown to correlate with disease activity of ibd as assessed by standard disease activity indices [4, 6] . however, the long half-life (5 days) diminishes its usefulness in practice [6] . therefore, orosomucoid does not appear to be a useful marker for screening health populations or for distinguishing between patients with inflammatory versus functional disorders [1] . the most prominent member of anti-glycan antibodies is anti-saccharomyces cerevisiae antibodies (asca) [8] , which are formed both as igg and iga antibodies [9] . they are believed to interact with mannose residues on mannan in the cell walls of s. cerevisiae. more specifically, the major antigen targeted with asca antibodies is a mannan, a cell wall glycoprotein, the 200 kda phosphopeptidomannan (ppm), of the common baker's or brewer's yeast saccharomyces cerevisiae [1, 7, 8] . in particular, the su1 strain of s. cerevisiae used in beer brewing and mannotetraose has been labeled as the most vital polysaccharide epitope within ppm [8] . the production of these antibodies is poorly understood [9] . therefore, three theories have been proposed regarding the widespread distribution of oligomannosides: the first theory is that dietary yeasts or yeasts that colonize the digestive tract activate the production of asca antibodies. the second theory concerns the epitopes shared by other microorganisms (mycobacterium species). the third one assumes that there are structural homologies between s. cerevisiae oligomannosides and oligomannosides expressed on human glycoconjugates as autoantigens or neoautoantigens [8] . asca are widely used as biomarkers for the discrimination among patients with cd versus those with uc [7, 8] . increased titers of asca were reported to identify cd with high levels of specificity (96-100%) but low sensitivity (approximately 50%), while both iga and igg antibodies are formed [7, 8] . indirect immunofluorescence (iif) and standardized enzyme-linked immunosorbent assays (elisa) are the two main methods used for the detection of these antibodies [8] . moreover, the titers of these antibodies do not seem to correlate with disease activity, and asca levels appear to be stable for long periods. in fact, patients with cd have been reported to have still high rates of asca, though their last outbreak of cd was 20 years ago and their markings on gastroscopy, colonoscopy, and histology were normal [9] . interestingly, the results vary between different populations, as the sensitivity of asca iga is lower in japanese and chinese cd patients when compared to caucasian cd patients [1] . this observation suggests that several distinct genetic determinants and/or environmental risk factors may influence the asca response [1] . yet the question still remains, do high rates of asca antibodies indicate a predisposition to ibd and are they genetic markers or not? numerous studies have been performed; in one study, asca antibodies were detected in 20-25% of healthy first-degree relatives of patients with cd. another study showed detectable levels of asca in 31% of patients long before they were diagnosed with cd. for them the maximum frequency of antibodies was recorded 36 months before diagnosis [9] . these studies indicate that asca growth happens before or during early stages of disease. hence, we assume that asca are likely produced in the context of early development of the disease, rather than as genetic markers in childhood. further study needs to be done in order to conclude whether asca antibodies serve as indicators of future disease. for now, the initial event leading to ifpe is still unclear, but it is of great interest [9] . anti-neutrophil cytoplasmic antibodies (ancas) are a group of autoantibodies, mainly of the igg type, against antigens in the cytoplasm of neutrophil granulocytes (the most common type of white blood cell) and monocytes. they are detected during a blood test in a number of autoimmune disorders but are classically associated with small-vessel systemic vasculitis, so called anca-associated vasculitides, such as wegener granulomatosis, churg-strauss syndrome, microscopic polyangiitis, and its renal-limited variant (pauci-immune necrotizing and crescentic glomerulonephritis) [6, 8, 10] . in addition, ancas are found in other chronic inflammatory disorders, most notably in rheumatoid arthritis and in uc [1] . serum levels of anca are used for the purposes of diagnosis and prognosis and in monitoring of inflammatory activity [1, 8] . in vasculitis, anca antibodies target different proteins, usually located in the lysosomes of monocytes and in the azurophilic granules of neutrophils. the most commonly employed method in use as a screening method for ancas is indirect immunofluorescence (iif) on normal peripheral blood neutrophils [8] . but for diagnostic purposes, enzyme-linked immunosorbent assay (elisa) is used in laboratories to detect ancas [8, 10] . according to iif, there are two types of fluorescence pattern: cytoplasmic granular with central interlobular accentuation (canca) and fine homogenous, rim-like staining of the perinuclear cytoplasm (or rim-accentuated fluorescence of the nuclei) designated as the panca pattern. various target antigens of atypical panca have been intensively studied in ibd patients, and so far results have been conflicting regarding their potential use in clinical practice as their sensitivity and specificity are less than 60% [8] . it is likely that the target antigen for uc-related atypical panca is a complex conformational epitope which comprises the nuclear proteins histone h1, hmg-1, and hmg-2. nevertheless, since the target antigen for uc-associated panca is yet unrecognized, sensitive and specific solidphase methods cannot be developed [8] . the sensitivity of the iif assay for uc reaches the 87.2% [8] . acca, alca, and amca (acca, anti-chitobioside carbohydrate iga antibody; alca, anti-laminaribioside carbohydrate igg antibody; amca, anti-mannobioside carbohydrate igg antibody) are novel anti-glycan antibodies to sugars on the surface of microorganisms. various studies have taken place that have associated alca and acca with cd [6] . interestingly, in a study with cd patients, a rate of 17-28% alca and acca was found, while the sensitivity ranged between 34 and 44% in cd patients who were asca negative [6, 7] . asma antibodies also seemed to be positive in 24% of patients with cd who were negative for asca and had a lower sensitivity but higher specificity when compared with asca. moreover, acca and alca antibodies have similarly exhibited low sensitivity but relatively high specificity in cd patients, compared to patients with uc [6, 7] . other studies have shown that the combination of gasca, panca, and alca is more accurate than each individual test separately in distinguishing individuals with ibd (uc or cd) from healthy controls [7] . ompc is a major outer-membrane protein, porin c isolated from escherichia coli [1, 8] . adherent-invasive e. coli has been found in ileal cd lesions, and ompc is necessary for these organisms to thrive in the gastrointestinal tract [6] . an excessive secretion of ompc antibodies has been recently reported, with elisa assay, mainly in cd patients (55%), while it was insignificant in uc patients and in healthy subjects (5-11% and 5%, respectively) [1, 8] . originally, this protein was identified as a panca cross-reactive antigen using the library of colonic bacteria [8] . anti-ompc may also be of value in aiding diagnosis of asca negative cd patients [1] . flagellin is a protein that arranges itself in a hollow cylinder to form the filament in bacterial flagellum. it is the principal substituent of bacterial flagellum and is present in large amounts on nearly all flagellated bacteria. flagellin is a common bacterial antigen which plays a vital role in mucosal immune responses, as it is present in most motile bacteria in the gastrointestinal tract and is highly antigenic [8] . antiflagellin antibodies can be directed against flagellin (anti-cbir1), pseudomonas fluorescens-associated sequence i-2 (anti-i2), and flagellins a4-fla2 and fla-x [6] . cbir1 has been identified as an immunodominant colitogenic antigen which was initially identified in the enteric flora of mice and has the ability to induce colitis in immunodeficient mice [6, 8] . cbir1 is closely related to flagellin from butyrivibrio, roseburia, thermagota, and clostridium species and appears in the clostridium subphylum cluster xiva of gram-positive bacteria [1] . cbir1 has been measured in human sera with the elisa assay and has shown a high anti-cbir1 igg reactivity (50%) in cd patients while has only exhibited minor reactivity in uc patients (5-11%) or other inflammatory git diseases [6, 8] . the sequence i2 was discovered in 2000, and this dna was homologous to the ptxr and tetr bacterial transcription factor family, which was isolated from cd colonic lesional mucosa [6, 8] . this suggests that the microorganism expressing the i2 gene product might be related to cd pathogenesis. this sequence derives from pseudomonas fluorescens [8] . studies using elisa assays have shown 30-50% iga seroreactivity against i2 in cd, 10% in uc 36-42% in indeterminate colitis, 19% in patients with other inflammatory gastrointestinal diseases, and 5% in healthy controls [1, 6, 8] . thus, the presence of anti-i2 seems to correlate with small bowel perforating disease [1] . flagellins a4-fla2 and fla-x are newly identified flagellins. according to data some cd patients are seropositive to them. a 2-year study of 252 patients with cd was carried out which indicated that 76% of these patients had small bowel cd and 59% had antibodies to a4-fla2 and 57% to fla-x. incidentally in a cross-sectional study, antibodies to flagellin a4-fla2 and fla-x were found in 29% and 26% of ibs patients, respectively [6] . anti-flagellin antibodies need further refinement before they can be used in ibd clinical practice and routine diagnostics. the cytokines are intercellular signaling polypeptides produced by activated cells. in patients with active ibd, the expression of proinflammatory cytokines is markedly increased in the intestinal mucosa, but this increase is not always translated in higher serum concentrations. some cytokines used as biomarkers are various interleukins (il-6, il-10, il-17, il-23) and tumor necrosis factor (tnf) and its receptor [1, 4] . interleukins are detected in blood serum, and the most common noninvasive method of detecting them is elisa (enzyme-linked immunosorbent assay), a widespread form of biochemical analytical test which uses a subtype and a heterogeneous solidphase enzyme immunoassay for the detection of a substance, typically an antigen, in a liquid sample [11] . some of the emerging interleukins, their sources, and biological activity are presented below [ table 2 ]. tumor necrosis factor a (tnfa) is produced by activated macrophages and monocytes. serum levels of tnfa are usually increased in patients with active ibd, though they are not consistently elevated [4] . hence, tnfa determination is of limited utility as a marker of disease activity in ibd patients [4] . nowadays, biological therapies targeting tnfα have significantly improved the management of ibd refractory to conventional therapies, thus, turning tnfα into a crucial mediator of this abnormal immune response [11] . the most widely used anti-tnfα agents are infliximab and adalimumab. infliximab is the best studied anti-tnfα agent and is currently approved in the european union for adults and children with cd and adults with uc [11] . in cd and uc, infliximab has confirmed efficacy in adults with benefits observed in both clinical remission and mucosal healing. it has also shown similar effectiveness in children with cd [11] . evidence suggests that early treatment with infliximab may improve the natural course of the disease [11] . other calprotectin is a 36 kda calcium-and zinc-binding protein that represents 50-60% of cytosolic proteins in granulocytes [6, 7] . it has antimicrobial effects and is stable in feces for 1 week [7] . calprotectin is measured by enzyme-linked immunosorbent assays (elisa), and its concentration is an indirect measure of neutrophil infiltrate in the bowel mucosa [6, 7] . it is released with cell death or activation, making it a sensitive marker of inflammation [6] . various studies identify calprotectin as a sensitive marker of activity in cd, which also correlates well with endoscopic and histological activity in uc [4, 7] . increased levels of fecal calprotectin had a positive predictive value of 81% and a negative-predictive value of 90% for relapse of uc. in patients with cd, the positive predictive value was 87%, and the negativepredictive value was 43% [6, 7] . its sensitivity and specificity of relapse in both cd and uc ranges approximately at 90% and 83%, respectively. additionally, the sensitivity and specificity for identification of ibd in adults were 93% and 96%, while, in children, the test confirmed 92% and only 76%, respectively [4, 6, 7] . despite calprotectin's high sensitivity and specificity, it ought not to be considered as a specific marker of inflammation, since increased levels are also found in neoplasia, polyps, microscopic colitis, allergic colitis, active celiac disease, infections, nonsteroidal anti-inflammatory enteropathy, increasing age, etc. [1, 6] . relatively high levels of calprotectin are noticed in the stools of normal individuals, and this data is compatible with the hypothesis that in normal individuals most circulating neutrophils migrate through the mucosal membrane of the gastrointestinal tract wall and thereby terminate their circulating life [1] . another fecal biomarker of inflammation is lactoferrin, an iron-binding glycoprotein found in neutrophil granules, which possesses antimicrobial properties [4, 6] . fecal lactoferrin is easily quantified using an elisa specific for human lactoferrin; it is resistant to freeze-thaw cycles and degradation, facilitating its use as a laboratory test [1] . multiple studies estimated that the lactoferrin test identified patients with ibd with a mean sensitivity of 80% and specificity of 82% [7] . furthermore, the protein's concentration increases in active ibd, compared to inactive ibd with specificity between 85 and 90%. fecal lactoferrin levels may rise significantly prior to a clinically evident relapse and thus may be a good marker to predict subsequent ibd flares [4] . over the past decade, there has been a renewed enthusiasm for developing noninvasive serum markers or tests to assess the presence and severity of chronic inflammation and fibrosis in chronic liver disease. although a single marker or test has lacked the necessary accuracy to predict fibrosis, different combinations of these markers or tests have shown encouraging results [12] . for hepatitis b virus, the level of hbv dna in serum or plasma probably reflects the replicative activity of hbv. various techniques for detection of hbv dna have been developed, including hybridization assays and pcr. for hepatitis c virus, the introduction of the approved immunoassay eia has reduced the incidence of hcv transmission via blood transfusion. another test available for hcv is an immunoblot assay (riba-2). both methods are of limited use, however, because a period of several weeks separates infection and seroconversion. recently, a genetic polymorphism near the il28b gene, encoding ifn-lambda-3, has been reported to be associated with an approximately twofold change in response to treatment [12] . serum proteomics are of importance for biomarker research in nonalcoholic fatty liver disease (nafld) but have not been implemented in clinical practice [13] . recently, 4-hydroxynonenal-protein adducts have been suggested as a reliable biomarker of lipid oxidation in liver diseases [14] . the current "gold standard" for liver cirrhosis detection is an invasive, costly, often painful liver biopsy. therefore, there is a need for biomarkers that could obviate biopsy in cirrhosis patients. among the noninvasive alternatives to liver biopsy, several studies have demonstrated the predictive value and a better benefit-to-risk ratio than biopsy of five combinations of simple serum biochemical markers i.e., fibromax in patients at risk of chronic liver diseases such as patients with chronic hepatitis b or c: fibrotest for the quantitative assessment of fibrosis in patients with chronic hepatitis or drug-induced liver damage; steatotest for the quantitative assessment of steatosis in fatty liver disease; actitest for the quantitative assessment of necroinflammatory activity in chronic viral hepatitis c and b and nash test for the categorical diagnosis of nonalcoholic steatohepatitis; and ashtest for the quantitative assessment of alcoholic steatohepatitis (known in the usa as hcv fibrosure, hbv fibrosure, ash fibrosure, and nash fibrosure) [15] . activin is a cytokine, which belongs to the transforming growth factor-β superfamily and is released rapidly into the circulation during inflammation [16, 17] . studies suggest an involvement of activin a not only in fibrosis but also in lipid accumulation [16] . therefore it is used as a diagnostic marker of clinical inflammation and probably plays a therapeutic role [17] . follistatin is an activin-binding protein. although elevated follistatin can be detected both in tumor tissue and in the peripheral blood, it has no benefit as surveillance biomarker for hcc development in patients with alcoholic and nonalcoholic liver disease because of its already elevated levels in the underlying liver pathologies [16] . εlevated serum concentrations) of t β4 might be a defense mechanism counteracting ongoing inflammation and fibrogenesis [18, 19] . crp is an acute-phase protein produced mainly by hepatocytes. it is produced as an acute-phase reactant predominantly in the liver, in response to a variety of acute and chronic inflammatory conditions, and is an important component of the innate immune system [1, 4, 6] . the hepatic inflammatory response of crp to injury and the production of proinflammatory cytokines are the main factors driving stellate cell activation and fibrogenesis [20] . serum high-sensitivity crp (hs-crp) is measured by immunoturbidimetry or by multiplex enzyme-linked immunosorbent assay (elisa)-based assays [20, 21] . interleukin-6 (il-6) is a proinflammatory cytokine derived from the adipose tissue. il-6 induces secretion of crp in the liver and, hence, may contribute to hepatocarcinogenesis [22] . tumor necrosis factor-alpha (tnf-α) is a proinflammatory cytokine, and emerging data leads to the conclusion that serum tnf-α levels could be used as a sensitive predictor of liver inflammation and even hepatic malfunctions [23, 24] . adiponectin is a protein, secreted from adipose tissue. high concentrations of adiponectin were associated with higher risk of hepatocellular carcinoma [25] . all these biomarkers are not used in the routine clinical practice but are expected to be integrated in future algorithms of diagnosing and treating patients with cirrhosis. regarding biomarkers of chronic inflammatory conditions in neurology, most studies have concentrated on the discovery, characterization, and validation of several highly promising individual biomarkers, but their impact on different disease stages has hardly been extensively investigated. one of the primary goals of future studies on biomarkers should therefore be the evaluation and validation of given markers according to their impact on diagnosis of subjects at risk, differential diagnosis at early clinical stages, and predication and description of disease course. future research should also focus on the development and validation of cost-effective and broadly available high-throughput technologies for biomarker quantification, as this seems the only way to address the need for highly accurate diagnosis and sufficient supervision of therapeutic strategies. multiple sclerosis (ms) is the most important chronic inflammatory disease of the central nervous system (cns) with a complex pathophysiological course that includes inflammation, demyelination, axonal damage, and repairing [26] . ms is characterized by heterogenous genetic backgrounds and immunopathogenetic subtypes, which are reflected in variable clinical disease courses and unpredictable therapeutic effects. therefore it seems more credible that a panel of different biological markers, rather than a single antibody or other biological marker, should have to be discovered to reflect the various stages of inflammation, demyelination, axonal degeneration, and remyelination [27] . despite the progress in new technologies (such as dna microarrays, real-time polymerase chain reaction (pcr), multicolor flow cytometry) and the advances in the knowledge of the ms pathogenesis, the body of scientific evidence obtained so far cannot support the existence of reliable biological markers. most of the peripheral blood markers under consideration are of little reproducibility, while biological markers in the cerebrospinal fluid (csf) are of little utility due to inability of repeated sampling [28] . biomarkers of disease activity in ms could help in predicting the disease course and treatment response, thus providing relapse monitoring, treatment guidance, and long-term outcome improvement. unseparated blood is used both for flow cytometry analysis of cellular subpopulations and for pcr studies. after coagulation, serum can be used for the measurement of soluble markers, such as antibodies and cytokines, while after separation procedures of uncoagulated samples, different cellular populations can be used for functional studies [29] . numerous cytokines (tnfa, il-12, il-17, il-23, inf-γ), cell surface markers, adhesion and migration markers, antibodies, and other markers of tissue damage have been explored as serum or csf biomarkers for disease activity, but none of them has so far the necessary validated reliability for widespread clinical use [30, 31] . this could probably be attributed to the fact that ms is not a systemic disease and therefore serum molecules might be not able to depict the immunological changes that take place in the cns. autoantibodies against myelin (anti-mog, anti-mbp, anti-plp, anti-gaga4) have been thoroughly studied, but none of them has so far demonstrated convincingly a ms-specific antibody response to a certain cns target antigen. moreover, most of the antibodies, detected in ms, are also found in other disease conditions and, to a lower extent, in healthy controls. among the aforementioned antibodies, myelin oligodendrocyte glycoprotein (mog) appears to be the most promising marker. mog was initially identified as a dominant target antigen for demyelinating antibodies in experimental autoimmune encephalomyelitis, and its role in the pathogenesis of ms still remains unresolved and controversial [32] . to date, 24s-hydroxycholesterol is the only promising biomarker related to neuronal damage in peripheral blood. 24s-hydroxycholesterol is a cholesterol metabolite specific to the brain, formed by the catalytic activity of the cytochrome p450 enzyme, and thus could serve as a marker for changes in brain cholesterol turnover caused by demyelination or neurodegeneration [33] . a moderate correlation was found between the numbers of apolipoprotein e4 (apoe4) alleles and ms disease progression, while the concentration of serum apoe was not found to be consistently abnormal in all groups of ms patients. apart from lipid transport and cholesterol homoeostasis, evidence suggests that apoe is also involved in the blood-brain barrier maintenance and in oxidative stress protection [33] . serum fluctuations of β2 microglobulin (β2-mg), the 12 kda light chain of the class i major histocompatibility complex (mhc-i) on the surface of many cells, and neopterin, a low molecular mass molecule that is synthesized from guanosine triphosphate, have been found to be good indicators of treatment effect but not of disease activity in ms. during the natural course of ms, β2-mg was found to be stable over time, and although urinary excretion of neopterin was found to be higher during clinical relapses, neopterin blood levels were not found to correlate with clinical and mri measurements. furthermore, both neopterin and β2-mg are unstable and rapidly eliminated by the kidneys, and thus these molecules can be easily detected in urine specimens [34] . urine collection is a simple and noninvasive process; however, disabled multiple sclerosis patients often have chronic urinary tract infections or asymptomatic bacterial bladder colonization-due to bladder instability-which both can negatively affect the urine biomarker results. currently, measurable urine biomarkers include neopterin, nitrate/nitrite, prostaglandin metabolites, b2-mg, immunoglobulin (ig) light chains, interleukins (il-1, il-2, sil-2r, il-6, il-8), and myelin basic protein (mbp)-like material [29] . finally, possible biomarkers in cerebrospinal fluid (csf) that reflect key pathological processes of ms in inflammation, immune th1 dysfunction, demyelination, oxidative stress, remyelination, and neuroaxonal damage are being investigated [35] . currently, the main role of csf examination in ms is to support the diagnosis of ms and to exclude other diagnoses. the most useful markers are currently the presence of two or more igg oligoclonal bands (ocbs), as well as an elevated igg index. however, numerous other csf biomarkers are under investigation, including kappa free light chains (kflc), anti-myelin antibodies, svcam-1 (soluble vascular cell adhesion molecule-1), and 24s-hydroxycholestrol [36] . at present none of them, except for the ocbs, fulfills the criteria of applicability in clinical practice and should therefore be further investigated and validated in future studies [37] . as for csf oligoclonal banding test, it is estimated to have sensitivities between 69 and 91% with specificities between 59 and 94% for the diagnosis of ms, and when combined with mri studies, both sensitivity (56-100%) and specificity (53-96%) are enhanced [38] . other biomarkers that are currently used in clinical practice in the diagnosis or treatment of ms are mentioned in table 3 . chronic inflammatory demyelinating polyneuropathy (cidp) is an autoimmune demyelinating disease of the peripheral nervous system. due to the high clinical heterogeneity and lack of a specific confirmatory biomarker, at least 14 different sets of diagnostic criteria (with varying sensitivities) have been proposed so far. ancillary diagnostic tests include the measurement of cerebrospinal fluid protein levels, nerve biopsy, electrodiagnostic testing, and treatment response [39, 40] . high titers of igm antineural antibodies to myelin-associated glycoprotein (mag), sulfatide, and gangliosides (gm1, gm2, gd1a, gd1b) are highly predictive of a chronic immune-mediated neuropathy; however, they are not specific and thus cannot be strictly associated with a definite clinical syndrome [41] . transthyretin (ttr)-a haptoglobin isoform-was highlighted in a small pilot study of csf proteome analysis in patients with cidp as a promising marker that warrants further evaluation [42] . the heterogeneity in therapeutic responses to immunoglobulin (ivig), steroids, or plasmapheresis necessitates the need for identifying biomarkers to determine the most suitable therapy and to monitor the therapeutic response in patients with cidp [43] . potential biomarkers that may help to guide therapy and treatment response with ivig include the alterations in transient axonal glycoprotein-1 (tag-1) and measurement of fcγ riib density on b cells, following infusion of ivig [40] . skin involvement is of major prognostic value in systemic sclerosis (ssc) and often the primary outcome in clinical trials. nevertheless, an objective, validated biomarker of skin fibrosis is lacking. optical coherence tomography (oct) is an imaging technology providing high-contrast images with 4 μm resolution, comparable with microscopy ("virtual biopsy") [44] . it has been also suggested that levels of adiponectin, a marker for ppar-gamma activity, correlate with skin fibrosis in systemic sclerosis [45] . the deamidated gliadin peptides (dgp) cross-linked to human tissue transglutaminase (ttg) comprise a novel neo-epitope structure (neo-ttg) for serological screening of celiac disease (cd). neo-epitope tissue transglutaminase autoantibodies have been suggested as a useful biomarker of the gluten-sensitive skin disease called dermatitis herpetiformis [46] . oxidative stress was implicated in the psoriasis disease development and may damage dna leading to keratinocytes cell death. according to an interesting study, serum 8-ohdg levels could be used as good biomarker for the early diagnosis of psoriasis and its management [47] . other serum biomarkers for psoriasis are ykl-40 (chitinase 3-like-1), which has been suggested as a biomarker for psoriasis vulgaris and pustular psoriasis, and beta-defensin-2 protein as a serum biomarker for disease activity in psoriasis which reaches biologically relevant concentrations in lesional skin [48] . of interest, plasma levels of transforming growth factor (tgf)-beta1, tissue inhibitors of metalloproteinases (timp)-1, matrix metalloproteinase (mmp)-1, and interleukin(il)-18, when analyzed separately, demonstrate an association with psoriasis severity and treatment efficacy [49] . finally, soluble tumor necrosis factoralpha receptor type 1 has been suggested as a biomarker of response to phototherapy in patients with psoriasis [50] . several cytokines/chemokines, especially in breast milk, are potential biomarkers for development of atopic dermatitis in early infancy [51] . recently, urinary eosinophil protein x has been suggested as a novel inflammatory biomarker that identifies children at risk of developing atopic disease [52] . graft-versus-host disease (gvhd), the major complication of allogeneic bone marrow transplantation, affects the skin, liver, and gastrointestinal tract. there are no plasma biomarkers specific for any acute gvhd target organ [53] . recently serum elafin has been suggested as a valuable biomarker of graft-versus-host disease of the skin [54] . in patients with polymyositis and dermatomyositis, kl-6, which is a mucin-like high-molecular-weight glycoprotein, has been suggested as a promising biomarker for use in clinical practice to assess clinical response to treatment [55] . autoimmune rheumatic diseases (ards) comprise a wide variety of chronic inflammatory disorders in which innate and adaptive immune responses lead to autoimmune-mediated tissue damage. while the etiology of ards remains unclear, multiple genetic, epigenetic, hormonal, and environmental influences appear to play a role in disease pathogenesis. the spectrum of their clinical manifestations is characterized by great diversity in terms of disease severity and the extent of organ involvement. most ards run a relapsing-remitting course, yet the pattern of continuously active disease is described in a considerable proportion of patients diagnosed with these chronic inflammatory illnesses. overall, ards affect approximately 5% of the population and result in substantial morbidity and increased mortality. furthermore, these diseases place a significant burden on public health with high financial costs [56, 57] . the identification of biomarkers that measure the underlying biologic processes in ards reliably and reproducibly has been recognized as a growing need in rheumatology, but given the complex pathogenesis of these disorders, heterogeneous clinical manifestations, and varying rates of disease progression among patients, it is reasonable to assume that a particular biomarker may reflect only one specific aspect, rather than all aspects of the disease course at any given time. therefore, some biomarkers provide prognostic information regarding severity of the disease, whereas others predict response to therapy optimizing risk/benefit assessment in individual patients. likewise, biomarkers might predict or quantify the risk of ards in individuals or populations, and others could establish or confirm the diagnosis of a specific ard. this section will only focus on biomarkers detected through blood tests that have established clinical utility in major ards. rheumatoid arthritis (ra) is an ard that primarily affects the joints and is associated with progressive functional disability, systemic complications, high socioeconomic costs, and a guarded prognosis. while the exact prevalence across the entire population is unknown, available data suggest that it affects about 1% of the population, making it one of the most common ards [58] . ra involves a complex interaction among genotype and environmental triggers, leading to a breakdown of immune tolerance with autoantibody production and synovial inflammation in a characteristic symmetric pattern. distinct mechanisms regulate inflammation and matrix destruction, including damage to bone and cartilage [59] . the diagnosis of ra remains clinical and is based on several criteria, including physical symptoms, joint radiographs, and serological tests [60] . recently, emerging data suggest that a preclinical period precedes the onset of clinically apparent ra, which is characterized by the presence of abnormalities in disease-related biomarkers. during this period many genetic and environmental risk factors are likely to act to initiate or propagate autoimmunity [61] . the main clinically useful biomarkers in patients with ra are rheumatoid factors (rf) and antibodies to citrullinated peptides (acpa) for diagnosis and prediction of functional and radiographic outcomes. these autoantibodies, when appearing during the preclinical period, may predict the disease onset [61] . other useful laboratory biomarkers are erythrocyte sedimentation rate (esr) and c-reactive protein (crp). susceptibility to ra is clearly defined by a pattern of inherited genes, with the human leukocyte antigen (hla)-drb1 locus being the most important biomarker, particularly in patients who are rf or acpa positive. however, other risk alleles that consistently aggregate functionally with immune regulation, including cytokine promoters and genes involved in t-cell stimulation, activation, and functional differentiation, contribute to susceptibility and disease severity [59] . rf is an antibody directed against the constant (fc) portion of igg. the key pathogenic markers in ra are igm and iga rfs. they are found in 75-80% of ra patients (table 4) at some time during the disease course. high titer igm rf is relatively specific for the diagnosis of ra in the context of a chronic symmetric polyarthritis and was, for decades, the only serologic criterion widely used in the diagnosis of ra. nevertheless, rfs may also be present in other ards and chronic infections (table 4) . rfs in ra patients are distinguished from rfs in healthy individuals in that they exhibit affinity maturation [62] . rf has little predictive value in the general population, because the disease prevalence is relatively low. rf may have some prognostic value with regard to disease manifestations and activity, as well as the severity of joint erosions. seropositive ra (i.e., disease associated with a positive rf test) is often correlated with more aggressive joint disease and is more commonly complicated by extra-articular manifestations than seronegative ra [62] . rheumatoid nodules and vasculitis occur almost exclusively in seropositive patients. a positive rf test at the initial evaluation is associated with more destructive joint pathology. it has also been proposed that the rate of formation of new erosions among seropositive patients correlates with the rf titer [62] . the presence of rf increases the likelihood of a clinically significant response to rituximab which is a monoclonal antibody against the cd20 antigen found on the surface of b lymphocytes after failure of tumor necrosis factor (tnf)-alpha inhibitor therapy. finally, the presence of rf, particularly at high titers, may antedate the clinical development of ra [61, 63] . acpa are directed against citrullinated protein epitopes and are detected by use of elisa for antibodies against synthetic cyclic citrullinated peptides. the sensitivity of acpa assays for ra varies from about 50 to 75%, depending upon the assay and study population, while specificity of acpa for ra is relatively high, usually >90% (table 4) . acpa-positive patients with early ra are at increased risk of progressive joint damage, while acpa testing may predict erosive disease more effectively than rf. the presence of acpa is also predictive of more rapid radiographic progression [62] . positive acpa testing also appears to predict an increased risk for radiographic progression in patients with early oligo-or polyarthritis who are igm rf negative. a decrease in acpa titers can be seen in patients treated effectively, particularly if treated early with nonbiologic or biologic disease-modifying antirheumatic drugs (dmards), but is less frequent and of a lesser magnitude than the decrease in igm rf. acpa may be detectable months or even years before the development of ra, and the proportion of individuals who are seropositive for acpa increases progressively until clinical onset of disease [61, 63] . finally, this biomarker is useful in the differential diagnosis of early polyarthritis, because of the relatively high specificity for ra of these autoantibodies. the presence of acpa is highly predictive of future development of ra with positive predictive values of >90% in most studies [61, 63] . the rate at which erythrocytes fall through plasma, the esr, depends largely upon the plasma concentration of fibrinogen and can be influenced by the size, shape, and number of red cells, as well as by other plasma constituents. thus, results may be imprecise and sometimes misleading. despite these shortcomings, an elevated esr in patients with early ra is predictive of greater radiographic joint damage in subsequent years despite treatment with conventional dmards. esr values tend to correlate with disease activity in ra as well as disease severity and may be useful for monitoring therapeutic response. crp has been recommended as an objective measure of disease activity in ra. unlike the esr, crp can be measured using stored serum samples; it is independent of the hemoglobin concentration and can be performed in automated serum analyzers. radiographic damage is significantly more likely to progress when crp and esr are elevated, irrespective of the presence or absence of rf and irrespective of therapeutic intervention. elevations of both esr and crp are stronger indications of radiologic progression than crp alone. however, a wide variation in the relationship between the degree of radiographic change and cumulative crp was noted in several studies between patients, particularly those with low crp levels. cytokines are small proteins that regulate the immune system and participate in intercellular communications. there is more information on the role of cytokines in ra than in any other ard which has led to clinical trials with several novel agents designed to interrupt the cytokine network of this disease. anti-cytokine therapy has shown clear evidence of clinical efficacy, especially with tnf-alpha-directed therapeutic approaches. overexpression of certain cytokines, such as il-2, il-6, il-8, il-17, il-21, tnf-α and granulocyte-macrophage colony-stimulating factor have been reported in ra. however, discrepancy in the results of several studies, differential cytokine levels in sera and synovial fluid, analytical variability among immunoassays, and certain limitations of cytokine measurements [64] preclude their use as reliable biomarkers in everyday clinical practice. systemic lupus erythematosus (sle) is the most heterogeneous ard with more than 100 autoantibodies found in patients and a disease spectrum ranging from mild symptoms to life-threatening multi-organ manifestations. the hallmark characteristics of sle, including production of autoantibodies, deposition of immune complexes in tissues, and excessive complement activation, are generally thought to be consequences of immune dysregulation. owing to its complex etiology and pathogenesis, diverse clinical presentation, and unpredictable course, sle remains one of the greatest challenges in the field of rheumatology [65] . the genome-wide association study (gwas) approach has accelerated the discovery of genetic variations contributing to sle. over 50 loci associated with sle susceptibility have been identified, and most encode gene products participating in the key pathways relevant to sle pathogenesis, including those encoding the early components of the complement system, cytokines, chemokines, loci that mediate signaling transduction in b and t cells, as well as variants implicated in immune complex clearance. yet, dysregulation of type i interferon (ifn) appears as a central driver of sle pathogenesis [66, 67] . variants of fcgr2a also represent significant risk factors for the disease, whereas the fcg3a-v/f158 polymorphism has a substantial impact on the development of lupus nephritis [68, 69] . individuals who develop sle appear to have an initial preclinical stage of "benign autoimmunity" which is characterized by antinuclear antibody (ana) positivity and detection of several autoantibodies (anti-ro/ssa, anti-la/ssb, anti-phospholipid). this phase develops into a more aggressive stage of "pathogenic autoimmunity" characterized by the presence of antibodies targeting double-stranded dna (dsdna), the smith (sm) antigen, and ribonucleoproteins (rnp) that in turn rapidly evolves into clinically apparent disease and tissue inflammation [63] . while certain biomarkers such as anti-dsdna and anti-sm are elevated relatively shortly before sle onset, the relationship with preclinical autoimmunity remains uncertain for anas which have been shown to be present at titers of >1/40 in up to 27% of subjects, most of whom will never develop sle or other ard [63] . although the american college of rheumatology (acr) developed classification criteria for sle (published in 1982 and revised in 1997), these criteria are often cited to support a lupus diagnosis; yet this approach is problematic in clinical practice. traditionally, determination of autoantibodies (table 4 ) such as ana, antiextractable nuclear antigen antibodies (anti-ro/ssa, anti-la/ssb, anti-rnp, and anti-sm), and anti-dsdna is used in diagnosing and monitoring sle. anas are present in virtually all sle patients, whereas anti-dsdna and anti-sm are highly specific for the disease (table 4) . nevertheless, there are considerable drawbacks to the use of these immunologic markers [65] . recently, cell-bound complement activation products have been proposed as more sensitive biomarkers for sle diagnosis compared to other autoantibodies [67] , but larger studies are needed to provide a more definite confirmation of their validity. currently, disease activity in sle is frequently assessed using composite indices which include a variety of clinical and laboratory parameters. laboratory measures of complement and autoantibodies are components of most disease indices. several studies have been conducted to identify the associations of various autoantibodies, particularly anti-dsdna and complement proteins (including c3 and c4 as well as activation products) with sle disease activity. the results, however, are inconsistent, and such uncertainty may also confound the assessment of disease activity with the widely used disease indices. therefore, the value of conventional tests measuring serum complement and autoantibodies, as biomarkers for sle disease activity, is being revisited. several candidate biomarkers have emerged recently; alteration of the b cell subpopulation (namely, the increase in cd27 high plasma cells), cell-bound complement activation products, ifn-inducible genes expression and/or serum levels of ifn-inducible chemokines, serum levels of b lymphocyte stimulator, cytokines (il-6, il-10, il-16, and il-18), and anti-nucleosome antibodies may be valuable biomarkers for monitoring disease activity [67] . sle can affect practically any tissue and organ. nevertheless, not all organs are affected simultaneously, and involvement of a specific organ will not necessarily be manifested in the same manner in all patients. lupus patient care would benefit immensely from biomarkers that could determine and/or predict organ-specific disease. among the numerous manifestations of sle, renal involvement is one of the most common, and it continues to cause significant morbidity and mortality. laboratory biomarkers such as creatinine clearance, proteinuria, urine sediment, serum c3 and c4, and anti-dsdna have for decades been used to follow the onset, course, and severity of lupus nephritis [65] , yet, it is generally recognized that these measurements are inadequate. current efforts are focused on identification of more sensitive and specific biomarkers to diagnose and monitor renal disease in sle. anti-nucleosome antibodies may be more sensitive and offer better diagnostic performance than anti-dsdna for active disease, especially nephritis, in sle patients. other recent studies demonstrate a strong correlation between the presence of anti-c1q antibodies and lupus nephritis and suggest that anti-c1q determination may serve as a biomarker to monitor renal involvement and/or predict renal flares. other promising biomarkers include the serum neutrophil gelatinase-associated lipocalin which might predict renal relapses, yet longitudinal studies of adult lupus are needed [67] . many patients with sle experience a wide range of neuropsychiatric (np) manifestations that result predominantly from immune-mediated damage of the central nervous system (cns). while npsle is a common manifestation, its diagnosis is extremely difficult due to lack of reliable biomarkers. because autoantibodies are clearly involved in tissue damage in other organs, autoantibodies reactive to cns antigens naturally become the focus of the investigation of npsle pathogenesis and the pursuit for reliable biomarkers. stroke in patients with sle is likely to reflect thrombosis due to antiphospholipid antibodies. in addition, recent studies provide convincing evidence that a subset of anti-dsdna antibodies cross-react with a pentapeptide consensus sequence that is present in the extracellular domain of the n-methyl-d-aspartate (nmda) receptor. nmda receptors bind the neurotransmitter glutamate expressed by neurons throughout the forebrain and particularly at the hippocampus. if such cross-reacting autoantibodies enter the brain upon transient breach of the blood-brain barrier, they may bind to antigens expressed in different regions of the brain and induce non-inflammatory neuronal injury resulting in various neurologic and psychological changes [67] . antibodies to ribosomal p proteins (anti-p) were initially considered extremely promising biomarkers for the diagnosis of lupus psychosis and of mood disorders. yet, it was demonstrated that anti-p antibody testing has negligible diagnostic utility for npsle overall or for these particular manifestations [70] . testing for anti-p antibody is not useful in excluding disease-mediated psychosis or mood disorder with enough certainty, since more than 60% of cases are false negative. also, a false-positive rate of ∼20% militates against the dependence on this laboratory test for diagnosing psychiatric disorders in lupus patients [70] . vasculitis comprises a heterogeneous group of ards that is characterized by inflammatory destruction of blood vessels which become liable to occlude or rupture. depending on the size, distribution, and severity of the affected vessels, vasculitis can result in clinical syndromes that vary in severity from a minor self-limited rash to a life-threatening multisystem disorder. the most important primary systemic vasculitis syndromes are granulomatosis with polyangiitis (gpa, formerly known as wegener's granulomatosis) and microscopic polyangiitis (mpa) which are associated with circulating autoantibodies to neutrophil cytoplasmic antigens (anca). it has been suggested that they be grouped together as anca-associated vasculitis because of their histologic similarities, the absence of immune deposits in involved tissues, the potential contribution of anca to their pathogenesis, and their similar responses to immunosuppressive therapy. renal involvement occurs in 70% of affected patients and is manifested as rapidly progressive glomerulonephritis; it results in either death or end-stage renal failure within 2 years in more than 40% of patients. mpa and gpa were once considered life-threatening ards, but immunosuppressive therapy has substantially improved the survival of affected patients [71, 72] . the most useful biomarkers for diagnosis of anca-associated vasculitis include antibodies to proteinase-3 (pr3) or myeloperoxidase (mpo) which are highly specific for gpa and mpa, respectively (table 4) . inflammatory markers such as esr and crp exhibit only modest sensitivity for active early vasculitis in untreated patients [71, 72] . all of these biomarkers are problematic for use in assessing disease activity in patients with established diagnoses. many relapses of gpa or mpa are accompanied by increases in anti-pr3 or anti-mpo titers; yet these markers do not appear to have the same predictive value that they do before treatment, making their use controversial. laboratory biomarkers such as creatinine clearance, proteinuria, and urine sediment are thought to possess very high sensitivity for active glomerulonephritis, that is, a normal urinalysis is regarded as ruling out glomerulonephritis. similarly, the presence of red blood cell casts is regarded as having high specificity, albeit low sensitivity for glomerulonephritis. nevertheless, once renal damage has occurred due to glomerulonephritis, proteinuria, hematuria, and even red blood cell, casts may persist without evidence of progressive kidney disease. among many markers of inflammation, angiogenesis, tissue damage, and repair that were measured in gpa and mpa patients, the most promising include mmp-3, tissue inhibitor of metalloproteinase-1, and cxcl13 (b-cell attracting chemokine 1) [71, 72] . metabolomics is a novel and powerful technique for studying biological systems that allows for the analysis of the specific response of organisms to environmental stimuli. this new approach belongs, along with genomics, proteomics, and transcriptomics, to the family of "-omics" sciences. it is based on non-hypothesis-driven scanning platforms for identifying biomarkers and profiling the patients. thus, the fundamental rationale in metabolomics is that perturbations caused by a disease in a biological system will lead to correlated changes in concentrations of certain metabolites. this procedure has become feasible only recently with the advent of new high-throughput technologies, including mass spectrometry and nuclear magnetic resonance. comparison of patients with active ra and those in remission provided different baseline metabolic profiles, suggesting that efficacious treatment may affect biological changes for improved metabolic profiles. in sle, the disease influences the metabolite profile and particularly energy, amino acid, lipid, and purine metabolism [73] . proteomic approaches include gel-based methods such as two-dimensional difference gel electrophoresis (2d dige) and modern mass spectrometric techniques to identify encoded proteins that may better reflect cell function and disease. most profiling studies in ards have been conducted in ra. using high-throughput mass spectrometric techniques, these studies have uncovered about 33 different proteins that are differentially expressed in ra, of which 3 (serum amyloid a, superoxide dismutase, and triose phosphate isomerase) are of particular interest as their elevations in plasma have been reported in multiple studies. proteomics can also yield insights into the molecular pathways impacted by therapy. a recent example is the identification of the nuclear factor-kappa b pathway as being differentially expressed in ra patients treated with anti-tnf-alpha agents [74] . transcriptomic profiling using dna microarrays has been applied to the study of several ards. the analysis of antibody repertoires with antigen microarrays found that those ra patients who had antibodies to citrullinated peptides in which peptidylarginine has been converted to peptidylcitrulline were more prone to develop severe disease. in another sle study, the investigators developed antigen microarrays including several sle-related antigens, and they found clusters of antibody reactivity associated to glomerulonephritis and overall disease activity [75] [76] [77] . biomarker discovery and development is a rapidly growing field in rheumatology, which is nevertheless fraught with several limitations. important amendments to the translational research enterprise are necessary to address these challenges. hence, imperative alterations should include rigorous design for studies of biomarkers that would allow valid interpretation of their clinical utility. in particular, for studies of biomarkers for diagnosis, greater attention to potential confounders would help ensure accuracy. when examining biomarkers of disease activity, emphasis should be placed on evaluating these markers longitudinally. in research regarding biomarkers of prognosis, longitudinal design and the use of validated outcome measures would provide reliable results in order to enable use in clinical decision-making process of novel biomarkers that outperforms currently available tests. finally, the development of novel high-throughput assays might hold tremendous potential for shaping how ards are diagnosed, prognosticated, and managed clinically over the coming years. personalized medicine is the best way to integrate new biotechnologies into medicine for improving the understanding of pathogenesis of diseases and management of patients. development of personalized medicine is closely linked to biomarkers, which may serve as the basis for diagnosis, drug discovery, and monitoring of diseases. biomarker discovery is an ongoing process, with translation being tested de novo in every single study, providing us with the opportunity to revise our knowledge of the complex scheme of human physiology and pathophysiology. an ideal biomarker must be specifically associated with a particular disease or disease state and be able to differentiate between similar physiological conditions. a rapid, simple, accurate, and inexpensive detection of the relevant marker should be available, together with a measurable and standard baseline as a reference point. in the active search for new biomarkers, many potential candidates can be considered side by side, allowing many failures but a few winners. the traditional identification of biomarkers as an observational side product of clinical practice is increasingly turning into an industrialized process of biomarker discovery, supported by standardized paradigms of biomarker validation and translation from bench to bedside. in recent years, significant advances in genomics increasingly impact disease detection, prognosis, prediction, and efficient patient stratification. moreover, genomic biomarkers greatly influence the development of personalized medicine by providing treatments adapted to the genetic characteristics of the individual patient's disease. biological markers in inflammatory bowel disease: practical consideration for clinicians quantitative proteomic approaches in biomarker discovery of inflammatory bowel disease: biomarkers for ibd and colitic cancer new serological biomarkers of inflammatory bowel disease review article: biological activity markers in inflammatory bowel disease intestinal microbiota: a source of novel biomarkers in inflammatory bowel diseases? biomarkers in inflammatory bowel disease: current practices and recent advances the utility of biomarkers in the diagnosis and therapy of inflammatory bowel disease asca: genetic marker, predictor of disease, or marker of a response to an environmental antigen? advanced atlas of autoantibody patterns cytokine networks in ulcerative colitis proinflammatory cytokines and il-10 in inflammatory bowel disease and colorectal cancer patients proteomic analysis of liver diseases: molecular mechanisms and biomarker discovery serum proteomics for biomarker discovery in nonalcoholic fatty liver disease 4-hydroxynonenal-protein adducts: a reliable biomarker of lipid oxidation in liver diseases fibromax: towards a new universal biomarker of liver disease? activins and follistatins: emerging roles in liver physiology and cancer activin and related proteins in inflammation: not just interested bystanders from proteomic multimarker profiling to interesting proteins: thymosin-β4 and kininogen-1 as new potential biomarkers for inflammatory hepatic lesions thymosinbeta4: a novel assessed biomarker of the prognosis of acute-on-chronic liver failure patient? obesity and steatosis influence serum and hepatic inflammatory markers in chronic hepatitis c biomarkers of inflammation and coagulation are associated with mortality and hepatitis flares in persons coinfected with hiv and hepatitis viruses inflammatory and metabolic biomarkers and risk of liver and biliary tract cancer obesity, inflammation, and liver cancer soluble inflammatory markers as predictors of hepatocellular damage and therapeutic response in chronic hepatitis c serum adiponectin level and different kinds of cancer: a review of recent evidence immunological markers in multiple sclerosis: tackling the missing elements multiple sclerosis: disease biomarkers as indicated by pathophysiology biological markers of the inflammatory phase of multiple sclerosis development of biomarkers in multiple sclerosis biomarkers of disease activity in multiple sclerosis body fluid biomarkers in multiple sclerosis development of biomarkers for multiple sclerosis as a neurodegenerative disorder biological markers in csf and blood for axonal degeneration in multiple sclerosis beta-2 microglobulin and neopterin as markers of disease activity in multiple sclerosis cerebrospinal fluid biomarkers in multiple sclerosis analyses of cerebrospinal fluid in the diagnosis and monitoring of multiple sclerosis biological indicators of the neurodegenerative phase of multiple sclerosis accuracy of diagnostic tests in multiple sclerosis-a systematic review current diagnosis of cidp: the need for biomarkers biomarkers in the diagnosis and therapy of cidp: proceedings of the biomarkers faculty meeting how useful are antineural igm antibodies in the diagnosis of chronic immune-mediated neuropathies? candidate biomarkers of chronic inflammatory demyelinating polyneuropathy (cidp): proteome analysis of cerebrospinal fluid potential biomarkers for monitoring therapeutic response in patients with cidp virtual skin biopsy by optical coherence tomography: the first quantitative imaging biomarker for scleroderma levels of adiponectin, a marker for ppar-gamma activity, correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker? neo-epitope tissue transglutaminase autoantibodies as a biomarker of the gluten sensitive skin disease-dermatitis herpetiformis studies on serum 8-hydroxy guanosine (8-ohdg) as reliable biomarker for psoriasis ykl-40 (chitinase 3-like-1) as a biomarker for psoriasis vulgaris and pustular psoriasis plasma tgf-beta1, timp-1, mmp-1 and il-18 as a combined biomarker of psoriasis activity soluble tumour necrosis factor-alpha receptor type 1 as a biomarker of response to phototherapy in patients with psoriasis cytokine biomarker candidates in breast milk associated with the development of atopic dermatitis in 6-month-old infants urinary eosinophil protein x: a novel inflammatory biomarker that identifies children at risk of developing atopic disease elafin is a biomarker of graft-versus-host disease of the skin plasma transforming growth factor beta1 as a biomarker of psoriasis activity and treatment efficacy kl-6: a serological biomarker for interstitial lung disease in patients with polymyositis and dermatomyositis estimates of the prevalence of arthritis and other rheumatic conditions in the united states. part i estimates of the prevalence of arthritis and other rheumatic conditions in the united states. part ii the lifetime risk of adult-onset rheumatoid arthritis and other inflammatory autoimmune rheumatic diseases the pathogenesis of rheumatoid arthritis rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative can rheumatoid arthritis be prevented? autoantibodies in rheumatoid arthritis: rheumatoid factors and anticitrullinated protein antibodies pathogenesis and prevention of rheumatic disease: focus on preclinical ra and sle cytokines as biomarkers in rheumatoid arthritis systemic lupus erythematosus advances in understanding the role of type i interferons in systemic lupus erythematosus biomarkers for systemic lupus erythematosus role of the fcgamma receptor iia polymorphism in susceptibility to systemic lupus erythematosus and lupus nephritis: a meta-analysis fc gamma riiia-sle meta-analysis investigators. the fc gamma riiia-f158 allele is a risk factor for the development of lupus nephritis: a meta-analysis accuracy of anti-ribosomal p protein antibody testing for the diagnosis of neuropsychiatric systemic lupus erythematosus: an international meta-analysis biomarkers in vasculitis serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ancaassociated vasculitis metabolomics in rheumatic diseases: the potential of an emerging methodology for improved patient diagnosis, prognosis, and treatment efficacy proteomics in rheumatology: the dawn of a new era antigen microarrays for the study of autoimmune diseases serological markers of inflammatory bowel disease il-6: from its discovery to clinical applications key: cord-0016212-111rdyo2 authors: schmidlin, patrick r.; dehghannejad, mandana; fakheran, omid title: interleukin-35 pathobiology in periodontal disease: a systematic scoping review date: 2021-03-20 journal: bmc oral health doi: 10.1186/s12903-021-01515-1 sha: 895b65fe6782b4527255f0a0915bd2d1dd45f237 doc_id: 16212 cord_uid: 111rdyo2 background: interleukin (il)-35 is a novel anti-inflammatory cytokine that is produced by regulatory t cells. il-35 mediates immunological functions and plays a protective role in several diseases such as asthma and rheumatoid arthritis. however, the role of il-35 in gingivitis and periodontitis remains unclear. the aim of this study was to systematically review the literature and collecting the available evidence regarding the role of il-35 in pathogenesis of periodontal disease. methods: a systematic search of electronic databases including medline, google scholar, cochrane library, web of science, and scopus was conducted in november 2020 to identify studies addressing the interleukin-35 pathobiology in periodontal disease. the identified studies were subjected to pre-identified inclusion criteria. the retrived papers were assessed by the authours independently and consensus was reached in cases where disagreement occurred. articles written in languages other than english, case reports, letters to editors, conference abstracts, theses, and dissertations were excluded from the review. results: a total of 176 possibly relevant articles were identified through the search strategy. finally, 15 papers which met the criteria of eligibility were included in this review by consensus. the included articles were classified based on their design and level of evidence.three subclinical study, ten cross sectional investigation and two randomized clinical trials constituted the final set of studies in this review. at preclinical level, il-35 showed inhibitory characteristics regarding alveolar bone resorption of animal periodontitis models. the results of observatory human studies confirmed the presence of high levels of il-35 in saliva, gcf, serum, and gingival biopsies of patients suffering from inflammatory periodontal disease. moreover, two included clinical trials showed that non-surgical periodontal therapy could downregulate il-35 production in chronic periodontitis patients. conclusion: interleukin-35 has an undeniable role in pathobiology of inflammatory periodontal disease. further well-controlled studies are needed to better elucidate the functional pattern of il-35 in pathogeneisis of gingival and periodontal disease. supplementary information: the online version contains supplementary material available at 10.1186/s12903-021-01515-1. studies revealed that periodontitis is the sixth most common disease globally. the prevalence of periodontitis is high, and approximately 10% of the global population has been affected by severe periodontitis [1] . as such, it affects the tooth supporting tissues leading to attachment loss and-if left untreated-to tooth loss. it represents a multifactorial disease primarily caused by pathogenic bacteria, which triggers and maintains a host-mediated process [2, 3] . the interplay between the imbalance within the microbial community and a dysregulated host immune response-known as dysbiosis-including some systemic and environmental modifying risk-factors leads to destruction of periodontal tissues, i.e., the periodontal ligament and alveolar bone [4] . several studies have documented that cell-produced cytokines play a critical role in this process [5, 6] , as they up-and down-regulated the local cell environment. different types of cells, such as fibroblasts, macrophages, and lymphocytes secrete a plethora of cytokines, especially during the active phases of periodontitis. among the first cytokines discovered within the context of periodontal inflammation were the interleukin-1 (il-1), il-6, and tumor necrosis factor (tnf) families [7] . these cytokines are primarily recognized as proinflammatory cytokines exhibiting mainly pleiotropic effects on lymphocyte promotion and tissue destruction [8] . further studies have documented some important related cytokines such as il-1 receptor antagonist (il-1ra), il-10, il-4, il-11, and transforming growth factorbeta [tgfb] with anti-inflammatory functions within the destructive processes [9] . it has been shown that both pro-inflammatory and anti-inflammatory cytokines play a critical role in the pathogenesis of periodontitis, thereby influencing destruction, repair, and remodeling of periodontal tissues [10, 11] . in this context, il-35 is a rather newly discovered cytokine with shown antiinflammatory capacities [12] . it is mainly produced by regulatory t cells (treg) and formed from a heterodimer of il-12p35 subunits and epstein-barr virus induced gene 3 (ebi3) [13] . the binding of il-35 and its receptors, il-12rβ2, gp130, or a heterodimer of il-12rβ2:gp130 activates the janus kinase-signal transducer and activator of transcription (jak-stat) pathway and induces immunosuppression [14] .in this regard, in a gene polymorphism analyses study among japanese subjects, the authors reported that the frequencies of variant alleles of il-12rβ2 were significantly higher in aggressive periodontitis patients as compared with healthy controls or chronic periodontitis patients. it has been documented that il-35 mainly suppresses proliferation of t cells by blocking mitosis in g1 phase without provoking apoptosis [15, 16] . it can also induce the development of il-35-producing t cells (itr35), which are subsets of regulatory t cells (treg) [16] and inhibits il-17 production by inhibiting t-helper-17 (th17) cell induction and, thus, plays a protective role in several diseases, whose pathogenesis is closely associated with the th17/treg imbalance, such as experimental colitis, asthma, inflammatory bowel disease, and rheumatoid arthritis [17] [18] [19] [20] . il-35 is also secreted by b cells and induces the conversion of human b cells into breg cells to inhibit antimicrobial immunity through production of il-35. this cytokine may also play a crucial role in the immune suppression function of breg cells [21, 22] . moreover, a recently published experimental study's findings revealed that il-35 prevents bone loss in rheumatoid arthritis disease [23] . il-35 has also been detected in periodontal tissues, gingival crevicular fluid (gcf) of different types of periodontal disease [24] [25] [26] and some evidence supports its important role in the pathogenesis of periodontitis [27, 28] . the itr35 cells also negatively regulate various immune responses mediated by th1, th17, and cytotoxic t lymphocyte cells. it has been documented that all of these immune cells are involved in pathogenesis of periodontitis [12, [29] [30] [31] [32] . previously it has been documented that through the production of il-10, il-35, and transforming growth factor b (tgf-b), breg cells suppress immunopathology by prohibiting the expansion of pathogenic t cells and other pro-inflammatory lymphocytes [33] . recently the results of a human study approved that the frequency of breg cells was positively correlated to serum levels of il10, il35, and tgfβ1 in peripheral blood samples of periodontitis patients [34] . periodontitis may be viewed as an infectious disease with a number of specific characteristics [35] . pathogens of the subgingival microbiota can interact with host tissues and activate related immune responses. recent studies suggested that il-35 may promote infectious tolerance in some ways [21, [36] [37] [38] . in this regard the results of an animal study showed that the mice which did not express il-35 displayed a strikingly improved resistance to infection with the intracellular bacterial pathogen salmonella typhimurium [37] . these experimental samples showed superior containment of the bacterial growth and prolonged survival both after primary infection and upon secondary challenge after vaccination, compared to control group. this ability of il-35 should be also considered in the way of describing the role of il-35 in pathogenesis of periodontal disease. to the best of our knowledge, there is no systematically collected evidence in the literature in this regard, especially in the field of periodontology. hence, this study aimed to systematically review the existing literature regarding the role of il-35 in pathogenesis of periodontal disease. a literature review using a scoping review approach, which is commonly used to elaborate and better understand the available evidence on a given topic, identify gaps in the literature and assess the need for further research [39] . arksey and o'malley explained the required methodological framework stages for a scoping review [39] and served as the methodological basis for this work. the main modified focus question of this study was as follows: ''what is known from the available literature about the role of il-35 in the pathogenesis and controlling of periodontal disease?'' . our literature search applied a wide range of computerized databases, including medline, google scholar, cochrane library, web of science, and scopus. the following search terms included relevant keywords in various forms and combinations are used in the study: periodontitis, gingivitis, and interleukin-35 and interleukin-35 receptors (il-12rβ2, gp130). full details of the search strings used in this review are shown in additional file 1. it should be noted that the search syntax was adapted to each database based on their specific instructions. retrieved titles and abstracts obtained from the search, along with the full texts (if necessary), were assessed by two independent reviewers (of and md). any inconsistencies in this regard were solved by a constructive discussion. after completing this initial search, the reference lists of the selected articles were also reviewed to identify additional relevant studies not found during the original electronic search. all letters, narrative reviews, and duplicate articles were excluded. the search strategy was not restricted by the publication date. hence, all relevant evidence that met the inclusion criteria by november 20, 2020, was assessed. the process of selection articles for the final review is illustrated in fig. 1 . two authors (of and md) searched the databases independently. the two sets of articles were then compared. disagreements were resolved through discussion or, if necessary, by including a third researcher (ps) to make the final decision. duplicate articles were also excluded from the review. one researcher (of) extracted the data, and another author (ps) checked its accuracy and completeness. the final set of selected articles and the relevant data based on the study research question is summarized in table 1 . articles written in languages other than english, studies focusing on medically compromised individuals, case reports/case series, letters to editors, opinions, conference abstracts, commentaries, theses, and dissertations were excluded from the review. a total of 176 possibly relevant articles were identified through the search strategy. after completing the screening titles and abstracts and eliminating duplicates, 23 studies were retrieved, and their full-text versions were collected for further assessment. a manual search for the reference lists of the 23 studies revealed no additional qualifying paper. out of these 23 studies were subjected to full article review eligibility, and eight articles were excluded. the reasons for excluding full-text articles are as follows: the english versions of the full-text articles were not available (n = 3), the articles were not suitable for our research question (n = 2), and articles consisting of literature reviews without any original data (n = 3). finally, 15 articles were found to be eligible and could be included in this review. additional details on the data search are shown in the flowchart (fig. 1) . we classified and presented the results of fifteen articles included in this review based on their design and level of evidence [40] . in the first subchapter, we presented the results of two in vitro cell studies and one animal (preclinical) study. in the second subchapter, including the most articles, we described the results of ten crosssectional studies. finally, in the last subchapter, the results and outcomes of two human randomized control trials (rct) regarding the effect of non-surgical periodontal treatment on levels of il-35 were presented. the detailed characteristics of included studies are summarized in table 1 . in the first in vitro cell study by shindo et al., who used human periodontal ligament cells stimulated with il-17a [41] . in this research, the authors reported that il-35 produced from regulatory t cells might inhibit progression of periodontitis by decreasing il-17a-induced levels of il-6 and il-8. another in vitro cell study, using cultured mouse monocyte line raw cells, osteoclastogenesis function of il-35 was assessed [42] . they stimulated rankl-treated raw cells with or without il-35 to determine whether il-35 affects rankl-dependent osteoclastogenesis. the results showed that il-35 and rankl induced osteoclastogenesis synergistically. in the last study in this category, an interventional animal study was conducted on mice with ligature-induced periodontitis [43] . in this study, the researchers evaluated the effect of locally or systemically administered il-35 on alveolar bone resorption measured by micro-computed tomography and scanning electron microscopy. the results of this project revealed that il-35 inhibited alveolar bone resorption in periodontitis mice. furthermore, il-35 induced less detection of th17 lymphocytes and down regulation of th17-related cytokines, along with higher detection of treg lymphocytes and upregulation of treg-related cytokines in periodontitis-affected tissues. the authors of this article also reported that in periodontitis-affected mice treated with il-35, the mrna and protein levels of rankl were significantly lower and conversely the expressed and secreted levels of opg were significantly higher comparing to mice without il_35 treatment. ten of the studies included in this review were mainly association studies with a cross-sectional design. almost all of these studies compared the level of il-35 in various tissue samples, such as peripheral blood, serum, gingival crevicular fluid (gcf), and periodontal tissue biopsies in periodontitis patients with a healthy control group. among these, nine studies were conducted among systemically healthy individuals, and only one study was conducted among patients with type 2 diabetes mellitus. the study results among patients with diabetes showed that neither type 2 diabetes mellitus nor chronic periodontitis differentially has affected serum levels of il-35 [44] . however, the results of all the studies among systematically healthy individuals showed that the levels of il-35 were higher in tissue samples of periodontitis patients comparing to non-infected samples. in this regard, eight studies focused on chronic types of periodontal disease, including gingivitis and periodontitis. however, kalburgi et al. examined il-35 mrna expression in gingival tissue samples of patients with aggressive periodontitis, patients with chronic periodontitis, and healthy controls using reverse transcriptase polymerase chain reaction (rt-pcr). the results showed that il-35 mrna expression was highest in subjects with chronic periodontitis compared to the subjects with aggressive periodontitis and the least seen in healthy patients [26] . among the included studies, only one paper reported that il-35 elevated level was also associated with the severity of chronic periodontitis [45] . unfortunately, due to substantial heterogeneity in sampling methods, severity of disease, and laboratory tests used in these observational studies, the post hoc analysis such as meta-analysis was not feasible [46] . only two human interventional studies on il-35 (patho) biology in periodontal disease were identified [47, 48] . both were randomized controlled clinical trials and assessed the effect of non-surgical periodontal treatment (nspt) on il-35 level among patients with chronic periodontitis. the first study was conducted among forty patients with chronic periodontitis who were randomly divided into two equal groups [48] . the control group received scaling root planning (srp), and the test group treated with srp followed by er, cr: ysgg laser. the results of this trial showed that all clinical parameters and gcf levels of immunological factors (il-1β, il-6, il-35) significantly reduced in both groups compared with the baseline (p < 0.05), but no significant change was detected among the groups. in the second study, 60 participants were divided into three equal groups, including healthy, gingivitis, and chronic periodontitis patients [47] . only the subjects in periodontitis group underwent srp treatment completed in two sessions within 24 h in accordance with the quirynen's one stage, full mouth debridement protocol. the study findings revealed that the mean il-35 concentration in gcf samples of patients with chronic periodontitis was significantly higher than the other groups at baseline. furthermore, the results confirmed a significant reduction in il-35 concentration of gcf samples obtained from patients with periodontal disease six weeks after nspt. it should be noted that this reduction of il-35 concentration was consistent with the decrease in clinical parameters, including periodontal pocket depth (ppd), clinical attachment level (cal), plaque index (pi), gingival index (gi) and bleeding index (bi) in patients with periodontal disease. as there is a high level of heterogeneity in periodontal disease definitions, treatment protocols, and laboratory tests used in these studies, it was not appropriate to apply meta-analysis. il-35 has been introduced as an anti-inflammatory cytokine in various diseases and conditions, such as experimental colitis, allergic asthma, and collageninduced autoimmune arthritis [15, 17, 49] . il-35 isrelatively spoken-a newcomer among suppressive cytokines [12] . the results of many studies showed the role of th1 and th17 cells and their cytokine profiles in periodontal disease [31, [50] [51] [52] [53] . based on the results of various studies, abnormal cellular immunity occurs in chronic periodontitis and elevated peripheral th17 and th1 cells might be simultaneously associated with the development of periodontal disease [31, 50] . it is interesting that il-35 inhibits th17 and th1 cells and may play a protective role in periodontal disease [22, 54] . in this study, we tried to collect all available data in the literature about the role of il-35 in pathobiology of periodontal disease. an in-depth analysis of the role of il-35 could provide novel avenues for diagnosing, monitoring, and treating periodontitis. in vitro and preclinical animal studies provided critical information about the function of il-35 and the interaction of other immunological factors with this newly found agent. a study by shindo et al. suggested an anti-inhibitory action for il-35 in pathogenesis of periodontitis [41] and reported that il-35 could inhibit the production of inflammatory cytokines such as il-6 and il-8. in this line, the results of an interventional animal study also considered an inhibitory effect for il-35 in pathogenesis of alveolar bone resorption [43] . another in vitro cell study suggested that il-35 may also induce osteoclastogenesis by a synergistic correlation with rankl [42] . it should be noted that in this study, the researchers just evaluated one type of cells (raw264). however as they declared in their report, bone marrow derived-macrophages include many cell populations, mainly lymphocytes, which may affected by il-35 in various manners [42] . in this regard, the results from the cross-sectional and rcts were more consistent. all of these studies, except one conducted on patients with diabetes, confirmed the higher levels of il-35 in tissue samples of patients with periodontal disease. in only one study evaluating serum levels of il-35 in patients with periodontal disease, included type 2 diabetic individuals with a control group, the results were inconsistent with the others [44] . the authors of this study reported that, despite significant associations of serum concentration of il-35 with certain periodontal and inflammatory indices, neither type 2 diabetes mellitus nor chronic periodontitis affected the serum levels of these cytokines differently. this contradictory result may be related to the evaluation of this biomarker in the serum, not with gcf or a gingival biopsy. in this way, the results of two interventional randomized clinical trials proved that treatment of chronic periodontitis and controlling periodontal tissues' inflammation could reduce the concentration of il-35 in gcf. the exact function of il-35 in etiopathogenesis of periodontal disease is not clear, but it is suggested to be produced by treg cells as negative feedback regulation [12] . previous research has revealed that in chronic periodontitis, the expression of treg cells is significantly increased compared to gingivitis and healthy condition [55, 56] . these cells are recruited by immune system for arresting tissue destruction and containing the disease. hence, a reduction in inflammatory load reduces the expression of treg cells. therefore, il-35 levels in gcf also decrease. this may explain the significant reduction in gcf il-35 levels in chronic patients with periodontal disease after nspt was performed in two included rcts [47, 48] . the results of the included studies in this review do not contradict previous reports on the protective role of il-35 in pathogenesis of inflammatory diseases. loss of il-35 activity in animal models leads to worsening and progression of various inflammatory diseases such as encephalomyelitis [57, 58] . in this regard, samples lacking functional il-35 showed greater severity of the disease [59, 60] . conversely, when il-35 was administered as a therapeutic agent in collagen-induced rheumatoid arthritis-affected mice, a significant reduction of pathological inflammation was observed [20] . in this scope, there is only one study in periodontology conducted by cafferata et al. examining the effect of il-35 administration on the alveolar bone resorption in periodontitis mice [43] . they concluded that il-35 is beneficial in the regulation of periodontitis. particularly, il-35 inhibited alveolar bone resorption, and this inhibition was closely associated with modulation of the periodontal th17/treg imbalance. most of the studies included in this review had a crosssectional design (level iii) and thus indicated an overall low level of evidence to prove the definitive role of il-35 in periodontitis [40] . further longitudinal and controlled clinical trials are required to clarify the detailed functional pattern of il-35 in various periodontal diseases. future research in the il-35 field could develop new innovative treatment methods to control periodontal. in summary, these scoping review results confirmed the presence of high levels of il-35 in saliva, gcf, serum, and gingival biopsies of patients with clinically diagnosed inflammatory periodontal disease. elevated levels of il-35 in periodontal tissues might be associated with the severity of periodontitis. at preclinical level, il-35 showed inhibitory characteristics regarding alveolar bone resorption of periodontitis models. moreover, non-surgical periodontal therapy could downregulate il-35 production in patients with chronic periodontal disease. global epidemiology of dental caries and severe periodontitis-a comprehensive review an appraisal of the role of specific bacteria in the initial pathogenesis of periodontitis effect of vitamin d level on periodontal treatment outcomes: a systematic review the nexus between periodontal inflammation and dysbiosis. frontiers in immunology the cytokine network involved in the host immune response to periodontitis cytokines that promote periodontal tissue destruction regulation of cytokine signaling and inflammation revisiting the page & schroeder model: the good, the bad and the unknowns in the periodontal host response 40 years later perspective of cytokine regulation for periodontal treatment: fibroblast biology inflammatory mediators in the pathogenesis of periodontitis mechanisms of bone resorption in periodontitis interleukin-35: expanding its job profile epstein-barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin possible roles of il-12-family cytokines in rheumatoid arthritis the inhibitory cytokine il-35 contributes to regulatory t-cell function il-35-mediated induction of a potent regulatory t cell population interleukin-35 mediates mucosal immune responses that protect against t-celldependent colitis the possible role of the novel cytokines il-35 and il-37 in inflammatory bowel disease interluekin-35 in asthma and its potential as an effective therapeutic agent il-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory t cells and suppression of th17 cells il-35-producing b cells are critical regulators of immunity during autoimmune and infectious diseases interleukin-35 induces regulatory b cells that suppress autoimmune disease interleukin-35 upregulates opg and inhibits rankl in mice with collagen-induced arthritis and fibroblast-like synoviocytes level of interleukin-35 in gingival crevicular fluid, saliva, and plasma in periodontal disease and health increased expression of interleukin (il)-35 and il-17, but not il-27, in gingival tissues with chronic periodontitis expression profile of il-35 mrna in gingiva of chronic periodontitis and aggressive periodontitis patients: a semiquantitative rt-pcr study il-35 may maintain homeostasis of the immune microenvironment in periodontitis il-37-and il-35/il-37-producing plasma cells in chronic periodontitis inhibited interleukin 35 expression and interleukin 35-induced regulatory t cells promote type ii innate lymphoid cell response in allergic rhinitis the role, involvement and function (s) of interleukin-35 and interleukin-37 in disease pathogenesis th17 and th1 lymphocytes are correlated with chronic periodontitis immune response mediated by th1/il-17/caspase-9 promotes evolution of periodontal disease regulatory b cells: origin, phenotype, and function cd19+ cd24hi cd38hi regulatory b cells and memory b cells in periodontitis: association with pro-inflammatory and anti-inflammatory cytokines periodontitis: facts, fallacies and the future infectious tolerance as seen with 2020 vision: the role of il-35 and extracellular vesicles treg-cell-derived il-35-coated extracellular vesicles promote infectious tolerance montoya riveros a: t regulatory cells-derived extracellular vesicles and their contribution to the generation of immune tolerance scoping studies: towards a methodological framework the levels of evidence and their role in evidence-based medicine interleukin (il)-35 suppresses il-6 and il-8 production in il-17a-stimulated human periodontal ligament cells il-35 and rankl synergistically induce osteoclastogenesis in raw264 mouse monocytic cells interleukin-35 inhibits alveolar bone resorption by modulating the th17/treg imbalance during periodontitis serum levels of interleukin-23 and 35 in patients with and without type 2 diabetes mellitus and chronic periodontitis elevated interleukin (il)-35-related scd14 but not il-23 is associated with the severity of chronic periodontitis applicable or non-applicable: investigations of clinical heterogeneity in systematic reviews association of human interleukin-35 level in gingival crevicular fluid and serum in periodontal health, disease, and after nonsurgical therapy: a comparative study clinical and biochemical effects of erbium, chromium: yttrium, scandium, gallium, garnet laser treatment as a complement to periodontal treatment airway inflammation and ige production induced by dust mite allergenspecific memory/effector th2 cell line can be effectively attenuated by il-35 t-helper cells in the etiopathogenesis of periodontal disease: a mini review effect of non-surgical periodontal therapy on the levels of th17/th1/th2 cytokines and their transcription factors in chinese chronic periodontitis patients calcitriol inhibits osteoclastogenesis in an inflammatory environment by changing the proportion and function of t helper cell subsets (th2/th17) induced experimental periimplantitis and periodontitis: what are the differences in the inflammatory response? experimental periimplantitis and periodontitis in dogs convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? elevated interleukin-35 suppresses liver inflammation by regulation of t helper 17 cells in acute hepatitis b virus infection regulatory t-cells infiltrate periodontal disease tissues regulatory t cells attenuate experimental periodontitis progression in mice epstein-barr virus-induced gene 3 negatively regulates neuroinflammation and t cell activation following coronavirus-induced encephalomyelitis increased th17 and regulatory t cell responses in ebv-induced gene 3-deficient mice lead to marginally enhanced development of autoimmune encephalomyelitis interleukin 35: critical regulator of immunity and lymphocyte-mediated diseases association between serum interleukin-35 levels and severity of acute pancreatitis evaluation of salivary cytokines and vitamin d levels in periodontopathic patients effect of interleukin (il)-35 on il-17 expression and production by human cd4+ t cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. 1 the online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12903-021-01515-1.additional file 1. full details of the search protocol used in this systematic review. the datasets supporting the conclusions of this article are included within the article and its additional file. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord-0021311-hnmpfc10 authors: lv, xiaolan; wang, liming; zou, xiaorong; huang, shigao title: umbilical cord mesenchymal stem cell therapy for regenerative treatment of rheumatoid arthritis: opportunities and challenges date: 2021-09-15 journal: drug des devel ther doi: 10.2147/dddt.s323107 sha: 81bac609ee0edcc4ca1b18126a2b14a86d05654e doc_id: 21311 cord_uid: hnmpfc10 rheumatoid arthritis (ra) is an autoimmune disease of unknown etiology with a high rate of disability. traditional treatments for ra remain a challenging issue. for example, nonsteroidal anti-inflammatory drugs (nsaids) have no therapeutic effects on joint destruction, and the prominent side effects include gastrointestinal symptoms. ra is characterized by recurrence and bone attrition. therefore, regenerative medicine and the use of umbilical cord mesenchymal stem cell (uc-msc) therapies have recently emerged as potential options. uc-mscs are multifunctional stem cells that are present in neonatal umbilical cord tissue and can differentiate into many kinds of cells, which have broad clinical application prospects in the tissue engineering of bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, and myocardium. moreover, uc-mscs have advantages, such as convenient collection of materials and no ethical disputes; thus, these cells have attracted increasing attention from researchers. however, there are few clinical studies regarding uc-msc therapy for ra. in this paper, we will review traditional drugs for ra treatment and then focus on uc-msc therapy for ra, including preclinical and clinical uc-msc applications for ra patients in the context of regenerative medicine. finally, we will summarize the challenges and perspectives of uc-mscs as a potential therapeutic strategy for ra. this review will help to design and discover more potent and efficacious treatments for ra patients and aid in advancing this class of cell therapy. rheumatoid arthritis (ra) is a systemic, inflammatory autoimmune disease that mainly involves peripheral facet joint disease, 1 which is the main pathological feature, as well as joint synovial cell proliferation, inflammatory cell infiltration, and pannus. 2 to date, traditional ra treatment mainly involves reducing the symptomatic inflammatory reaction and sequelae; 3 conventional treatment cannot satisfy the clinical requirement of achieving a curative effect. 4 therefore, exploring more effective and safer treatment options is increasingly important. mesenchymal stem cells (mscs) are derived from early-development mesoderm pluripotent stem cells and have a high degree of self-renewal and multidirectional differentiation potential; mscs widely exist in a variety of human tissues and can be cultured in vitro. under specific conditions, mscs can differentiate into osteoblasts, nerve cells, adipose cells, muscle cells, and cardiomyocytes. these cells have great application value in tissue engineering repair and in cell replacement therapy. 5 first, mscs can differentiate into bone and cartilage. second, these cells also inhibit immunity, alleviate inflammation, resist fibrosis, and promote vascular repair. umbilical cord mscs (uc-mscs) are derived from human umbilical cord blood and have msc characteristics and functions. therefore, uc-mscs offer the hope of a new treatment option for ra. in recent years, the use of uc-mscs to treat rheumatic immune diseases has garnered increasing attention, [6] [7] [8] [9] and the promising results of preclinical studies suggest that uc-mscs may be a therapeutic method for regenerative medicine. 7, 10, 11 however, there have been few studies regarding the effects of uc-mscs on patients with ra who had recurrent symptoms after long-term treatment with regular agents in the clinic. however, there is still a pressing need for examining efficiency and safety considerations of uc-mscs in clinical investigations. this review aims to summarize the current preclinical experimental and clinical studies of ra treatment with uc-mscs, examining the opportunities and challenges in research development progress. according to the development time and principles, the current drugs for treating ra are divided into four generations: the first generation is nonsteroidal anti-inflammatory drugs (nsaids); the second generation is glucocorticoids; the third generation is disease-changing drugs (slow-acting antirheumatic drugs, saards); and the fourth generation are early biologic agents dominated by tumor necrosis factor (tnf)-α inhibitors. the following are the descriptions of the difficulties of these four drug types in ra treatment. first, nsaids 12 are the first-generation treatment for ra and represent drugs such as aspirin and diclofenac. these drugs inactivate cyclooxygenase (cox)-1 and cox-2 mainly through acetylation. 13 nsaids have no therapeutic effect on joint destruction, and the prominent side effects are gastrointestinal symptoms. in recent years, newly marketed specific cox-2 inhibitors, such as meloxicam and celecoxib, have not affected cox-l due to their specific inhibition of cox-2. 14 thus, this treatment can avoid the traditional gastrointestinal side effects of nsaids. second, disease-modifying antirheumatic drugs (dmards) 15 or saards are third-generation therapies for ra. this class of drugs includes antimalarial drugs, gold preparations, and cytotoxic drugs. dmards mainly control the development of joint disease by reducing synovial inflammation and even play a role in repair, but most of these agents have serious side effects, such as vomiting, skin rash, leukocyte reductions, and liver and kidney function damage. third, glucocorticoids are the secondgeneration treatment for ra. 16 the mechanism of action is that glucocorticoids bind to glucocorticoid receptors and reach the nucleus, lowering the activity of nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) and reducing the production of proinflammatory cytokines to effectively reduce inflammation. these drugs cannot block the progression of ra or joint destruction, and longterm use can induce side effects such as infection, cortical hyperfunction, osteoporosis and hypertension. however, small doses and short courses of treatment can reduce symptoms through anti-inflammatory and anticleric effects. finally, early biological agents, mainly tnf-α inhibitors, are the fourth generation of drugs for the treatment of ra. 17 because of the advantages of high pharmacological selectivity and low toxicity and side effects, biologics will have wide application prospects. among the biologics used for the treatment of ra, the most clinically studied are tnf-α inhibitors. three tnf-α inhibitors, etanercept, infliximab and adalimumab, are currently approved for the treatment of ra. 18 the most common adverse reactions are injection-site reactions and infection. infliximab, developed by centocor, is used for the treatment of patients with early ra, 19 and the common adverse reactions are infection and allergic reactions in some patients. 20 adalimumab, developed by abbott, is the first human tnf antagonist and has adverse reactions such as nasopharyngitis and upper respiratory tract infection, and patients are prone to tuberculosis recurrence. 14, 21 in addition, compared with the use of methotrexate (mtx; 30.9%), the risk of opportunistic infections and invasive melanoma increased by 50% in patients treated with tnf antagonists (40.1%) compared with those treated with tnf antagonists. 22 these findings limit the wide application of biological agents to some extent. mscs are multipotent cells derived from the heart, peripheral blood, umbilical cord blood, muscle, lung, trabecular bone, intestinal tract, kidney, liver, pancreas, synovium, skin, and even the brain and can be a promising form of regenerative medicine. 23 in recent years, mscs have been widely used in the treatment of various diseases. 24 in china, mscs have been used to treat some clinically refractory diseases, such as spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus, systemic sclerosis, crohn's disease, stroke, diabetes, diabetic foot, and cirrhosis, among other conditions. [25] [26] [27] clinical practice shows that mscs are effective treatments for many diseases, such cotransplantation with hematopoietic stem cells, the enhancement of hematopoietic function, and implantation with hematopoietic stem cell grafts to treat graft-versus-host disease (gvhd). 24 in clinical research, the main types of mscs are bone marrow-derived mscs (bm-mscs), adipose-derived mesenchymal stromal cells (ad-mscs) and uc-mscs. ra is an inexorably progressive lung disease of unknown origin. 28, 29 the prognosis is poor, limited treatment options are available, and the median survival remains just 3-5 years. 30 despite the use of pirfenidone and nintedanib for the treatment of ra, mscs have potential as therapies for ra. 3 in recent years, based on the biological characteristics of mscs, much research has been done on the role of mscs in autoimmune diseases. 31 in particular, mscs can reduce the incidence of such diseases, especially ra, can also reduce inflammation after disease onset and can delay further disease development. 32 mscs have immunoregulatory roles, regulating the expression of cytokines by t cells, b cells, dendritic cells (dcs), and natural killer (nk) cells. 33, 34 however, ra is mainly caused by autoimmune disease mediated by t cells and involves b cells, cytokines, apoptosis, proteases, and other factors. 35 therefore, the use of mscs to intervene in the pathological basis of ramediated inflammation has become a feasible method. nearly 100 studies have been conducted to establish experimental models for msc treatment of ra. in most experimental models, the efficacy of msc treatment was demonstrated by significantly reducing the induction and progression of experimental arthritis. the most widely used model is the collagen-induced arthritis (cia) model. 36 these promising models pave the way for msc treatment of human ra disease. currently, nine clinical trials are active, and as a consequence, the clinical data are not publicly available, although the mscs are known to have included 50% uc-mscs, 25% ad-mscs, and 25% bm-mscs. 9 compared with bm-mscs and ad-mscs, uc-mscs have the following advantages: cell surface expression of major histocompatibility complex i (mhc i) is lower, and mhcii is not expressed, so uc-mscs seldom cause immune rejection. additionally, the growth environment of uc-mscs more pure than that of other mscs, these cells have stronger abilities to proliferate and differentiate and are easy to culture in vitro. furthermore, uc-mscs regulation of the immune response uc-mscs mediate the differentiation, proliferation, and activation of t cell subsets in various ways and inhibit b cell proliferation and differentiation, as well as the maturation of dcs and nk cell activity to achieve various immunomodulatory effects. 6 therefore, uc-mscs may be a feasible method to intervene in the pathological basis of ra-associated inflammation. recent studies on the treatment of ra suggest that uc-mscs suppress the various inflammatory effects of fibroblast-like synoviocytes (flss) and t cells in vitro and attenuate the development of cia in vivo. 38 mscs have low immunogenicity and express mhc-i molecules but not mhc-ii molecules and costimulatory molecules such as b7-1, b7-2, cd40, cd40, and fas ligand. mscs are not restricted by mhc molecules, so they play an immunomodulatory role invivo. 39 researchers 40 have demonstrated that uc-mscs have an immunomodulatory effect on allogeneic t lymphocytes and can inhibit the proliferation and changes in t lymphocytes stimulated by other cell subsets and cytokines secreted by t cells in the context of immune responses. the effects of uc-mscs on the immune regulation of th17 cells in ra patients have been reported by wang et al. 41 the results showed that uc-mscs could significantly reduce the numbers of th17 cells and the expression of cytokines and reduce the expression of ror-γt and serum levels il-17, and there was a good correlation with the dose. uc-mscs were observed in vitro in a mixed cell culture with peripheral blood mononuclear cells (pbmcs) from ra patients. in vitro, the th17 level was downregulated in the highly active group, but no similar regulation was observed in the normal and mildly active groups, suggesting that the immunoregulatory effect of uc-mscs on th17 cells may be regulated by other factors. gu et al, 42 concluded that uc-mscs could increase the peripheral blood treg proportion in cia rats and inhibit secretion by th17 cells and the activity of neutrophils. uc-mscs can also decrease the release of proinflammatory factors and induce immune reconstruction, in addition to reducing the immune inflammatory reaction. therefore, uc-mscs may provide a new way to treat ra. another intriguing feature of mscs is that they can escape immune recognition and inhibit immune responses and could become a potential tool for immunomodulatory cell therapy in immune-mediated diseases. 43 injury repair uc-mscs have strong proliferative abilities and multidirectional properties and can differentiate into adult cells in vivo or in vitro under appropriate environments. 44 uc-mscs have the capacity for tissue repair and regeneration. uc-mscs secrete a range of growth factors, such as hepatocyte growth factor (hgf), vascular endothelial growth factor (vegf), stromal cell-derived factor-1 (sdf-1), keratinocyte growth factor (kgf), fibroblast growth factor (fgf), and insulin-like growth factor-1 (igf-1), to facilitate proliferation and tissue damage repair. 45, 46 mscs have the potential to be pluripotent and capable of giving rise to a variety of cell types. the properties of various factors, growth factors, and extracellular vesicles can be transmitted. 47 nf-κb and other cellular signaling pathways can be affected to treat diseases. 48 after intravenous injection, uc-mscs can differentiate into alveolar epithelial cells and pulmonary vascular endothelial cells. 49 additionally, uc-mscs can play a role in injury repair. there is evidence that angiopoietin-1 (ang-1) and angiopoietin-2 (ang-2) are secreted with kgf to repair damage. mscs have homing abilities, can migrate to injured sites, and can differentiate into local components of injured sites and help tissue regeneration by secreting chemokines, cytokines, and growth factors 45. hence, uc-mscs can be widely used in articular cartilage repair. 3930 costimulatory molecules associated with t cell activation. 56 uc-mscs significantly reduce inflammation by extensively downregulating inflammatory products or cytotoxic mediators that act on immune cells and increase the level of the anti-inflammatory cytokine il-10. 57 in addition to suppressing the immune response, uc-mscs induce the production of il-10-secreting regulatory t cells (tregs) during local inflammation. il-10, a characteristic treg cytokine, plays an important role in controlling autoantigen-responsive t cells in vivo, and these tregs inhibit the effects of effector t cells on antigens. uc-mscs extensively reduce cytokines and chemokines and directly activate macrophages. 58 greish et al 59 used uc-mscs to treat an ra rat model induced with complete freund's adjuvant (cfa), and the results showed that the group of rats that received hematopoietic stem cells (hscs) and the group of rats that received uc-mscs both showed improvements in arthritis symptoms after 21 days of treatment, and arthritis symptoms completely disappeared after 34 days. the mean diameter in the uc-msc group was only half that of the control group and of the mtx group, and that of the uc-msc group was 0.6 mm smaller than that of the hsc group. serum levels of tnf-α, ifn-γ, and il-1 in the uc-msc group were significantly decreased compared with those of the control group and the mtx group, while the expression of il-10 was increased. hematoxylin-eosin (he) staining showed that uc-mscs could significantly reduce leukocyte infiltration and synovial tissue hyperplasia in the articular cavity. masson staining showed edema in the intrasynovial vessels in almost all arthritic rats, with vacuoles forming in the vessel walls, ultimately leading to hemorrhagic necrosis. additionally, the uc-msc group also showed a reduction in extensive fibrosis of the joint space, which verified that uc-mscs could improve the symptoms of cfa-induced ra, and the mechanism of action may be through regulating the expression of cytokines and improving pathological changes. at present, many universities, medical institutions, scientific research institutions, and enterprises have launched dozens of msc treatments for ra disease. related clinical studies are proceeding. by july 2020, according to clinicaltrials.gov, an international clinical trial registry, multiple clinical trials of uc-mscs for the treatment of ra have been recorded. there have been as many as six application cases, which have been organized by domestic units and korean affiliates, using uc-mscs either alone or combined with dmards. the interventions, outcomes, measures, and study designs were analyzed. information on the clinical trial phase is shown in table 1 . domestic research is mostly included. there was one clinical trial abroad with applicants from korea (nct03618784). the study designs were similar to the domestic studies, and the evaluation included erythrocyte sedimentation rate (esr), korean health assessment questionnaire (khaq), clinical disease activity index (cdai), 100 mm pain vas and changes in cytokines. in most clinical studies, uc-mscs extensively reduced cytokines and chemokines and directly activated macrophages. 8 the pathogenesis of ra and its immune mechanisms are related to cytokine disorders. uc-mscs are widely available, and their immune rejection is low. based on preclinical studies, uc-mscs have very good immune regulation and are able to repair tissue damage. therefore, the clinical application of cell therapy for ra has achieved a certain effect. many national clinical trials (ncts) registered on clinicaltrials.gov are shown in table 1 . most of the studies are from china, and only two furestem-ra inj trials are from korea, the latter of which evaluated general indices such as acr20/50/70, eular, das28-esr, and khaq and detected changes in cytokines (tnf-α, il-1β, il-4, il-6, il-8, il-10, il-13, il-17a, il-21, and il-22). 60 in south korea, a multicenter randomized double-blind parallel placebocontrolled phase i/iia ra clinical trial (nct03618784) administered allogeneic uc-mscs (named furestem-ra inj) to 33 ra patients. clinical and safety parameters were monitored for one month following the infusion of uc-mscs. no serious adverse events or major abnormalities in serum chemical or hematologic profiles were observed. early clinical studies of the use of uc-mscs in ra patients have shown promising safety profiles and could significantly improve symptoms and repair damaged tissue. 7 patients met the acr70 criteria, and the das 28 score was less than 2.6. the levels of complement c3 and c4, routine blood tests, liver and kidney function, and serum immunoglobulin levels showed no significant changes before and after treatment. no serious adverse effects occurred during the treatment period, and so uc-mscs were considered to exhibit immune regulation, prevent the release of inflammatory mediators, reduce soft tissue injury and play a role in other important effects that could alleviate clinical ra symptoms. thus, safety has additionally, we conducted prospective phase i/ii studies in 10 juvenile idiopathic arthritis (jia) children 56 less than 17 years of age and 64 ra patients 7 aged 18 to 64 years. the patients were treated with a 40 ml of a uc-msc suspension product (2 × 10 7 cells/20 ml) via intravenous injection. the esr, c reactive protein (crp) levels, and rheumatoid factor (rf) levels at 1 year and 3 years after treatment and anti-ccp at 3 years after treatment were significantly lower than those pretreatment (p < 0.05). one year and three years after treatment, the haq and das28 decreased in comparison to the pretreatment levels (p < 0.05). overall, uc-msc therapy can be a safe, effective, and feasible therapeutic option for ra patients. liu and colleague showed that uc-mscs have therapeutic potential in the treatment of ra. the results showed that uc-mscs suppressed the various inflammatory effects of flss and t cells. in addition, the immunosuppressive activity of uc-mscs could be prolonged by the participation of tregs. umbilical cord blood and tissue are ideal sources of mscs because they can be obtained by noninvasive means more easily than from bone marrow. 60 these cells have a lower relative risk of viral contamination and can be cryopreserved for autologous transplantation. however, the success rate of cultured mscs from umbilical cord blood is not high, with one study suggesting only 6%, while the success rate of cultured mscs from umbilical cord blood can reach 100%. therefore, in recent years, the umbilical cord has become an important source of mscs. although the physiological characteristics and mechanisms of action of mscs have been deeply studied, there are still many challenges to be solved in the treatment of ra. at present, there is no unified plan for stem cell transplantation to treat this disease, such as pretransplantation medication, transplanted cell numbers, and the optimal timing of treatments. the safety of the treatment and the suitable population still need long-term follow-up. genetic stability is affected by donors, tissue sources, and culture conditions during amplification, storage conditions, and the time of passage. 62, 63 stultz et al, 64 examined the chromosomal stability of bm-mscs from different donors and algebraically analyzed the results. the results showed that there were more abnormal karyotypes in the third generation than in the 5th and 7th generations, and the abnormal karyotypes gradually decreased with increasing generations through genetic modeling of inheritance. according to different donor ages in the elderly and young groups, the algebraic model showed that donors from the elderly group were characterized mainly by chromosome translocation, while donors from the young group were characterized by aneuploidy, but in both the elderly group and the young group, there were no significant differences in chromosomes; chromosomal abnormality occurred mainly in the early stages of the algebraic model, and with more passage times, there were more mature and stable the chromosomes. duarte and colleagues 65 compared pre-and post cryopreservation of the human umbilical vein to assess the influence of chromosome status on mscs and found that nonclone chromosome aberration was detected after frozen storage in liquid nitrogen, and the three short arms of a chromosome were inverted when the cells were stored for two months. at present, the mscs used to study regeneration technology mainly include bm-mscs and uc-mscs. 7, 10, 50 cord blood is from the umbilical cord near the fetal side of the placenta. this blood is rich in stem cells and progenitor cells and has the potential for multidirectional differentiation. there is no ethical problem in treatments with cord blood stem cells. china is a populous country with abundant availability of umbilical cord blood. due to the lack of understanding and a scientific research platform, the development of umbilical cord blood has been neglected. bm-mscs have more sufficient sources and are more convenient to collect than other types of mscs and do not express mhc ii molecules, so they cannot identify various antigenic mechanisms. due to a lack of immune activation in two common stimulatory signal transduction pathways and immune tolerance, the incidence of gvhd is low. 66 there are also primitive cells, such as endothelial progenitor cells, in umbilical cord blood, which are beneficial to vascular reconstruction and to improving blood supply at the site of injuries, as such cells play an auxiliary role in repair. compared with bm-mscs, uc-mscs are more primitive and have stronger amplification abilities. the application of uc-mscs does not involve ethical issues, and obtaining umbilical cord blood from newborns does not cause any harm to newborns. because umbilical cord blood is protected by the placental barrier, it is less likely to cause viral infections during transplantation than bone marrow transplantation. moreover, cord blood stem cells are extremely pure. cord blood is easy to obtain and is an available resource. uc-msc therapy involves no ethical issues and no tissue antigenicity and has a simple transplantation route, obvious and reliable efficacy, and no adverse side effects. uc-mscs can promote the directional activation of stem cells and accelerate nerve function repair. a large number of studies have confirmed that uc-mscs play a role in ra disease, but their mechanism remains unclear. there are challenges in the use of uc-msc therapy in clinical trials. for example, a multiple center randomized controlled trial (rct) needs to be performed; a large sample size and clinical trials need to be constructed to verify the therapeutic effect; there is no unified standard for the optimal dose, optimal time, treatment frequency, approach, treatment conditions and indications of uc-msc therapy for ra; and whether there will be other potential side effects is a problem that needs to be observed and solved in future research. in summary, uc-mscs are studied not only in the laboratory but also in the clinic. all studies showed excellent efficacy, especially those utilizing uc-mscs, which are a potential feasible method of ra treatment. uc-mscs show good efficacy and tolerability in ra patients and have emerged as a promising alternative in the management of ra. the pathogenesis of ra is related to disorders of immune mechanisms and cytokines. uc-mscs are widely available and have low immunogenic responses, and the limitations, such as the lack of traditional stem cell sources, allogeneic rejection, and ethics, have been overcome. as uc-mscs have achieved a certain efficacy and few side effects in clinical practice, they are worthy of further large-sample rcts for future evaluation. abbreviations bm, bone marrow; bm-mscs, bone marrow mesenchymal stem cells; ra, rheumatoid arthritis; uc-mscs, umbilical cord mesenchymal stem cells; das 28, disease activity score 28-joint; ae, adverse events; esr, erythrocyte sedimentation rate; rf rheumatoid factor; dmards, drug rheumatoid arthritis with disease-modifying drugs; sae, serious adverse events; das28-crp, disease activity score 28-joint count using c reactive protein; haq-di, health assessment questionnaire disability index; sdai, simplified disease activity index; anti-ccp, anti-cyclic citrullinated peptide; hgb, hemoglobin; cia, collagen-induced arthritis; flss, fibroblast-like synoviocytes; fvc, forced vital capacity; acr, american college of rheumatology. this research was supported by the guangxi zhuang autonomous region self-funded project (no: z20190359) and the project of liuzhou science and technology bureau (no: 2018bj10502). the epidemiology of established rheumatoid arthritis pathogenesis of bone erosions in rheumatoid arthritis a systematic review and metaanalysis of antirheumatic drugs and vaccine immunogenicity in rheumatoid arthritis the preclinical stages of ra: lessons from human studies and animal models autophagy and stem cells: self-eating for self-renewal targeting mesenchymal stromal cells/pericytes (mscs) with pulsed electromagnetic field (pemf) has the potential to treat rheumatoid arthritis efficacy and safety of umbilical cord mesenchymal stem cell therapy for rheumatoid arthritis patients: a prospective phase i/ii study human umbilical cord mesenchymal stem cell therapy for patients with active rheumatoid arthritis: safety and efficacy mesenchymal stem/stromal cells for rheumatoid arthritis treatment: an update on clinical applications mesenchymal stem cells (mscs) as a potential therapeutic strategy in covid-19 patients: literature research cord blood-derived cytokine-induced killer cellular therapy plus radiation therapy for esophageal cancer: a case report paracetamol versus nonsteroidal antiinflammatory drugs for rheumatoid arthritis deficiency of either cyclooxygenase (cox)-1 or cox-2 alters epidermal differentiation and reduces mouse skin tumorigenesis adalimumab with or without methotrexate in juvenile rheumatoid arthritis tapering biologic and conventional dmard therapy in rheumatoid arthritis: current evidence and future directions ofatumumab, a second-generation anti-cd20 monoclonal antibody, for the treatment of lymphoproliferative and autoimmune disorders comparative evaluation of the effects of treatment with tocilizumab and tnf-α inhibitors on serum hepcidin, anemia response and disease activity in rheumatoid arthritis patients serious infection during etanercept, infliximab and adalimumab therapy for rheumatoid arthritis: a literature review predictors of joint damage in patients with early rheumatoid arthritis treated with high-dose methotrexate with or without concomitant infliximab: results from the aspire trial combination of infliximab and methotrexate therapy for early rheumatoid arthritis: a randomized, controlled trial clinical response to adalimumab: relationship to anti-adalimumab antibodies and serum adalimumab concentrations in rheumatoid arthritis rheumatoid arthritis, anti-tumour necrosis factor therapy, and risk of malignant melanoma: nationwide population based prospective cohort study from sweden mesenchymal stem cells in homeostasis and systemic diseases: hypothesis, evidences, and therapeutic opportunities the multi-functional roles of menstrual blood-derived stem cells in regenerative medicine mesenchymal stem cells-derived exosomes: a possible therapeutic strategy for osteoporosis roles of mesenchymal stem cells in spinal cord injury clinical efficacy and safety of mesenchymal stem cells for systemic lupus erythematosus one year in review 2017: novelties in the treatment of rheumatoid arthritis one year in review 2019: novelties in the treatment of rheumatoid arthritis future therapeutic targets in rheumatoid arthritis? human gingival tissue-derived msc suppress osteoclastogenesis and bone erosion via cd39-adenosine signal pathway in autoimmune arthritis mesenchymal stem cells improve rheumatoid arthritis progression by controlling memory t cell response cell therapies for refractory rheumatoid arthritis combination therapy of mesenchymal stromal cells and interleukin-4 attenuates rheumatoid arthritis in a collagen-induced murine model generation of mesenchymal stem cells from human embryonic stem cells in a complete serum-free condition immunomodulatory properties of mesenchymal stem cell in experimental arthritis in rat and mouse models: a systematic review concise review: mesenchymal stem cells: from roots to boost therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis transforming growth factor-β2 downregulates major histocompatibility complex (mhc) i and mhc ii surface expression on equine bone marrow-derived mesenchymal stem cells without altering other phenotypic cell surface markers biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys the allogeneic umbilical cord mesenchymal stem cells regulate the function of t helper 17 cells from patients with rheumatoid arthritis in an in vitro co-culture system mesenchymal stem cells: immunomodulatory capability and clinical potential in immune diseases induction of mesenchymal stem cell differentiation in the absence of soluble inducer for cutaneous wound regeneration by a chitin nanofiber-based hydrogel mesenchymal stem cell migration and tissue repair effect of fibronectin, fgf-2, and bmp4 in the stemness maintenance of bmscs and the metabolic and proteomic cues involved tissue source determines the differentiation potentials of mesenchymal stem cells: a comparative study of human mesenchymal stem cells from bone marrow and adipose tissue directed differentiation and paracrine mechanisms of mesenchymal stem cells: potential implications for tendon repair and regeneration enhanced effect of tendon stem/progenitor cells combined with tendon-derived decellularized extracellular matrix on tendon regeneration drug design bone marrow-derived stem cells in wound healing: a review multipotent adult progenitor cells: their role in wound healing and the treatment of dermal wounds stem cells in cutaneous wound healing mesenchymal stem cells and skin wound repair and regeneration: possibilities and questions transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization stromal progenitor cell therapy corrects the wound-healing defect in the ischemic rabbit ear model of chronic wound repair regulatory effect of mesenchymal stem cells on t cell phenotypes in autoimmune diseases comparing the immunomodulatory properties of bone marrow, adipose tissue, and birth-associated tissue mesenchymal stromal cells umbilical cord-derived mesenchymal stem cell extracts reduce colitis in mice by re-polarizing intestinal macrophages human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model intravenous infusion of umbilical cord blood-derived mesenchymal stem cells in rheumatoid arthritis: a phase ia clinical trial cervus and cucumis peptides combined umbilical cord mesenchymal stem cells therapy for rheumatoid arthritis cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis limited acquisition of chromosomal aberrations in human adult mesenchymal stromal cells chromosomal stability of mesenchymal stromal cells during in vitro culture chromosomal characterization of cryopreserved mesenchymal stem cells from the human subendothelium umbilical cord vein differentiation of human umbilical cord-derived mesenchymal stem cells into hepatocytes in vitro drug design, development and therapy drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are a feature of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system the authors report no conflicts of interest in this work. key: cord-0021655-m7q1m8ow authors: choquette, denis; chan, jonathan; bardi, mohammad; whiskin, carolyn; torani, gabriel; smith, brennan k.; sihota, aaron title: monitoring the transition of patients on biologics in rheumatoid arthritis: consensus guidance for pharmacists date: 2021-09-14 journal: pharm pract (granada) doi: 10.18549/pharmpract.2021.3.2377 sha: 1edb7523bdcd0ef17c5db08ecfa6539b1d6a7270 doc_id: 21655 cord_uid: m7q1m8ow background: recent approvals for novel agents such as the small molecule janus kinase inhibitors (jaki), combined with the advent of biosimilars has widened the gamut of available therapeutic options in the treatment of rheumatoid arthritis (ra). this combined with the introduction of mandatory nonmedical switches to biosimilars in some jurisdictions by both public and private payors has led to a significant increase in the volume of therapeutic changes for patients. pharmacists are well positioned to ensure effective and safe transitions, however there is a significant unmet need for objective and subjective clinical guidance around therapy as well disease state monitoring in ra that facilitates best practices throughout the patient journey. objective: in this paper we aim to create a consensus derived monitoring algorithm for pharmacists to facilitate best practices throughout therapeutic transitions from originator biologic to other originator biologics, biosimilars, and janus kinase inhibitors in ra. methods: the nominal group technique (ngt) was used to understand if consensus could be found among the participants. clinically relevant questions were developed to capture solutions to the identified unmet need. the faculty considered the questions as individuals, and privately generated answers/ideas. after discussion and consideration, the participants ranked the ideas and established a consensus. results: based on the outcome of the consensus discussions, an algorithm was created to help guide pharmacists through therapeutic transitions in ra. the tool covers important topics such as pre-transition considerations, avoiding the nocebo effect for biosimilars, specific considerations for each drug or class, monitoring efficacy, and when to refer. conclusions: new classes of anti-rheumatic drugs including jaki, along with the introduction of biosimilars are presenting more opportunity for therapeutic changes and monitoring in patients with ra. we hope our evidence-based consensus derived guidance tool will assist frontline pharmacists in supporting their patients to a successful therapeutic transition in ra. biologic drugs for the treatment of rheumatoid arthritis (ra) and other inflammatory conditions have been widely used for over 20 years. 1 the first tumor necrosis factor alpha (tnf-α) inhibitor, infliximab, was approved by the fda in 1998. since then, many other biologics including those with novel mechanisms of action have entered the ra market. in addition, the recent approvals of the small molecule of the janus kinase inhibitors class (jaki) provide patients with targeted oral therapeutic options. there are many reasons why a rheumatologist and their patient may decide to change ra pharmacotherapy during the course of disease progression. most often this is due to no response at all or inadequate response or adverse reactions/intolerance to their current drug therapy. patients not responding to therapy may be categorized as primary or secondary non-responders, the former due to initial complete lack of response, and the latter due to loss of responsiveness over time. 2 eular (the european league against rheumatism) recently updated their recommendations for the treatment of ra in 2019. 3 they recommend patients who fail a tnf-a inhibitor should switch to a different mechanism of action, rather than trying another tnf-a inhibitor within class. increasingly, some patients with ra are also being required to change therapy for non-clinical reasons. typically, due to payor formulary constraints (often based on cost), some canadian provinces are mandating a non-clinical transition from biologic originator to biosimilar. 4,5 biosimilars (or "subsequent-entry biologics") are akin to the biologic medicines' equivalent of chemical generics. 6 the costsavings associated with biosimilars may ultimately improve patient access to biologics and other valuable medicines. [7] [8] [9] there is a growing body of evidence that suggests switching to a biosimilar is safe and effective, however the very faint theoretical potential of developing immunogenicity along with the impact of the nocebo effect can create confusion monitoring the transition of patients on biologics in rheumatoid arthritis: consensus guidance for pharmacists for patients. [10] [11] [12] [13] [14] healthcare professionals must exercise their own clinical judgement when considering the suitability of transitions on a patient-by-patient basis. 15 considering the scenarios above, the pharmacist can expect to see more patients with ra undergoing complex therapeutic changes. pharmacists are well positioned to guide and assist patients with appropriate clinical education as well as complete administrative tasks that can facilitate a smooth transition. pharmacists typically follow patients more frequently and have more touchpoints than their rheumatologist does post-transition, so are ideally placed to flag any early safety or efficacy issues. this is especially the case with refilling medications which represent an opportunity to engage and monitor a patient. there is currently limited practical guidance available for pharmacists who are managing transitions from originator biologic to another originator biologic, biosimilar, or jaki. as biosimilars and novel mechanisms of action continue to enter the marketplace, there is an unmet need for pharmacist guidance on the key roles they can play during the therapeutic transition. a review of the literature yields a paucity of guidance, and other authors have highlighted this need when drawing their conclusions. [16] [17] [18] in this paper we aim to create a monitoring algorithm for pharmacists to facilitate best practices throughout therapeutic transitions to biologics and jakis in ra. it should be noted that although we have developed our guidance in the context of pharmacy practice in canada, we believe it should still provide value regardless of clinical setting or jurisdiction. we encourage the reader to exercise best judgement when applying our guidance to their local regulatory policies. a group of experts was gathered to address an unmet need for pharmacists to better understand the impact of patients being transitioned to new ra therapies, and how they can best support those patients and the rheumatologists. the nominal group technique was used to establish a consensus among the group. a facilitator and assistant were appointed to moderate all group discussions and voting rounds. the multidisciplinary participant committee was selected to represent both pharmacists and rheumatologists in multiple practice settings and geographies across canada with clinical experience in assisting patients with inflammatory arthritis and related sequalae. upon agreement to participate, the participants met virtually to discuss scope and a suitable methodology to find consensus. the nominal group technique (ngt) was used to understand if consensus could be found among the participants. ngt is a structured variation of a small group discussion that lends itself to finding a consensus especially when there is a goal of prioritizing free-structured ideas. the ngt was performed according to the methodology of mcmillan et al. 21 some of the advantages of the ngt that made it particularly suitable for this application include: effective for smaller groups; balances the influence of individuals; allows for a greater number of ideas to be considered; allows the group to prioritize ideas democratically. 22 the first step was alignment on the clinical need of the community pharmacist. this was achieved during a series of structured, moderated participant discussions in the virtual setting utilizing zoom software (san jose, usa). the methodological framework used was grounded theory. 23 a list of clinically relevant questions was generated; some of which were discarded as out of scope, and some kept. the complete list, including outcome and rationale are included in table 1 . the refined list progressed to the voting round of the ngt. the clinical questions that were identified fell into three categories: 1. considerations prior to transition to another originator biologic therapy, biosimilar or jak 2. considerations during transition to another originator biologic therapy, biosimilar or jak 3. considerations after transition to another originator biologic therapy, biosimilar or jak once the clinical questions were established, the formal ngt could begin ( figure 1 ). step two was a silent generation of ideas where the participants considered the questions as individuals, and privately generated answers/ideas. step three was a round robin, where each question was discussed as a group, along with a comprehensive list of ideas that had been generated in the previous step. after discussion and consideration, the participants ranked the ideas using the online polling tool, mentimeter (stockholm, sweden). 24 once all questions had been addressed, the group in realtime discussed the results and established a consensus. when ranking the questions, the group agreed that the top three ideas would be selected as their consensus. in cases where ranking was deemed inappropriate, or if the group felt all ideas were equally important, they abstained from voting and agreed that a comprehensive list was the best outcome. the results of the iterations of voting and discussion were developed into a clinical decision-making monitoring tool ( figure 2 ). the below commentary highlights where the participants believe they could find (or not find) a consensus and establish a recommendation for pharmacists. a full list of questions and responses, including voting results can be found in table 2 and table 3 . question 1: how can pharmacists reduce the risk of a nocebo effect? the nocebo effect is defined as a negative effect of a pharmacological or non-pharmacological medical treatment that is induced by patients' expectations, and that is unrelated to the physiological action of the treatment. 25 the most important factor in reducing the risk of a nocebo effect is for the pharmacist to demonstrate confidence in the transition using positive verbal language. the next most important factor is using positive body language. and the third most important factor is consistent messaging from all healthcare providers. the participants did not see the value of using a validated tool to screen and assess patient attitudes towards the therapeutic transition as a proxy for potential nocebo. in what circumstances should the pharmacist refer back to the rheumatologist prior to transition? before transition, a pharmacist must receive a new prescription. however, if the pharmacist believes there is a significant safety, efficacy, or other concern that may lead to a poor outcome, they should discuss with the prescribing rheumatologist prior to the transition. the participants felt it would be impractical to reach a consensus on this question as there are many factors that are specific to an individual patient or circumstance. depending on severity or seriousness, any factor could be deemed important enough to warrant a referral or at least a query to the rheumatologist. the pharmacist should exercise their own professional judgement. appropriate. all responses were deemed important and the group agreed that providing a complete list of considerations would be more valuable than prioritizing. question 5: what tools should pharmacists use to assess disease activity? the participants voted on this question and the most important tool was found to be a visual analog scale (vas) to assess pain/fatigue/quality of life. the next two most important tools to consider are the rapid3 tool and the likert scale. the health assessment questionnaire (haq) and a general questioning approach were deemed less important. question 6: when should a pharmacist refer for earlier than scheduled follow up with the rheumatologist? question 6 was not ranked as the participants agreed that similar to question 2, the seriousness and severity of the individual factors (infection, repeated flare ups, surgery, live vaccinations etc.) are more important than the factors themselves. they noted however that, as a rule, unmanaged flares that are significantly impacting on health-related quality of life (hrqol) or functioning should be referred for early follow up. they also noted that the pharmacist is well positioned to monitor and resolve issues relating to adherence and safety. question 7: when to refer to rheumatologist for safety? there was a lengthy list of situations where a referral for safety concerns was warranted. the participants agreed that all were legitimate and for that reason it was not appropriate to rank importance. the entire list should be included in no specific order. based on the outcome of the consensus discussions, an algorithm was created to help guide pharmacists through therapeutic transitions in ra (figure 2 ). based on our results, we have created a tool to guide pharmacists through the transition process, highlighting areas where they can have the most positive impact. pre-transition considerations include being aware of possible nocebo effects for biosimilars. although a biosimilar and its reference product have an identical amino acid sequence, subtle differences in post-translational modifications that occur during the manufacturing process in living organisms can be expected. 26 for this reason, there are occasional reservations among the medical and patient communities regarding the transition from originator products to biosimilars when a patient is otherwise stable, in lowdisease activity or in boolean remission. 27 these reservations can manifest a nocebo response in the patient. 14, 25, 28 however, a 2018 consensus document by the task force on the use of biosimilars to treat rheumatological diseases states that the currently available scientific evidence indicates that a single switch from a reference to a biosimilar is safe and effective. 29 additionally, clinical trials assessing switch from a biooriginator to a biosimilar have not demonstrated any loss of efficacy, increase in adverse events, or increased immunogenicity. 11, 12 a recent systemic review of switching between originator biologics and biosimilars confirms this further. 30 finally, it should also be noted that posttranslational modifications can also occur between batches of the same originator biologic, so this phenomenon is not a unique characteristic of biosimilars. the participants noted the importance of avoiding the nocebo effect by using positive verbal and body language and aligning with consistent messaging from all health care professionals to the patient. patients should receive simple and understandable language, as well as a tailored approach when talking to clinicians. some patients will require more information, other will not. it is the task of all clinicians (nurse, physicians, pharmacist) to assess the individual patient needs. on the one hand, one wants to avoid giving too much information and creating doubts. on the other hand, clinicians should also avoid giving too little information. 30, 31 other clinical considerations prior to the transition are mechanism of action specific. the most pertinent clinical concerns that the pharmacist should be aware of are listed for each of the mechanism of action classes. [32] [33] [34] [35] [36] a "before starting" checklist for the pharmacist is included -a convenient reminder of the most important administrative tasks that should be completed to ensure a smooth transition to the new therapy. the pharmacist is well positioned to assist with transition to the new patient support program (psp), an area noted for commonly causing distress for patients and uncertainty for rheumatologists. a validated tool to assess patient attitude 0 1 0 0 2 0 9 5 share data showing equivalence 0 0 0 4 1 0 14 4 use positive body language 1 3 1 1 0 0 28 2 attitudes questionnaire 0 0 0 0 1 1 3 a consensus was reached regarding monitoring the patient for signs of primary non-response. the pharmacist is likely going to interact with the patient for medication refills before the scheduled three to six-month follow-up appointment with the rheumatologist. although a lack of peak-response early in treatment is not particularly concerning, a series of uncontrolled or frequent flare ups as compared to baseline after 3 months of therapy would be cause for immediate referral. there was strong agreement among the participants that the pharmacist should monitor for changes in symptoms such as pain, fatigue, and quality of life using a simple visual tool such as the visual analog scale (vas). the rapid3 questionnaire and likert scale tools ranked second and third respectively. vas and likert responses are highly correlated and yield similar precision. since likert responses are easier to administer and interpret, some pharmacists may find it preferable. 37 rapid3 is a validated tool for ra so some may prefer its specificity and greater accuracy. 38 the final section of the clinical monitoring tool developed lists scenarios for early referral to the rheumatologist. although the list could never be exhaustive, there was unanimous agreement that the most common issues have been captured. as previously mentioned, uncontrolled and frequent flares as compared to patient baseline are a cause for concern and should be referred. but it should be noted that flares are a common and expected feature of ra, even in well controlled patients. depending on the setting, pharmacists may be able to help their patients manage flares, using non-steroidal anti-inflammatory drugs (nsaids), a short course of steroids, or titration of other anti-rheumatic agents. our aim was to create a concise quick reference tool that would be practically useful to a pharmacist in any setting. it serves as both a reference and a checklist and can be applied to any situation where a patient is transitioning from one ra therapy to another. the tool is easy to use with logical progression from pre-transition considerations to post-transition monitoring. it answers the identified unmet need for pharmacists, specifically how to talk to patients about biosimilars, when to flag a potentially inappropriate transition, and how to monitor for efficacy. the tool could be used in hospital, community, or specialty settings. as it is all contained on one page, we would suggest it can be printed and posted in a dispensary or made available on a dispensary computer for quick reference when preparing to counsel a patient. depending on local policies and practice, it could also be converted and used as a checklist. new classes of anti-rheumatic drugs including jaks, along with the introduction of biosimilars are presenting more opportunity for therapeutic changes in patients with ra. these changes are often complex due to the nature of the drugs, along with an associated administrative burden. pharmacists are well positioned to manage the transition and be an advocate for the patient. we hope our novel clinical guidance and monitoring tool will assist pharmacists in supporting their patients to a successful therapeutic transition and allow for a greater role in their overall care. immune-mediated inflammatory diseases (imids) and biologic therapy: a medical revolution treatment options in patients with rheumatoid arthritis failing initial tnf inhibitor therapy: a critical review eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update biosimilars faq for prescribers biosimilars: what clinicians should know real-world observational study of biosimilars in inflammatory arthritis treatment: a systematic literature review potential cost-savings from the use of the biosimilars filgrastim, infliximab and insulin glargine in canada: a retrospective analysis efficacy and safety in the continued treatment with a biosimilar drug in patients receiving infliximab: a systematic review in the context of decision-making from a latin-american country switching reference medicines to biosimilars: a systematic literature review of clinical outcomes switching from originator infliximab to biosimilar ct-p13 compared with maintained treatment with originator infliximab (nor-switch): a 52-week, randomised, double-blind, non-inferiority trial multiple switches between gp2015, an etanercept biosimilar, with originator product do not impact efficacy, safety and immunogenicity in patients with chronic plaque-type psoriasis: 30-week results from the phase 3, confirmatory egality study switching from originator biological agents to biosimilars: what is the evidence and what are the issues non-pharmacological effects in switching medication: the nocebo effect in switching from originator to biosimilar agent fda. biosimilar and interchangeable biologics: more treatment choices a survey of global biosimilar implementation practice conducted by the international society of oncology pharmacy practitioners biosimilar monoclonal antibodies: a canadian regulatory perspective on the assessment of clinically relevant differences and indication extrapolation subsequent entry biologics in canada: current state of the science nominal group technique (ngt): nominal brainstorming steps nominal group technique: an effective method for obtaining group consensus how to use the nominal group and delphi techniques gaining consensus among stakeholders through the nominal group technique the blackwell encyclopedia of sociology the nocebo effect: a clinical challenge in the era of biosimilars switching to biosimilars: current perspectives in immune-mediated inflammatory diseases the arthritis society. the arthritis society position on biosimilars treatment outcomes with biosimilars: be aware of the nocebo effect consensus-based recommendations for the use of biosimilars to treat rheumatological diseases the efficacy, safety, and immunogenicity of switching between reference biopharmaceuticals and biosimilars: a systematic review informing patients about biosimilar medicines: the role of european patient associations abbvie corporation monitoring the transition of patients on biologics in rheumatoid arthritis: consensus guidance for pharmacists response relationship of vas and likert scales in osteoarthritis efficacy measurement rapid3, an index to assess and monitor patients with rheumatoid arthritis, without formal joint counts: similar results to das28 and cdai in clinical trials and clinical care the authors would like to acknowledge the assistance of andrew o'reilly of pinewood digital consulting in developing the manuscript. key: cord-0022253-6gvn6tzb authors: gebretsadik, achamyelesh; taddesse, fiker; melaku, nebiyu; haji, yusuf title: balneotherapy for musculoskeletal pain management of hot spring water in southern ethiopia: perceived improvements date: 2021-10-15 journal: inquiry doi: 10.1177/00469580211049063 sha: 63297a451b64a4696eb6ae24f479464f6f315b83 doc_id: 22253 cord_uid: 6gvn6tzb background: balneotherapy and hydrotherapy offer interesting treatment alternatives and are commonly used as additional interventions in the management of musculoskeletal disorders and pain management. therefore, the aim of this study was to assess the effect of balneotherapy on musculoskeletal disorder pain and its perceived improvement among users of hot spring water in southern ethiopia. methods: a single-arm cohort study and convenient sampling method were used to select 1337 study participants from four hot springs in southern ethiopia. a structured questionnaire, a physical examination, and laboratory blood tests were used to collect data. data were entered using epi data and transferred to spss 25 for cleaning and analysis. descriptive analysis was made. results: a total of 1279 participants were included in the study, giving a response rate of 96%. the majority of these patients have multiple health problems. of all, 1137 (88.9%) of the patients were visiting the hot springs for joint pain followed by muscle pain 669 (52.2). out of all cases of joint pain, 132 (11.6%) were clinically diagnosed with rheumatoid arthritis, and 5.3% were confirmed as having the disease based on a laboratory test. of the total number of study participants, 1064 (83.2%) reported complete relief from the complaints they had at the start of the bath. conclusions: hot spring baths for three and more days have significant therapeutic effects on patients with musculoskeletal disorders, including rheumatoid arthritis. physicians who are currently working in the area of diagnosis and treatment of patients in government and public facilities of the southern region should consider hot spring bath treatment for those patients with complaints of musculoskeletal pain, nonspecific arthritis, and rheumatoid arthritis. a hot spring bath is beneficial for everyone because it is a natural treatment with few side effects and a low cost. what do we already know about this topic? there is no adequate information about the use of hot springs bath. balneotherapy was not well understood by medical staff as an alternative therapy. • how does your research contribute to the field? most patients with chronic musculoskeletal conditions traditionally sought a solution to their problem. it is good to provide information about thermal baths at healthcare facilities. • what are your research's implications toward practice? fortunately, our community believes that hot spring bath is a natural remedy. however, the modern medicine not practically integrated with that of the traditional therapy. it is useful if a physician prescribe a hot spring bath as a supportive treatment alternative for the management of specific and nonspecific musculoskeletal disorder pain. this is accessible and affordable for most people with lower economic status. creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 1 the term balneotherapy is seated immersion or spa-therapy. often, natural mineral or thermal waters are used for bathing, drinking, and inhalation. 1 balneotherapy and hydrotherapy offer interesting treatment alternatives and are commonly used as additional interventions in the management of musculoskeletal disorders and pain management. 2 musculoskeletal conditions, also called musculoskeletal disorders, consist of conditions where a part of the musculoskeletal system is injured or affected over time. symptoms include pain, function, or discomfort in the bones, joints, muscles, or surrounding structures, which can be acute or chronic, focal or diffuse. the most frequent musculoskeletal disorders are osteoarthritis, fibromyalgia, rheumatoid arthritis, and low-back pain. 3 nowadays, when warm water can be easily obtained from springs in any country, the question of whether this warm mineral water is of any medical importance arises. for this reason, physico-chemical analyses are the first step in order to discover these characteristics. 4 hydrotherapy, using water and heat together to treat conditions, relieve pain, and increase muscular power and a range of joint movement, has been a mainstay of managing many medical conditions. these include arthritis, neurological conditions, and rehabilitation from sports injuries. 5 balneotherapy can be used to specifically improve: strength and/or prevent muscle wastage, posture, flexibility and/or prevention of contracture (shortening and hardening of muscles, tendons, or other tissue); ability to perform normal everyday activities, including walking, exercise and work tolerance, cardiovascular fitness and balance, and reaction times. 5 studies have reported a faster rate of improvement in diseases in patients using drugs with balneotherapy than drugs alone for some of the above-mentioned diseases, which in turn reduces the duration of drug exposure. 6, 7 the therapeutic outcome of water comes from its mechanical, thermal, and chemical effects that allow the patient to mobilize joints and strengthen muscles with minimal discomfort mechanically, vasodilatation by the higher temperature, and healing of the skin chemically with the help of minerals where, under certain conditions, resorption is possible. 8, 9 the common elements/minerals that are said to have therapeutic properties include calcium, magnesium, sodium, potassium, sulfur/sulfate, and bicarbonates. however, despite the fact that people in ethiopia have traditionally used hot springs for therapeutic purposes, no study has reported the effect of balneotherapy on the management of musculoskeletal disorders and pain management. therefore, the aim of this study was to assess the therapeutic properties of the selected hot springs for the management of musculoskeletal disorders in the southern part of the region. the study was conducted in bersiso (dilla district), yirgalem (yirgalem town), burkitu (hawassa city), and wondo (wondo district) hot water springs in the southern nation national people's region (snnpr) in 2019. the hot springs were selected randomly. the study population consisted of adults aged 18 and above who used immersion or bath at the selected hot springs in snnpr during the study period. the study was conducted from march to october 2019. a single-arm prospective cohort design was used to assess musculoskeletal complaints and observe patients' improvement in their musculoskeletal system complaints after utilization of hot spring water for at least 3 days. participants were asked about their health status at the beginning of the hot spring immersion and followed for 3-10 days as per their willingness to stay and again asked about their health status at the end of the hot spring bath. the sample size for the study was determined using a double population proportion formula. 80% power, 95% confidence level, aor 2.49, and a 1:1 ratio were considered. 608 was the calculated sample multiplied by a design effect of 2 and a 10% nonresponse rate. the total sample size required for the study was 1337 persons. 10 to include the study subjects, a convenient sampling technique was used. based on the client's age and the availability of interesting health problems, assigned study subjects to the four hot springs in a proportional manner. an average of 334 samples were allocated to each of the four sites. blood samples were collected, if the duration of the symptoms was greater than 2 weeks and 2-10 large joints or 1-3 small joints were involved. as a result, 132 blood samples were collected from the four hot springs sites studied. inclusion: clients who were using the hot spring for healthrelated problem improvements and wanted to stay for at least 3 days to follow the change in signs and symptoms. exclusion: clients under the age of 18, those clients who came for recreational purposes, and pregnant women were also excluded. socio-demographic factors, type and duration of illness, and improvement in signs/symptoms of diseases. a questionnaire that includes the signs, symptoms, and laboratory investigations needed to diagnose these specific diseases with the socio-demographic characteristics of the respondents was developed. the tool was pretested on 5% of respondents from other hot springs (alaba site) and then modified as needed. the data collection team was selected based on educational status (medical degree holders) and proficiency in the local languages, sidamu afo and amharic. the data collectors and supervisors received 2 days of training prior to data collection. furthermore, one laboratory technician was assigned to each target site to collect blood samples, which were then properly stored and transported to the nearest hospital for laboratory testing. examination was done for all patients enrolled in this study and who had signs and symptoms of rheumatic arthritis by the data collectors (physicians). balneotherapy use: immersion of the body in the hot springs fully or partially for a minimum of half hour a day for at least three days was considered as a user. this test measures the level of rf, which acts as an antibody against gamma globulins in the blood. and, we are interpreting the results: a person who tests positive for rf could have ra. however, doctors cannot conclude this from an rf test alone, as several other conditions can increase the amount of rf in the body, including gout. similarly, a negative rf test result is insufficient evidence to confirm that a person does not have ra. erythrocyte sedimentation rate is also called esr or "sed rate". this test measures how fast red blood cells cling together, fall, and settle (like sediment) at the bottom of a glass tube over the course of an hour. the higher the sedimentation rate, the greater the amount of inflammation is. an elevated esr can be caused by a variety of conditions, including infection or anemia. the criteria established by the american college of rheumatology (acr) board of directors and the european league against rheumatism (eular) executive committee were used to diagnose rheumatoid arthritis. a total score of 6 or greater from the individual scores in four domains: number and site of involved joints (score range 0-5), serologic abnormality (score range 0-3), elevated acute-phase response (score range 0-1), and symptom duration (2 levels; range 0-1). finally, the presences of rf and/or increased esr, together with the symptoms typical of ra, make it more likely that a person has ra. (supplementary table) . the data were entered using epi data software version 3.1 and transferred to spss software version 25 for analysis. data cleaning, recoding, and analysis were done using spss. descriptive statistics such as proportion mean and standard deviation measurements were used to assess the types of musculoskeletal complaints in patients using balneotherapy, and the data were coded by identifying those symptoms that were improved/not improved after using the hot spring immersion and by considering the time factor. a total of 1337 patients were enrolled in the study for the initial assessment. however, 1279 patients who had a different chronic illness were interviewed for the second time in the study, and had a response rate of 96%. the average age of the patients was 44.1 years 16.5 months. of the 867 users, 6 (67.8%) were from rural areas, 1042 (81.5%) were married, 499 (39%) could not read or write, and 500 (39.1%) were farmers. table1. four hundred seventy-three (37%) participants visit the hot spring sites on a yearly basis, with 337 (26.3%) visiting when necessary. overall, the mean duration of stay at the site of the hot springs was 4 ± 3 days. table 2 . the majority of the patients had multiple health problems. of all, 1137 (88.9%) of the patients were visiting the hot springs for joint pain followed by muscle pain 669 (52.2) (see table 3 ). patients who visited the hot springs were self-referred by 590 (46.1%), previous users by 395 (30.9%), relatives or friends by 289 (22.6%), and traditional healers by 5 (4%).1120 (87.6%) of the patients reported that the duration of the illness was less than one year. more than two-thirds of the 1040 (81.3%) patients had not sought any help before coming to the hot spring site and 237 (18.7%) of the patients had sought help for their illness. of these, 221 (93.3%) sought modern medicine and 16 (6.7%) sought balneotherapy. a total of 1137 (88.9%) patients reported joint pain. of these, 489 (43%) had joint stiffness, of which 376 (76.9%) complained of morning stiffness. of the total patients who complained of joint pain, 383 (33.7%) had tenderness (pain during touch over the joint), 141 (11%) had joint swelling with a mean length of 6.6 ± 3.2 cm and a mean width of 6.7 ± 6.4 cm, and 45 (3.9%) of the patients had a skin lesion over the joint. more than half of the patients, 640 (56.3%), reported the symptoms started less than 6 weeks. a total of 669 (52.3%) of the patients have also reported generalized muscle pain. knee (619, 54.4%) was the most commonly affected joint, followed by the hip (324, 28.5%)( see table 3 ). all 132 (10.3%) patients who got a score of 2 points and more (had complained of one or more joint pain for the duration of six or more weeks) were clinically suspected of rheumatoid arthritis, and then a blood sample was sent for serologic tests, namely, rf and esr. all of the patients consented verbally, and a blood sample was taken for a laboratory examination. the majority of 125 (94.7%) of the laboratory results were negative. 7/132 (5.3%) were positive for rf and 6/132 (4.5%) were positive for esr, with 3 of the blood samples being highly positive and 4 being low positive. therefore, 7 (5.3%) who got a score of six or more were considered to have confirmed rheumatoid arthritis. a total of 1279 patients were re-interviewed about their progress and the effect of the hot bath after 3 days of minimal use. of the total study participants who used balneotherapy, 1064 (83.2%) reported being completely relieved from the complaints they had, 117 (9.1%) were partially improved and 98 (7.7%) of the patients had no change (see table 4 ). to the best of our knowledge, this is the first study in the area which assessed the therapeutic properties of selected hot springs among patients who were visiting the site. accordingly, the majority of the patients had multiple health problems. of all, 1137 (88.9%) of the patients were visiting the hot springs for joint pain and muscle pain, 669 (52.2%). out of all cases of joint pain, 132 (11.6%) were clinically diagnosed with rheumatoid arthritis, and 5.3% were confirmed as having the disease based on a laboratory test. of the total number of study participants, 1064 (83.2%) reported complete relief from the complaints they had at the start of the bath. our study shows that the predominant users of the hot spring bath were patients with joint problems like joint pain, stiffness, and swelling. this is consistent with a systematic review on balneotherapy in medicine, which cites the united states for the beneficial effects of spa therapy on a variety of diseases and conditions. 11, 12 the study showed that patients with musculoskeletal pain were among the dominant users of the hot springs bath. this is because the patients were referred to them by the previous users to visit the site of the hot springs. the hot spring bath's effectiveness is communicated to those who benefit from it. the finding contradicts studies conducted in the united states, which found that using a hot spring bath for nonspecific joint pain resulted in a significant reduction in pain intensity. 12, 13 it has been reported that balneotherapy exerts some beneficial effects on the immune system likely due to its chemical, thermal, and mechanical properties. 14 patients with rheumatoid arthritis were also among the patients who visited the hot spring site for a hot spring bath. in addition, the majority of cases reported perceived relief of joint pain and swelling. this is because studies have confirmed that a hot bath increases muscle relaxation, reduces joint load, and increases hemodilution and diuresis. 13, 15 in addition, studies suggested that balneotherapy results in increased hormone production and effect on immune system. [14] [15] [16] patients with muscle pain are also frequent users of hot spring baths, and they report pain relief after at least 3 days of use. hot spring water baths have also been shown in studies to significantly reduce pain. 12, 13 this is due to the heat or the ionic elements of the water. 13, 17 conclusion hot spring baths for three and more days have significant therapeutic effects among patients with musculoskeletal disorders, including rheumatoid arthritis. physicians who are currently working in the area of diagnosis and treatment of patients in government and public facilities of the southern region should consider hot spring bath treatment for those patients with complaints of musculoskeletal pain, nonspecific arthritis, and rheumatoid arthritis. a hot spring bath is beneficial for everyone because it is a natural treatment with few side effects and a low cost. further study might be useful to have a concrete knowledge in wider area and the entire aspects of the balneotherapy. the data used to support the findings of this study are available from the corresponding author upon request. an internet survey of 2,596 people with fibromyalgia a proposal for a worldwide definition of health resort medicine, balneology, medical hydrology and climatology epidemiology of the rheumatic diseases thermal balneotherapy in antsirabe-madagascar: water analysis and its applications in an african context wet areas. guide to hydrothermal spa and wellness development standards. what you need to know before building. global wellness institute balneology, mineral water, and spas in historical perspective pharmacoepidemiology book: principles and practices. mcgraw-hill observations on the effect of immersion in bath spa water some clinical experience with the springs at zohar on the shore of the dead sea prevalence and associated factors of musculoskeletal disorders among cleaners working at mekelle university, ethiopia balneotherapy in medicine: a review anti-inflammatory effect as a mechanism of effectiveness underlying the clinical benefits of pelotherapy in osteoarthritis patients: regulation of the altered inflammatory and stress feedback response impact of experience on emotional wellbeing and loyalty immunological events, emerging pharmaceutical treatments and therapeutic potential of balneotherapy on osteoarthritis balneotherapy and human immune function in the era of covid-19 from in vitro research to real life studies: an extensive narrative review of the effects of balneotherapy on human immune response the biologic aspects of hydrotherapy the authors express great thanks to the hawassa university, research and technology transfer directorate office for their financial support. i express heartfelt gratitude to mrs simegne serka for her unreserved encouragement and constructive guidance and to data collectors, laboratory workers, and patients who participated in this study for their willingness to share their health status. all authors declare that the submitted work has not been published before (neither in english nor in any other language) and that the work is not under consideration for publication elsewhere. all authors contributed to the conceptualization, methodology, writing original draft preparation, and review and editing work of this manuscript. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. ethical clearance was secured from the institutional review board (irb) of the college of medicine and health sciences, hawassa university. the reference number is irb/041/19. the respondents' verbal consent was obtained before the data were collected. before collecting the blood samples from those who are eligible, the purpose and benefits of the study are clearly described. after doing so, their permission was obtained. furthermore, permission was granted by the owners of the hot springs. achamyelesh gebretsadik  https://orcid.org/0000-0002-0060-2103 supplemental material for this article is available online. key: cord-0027854-9a3t6vf7 authors: tang, yujun; li, haichang; huang, lin; wang, qiao; han, yongmei; wu, huaxiang; su, xiao; hou, xiujuan; huang, chuanbing; lin, changsong; tao, qingwen; tang, jinyang; cao, wei; xie, zhijun; wen, chengping title: yunpi qufeng chushi formula for pre-rheumatoid arthritis: study protocol for a multiple-center, double-blind, placebo-controlled randomized controlled trial date: 2022-02-14 journal: front pharmacol doi: 10.3389/fphar.2022.793394 sha: c3b2eb4c7b36c77b41a6038e51bbb64b0c9390e8 doc_id: 27854 cord_uid: 9a3t6vf7 introduction: rheumatoid arthritis (ra) is an autoimmune disease characterized by progressive bone erosion on diarthrodial joints. ra patients usually experienced three stages before final diagnosis: the health period, the pre-clinical period (immune response exists without clinical symptoms), and the pre-ra period (immune response exists with mild inflammatory manifestation). presently, there is seldom guidance referring to early intervention which is a benefit for stable disease conditions and low morbidity. prophylactic treatment is a major feature of traditional chinese medicine (tcm). in this present study, a multi-center, double-blind, placebo-controlled clinical trial is carried out to evaluate both efficacy and safety in preventing ra progression on yunpi qufeng chushi formula (yqcf). method: the multi-center, double-blind, placebo-controlled clinical trial is conducted in 13 hospitals nationwide. a total of 390 patients ages between 18 and 70 will be recruited in the trial. they will be randomly assigned to the intervention group (yqcf) and placebo group. the follow-up visit will be taken every 3 months from baseline to 1 year. diagnosis, disease activity scores, clinical disease activity index (cdai), simplified disease activity index (sdai), tcm syndrome scores, and safety assessments will be recorded at every visit. joint color doppler ultrasound, health assessment questionnaire-disability index (haq-di), and functional assessment of chronic illness therapy-fatigue (facit-f) will be recorded at baseline and the last visit. discussion: this work will provide evidence of yqcf in preventing ra progression. however, whether early intervention would benefit the controlling ra disease still needs a long-term follow-up. ethics and dissemination: protocol version 2 (201910-1). this research was approved by the medical ethics committee of zhejiang chinese medical university (2019-045). results will be published in a peer-reviewed academic journal. trial registration numbers: http://www.chictr.org.cn/index.aspx, chictr1900024166. rheumatoid arthritis (ra) is an autoimmune disease characterized by progressive bone erosion on diarthrodial joints. the global incidence of ra is about 0.5-1% (firestein et al., 2020) . although the pathogenesis of ra is not fully understood, genes, infection, and the environment have been demonstrated to be involved. ra patients usually experienced three stages before final diagnosis, namely the health period, the pre-clinical period (immune response exists without clinical symptoms), and the pre-ra period (immune response exists with mild inflammatory manifestation), and sometimes making a definite diagnosis is time-consuming . ra management emphasized the early diagnosis and targeted therapy smolen et al., 2018) which can slow down disease progression and avoid irreversible disability. however, there is little guidance to make the detailed suggestion for pre-clinical and pre-ra patients (fraenkel et al., 2021) . to date, many trials have tried to answer the definition and management of pre-clinical ra and pre-ra (lard et al., 2001; nell et al., 2004; van dongen et al., 2007; lukas et al., 2011; wevers-de boer et al., 2012; van aken et al., 2014; stamm et al., 2018; gerlag et al., 2019) . for example, van dongen et al. (2007) found that mtx was beneficial in postponing the diagnosis of ra (acr 1987 criteria), and retarding radiographic joint damage in undifferentiated arthritis (ua) patients. lukas et al. (2011) showed that early initiation of dmard therapy reduces the radiological progression of 12 months. in all, the decision supported that pre-ra or pre-clinical ra patients benefit from early initiation of dmard. prophylactic treatment is a major feature of traditional chinese medicine (tcm). in the recent pandemic of sars-cov-2, the combination of western medicine and tcm had better performance on preventing disease transformation than western medicine only (li et al., 2020; wu et al., 2021) . to date, there is no tcm-related trial aimed at preventing ra onset. nonspecific musculoskeletal (msk) symptoms with positive anticyclic citrullinated peptide (anti-ccp) antibodies patients have a high risk of developing into ra in 12 months (rakieh et al., 2015) . besides, some anti-ccp negative arthralgia patients also have a high risk of developing ra (van steenbergen et al., 2017) . thus, we would conduct a multi-center, double-blind, placebo-controlled randomized trial recruiting both anti-ccp positive and negative patients, to demonstrate the efficacy and safety of yunpi qufeng chushi formula (yqcf) in preventing the development of ra. the a total of 390 participants aged 18-70 will be recruited. inclusion and exclusion criteria are presented as follows. to date, the definition (or criteria) of pre-ra is controversial. we propose our definition based on the trials mentioned above. for the patients with positive anti-ccp antibodies: at least 1 joint involvement (any swollen or tender joint on examination) and do not meet any ra classification criteria [acr 1987 revised criteria (arnett et al., 1988) and acr/eular 2010 criteria (aletaha et al., 2010) ]. for the patients with negative anti-ccp antibodies: meet with at least 5 parameters according to the eular definition of arthralgia suspicious for progression to ra (van steenbergen et al., 2017) (details available in table 1 ). (1) meet the definition which we proposed of pre-ra. (2) the course of disease is between 6 weeks and 6 months. (3) meet with the diagnostic criteria of tcm syndrome of spleen deficiency and wind-damp blockade syndrome (available in table 2 ). (4) the men and women patients are aged 18-70 years. (5) patients provided with informed consent and signed agreement. (1) any dmards, including conventional synthetic dmards (csdmards), targeted synthetic dmards (tsdmards), and biological dmards (bdmards), have been used for treatment within 2 months before enrollment. further explanation: a patient is undertaking some dmards and is willing to take part in the study, they can stop the dmards for 2 months and subsequently recheck the necessary examination to take part in the trial. (2) patients have recently taken glucocorticoids, such as prednisone. (3) with a history of severe organ diseases or mental diseases. (4) patients are allergic to the drugs involved in the study protocol or have contraindications. subjects will be randomly allocated to either the intervention group or the control group through a central randomization system. treatment will continue for 48 weeks. the visit time will be set at 0 weeks ( nonsteroidal anti-inflammatory drugs (nsaids) will be allowed to be prescribed when the patients suffer from pain. damars and glucocorticoids will not be allowed in the study. the untaken drug will be returned. mild and moderate adverse events (aes) will change the composition of yqcf granules according to the relative granules aes. for example, sinomenium acutum (thunb.) rehder and e.h. wilson will be removed because it would cause cutaneous pruritus. severe aes will stop the intervention and the patient will withdraw. 2.6.1 primary outcomes 2.6.1.1 ra transition rate definition: pre-ra patients who fulfill any criteria of ra classification at any time will be defined as "ra transition." survival time will be recorded as the number of days between the date of informed consent and the date of transition. 3. changes in clinical disease activity index (cdai) and simplified disease activity index (sdai): measurement will be performed at every visit. the evaluation methods were described carefully in a former study (aletaha and smolen, 2005) . 4. changes in disease activity score 28 (das28): measurement will be performed at every visit. the evaluation methods were described carefully in a former study (prevoo et al., 1995) . 5. changes in tcm syndromes efficacy score scale: tcm syndromes efficacy score ranges from from 0 to 45. measurement will be performed at every visit. 6. changes in health assessment questionnaire-disability index (haq-di): measurement will be performed at baseline and week 48. the evaluation methods are available at khanna et al. (2005) . 7. changes in facit-fatigue score scale: measurement will be performed at baseline and week 48. the evaluation methods are available in the yellen et al. (1997) study. 8. changes in color doppler ultrasound semi-quantitative score of joints. measurement will be performed at baseline and week 48. we consider an ultrasound score based on 7 joints of the clinically dominant hand and foot evaluation methods (backhaus et al., 2009 ). 9. changes in crp, esr, rf, and anti-ccp. crp and esr will be performed at every visit. rf and anti-ccp will be performed at baseline and week 48. aes will be monitored for 48 weeks from the time when the subject signed the informed consent and enrolled in the trial to the last follow-up. especially, visit 2 (4 weeks ± 2 days) is set to monitor the aes. the description of severity (grades) and system organ class (soc) of aes will follow the common terminology criteria for adverse events (ctcae) version 5.0 (services, 2017). the aes would be coded using the current version of the medical dictionary for regulatory activities (meddra). the relationship between aes and intervention will be judged according to the adverse drug reaction report and testing manual published by the department of drug safety supervision, state food and drug administration of the people's republic of china (guan et al., 2008) . the study will last 3 years: from july 2019 to july 2022. the timeline and study procedure are available in table 3 and figure 1 . based on the primary outcome, we use the model of "tests for two proportions" in pass 15.0. alternative hypothesis: twosided. test type: z-test (unpooled). power was set at 0.9 and alpha was set at 0.05. the intervention group and control group were allocated a 1:1 ratio. the effect size of the treatment group (p1): based on the results of our former small sample of observational study (results unpublished), about 53.26% of patients would transit to ra after 1 year of tcm treatment. the effect size of the control group (p2): without any medication interventions, about 70% of early-stage ua patients with acpas meet the 1987 acr criteria 1-year follow-up (firestein et al., 2016) . we would need 173 subjects in each group. predicting a 10% dropout rate, the subjects rise to 385. considering there were 13 centers that took part in the studies, the final subject was set at 390 (30 subjects per center). for achieving adequate participant enrolment: 1) we placed the recruitment advertisement in the hospital outpatient waiting hall; 2) we published the announcement in a wechat individual official account (one of the most popular social networking software in china); and 3) we referred patients who meet inclusion criteria in cooperation with primary care units. drug clinical trial data management system [abbreviated as electronic case report form (ecrf) system)] developed by nanjing haitai medical information system co., ltd. is a secure web application for building and managing online surveys and databases. sequence generation: a multicenter block random method was used. the random number of multicenter groups was generated through the corresponding randomization program of sas software (version 9.4). the relevant parameters and results of the sas randomization program were saved by zhejiang chinese medical university (study design unit). the sequence was uploaded to the ecrf system and researchers took part in the study retrieve sequence for recruiting patients. zhijun xie (one of the designers), outcome assessors, and workers in the drug manufacturing center are not masking (participants and investigators are blinding). in the real practice of tcm diagnosis and treatment, tcm doctors usually prescribe 14-d to 28-d treatment based on the patients' symptoms and tcm patents. thus, the researchers would upload the prescriptions and patient express information to zhijun xie for simulating a real tcm situation. subsequently, zhijun xie would upload patient express information and which drug (placebo or yqcf, including drug compatibility) the manufacture center will process. the product would be delivered to the patients directly based on their express information. all the participants who save the patient personal privacy information strictly abide by the confidentiality regulations. data will be collected in the paper and electronic crf via the clinic and the internet (using the ecrf system), respectively. paper crf will be delivered to clinical centers. training and support for using the ecrf system will be made available to researchers by the zhejiang chinese medical university. for promoting participant retention and complete follow-up, firstline investigators will inform the patients when they should go to the hospital to complete follow-up via telephone and wechat. two independent investigators will check the data values. the final ra transition rate will be performed by pearson's chisquare or fisher's exact chi-square tests. in addition, ra transition survival analysis will be performed by kaplan-meier survival analysis. binary logistic regression is utilized to investigate the multiple variables which can affect ra transition rate. the variables include sex, age, anti-ccp and rf state, family history, das28, continuous nsaids use, and yqcf use. per-protocol (pp) analysis and intention-to-treat (itt) analysis will be applied in chi-square tests, survival analysis, and binary logistic regression. all the secondary outcomes will be summarized in mean ± sd and analyzed by repeated measurement analysis of variance. in addition, scores of das28, cdai, and sdai will be classified as disease remission, low disease activity, moderate disease activity, and high disease activity according to the relevant definition (prevoo et al., 1995; aletaha and smolen, 2005) . the categorical data will be performed by chi-square tests by row sums (different disease activities) and column sums (groups). safety outcomes in itt data will be summarized according to the different soc with ctcae grades. every category of aes will be performed by chi-square tests by row sums (different ctcae grades) and column sums (groups). in addition, we would screen out the yqcf-unrelated aes to validate the safety of yqcf. the data will be summarized and analyzed as the methods mentioned above. c c c c c combined drugs*** c c adverse event*** c c c c c distribution of medicine by express mail c c c c c end of study summary c *record medications that were used and discontinued prior to the study. drugs that were not discontinued prior to the study and entered the treatment period should be recorded in "combined drugs". **only liver function needs to be tested at 2 weeks. ***observe continuously throughout the study and record when it occurs. the subgroup analysis of the primary outcome and the secondary outcome will be based on anti-ccp positive or negative. zhejiang chinese medical university led a steering committee and data monitoring committee (dmc) which is composed of experienced clinical researchers and academic investigators in this field. the committee will carry every month online and half a year of on-site auditing. the committee will oversee the data and safety of the study and will conduct primary outcome interim analyses using lan-demets with o'brien fleming to oversee the efficacy and safety when it reaches 50% of the recruitment. moreover, the committee would assess the status and quality of the study, such as the rate of recruitment and withdrawal. overall, the ygcg is relatively safe, and the aes are mild. every patient and their family members could report any aes/conditions to the investigators via wechat and the investigators would immediately take action. this trial has been approved by the zhejiang chinese medical university ethics committee under number 2019-045. approval has been obtained from the local institutional review boards at all participating centers. the results of rct will be published in peer-reviewed journals. all subjects were required to obtain permission to publish the results of the study and to ensure anonymity and confidentiality. all data will be processed under the rules of the government and law. all researchers will guarantee the anonymity of patients and will not disclose the names of patients in forms, reports, or articles unless required by law. only authorized individuals can obtain patient information. nowadays, more and more evidence points out the importance of early diagnosis and treatment of ra smolen et al., 2020) . however, there is a lack of treatment schemes for pre-ra patients. the trial is the first multi-center, double-blind, placebocontrolled rct carried out in china to evaluate the tcm in stopping the pre-ra progression. it will provide evidence of tcm in disease prevention of ra. in this study, we also focus on health-related assessment (such as haq-di and facit-fatigue) and ultrasound changes in pre-ra patients. based on the trial, we will conduct an observational study to evaluate the economic benefit, efficacy, and safety of our treatment scheme as well. in this study, we propose our definition of pre-ra. studies have shown that anti-ccp antibody-positive makes a great contribution to the development of ra and many of the arthralgia individuals with anti-ccp antibody-positive would progress to ra in the first year (rantapää-dahlqvist et al., 2003; van steenbergen et al., 2017) . anti-ccp antibodynegative individuals were likely to progress to other rheumatism diseases instead of ra (hiura et al., 2013) . however, anti-ccp antibody-negative individuals who meet the eular definition have a high risk of developing ra . thus, we recruit anti-ccp antibody positive and negative individuals to provide accurate evidence in the treatment of pre-ra. moreover, anti-ccp antibody status would affect the efficacy of dmards in pre-ra. for example, methotrexate could not stop the anti-ccp-negative individuals to progress to ra (van aken et al., 2014) . tcm may prevent pre-ra progression by targeting multiple molecules regardless of the anti-ccp antibody status. in this study, the following measures have been taken for diagnosis and differential diagnosis of pre-ra: according to the ra classification criteria, joint involvement, up to 5 scores and only 1 point away from the ra diagnostic threshold, is defined as: any swollen or tender joint on examination, which may be confirmed by imaging evidence of synovitis (aletaha et al., 2010) . the exact joint involvement assessment is critical in pre-ra diagnosis. both ultrasound and mri are effective techniques to evaluate joint inflammation. however, in view of the advantages of low cost and convenience, ultrasound may be a better choice in pre-ra diagnosis (xu et al., 2017) . other antinuclear antibodies (ana) also would be detected at baseline for diagnosing other rheumatoid diseases. this study also has certain limitations. the sample size and trial duration are calculated based on the anti-ccp-positive individuals. anti-ccp level not only affects the efficacy of dmards but also is related to the onset time of arthritis (burgers et al., 2017) . anti-ccp-positive individuals are more likely to develop arthritis and ra as compared to anti-ccp-negative individuals. more subjects and longer follow-up visits are needed in prognosis analysis. the guidelines published by acr and eular stated that an early treat-to-target (t2t) strategy will improve outcomes, such as bone erosions (smolen et al., 2020; fraenkel et al., 2021) . however, due to the limited observation time, we cannot answer whether the use of tcm schemes can achieve better outcomes. the studies involving human participants were reviewed and approved by the zhejiang chinese medical university committee (2019-045). the patients/participants provided their written informed consent to participate in this study. yt: parts of the study design, manuscript drafting. hl: manuscript drafting, additional files preparation. hl, qw, yh, hw, xh, qt, jt, cl, and xs: study design discussion. wc: manuscript review. zx: manuscript review and study design. cw: manuscript review, study design, and project funding. all authors approved the final submitted version of this protocol. zhejiang nature science foundation (lq20h270006) rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative the simplified disease activity index (sdai) and the clinical disease activity index (cdai): a review of their usefulness and validity in rheumatoid arthritis diagnosis and management of rheumatoid arthritis: a review the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis evaluation of a novel 7-joint ultrasound score in daily rheumatologic practice: a pilot project differences in the symptomatic phase preceding acpa-positive and acpa-negative ra: a longitudinal study in arthralgia during progression to clinical arthritis 2016 update of the eular recommendations for the management of early arthritis firestein & kelley's textbook of rheumatology-e-book kelley and firestein's textbook of rheumatology e-book 2021 american college of rheumatology guideline for the treatment of rheumatoid arthritis effects of b-cell directed therapy on the preclinical stage of rheumatoid arthritis: the prairi study evaluation criteria of adr case report the diagnostic utility of matrix metalloproteinase-3 and high-sensitivity c-reactive protein for predicting rheumatoid arthritis in anticyclic citrullinated peptide antibody-negative patients with recent-onset undifferentiated arthritis responsiveness of the sf-36 and the health assessment questionnaire disability index in a systemic sclerosis clinical trial early versus delayed treatment in patients with recent-onset rheumatoid arthritis: comparison of two cohorts who received different treatment strategies network analysis indicating the pharmacological mechanism of yunpi-qufeng-chushi-prescription in prophylactic treatment of rheumatoid arthritis the role played by traditional chinese medicine in preventing and treating covid-19 in china favorable effect of very early disease-modifying antirheumatic drug treatment on radiographic progression in early inflammatory arthritis: data from the étude et suivi des polyarthrites indifférenciées récentes (study and followup of early undifferentiated polyarthritis) benefit of very early referral and very early therapy with disease-modifying anti-rheumatic drugs in patients with early rheumatoid arthritis modified disease activity scores that include twenty-eight-joint counts. development and validation in a prospective longitudinal study of patients with rheumatoid arthritis predicting the development of clinical arthritis in anti-ccp positive individuals with non-specific musculoskeletal symptoms: a prospective observational cohort study antibodies against cyclic citrullinated peptide and iga rheumatoid factor predict the development of rheumatoid arthritis common terminology criteria for adverse events (ctcae) version 5.0 rheumatoid arthritis eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update induction of sustained remission in early inflammatory arthritis with the combination of infliximab plus methotrexate: the dinora trial efficacy of methotrexate treatment in patients with probable rheumatoid arthritis: a double-blind, randomized, placebo-controlled trial frontiers in pharmacology | www.frontiersin.org remission induction therapy with methotrexate and prednisone in patients with early rheumatoid and undifferentiated arthritis (the improved study) traditional chinese medicine as a complementary therapy in combat with covid-19-a review of evidence-based research and clinical practice comparison of the clinical effectiveness of us grading scoring system vs mri in the diagnosis of early rheumatoid arthritis (ra) measuring fatigue and other anemia-related symptoms with the functional assessment of cancer therapy (fact) measurement system the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2022.793394/ full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.copyright © 2022 tang, li, huang, wang, han, wu, su, hou, huang, lin, tao, tang, cao, xie and wen. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0006665-of0x1h34 authors: ghezzi, pietro; cerami, anthony title: tumor necrosis factor as a pharmacological target date: 2005 journal: mol biotechnol doi: 10.1385/mb:31:3:239 sha: 7c506ebb4a4c74b60b7b21616ad5c7f7b14b2e22 doc_id: 6665 cord_uid: of0x1h34 as indicated by its name, tumor necrosis factor (tnf), cloned in 1985, was originally described as a macrophage-derived endogenous mediator that can induce hemorrhagic necrosis of solid tumors and kill some tumor cell lines in vitro. unfortunately, its promising use as an anticancer agent was biased by its toxicity, which was clear soon from the first clinical trials with tnf in cancer. almost at the same time tnf was being developed as an anticancer drug, it became clear that tnf was identical to a mediator responsible for cachexia associated with sepsis, which was termed cachectin. this research led to the finding that tnf is, in fact, the main lethal mediator of sepsis and to the publication of a huge number of articles showing that tnf inhibits the toxic effects of bacterial endotoxins, which are now described as systemic inflammatory response. although the clinical trials with anti-tnf in sepsis have not been successful thus far, undoubtedly as a result of the complexity of this clinical setting, these studies ultimately led to the identification of tnf as a key inflammatory mediator and to the development of anti-tnf molecules (soluble receptors and antibodies) for important diseases including rheumatoid arthritis and crohn’s disease. on the other side, the mechanisms by which tnf and related molecules induce cell death have been studied in depth, and their knowledge might, in the future, suggest means of improve the therapeutic index of tnf in cancer. the 1970s and 1980s were the golden age of cytokines when the biochemical nature of several soluble mediators was clarified. cellular immunologists then identified macrophage-derived mediators that activate lymphocytes (laf, lymphocyte-activating factor) and lymphocyte-derived mediators that activate macrophages (maf, macrophage-activating factor). these molecules are in the class of mediators defined as growth factors, which include hematopoietic growth factors (that now retain those names: g-csf, gm-csf, epo), and interferons (described as antiviral factors in the late 1950s). one particularly active field was that of the research of soluble mediators that could kill tumor cells or boost anticancer defense. along this line, earlier studies focused on a lymphocyte-derived cytotoxin termed lymphotoxin (lt), and led to the discovery of a serum factor capable of inducing hemorrhagic necrosis of tumors in vivo and killing of tumor cells in vitro. this factor was termed tumor necrosis factor (tnf) and shown to be mainly a macrophage product, as opposed to lt. in 1985 several groups reported the cloning of human and mouse tnf and the ability of recombinant tnf to induce hemorrhagic necrosis of tumors in mice. it would have not been easy, 15 yr ago, to predict that the main clinical application of the discovery and characterization of tnf would have consisted in the administration of anti-tnf molecules for the therapy of rheuma-molecular biotechnology volume 31, 2005 toid arthritis and crohn's disease. in fact, tnf turned out to be a key pathogenic mediator with pleiotrophic activities, and its history and road, from immunity to inflammation, very similar to that of interleukin (il)-1. also unexpected is the fact that the characterization of the inflammatory action of tnf stemmed from studies on models of sepsis. studies on the molecular basis of cachexia associated with sepsis led to the finding that macrophages activated with endotoxin, used to reproduce settings of septic shock, release a factor that is cachectogenic in vivo and inhibits lipogenesis in cultured adipocytes. we termed this factor cachectin and, when we purified it, found that it was identical to tnf. these earlier studies pointed out a pathogenic role for tnf in sepsis and inflammation, confirmed by earlier clinical trials with recombinant tnf in cancer patients showing toxicity in phase i and ii studies. the first studies of neutralization of endogenous tnf have shown that this cytokine is a lethal mediator associated with toxicity of endotoxin (1) and septic shock induced by live bacteria (2). these studies have pointed at the possible use of anti-tnf antibodies in the therapy of septic shock. however, the clinical trials conducted so far have not indicated a clear efficacy of anti-tnf in septic shock. after a period of great enthusiasm, during which septic shock was considered the prototypic cytokine-mediated disease, the complexity of this pathological condition, which is associated with other diseases such as cancer, trauma, or burn injury scared most of the big pharmaceutical companies from pursuing this line. some attempts have been made toward a narrower definition of the component of septic shock, including acute respiratory distress syndrome (ards) and multiple organ failure (mof), in which cytokines play an important role. in 1992 the american college of chest physicians and the society of critical care medicine consensus conference introduced the term systemic inflammatory response syndrome (sirs) (3). despite these difficulties, the scientists working in the field of cytokine and inflammation have continued using the models of lipopolysaccharide (lps) toxicity as a means of inducing a systemic inflammatory response (to stick to the above-mentioned definition). the studies on the role of tnf and il-1 in septic shock have stimulated a large number of studies on these cytokines in several models of inflammation. these included demonstration of inflammatory effects of tnf administration in various models in vivo and in vitro, as well as reports of increased tnf production in patients or animal models of diseases including rheumatic diseases. soon after their characterization, cytokines were suggested to be involved in arthritis. the same year tnf was cloned, dayer et al. (4) and saklatvala (5) reported that tnf was able to induce prostaglandin and collagenase production by synovial cells and stimulate resorption in cartilage, and suggested its pathogenic role in rheumatoid arthritis. the development of anti-tnf antibodies was the first strategy to inhibit tnf (discussed previously). anti-tnf antibodies, then soluble tnf receptors (discussed later), and, more recently, il-1ra, are now approved drugs for the therapy of rheumatoid arthritis or crohn's disease. retrospectively, one can say that the models of lps toxicity in vivo have been predictive of an anti-inflammatory action in diseases where inflammation is induced in the absence of sepsis. it is impossible to cite all the molecules that have been shown to inhibit tnf production or action. from the perspective of basic research and immunopathology, the endogenous inhibitors of tnf are particularly interesting, but also indicates inhibitors of tnf production of possible pharmacological interest. this was particularly evident molecular biotechnology volume 31, 2005 for one inhibitor, soluble tnf receptor (stnfr), that is now a widely used anti-tnf drug. as early as 1988 seckinger et al. reported the existence of a tnf inhibitor in human urine (6), soon identified as a soluble form of the tnf receptor (7,8) . as a consequence of these studies, administration of recombinant soluble tnf receptor, both the native molecule and the engineered fc fusion protein, developed to increase the plasma half-life, were tested in models of disease and are at the basis of the current use of these molecules in patients with inflammatory diseases. glucocorticoids have been the first reported inhibitors of tnf production (9). their action is mediated by the glucocorticoid receptor and reversed by gc receptor antagonist mifepristone (10). it should be noted that other steroids, namely neurosteroids, inhibit tnf production by a glucocorticoid receptor-independent mechanism (11). endogenous glucocorticoids probably represent the most important feedback system to limit tnf production, as demonstrated by the augmentation of tnf-mediated endotoxic shock in adrenalectomized mice, known for a long time (12,13) , and by similar results obtained with mifepristone (14,15). it is now recognized that tnf increases serum glucocorticoid levels through the activation of the hypothalamus pituitary adrenal axis (16). the inhibitory effect of the phosphodiesterase inhibitors on tnf production has been described soon after the discovery of tnf. in particular, pentoxifylline and rolipram have been widely used to inhibit tnf production in several animal models. their inhibition is mediated by the increase in intracellular cyclic amp (camp). likewise, other agents that augment intracellular camp (particularly prostaglandin e 2 ) inhibits tnf production. in fact, prostaglandin e 2 is another very important feedback inhibitor of tnf (and other cytokines) production because inhibitors of prostaglandin synthesis (cyclooxygenase inhibitors also known as nonsteroidal anti-inflammatory drugs) augment cytokine production in most models ranging from in vitro systems (17) to human volunteers injected with lps and in vivo (18,19) . this effect demonstrates that prostaglandin e 2 endogenously produced during inflammation effectively switches off tnf synthesis. based on these findings, several clinical trials have been initiated using phosphodiesterase inhibitors to augment intracellular camp, such as rolipram or pentoxifylline. clearly, this approach is not specific for tnf. interleukin-10 and il-4 are the prototypic "anti-inflammatory cytokines" and inhibit tnf production in vitro and in vivo (20) (21) (22) . this effect was later demonstrated with il-13 (23) and other cytokines of the so-called il-10 family (24). these anti-inflammatory cytokines (according to pubmed this term was actually used first for il-4; see ref. 22) are being investigated as possible antiinflammatory drugs. studies by borovikova et al. (25) and tracey (26) using vagotomized animals or electrical stimulation of the vagus nerve have shown that efferent activity in the nerve inhibits tnf production and has anti-inflammatory actions. this pathway has been termed the cholinergic anti-inflammatory pathway, as inhibition of tnf synthesis is mediated by acetylcholine acting on nicotinic bungarotoxin-sensitive acetylcholine receptors on macrophages. this finding provides a new means of inhibiting tnf production by electrical or chemical methods. it is of no surprise that the effectiveness of recombinant proteins acting as tnf inhibitors has prompted the research of small molecular weight drugs to act as inhibitors of tnf production and that might be administered orally. volume 31, 2005 several classes of drugs have been reported to act in this context, but none, as far as we know, are specific for tnf. in particular, no small tnf receptor antagonists have been described to the best of our knowledge. following is a (partial) list of drugs, or classes of drugs, that reportedly inhibit either tnf production or tnf action and that have been shown to be efficacious in animal models of inflammation. nuclear factor (nf)-κb is a transcription factor implicated in the expression of several inflammatory genes including tnf, and it is regarded as a major pharmacological target for anti-inflammatory drugs. a long list of well-known molecules were reported to inhibit nf-κb, including antioxidants (27), glucocorticoids (28), aspirin, and salicylates (29). the tnf-α is synthetized as a membraneanchored translation product. it is processed to mature tnf, which is then released, by tnf-αconverting enzyme (tace), a membrane protease in the class of the adam proteases (that contain a disintegrin and a metalloprotease domain). inhibitors of tace are thus potential anti-tnf molecules and some have been shown active in various animal models of tnf-mediated pathologies (30). this old drug was shown to inhibit tnf production in 1991 (31,32) . it is now considered for rheumatoid arthritis and crohn's disease, and the search for analogs without its teratogenic properties is actively pursued. unlike the camp pathways, the p38mapk pathway has been identified only after the discovery of tnf and il-1. this kinase was originally described by lee et al. (33) . by studying the mechanism of action of a new series of compounds acting as cytokine synthesis inhibitors they identified p38 mapk by photoaffinity label-ing. this kinase was soon identified as a key step in the pathway leading to cytokine production and action. another compound that was described as an inhibitor of tnf production and was then found to act probably by inhibiting p38mapk is the guanylhydrazone cni1493 (34,35) . cni1493 showed promising activity in patients with crohn's disease (36). although this short historic overview was focused on the pro-inflammatory actions of tnf and anti-tnf strategies, it is important to remember that tnf is also a key mediator in host defense and innate immunity. these are probably exemplified by the increased incidence of infections in patients with arthritis given anti-tnf molecules, an observation that is now incorporated in the prescription information for these drugs, advising to avoid use in patients with underlying sepsis (see prescription information). although this finding was not totally unexpected (other drugs used for the therapy of rheumatoid arthritis, methotrexate and glucocorticoids, are, by definition, immunosuppressive drugs), it reinforces the animal data showing that tnf is a key molecule in the innate immunity to infection. nevertheless, the successful therapeutic applications with anti-tnf molecules for a variety of diseases stresses the deleterious effects of its overproduction. passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin anti-cachectin/tnf monoclonal antibodies prevent septic shock during lethal bacteraemia definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis cachectin/tumor necrosis factor stimulates collage-molecular biotechnology nase and prostaglandin e2 production by human synovial cells and dermal fibroblasts tumour necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage a human inhibitor of tumor necrosis factor alpha characterization of a tumor necrosis factor alpha (tnf-alpha) inhibitor: evidence of immunological cross-reactivity with the tnf receptor two tumor necrosis factor-binding proteins purified from human urine. evidence for immunological cross-reactivity with cell surface tumor necrosis factor receptors control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance antagonist effect of ru 486 on transcription of glucocorticoid-regulated genes a glucocorticoid receptor-independent mechanism for neurosteroid inhibition of tumor necrosis factor production role of adrenal cortex in physiological processes studies on mechanism of chloropromazine protection against brucella endotoxin the influence of a novel glucocorticoid antagonist on endotoxin lethality in mice strains effect of ru 38486 on tnf production and toxicity synergistic roles of interleukin-6, interleukin-1, and tumor necrosis factor in the adrenocorticotropin response to bacterial lipopolysaccharide in vivo prostaglandin e2 regulates macrophage-derived tumor necrosis factor gene expression pretreatment with ibuprofen augments circulating tumor necrosis factor-a, interleukin-6, and elastase during acute endotoxemia detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia interleukin-10 (il-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of il-10 produced by monocytes il-4 suppresses cytokine gene expression induced by ifn-gamma and/or il-2 in murine peritoneal macrophages effects of il-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. comparison with il-4 and modulation by ifngamma or il-10 viral and cellular interleukin-10 (il-10)-related cytokines: from structures to functions vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin the inflammatory reflex intracellular thiols regulate activation of nuclear factor kappa b and transcription of human immunodeficiency virus immunosuppression by glucocorticoids: inhibition of nf-kappa b activity through induction of i kappa b synthesis inhibition of nfkappa b by sodium salicylate and aspirin tace and other adam proteases as targets for drug discovery thalidomide selectively inhibits tumor necrosis factor alpha production by stimulated human monocytes thalidomide exerts its inhibitory action on tumor necrosis factor alpha by enhancing mrna degradation a protein kinase involved in the regulation of inflammatory cytokine biosynthesis suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone the critical role of p38 map kinase in t cell hiv-1 replication inhibition of stress-activated map kinases induces clinical improvement in moderate to severe crohn's disease key: cord-0009885-r2sz2dm9 authors: holmdahl, rikard; vingsbo, carina; hedrich, hans; karlsson, mikael; kvick, christina; goldschmidt, tom j.; gustafsson, kenth title: homologous collagen‐induced arthritis in ratg and mice are associated with structurally different major histocompatibility complex dq‐like molecules date: 2005-11-17 journal: eur j immunol doi: 10.1002/eji.1830220220 sha: 8936065192a304b6c8ac8fd0b2e1bbe66327369a doc_id: 9885 cord_uid: r2sz2dm9 collagen‐induced arthritis (cia) in rats, induced with homologous type ii collagen (cii), is a genetically more restricted disease and has better resemblance to rheumatoid arthritis by its chronic disease course, than cia induced with heterologous cii. the da strain is highly susceptible to cia induced with homologous cii, while the lewis strain is resistant. (daxlew)f(1) is susceptible and backcrossing to lewis reveals a close, but not complete, association of both arthritis and cii responsiveness to the rt1(a) haplotype. analyses of congenic strains on da and lewis backgrounds suggest that expression of a major histocompatibility complex class ii b(a) molecule, encoded from the rt1b(a) locus, is associated with arthritis susceptibility and cii responsiveness. the second exons coding for the first domains of the α and β chains of both the rt1(a) and rt1(1) haplotypes were sequenced and the deduced amino acid sequences compared with the corresponding molecule associated with susceptibility to cia in the mouse (h‐2 a(q)). the sequences of the respective alleles revealed no obvious structural homology explaining the extensive similarities in the development of chronic autoimmune arthritis. instead, this finding implies that different trimolecular constituents (i.e. class ii,t cell receptor, and cii peptides) may yield an antigen presentation event that is able to trigger a similar autoaggressiveness in the two rodent species. rheumatoid arthritis (ra) is a disease of uncertain etiology although recent data suggest a t cell-dependent autoimmune inflammatory attack on joints to be a crucial event in the disease. one of the most exciting recent findings is the genetic association of ra with certain mhc haplotypes. it was recently suggested that classical, severe, ra is closely associated with class i1 molecules sharing structures in the third and fourth hypervariable region of the drb chain [ 1-31. this is the hitherto best-defined genetic association of the disease and supports an important role of antigen specific recognition by autoreactive tcells. however, it has not yet been possible to demonstrate which self peptides, or which autoreactivet cells, are of importance.these interactions, and their importance for the development of arthritis, need to be studied in vivo and for this purpose animal models are useful. the only model for ra in which susceptibility to arthritis has reproducibly been shown to be mhc associated is the type i1 collageninduced arthritis (cia). in the mouse, immunization with type i1 collagen (cii) of heterologous or homologous [7] origin leads to arthritis only in h-2'1 and h-2' haplotype strains. the ability to respond to homologous cii was found to be critical for arthritis susceptibility since immunization with heterologous cii induced a strong immune response to cii in mouse strains with other h-2 haplotypes, whereas immunization with homologous cii evoked an immune response to cii only in h-2s and h-2' strains. similarly to ra, small structural differences between class i1 molecules determine arthritis susceptibility, as demonstrated by the finding that only four amino acid differences located in the 85,86,88 and 89 position of the ag chain of q, as compared with p, determine cia susceptibility and immune responsiveness to cii [8] [9] .the rat mhc has been termed rt1. the rat homologues of the mouse h-2 class i1 genes, a,, ap, e, and eg (corresponding to the human dqa, dqb, dra and drb), have been termed rt1-b,, bg, d, and dg, respectively. in the rat, the mhc association has been less clear than in the mouse. immunization with heterologous cii leads to the highest incidence of arthritis and the strongest anti-cii immune response in strains with rtiu, rtll and rtla haplotypes, although no haplotype confers resistance to arthritis [lo-111. analysis of the mhc association of cia induced with homologous cii in rats is needed for three reasons. first, immunization with homologous cii may induce a disease more similar to ra with a chronic development of arthritis [12] . second, the disease induced with homologous cii is critically dependent on the activation of autoreactive t cells, whereas in the disease induced with heterologous cii the disease is dependent on the activity of tcells reactive with heterologous cii [13] . third [14] . for induction of cia, native cii were disolved in 0.1 m acetic acid at 4 "c and emulsified 1 : 1 on ice with ifa (difco, detroit, mi) to a final concentration of 0.5 mg/ml. rats were injected intradermally in the base of the tail with 300 pl of the emulsion. arthritis development was followed for approximately 4 months after immunization by inspections using a macroscopic scoring system for the four paws ranging from 0 to 4 (1 = swelling and/or redness of one toe or finger joint, 2 1 two joints involved, 3 = more than two joints involved, and 4 = severe arthritis in the entire paw with functional gait and grip impairment). the maximal score obtained during the disease course for each rat was determined. to ascertain that only severity, and not incidence, is reflected, the mean value of the maximal scores of arthritic rats only are shown. sera were collected individually and stored at -20 "c until assayed. for the quantification of cii-reactive autoantibodies in serum, a modified elisa was used [6] . micro-elisa plates (dynatech, plochingen, frg) were coated overnight at 4°c with 10 pg/ml of native rat cii in phosphate-buffered saline. all tests were carried out in duplicates, and the standard deviations did not exceed 10%. the amount of bound antibody was estimated after incubation with a goat anti-rat igg (h+l) affinity-purified antibody conjugated to alkaline phosphatase (jackson immunoresearch laboratories, west groove, pa). the subsequent quantification of bound enzyme was performed with a p-nitrophenol-containing substrate buffer in a titertek multiscan (flow labs., irvine, scotland) spectrophotometer. to estimate the amount of cii-reactive antibodies present in the serum samples, affinity-purified rat c11-reactive antibodies were used as standards. genomic dna was prepared from liver tissue of da and lewis rats as described earlier in detail [8, 9] .the following primers were synthesized on an applied biosystems 380a (warrington, gb) oligonucleotide synthesizer and used for amplification of exon 2 encoding the bg chain first domain; la3 : 5'-atggatccgtccgtccgcagggcattt-3', r1: 5'-cccgaattcgcgctcaccaagccgccg-3'. the following primers were used for the amplification of exon 2 coding for the b, chain first domain; r2 : 5'-cgcg-aattcagggtggtgagcacgtac-3'. the polymerase chain reaction (pcr) amplifications were carried out using 1 pg of genomic dna, oligonucleotide primers at a concentration of 1 p~ and 2.5 units taq polymerase (perkin-elmer, cetus, emeryville, ca) in a final volume of 100 pl. the amplified products were cloned into m13mp18 and/or m13mp19 and the nucleotide sequence determined using the chain termination method [15] .the sequence of at least three different m13 clones were determined, in order to detect pcr misincorporations. gatcccacctaaattcctcagcc-3', r3 : 5'-ccggincidences of arthritis were analysed by their proportionate group frequencies (two-tailed x2 test with continuity correction) and the mann whitney u-test was used for analysis of arthritic scores. antibody levels were analyzed by the two-tailed student's t-test. the da rat strain develops chronic arthritis after immunization with homologous rat cii emulsified in ifa (the siveness and arthritis development. a comparison with the lewis rats, which are resistant to arthritis but develop low levels of anti-cii autoantibody levels, shows an influence of non-mhc background genes. the development of arthritis in 1/1 rats did not correlate with development of an anti-cii autoantibody response, in fact, some of these rats had no detectable (< 1 pg/ml) anti-cii antibodies in serum. to investigate the role of the class i1 region and whether (table 4 ). not unexpectedly, the association is with the class ii region since the da.ll strain, carrying non-a alleles flanking the class i1 region, was as highly susceptible as the da rat. furthermore, it is likely that the susceptibility is associated with the mhc class 11 molecule encoded from the b locus since the lewlc rat,which was resistant to arthritis, carries the c haplotype known to express a d molecule indistinguishable from that of the a haplotype [16, 171. the congenic strains carrying other rt1 haplotypes were low responders to cii except the u haplotype which developed a strong anti-cii autoantibody response but no arthritis. one rat of the f haplotype developed arthritis, althoughvery late after immunization, and this single rat developed a relatively high antibody response to cii. a lewis rat developed mild arthritis in a proximal interphalangeal joint, late after immunization, but did not develop a cii autoantibody response. taken together, these findings suggest that self cii responsiveness and development of arthritis after immunization with homologous cii are associated with expression of a b" molecule whereas other rt1 haplotypes confer weak and variable responses. r. holmdahl we have earlier shown that cia in mice is associated with expression of an aq molecule, both when induced with rat cii or with homologous mouse cti. since the a molecule in the mouse corresponds to the b molecule in the rat, and since in both species chronic arthritis can be induced with homologous cii 112, 181, we wanted to compare the structures of these molecules. for this purpose we sequenced the second exons, encoding the first domains of and ap first domains from mice and rats. the aq molecule has earlier been showed to be associated with development of cia induced with heterologous or homologous cii while the ap molecule is associated with resistance against disease [7] . positions postulated to be involved in the abs [19] are boxed. the b, and be molecules, of the susceptible rtla haplotype and the resistant rtll haplotype (fig. 1) . the deduced amino acid sequences showed a number of differences between the two haplotypes (fig. 2) . several of these differences were found in the postulated antigen-binding site (abs) [19] .thus, of a total of 16 abs residues in the @ chains, 4 were different between the haplotypes. the corresponding figures for the a chains were 8 differences of a total of 20 abs residues. an alignment of the respective rat a and @ chain amino acid sequences with the corresponding mouse a molecule sequences showed no obvious structural homology which could explain the similarities in responsiveness to cii in the two species. instead, of the 20 m chain abs residues, 8 differed in all comparisons between the species. in the chains, of the 16 abs residues, 11 differed between the non-susceptible rt1 b1 and h-2 ap, whereas 10 out of 16 residues differed, in the abs, between the susceptible rt1 b" and h-2 as haplotypes. in fact, the susceptible rat ba molecule was found to share the sequence in the critical region, position 85-89 in the ap chain, with the mouse ap molecule, which is associated with resistance to cia in the mouse model [8] . in this report we show that the development of cia and an autoimmune response in rats, after immunization with homologous cii, are associated with the class 11 genes of the rt1" haplotype. this is different from the mhc association of arthritis susceptibility after immunization with heterologous cii where also the 1 and u haplotypes have been found to be highly susceptible to arthritis. arthritis induced with homologous cii provides several similarity with the role of tnf genes for susceptibility to murine lupus [28] . advantages as a model for ra and for studies of autoimmune arthritis compared with arthritis induced with heterologous cii. it is a chronic disease [12] in which the autoimmune disease process has been shown to be driven by alp receptor-expressing tcells [20] and in which a tcell-dependent anti-cii autoantibody production is induced. cia induced with heterologous cii is a more self-limited disease dependent on anti-cii antibody production. immunization with heterologous cii triggers predominantly t cells reactive with heterologous cii and a strong b cell activation leading to anti-cii antibody production. in fact, it has been suggested that the pathogenic effector mechanisms are antibody mediated and do not involve cii autoreactive t cells . the earlier described association of several rt1 haplotypes may reflect their importance as restriction elements for heteroreactive tcells and does not necessarily imply a critical role for triggering of autoreactive t cells. we have analyzed earlier the immune response to heterologous and homologous cii in detail in the mouse and shown that immunization with heterologous cii induces cii-specific proliferation of tcells with no cross-reactivity to homologous cii [25] . in addition, a strong autoantibody response to cii, but no arthritis, develops after immunization with heterologous cii in mouse strains with certain h-2 haplotypes [7] , parallelling the presently described situation in the rat. in both mice and rats, after immunization with homologous c11, the class i1 dq-like molecule seems to be used as the restriction element for cii-reactivet cells of critical importance for the development of cia and a cii autoantibody response. however, a structural comparison of the a and first domains of the rat bd and the mouse aq molecules did not reveal any apparent homology which could argue for a shared structural interaction as an explanation for the development of chronic arthritis after immunization with homologous cii in the two species. this is in line with a structural comparison with class i1 genes associated with ra which yields even less similarities since the restriction clement is the dr and not the d q molecule. it follows that structural dissimilarities between mice and rats, or between rodents and humans, do not exclude similarities in the pathogenesis and etiology. thus, our data indicate a situation where, in different species, different cii peptides along the highly conserved cii molecule, are recognized by different t cell receptors in the trimolecular antigen presentation event leading to similar disease development. however, existence of shared structural motifs in the class i1 molecules which allow binding and presentation of collagen peptides, as an explanation for the mhc association of ra andd cia, cannot be excluded and structural similarities between the mouse aq vs. dr4 and rat d u vs. dr4 have earlier been highlighted [13, 26] .the latter cases are of interest since in the present investigation we found that the lewislw (rtiu) developed a strong autoantibody response after immunization with homologous cii. however, we could not find any sign of arthritis in this strain. a possible explanation could be that a different functional subtype of tcells are preferentially activated by cii in conjunction with a class i1 molecule as has been shown for the immune response to type iv collagen in mice [27] . another explanation might be that non-class i1 genes flanking the mhc or within the complex and differing between the congenic strains, influence the susceptibility to arthritis, but not the anti-cii autoimmune response, in the mhc class i1 association of cia susceptibility after immunization with homologous cii was surprisingly restricted, compared with the cia induced with heterologous cii. however, in several other haplotypes a weak but significant autoantibody response developed, and in rats with rtl' and rtlf a few animals developed arthritis. this putative responsiveness is dependent also on the non-mhc background genes as is clearly seen in the daxlewis backcross experiment and by the comparison of da and lewla strains. in the backcross experiments several lew(daxlew) rt1' homozygous rats developed arthritis without detectable anti-cii autoantibodies in serum. we have obtained similar findings in the development of cia in certain mouse strains induced with homologous cii and this supports the conclusion that cia may develop without the participation of arthritogenic antibodies, although it does not exclude a critical role of b cell activation to cii as being important for the chronicity of the disease, as has been earlier discussed [13] .the above findings do also emphasize the influence of non-mhc genes in the development of arthritis. the same genes may be of importance also in the development of arthritis after injection of non-immunogenic adjuvant oils since da, but not lewis or (dax-lew)f1 rats, are susceptible to this oil adjuvant-induced arthritis [29] .thus, the rat provides us with disease models, induced without involvement of non-self immunogens, in which the genetic contribution of both mhc and non-mhc genes to the susceptibility for autoimmune arthritis can be analyzed and compared with analyses of the genetic contribution to ra. received october 4, 1091 proc. nutl. acad. sci nucleic acids res 170: 2135. press key: cord-0001828-navjkevd authors: zhang, xiaoqing; liu, sibo; li, shentao; du, yuxuan; dou, yunpeng; li, zhanguo; yuan, huihui; zhao, wenming title: designation of a novel dkk1 multiepitope dna vaccine and inhibition of bone loss in collagen-induced arthritic mice date: 2015-05-05 journal: biomed res int doi: 10.1155/2015/765490 sha: a3a2b5d607691f30dd1b1baeca0e41817e2fde86 doc_id: 1828 cord_uid: navjkevd dickkopf-1 (dkk1), a secretory inhibitor of canonical wnt signaling, plays a critical role in certain bone loss diseases. studies have shown that serum levels of dkk1 are significantly higher in rheumatoid arthritis (ra) patients and are correlated with the severity of the disease, which indicates the possibility that bone erosion in ra may be inhibited by neutralizing the biological activity of dkk1. in this study, we selected a panel of twelve peptides using the software dnastar 7.1 and screened high affinity and immunogenicity epitopes in vitro and in vivo assays. furthermore, we optimized four b cell epitopes to design a novel dkk1 multiepitope dna vaccine and evaluated its bone protective effects in collagen-induced arthritis (cia), a mouse model of ra. high level expression of the designed vaccine was measured in supernatant of cos7 cells. in addition, intramuscular immunization of balb/c mice with this vaccine was also highly expressed and sufficient to induce the production of long-term igg, which neutralized natural dkk1 in vivo. importantly, this vaccine significantly attenuated bone erosion in cia mice compared with positive control mice. these results provide evidence for the development of a dna vaccine targeted against dkk1 to attenuate bone erosion. rheumatoid arthritis (ra), a chronic symmetrical autoimmune disease, is characterized by synovial inflammation and proliferation accompanied by cartilage erosion and bone loss [1] . more than one-third of patients eventually experience employment disability and lower quality of life because of this disease, which is largely responsible for the high socioeconomic burden of ra [2] . furthermore, mortality rates in ra patients are higher than in the healthy population [3] . the aim of ra treatment is the achievement of remission [4] , but many ra patients who are judged by their consulting rheumatologist to be in remission after receiving conventional therapy still show structural deterioration [5] . therefore, the long-term goals of treatment are to prevent joint destruction and the comorbidities of the disease [3] . a number of reports have suggested that modern therapy does not inhibit joint damage satisfactorily despite achieving clinical remission [6] . therefore, the development of effective agents to inhibit bone erosion in ra patients is urgent. the canonical wnt signaling pathway promotes bone formation not only by stimulating the differentiation of osteoblasts, increasing the growth rate of osteoblasts and reducing their apoptosis but also by inhibiting osteoclastogenesis [7] . the canonical wnt signaling pathway is triggered by the association of secretory wnt with its frizzled (fz) receptors and low density lipoprotein receptor-related protein 5/6 (lrp5/6) on the cell surface. this stabilizes -catenin, which is eventually translocated into the nucleus, and activates the tcf/lef-mediated transcription of target genes that elicit a variety of effects, including the induction of osteoblast differentiation and proliferation [8] . dickkopf-1 (dkk1), a soluble and natural inhibitor of the canonical wnt signaling pathway, may play an active role in inhibiting osteogenesis by binding the ligands of wnt proteins [9, 10] . in addition to lpr5/6, dkk1 was also found to bind to kremen, another cell surface coreceptor, forming a ternary complex that is rapidly endocytosed, resulting in the depletion of cell surface lrp5/6 [11] . studies have demonstrated that the levels of dkk1 in serum were significantly higher in ra patients and were correlated with the severity of the disease [12] . in a mouse model of ra, treatment with an anti-dkk1 antibody has attenuated bone erosion [9] . therefore, dkk1 may be a promising therapeutic target for ra bone loss. over the last 20 years, great progress has been made in developing dna vaccines [13] . dna vaccines have many advantages over conventional chemical agents, biological agents, and protein vaccines in the treatment of diseases. first, plasmid preparation is rapid and cost effective and does not suffer from problems such as improper protein folding. plasmid dna is highly stable and flexible, allowing for the modification of plasmid sequences [14] . in addition, the antigen presenting cells (apcs) process and present the epitopes from antigens on mhc i and ii molecules, thereby inducing both humoral immunity and cellular immunity. active immunotherapy is feasible to reverse the disorders of autoimmune diseases. dna vaccines have proven effective in animal models, including ra, crohn's disease, systemic erythematosus lupus (sle), and infectious diseases [15] and have been extensively evaluated in humans. the advancements of bioinformation software and molecular immunology promoted the development of dna vaccine [16] . currently, 72 phase i, 20 phase ii, and 2 phase iii clinical trials have been identified [17] . in this study, we selected a panel of twelve peptides using the software dnastar 7.1 and screened high affinity and immunogenicity epitopes in vitro and in vivo assays. then, we optimized four b cell epitopes and designed a novel dkk1 multiepitope dna vaccine, determined its immunogenicity, and evaluated its protective effects. our data demonstrated that this dna vaccine ameliorated bone erosion significantly in mice with collagen-induced arthritis (cia). mice. cos7 cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with 5% fetal bovine serum (fbs) in a humidified 5% co 2 incubator at 37 ∘ c. six-week-old female balb/c (h-2d) mice and 5-weekold male dba/1 mice (a cia-susceptible mouse strain, h-2q) were both purchased from hfk biotechnology co. ltd. all mice were maintained in a specific pathogen-free environment. all animal experiments were performed according to the guidelines of the animal care and use committee of capital medical university. preparation of the dna vaccine. b cell epitopes in the amino acid sequence of human dkk1 were analyzed using the software dnastar 7.1. the separated epitopes were synthesis from invitrogen (life technologies, california, usa). subsequently, the indirect elisa was utilized to detect the affinity of separated epitopes. simply, 96-well plates were coated with peptides (1 g/ml) overnight at 4 ∘ c. the dkk1 polyclonal antibodies (diluted 1 : 1000, 200 l, r&d systems, minneapolis, mn, usa) were added to wells and incubated overnight at 4 ∘ c. after washing, the hrp conjugate goat anti-human dkk1 secondary antibody (100 l, diluted 1 : 2000, southern biotech, birmingham, al, usa) was added and the plates were incubated for 1 h at 37 ∘ c. then, the peptides were immunized balb/c mice and the immunogenicity was measured as previously described [18] . the reactions were stopped by the addition of 50 l of stop solution (r&d systems), and od450 readings were measured with an elisa plate reader. to reduce the bioactivity of dkk1 vaccine, the peptides (100 g/injection, intraperitoneally) were applied for a week. the tibias were collected and an acid phosphatase kit (sigma-aldrich, st. louis, mo, usa) was used to demonstrate osteoclasts [19] . four fragments in the dkk1 sequence (110-144aa, 153-181aa, 182-216aa, and 228-253aa) with high affinity and immunogenicity were selected to construct the multiepitope dna vaccine. the dna sequences encoding four amino acids (r203, h204, k211, and r236) in dkk1 were mutated to glutamate to reduce the biological activity of dkk1 [20] . aay spacers between two adjacent epitope fragments were used to link the four selected epitopes. a th2 cell epitope was added at both sequence termini. finally, the signal peptide of human dkk1 was added at the n-terminus of the sequence. the synthetic nucleotide sequence, named dp, was incorporated into the expression vector pcmv6-xl5 using a standard dna recombination procedure, resulting in the recombinant plasmid pcmv-dp. plasmids for immunization were extracted and purified from transformed escherichia coli strain dh5 using an endotoxin-free plasmid extraction kit (roche, mannheim, germany) according to the manufacturer's instructions. the purified plasmids were adjusted to a concentration of 1 mg/ml in sterile saline and stored at −80 ∘ c. cos7 cells were cultured in a 6-well tissue culture plate until the cells reached approximately 60% to 80% confluence. the cells were transfected with the purified plasmid dna using turbofect in vitro transfection reagent (fermentas, thermoscientific, pittsburgh pa, usa) according to the manufacturer's instructions. briefly, 4 l of plasmid dna (1 mg/ml) was diluted in 390 l of serum-free dmem. turbofect reagents (6 l) were added to the diluted dna. after immediate mixing and incubation for 20 min at room temperature, 400 l of the mixture was distributed dropwise into cultured cos7 cells in a 6-well plate. the transfected cells biomed research international 3 were cultured for 72 h. cell lysates and culture supernatants were collected for further analyses. lysates. the proteins in the cos7 cell lysates were separated on a 12% sds-page gel and transferred onto a nitrocellulose membrane by electroblotting. the membrane was blocked with 5% nonfat dry milk in tbst (tbs with 0.05% tween-20) for 1 h at room temperature and then incubated with a polyclonal rabbit anti-dkk1 antibody (1 : 1000, millipore) in tbst containing 0.25% bovine serum albumin overnight at 4 ∘ c. after washing three times with tbst, the membrane was incubated with horseradish peroxidase-(hrp-) conjugated goat anti-rabbit igg (1 : 10000, protein-tech group, chicago, usa) for 1 h at room temperature. after washing three times, the membrane was exposed to a supersignal west pico stable peroxide solution (pierce, thermoscientific, pittsburgh, pa, usa). the protein secreted in the cell culture supernatant was detected with elisa. briefly, 96-well plates were coated with a goat anti-human dkk1 antibody (200 ng/ml, r&d systems, minneapolis, mn, usa) overnight at 4 ∘ c. the cell culture supernatant (200 l) was added to the wells and was incubated overnight at 4 ∘ c. after incubating with 100 l of a goat anti-human dkk1 antibody (50 ng/ml, r&d systems) for 2 h at 37 ∘ c, 100 l of a working dilution of streptavidin-hrp was added to each well. color was developed by the addition of a substrate solution of orthophenylene diamine (opd) for 20 min at 37 ∘ c. the reaction was stopped by the addition of 50 l of 2 m h 2 so 4 . the absorbance was read at 450 nm by a microplate reader (thermomax technologies). in vivo. balb/c mice were injected intramuscularly with 100 g of plasmid pcmv-dp or empty vector pcmv. seven days later, the injected muscles were surgically removed and frozen sections were prepared. the sections were dried for 45 min at room temperature, fixed with anhydrous acetone, and then incubated with 0.05% h 2 o 2 for 20 min to quench the endogenous peroxidase. after blocking with 5% horse serum, the sections were incubated with a goat antihuman dkk1 antibody (5 g/ml, r&d systems) overnight at 4 ∘ c. the next day, the sections were incubated with a hrp-conjugated donkey anti-goat igg (1 : 1000, proteintech group) for 1 h at room temperature. the positive signals were detected with 3, 3 diaminobenzidine (dab, r&d systems). for dna immunization experiments, 6-week-old female balb/c mice ( = 6) were injected intramuscularly (i.m.) with plasmid pcmv-dp three times at weeks 0, 2, and 4. mice ( = 6) immunized with empty vector pcmv served as negative controls. intramuscular injection of plasmid dna followed by electroporation (dna + ep) was performed as previously described [21] . briefly, 100 g of plasmid pcmv-dp was injected intramuscularly in one tibialis anterior muscle using a 27-gauge needle. immediately after the injection, electroporation with 6 electric pulses was applied through a pair of silver electrodes spaced 3 mm apart covering the i.m. injection site. the electric pulses were 50 ms in duration and 1 s apart at a voltage of 60 v (i.e., 200 v/cm). from the first injection, mouse serum samples were collected at 2-week intervals and stored at −80 ∘ c. assay. the antibodies against human dkk1 were evaluated using elisa. briefly, 96-well microtiter plates were coated with 100 l of recombinant human dkk1 proteins (200 ng/ml, r&d systems) and incubated overnight at 4 ∘ c. after washing three times with pbst (pbs with 0.05% tween-20), the plates were blocked with 200 l of 1% bovine serum albumin (bsa) in pbst for 2 h at 37 ∘ c. after washing, 100 l of diluted serum (1 : 200) was added to each well. then, the plates were incubated for 2 h at 37 ∘ c and washed five times with pbst. hrp-conjugated goat anti-mouse igg secondary antibodies (100 l) (1 : 10000, proteintech group) were added to each well followed by incubation for 1 h at 37 ∘ c. next, the plates were washed five times with pbst. after adding the substrate solution and stop solution, the absorbance was read at 450 nm by a microplate reader (thermomax technologies). the end-point titer of antibody was determined in the same way. the serum samples were serially diluted from 1 : 200 to 1 : 12800. mouse model. dba/1 mice ( = 6) were immunized with plasmid pcmv-dp three times at 2-week intervals as described above. control mice ( = 6) were immunized with an equal amount of empty vector pcmv. one week after the final immunization, all mice were injected intradermally at the base of the tail with 100 g of bovine c ii emulsified with complete freud's adjuvant (cfa) containing 4 mg/ml of heat-killed mycobacterium tuberculosis. on day 21, the animals were given a booster injection with 100 g of bovine c ii dissolved in incomplete freud's adjuvant (ifa) [22] . animals were observed and recorded every 3 days after disease onset. clinical scores were assigned to evaluate the disease severity as follows: 0: no signs of arthritis; 1: swelling and/or redness of one paw or one digit; 2: two joints involved; 3: three or more joints involved; and 4: severe arthritis of the entire paws and digits. each limb was graded independently, resulting in a maximal clinical score of 16 per affected animal [23] . to determine the protective effects of the dna vaccine, 40 days after the challenge with bovine c ii, a micro-ct-200 system (aloka latheta laboratory, japan) was employed to detect the bone mineral density (bmd) and the degree of bone erosion after the mice were anaesthetized with chloral hydrate (10%). x-ray images were analyzed by reconstructed 3d quantitative analyses using the software vgstudio max 2.0. ethylenediaminetetraacetic acid (edta) for 6 weeks, and then dehydrated and embedded in paraffin. sections were cut along the longitudinal axis and stained with toluidine blue (tb) as previously described [18] . analysis. data were analyzed using the software spss (version 16.0) and presented as the mean ± sem. differences between two groups of mice were compared using student's -test. a value less than 0.05 was considered to be statistically significant. according to the analysis of the potential b cell epitopes in human dkk1 by the epitope prediction software dnastar 7.1, a panel of twelve peptides fragments was synthesized (figure 1(a) ). in addition, to select the high affinity and immunogenicity of synthesis peptides, the titers of peptides were determined and the peptides of grey-blue columns were candidates to design dna vaccine (figures 1(b)-1(c) ). furthermore, to decrease the pathological functions of dkk1 in peptides, the osteoclast-forming assay was performed. compared with controls, no significant osteoclastogenesis was observed in peptides-treatment group (figure 1(d) ). the synthetic nucleotide sequence with muted four amino acids encoding the dna vaccine was cloned in the eukaryotic expression vector pcmv6-xl5 (figure 2 ). in vivo. the expression of the multiepitope dna vaccine in vitro was evaluated in cos7 cells. elisa showed that the multiepitope dna vaccine recombinant protein of dkk1 was secreted abundantly and was recognized by its polyclonal antibodies (figure 3(a) ). in addition, western blotting also showed that cos7 cells transfected with plasmid pcmv-dp highly expressed the recombinant protein (figure 3(b) ). to assess the expression of the target protein in vivo, the injected muscles of mice were further stained with an anti-dkk1 antibody. the expression of recombinant protein in the dkk1 dna vaccine was much higher in the muscles at the injected site than in the control group (figure 3(c) ). due to these observations, we concluded that the multiepitope gene was expressed in eukaryotes both in vitro and in vivo. a specific anti-human dkk1 antibody was identified in balb/c mice immunized with plasmid pcmv-dp. the serum igg titer began to increase as early as 4 weeks after the primary immunization and reached its peak at 6 weeks (figure 3(d) ). the end-point titer of the specific antibody was also determined to evaluate its neutralizing effects in vitro (figure 3(e) ). although the end-point titer of anti-dkk1 was only 1 : 1600, the specific antibody existed persistently in the serum up to 6 months after immunization. mice. dba/1 mice were injected with bovine c ii twice at 3-week intervals after immunization. the signs of arthritis appeared around day 24 after injection with bovine c ii and continued to develop at later time points. the disease incidence was 83% in prevaccinated mice and 100% in the positive control mice on day 39. paw erythema and swelling also became more severe with time in control mice than the dkk1 vaccine group (figures 4(a) and 4(b) ). in addition, the infiltration of inflammatory cells was reduced in the vaccine-immunized mice compared with the controls (figure 4(c) ). we further evaluated the amelioration of arthritic bone, and the joint structure was well conserved in the vaccinated mice compared with the positive control mice as demonstrated by microcomputer tomography (ct) scanning (figure 4(d) ). the total bone mineral density (bmd) of the mice in the vaccinated groups was significantly higher than that in the positive control group (figure 4(e) ). the results of tb staining showed that the degree of overall destruction of cartilage was reduced markedly in the arthritic paws of vaccinated mice compared with the positive controls (figure 4(f) ). the results of micro-ct scanning and tb staining demonstrated that the dna vaccine attenuated bone erosion in cia mice. dna vaccines, also termed nucleic acid vaccines or gene vaccines, were first described in the 1990s when wolff found that dna could be taken up and expressed by mouse skeletal muscle cells in vivo [24] . it is generally thought that the gene of interest in a eukaryotic expression vector is translated into antigen protein using the cellular expression system of the host. subsequently, the antigen protein stimulates the host to generate an immune response. compared with traditional vaccines, the greatest advantage of dna vaccines is their ability to elicit both humoral immunity and cellular immunity. simultaneously, they are simple to prepare, easy to deliver, and relatively safe to apply. therefore, dna vaccination is a promising new technology for the prevention and therapy of diseases, such as infectious diseases, autoimmune diseases, and cancers. in this study, we constructed a dna vaccine targeting human dkk1, a major contributor to bone loss in ra. we expected to use this vaccine to inhibit bone erosion in a ra mouse model through the blockade of the biological activity of dkk1. we have adopted optimization strategies to improve the antigen expression and the immunogenicity of the dna vaccine, which are discussed below. to produce specific antibodies, b cell epitopes of dkk1 were the first choice to construct the dna vaccine. to increase the titer of specific antibodies, we combined the predictions of the software dnastar 7.1 and the affinity and immunogenicity assays. to reduce their potential bone resorption of natural dkk1, the synthesis epitopes were injected to balb/c and no significant osteoclastogenesis was observed. these data demonstrated that these separated epitopes were safe as a vaccine utilized in vivo. to increase the immunogenicity of the dna vaccine as expressed in the host, the key binding sites of dkk1 (r203, h204, k211, and r236) to lrp5/6 and kremen were mutated. at the same time, the mutated protein expressed in vivo enhanced the immunogenicity of the vaccine [20] . to facilitate the epitope processing, the four selected epitopes were separated from one another with aay spacers [25] . aays expressed inside the selected epitopes were easily recognized by proteasomes, and the full-length protein was more easily processed and presented in vivo. dkk1 is expressed naturally in the body. to further increase the immunogenicity of the recombinant dna vaccine, the pan dr t helper epitope (padre) was added to enhance antibody responses [26] . finally, the signal peptide for human dkk1 was added at the n-terminus of the dna vaccine to facilitate the extracellular secretion of the protein antigen. the dna vaccine delivery method is one of the most important factors that affect immunization efficiency. the methods for dna vaccine delivery currently used include intramuscular, intraperitoneal, intravenous, intradermal, subcutaneous injection, and others. the delivery method for the plasmid directly affects the uptake of the gene and further affects the expression of the target protein. intramuscular injection is the earliest and simplest method for dna delivery. however, the majority of the administered plasmid dna was blocked by perimysia of the skeletal muscles, where most of the dna was degraded by dnase i in the plasma. less than 1% of the dna actually entered the nucleus [27] . recently, electroporation has been used for dna vaccine delivery and has proven to be a better dna delivery method. in this study, intramuscular injection of plasmid dna followed by electroporation (dna + ep) was employed. the dc electric current disrupts cell membranes for a short time, allowing the plasmid dna to cross the membranes into the nucleus [28] . in addition, the electric current causes cell destruction and a local inflammatory reaction. a large number of inflammatory cells, including macrophages and other antigen-presenting cells, infiltrated the injected muscles, which enhanced the efficiency of antigen presentation. in our study, the highly specific igg antibodies in serum were induced as early as 4 weeks, which also demonstrated the efficiency of the electroporation of the dna vaccine. dkk1, a negative regulator of the canonical wnt signaling pathway, plays an important role in ra bone destruction. inhibition of its biological functions can attenuate bone destruction in ra [9] . our results showed that the recombinant human dkk1 multiepitope dna vaccine induced specific antibodies that effectively neutralized the biological activities of dkk1 and attenuated bone destruction in cia mice. this study may provide a potential therapy for ra and other bone loss diseases. biologics-based therapy for the treatment of rheumatoid arthritis my treatment approach to rheumatoid arthritis current understanding of rheumatoid arthritis therapy improving the routine management of rheumatoid arthritis: the value of tight control an explanation for the apparent dissociation between clinical remission and continued structural deterioration in rheumatoid arthritis progression of radiologic damage in patients with rheumatoid arthritis in clinical remission secreted antagonists of the wnt signalling pathway reconstitution of a frizzled8⋅wnt3a⋅lrp6 signaling complex reveals multiple wnt and dkk1 binding sites on lrp6 dickkopf-1 is a master regulator of joint remodeling the emerging role of dickkopf -1 in bone biology: is it the main switch controlling bone and joint remodeling novel mechanism of wnt signalling inhibition mediated by dickkopf-1 interaction with lrp6/arrow circulating dickkopf-1 is correlated with bone erosion and inflammation in rheumatoid arthritis the future of human dna vaccines technologies for enhanced efficacy of dna vaccines fda guidance on prophylactic dna vaccines: analysis and recommendations a systems framework for vaccine design dna vaccines: ready for prime time? therapeutic role of a vaccine targeting rankl and tnf-on collagen-induced arthritis arctigenin suppresses receptor activator of nuclear factor b ligand (rankl)-mediated osteoclast differentiation in bone marrow-derived macrophages characterization of the kremen-binding site on dkk1 and elucidation of the role of kremen in dkk-mediated wnt antagonism in vivo plasmid dna electroporation resulted in transfection of satellite cells and lasting transgene expression in regenerated muscle fibers collageninduced arthritis role of glucocorticoid-induced tnf receptor family gene (gitr) in collagen-induced arthritis direct gene transfer into mouse muscle in vivo a chimeric multi-epitope dna vaccine elicited specific antibody response against severe acute respiratory syndrome-associated coronavirus which attenuated the virulence of sars-cov in vitro potent immunogenic short linear peptide constructs composed of b cell epitopes and pan dr t helper epitopes (padre) for antibody responses in vivo immunization of non-human primates with dna vaccines direct visualization at the single-cell level of electrically mediated gene delivery the authors declare no conflict of interests. xiaoqing zhang, huihui yuan, and wenming zhao conceived and designed the experiments; xiaoqing zhang and sibo liu performed the experiments; yuxuan du and yunpeng dou analyzed the data; shentao li and zhanguo li contributed reagents/materials/analysis tools; xiaoqing zhang and huihui yuan wrote the paper. xiaoqing zhang and sibo liu contributed equally to this work. key: cord-0024589-8b6a30a8 authors: aljurbua, faisal i.; selaihem, ahmed; alomari, naif a.; alrashoud, abdualziz m. title: a cross-sectional study on generalized anxiety disorder and its socio-demographic correlates among the general population in saudi arabia date: 2021-11-05 journal: j family med prim care doi: 10.4103/jfmpc.jfmpc_847_21 sha: 1e24e13251d75e046915fd85b1200dcacc06c2ea doc_id: 24589 cord_uid: 8b6a30a8 background: generalized anxiety disorder (gad) is often described than define as a psychological illness that is characterized by excessive worry. little attention has been given to anxiety disorders by the medical community in saudi arabia. this study was carried out to screen for gad among adults and determine the correlation of anxiety disorder with other comorbidities. methodology: this study was a cross-sectional observational study carried out among adults aged 18 years and above in the general population in saudi arabia. the people were screened by using an arabic validated version of general anxiety disorder-7 (gad-7) questionnaire that was sent as a google link via emails or different social media (twitter, facebook, whatsapp, and telegram groups) of the general population. the study was approved by the hospital research committee and the institutional review board (hlri-05-apr21-01). results: we collected 338 participants in response to our questionnaire where 60.7% of them were females and 54.5% were aged between 25 and 34 years old. hypersensitivity, rheumatoid arthritis (ra), hypertension and diabetes mellitus (dm) were the main medical conditions represented by 7.6%, 4.8%, 3.7%, and 3.4%. according to gad-7 questionnaire, prevalence of any degree of anxiety was 62.1% where 33.1% of the total sample had mild anxiety, 15.7% had a moderate degree of anxiety and 13.3% had severe anxiety. anxiety was related to age, residency, occupation, and some medical conditions. conclusion: we found high prevalence of anxiety among our population which was the highest among younger participants, and students. moreover, we found that prevalence and severity of anxiety were higher in patients with chronic conditions and depressed patients. introduction use, and obesity. they may have a history of trauma or family member with gad. [3] although the high prevalence of gad and the burden of untreated patients including but not limited to missing working days, unemployment support, emergency visits because of somatic manifestation. in the united states, costs associated with anxiety disorders were $46.6 billion, 31.5% of total mental illness expenditures. less than one-quarter of the costs related to anxiety disorders were for direct medical treatment; over three-quarters were attributable to lost or reduced productivity. prompt detection and treatment substantially reduce the economic and social burden of these common and often crippling disorders. [4] moreover, little attention has been given to anxiety disorders by the medical community in saudi arabia. mental illness is often ignored as a severe illness in middle east societies. [5] therefore, depending on the rate of prevalence, this study will likely raise awareness of the seriousness of the disease and possibly influence policy change in saudi arabia regarding the plan of care for gad patients. in cross-sectional community-bases study which was conducted by alzahrani et al. [6] in rehabilitations centers at majmaah and sharqa citites, the authors discussed the prevalence of gad and major depression in health-care givers which affirmed that the predominance of psychosomatic disarray was very low. nationality, gender, marital status, educational level, profession, and income were not considerably linked with psychosomatic disorders. in another study published in 2020 among pharmacy students, samreen s et al. [7] used a cross-sectional design, using paper-based self-generated questionnaires. the general anxiety disorder-7 (gad-7) scale was used to measure and sort anxiety among the study providers. this study's results brought the fact to the front that half of the pharmacy students underwent anxiety occurrence during their study period in university. a study carried out in different regions of saudi arabia to know the prevalence of anxiety in general youth found 53 (11%) had mild gad indications, 268 (55.8%) had reasonable, and 39 (8.1%) had tremendously severe gad symptoms. more than half the youth in this study had shown indications of gad. [8] the cultural background becomes an important paradigm together with gender and impact of gad on individuals and society for assessing the prevalence of gad among the adult population. the previous researches carried out in different cities of saudi arabia focused mainly on anxiety and psychosomatic symptoms in specific populations like students and healthcare workers. there is severe lacking of studies that quantify the anxiety in the general population, its correlation with socio-demographic variables and other preexisting comorbidities. in saudi arabia, anxiety and depression screening is the cornerstone of primary care and family physicians' practice. this is due to the patient longitudinal follow-up of chronic disease in the primary health centers especially diabetes, hypertension, and hypothyroidism. counseling and primary care services can help ensure that patients get the mental health care they need. this study was carried out to screen for gad among adults and determine the correlation of anxiety disorder with other co-morbidities. this study was a community-based study targeting people living in saudi arabia through online social media (twitter, facebook, whatsapp, and telegram groups). individuals aged above 18 years of both genders were included in the study on their consent to participate in the study. however, individuals aged less than 18 years and already on treatment for gad were excluded from the study. this was a cross-sectional observational study. the sample size is calculated based on the prevalence of anxiety of 10% [9] by using the formula of z2 pq/d2 with an error of 5%. and sample was estimated to be 138. a non-probability sampling (convenience sampling) as the sample was collected from easily accessible participants with no randomization through online social media. this study was carried out among adults aged 18 years and above in the general population in saudi arabia. the people were screened for generalized anxiety by using the gad-7 questionnaire. the arabic validated version of gad-7 was sent as a google link via emails or different social media (twitter, facebook, whatsapp, and telegram groups) of the general population. the questionnaire started with a statement to express the participant's consent to be included in this study in the google link. the questionnaire consisted of two sections; the first section included questions related to socio-demographic correlations such as age, gender, and employment status. it is also included questions to know if the respondents are suffering from diabetes mellitus, hypertension, other chronic diseases, and any psychological disease. the second section of the questionnaire consisted of 7 items gad-7 "in arabic" to screen for any anxiety disorder in the general population. the score was determined by applying scores of 0, 1, 2, and 3 to the "not at all," "several days," "more than half the days," and "almost every day" response groups, respectively. total scores of 5, 10, and 15 were recorded for mild, moderate, and extreme anxiety as the reference points. data were retrieved on an excel file and analyzed using ibm spss statistics 24.0. mean and sd were estimated for quantitative variables. frequencies and percentages were used for qualitative variables. pearson chi-square and fisher exact test were applied to observe associations between qualitative variables. a value of p < 0.05 was considered statistically significant. the questionnaire started with a statement to express the participant's consent to be included in this study in the google link. all records were kept confidential, no names or any sort of identification were asked in the link as anonymity was maintained. the research was conducted only after getting the institutional review board (irb) from king saud medical city research center (hlri-05-apr21-01). in this study, we collected 338 participants in response to our questionnaire where 60.7% of them were females and 54.5% were aged between 25 and 34 years old. moreover, 52.7% of participants indicated that they were from central region, whereas 15.1% were from southern region. after calculated bmi from reported height and weight, we found that prevalence of underweight, overweight and obesity in our study were 7.6%, 28% and 28% respectively. moreover, 64.5% of participants that they are worker and 14.8% as smoker [ table 1 ]. moreover, 69.1% of the sample indicated that they had no medical comorbidity; however, hypersensitivity, rheumatoid arthritis (ra), hypertension and diabetes mellitus (dm) were the main medical conditions represented by 7.6%, 4.8%, 3.7% and 3.4% [ figure 1 ]. furthermore, 75.4% of participants indicated having no psychological condition or not diagnosed with psychological condition; however, 12.7% indicated having anxiety, 9.9% depression and 1.9% bipolar conditions [ figure 2 ]. according to gad-7 questionnaire, prevalence of any degree of anxiety was 62.1% where 33.1% of total sample had mild anxiety, 15.7% had moderate degree of anxiety and 13.3% had severe anxiety [ figure 3 ]. in [table 2 ], we showed the relation between demographic factors of participants and level of anxiety as interrupted from gad-7 questionnaire. we found that anxiety were more prevalent in females than males where 35.1% of females compared with 42.1% of males had no anxiety, whereas 15.6% of females had severe anxiety compared with 9.8% of males; however, there is no significant difference between the two genders. moreover, we found a significant difference between participants considering degree of anxiety depending on their age categories where it seems that prevalence of anxiety was higher in younger participants who had more severe anxiety than older participants (p = 0.00) where almost 85% of participants of age category (18-24) had a degree of anxiety. moreover, we found that the higher prevalence of anxiety was in western region and southern region (83.7% and 76.5%) (p = 0.015). we did not find a significant difference depending bmi; however, anxiety was more prevalence and more severe in underweight and obese participants. moreover, we found that prevalence of anxiety was significantly higher in students than workers; however, participants who did not work show more severe anxiety. prevalence and severity of anxiety were higher in smokers; however, this is not significant. moreover, we found that prevalence and severity of anxiety is related to some medical conditions. as shown in [ table 3 ], prevalence of anxiety was significantly higher in patients with colon conditions, lupus erythematosus, heart conditions and thyroid dysfunction with 100%, 77.8%, 75%, and 72.7%, whereas patients of anemia represented the highest severity of anxiety (33.3%). patients with depression and anxiety showed significant higher prevalence of anxiety (88.9% and 89.1%). in our study, we aimed to assess the prevalence and severity of anxiety among public population in saudi arabia as well as to determine if any relationship exists between gad and other co-morbidities like diabetes, hypertension, and other chronic conditions. in this study, the prevalence of any degree of anxiety according to gad-7 tool was 62.1% where 33.1% of total sample had mild anxiety, 15.7% had moderate degree of anxiety and 13.3% had severe anxiety. in a study conducted by salari n, the authors found that prevalence of anxiety among public population was 31.9%, [10] whereas study of maroufizadech s which conducted among public population in iran, the authors found that prevalence of anxiety was 61.8% where 31.7% of total population had mild anxiety, 18.1% had moderate anxiety and 12.1% had severe anxiety which is similar to our results. [11] moreover, study of hajure m among chronic medical patients showed that prevalence of anxiety was 61.8% [12] and study of samreen s found that prevalence of anxiety among students was 49% where 25.9% had mild anxiety, 14.1% had moderate anxiety and 8.8% had severe anxiety. [7] moreover, we found that prevalence of anxiety is higher among females than males and percent of females who had severe anxiety was higher than percent of males; however, we could not find a significant difference. women are more vulnerable to stress and post traumatic stress disorder than men. [13, 14] in previous studies, the prevalence of anxiety is shown to be higher in women than in men. [9, 11, [14] [15] [16] considering age, we found that prevalence and severity of anxiety were significantly higher in younger participants than older participants where 85% of population aged between 18 and 24 had anxiety and 65% of population aged between 35 and 44. similar results were reported by maroufizadech s who reported that prevalence was higher in young and middle-aged populations, [11] and study of salari n who reported that the highest prevalence of anxiety among population because of covid-19 was among age group of 21-40 years, [10] as well as some other studies. [17, 18] this could be explained as young population have many stressors that affect their anxiety level as they worry about educational progress, need for work, preparing new families and building their own future. according to our results, the prevalence of underweight, overweight and obesity in our study were 7.6%, 28% and 28% respectively and there is no significant relation between bmi of participants and prevalence of anxiety. in study of alqarni s, almost half of the students (51.5%) have normal weight; and 23.1% and 3.8% are overweight and obese respectively, whereas 19.2% of the respondents are underweight. [19] moreover, study of samreen s confirms our results about the non-significant effect of obesity on anxiety. [7] moreover, it was found that prevalence of anxiety was higher in students and not working participants. considering the relation between comorbidity of participants and anxiety, we discussed the effect of chronic conditions and psychological conditions. the most prevalent conditions among our population were hypersensitivity, ra) hypertension and dm. moreover, we found a significant difference between patients with these conditions considering degree of anxiety. prevalence of anxiety was significantly higher in patients with colon conditions, lupus erythematosus, heart conditions and thyroid dysfunction with 100%, 77.8%, 75%, and 72.7%, whereas patients of anemia represented the highest severity of anxiety (33.3%). similar results were reported by peltzer k who found that 19.7% of patients with stomach and intestinal diseases had anxiety, whereas 22% of patients with cardiovascular conditions, 19.6% of arthritis patients had anxiety. [20] yohannes et al. [21] in a systematic review of studies published between 1994 and 2009, found that patients with chronic conditions, and especially patients with obstructive lung disease and cardiovascular diseases can have particularly high rates of anxiety and depression, which had a negative effect on increasing mortality rates and decreasing the quality-of-life levels. considering psychological conditions, we found that depressed patients showed more severe anxiety which is in consistency with other studies. [1, [22] [23] [24] [25] this study had some limitations; the first limitation was the use of social media to collect responses which lead to that most of participants were mainly of young age group. however, we used validated questionnaire to assess all variables, these scales aimed for screening rather than diagnosis therefore, they may overestimate the true prevalence. furthermore, participants may have been untruthful and biases that cannot be excluded. we found high prevalence of anxiety among our population which was the highest among females, younger participants, students, not-working population and smokers; however, some significance could be found. moreover, we found that prevalence of anxiety and severity of anxiety were higher in patients with chronic conditions and depressed patients therefore, there is a need for assessing anxiety among these population in order to reduce the impact of anxiety on quality of life. more investigations should be conducted to find the relation between comorbidity of patients and level of anxiety. nil. there are no conflicts of interest. prevalence and influencing factors of anxiety and depression symptoms in the first-line medical staff fighting against covid-19 in gansu prevalence of mental disorders and the use of mental health services among the adult population in united arab emirates massachusetts general hospital comprehensive clinical psychiatry economic costs of anxiety disorders the life of pii: developing a positive islamic identity prevalence of generalized anxiety disorder and major depression in health-care givers of disabled patients in majmaah and shaqra cities, kingdom of saudi arabia prevalence of anxiety and associated factors among pharmacy students in saudi arabia: a cross-sectional study prevalence of generalized anxiety disorder among saudi youth during covid-19 pandemic in saudi arabia prevalence of depression, anxiety, and stress among the general population in saudi arabia during covid-19 pandemic prevalence of stress, anxiety, depression among the general population during the covid-19 pandemic: a systematic review and meta-analysis prevalence of anxiety and depression in general population of iran during the covid-19 pandemic: a web-based cross-sectional study depression, anxiety and associated factors among chronic medical patients amid covid-19 pandemic in risk factors for post-injury mental health problems the psychological and mental impact of coronavirus disease 2019 (covid-19) on medical staff and general public -a systematic review and meta-analysis prevalence and socio-demographic correlates of psychological health problems in chinese adolescents during the outbreak of covid-19 epidemic of covid-19 in china and associated psychological problems generalized anxiety disorder, depressive symptoms and sleep quality during covid-19 outbreak in china: a web-based cross-sectional survey a review of prevalence of obesity in saudi arabia anxiety and depressive features in chronic disease patients in cambodia, myanmar and vietnam depression and anxiety in chronic heart failure and chronic obstructive pulmonary disease: prevalence, relevance, clinical implications and management principles prevalence of generalized anxiety disorder and its related factors among infertile patients in iran: a cross-sectional study factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019 depression, anxiety, stress levels of physicians and associated factors in covid-19 pandemics prevalence of depression, anxiety, and insomnia among healthcare workers during the covid-19 pandemic: a systematic review and meta-analysis anxiety and depression in covid-19 survivors: role of inflammatory and clinical predictors key: cord-0024570-ibo1bxxw authors: chen, conan; mao, yixiang; falahpour, maryam; macniven, kelly h.; heit, gary; sharma, vivek; alataris, konstantinos; liu, thomas t. title: effects of sub-threshold transcutaneous auricular vagus nerve stimulation on cerebral blood flow date: 2021-12-15 journal: sci rep doi: 10.1038/s41598-021-03401-w sha: 1b44495d7083605eaca5521aace42a10f1f1fecb doc_id: 24570 cord_uid: ibo1bxxw transcutaneous auricular vagus nerve stimulation (tavns) has shown promise as a non-invasive alternative to vagus nerve stimulation (vns) with implantable devices, which has been used to treat drug-resistant epilepsy and treatment-resistant depression. prior work has used functional mri to investigate the brain response to tavns, and more recent work has also demonstrated potential therapeutic effects of high-frequency sub-threshold tavns in rheumatoid arthritis. however, no studies to date have measured the effects of high-frequency sub-threshold tavns on cerebral blood flow (cbf). the objective of this study was to determine whether high-frequency (20 khz) sub-threshold tavns induces significant changes in cbf, a promising metric for the assessment of the sustained effects of tavns. arterial spin labeling (asl) mri scans were performed on 20 healthy subjects in a single-blind placebo-controlled repeated measures experimental design. the asl scans were performed before and after 15 min of either sub-threshold tavns treatment or a sham control. tavns induced significant changes in cbf in the superior posterior cerebellum that were largely localized to bilateral crus i and crus ii. post hoc analyses showed that the changes were driven by a treatment-related decrease in cbf. fifteen minutes of high-frequency sub-threshold tavns can induce sustained cbf decreases in the bilateral posterior cerebellum in a cohort of healthy subjects. this study lays the foundation for future studies in clinical populations, and also supports the use of asl measures of cbf for the assessment of the sustained effects of tavns. . in contrast, measures of cerebral blood flow (cbf), a well-defined physiological quantity, can be used to characterize changes in brain physiology occurring over periods ranging from seconds to years 18 , and therefore constitute a promising metric for the assessment of the sustained effects of vns. in prior work, both spect and pet have been used to characterize the cbf response to vns applied using implantable devices in patients with either major depression (md) or epilepsy. in md patients, spect has been used to demonstrate regionally dependent increases and decreases in cbf due to 4 to 10 weeks of chronic vns 19, 20 . in a pet study with md patients, conway et al. 21, 22 reported regionally dependent increases and decreases in the evoked cbf response to short duration (90 s) vns. for epilepsy patients, henry et al. 23, 24 reported pet-based cbf increases and decreases in the evoked cbf reponse to 30 s of vns, with the effects diminishing after 12 weeks of chronic vns. in addition, two earlier pilot studies 25, 26 reported vns evoked changes in pet cbf measures. to the best of our knowledge, no studies to date have examined the cbf response to tavns. in this study, we used arterial spin labeling (asl) mri to measure changes in cbf induced by 15 min of tavns in healthy subjects, employing a high-frequency 20 khz stimulus similar to that employed by marsal et al. 11 . in contrast to spect and pet, asl mri does not require the injection of radioactive tracers, making it especially suitable for repeated measures in a healthy population 27, 28 . furthermore, in this study the tavns is applied at a sub-threshold level, such that subjects cannot readily determine if they are receiving the treatment or a placebo. in contrast to prior studies where the perceptible sensations associated with vns may have complicated the interpretation of the results, the sub-threshold stimulus used in this study removes somatosensory confounds and is therefore compatible with a single-blind placebo-controlled design. the goal of this study was to determine whether a single course of tavns with duration similar to that of the daily dose employed in 11 could lead to detectable changes in cbf. overview. we used a single-blind placebo-controlled experimental design. twenty seven healthy subjects were enrolled in this study after providing informed consent (14 females and 13 males, aged 19-40 years). the study was approved by the ucsd institutional review board (irb), and informed consent was obtained from all participants. all experiments were performed in accordance with relevant guidelines and regulations. figure 1 shows the overall experiment structure. each subject participated in a control session and a treatment session, with the order randomized across subjects. the sessions were separated by at least one day to avoid tavns carryover effects. all scanning visits consisted of a pre scan section, a stimulation (or sham) section, and a post scan section. for the first 6 subjects, the subjects wore the stimulation earpieces throughout the entire session, and the stimulation section took place while they remained lying inside the scanner. to improve the subject experience, the protocol was slightly modified for the subsequent 21 subjects. these 21 subjects wore regular earplugs during the mri scans, came out of the scanner for the stimulation section, and then returned to the scanner with regular earplugs to finish the post scan section. mri scans. scans were acquired on a ge discovery mr750 3.0 t system. pre and post scan sections consisted of (1) a high-resolution anatomical scan (mprage, resolution = 1mm 3 , 208 slices, fov = 25.6 cm, tr = 2500 ms, te = 2.92 ms, fa = 8 deg), (2) resting-state bold fmri scans, and (3) arterial spin labeling (asl) scans (2d pseudo-continuous asl (pcasl), single-shot spiral, 100 repetitions, resolution = 3.75 mm × 3.75 mm, slice thickness = 6 mm, matrix size = 64 × 64 × 24, fov = 24 cm, te = 3.2 ms, tr = 4300 ms, labeling duration = 1800 ms and post-labeling delay = 1800 ms) 28 . only the anatomical and asl scans are analyzed here. the resting-state bold scans will be analyzed in a separate paper. during all asl scans, subjects rested with their eyes open and gently focused on a fixation point. www.nature.com/scientificreports/ tavns. tavns was delivered using custom-fit ear pieces designed by nēsos® (redwood city, ca). a bipolar configuration was used and two circular (radius 0.3 cm) hydrogel electrodes were placed on the ear piece surface interfacing with the cymba concha, with a contact area of 0.28 cm 2 for each electrode. alcohol wipes were used to clean the cymba concha prior to inserting the ear pieces. stimulation was applied to the left ear, targeting vagal afferents in the cymba concha based on known vagus nerve anatomy 8, 29 . for all visits, the stimulation (or sham) section took place between the pre and post scan sections. on both scan days, a sensation test was performed to determine a subject's threshold for perceiving the stimulation, which entailed stimulating at 1.0 ma and increasing by increments of 0.5 ma (up to a maximum of 5.0 ma). criteria for perceiving the stimulation include sensations of stinging, pricking, or warmth. this procedure was repeated until the subject reported feeling the stimulation at the same level three times. the sensation test was followed by a 15-min stimulation period. the stimulation was delivered continuously at 20 khz, using biphasic square waves with pulse width of 20us. the stimulation amplitude was set at 75% of a subject's perceptual threshold (1.5-3.8 ma; impedance 3-9.5 kω at 20 khz) on the "active" stimulation day and was set to zero on the control day ("sham" condition). the order of "active" and "sham" stimulation days was randomized across subjects. to verify that the subthreshold stimulation did not cause any local heating, we used a dtt-1372 thermometer (ames instruments tm , harbor freight tools usa. inc., calabasas, ca) to measure temperature changes over a 15-min period in both the active and sham conditions for one of the subjects. the probe was placed on the medial surface of the external ear, directly across from the earpiece that was positioned at the cymba concha on the lateral surface of the external ear. minimal temperature increases were observed for active (0.20 °c) and sham (0.25 °c) conditions with no significant difference (p = 0.46) in the slope of the changes over the 15-min period. since the stimulation was delivered at 75% of perceptual threshold, subjects were blinded to whether they were undergoing the sham or stimulus treatment. at the end of their second session, all subjects were asked whether they could identify their control and treatment sessions across their two visits. data processing and analysis. data from the asl scan were volume registered and quantified into physiological cbf units (ml/100 g/min) via local tissue correction with a proton-density scan acquired during the same scan section 30 . the score algorithm 31 was used on a per-slice basis to censor outlier timepoints in the cbf time series before computing the average cbf map. using afni software 32 , the high-resolution anatomical scans were transformed into mni space, and the cbf volumes were then warped to mni space using the transformation matrices from the anatomical transformation, with a final voxel resolution of 4mm 3 . for group analysis in mni space, post minus pre cbf difference maps (termed "post-pre" for the remainder of the manuscript) were computed for control and treatment sessions. a two-sided paired t-test was performed for the main contrast (post-pre) treatment − (post-pre) control on a per-voxel basis using afni 3dttest++. this contrast is equivalent to the interaction effect for a two-way (post, pre by treatment, control) repeated measures anova. after setting a primary voxelwise threshold, spatial clustering thresholds were used to control the family-wise error rate (fwer) associated with detecting clusters of supra-threshold t-values across the 27,168 voxels in the mni brain mask 33 . for a specified voxelwise threshold, a permutation-based method (as implemented in afni 3dttest++ with the -clustsim option) was used to estimate the false positive rates associated with detecting clusters with varying cluster-size thresholds. additional details and background on this approach are provided in cox et al. 2017 33 . a voxelwise threshold of p < 0.01 and a cluster size threshold of 22 voxels (clusters defined with voxel faces touching) were used to assess results at a fwe-corrected p < 0.05 level. post-hoc two-sided paired t-tests were also performed for each session (control and treatment), and were assessed at a voxelwise threshold p < 0.01 and a fwe-corrected p < 0.05 level (using cluster size thresholds of 35 and 51 voxels for the control and treatment contrasts, respectively). in addition, post-hoc analyses were performed using the mean cbf values of any significant clusters found from the main contrast of (post-pre) treatment − (post-pre) control . finally, subjects that were randomized to a session order of control followed by treatment were compared to subjects randomized to treatment then control. a post-hoc two-sided two-sample t-test was performed between these two groups using the mean cbf values of any significant clusters found from the main contrast. participants and stimulation. out of twenty-seven total subjects, two dropped out after their first session, due in part to the covid-19 related suspension of research activities during the course of the study. the remaining twenty-five subjects completed both the control and treatment sessions. five subjects were excluded from the study due to technical errors or severe image artifacts in at least one of their cbf scans, leaving twenty total subjects included in the analysis. these twenty subjects received a mean stimulation amplitude of 3.24 ± 0.56 ma (min = 1.5 ma, max = 3.8 ma). the control and treatment sessions were matched to take place at the same time each day whenever possible, with an average time-of-day difference of 0.9 ± 1.32 h (min = 0 h, max = 4 h). when asked whether they could identify the control and treatment sessions across their two visits, sixteen of the twenty subjects responded that they could not guess. of the remaining four subjects who indicated they could identify their sessions, three guessed correctly and one guessed incorrectly. overall, an average of 24.81 ± 6.93 min elapsed from the end of the stimulation to the start of the post section asl scan for these subjects. group-level whole-brain analysis. for the main contrast shown in fig. 2a figure 2b shows no significant cbf changes in the control session, while fig. 2c shows a left hemispheric cerebellar cluster (69 voxels) and a right hemispheric cerebellar cluster (74 voxels) with significant cbf decreases in the treatment session. we used the probabilistic atlas of the cerebellar lobules as defined in 34, 35 post-hoc analyses of the mean cbf values from the main contrast cluster are shown in fig. 3 . the cluster shows a significant decrease in cbf related to treatment (p < 0.001, −15.90%) alongside a statistically insignificant increase in the control session (p = 0.19, + 4.02%). consistent with the maps shown in fig. 2 , the post-hoc analyses indicate that the contrast of interest mainly reflects significant treatment-related decreases in cerebellar cbf. post-hoc analysis of session-ordering effects in the main cluster is shown in fig. 4 . there was not a significant difference in the main contrast values between the two session orders (p = 0.54), indicating that the main contrast effect is not driven by either randomization order. our findings reveal a significant bilateral decrease in cerebellar cbf as a result of 15 min of sub-threshold tavns, reflecting physiological changes that persisted for many minutes after the treatment. these effects were observed using a stimulus paradigm and duration similar to those of the daily dose employed in the recent open label clinical trial of 11 . in contrast to the current findings, prior studies have reported both unilateral 26 and bilateral 21,23,24 increases in cerebellar cbf. however, there are several key methodological differences that are worth noting. first and foremost, the prior studies examined evoked cbf changes in response to the concurrent application of 30 to 90 s of vns, whereas the current study assessed resting-state cbf measures obtained before and after the application of tavns. thus, the prior findings are limited to characterizing the acute cerebellar cbf response to vns, while the current findings offer evidence of a sustained decrease in cerebellar cbf in reponse to tavns. second, vns consists of an implantable device targeting the cervical vagus nerve, while tavns is non-invasive and applied to a branch originating from the superior ganglion of the vagus nerve. this difference in neural targets may be expected to produce different physiological responses. third, the use of a sub-threshold high frequency (20 khz) fig. 3 . on the left are subjects randomized to group a; on the right are subjects randomized to group b. a two-sided two-sample t-test was performed between the two groups, indicating no significant difference in main effect (p = 0.54) between the two session orders. www.nature.com/scientificreports/ stimulus in the current study enabled the use of a single-blind placebo controlled design, in contrast to the openlabel design with the super-threshold low frequency (20 to 30 hz) stimuli employed in prior studies. the placebo control in the current study enabled a clean isolation of the direct vagal stimulation effects from those associated with stimulus-associated sensations and responses, a separation that was not possible with prior designs. in addition, there are likely to be frequency dependent neurophysiological mechanisms that lead to differences in the effects between the high (20 khz) versus low (20 to 30 hz) frequency stimulus paradigms. finally, the current findings were obtained in healthy subjects who were naïve to tavns, whereas the prior results characterized the responses in md or epilepsy patients at a time point that was hours to months after the commencement of continuous vns therapy. thus, the current study provides critical information about the effect of tavns on the healthy brain that can serve as a baseline for the future assessment of effects in clinical populations. stimulus-induced changes in cerebellar activity have also been observed with bold fmri, but the experimental methods and results greatly vary across studies with reports of both evoked activations in healthy subjects 1, 15, 16 and epileptic patients 36 and deactivations in healthy subjects 2 and md subjects 37, 38 . as the bold signal exhibits a complex dependence on neural, vascular, and metabolic factors, a definitive link between observed bold changes and underlying cbf changes cannot be established without additional experimental measures 39 . however for most conditions, task-evoked increases and decreases in the bold signal are generally thought to reflect concomitant cbf increases and decreases. thus, while the fmri findings cannot resolve whether vns induces increases or decreases in cerebellar cbf, they offer general support for the potential of vns to modulate cbf levels in the cerebellum. in this study, significant cbf changes were limited to the cerebellum. the prior pet and spect studies reported cbf changes in additional brain regions, although the results were highly variable across studies. as noted above, a number of these studies considered the evoked cbf response as compared to the sustained response reported here. two spect studies examined the effects of 4 to 10 weeks of chronic vns 19, 20 and reported increases and decreases in cbf in multiple brain regions, but there were differences between the two studies in the regions that were identified and neither study reported cerebellar cbf changes. in light of the variability observed in prior studies and the significant methodological differences discussed above, discrepancies between the prior and current findings are to be expected. in particular, the interpretation of the changes observed in the prior studies typically discussed the potential modulation of activity in regions that were thought to be associated with the disease state. these brain regions would not necessarily show an effect in healthy subjects. nevertheless, a future study examining the effects of sub-threshold tavns over a longer period (e.g. weeks) would be of interest to determine if cbf changes can be detected in areas beyond the cerebellum. while the cerebellum has traditionally been viewed as a motor control region, a wealth of evidence suggests that it also plays important roles in cognitive, affective, and social processing [40] [41] [42] . in particular, the cerebellar regions crus i and crus ii, which exhibited the greatest overlap with significant cbf changes in this study, are considered to be primarily associated with non-motor functions, such as working memory, executive function, language processing, social mentalizing, and emotional cognition 41, 43, 44 . interestingly, the cerebellum has a purported role in conditions for which tavns has beneficial effects. for example, cerebellar dysfunction has been implicated in major depressive disorder (mdd) 45 , and a preliminary clinical study found that a multi-week regime of tavns (relative to sham) can alleviate depression severity as assessed via the hamilton depression scale in mdd patients 5 . tavns-induced cerebellar activation has also been reported in an fmri study of mdd patients, though individual differences in activation were not related to clinical outcomes 46 . furthermore, tavns has shown promise as a treatment for migraine 13 , another disorder that has been linked to functional and structural alterations within the posterior cerebellum, namely crus i and crus ii 47, 48 . finally, a recent study found that a 12-week regime of tavns treatment (with parameters similar to those used in this study) improved symptoms for patients with rheumatoid arthritis (ra) 11 . reduced gray matter of the cerebellum has been associated with higher levels of peripheral inflammation in ra patients 49 . together, these findings suggest that the link between tavns therapeutic efficacy and cerebellar function should be further explored. there are limitations to the study that would be important to address in future follow-up work. first, the analysis required that all four asl scans (pre control , post control , pre treatment , and post treatment ) were free from artifacts, as issues in any run could affect the main contrast. however, this strict approach makes the study sensitive to any issues like subject motion that can cause artifacts in the cbf maps, leading us to exclude five out of twentyfive subjects, even though a much lower fraction (10 out of 100) of the scans exhibited artifacts. for future work using asl to probe tavns effects, one improvement would be to use asl sequences with higher snr, such as 3d pcasl with background suppression to reduce motion sensitivity 50, 51 . the increased snr can be used to shorten the scan lengths while maintaining similar image quality, mitigating the risk of motion associated with longer scan times. the higher snr also increases the ability to detect cbf changes in a group analysis, so that fewer subjects would be needed to detect the same effects, increasing the study robustness to subject exclusions. second, there is a small possibility of a thermo-regulatory vascular confound in response to local energy deposition from tavns. we believe this to be unlikely, as no subjects reported sensations of heating at the stimulation site. furthermore, an in-situ thermal test of the device found minimal (< 0.3 °c) changes in temperature over a 15-min period in both active and sham conditions. however, we cannot completely rule out a vascular response that quickly dissipates heat and could prevent any detection of warmth despite an underlying change in neural response. in this study, the tavns was applied at the cymba concha, which is 100% innervated by the auricular branch of the vagus nerve 8 . however, given the overlapping innervation by the greater auricular nerve and the auriculotemporal nerve of auricular regions proximal to the cymba concha 8 , it is not possible to completely rule out the effects on cbf through these pathways. for example, the auriculotemporal nerve lies within the mandibular branch of the trigeminal nerve, and prior work indicates that stimulation of the trigeminal nerve can cause changes in cbf 52 . in conclusion, the present results indicate that 15 min of high-frequency sub-threshold tavns can induce sustained cbf decreases in the bilateral posterior cerebellum in a sample of healthy subjects. further work is needed to determine if similar changes can be observed in clinical populations and to assess if the measures can provide greater insight into the mechanisms underlying treatment outcomes. in addition, future research should also address how stimulation parameters such as frequency, duration, and electrode location impact the response. neurophysiologic effects of transcutaneous auricular vagus nerve stimulation (tavns) via electrical stimulation of the tragus: a concurrent tavns/fmri study and review a novel transcutaneous vagus nerve stimulation leads to brainstem and cerebral activations measured by functional mri bold fmri deactivation of limbic and temporal brain structures and mood enhancing effect by transcutaneous vagus nerve stimulation transcutaneous vagus nerve stimulation modulates default mode network in major depressive disorder effect of transcutaneous auricular vagus nerve stimulation on major depressive disorder: a nonrandomized controlled pilot study modulation of brainstem activity and connectivity by respiratory-gated auricular vagal afferent nerve stimulation in migraine patients investigational treatment of rheumatoid arthritis with a vibrotactile device applied to the external ear the nerve supply of the human auricle current directions in the auricular vagus nerve stimulation i-a physiological perspective neural networks and the anti-inflammatory effect of transcutaneous auricular vagus nerve stimulation in depression non-invasive vagus nerve stimulation for rheumatoid arthritis: a proof-of-concept study treating depression with transcutaneous auricular vagus nerve stimulation: state of the art and future perspectives treatment of chronic migraine with transcutaneous stimulation of the auricular branch of the vagal nerve (auricular t-vns): a randomized, monocentric clinical trial transcutaneous auricular vagus nerve stimulation at 1 hz modulates locus coeruleus activity and resting state functional connectivity in patients with migraine: an fmri study non-invasive access to the vagus nerve central projections via electrical stimulation of the external ear: fmri evidence in humans optimization of transcutaneous vagus nerve stimulation using functional mri arterial spin labeling perfusion fmri with very low task frequency measurement of cerebral perfusion with arterial spin labeling: part 1 changes in regional cerebral blood flow by therapeutic vagus nerve stimulation in depression: an exploratory approach chronic vagus nerve stimulation for treatment-resistant depression increases regional cerebral blood flow in the dorsolateral prefrontal cortex cerebral blood flow changes during vagus nerve stimulation for depression brain blood-flow change with acute vagus nerve stimulation in treatment-refractory major depressive disorder brain blood flow alterations induced by therapeutic vagus nerve stimulation in partial epilepsy: i. acute effects at high and low levels of stimulation brain blood-flow alterations induced by therapeutic vagus nerve stimulation in partial epilepsy: ii. prolonged effects at high and low levels of stimulation regional cerebral blood flow in man manipulated by direct vagal stimulation vagus nerve stimulation activates central nervous system structures in epileptic patients during pet h2150 blood flow imaging applications of arterial spin labeled mri in the brain recommended implementation of arterial spin-labeled perfusion mri for clinical applications: a consensus of the ismrm perfusion study group and the european consortium for asl in dementia functional and chemical anatomy of the afferent vagal system the cerebral blood flow biomedical informatics research network (cbfbirn) database and analysis pipeline for arterial spin labeling mri data structural correlation-based outlier rejection (score) algorithm for arterial spin labeling time series afni: software for analysis and visualization of functional magnetic resonance neuroimages fmri clustering in afni: false-positive rates redux a probabilistic mr atlas of the human cerebellum imaging the deep cerebellar nuclei: a probabilistic atlas and normalization procedure bold fmri activation induced by vagus nerve stimulation in seizure patients acute vagus nerve stimulation using different pulse widths produces varying brain effects serial vagus nerve stimulation functional mri in treatment-resistant depression an introduction to normalization and calibration methods in functional mri cerebellar functional anatomy: a didactic summary based on human fmri evidence the cerebellum and cognition triple representation of language, working memory, social and emotion processing in the cerebellum: convergent evidence from task and seed-based resting-state fmri analyses in a single large cohort functional topography of the human cerebellum early cortical biomarkers of longitudinal transcutaneous vagus nerve stimulation treatment success in depression functional and structural alterations in the migraine cerebellum cerebellar involvement in migraine a multi-modal mri study of the central response to inflammation in rheumatoid arthritis comparison of pasl, pcasl, and background-suppressed 3d pcasl in mild cognitive impairment comparison of 2d and 3d single-shot asl perfusion fmri sequences trigeminal nerve control of cerebral blood flow: a brief review this work was supported by a research grant from nēsos corporation, redwood city, ca. g.h., k.m., m.f., and t.l. designed the experiment. c.c. and t.l. wrote the main manuscript text. c.c. performed the processing and analysis of data and prepared all figures. y.m., c.c. and m.f. collected the data used in the study. v.s. provided hardware support for the study. g.h., k.m., k.a. and t.l. provided interpretation of the data. all authors reviewed the manuscript. funding for this study was provided by an unrestricted grant from nēsos® (redwood city, ca), which also provided hardware support for this study. kelly h. macniven and gary heit report consultant fees from nēsos. konstantinos alataris, gary heit and vivek sharma report equity holdings in nēsos. konstantinos alataris and vivek sharma are current employees. thomas liu reports grants from nēsos. all other authors declare no competing interests. correspondence and requests for materials should be addressed to c.c. or t.t.l.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. key: cord-0025788-0mz5umtn authors: fiehn, christoph title: biologikatherapie von rheumatoider arthritis und spondyloarthritiden date: 2022-01-14 journal: internist (berl) doi: 10.1007/s00108-021-01248-x sha: 42d0e09b0f7f9e1479995aa781c0055fb0da0bb4 doc_id: 25788 cord_uid: 0mz5umtn biologics are an integral part of modern strategies for treatment of rheumatoid arthritis (ra) and spondylarthritis (spa), including psoriatic arthritis (psa). biologics are biotechnologically produced proteins that have inhibiting effects on humoral and cellular components of rheumatic inflammation. substance classes used in rheumatology are tumor necrosis factor (tnf)-alpha, interleukin (il)-1, il‑6, il-12, il-17 and il-23 inhibitors effective against cytokines as well as the t lymphocyte activation inhibitor abatacept and the b lymphocyte-depleting rituximab. there are clear recommendations for the use of biologics for ra patients inadequately responding to one or more conventional synthetic disease-modifying antirheumatic drugs and for ankylosing spondylitis (as) and nonradiographical axial spa patients with an inadequate response to at least two nonsteroidal antirheumatic drugs. for psa the recommended use depends on the most prominent manifestations in each case. treatment with biologics should follow the treat to target principle, with a defined and validated treatment target. treatment in cases of ra and spa should target remission or at least a low or minimum disease activity. the safety of treatment with biologics has been intensively investigated. there are very specific contraindications for individual substance classes with a focus on an increased risk of infections. the standard procedure before starting treatment with biologics includes the exclusion of latent tuberculosis and hepatitis b. the tnf-alpha inhibitors have a protective effect with respect to myocardial infarction, stroke and venous thromboembolism. interleukin-6-inhibitoren · abatacept · rituximab · interleukin-17-inhibitoren · interleukin-12und interleukin-23-inhibitoren -therapieziele -sicherheitsaspekte der therapie mit bd-mard infektionsrisiko · kardiovaskuläre protektion · malignome -diskussion biologika sind ein wichtiger bestandteil moderner therapiestrategien zur behandlung von rheumatoider arthritis (ra) und spondyloarthritiden (spa) inklusive der psoriasisarthritis (psa). biologika sind biotechnologisch hergestellte proteine, die inhibitorische wirkungen auf humorale und zelluläre komponenten der rheumatischen entzündung haben. in der rheumatologie eingesetzte substanzklassen sind die gegen zytokine gerichteten tumor-nekrose-faktor(tnf)-α-, interleukin(il)-1-, il-6-, il-12-, il-17-und il-23-inhibitoren sowie der hemmer der t-lymphozyten-aktivierung abatacept und das b-lymphozyten-depletierende rituximab. für den einsatz von biologika existieren empfehlungen, die bei der ra das inadäquate ansprechen auf ein oder mehrere konventionelle synthetische "disease-modifying antirheumatic drugs" und bei der ankylosierenden spondylitis und der nichtröntgenologischen axialen spa das inadäquate ansprechen auf mindestens 2 nichtsteroidale antirheumatika vorsehen. bei der psa hängt der empfohlene einsatz von der jeweiligen im vordergrund stehenden manifestation ab. eine biologikatherapie sollte nach dem treat-to-target-prinzip erfolgen, also mit einem definierten und validierten therapieziel. dies ist bei der ra und den spa jeweils entweder die remission oder aber die niedrige bzw. minimale krankheitsaktivität. die sicherheit der biologikatherapie wurde intensiv untersucht. für die einzelnen substanzklassen existieren sehr spezifische kontraindikationen mit dem fokus auf einem erhöhten infektionsrisiko. der ausschluss einer latenten tuberkulose wie auch einer hepatitis b gehört zum standard vor beginn einer biologikatherapie. tnf-α-hemmer haben einen protektiven effekt in bezug auf myokardinfarkt, schlaganfall und venöse thromboembolien. antirheumatika · tumor-nekrose-faktor-inhibitoren · interleukininhibitoren · psoriasisarthritis · ankylosierende spondylitis in der rheumatologie haben biologika ungefähr seit dem jahr 1999 nach und nach fortschritte in der behandlung bewirkt, die vorher kaum für möglich gehalten wurden. dies betraf als erstes die rheumatoide arthritis (ra). im weiteren wurden biologika jedoch auch bei den spondyloarthritiden (spa) erprobt und zugelassen, zu denen man nicht nur die ankylosierende spondylitis (as), sondern auch die davon abgegrenzte nichtröntgenologische axiale spa (nr-axspa) sowie die psoriasisarthritis (psa) zählt. bei der ra und der spa werden die biologika zu den krankheitsmodifizierenden medikamenten ("disease-modifying antirheumatic drugs" [dmard]) gerechnet. im gegensatz zu den konventionellen synthetischen dmard (csdmard) wie methotrexat (mtx), leflunomid und sulfasalazin (ssz) werden diese als biologische dmard (bdmard) bezeichnet. die bezeichnung bdmard wird daher in der vorliegenden arbeit verwendet. bdmard sind wichtige, aber auch hochpreisige ergänzungen der konventionellen therapien. unter anderem existieren daher für alle oben genannten erkrankungen klare empfehlungen, wann diese eingesetzt werden sollen (. tab. 1). zu beginn der therapie einer rheumatoiden arthritis wird nicht empfohlen bei der ra ist der leitliniengemäße einsatz von bdmard möglich und empfohlen, wenn nach ungenügendem ansprechen auf eine, beim fehlen von prognostisch ungünstigen faktoren auch auf zwei therapiestrategien mit csdmard eine therapieänderung indiziert ist [5, 6] . das cs-dmard der ersten wahl ist dabei mtx, das [7] und wird daher nicht empfohlen. bei der entzündlich-aktiven psa mit im vordergrund stehender arthritis wird ebenfalls mindestens eine csdmard-therapie vor beginn der bdmard-therapie empfohlen [8, 9] . auch hier wird meist mtx gegeben, wobei die datenlage dafür weniger solideals beider raist [8] [9] [10] .ausnahme davon sind jedoch patienten mit im vordergrund stehender enthesitis oder axialem befall [8] und in den ganz neuen empfehlungen der group for research and assessment of psoriasis and psoriatic arthritis (grappa) auch patienten mit daktylitis [9] . hier sind csdmard unwirksam und die gabe von bdmard ist bereits möglich, wenn nichtsteroidale antirheumatika (nsar) oder im fall der enthesitis unter umständen eine lokale glukokortikoidinjektion therapeutisch nicht ausreichen [8] [9] [10] . ähnlich ist es bei der schweren und aktiven nr-axspa oder as. hier sollten 2 nsar in ausreichender dosis gegeben worden sein. die csdmard, und dann auch nur ssz, spielen hier nur bei der peripheren arthritis eine rolle [11] . tnf-α-inhibitoren waren die ersten und sind heute immer noch die am häufigsten gegebenen bdmard in der rheumatologie. fünf substanzen sind zugelassen, davon 4 monoklonale antikörper (adalimumab, certolizumab pegol, golimumab und infliximab) und ein lösliches rezeptorkonstrukt (etanercept; . tab. 2). pharmakologisch nimmt certolizumab pegol eine sonderstellung ein, da die pegylierung die plazentagängigkeit stark reduziert. deswegen wird dieses präparat bei kinderwunsch oder schwangerschaft präferenziell angewendet. golimumab und infliximab sind bei der ra nur in kombination mit mtx zugelassen. » bei der rheumatoiden arthritis sollten dmard möglichst mit methotrexat kombiniert werden bei der ra werden in einer höheren rate als bei der spa anti-drug-antikörper gegen die bdmard gebildet [12] . die rate unterscheidet sich bei den verschiedenen anti-tnf-α-therapien, wobei sie bei adalimumab und infliximab am höchsten ist [12] . in der leitlinie zur behandlung der ra mit dmard wird daher wann immer möglich die kombination mit mtx empfohlen [5] . dies reduziert die anti-drug-antikörper-bildung signifikant, dafür sind 10 mg mtx pro woche ausreichend [13] . bei der psa, nr-axspa und as hat die kombination der bdmard mit mtx keinen zusätzlichen klinischen nutzen gezeigt und wird daher nicht allgemein empfohlen [8] [9] [10] [11] . der interleukin(il)-1-rezeptor-antagonist anakinra ist zur behandlung der ra zugelassen. wegen einer im verhältnis schwächeren wirkung wird er jedoch nicht mehr empfohlen [5] . er hat in der rheumatologie jedoch beim morbus still des erwachsenen und beim familiären mittelmeerfieber als zugelassene indikation einen festen stellenwert, der il-1-inhibitor canakinumab zusätzlich auch noch bei der arthritis urica. die anti-il-6-rezeptor-antikörper tocilizumab und sarilumab sind beide zur behandlung der ra zugelassen und effektiv. klinische studien haben für die monotherapie ohne den kombinationspartner mtx eine gute wirksamkeit gezeigt [14, 15] , sodass eine kombination mit mtx bei den il-6-inhibitoren nicht notwendig ist [5] . für tocilizumab existiert in der rheumatologie außerdem eine zulassung zur behandlung der riesenzellarteriitis, nicht jedoch für die spa inklusive der psa. abatacept ist ein hemmer der t-lymphozyten-kostimulation und ein fusionsprotein aus "cytotoxic t-lymphocyte-associated protein 4" (ctla-4) und einem immunglobulinanteil. es ist für die behandlung der ra in kombination mit mtx zugelassen und wird in dieser indikation breit eingesetzt. kürzlich erfolgte auch eine zulassung für die psa, abatacept wird aber wegen seiner allenfalls minimalen wirkung auf die kutane psoriasis oder axiale manifestationen bisher nur selten bei psa eingesetzt. rituximab ist ein monoklonaler anti-cd20-antikörper mit depletierender wirkung auf tab. 3 definitionen der behandlungsziele der therapie mit biologischen (und "targeted synthetic") dmard nach dem treat-to-target-prinzip rheumatoide arthritis behandlungsziel remission oder niedrige krankheitsaktivität [ ustekinumab ist ein il-12/il-23-inhibitor, guselkumab hemmt il-23. beide sind in der behandlung der psa wirksam und dafür zugelassen. therapien mit bdmard sollten nach dem treat-to-target(t2t)-prinzip erfolgen, also mit einem definierten therapieziel, meist der remission oder einer niedrigen bzw. minimalen krankheitsaktivität. dazu ist die nutzung validierter assessment-scores notwendig. für die entsprechenden entitäten sind die therapieziele und die mit den scores erfassten kategorien in . tab. 3 aufgelistet. wird das therapieziel nicht erreicht, sollte die therapie entsprechend angepasst werden, was meist den wechsel der medikation, unter umständen aber auch die ergänzung von mtx bedeutet. sicherheitsaspekte der therapie mit bdmard bdmard wurden seit ihrer klinischen einführung international sehr intensiv auf mögliche sicherheitssignale untersucht. in deutschland haben die vom deutschen rheuma-forschungszentrum in berlin geleiteten register "rheumatoide arthritis: beobachtung der biologika-therapie" (rabbit) für die ra und rabbit-spa für die spa die studiengenerierten daten zur sicherheit der bdmard ergänzt [4] . das risiko von infektionen unter bd-mard-therapien ist auf das 1,6-fache im vergleich zu patienten unter csdmard-behandlung erhöht [16] . dieses risiko ist jedoch sehr stark von den komorbiditäten und einer begleitenden glukokortikoidtherapie abhängig. letztere wird durch die bdmard-therapie positiv beeinflusst, sodass der gesamteffekt einer bdmard-therapie auf das infektionsrisiko positiv sein kann. auch ist das risiko des auftretens einer sepsis oder des versterbens nach schwerwiegender infektion unter bdmard im vergleich zu einer csdmard-therapie erniedrigt [17] . das individuelle risiko kann mit einem score (https:// www.biologika-register.de) abgeschätzt werden, der diese faktoren einberechnet [18] . zu beginn der tnf-α-hemmer-ära fielen vermehrt reaktivierungen von tuberkulosen (tbc) auf, wobei das risiko unter monoklonalen antikörpern gegen tnf-α höher war als unter etanercept [19] . daher wurden neben dem screening auf hepatitis b zelluläre tests auf latente tbc ("enzyme-linked immunospot assay" oder interferon-γ-release-assay) sowie eine bildgebung des thorax vor beginn aller therapien mit bdmard oder jak-inhibitoren zum standard. patienten mit nachweis einer latenten tbc müssen mit einer isoniazid-oder rifampicinprophylaxe behandelt werden. der il-6-inhibitor tocilizumab ist mit darmperforationen assoziiert [20] , weswegen eine divertikulitis in der vorgeschichte eine relative kontraindikation für il-6-inhibitoren ist. sowohl monoklonale anti-tnf-α-inhibitoren als auch rituximab gehen mit einer 1,6-fach erhöhten rate an herpes zoster im vergleich zu csdmard einher [4] . bei jak-inhibitoren ist die rate mit 3,7 allerdings noch deutlich höher. bei allen diesen therapien ist daher die impfung gegen herpes zoster mit dem dafür verfügbaren totimpfstoff empfohlen. bei rituximab ist es besonders wichtig, dass dies möglichst vor beginn der therapie erfolgt, da das medikament die impfantwort beeinträchtigen kann [5] . in der severe-acute-respiratory-syndrome-coronavirus-2(sars-cov-2)-pandemie hat sich gezeigt, dass bdmard in unterschiedlicher weise den verlauf der "coronavirus disease 2019" (covid19) beeinflussen. während tnf-α-inhibitoren mit einem positiven verlauf im vergleich zum csdmard mtx assoziiert waren, ging das b-lymphozyten-depletierende rituximab, wie auch jak-inhibitoren, mit schweren verläufen einher [21] . für tnf-α-inhibitoren ist belegt, dass sie im vergleich zu csdmard eine protektive wirkung hinsichtlich des auftretens von myokardinfarkt und schlaganfall [22, 23] inzwischen sind bdmard wohl die wichtigsten bausteine der therapie von ra und spa. die schon jahrzehntelange erfahrung mit dieser medikamentengruppe hat gezeigt, dass bdmard anhaltend wirksame und bei beachtung von kontraindikationen auch sichere substanzen sind. in den entsprechenden leitlinien der fachgesellschaften [5, 6, 8, 9] wurden den bdmard jeweils feste indikationen in einem schrittweisen therapiealgorithmus zugeordnet. zum auswertungszeitraum bis 2016 waren in der kerndokumentation der deutschen rheumazentren 27 % der patienten mit ra, 30 % der patienten mit psa und 50 % der patienten mit as unter bdmard-behandlung [29] . ob die bdmard dabei ihre volle wirkung auf die jeweiligen therapieziele erreichen, hängt aber ganz wesentlich davon ab, dass ihr einsatz konsequent entsprechend den t2t-prinzipien erfolgt und der patient eine frühe diagnose und therapie erhält. hier liegen die wesentlichen herausforderungen für die zukunft. pathogenetic insights from the treatment of rheumatoid arthritis toward a cytokine-based disease taxonomy targeted treatments for rheumatoid arthritis 2. novel treatment strategiesinrheumatoidarthritis risikoprofil rheumatologische basistherapie -ein update aus dem rabbit-register s2e-leitlinie: behandlung der rheumatoiden arthritis mit krankheitsmodifizierenden medikamenten eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update initiating tocilizumab, with or without methotrexate, compared with starting methotrexate with prednisone within step-up treatment strategies in early rheumatoid arthritis: an indirect comparison of effectiveness and safety of the u-act-early and camera-ii treat-to-target trials eular recommendations for the management of psoriatic arthritis with pharmacological therapies: 2019 update the group for research an assessment of psoriasis and psoriatic arthritis (grappa) treatment recommendations 2021 therapie der psoriasisarthritis. gibt es eine differenzialindikation? evidenzbasierte empfehlungen zur diagnose und therapie der axialen spondyloarthritis: s3 leitlinien der deutschengesellschaftfürrheumatologie(dgrh) in kooperation mit der arbeitsgemeinschaft der medizinsich-wissenschaftlichen fachgesellschaften (awmf) comparative immunogenicity of tnf inhibitors: impact on clinicalefficacyandtolerabilityinthemanagement of autoimmune diseases. a systematic review and meta-analysis efficacy and safety of ascending methotrexate dose in combination with adalimumab: the randomised concerto trial tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind efficacy and safety of sarilumab monotherapy versus adalimumab monotherapy for the treatment of patients with active rheumatoid arthritis (monarch): a randomised, double-blind, parallelgroup phase iii trial treatment benefit or survival of the fittest: what drives the time-dependent decrease in serious infection rates under tnf inhibition and what does this imply for the individual patient impact of treatment with biologic dmards on the risk of sepsis or mortality after serious infection in patients with rheumatoid arthritis validation of the rabbit risk score for serious infections drug-specific risk of tuberculosis in patients with rheumatoid arthritis treated with anti-tnf therapy: results from the british society for rheumatology biologics register (bsrbr) risk for lower intestinal perforations in patients with rheumatoid arthritis treated with tocilizumab in comparison to treatment with other biologic or conventional synthetic dmards tnfi is associated with positive outcome, but jaki and rituximab are associated with negative outcome of sars-cov-2 infection in patients with rmd comparative risk of cardiovascular events with biologic and synthetic disease-modifying antirheumatic biologics are an integral part of modern strategies for treatment of rheumatoid arthritis (ra) and spondylarthritis (spa), including psoriatic arthritis (psa). biologics are biotechnologically produced proteins that have inhibiting effects on humoral and cellular components of rheumatic inflammation. substance classes used in rheumatology are tumor necrosis factor (tnf)-alpha, interleukin (il)-1, il-6, il-12, il-17 and il-23 inhibitors effective against cytokines as well as the t lymphocyte activation inhibitor abatacept and the b lymphocyte-depleting rituximab. there are clear recommendations for the use of biologics for ra patients inadequately responding to one or more conventional synthetic disease-modifying antirheumatic drugs and for ankylosing spondylitis (as) and nonradiographical axial spa patients with an inadequate response to at least two nonsteroidal antirheumatic drugs. for psa the recommended use depends on the most prominent manifestations in each case. treatment with biologics should follow the treat to target principle, with a defined and validated treatment target. treatment in cases of ra and spa should target remission or at least a low or minimum disease activity. the safety of treatment with biologics has been intensively investigated. there are very specific contraindications for individual substance classes with a focus on an increased risk of infections. the standard procedure before starting treatment with biologics includes the exclusion of latent tuberculosis and hepatitis b. the tnf-alpha inhibitors have a protective effect with respect to myocardial infarction, stroke and venous thromboembolism. antirheumatic agents · tumor necrosis factor inhibitors · interleukin inhibitors · arthritis, psoriatic · spondylitis, ankylosing drugs in patients with rheumatoid arthritis: a systematicreviewandmeta-analysis. key: cord-0016676-1c94qcm0 authors: wang, ziyu; li, zijian; wang, guojue; sun, ying; yuan, yuanyang; nie, hong title: salubrinal alleviates collagen-induced arthritis through promoting p65 degradation in osteoclastogenesis date: 2021-03-28 journal: int j mol sci doi: 10.3390/ijms22073501 sha: ad11973166abbf20df0c0ab2fa8825b915f4168d doc_id: 16676 cord_uid: 1c94qcm0 rheumatoid arthritis (ra) is a complex systemic autoimmune disorder that primarily involves joints, further affects the life quality of patients, and has increased mortality. the pathogenesis of ra involves multiple pathways, resulting in some patients showing resistance to the existing drugs. salubrinal is a small molecule compound that has recently been shown to exert multiple beneficial effects on bone tissue. however, the effect of salubrinal in ra has not been clearly confirmed. hence, we induced collagen-induced arthritis (cia) in dba/1j mice and found that salubrinal treatment decreased the clinical score of cia mice, inhibiting joint damage and bone destruction. furthermore, salubrinal treatment downregulated osteoclast number in knee joint of cia in mice, and suppressed bone marrow-derived osteoclast formation and function, downregulated osteoclast-related gene expression. moreover, salubrinal treatment inhibited rankl-induced nf-κb signaling pathway, and promoted p65 degradation through the ubiquitin-proteasome system, further restrained rankl-induced osteoclastogenesis. this study explains the mechanism by which salubrinal ameliorates arthritis of cia in mice, indicating that salubrinal may be a potential drug for ra, and expands the potential uses of salubrinal in the treatment of bone destruction-related diseases. rheumatoid arthritis (ra) is a chronic progressive autoimmune disorder that typically causes cartilage structural damage, synovial inflammation, and bone erosion, resulting in swelling and destruction of the joints and corresponding disability [1] . bone loss in ra results from excessive osteoclast activation and blunted bone formation, which is correlated with disease severity and functional disturbance [2] . conventional synthetic disease-modifying antirheumatic drugs (dmards) alleviate disease progression in some ra patients, although a comprehensive perspective of the mechanisms of action of these drugs remains elusive [3] . biologic dmards (bdmards) represented by tnf inhibitors have revolutionized the treatment landscape for ra in the past few decades [4] . however, several clinical studies have shown that refractory disease still presents a significant clinical challenge to multiple bdmards [5] . in the past few years, the oral small-molecule inhibitors, such as janus kinase inhibitors (such as tofacitinib) and nuclear factor (nf)-κb inhibitors (such as iguratimod), have improved treatment outcomes in patients with ra by interfering with various intracellular signaling pathways upon which immune functions converge [6, 7] . thus, these drugs have been shown to have efficacies similar to those of bdmards in the treatment of ra. bone homeostasis is regulated by osteoclast-mediated bone resorption and osteoblastmediated bone formation [8] . osteoclasts are the only cells responsible for bone resorption in vivo that are derived from hematopoietic precursor cells. macrophage-colony stimulating factor (m-csf) and receptor activator of nuclear factor-κb ligand (rankl) are int. j. mol. sci. 2021, 22, 3501 2 of 12 required for osteoclast proliferation, differentiation, and activation. m-csf supports osteoclast precursors' survival and proliferation by binding to m-csf receptor c-fms on osteoclast precursors. meanwhile, rankl binds to its receptor rank to trigger multiple signaling transduction pathways in osteoclast precursors, promoting the differentiation of osteoclast precursors into osteoclasts. then, mature osteoclasts experiencing cytoskeletal rearrangements and spreading in a c-src dependent manner in response to m-csf and rankl [9] [10] [11] , finally adhere to bone matrix as multinucleated osteoclasts. acid and lytic enzymes are secreted by rankl-activated multinucleated osteoclasts to degrade bone matrix in a specialized compartment, and then can cause cartilage and bone damage in vivo. in ra, osteoclasts show enhanced differentiation capacity and increased activity related to direct intercellular interactions, and the systemic effects of inflammatory cytokines and autoantibodies involved in ra [12] . furthermore, osteoclasts are much more than bone decomposers. increasing evidence indicates that several pathogens use osteoclasts as a host cell to reproduce and escape, finally causing bone damage [13] . thus, osteoclasts are also a new cell target for bone defects caused by infections. salubrinal is a small compound with a molecular weight of 479.81 that rescues rat pc12 cells from endoplasmic reticulum stress-induced apoptosis by inhibiting the dephosphorylation of phospho-eif2α via control of serine/threonine protein phosphatase 1 and growth arrest-and dna damage-induced transcript 34 [14] . through regulation of the eif2α-mediated signaling pathway, salubrinal exerts various beneficial effects on bone metabolism, including the inhibition of matrix metalloproteinase (mmp) 13 expression and activity in chondrocytes via the inactivation of p38 and nf-κb signaling pathways [15] and activation of activating transcription factor 4 translation by elevation of eif2α phosphorylation, and hence promotes osteoblastogenesis and bone regeneration [16] . moreover, salubrinal suppresses bone resorption by inhibiting rankl-induced osteoclastogenesis through inactivation of nuclear factor of activated t-cells, cytoplasmic, calcineurin dependent 1 (nfatc1) [17] . briefly, salubrinal has potential to be a candidate drug in skeletal system disease by promoting osteoblastogenesis, inhibiting osteoclastogenesis, and suppressing chondrocytes activity. it was reported that salubrinal could suppress inflammation in anti-collagen antibody-induced arthritis (caia) by inhibiting dual-specificity phosphatase 2 [18] . however, the effect and mechanism of action of salubrinal in ra have not been clearly confirmed. therefore, in this study, we first investigated the potential therapeutic effects of salubrinal in a mouse model of collagen-induced arthritis (cia), a classical arthritis model that resembles human ra. we further explored the mechanism by which salubrinal inhibits osteoclast formation. we hope to thus find a new treatment strategy for ra and other diseases with osteoclast-related bone destruction. first, we established a cia model in dba/1j mice. daily salubrinal (2 mg/kg) treatment was started on day 21. the results showed that salubrinal markedly attenuated the severity of arthritis, but did not affect the body weights of mice ( figure 1a ). additionally, decreased synovium inflammation and joint damage were observed in the cia mice treated with salubrinal ( figure 1b) . furthermore, we observed that salubrinal reduced the degree of bone destruction through micro-ct and three-dimensional reconstruction of the ankle joints and paws ( figure 1c ). as osteoclasts are the only cells responsible for bone resorption in vivo, we next evaluated the effects of salubrinal on osteoclast formation in the knee joints of cia mice by tartrate-resistant acid phosphatase (trap) staining. our results showed that the number of osteoclasts decreased after treatment with salubrinal ( figure 1d ). overall, these data showed that salubrinal inhibited joint damage, thereby alleviating the clinical symptoms of cia in mice. results showed that the number of osteoclasts decreased after treatment with salubrinal ( figure 1d ). overall, these data showed that salubrinal inhibited joint damage, thereby alleviating the clinical symptoms of cia in mice. to further investigate the effects of salubrinal on osteoclastogenesis in vitro, bone marrow cells were separated and differentiated into osteoclasts by stimulation with m-csf and rankl. we found that salubrinal decreased osteoclast number in a dose-dependent manner, as indicated by trap staining (figure 2a ). next, we investigated the effects of salubrinal on osteoclast function using bone resorption assays. the results showed that salubrinal inhibited hydroxyapatite coating-removal (as a surrogate for bone resorption) mediated by osteoclasts in a dose-dependent manner ( figure 2b ). consistent with these results, genes related to osteoclast formation and function (such as nfatc1, ctsk, etc.) were also downregulated by salubrinal in a dose-dependent manner ( figure 2c ). thus, our findings showed that salubrinal could suppress osteoclast formation and function. to further investigate the effects of salubrinal on osteoclastogenesis in vitro, bone marrow cells were separated and differentiated into osteoclasts by stimulation with m-csf and rankl. we found that salubrinal decreased osteoclast number in a dose-dependent manner, as indicated by trap staining (figure 2a ). next, we investigated the effects of salubrinal on osteoclast function using bone resorption assays. the results showed that salubrinal inhibited hydroxyapatite coating-removal (as a surrogate for bone resorption) mediated by osteoclasts in a dose-dependent manner ( figure 2b ). consistent with these results, genes related to osteoclast formation and function (such as nfatc1, ctsk, etc.) were also downregulated by salubrinal in a dose-dependent manner ( figure 2c ). thus, our findings showed that salubrinal could suppress osteoclast formation and function. the rankl-induced nf-κb signaling pathway is a vital pathway involved in osteoclastogenesis and osteoclast function [19] . therefore, we investigated the effects of salubrinal on the rankl-induced nf-κb signaling pathway in osteoclast precursors by western blotting. we found that salubrinal treatment decreased the resynthesis abundance of iκbα and downregulated the protein level of p65, a key transcription factor of iκbα in the nf-κb pathway ( figure 3a ). moreover, we found that after rankl stimulation, p65 abundance was decreased in the cytoplasm and nucleus of osteoclast precursors by salubrinal treatment ( figure 3b ). this result was further confirmed using immunofluorescence technique ( figure 3c ). in addition, we found that salubrinal inhibited rankl-induced nf-κb signaling pathway transcriptional activity, similar to the effects of the nf-κb inhibitor bay-11 ( figure 3d ). according to the above results, we deduced that salubrinal inhibited the rankl-induced nf-κb signaling pathway by decreasing p65 protein level. further we found salubrinal treatment in vivo also decreased p65 expression in the the rankl-induced nf-κb signaling pathway is a vital pathway involved in osteoclastogenesis and osteoclast function [19] . therefore, we investigated the effects of salubrinal on the rankl-induced nf-κb signaling pathway in osteoclast precursors by western blotting. we found that salubrinal treatment decreased the resynthesis abundance of iκbα and downregulated the protein level of p65, a key transcription factor of iκbα in the nf-κb pathway ( figure 3a ). moreover, we found that after rankl stimulation, p65 abundance was decreased in the cytoplasm and nucleus of osteoclast precursors by salubrinal treatment ( figure 3b ). this result was further confirmed using immunofluorescence technique ( figure 3c ). in addition, we found that salubrinal inhibited rankl-induced nf-κb signaling pathway transcriptional activity, similar to the effects of the nf-κb inhibitor bay-11 ( figure 3d ). according to the above results, we deduced that salubrinal inhibited the rankl-induced nf-κb signaling pathway by decreasing p65 protein level. further we found salubrinal treatment in vivo also decreased p65 expression in the knees of cia mice ( figure 3e ). overall, these data suggested that salubrinal might inhibit the rankl-induced nf-κb signaling pathway by downregulating p65 abundance. knees of cia mice ( figure 3e ). overall, these data suggested that salubrinal might inhibit the rankl-induced nf-κb signaling pathway by downregulating p65 abundance. to determine whether salubrinal impaired osteoclastogenesis by downregulating p65 protein level, we designed two pairs of sirna oligonucleotides specific for p65 mrna and used them to transfect raw264.7 cells, resulting in p65 knockdown ( figure 4a ). after p65 knockdown, the mrna expression levels of trap, matrix metalloproteinase 9 (mmp-9), and cathepsin k (ctsk) gene were significantly reduced and comparable with salubrinal treatment, whereas osteoclast-associated receptor (oscar) gene expression levels were partly decreased, and nfatc1 expression levels were not significantly influenced. however, the expression levels of both genes were obviously decreased after salubrinal treatment ( figure 4b ). these results indicated that knockdown of p65 had an effect similar to that of salubrinal treatment in the regulation of osteoclast-regulated gene expression. to determine whether salubrinal impaired osteoclastogenesis by downregulating p65 protein level, we designed two pairs of sirna oligonucleotides specific for p65 mrna and used them to transfect raw264.7 cells, resulting in p65 knockdown ( figure 4a ). after p65 knockdown, the mrna expression levels of trap, matrix metalloproteinase 9 (mmp-9), and cathepsin k (ctsk) gene were significantly reduced and comparable with salubrinal treatment, whereas osteoclast-associated receptor (oscar) gene expression levels were partly decreased, and nfatc1 expression levels were not significantly influenced. however, the expression levels of both genes were obviously decreased after salubrinal treatment ( figure 4b ). these results indicated that knockdown of p65 had an effect similar to that of salubrinal treatment in the regulation of osteoclast-regulated gene expression. finally, we induced p65-knockdown raw264.7 cells to differentiate into osteoclasts and found that osteoclast formation was significantly suppressed, comparable to the levels achieved with salubrinal treatment ( figure 4c ). taken together, these findings indicated that salubrinal inhibited osteoclast formation by downregulating p65 protein level. finally, we induced p65-knockdown raw264.7 cells to differentiate into osteoclasts and found that osteoclast formation was significantly suppressed, comparable to the levels achieved with salubrinal treatment ( figure 4c ). taken together, these findings indicated that salubrinal inhibited osteoclast formation by downregulating p65 protein level. osteoclast numbers were quantified by trap staining after induction by rankl (100 ng/ml) for 4 days, salubrinal (10 µm) was added for the last 3 days. data are shown as means ± sem. *p < 0.05, **p < 0.01, ***p < 0.001. to study the mechanism involved in the decrease in p65 protein level induced by salubrinal treatment, we used chx chase experiments to check how salubrinal affected p65 total protein abundance. after using chx to inhibit protein synthesis, p65 abundance was still downregulated by salubrinal ( figure 5a ), indicating that salubrinal decreased p65 abundance by promoting p65 degradation. additionally, treatment with the proteasome inhibitor mg132 and the autophagy inhibitor cq showed that salubrinal promoted p65 degradation through the ubiquitin-proteasome system ( figure 5b ) rather than , and ctsk gene were detected by qpcr after induction by rankl (100 ng/ml) for 24 h, salubrinal (10 µm) was added together with rankl for 24 h. (c) osteoclast numbers were quantified by trap staining after induction by rankl (100 ng/ml) for 4 days, salubrinal (10 µm) was added for the last 3 days. data are shown as means ± sem. * p < 0.05, ** p < 0.01, *** p < 0.001. to study the mechanism involved in the decrease in p65 protein level induced by salubrinal treatment, we used chx chase experiments to check how salubrinal affected p65 total protein abundance. after using chx to inhibit protein synthesis, p65 abundance was still downregulated by salubrinal ( figure 5a ), indicating that salubrinal decreased p65 abundance by promoting p65 degradation. additionally, treatment with the proteasome inhibitor mg132 and the autophagy inhibitor cq showed that salubrinal promoted p65 degradation through the ubiquitin-proteasome system ( figure 5b ) rather than the autophagy-lysosome pathway ( figure 5c ). taken together, our results indicated that salubrinal mediated p65 degradation is dependent on the ubiquitin-proteasome system. the autophagy-lysosome pathway ( figure 5c ). taken together, our results indicated that salubrinal mediated p65 degradation is dependent on the ubiquitin-proteasome system. in this study, we demonstrated that salubrinal could alleviate the clinical symptoms of cia in mice by reducing bone erosion and joint destruction. moreover, salubrinal inhibited the rankl-induced nf-κb signaling pathway by promoting p65 degradation via the ubiquitin-proteasome system, further suppressing rankl-induced osteoclastogenesis. previous studies on salubrinal have focused on its neuroprotective effects against neurotoxic substances, demonstrating that this drug can mediate neurological disorders via inhibition of eif2α dephosphorylation [20] . however, in recent studies, salubrinal was shown to exert multiple beneficial effects on skeletal tissues, including effects on chondrocytes, osteoclasts, and osteoblasts. although it was reported that salubrinal could suppress inflammation in anti-collagen antibody-induced arthritis (caia) by inhibiting dualspecificity phosphatase 2 [18] , the caia model does not fully simulate the pathogenesis of ra because caia mice exhibit arthritis triggered by passive immunity rather than active immunity. in this study, we proved that salubrinal could alleviate the clinical symptoms of cia in mice by reducing bone erosion and joint destruction. furthermore, we observed that salubrinal decreased osteoclast number in the joint of cia mice. osteoclasts are specialized cells derived from the monocyte/macrophage hematopoietic lineage that adhere to the bone matrix and then secrete acid and lytic enzymes that can cause cartilage and bone damage in ra [21] . two cytokines, rankl and m-csf, are both necessary for osteoclastogenesis. these proteins act together to induce the expression of genes related to osteoclast differentiation and function, leading to the development of mature osteoclasts [22] . in our study, we used rankl to induce osteoclastogenesis and found that salubrinal decreased rankl-induced bone marrow-derived osteoclast number and function and reduced the expression levels of various genes, including nfatc1, trap, oscar, and in this study, we demonstrated that salubrinal could alleviate the clinical symptoms of cia in mice by reducing bone erosion and joint destruction. moreover, salubrinal inhibited the rankl-induced nf-κb signaling pathway by promoting p65 degradation via the ubiquitin-proteasome system, further suppressing rankl-induced osteoclastogenesis. previous studies on salubrinal have focused on its neuroprotective effects against neurotoxic substances, demonstrating that this drug can mediate neurological disorders via inhibition of eif2α dephosphorylation [20] . however, in recent studies, salubrinal was shown to exert multiple beneficial effects on skeletal tissues, including effects on chondrocytes, osteoclasts, and osteoblasts. although it was reported that salubrinal could suppress inflammation in anti-collagen antibody-induced arthritis (caia) by inhibiting dual-specificity phosphatase 2 [18] , the caia model does not fully simulate the pathogenesis of ra because caia mice exhibit arthritis triggered by passive immunity rather than active immunity. in this study, we proved that salubrinal could alleviate the clinical symptoms of cia in mice by reducing bone erosion and joint destruction. furthermore, we observed that salubrinal decreased osteoclast number in the joint of cia mice. osteoclasts are specialized cells derived from the monocyte/macrophage hematopoietic lineage that adhere to the bone matrix and then secrete acid and lytic enzymes that can cause cartilage and bone damage in ra [21] . two cytokines, rankl and m-csf, are both necessary for osteoclastogenesis. these proteins act together to induce the expression of genes related to osteoclast differentiation and function, leading to the development of mature osteoclasts [22] . in our study, we used rankl to induce osteoclastogenesis and found that salubrinal decreased rankl-induced bone marrow-derived osteoclast number and function and reduced the expression levels of various genes, including nfatc1, trap, oscar, and ctsk, which are the markers of the osteoclast lineage and are responsible for osteoclast function [23] [24] [25] . among these genes, nfatc1 is the first to receive rankl signals. rankl-induced osteoclast differentiation is divided into three stages. first, rankl binds to rank, resulting in the recruitment of traf6 and leading to the activation of the nf-κb signaling pathway. nf-κb and nfatc2 are then recruited to the nfatc1 promoter at an early stage. second, activated nfatc1 binds to its own promoter with activating protein-1 (ap-1), leading to the autoamplification of nfatc1. in this stage, ap-1 is critical for nfatc1 autoamplification. third, a number of osteoclast-specific genes, including ctsk, trap, and mmp-9, are activated by nfatc1 and other transcriptional factors. therefore, the nf-κb and ap-1 signaling pathways are vital in rankl-induced osteoclast differentiation, and inhibition of ikkβ and ikkα in rankl-induced bone marrow-derived osteoclast precursors could suppress osteoclastogenesis and prevent inflammatory bone destruction [26] . previous studies have indicated that salubrinal can inhibit rankl-induced ap-1 signaling in raw264.7 cells [17, 27] . here, we showed that salubrinal inhibited the rankl-induced nf-κb signaling pathway by downregulating p65 protein abundance in bone marrow-derived osteoclast precursors. furthermore, after p65 knockdown, ranklinduced osteoclastogenesis was significantly suppressed. p65 knockdown also decreased trap, mmp-9, and ctsk expression levels, which were comparable to those under salubrinal treatment. however, nfatc1 expression levels were not significantly affected by p65 knockdown, and oscar expression levels were partly decreased by p65 knockdown, whereas salubrinal significantly inhibited the expression of the both genes. we speculate that this may be because p65 is recruited to the nfatc1 promoter at an early stage, as described above. ap-1 facilitates the autoamplification of nfatc1, and the transcription of oscar is mainly controlled by nfatc1 [25] . thus, p65 knockdown can have early inhibitory effects on nfatc1 transcription, whereas nfatc1 can still undergo autoamplification without inhibition of ap-1. however, salubrinal inhibited both the nf-κb and ap-1 pathways. therefore, the effects of salubrinal on suppression of rankl-induced osteoclastogenesis relied on the inhibition of both the nf-κb and ap-1 pathways. notably, salubrinal promoted p65 degradation through the ubiquitin-proteasome system rather than the autophagy-lysosome pathway. additionally, we found that salubrinal could directly bind to p65, which may influence the binding of p65 to the proteasome and is responsible for promoting p65 degradation (data not shown). nf-κb is a critical transcription factor that regulates multiple immune and inflammatory responses, and the most abundant form of nf-κb in the classical pathway is the heterodimer of p50 and p65 [28] . an increase in activated p65 levels triggers the overactivation of downstream effector pathways that are involved in many autoimmune diseases [29] . hence, the nf-κb p65 signaling pathway is essential for drug discovery. moreover, p65 degradation has been suggested as a mechanism that controls the strength and duration of nf-κb activation [30] . thus, our findings highlight the potential for targeting p65 using salubrinal, suggesting promising applications of salubrinal in the clinical setting. taken together, our findings demonstrated that salubrinal could serve as an efficient therapeutic drug for cia by inhibiting osteoclast formation and function. our results also clarified the potential mechanisms, showing that salubrinal suppressed ranklinduced nf-κb signaling by downregulating p65 protein abundance via promotion of p65 degradation by the ubiquitin-proteasome system. our findings established a solid foundation for the application of salubrinal as a potential treatment for ra and expanded the potential uses of salubrinal in the treatment of bone destruction-related diseases. eight-week-old male dba/1j mice and six-week-old male c57bl/6 mice (shanghai slac laboratory animal co., ltd., shanghai, china) were maintained under pathogenfree conditions at shanghai jiao tong university school of medicine. all experimental procedures were performed in accordance with the guidelines of the animal care and use committee. salubrinal and mg132 were purchased from selleck chemicals (houston, tx, usa). bay11 was purchased from beyotime biotechnology (shanghai, china), and cycloheximide (chx) was purchased from medchemexpress (monmouth junction, nj, usa). chloroquine (cq) was purchased from sigma-aldrich (st. louis, mo, usa). to establish the cia model, eight-week-old male dba/1j mice were intradermally injected with cii (150 µg/mouse; chondrex, redmond, wa, usa) mixed with freund's complete adjuvant (sigma-aldrich, st. louis, mo, usa) in the tail on day 0. on day 21, mice were injected with cii (75 µg/mouse) mixed with freund's incomplete adjuvant (sigma-aldrich, st. louis, mo, usa) as a booster. on the day of the booster injection, the mice were randomly divided into two groups, and intraperitoneally injected with salubrinal (2 mg/kg, stocked in dimethyl sulfoxide, further dissolved in phosphate-buffered saline (pbs)) or with dimethyl sulfoxide (dissolved in pbs), respectively. mice were observed daily and scored for disease severity as previous describe [31] . cia mice were sacrificed on day 37 after the first immunization. the hind legs were fixed, and three-dimensional micro-ct analysis was performed. micro-ct scanning was performed using a skyscan1176 instrument as previous described [32] . three-dimensional microstructural image data were analyzed using ct vox software (skycan). alternatively, samples were decalcified for hematoxylin and eosin staining and p65 immunohistochemical staining. bone marrow-derived cells were separated from the tibiae and femora of six-week-old male c57bl/6 mice, induced using α-mem medium (thermo fisher scientific, waltham, ma, usa) containing 50 ng/ml m-csf (peprotech, rocky hill, nj, usa) for 3 days to differentiate into osteoclast precursors, and then induced into mature osteoclasts by treatment with 30 ng/ml rankl (r&d systems, minneapolis, mn, usa) and 50 ng/ml m-csf for another 4 days. trap staining was performed using a leukocyte acid phosphatase (trap) kit (sigma-aldrich, st. louis, mo, usa) according to the manufacturer's instructions. briefly, slides were fixed by immersion in fixative solution for 30 s, trap staining solution was prepared, and the slides were incubated for 1 h at 37 • c in the dark. cells were observed using a light microscope, and osteoclasts are defined as trap-positive cells containing more than three nuclei. osteoclast bone-resorption ability was measured using hydroxyapatite-coated os-teoassay plate resorption assays. bone marrow-derived cells were seeded in 24-well osteoassay plates and induced with 50 ng/ml m-csf for 3 days to generate osteoclast precursors, then treated with 30 ng/ml rankl and 50 ng/ml m-csf for another 4 days. cells were removing using sodium hypochlorite solution. the absorption pit aera were observed using a light microscope. resorption pit aeras were quantified using imagej software (nih, bethesda, md, usa). bone marrow-derived osteoclasts or raw264.7 cells were lysed in trizol (life sciences, grand island, ny, usa) to extract total rna. cdna was synthesized using the prime script rt master mix kit (takara, kusatsu, shiga, japan). the expression levels of osteoclastogenesis-related genes, nfatc1, trap, oscar, mmp-9, and ctsk were measured using qpcr with power sybr green master mix (life sciences, grand island, ny, usa), and the mrna expression of the above target genes were normalized to the mrna levels of β-actin. the primer sequences used are listed in table 1 . for subcellular fractionation, nuclear and cytoplasmic fractions were obtained using ne-pertm nuclear and cytoplasmic extraction reagents (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's instructions. for traditional western blotting, cells were lysed with radio-immunoprecipitation assay buffer. after protein quantification, the samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels and transferred to nitrocellulose membranes. the membranes were then blocked with 5% bovine serum albumin (bsa) in tbst and sequentially incubated with primary antibodies and horseradish peroxidase-linked secondary antibodies. the protein bands were visualized by enhanced chemiluminescence (thermo fisher scientific, waltham, ma, usa) and quantified by densitometry analysis using imagej software (nih, bethesda, md, usa). the primary antibodies were used as follows: p-iκbα (cell signaling technology, beverly, ca, usa); iκbα (cell signaling technology, beverly, ca, usa); p-p65 (cell signaling technology); p65 (cell signaling technology, beverly, ca, usa); β-actin (cell signaling technology, beverly, ca, usa); lamin b (proteintech, shanghai, china); gapdh (cell signaling technology, beverly, ca, usa). bone marrow-derived cells were cultured on coverslips, seeded in 24-well plates and induced with 50 ng/ml m-csf to generate osteoclast precursors. the cells were then stimulated with 30 ng/ml rankl for 30 min and fixed in paraformaldehyde for 15 min. triton-x-100 was used to permeabilize the cell membrane. sections were then blocked in 5% bsa for 1 h and incubated with anti-p65 antibodies (cell signaling technology, beverly, ca, usa) at 4 • c overnight in the dark. the cells were then incubated with secondary antibodies labeled with alexa fluor 488 (thermo fisher scientific, waltham, ma, usa) and 4 ,6-diamidino-2-phenylindole (thermo fisher scientific, waltham, ma, usa). p65 location was observed using a confocal microscope. ) and a multicell plate luminometer according to the manufacturer's instructions cell-derived osteoclast induction and sirna transfection two pairs of sirnas were designed against mouse p65 using following sirna sequences: sirna1 sense, 5 -ggaccuaugagaccuucaatt-3 and sirna1 antisense, 5 -uugaaggucucauaggucctt-3 ; sirna2 sense, 5 -ccauggaguuccaguacuutt-3 and sirna2 antisense, 5 -aaguacuggaacuccaugggc-3 . raw264.7 cells were transfected with sirna using attractene transfection reagent rheumatoid arthritis osteoblast-osteoclast interactions the early history of antirheumatic drugs reflections on 'older' drugs: learning new lessons in rheumatology defining refractory rheumatoid arthritis jak inhibition as a therapeutic strategy for immune and inflammatory diseases small molecule inhibitors in the treatment of rheumatoid arthritis and beyond: latest updates and potential strategy for fighting covid-19 osteoclast differentiation by rankl and opg signaling pathways colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts evidence for a functional association between phosphatidylinositol 3-kinase and c-src in the spreading response of osteoclasts to colony-stimulating factor-1 the ligand for osteoprotegerin (opgl) directly activates mature osteoclasts how autoantibodies regulate osteoclast induced bone loss in rheumatoid arthritis maridonneau-parini, i. the osteoclast, a target cell for microorganisms a selective inhibitor of eif2α dephosphorylation protects cells from er stress chondroprotective effects of salubrinal in a mouse model of osteoarthritis enhancement of osteoblastogenesis and suppression of osteoclastogenesis by inhibition of de-phosphorylation of eukaryotic translation initiation factor 2 alpha suppression of osteoclastogenesis through phosphorylation of eukaryotic translation initiation factor 2 alpha salubrinal acts as a dusp2 inhibitor and suppresses inflammation in anti-collagen antibody-induced arthritis current understanding of rank signaling in osteoclast differentiation and maturation experimental evidence shows salubrinal, an eif2α dephosphorylation inhibitor, reduces xenotoxicant-induced cellular damage recent advances in osteoclast biology osteoclast differentiation and activation tartrate-resistant acid phosphatase (trap) and the osteoclast/immune cell dichotomy osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation the molecular understanding of osteoclast differentiation selective inhibition of nf-kappa b blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo in vitro and in silico analysis of an inhibitory mechanism of osteoclastogenesis by salubrinal and guanabenz mechanisms of nf-kappab p65 and strategies for therapeutic manipulation nf-κb as a therapeutic target in inflammatory-associated bone diseases degradation of promoter-bound p65/rela is essential for the prompt termination of the nuclear factor kappab response collagen-induced arthritis guidelines for assessment of bone microstructure in rodents using micro-computed tomography the data presented in this study are available in the article. the authors have declared that no competing interest exists. key: cord-0002395-9a163ad7 authors: zhang, li; wang, jin; xu, aizhang; zhong, conghao; lu, wuguang; deng, li; li, rongxiu title: a rationally designed tnf-α epitope-scaffold immunogen induces sustained antibody response and alleviates collagen-induced arthritis in mice date: 2016-09-22 journal: plos one doi: 10.1371/journal.pone.0163080 sha: b11cf879da2036d92fbdbbca07fd8cb7f0416e5f doc_id: 2395 cord_uid: 9a163ad7 the tnf-α biological inhibitors have significantly improved the clinical outcomes of many autoimmune diseases, in particular rheumatoid arthritis. however, the practical uses are limited due to high costs and the risk of anti-drug antibody responses. attempts to develop anti-tnf-α vaccines have generated encouraging data in animal models, however, data from clinical trials have not met expectations. in present study, we designed a tnf-α epitope-scaffold immunogen dtnf7 using the transmembrane domain of diphtheria toxin, named dtt as a scaffold. molecular dynamics simulation shows that the grafted tnf-α epitope is entirely surface-exposed and presented in a native-like conformation while the rigid helical structure of dtt is minimally perturbed, thereby rendering the immunogen highly stable. immunization of mice with alum formulated dtnf7 induced humoral responses against native tnf-α, and the antibody titer was sustained for more than 6 months, which supports a role of the universal cd4 t cell epitopes of dtt in breaking self-immune tolerance. in a mouse model of rheumatoid arthritis, dtnf7-alum vaccination markedly delayed the onset of collagen-induced arthritis, and reduced incidence as well as clinical score. dtt is presumed safe as an epitope carrier because a catalytic inactive mutant of diphtheria toxin, crm197 has good clinical safety records as an active vaccine component. taken all together, we show that dtt-based epitope vaccine is a promising strategy for prevention and treatment of autoimmune diseases. tnf-α is a pleiotropic pro-inflammatory cytokine playing pivotal roles in both physiological and pathological processes [1] . the primary function of tnf-α is to regulate immune cells in inflammation as well as in protective immune responses against a variety of infectious pathogens. as a master regulator of pro-inflammatory cytokines such as il-1β, il-6 and gm-csf [2] , over expression of tnf-α causes a variety of chronic inflammatory diseases including rheumatoid arthritis, crohn's disease, psoriatic arthritis, ankylosing spondylitis and psoriasis, and so on [3] . blockade of tnf-α activities with monoclonal antibodies (e.g. infliximab, adalimumab) as well as a receptor-immunoglobulin fusion protein (e.g. etanercept) [3] [4] [5] significantly improved clinical outcomes, in particular, rheumatoid arthritis. nevertheless, all tnf-α biological inhibitors that have been approved for clinical uses are limited in practice due to both high manufacture costs and the risk of anti-drug antibodies (adas) response [6, 7] . it is therefore imperative to develop novel strategies to circumvent these shortcomings. active immunization against tnf-α has been intensively investigated as an alternative approach to address these limits [8] . so far, tnf-α vaccines based on the whole molecule such as tnf-k and tnf autovaccine have shown remarkable results in animal studies [9, 10] . however, these vaccine strategies are not successful in the human trials. tnf-k is a conjugate mixture of tnf-α and klh. the bioactivity of the cytokine is inactivated by formaldehyde treatment. tnf autovaccine comprises two recombinant tnf-α proteins with specific peptides replaced by a cd4 t cell epitope from tetanus toxin. in both cases, the elicited antibody responses against the endogenous molecule were weak while humoral responses against the immunogens or partially denatured tnf-α were strong, suggesting that the epitopes are compromised during production processes [11, 12] . thus a rational design is necessary to generate a stable structure feasible to manufacture. since tnf-α is a potent cytokine, a whole molecule immunogen need inactivated either by chemical modification such as formaldehyde treatment [10] or site directed mutagenesis [13] . however, both approaches unavoidably compromise the immunogenicity of the immunogen. therefore, a peptide epitope based vaccine design is a preferred choice. work from capini et al have shown that a cyclic tnf-α epitope peptide (aa 80-96) conjugated to klh elicits a stronger neutralizing antibody response than the linear counterpart, which suggests that stabilizing the conformation of the peptide epitope is a key criterion in the vaccine design [14] . here, we developed an epitope-scaffold immunogen against tnf-α, in which the conformation of the epitope peptide tnf-α aa 80-97 is stabilized by transplantation onto a scaffold molecule, a transmembrane domain of diphtheria toxin (dtt). we assessed the immunogenicity of the vaccine against native tnf-α in mice as well as the therapeutic efficacy in collagen-induced arthritis mouse model. our results showed that dtt-based epitope-scaffold vaccine is a promising strategy for prevention and treatment of autoimmune diseases. for immunization, we used 54 balb/c mice (females, aged 6-7weeks), 6 mice per experimental group. for vaccine efficacy in cia mouse model, we used 19 dba/1j mice (males, aged 6 weeks), 9 mice in the dtnf7 group, and 10 mice in the dtt control group. all mice were purchased from slac laboratory animal centre (shanghai, china) and kept in specific pathogen-free conditions in shanghai jiao tong university animal center. all animal studies were performed in accordance with institutional guidelines and with approval by the institutional animal care and use committee of shanghai jiao tong university. euthanasia was carried out using co 2 according to american veterinary medical association guidelines. the mice were inspected on a daily-basis for signs of ill health and distress. we euthanized the animals at the end of the experiments since there was no mice that reached humane endpoints including weight loss by 20% or more, severe lameness, dyspnea, ruffled fur, weakness, dehydration, or a hunched appearance, blistering at the injection site [15] . various number of amino acid residues at position 89-96 on diphtheria toxin t-domain (dtt, aa 202-378 of dt) corresponding to aa 290-297 of dt were replaced by the tnf-α epitope using discovery studio 3.5. the resulting epitope-scaffold proteins denoted as dtnf proteins were named dtnf1 to dtnf7. in dtnf1 to 6, amino acid residues corresponding to dtt aa 95-96, aa 94-96, aa 93-96, aa 92-96, aa 91-96, aa 90-96 were replaced with tnf-α epitope vsrfaisyqekvnlls a (aa 80-96), respectively. in dtnf7, aa 89-96 were replaced with tnf-α epitope vsrfaisyqekvnlls av (aa 80-97). the inter residue distances (td and dd) were calculated in swiss-pdb viewer 4.0.1 based on the x-ray crystal structures of mouse tnf-α and diphtheria toxin (pdb entry 2tnf, 1.4 å and 1mdt, 2.4å). td (å) stands for the interatomic distance between valine 80 to serine 96 or valine 97 of tnf-α. dd (å) stands for the interatomic distance between end residues of the replaced segment. the interatomic distance is calculated using the coordinates of the nitrogen atom of the alpha-amino group in n-terminal end residue and the carbon atom of the alpha-carboxy group in c-terminal end residue. the proteins were expressed and purified to homogeneity according to hatem a. elshabrawy [16] . the dna encoding dtnf with prescission protease cleavage site was generated by overlapping pcr, and digested with bamhi and xhoi, cloned into pgex6p-1. the vector dna was transformed into bl21 cells. protein expression was induced with 1mm iptg (isopropyl-d-thiogalactopyranoside) when the culture reached od = 0.6. after culturing for additional 12 h, the cells were pelleted by centrifugation, resuspended in pbs, sonicated and the debris was removed by centrifugation. the supernatant was applied to gst-column (ge healthcare) according to manufacturer's instruction. gst tag was cleaved with prescission protease overnight at 4°c, and removed by gst affinity column. the resulting dtnf proteins were further purified through superdex g75 chromatography. immunogenicity of dtnf proteins 50 μg of purified dtt or dtnf proteins mixed with aluminum hydroxide adjuvant (invivo-gen) were injected into female balb/c mice (6-7 weeks old) intradermally followed by three boost injections with 30 μg, 20 μg, 20 μg proteins plus aluminum hydroxide adjuvant on day14, 28, 42. blood samples were draw by retro-orbital sinus puncture after anesthesia by isoflurane inhalation on day 49. anti-tnf-α and anti-dtt ab titer and igg subclasses were measured by elisa. differential scanning calorimetry (dsc) 0.5 mg/ml dtnf proteins were degassed and the protein unfolding events were recorded on a vp-capillary differential scanning calorimeter (microcal, llc) between 35 and 100°c at a scan rate of 1°c /min and under a constant pressure of 30 psi. the data were analyzed using origin according to manufacturer's instruction. the cd spectra (195-250 nm) were recorded on jasco j-815 in 1-mm quartz cell at 25°c. protein concentrations were 0.230 mg/ml. the tertiary structure of dtnf proteins were modeled in modeller9v7 based on the x-ray crystal structure of dt and the mouse tnf-α mentioned above, and validated by procheck [17] . md simulations were performed as previously described using desmond 3.0 in default settings and lasted for 300 ns [18] . the protein structures were visualized using pymol 0.99 software. cia was induced and the arthritis score were assessed according to wei tang [19] . briefly, 2 mg/ml chicken type ii collagen (chondrex) mixed with an equal volume of cfa (4mg/ml mycobacterium tuberculosis h37ra) in a tissue homogenizer. on day 0, six-week-old dba/1j mice were anesthetized by isoflurane inhalation according to iacuc, and injected intradermally with 100 μl of the resulting emulsion at the base of the tail. on day 14, the experimental group mice were injected intradermally with 50 μg dtnf7 formulated with alum, and the control group mice were injected with alum and dtt. on day 28 and 42, the mice were boosted with 25 μg immunogen formulated with alum. mice were monitored on a daily basis for arthritis incidence and evaluated two or three times per week for severity. each paw was scored using a 0 to 4 scoring system. the paw scores were summed to yield individual mouse scores, with a maximum score of 16 for each mouse. score 0, normal; 1, mild swelling confined to the tarsal bones or ankle joint; 2, mild swelling extending from the ankle to the tarsal bones; 3, moderate swelling extending from the ankle to the metatarsal joints; and 4, severe swelling encompassing the ankle, foot and digits, or ankylosis of the limb. for histologic analysis by hematoxylin-eosin staining and safranin o staining, mice were sacrificed on day 56, and hind paws were used. sera were diluted 4-fold and the concentration of cytokines (il-1β, il-6, ifn-γ, il-10, il-17 and tnf-α) were measured using luminex multiplex technology (bio-rad laboratories) according to manufacturer's instruction. the means ± sem or means ± se were calculated in prism 6.0 (graphpad) from three to ten replicates. statistical comparisons were performed using t-test, where ã p 0.05, ãã p 0.01, or ããã p 0.001 was considered significant. the molecular design of a tnf-α epitope-scaffold immunogen nagahira k et al have shown that a neutralizing monoclonal antibody 3b10 binds tnf-α at position 81-88 [20] . when conjugated to klh, the linear epitope peptide (aa 80-96) elicits weak antibody response against the native tnf-α [21] whereas the cyclic form induced stronger antibody response, indicating that a stable conformation is preferred for immunogenicity. in the crystal structure of mouse tnf-α (mtnf-α, pdb 2tnf), residues 80 to 96 form a β-hairpin structure (fig 1a) , and the interatomic distance between the end residues (aa80 and aa96) is 8.76å. in search for a scaffold for this epitope, we found that diphtheria toxin t-domain (dtt, aa 202-378 of dt) could be a good candidate for both conformational stabilization and adjuvanting. firstly, the function of t-domain is engaged in translocation of the catalytic domain of dt across the endosomal membrane in low ph-dependent manner [22] . thus there is no safety issue associated with this domain [23] . secondly, dtt has a rigid tertiary structure composed entirely of α-helices and loops ( fig 1b) . therefore, it potentially tolerates transplantation of a foreign peptide and renders the peptide a more stable conformation [24, 25] . moreover, dtt can be expressed in e.coli as a highly soluble protein and the cd spectrum of the purified dtt shows a typical helical conformation with two minima at 208 and 222nm (fig 1c) . this would substantially reduce manufacture costs. thirdly, dtt possesses four t help cell epitopes (aa 69-88, 119-138, 129-148, 149-168, corresponding to dt aa 271-290, 321-340, 331-350, and 351-370), each of which is recognized by human major histocompatibility complexes (hal) with population coverage of 88%, 71%, 82%, 70%, respectively [26] . as human mhc ii genes are highly polymorphic, a combination of universal th epitopes promises more population coverage. among people previously immunized with dt vaccine, the th epitopes in a dttbased vaccine induced a rapid recall response of cd4 memory t cell [27] . as shown in fig 1b, the dtt residues at positions 89-96 form a turn-helix-turn structure that is entirely surface-exposed. we speculated that this is a potential site for the epitope transplantation. since the interatomic distance between the end residues of the epitope peptide is 2.88å shorter than that of dtt aa 89 and 96, direct transplantation would potentially introduce conformational constraint to both dtt and the epitope. in order to identify appropriate transplantation that keeps the epitope conformation close to that of native molecule while dtt conformation is minimally perturbed, we generated seven epitope-scaffold molecules ( fig 1d) with difference of the interatomic distance between the epitope and the replaced segment (δd) varies in a range of -3.34 å to +3.68 å. as δd of dtnf3 and dtnf4 is close to each other because some of the replaced residues are at the turn of the helix, we choose dtnf4 for further experiments. dtnf7 is generated by replacing dtt 89-96 with the epitope peptide encompassing tnf-α residues 80-97. the inclusion of one more residue in the epitope peptide increases td to 12.11 å and reduces the δd to -0.47 å. dtnf proteins were expressed in e. coli and purified to homogeneity. all of them are highly soluble at the concentration greater than 10 mg/ml in pbs ph 7.4, 25°c. their circular dichroism spectra are characteristic of α-helical conformation similar to that of dtt (fig 1c) . fig 2 shows that mice immunized with individual dtnf proteins generated high titers of antibody against dtt (fig 2a) indicating that the transplantations do not interfere with the antigenicity of dtt. however, dtnf7 elicited most robust antibody response against mouse and human tnf-α, whereas the rest dtnf proteins induced much weaker responses (fig 2b, fig 2c and 2d) , indicating that the epitope presentation is perturbed for most of the immunogens. no antibody response against native tnf-α was observed in mice immunized with dttpeptide conjugate d-tnf 80-96 , which is consistent with a previous report showing that this peptide in linear form conjugated to klh is a poor immunogen [14] . in dtnf7 immunized mice, over 98% of the antibodies on day 49 were igg1 subclass ( fig 2e) , which suggests that the immunoglobulin class switch has completed and the immune response is dominated by type 2 t helper cells. remarkably, the anti-tnf-α titer was sustained for more than six months with the level decreased slightly to 80% of the maximum response on day 223 (fig 2f) , suggesting that the b cell response is mediated by activated t helper cell. in comparison with conjugate vaccines targeting tnf-α, only transient antibody responses were observed due to lack of th-dependent b cell response [9, 10] . our data demonstrate that the universal th epitopes of dtt can help overcome this shortcoming. the sustained anti-tnf-α antibody response is beneficial to treatment of rheumatoid arthritis as excessive tnf-α is persistently expressed in patients. mice immunized with dtnf7 grew normally and their body weights, sizes of liver and spleen, and skin morphology showed no difference to control mice administered with pbs and dtt. no redness at injection sites were observed in dtnf7 immunized mice. these data implicate that the vaccine formulation is safe. further studies is warranted to exam whether the vaccination perturb the physiological function of the cytokine and the significance thereof. since δd values of dtnf1, 2, 5 and 6 are greater than that of dtnf7, the weaker antibody responses against tnf-α would indicate the conformational constraint caused by the transplantation affect the immunogenicity of the epitope. however, the weaker anti-tnf-α response of dtnf4 cannot be explained by the conformational constraint as δd value of dtnf4 is lower than that of dtnf7. it has been shown that induction of a robust antibody response is highly correlated with conformational stability of the immunogen [28] [29] [30] . therefore, the failure of dtnf1 to 6 to induce tnf-α-specific antibody response may be due to their overall conformation stabilities. to test this possibility, we performed dsc to look into the thermal stabilities of dtnf proteins and dtt. shown in fig 3a, the tm values of all dtnf proteins are lower than that of dtt. dtnf2 is the most unstable protein with tm value~9°c below dtt. the tm values of dtnf1 and 6 are decreased by~4°c, and those of dtnf4 and 7 decreased by~3°c. since dtnf4 and dtnf5 have tm values slightly higher than that of dtnf7, their poor immunogenicity cannot be explained by their thermal stabilities. to find out whether the stabilities of secondary structure elements could account for the immunogenicity of dtnf proteins, we performed molecular dynamics simulation. fig 4a shows that the conformations of the tnf-α epitopes in all dtnf proteins are stable and close to that of tnf-α except dtnf1 whose ser 81 adopts coil conformation instead of extended conformation. shown in fig 4e, the epitope of dtnf7 is entirely surface exposed. in contrast, conformational stability of the scaffold is perturbed to various degrees in all dtnf proteins with that of dtnf7 the least affected. the helix of dtt at position 97-101 is not stable in dtnf2, dtnf5 and dtnf6 (fig 4b) . particularly, the helix of dtnf2 is highly flexible and its rmsf is abruptly increased compared with that of dtt (fig 4c) . the residues at position 66-69 of dtnf1 also display higher degree of flexibility. the secondary structure of dtnf4 is stable and the epitope conformation is closed to that of tnf-α. however, its rmsfs at position 123-125 are decreased compared with that of dtt, indicating a more constrained conformation of the scaffold is unfavorable for the epitope presentation (fig 4d) . consistent with these observations, the solubility of the recombinant proteins varies after storage at 4°c for 10 days in pbs solution, and dtnf2 is especially unstable and becomes completely insoluble after the storage (fig 3b) . to investigate the therapeutic efficacy of dtnf7, we immunized mice with the purified protein 2-weeks after collagen administration using dtt as control (fig 5a and 5d ). compared with dtt treated mice, dtnf7 vaccination delayed the onset of arthritis by 15 days, reduced arthritis incident rate by 46% (fig 5b) , and markedly decreased the clinical arthritis score by 8 fold ( fig 5c) . as shown in fig 5e, symptoms including joint swelling and erythema were significantly improved. consistently, histological examination of hind paws demonstrated that dtnf7 vaccination reduced synovial inflammation, cartilage destruction, bone erosion, and immune cell infiltration (fig 6) . the body weights of mice immunized with dtnf7 were similar to those immunized with dtt ( fig 5f) . moreover, the serum levels of pro-inflammatory cytokines that persist in rheumatoid arthritis including interleukin il-1β, il-6 and il-10 were decreased, although the level changes had not reached statistical significance (fig 7) . of note, the level of tnf-α was not increased after dtnf7 vaccination. this is consistent to passive immunization with a tnf-α antibody in which the levels of tnf-α is not down-regulated after administration [31] . epitope peptide-based vaccines have been actively explored in the vaccine design by virtue of their targeting specificity [32, 33] . it potentially avoids allergenic and/or reactogenic effects associated with vaccines using whole organisms or large proteins [34] . however, an epitope peptide is inherently poor in immunogenicity [35] , and one of the challenges in the vaccine design is to identify an appropriate and safe carrier or scaffold for structural/chemical stability and adjuvanting [36] [37] [38] . so far, no epitope-peptide vaccine has reached the commercial market yet due to lack of efficacy and/or safety concern. in this study, we demonstrate that dtt can serve as a carrier or scaffold in vaccine design targeting self-molecules including tnf-α. we show that transplantation of a neutralizing epitope of mtnf-α into dtt at position 89-96 stabilizes the epitope in a native-like conformation while the scaffold conformation is minimally perturbed. the universal th epitope of the dtt facilitated a t helper cell-dependent tnf-α-specific b cell activation, which is critical for a long-lasting humoral response against tnf-α [10] . since the epitope peptide sequence is highly homologous between mouse and human, the transplantation strategy could be directly applied to human tnf-α vaccine design. our data also suggest that a successful vaccine design using dtt should avoid introduction of large conformational constrain to both the scaffold and peptide epitope. in addition, a stable secondary structure of the transplanted peptide is preferred as the transplanted segment of dtt is not a flexible loop. for a flexible peptide epitope, this could be achieved through engineering a disulfide bond in the epitope peptide [39, 40] . the length of the peptide should optimized to keep the span difference (δd) to a minimum. the solubility of the purified molecules after storage at 4°c in pbs is a good indication of its overall stability. the b fragment of dt (dtb) is composed of t-domain and receptor binding r-domain, and has been shown an effective vaccine carrier [41] . however, dtt is better than dtb with regard to antigen load. dtt possesses only one b cell epitope whereas dtb contains several highly immunogenic b cell epitopes in the r domain [42] . the b cell epitope of dt has been shown to suppress the immune response of certain conjugated haptens by competing dt primed helper t cells [43] . in case this happen to dtt-based vaccine, appropriate mutation(s) may be introduced to modify or eliminate the b cell epitope of dtt. collagen-induced arthritis (cia) is the most commonly used animal model to human rheumatoid arthritis, a chronical inflammatory autoimmune disease attacking joint. so far, tnf-α immunogens have only shown phylactic effects in cia mouse model. dtnf7 is the first vaccine to show therapeutic effect after collagen immunization and the onset of the arthritis. the plasma level of tnf-α is extraordinarily high in the cia model mice (2000 pg/ml) compared to that of tnf-α transgenic mice (9 pg/ml) [10] . consequently, we were unable to determine whether the anti-tnf-α antibodies elicited by dtnf7 immunization could neutralize bioactivity with l929 assay using diluted sera. measurements of the plasma levels of cytokines downstream of tnf-α such as il-1β and il-6 on day 28 and 56 were less in dtnf7 compared with dtt immunization in the cia mice. therefore detailed time course of more cytokine levels would reveal more insights in both the cia model and the therapeutic mechanisms of dtnf7 immunization. almost all known cytokines are present in the ra synovial fluid, among which tnf-α is the first cytokine proved to be effective therapeutic target [2] . in recent years, the immune-regulatory function of gm-csf has been shown to play crucial roles in driving the development of many autoimmune diseases [44] [45] [46] . gm-csf or its receptor-targeted therapies have been shown highly effective and remarkably safe in treatments of many autoimmune diseases, in particular, ra [47] . of note, antigen-specific regulatory t cells activated by therapeutics antagonizing tnf-α or gm-csf have been shown play major immune suppressive roles in ra and other autoimmune diseases [48, 49] . this mechanism also underlies immune tolerance of acaid (anterior chamber associated immune deviation) [50] . studies from shukkur m. farooq show that collagen type ii-specific acaid treg suppresses inflammation in cia [51] . therefore, it awaits further investigation to determine whether dtnf7 immunization modulate the function of collagen-specific treg in cia and whether the treg cells are functionally equivalent to acaid treg. tumor necrosis factor: a pleiotropic cytokine and therapeutic target anti-tnf therapy: past, present and future. international immunology tnf defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases nature medicine design of effective immunotherapy for human autoimmunity tumor necrosis factor antagonist mechanisms of action: a comprehensive review the antibody response against human and chimeric anti-tnf therapeutic antibodies primarily targets the tnf binding region. annals of the rheumatic diseases antidrug antibodies (adab) to tumour necrosis factor (tnf)-specific neutralising agents in chronic inflammatory diseases: a real issue, a clinical perspective. annals of the rheumatic diseases strategies for active tnf-alpha vaccination in rheumatoid arthritis treatment therapeutic antibodies elicited by immunization against tnf-a tnfalpha kinoid vaccination-induced neutralizing antibodies to tnfalpha protect mice from autologous tnfalpha-driven chronic and acute inflammation therapeutic vaccination with tnf-kinoid in tnf antagonist-resistant rheumatoid arthritis: a phase ii randomized, controlled clinical trial phase i study of tnf alpha autovaccine in patients with metastatic cancer immunochemical termination of self-tolerance active immunization against murine tnfalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection tumor necrosis factor: methods and protocols identification of a broadspectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay relative motions between left flipper and dorsal fin domains favour p2x4 receptor activation the growth factor progranulin binds to tnf receptors and is therapeutic against inflammatory arthritis in mice epitope mapping of monoclonal antibodies to tumor necrosis factor-a active immunization against murine tnf peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection ph-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin t-domain structural basis for lack of toxicity of the diphtheria toxin mutant crm197 chimeric glutathione s-transferases containing inserts of kininogen peptides: potential novel protein therapeutics the role of turns in the structure of an alpha-helical protein universal epitopes for human cd4+ cells on tetanus and diphtheria toxins generation of a universal cd4 memory t cell recall peptide effective in humans, mice and non-human primates antigen three-dimensional structure guides the processing and presentation of helper t-cell epitopes contribution of conformational stability of hen lysozyme to induction of type 2 t-helper immune responses a protein's conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins effects of treatment with a fully human anti-tumour necrosis factor alpha monoclonal antibody on the local and systemic homeostasis of interleukin 1 and tnfalpha in patients with rheumatoid arthritis. annals of the rheumatic diseases peptide-based synthetic vaccines recent advances in the molecular design of synthetic vaccines peptide vaccine: progress and challenges computational design of epitopescaffolds allows induction of antibodies specific for a poorly immunogenic hiv vaccine epitope. structure proof of principle for epitope-focused vaccine design elicitation of structure-specific antibodies by epitope scaffolds structural and computational biology in the design of immunogenic vaccine antigens insertion of foreign t cell epitopes in human tumor necrosis factor alpha with minimal effect on protein structure and biological activity conformational her-2/neu b-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities construction of an epitope vector utilising the diphtheria toxin b-subunit. fems microbiology letters perspectives of application of recombinant diphtheria toxin derivatives carrier priming or suppression: understanding carrier priming enhancement of anti-polysaccharide antibody response to conjugate vaccines. vaccine gm-csf: an immune modulatory cytokine that can suppress autoimmunity journal of interferon & cytokine research: the official journal of the international society for interferon and cytokine research granulocyte-macrophage colony-stimulating factor autoantibodies: a marker of aggressive crohn's disease. inflammatory bowel diseases the pathogenesis of rheumatoid arthritis. the new england journal of medicine granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory t cells interplay between tnf and regulatory t cells in a tnf-driven murine model of arthritis type ii collagen induces peripheral tolerance in balb/c mice via the generation of cd8+ t regulatory cells eye-mediated immune tolerance to type ii collagen in arthritisprone strains of mice we thank zhibing lin a , ye yu b for technical advices and assistance. key: cord-0029264-66kjhzwx authors: calderón espinoza, ivette; chavarria-avila, efrain; pizano-martinez, oscar; martínez-garcía, erika aurora; armendariz-borunda, juan; marquez-aguirre, ana laura; llamas-garcía, arcelia; corona-sánchez, esther guadalupe; toriz gonzález, guillermo; vazquez-del mercado, monica title: suicide risk in rheumatoid arthritis patients is associated with suboptimal vitamin d levels date: 2022-03-16 journal: j clin rheumatol doi: 10.1097/rhu.0000000000001823 sha: 3521dd5af56da92a5ac546b1c75527be222319e4 doc_id: 29264 cord_uid: 66kjhzwx rheumatoid arthritis (ra) patients might experience anxiety and depressive symptoms. deficient vitamin d levels may be a trigger for these conditions. the aim of this study was to determine the frequency of depression, anxiety symptoms, and suicidal risk or ideation in patients with ra associated with vitamin d serum levels. methods: in this cross-sectional study, we recruited ra patients older than 18 years, classified into 3 groups according to serum vitamin d levels: sufficient, ≥30 ng/ml; insufficient, 20–29 ng/ml; and deficient, <20 ng/ml. based on the self-reported plutchik and the hospital anxiety and depression scale, we evaluated the association of suicidal risk, depression, and anxiety with the vitamin d levels in ra and the rheumatoid arthritis quality-of-life questionnaire. results: we studied 72 patients with ra between january and october 2019. we found an inverse correlation between plutchik score and suicidal risk with inadequate vitamin d levels, but not with the hospital anxiety and depression scale. suicidal ideation was associated with a higher score on the rheumatoid arthritis quality-of-life questionnaire. conclusions: despite the high prevalence of depressive and anxiety symptoms in ra patients, a plutchik low correlation coefficient with inadequate serum levels of vitamin d was found. however, in the analysis of covariance, we were able to find that vitamin d levels remain associated with a reduction of suicide ideation. further studies are needed to identify a risk profile for early psychological interventions to improve the quality of life in ra patients. r heumatoid arthritis (ra) is one of the most prevalent chronic inflammatory autoimmune diseases that can cause disability if not diagnosed and treated on time. 1 besides the rapid remission goal that establishes the treat-to-target strategy in ra, we are not able to evaluate in a holistic way in our patients the impact of constant pain suffering, the psychological impact of a chronic treatment, changes in the quality of life, or self-perception that might contribute to the susceptibility of ra patients to anxiety and depressive disorders. 2, 3 in a previous study by our group, the biopsychosocial aspects of ra were evaluated, showing significantly lower levels of quality of life in patients with ra than in the general population, associated with polypharmacy. 4 a shared pathophysiological pathway between depression and chronic diseases such as ra is chronic inflammation, where proinflammatory cytokines and neurotransmitters produce alterations in behavior. altered levels of interleukin 6 and tumor necrosis factor α have been detected in patients with major depression. [5] [6] [7] in addition, suicidal ideation has been reported in approximately 6% of patients with ra, up to 2-fold compared with non-ra subjects. 8 vitamin d has a multitude of functions; the 2 main functions are classified based on the calcium relationship as calcemic and noncalcemic. the most important function of vitamin d is related to bone health, including mineralization, remodeling, and maintenance. 9 however, the function of vitamin d is not limited to bone metabolism, because the renal 25-hydroxyvitamin d-1α-hydroxylase enzyme (cyp27b1) has been identified in a large number of cells and tissues, such as the immune system and the central nervous system. 10 the association between vitamin d levels and depressive, anxiety, and suicidal ideation has been reported. 11, 12 recent studies reveal the importance of evaluating the suicide risk in vulnerable populations not related to ra, especially in latin american countries such as mexico, peru, and colombia with increased suicide cases reported. [13] [14] [15] [16] in mexico, the suicide mortality rate has grown from 1.13 in the 1970s to 5.31 in 2017. 13 notwithstanding in ra, the information seems to be oriented to emphasize the existence of depressive symptoms associated with the disease itself, [17] [18] [19] but the association of altered vitamin d levels as a possible triggering or additive factor to conceive depressive and anxiety symptoms including suicidal ideation in ra patients is scarce. 8, 11, 12 the aim of our study was to determine whether there is an association between depression, anxiety symptoms, and suicidal risk associated with vitamin d serum levels in patients with ra. this cross-sectional study was conducted between january and october 2019. all patients were recruited from the outpatient clinic of rheumatology at the hospital civil "dr. juan i. menchaca," guadalajara, mexico. all patients signed the informed written consent with the institutional review board of the hospital civil "dr. juan i. menchaca" with the approval of the secretaria de salud jalisco (register 0332/19 hcjim/2019). the research was conducted following the helsinki criteria last updated in 2013, fortaleza, brazil. the inclusion criteria were age >18 years and patients who fulfilled the 2010 classification criteria of the american college of rheumatology/european league against rheumatism. 20 the exclusion criteria were as follows: psychiatric disorders previously diagnosed; use of antidepressive drugs; comorbidities such as renal damage, liver failure, parathyroid disease, pregnancy, and osteoporosis; use of antiresorptive bone remodeling therapy; and use of glucocorticoids (>7.5 mg/d), antifungals, antiretroviral drugs, thiazides, valproate, phenobarbital, diuretics, and phenytoin. elimination criterion was as follows: patients who withdrew their informed consent. the patients were classified into 3 groups according to their vitamin d levels as follows: sufficient, ≥30 ng/ml; insufficient, 20-29 ng/ml; and deficient, <20 ng/ml. 21 the clinical activity of ra was evaluated using 2 scores: simplified disease activity index (sdai) 22 and clinical disease activity index (cdai). 23 the suicide risk was assessed by the spanish adaptation of the self-applied plutchik scale. this scale consists of 15 items with a score maximum of 15 points. high risk of suicide is considered higher than 6 points. this instrument is able to differentiate between suicidal intention and ideation in the past or in the present, and the evaluation of these items uses dichotomy answers (yes or no). the frequency of suicide ideation spans items 13 and 14, and frequency of suicide intention is on the 15 items of the plutchik scale. [24] [25] [26] [27] depression and anxiety were assessed by the hospital anxiety and depression scale (hads) score. this scale is made up of 2 subscales, one for anxiety and the other for depression; each subscale evaluates 7 items with scores from 0 to 3, and the standard cutoff point is ≥8 for both subscales. 28, 29 the sensitivity and specificity are shown greater than 80% according to a metaanalysis. 30 the mexican spanish language of this instrument has already been validated in the mexican population, showing consistency (cronbach α = 0.86) and good sensitivity and specificity (0.80). 31 functional capacity was evaluated using the health assessment questionnaire-disability index (haq-di), which evaluates the quality of life. using a likert scale from 0 to 3, it evaluates 8 aspects of daily life of the patient. a greater score implies more impact on the quality of life. [32] [33] [34] rheumatoid arthritis qualityof-life questionnaire (raqol) is composed of 30 elements, and its interpretation is that the higher score, the worse quality of life in ra. 32, 35 on the other hand, raqol contains 30 questions with dichotomic answers (yes/no), and the qualification is the result of the sum of yes answers (range, 0-30). a greater score implies more impact on the quality of life. anti-cyclic citrullinated peptide antibodies and rheumatoid factor were determined by enzyme-linked immunosorbent assay (axis-shield diagnostics ltd., dundee, scotland). erythrocyte sedimentation rate was measured using wintrobe's method and c-reactive protein by nephelometry. 36 vitamin d quantification was determined using the chemiluminescence immunoassay technique (liaison 25-oh vitamin d total assay, stillwater, mn). we used descriptive statistics including measures of central tendency and dispersion, categorical variables expressed as measures of absolute and relative frequency, and linear variables such as mean and sd or median and interquartile ranges, corresponding to the frequency distribution. the normality of the included variables was analyzed using the kolmogorov-smirnov test. groups were made according to the levels of vitamin d, depression, anxiety, suicidal ideation, and suicidal intention, which were compared among these groups. the hypothesis tests to evaluate linear variables were the student t test or the mann-whitney u test for independent samples or 1-way analysis of variance or the kruskal-wallis test. categorical variables were analyzed using the χ 2 test or fisher exact test. correlation analysis was carried out using the pearson (r) or spearman (ρ) coefficients according to the distribution and type of variables. variables with significant correlations were included in the construction of linear multivariate logistic regression models to predict depression and anxiety scores using clinical variables, disease activity, and vitamin d levels as predictive variables. in addition, the variables were dichotomized for the presence of depression, anxiety, and suicidal ideation and were included in the construction of multivariate binary logistic regression models to identify variables with predictive capacity for the presence of these conditions. the measure of association strength was the odds ratio and 95% confidence interval. an adjusted α error of less than 5% ( p < 0.05) at the 2 tails was considered significant. the statistical package stata se version 11.1 (statacorp llc, college station, tx) was used. the study included 72 patients with ra. most patients were women with clinically low to moderate active disease. the majority of ra patients were vitamin d deficient. the clinical and demographic variables evaluated are shown in the table 1 . when an additional analysis was made according to vitamin d levels classified as sufficient, ≥30 ng/ml; insufficient, 20-29 ng/ml; and deficient, <20 ng/ml, for all the variables assessed in the table 1 , only haq-di showed a difference ( p = 0.008) between deficient (1.2 ± 0.8) versus insufficient (0.6 ± 0.6) and sufficient (0.6 ± 0.7). frequency of depressive or anxiety symptoms was found in 88.9% by hads. at the same time, the frequency of suicidal risk was 40.3%, as reported through the self-applied plutchik scale. based on the same scale, frequency of suicide ideation was the hads scores according to anxiety and depression domains were neither associated nor correlated with vitamin d levels (figs. 1a-d) . notwithstanding, the plutchik scale score was associated and correlated with vitamin d levels ( p = 0.020) (figs. 1e, f) . the composite indices of disease activity sdai and cdai, as well as with the haq-di self-questionnaire scores used in this study, showed a low correlation with serum levels of vitamin d ( figs. 2a-c) . the haq-di and hads scores showed a correlation with raqol used in this study (figs. 3a-d). an association between raqol and suicide ideation was established in our patients (fig. 3b ). when performing a multivariate logistic regression analysis, a valid model for ideation was not obtained (supplementary data, http://links.lww.com/rhu/a420). in a linear regression analysis, vitamin d was the only variable that remained significantly associated with ideation, explaining 27.8% of the total variance, and also indicates that each nanogram of vitamin d reduces the value of the suicide ideation constant 2.24% (supplementary data, http:// links.lww.com/rhu/a420). to describe the associations between depressive and anxiety disorders, suicidal ideation, suicidal risk with vitamin d levels, and development of clinical trials related to vitamin d supplementation might help to develop a holistic approach in ra management. 37 in other words, supplementation with vitamin d could be used as another therapeutic step in the treat-to-target strategy for ra. an adequate and multidisciplinary management of depressive and anxious symptoms can improve a patient's health status and quality of life. our patients recruited for this study were low to moderately active according to sdai and cdai in approximately 60% associated with mild to moderate functional disability (table 1 ). this study also showed a high prevalence of depressive and anxious symptoms (approximately 90%), as evaluated by the hads score. in addition, the suicide risk in ra patients assessed by the selfreported plutchik scale was found in 6 of 10 patients included. these results are remarkably high compared with those in the literature using this instrument in different disorders 29, 38, 39 and in agreement with other authors. 38 notwithstanding the high prevalence of depressive and anxiety symptoms, we were able to demonstrate an association and a low correlation coefficient with plutchik score and inadequate serum levels of vitamin d (figs. 1e, f) . our study showed that vitamin d levels were inversely related to the sdai, cdai, and haq-di scores (fig. 2) . the importance of vitamin d supplementation has been debated in ra, and a recent meta-analysis showed that doses of vitamin d ≥50,000 iu seem to improve the quality of life and diminish the clinical disease activity index. 40 finally, higher scores of raqol correlated with a greater functional disability, anxiety, and depression (figs. 3a, c, d), and suicide ideation was associated with the raqol score (fig. 3b ). based on our results, patients with a hads score ≥8 were referred to the psychology service. when the plutchik scale score was ≥6, a referral to the psychiatric service was performed. vitamin d supplementation was started in all patients with insufficient or deficient levels, and diseasemodifying antirheumatic drug treatment with moderate or severe ra activity was adjusted when needed. this study described suicidal ideation and suicidal risk in patients with ra associated with inadequate vitamin d serum levels. if vitamin d is identified as an additional risk factor for maintaining the quality of life in ra, adequate levels of vitamin d could reduce the suicidal and ideation risk in this group of patients. our results suggest that further studies need to be carried out with a greater number of patients using other validated instruments that allow adequate and reliable screening of suicide, depression, and anxiety symptoms in ra patients in daily clinical practice. finally, we must be aware of the limitations of our study. first are the limitations inherent in cross-sectional studies like ours, such as: provide data from a single moment in time and it is impossible to make inferences of causality, mainly. second is the fact that the ra itself could trigger depression, anxiety, and even suicide ideation related to the complexity of the ra patient's life. third is the subclinical presence of these symptoms not registered through the instruments applied in this study. fourth is the impact of comorbidities such as diabetes mellitus or vascular disease that might influence the anxiety or depressive symptomatology or the existence of fibromyalgia not evaluated in this study. in conclusion, despite the high prevalence of depressive and anxiety symptoms in ra patients, a plutchik low correlation coefficient with inadequate serum levels of vitamin d was found. however, in the analysis of covariance, we were able to find that vitamin d levels remain associated with a reduction of suicide ideation. further studies are needed to identify a risk profile for early psychological interventions to improve the quality of life of ra patients. the pre-clinical phase of rheumatoid arthritis: from risk factors to prevention of arthritis affective disturbance in rheumatoid arthritis: psychological and disease-related pathways the relationship between anxiety, depression, suicidal ideation and quality of life in patients with rheumatoid arthritis disease activity score on 28 joints and polypharmacy are independent predictors for health-related quality of life evaluated by incavisa in patients with rheumatoid arthritis cytokine production and treatment response in major depressive disorder major depression is associated with significant diurnal elevations in plasma interleukin-6 levels, a shift of its circadian rhythm, and loss of physiological complexity in its secretion: clinical implications suicidal ideation among adults with arthritis: prevalence and subgroups at highest risk. data from the 2007-2008 national health and nutrition examination survey vitamin d and bone vitamin d metabolism: new concepts and clinical implications vitamin d deficiency and suicidal ideation: a cross-sectional study of 157,211 healthy adults the association between low vitamin d levels and suicide attempts in adolescents study of suicide burden of mortality in mexico characterisation of suicide in colombia evolution and differences of suicide rates in peru by gender and department the trend in mortality due to suicide in urban and rural areas of colombia prevalence of depression and anxiety in rheumatoid arthritis patients and their associations with serum vitamin d level association between depression and rheumatoid arthritis: two longitudinal follow-up studies using a national sample cohort the burden of depressive disorders in musculoskeletal diseases: is there an association between mood and inflammation? rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative evaluation, treatment, and prevention of vitamin d deficiency: an endocrine society clinical practice guideline a simplified disease activity index for rheumatoid arthritis for use in clinical practice acute phase reactants add little to composite disease activity indices for rheumatoid arthritis: validation of a clinical activity score the measurement of suicidality, aggressivity and impulsivity scale of psychological pain: spanish adaptation of the psychache scale in young adults escalas de evaluación de riesgo suicida en atención primaria validación de la escala de riesgo suicida de plutchik en población española validity and reliability of screening measures for depression and anxiety disorders in rheumatoid arthritis psychometric evaluation of the hospital anxiety and depression scale in mexican adults with ischemic and hypertensive cardiomyopathy revisión de estudios de la escala de ansiedad y depresión hospitalaria (ad) en muestras españolas propiedades psicometricas de la escala hospitalaria de ansiedad y depresión (hads) en una población de pacientes oncológicos mexicanos adaptation and validation of the rheumatoid arthritis quality of life scale (raqol) to mexican spanish spanish version of the health assessment questionnaire: reliability, validity and transcultural equivalency the health assessment questionnaire (haq) the reliability and construct validity of the raqol: a rheumatoid arthritis-specific quality of life instrument enalapril influence on arterial stiffness in rheumatoid arthritis women: a randomized clinical trial efficacy of oral vitamin supplementation in inflammatory rheumatic disorders: a systematic review and meta-analysis of randomized controlled trials detecting anxiety and depression among people with limited literacy living with chronic low back pain in nigeria: adaptation and validation of the hospital anxiety and depression scale prevalence and correlates of anxiety and depression in frontline healthcare workers treating people with covid-19 in bangladesh the effect of vitamin d supplementation on rheumatoid arthritis patients: a systematic review and meta-analysis key: cord-0018645-epplar05 authors: mate, gitanjali s.; kureshi, abdul k.; singh, bhupesh kumar title: an efficient cnn for hand x-ray classification of rheumatoid arthritis date: 2021-06-14 journal: j healthc eng doi: 10.1155/2021/6712785 sha: 62f2cfa0b5ddac6fc30f4c5a6db2f40bf766429b doc_id: 18645 cord_uid: epplar05 hand radiography (ra) is one of the prime tests for checking the progress of rheumatoid joint inflammation in human bone joints. recognizing the specific phase of ra is a difficult assignment, as human abilities regularly curb the techniques for it. convolutional neural network (cnn) is the center for hand recognition for recognizing complex examples. the human cerebrum capacities work in a high-level way, so cnn has been planned depending on organic neural-related organizations in humans for imitating its unpredictable capacities. this article accordingly presents the convolutional neural network (cnn) which has the ability to naturally gain proficiency with the qualities and anticipate the class of hand radiographs from an expansive informational collection. the reproduction of the cnn halfway layers, which depict the elements of the organization, is likewise appeared. for arrangement of the model, a dataset of 290 radiography images is utilized. the result indicates that hand x-rays are rated with an accuracy of 94.46% by the proposed methodology. our experiments show that the network sensitivity is observed to be 0.95 and the specificity is observed to be 0.82. joint inflammation is a state of spasms and pain in a human where two or more bones join together. arthritis results in excruciating pain, joint swellings, joint stiffness, and poor function in joints. healthy joints move naturally with the help tissue called the articular cartilage which is slippery and smooth. e tissues absorb pressure and shock which is caused by motion and pressure due to extreme stress on the joints where bones meet. is extreme pressure causes destruction of the tissues in the cartilage further resulting in arthritis. e only and primary cause of arthritis is the degradation of cartilage in the joints. e arthritis is generally classified in two types: (1) osteoarthritis and (2) rheumatoid arthritis. in osteoarthritis, cartilage and underlying bone degenerate which causes pain and stiffness. generally osteoarthritis is more common in middle age people. on the other hand, rheumatoid joint pain is an incendiary infection that influences joints. rheumatoid joint pain (ra) is an immune system condition and is quite possibly the most limit types of provocative joint pain. beforehand, bone misfortune from rheumatoid joint pain (ra) was considered irreversible. be that as it may, the presence of biologic specialists stayed away from the movement of bone misfortune related to ra [1] . e major imagery discoveries identified with ra advancement incorporate bone weakening, narrowing of joints, and periarticular osteoporosis, showing the annihilation of construction of the bones. specifically, the event of bone disintegration proposes functioning ra; therefore the assessment of bone disintegration is exceptionally significant for the underlying analysis and later assessment of ra patients. e procedures mentioned above are extremely tedious, in every case, since they require the assessment of all accessible joints and radiographs of the foot. furthermore, it is additionally hard for radiologists and other related medical practitioners to recognize the changes in the reports from the accessible foot x-beams on the grounds that the progressions are frequently unpretentious. e utilization of a pc technique for appraisal of ra in a quantitative degree is consequently prudent in medical field. in the past, a few methodologies were utilized like artificial neural network (ann), support vector machine (svm), and genetic algorithm (ga) strategies. in any case, these methodologies incorporate the computation of the adequacy highlights to order the injuries. profound learning has as of late acquired revenue in the space of speculation. by and large, the neural organization is classified as a convolution neural organization (cnn) or profound learning kind of neural organization. for visual imaging, the model of neural organization which has a profound learning as class is utilized generally. for the developing a natural spoken-language processing system cnn provides the tools for development. along with this cnn can be used in various areas like medical image analysis, audio and speech recognition, and computer vision. around the globe, it is estimated that there are approximately 35-70 million individuals suffering from ra. since there is no demonstrated solution for ra accessible yet, current medicines chiefly center around relief from excruciating pain, discomfort, irritation decrease, and easing back down or halting joint harm. to forestall irreversible joint harm, early location of ra is fundamental. for a successful clinical treatment, it is significant that the infection can be checked intently. joint harm appraisal close by radiographs is a much-of-the-time utilized technique for observing the movement of ra. e accompanying inquiries concerning ra have not yet been answered which enable us to further explore ra: (i) what causes ra? how does it begin? (ii) why does ra disturb the functioning of the joints? (iii) does ra come to an end in a human? if not, why? e inspiration driving such research is that the past related works of imagery investigation frameworks are not automized for the examination of ra as per factual properties like age factor and nonstop movement of ra action. ere is no mechanized framework for perceiving the seriousness of ra stage by seeing the image of influenced territory like hand and knee. such frameworks utilize a mix of image handling methods to deal with images and afterward measure the distance between the tibial and femoral bones as an authenticator of diagnosing the sickness. along these lines, there is a requirement for a canny rheumatoid joint inflammation framework that invigorates the human visual investigation that ordinarily analyzes the rheumatoid joint pain contingent upon certain highlights like the limited distance between the tibial and femoral bones and furthermore the bone prods. rheumatoid joint inflammation happens because of uncontrolled joint irritation, yet it is not right now known how and why this aggravation is set off. aside from this the third-stage patient does not recuperate. due to long period of treatment towards ra, inflammation in bones is created. e intricacy increases with respect to age factor and sometimes to x beam. an entire day is required for getting the treatment. so we ought to have some automized framework which will be useful specially towards conclusion and movement of ra. e existing ra calculations from essential consideration records depend on a manual determination of the most important codes [2] , which is to some degree emotional. id of patients from the essential consideration information base enduring with ra with precision and unwavering quality is an intriguing errand. e greater part of the connected ra seriousness evaluating examines centers around recognizing finger joints or distinguishing bone disintegration on the x-beam image dataset rather than ultrasound images. as of now, specialized progressions have prompted a significant improvement in image handling. regardless of specialized progressions and the accessibility of various devices and tests, like ultrasound, magnetic resonance imaging (mri), and conventional radiography (cr), cr stays the essential imaging device for ra. while investigating multiple joints by mri, the procedure takes an excessive lot of time and costs a lot consistently. ultrasound is a client subordinate method of imaging that can measure emanation and synovial changes. an aggravation might be utilized to play out a little, decay and bone rarefaction joint evaluation that is an extremely agonizing and consistently deteriorating flare-up. it might likewise cause various imperfections and utilitarian issues in the bones. such factors are in this manner generally imperative to identify in the beginning phases. all drugs, treatments, and procedures that have been undergone so far point just to lessening the manifestations and drawing out the movement of the infection. we can understand that the cases of ra are unsure and cures are not yet found in these painful cases. to understand and further study ra, the authors in [3, 4] propose using computerized joint location and evaluation of hand radiographs. e prime objective of this method is location of joint position and joint edge. besides, it is clarified by the edge time frame. e experiments were executed on five hand radiographical images of 16-bit grayscale with 2500 × 2000 pixels (0.1 × 0.1 mm). for this experiment and with manual joint outline, all the joints are comprehensively estimated. in [4] , the author has recommended a methodology using refreshed level set for programmed identification of the bone limit close by radiographs. e said strategy promises great extraction effectiveness. because the pixel powers of bone joints and different pixels are closer in certain areas, the method fails to function as expected with respect to the images. numerous conventional strategy studies like volumetric ultrasound and infrared imaging have detailed great execution in ra identification. profound learning is becoming normal in the imaging field. in [5, 6] , joint space narrowing estimation has been very much contemplated and accomplished a superb evaluation in ra development. e msgvf division calculation which is predominantly utilized for disintegration of bones and different cnn structures in [7] [8] [9] , was assumed a basic part as it presents a recognizable proof and assessment of ra. e production of the different cnn models to identify ra has been discussed in [8] [9] [10] which quickly gives us a new beginning for the examination of ra. experimenters have achieved great level in research using cnn for image processing with the aid of the learning networks, which adds a convolution and pooling design to a separate image database. in the diagnosis of ra, changing disease modifying antirheumatic drugs (dmards) and organic medications are utilized in [10] [11] [12] . as indicated by the writing, around 30-40% of ra patients [13] are not getting profited by exemplary first-line treatment which is mtx [14] . in 2019, a few significant logical advances were made in the clinical use of ml in rheumatology [15] [16] [17] [18] [19] [20] . cnn is utilized mostly in imaging undertakings [21] . e authors of [22] report the successful utilization of cnn to advance computerized scoring of the activity of joint inflammation infection on ultrasound images. e authors of [23] utilize x-beams of ra patients to prepare and foster a profound cnn for joint obliteration appraisal. for quick therapy, early finding of patients with ra in danger of creating ongoing agony is clinically fitting. lotsch et al. recommended that machine learning could be utilized to get ra useful boundaries from huge vault information, for instance, disease activity score-28, and that these boundaries could exhibit the creation and level of constant agony [24] . a current experimental calculation with an exactness of 94% [25] was outflanked by a calculation prepared on innocent outcomes. e troupe machine learning strategies have shown guarantee to date in the dependable id of ra and the savvy and precise evaluation of the illness [26, 27] . sumitra nair et al. [28] have proposed application depending on ai for investigation of emg images designs for ra. kernal lsk strategy is utilized for investigation of the appendage relocation in space [29] . in past examinations, numerous techniques for distinguishing injuries utilizing classifiers like a artificial neural network (ann), support vector machine (svm), and genetic algorithm (ga). as of late, profound learning has acquired revenue in the overall imaging field. in particular, numerous examinations have revealed a superior execution of the profound convolutional neural network (dcnn) compared to customary classifiers in image id [30] . cao and mills et al. have proposed towards quantitative assessment of ra utilizing volumetric ultrasound [31] . is calculation is used for sectioning (1) the 3d bone surface and (2) the 3d joint case district. ey broaden 2d bone extraction strategies to 3d and make calculation more vigorous to the force misfortune because of surface ordinary's confronting away from occurrence acoustic shafts. e separated bone surfaces combined with a joint-explicit anatomical model are utilized to introduce a coarse restriction of the joint case locale. e joint case division is refined iteratively using a probabilistic spot model. e highest quality level for deciding underlying joint harm is radiography. notwithstanding the wide utilization of radiography in joint assessment, pc supported indicative ultrasound frameworks have been created to arrange intra-and extra-articular pathologies with the likeness of joint space width appraisal [32] and joint aggravation assessment [33] . additionally, the semiquantitative ultrasound evaluation of synovitis seriousness assists with deciding the infection action of ra presented in [20, 34] . ra assessment is presented as an issue of image arrangement [35, 36] . in this investigation, two ra ultrasound image datasets were utilized for preparing of the organization. one of the datasets incorporate 662 images of patients suffering from ra in mcp joints, and the other incorporates 315 images of pip joint. e acquired examples were divided considering 80% data for training and the rest for test. a large number of ultrasound images do not prove to be sufficient for preparation of cnn and may lead to the major issue of overfitting [37] . us, for improving the capacity of organization speculation, information expansion was performed on preparing tests. in data increase, one can extend the preparation set by adjusting each class to protect the information name. e information expansion we took has the accompanying three strategies. for the development of this work, the images are collected from examined patients. data entails 290 radiograph images of different sizes. e images in the dataset have been processed at earlier stage with removal of nose and images are resized to 100 × 100. e images are then appropriately cropped to retain similar aspect ratio which was present in the images of the original datasets. in table 1 , we see the description of the dataset and table 2 shows us the spread of data. e dataset has been processed by resizing the images up to 100 × 100 and normalizing their values. e dataset was split into training dataset and testing dataset. e 75% images were used for training the model and 25% images are used for testing the model. to identify ra cases with specified outcomes over the other popular strategies, the presented work alters the fundamental design of the regulated cnn organization. e presented cnn model does not restrict the quantity of layers that can be utilized; it enhances the number to fulfill the issue's interest. moreover, middle convolution layers utilize different sizes of layers. figure 1 shows the proposed model's general gadget engineering. in the presented cnn the recently prepared image of size 28 × 28 expands the size of the layers alongside the initiating highlight. mean pooling is utilized for reducing, by two stages, the image created from the convolutional layers. further, the image is smoothed and taken care of by a method known as multilayer perception (mlp). here, the image is processed further to a layer that is totally associated. at long last, the sigmoid classifier gives us the likelihood of an image. standard neural organizations are adding to the issue of overfitting. en again, cnn snatches advantage of the neighborhood qualities of the images; for example it manages input pixels which are situated close as well as far. in this experimentation, cnn was delivered to perceive different images dependent on the properties of the image. e convolution network used in our experimentation is not only a profound neural organization with a few secret layers. it is a popular organization that copies how images are deciphered and recalled by the visuals. consequently, it is extremely difficult for specialists to get a handle on this idea at their first gathering. acknowledgment is the strategy for arranging images. in any case, preparing natural images straightforwardly to image acknowledgment does not create ideal execution. it is advisable that, to get an image contrast, preprocess the image. to further get quality image highlights, diverse image handling procedures are utilized. e information image is preprocessed. e element signals are removed and utilized within the arrangement of the information. e neural organization at that point receives the information of the image and afterward arranges it. e neural organization's component extractions include a few layers of convolutional and pooled sets. e convolution layer handles the image and rearranges it utilizing the activity of convolution. it is likewise viewed as a work of computerized calculations. e contiguous pixels into one single pixel were consolidated by utilizing pooling layer which eventually lessens the image measurement. meanwhile convnet is connected with two dimensional images, as the conduct of the convolution and pooling layers are easier to be understood in a 2d plane. is is an essential contrast among other neural organizations and convnets. convnet suggests the union of the grouping organization and extraction organization. e technique for readiness is utilized for trial computations of the loads of the two layers. e pooling layer has multiple layers of convolution followed by pooling arranged in a stack. e grouping network ordinarily utilizes a normal diverse neural organization for order. e convolution layer is utilized to makes new images which is called highlight maps. e capacity map stresses the surprising parts of the primary image. as opposed to the information layer, the convolution layer is fairly extraordinary by the way it measures input which does not use connection loads and a weighted sum; then again, this layer utilizes filters that change images. ese are called spatial channels. e way toward offering contribution to the image is from the convolution channels which give the capacity map. 2d networks are channels in the convolution layers. ey are typically accessible as 5 × 5 or 3 × 3 networks. most recent applications even utilize 1 × 1 channels. e convolution, as it stays on the two-dimensional plane, tends to be an extreme cycle to express in text. be that as it may, the understanding and computation measures are simpler than they appear. e interaction of convolution is the expansion of the climate items situated in the same places of the two networks. e qualities accessible in the basics of this element map, identical to the essential convolution activity, rely upon the image framework coordinates with the convolution channel, which is reused without change in measure until the component guide of the given channel is produced. toward the end, the element map is made when the convolution layer works the convolution channels on the info image. e skilled convolution channels choose the properties that are extricated in the convolution layer. us, contingent upon the convolution channel applied, the properties determined by the convolution layer changes. e component map that convolution channel creates is taken care of with the assistance of the initiation work before the yield layer is produced. e enactment highlight of the layer of convolution resembles that of the standard neural network. albeit the most extreme current applications utilize the relu highlights, alongside the sigmoid capacity and the tanh are also included. e generally utilized moving normal channel in the computerized signal preparing area might be a predominant type of convolution channel for appraisal. e measurements of the image are reduced by the layer of pooling as it presents a combination of adjoining pixels into one delegate worth of a specific image area. e technique of pooling is a standard technique as compared to other diverse image preparing frameworks. to choose the pooling pixel from the image and set the representative worth, neighboring pixels are selected from the framework, and the number of pixels consolidated unique in relation to the issue. regularly, an agent esteem is drawn regardless of the line or mean of the assigned pixels. at that point it could be a 2d cycle, the pooling layer measure is shockingly simple. in a numerical strategy, the pooling strategy can be suggested as a means of measure of convolution. e fluctuation from convolution layer is that the convolution channel is fixed, so the spaces of convolution are not covered. e suggested model given in the resulting area will rely upon this. e layer of pooling makes up for unpredictable and shifted antiquities somewhat. for example, the pooling layer may build a feline's prominence, which might be top centered inside the image. as the image size is getting diminished by the pooling stage, it is exceptionally important to improve the computational overload and deal with overfitting. layer. fully connected layers are considered once the pooling and convolution layers are added. in a completely connected layer, we connect neurons to all the arrangement of the past layer. simple lattice improvement is completed along with a predisposition balance. to decrease the number of limitations for managing overfitting issue, a dropout work is utilized inside the completely associated layer. e overfitting will be there when at least two neurons in completely connected layers often recognize similar qualities; neurons are said to have created cotransformation or codependence on one another. in such a circumstance, a dropout work is applied to diminish this intricacy by overlooking an inconclusive gathering of neurons at the hour of the preparation stage. one of the weaknesses of utilizing the dropout work is that to accomplish the ideal union stage, more emphasis is required. at last, to recognize the numerals accurately, a sigmoid classifier is utilized. loads need to change deliberately on account of beginning haphazardness in loads, and the cnn model should be executed once more. we consider the closeness to the expectations by the unmitigated crossentropy work and it is a somewhat micro methodology for estimating discrepancies and valid errors. figure 2 shows the framework of automatic classification of hand x-ray. e model for the picture order dependent on a neural organization of profound convolution is as follows: (1) input: input is a bunch of n pictures, where n is any large positive number; one of the k characterization labels is each picture mark. (2) . learning: this stage is normally called a classifier for preparing or a learning model. e assignment of this stage is to realize what each class resembles. (3) assessment: the classifier assigns marks to pictures that have not been seen to gauge the exactness of the classifiers. we liken the marks the classifier predicts with the picture's real names. ere is no doubt that the characterization marks the classifier predicts are viable with the genuine picture grouping names. when a piece for a specific component is characterized, the element is extricated by convolution tasks. for cnn, the quantity of highlights and the idea of a particular component are not characterized ahead of time. in this way, by means of the convolution interaction, no capacity is determined. in forward proliferation, convolution surmises a capacity and back-engendering endeavors to sequentially address the presumption. various portions after complete readiness are balanced out utilizing a mistake minimization strategy. convolution attempts to positively avoid every attribute that cannot be chosen until after this arrangement has been completed. in the proposed model, obscure hand x-beam qualities are divided by convolution layers, with 1 layer having 20 portions. e essential presence of each capacity is seen following the preparation. convolution layer is shown in subpart (a) of figure 3 and pool layer yield in subpart (b) of the same figure. for executing the cnn model, a matlab ® programming cloud service was utilized. e setup was established with a dataset of about 218 radiographical images of the handout of which 61 were standard and 120 had ra. at that point, 72 radiographs, comprising 32 standard and 40 ra tests, were used to test the system. figure 4(a) shows the original pictures, figure 4 it is evident that preparation exactness (blue line) and approval precision (dark line) approach each other during the preparation cycle. e loss subplot likewise shows that both the deficiency of preparing and the deficiency of approval are low and decline during the interaction. e proposed model reports improvement in accuracy as compared with cnn models previously executed. e popular matlab@2020 design deep learning app is utilized for doing this. our model is compared with other standard classifiers such as svm and ann and the proposed model presents a better classification accuracy. e determination of the classifier is the critical element that influences the programmed exactness of discovery of the hand x-beam dependent on highlight extraction by a similar strategy for misfortune work conducted by the machine. e result of a few classifiers on exactness of grouping is discussed in this article. table 3 gives the impact on characterization exactness of different standard classifiers. ese classifiers are linear svm and ann. e presented results show that the accuracy of the cnn classifier in the training phase and test set is higher than that of different classifiers which shows that a noteworthy accuracy is obtained through the presented method, i.e., by cnn. first and foremost, it requires a tremendous extra room, sets aside a ton of effort to gauge, and uses a great deal of computational force. because of this, all the put away preparing pictures needed to be contrasted. here in this article, the more focus is given on estimating execution much effectively than showing proficiency practically speaking. as a general rule, the cnn has certain limitations: while the training of the model takes a great deal of time, the arrangement of new test information is fast once the training of the model is complete. figure 6 shows the diagnostic accuracy graph for the algorithms. figure 7 shows the sensitivity and figure 8 shows the precision. e cnn lenet and cnn are compared to find out the accuracy. by training both networks it is found that cnn is better than lenet which is shown in figure 9 . journal of healthcare engineering 7 a decision of ra is made by assessing the lab reports and imaging especially hand x-beam. radiography is an effectively available imaging technique; the expert in surveying ra discoveries utilizing radiography is significant and simultaneously it is a ling tedious interaction. in the early treatment of ra finding permanent deformities found in ra can be forestalled. so mechanized technique for distinguishing ra becomes useful. in this examination we prepared and assessed cnn and cnn lenet model. likewise, the near examination is finished with svm and artificial neural organization grnn. we propose a framework that distinguishes ra without broad preprocessing of radiographic hand x-beam pictures. e proposed model zeroed in on the programmed characterization of hand x-beam for rheumatoid arthritis patients with the information picture dataset of 290 pictures. e proposed model presents a model accuracy of approximately 95%. is model is utilized to gain proficiency with the hand x-beam including consequently. e programmed recognition of the typical and unusual condition is tried effectively and furthermore confirmed with the specialist. e proposed model is contrasted and compared with diverse popular cnn techniques and reports improved accuracy. it is additionally contrasted and various classifiers. e proposed strategy includes improved exactness and accuracy. in future we desire to increase the dataset size with the prediction of phases of ra even before the transitional and serious stage. data are available on request to the corresponding author. figure 9 : accuracy: cnn and lenet. 125i-labeled gold nanorods for targeted imaging of inflammation how accurate are diagnoses for rheumatoid arthritis and juvenile idiopathic arthritis in the general practice research database? automatic joint detection in rheumatoid arthritis hand radiographs automatic quantification of joint space narrowing and erosions in rheumatoid arthritis hand radiograph analysis and joint space location improvement for image interpretation automatic detection of ra using hand x-ray automatic identification of bone erosions in rheumatoid arthritis from hand radiographs based on deep convolutional neural network automatic diagnosis of ra from hand radiographs using convolutional nueral network detection of ra from hand radiographs using cnn a study on deep machine learning algorithms for diagnosis of diseases diagnosis and treatment of inflammatory joint disease recommendations for the management of rheumatoid arthritis with synthetic and biological disease -modifying anti-rheumatic drugs long-term safety of methotrexate monotherapy in patients with rheumatoid arthritis: a systematic literature research insights into the treatment of rheumatoid arthritis: a paradigm in medicine machine learning to predict anti-tumor necrosis factor drug responses of rheumatoid arthritis patients by integrating clinical and genetic markers profiling of gene expression biomarkers as a classifier of methotrexate nonresponse in patients with rheumatoid arthritis comparative study of automated testing tools: test complete and quick test pro classification of the covid-19 infected patients using densenet201 based deep transfer learning a machine learning classifier for assigning individual patients with systemic sclerosis to intrinsic molecular subsets compendium of synovial signatures identifies pathologic characteristics for predicting treatment response in rheumatoid arthritis patients use of artificial intelligence in imaging in rheumatology-current status and future perspectives neural networks for automatic scoring of arthritis disease activity on ultrasound images development and validation of a deep-learning model for scoring of radiographic finger joint destruction in rheumatoid arthritis machine-learningbased knowledge discovery in rheumatoid arthritis-related registry data to identify predictors of persistent pain naive electronic health record phenotype identification for rheumatoid arthritis supplementing claims data with electronic medical records to improve estimation and classification of rheumatoid arthritis disease activity: a mach assessment of a deep learning model based on electronic health record data to forecast clinical outcomes in patients with rheumatoid arthritis e application of machine learning algorithms to the analysis of electromyographic patterns from ra arthritis of the finger joints: a comprehensive approach comparing conventional radiography, scintigraphy, ultrasound, and contrast-enhanced magnetic resonance imaging imagenet classification with deep convolutional neural networks towards quantitative assessment of ra using volerteic ultrasound e usefulness of computer-aided joint space analysis in the assessment of rheumatoid arthritis synovial pathology detected on ultrasound correlates with the severity of radiographic knee osteoarthritis more than with symptoms automated assessment of joint synovitis activity from medical ultrasound and power doppler examinations using image processing and machine learning journal of healthcare engineering 9 methods advances in neural information processing systems overfeat: integrated recognition, localization and detection using convolutional networks neural networks for classification: a survey key: cord-0034781-jsbca3xq authors: butnor, kelly j.; khoor, andras title: collagen vascular diseases and disorders of connective tissue date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_20 sha: 5060cbd289b48b4b2005376c2c18070830a80160 doc_id: 34781 cord_uid: jsbca3xq the collagen vascular diseases, also referred to as connective tissue diseases, are a diverse group of systemic inflammatory disorders thought to be immunologically mediated. the concept of collagen vascular disease began to take shape in the 1930s, when it was recognized that rheumatic fever and rheumatoid arthritis can affect connective tissues throughout the body.1,2 during the following decade, as conditions such as systemic lupus erythematosus (sle) and scleroderma came to be viewed as systemic diseases of connective tissue, the terms diffuse connective disease and diffuse collagen disease were proposed.3,4 during the same period, the designation of diffuse vascular disease was proposed for diseases such as scleroderma, polymyositis, sle, and polyarteritis nodosa, which featured widespread vascular involvement.5 with the realization that many of these entities can exhibit both systemic connective tissue manifestations and vascular abnormalities, the unifying designation of collagen vascular disease was introduced.6 the collagen vascular diseases, also referred to as connective tissue diseases, are a diverse group of systemic inflammatory disorders thought to be immunologically mediated. the concept of collagen vascular disease began to take shape in the 1930s, when it was recognized that rheumatic fever and rheumatoid arthritis can affect connective tissues throughout the body.l,2 during the following decade, as conditions such as systemic lupus erythematosus (sle) and scleroderma came to be viewed as systemic diseases of connective tissue, the terms diffuse connective disease and diffuse collagen disease were proposed. 3 ,4 during the same period, the designation of diffuse vascular disease was proposed for diseases such as scleroderma, polymyositis, sle, and polyarteritis nodosa, which featured widespread vascular involvement. 5 with the realization that many of these entities can exhibit both systemic connective tissue manifestations and vascular abnormalities, the unifying designation of collagen vascular disease was introduced. 6 pleuropulmonary manifestations are a significant source of morbidity and mortality for patients with collagen vascular disease. an estimated 1600 deaths result from pulmonary manifestations of collagen vascular disease each year in the united states, which accounts for nearly one quarter of all deaths related to interstitial lung disease. 7 the incidence of collagen vascular diseaserelated interstitial lung disease appears to be increasing, likely the result of improved recognition and enhancements in diagnostic imaging. 8 this chapter discusses pulmonary manifestations of collagen vascular diseases, pulmonary involvement by inflammatory bowel disease, primary biliary cirrhosis, and disorders of connective tissue that are not categorized as collagen vascular diseases. 722 general histopathologic patterns and approach to diagnosis while a few pulmonary lesions, such as rheumatoid nodules, are essentially pathognomonic of collagen vascular disease, the collagen vascular diseases are best considered a group of disorders with protean pulmonary manifestations. virtually any component of the lungs, from the large airways to the interstitium, can be affected by collagen vascular disease (table 20 .1). pulmonary involvement by collagen vascular disease can simulate a variety of primary diseases of the lung, in particular, the idiopathic interstitial pneumonias (see chapter 19 for a complete description of the idiopathic interstitial pneumonias). the successful recognition of collagen vascular disease-related interstitial lung disease, therefore, requires the careful integration of clinical and radiographic data. obtaining this information can often prove challenging. while a patient may have an established diagnosis of collagen vascular disease, such information is sometimes not communicated to the pathologist. in other cases, the patient may have rheumatologic symptoms or signs, but has not been fully evaluated for collagen vascular disease prior to lung biopsy. pulmonary disease can precede other manifestations of collagen vascular disease by months or even years, adding to the diagnostic challenge. 9 -11 it has been known for some time that collagen vascular diseases can mimic the histologic pattern of interstitial lung disease that is seen in idiopathic pulmonary fibrosis (ipf).12 for this reason, it is essential to exclude collagen vascular disease in patients who exhibit a pattern consistent with usual interstitial pneumonia (uip) on lung biopsy with a thorough rheumatologic evaluation and serologic studies such as antinuclear antibody (ana) and rheumatoid factor. it is particularly important for the pathologist to suggest the possibility of collagen vascular disease-related interstitial lung disease in cases with a uip pattern in which the clinical features are not entirely typical of ipf. such cases include patients younger than 50 years of age, never smokers, females, and those with atypical radiographic findings. collagen vascular diseaserelated interstitial lung disease with a uip-like pattern progresses more slowly and exhibits a more favorable prognosis than does idiopathic uip' 13 -17 the reason for the differences in behavior between these entities is unclear, but it has been noted that the collagen vascular disease-related uip-like pattern shows fewer fibroblastic foci than idiopathic uip ' 16 prior to the recognition of nonspecific interstitial pneumonia (nsip) (see chapter 19) , the uip pattern was the most frequently observed pathologic pattern of collagen vascular disease-related interstitial pneumonia. [18] [19] [20] [21] however, since its introduction, nsip has come to be recognized as the most common histologic pattern of interstitial lung disease in collagen vascular disease, accounting for 40% to 60% of cases of collagen vascular disease-related chronic interstitial lung disease in recent series. 22 -24 this is in contrast to the idiopathic interstitial pneumonias, in which the most common histologic pattern, uip, comprises three quarters of all cases. 24 although data are limited, the prognosis of nsip in the setting of collagen vascular disease appears to be similar to that for idiopathic nsip' 15 . 22 ,25-28 as with idiopathic fibrotic nsip, the fibrotic pattern of nsip in the setting of collagen vascular disease is associated with poorer survival than the cellular pattern of nsip ' 22 . 28 it is not uncommon for several histologic patterns, each mimicking one of the categories of idiopathic interstitial pneumonia, to coexist. alternatively, a mixture of overlapping patterns with histologic features that resemble several of the idiopathic interstitial pneumonias can occur. in addition to resembling the idiopathic interstitial pneumonias, collagen vascular disease may produce a pattern of interstitial lung disease that is difficult to categorize. in such cases, the recognition of subtle features associated with collagen vascular disease, such as follicular bronchiolitis or a bronchiolocentric distribution of interstitial pneumonia, may allow the pathologist to suggest collagen vascular disease as a possible etiology. patients with collagen vascular disease are often treated with immunosuppressive or cytotoxic drugs that predispose them to opportunistic infections. both drug toxicity and infection can mimic de novo collagen vascular disease-related interstitial lung disease. distinguishing between these entities can be difficult if not impossible in some cases (see chapter 22) . transbronchial biopsies are of limited utility in the evaluation of collagen vascular disease-related lung disease due to the small amount of tissue they provide. 29 . 3o bronchoalveolar lavage (bal) does playa role in the workup of patients with known or suspected collagen vascular disease inasmuch as its aids in the detection of pulmonary infections. 31 ,32 however, the pathologic assessment of interstitial lung disease associated with collagen vascular disease is best accomplished by open or video-assisted thoracoscopic wedge biopsy. in determining the most appropriate site, or preferable sites to biopsy, the same biopsy guidelines as have been established for suspected idiopathic interstitial lung disease apply. 33 the surgeon should be cautioned to avoid sampling the most severely affected areas, as tissue from such sites may show only nonspecific end-stage honeycomb fibrosis. 34 sampling less severely involved regions, as well as the transition between normal and affected parenchyma is likely to be most diagnostically useful (see chapter 1). when processing wedge biopsies from patients with an acute or subacute course, fresh tissue should be taken for microbiologic cultures if not already done so intraoperatively. the liberal use of histochemical stains for infectious organisms in biopsies from such patients can be diagnostically rewarding. in patients with known or suspected sjogren's syndrome, fresh tissue should be set aside for possible flow cytometric or immunohistochemical studies, given the relative frequency of lymphoproliferative disorders in such patients. rheumatic fever is a disease of presumed autoimmune etiology associated with antecedent throat infection with group a streptococcus. once common in the unites states and other western countries, rheumatic fever has virtually disappeared from developed countries during the last half-century. as a result, little has been published about its pulmonary manifestations. 35 rheumatic pneumanias have been the subject of sporadic reports describing both lobar and multilobar consolidation. 36 ,37 in 1966, massumi and legier 37 described three patients with active rheumatic carditis complicated by adult respiratory distress syndrome (ards) with a rapidly fatal course. autopsies were performed in two of their cases, which disclosed heavy lungs with organizing pneumonia, interstitial edema, and focal vasculitis. rheumatic heart disease remains a major health care problem in developing countries, where rheumatic mitral stenosis commonly affects juvenile age groups. lungs from cases of mitral stenosis exhibit pulmonary hypertension and prominent bronchial smooth muscle hypertrophy. 38.39 pulmonary hypertension in such cases manifests as moderate to marked medial hypertrophy of medium-sized branches of the pulmonary artery and dilatation lesions (see chapter 28). rheumatoid arthritis (ra) is a chronic systemic disease characterized by symmetric nonsuppurative arthritis of the small peripheral joints. it affects about 1 % of the general population worldwide. sir alfred garrod 40 first introduced the term rheumatoid arthritis in 1876 to distinguish it from gout. pulmonary involvement was first described by ellman and ball in 1948. 41 in autopsy studies, approximately half of patients with ra have evidence of active or remote pleuropulmonary disease. 42 ,43 although ra is more common in women than in men, rheumatoid lung disease is more frequently observed in men with long-standing rheumatoid factor-positive ra and subcutaneous nodules. 42 ,44 rheumatoid lung disease shows ethnic variations in prevalence; it seems to be more common in anglo-saxon than in mediterranean countries. 45 studies suggest that extraarticular disease is a major predictor of mortality in patients with ra. 46 rheumatoid arthritis can involve all major compartments of the lung and may lead to (1) airway disease, (2) diffuse alveolar hemorrhage, (3) interstitial lung disease, (4) pulmonary rheumatoid nodules, (5) vascular lesions, and (6) pleural disease. the prevalence of airflow obstruction in patients with ra is remarkably high, suggesting that airway disease may be a common form of lung involvement. 47 airway complications of ra include constrictive (obliterative) bronchiolitis, follicular bronchiolitis, bronchiectasis, and bronchocentric granulomatosis. 19.22.47-62 rheumatoid arthritis patients with constrictive bronchiolitis present with rapidly developing breathlessness, rales, and a high-pitched mid-inspiratory squeak.47 chest radiographs show distended but otherwise normallungs. 47 high-resolution computed tomography (hrct) reveals small nodular shadows in the centrilobular regions, patchy areas of low attenuation, and peribronchial thickening. 50 tests of lung function show airflow obstruction with air trapping. 47 in spite of treatment with antibiotics, bronchodilators, or corticosteroids, most patients die of respiratory failure in 5 to 18 months after the onset of pulmonary symptoms. 47 an association between constrictive bronchiolitis and penicillamine in ra patients has been reported. 48 it has been suggested that penicillamine may impair healing of bronchiolitis. the histologic features of constrictive bronchiolitis in ra are similar to those seen in patients without an underlying connective tissue disease ( fig. 20.1 ) (see also chapter 25) . follicular bronchiolitis is a common finding in patients with ra. tansey et al. 22 reviewed lung biopsies from patients with ra and respiratory symptoms. follicular bronchiolitis was the major histologic pattern in six patients (16%) and a minor pattern in five others (14%). the ra patients with follicular bronchiolitis have a chronic clinical course with a main complaint of productive cough.50 high-resolution ct reveals small centrilobular nodules variably associated with peribronchial nodules and ground-glass opacities. 5o ,56 histologically, coalescent reactive germinal centers are seen adjacent to airways.2o follicular bronchiolitis can be differentiated from lymphoproliferative disorders by the absence of tumefactive growth, rare lymphoepithelial complexes, and benign appearing follicles (see chapter 32) . 63 bronchiectasis can be a feature of rheumatoid arthritis and is often found in patients with severe long-standing nodular disease. productive cough, hemoptysis, and dyspnea are the most common respiratory symptoms. radiographic abnormalities in ra-associated bronchiectasis include bibasilar, diffusely increased, interstitial markings and focal infiltrates. recurrent pulmonary infections and respiratory failure are frequent and may be fatal (see chapter 5) . 60 bronchocentric granulomatosis has been reported in a few patients with ra. 61 ,62 the differential diagnosis in such cases includes mycobacterial and fungal infections as well as bronchocentric rheumatoid nodules. 64 obliteration of the airway lumen and epithelium is an important histologic feature of bronchocentric granulomatosis. this feature is helpful in distinguishing bronchocentric granulomatosis from other conditions that present with peribronchial granulomas. diffuse alveolar hemorrhage is a rare complication of ra. a clinicopathologic study of 34 patients with biopsyconfirmed diffuse pulmonary hemorrhage by travis prior to the description of nsip in 1994, uip was the most common pattern of interstitial lung disease in patients with ra, occurring in 1 % to 4% of cases. 76 . 77 however, if current american thoracic society/european respiratory society criteria for the diagnosis of idiopathic interstitial pneumonias are applied, nsip is more common than uip in ra (see chapter 19) .22.33 the relationship of dip and respiratory bronchiolitis to ra is uncertain, given that most reported patients are also smokers. 22 rare cases of rapidly fatal pulmonary fibrosis, clinically resembling hamman-rich syndrome or accelerated decline of idiopathic pulmonary fibrosis, have been described. 78 .79 histologically, these cases show a combination of uip pattern and diffuse alveolar damage. 79 open lung biopsy is the preferred method for evaluating ra -associated interstitial lung disease. 19 optic bronchoscopy and bal.32.80-82 the histopathologic patterns of interstitial lung disease in ra are generally considered indistinguishable from corresponding idiopathic interstitial pneumonias (discussed in detail in chapter 19) .33.83-85 however, there are some subtle features that, although not specific, may suggest a rheumatoid origin in cases with a uip-like pattern. 86 the reported prevalence of subcutaneous rheumatoid (necrobiotic) nodules varies greatly, ranging from 13.5% to 53%.87-89 pulmonary rheumatoid nodules, which can occur singly or as multiple nodules, are the most characteristic but least frequent type of lung lesions in ra. 90 93 walker and wright 90 reported only three cases (0.6%) in a series of 516 patients, while jurik et a1. 92 found only one case (0.3%) in a series of 309 patients. pulmonary rheumatoid nodules are most commonly found in men with advanced seropositive ra and subcutaneous nodules. however, pulmonary rheumatoid nodules can occasionally appear before the onset of arthritis.2o.91.93-97 solitary rheumatoid nodules may be confused with malignant tumors radiographically.98-loo however, pulmonary adenocarcinomas may also be associated with rheumatoid nodules. iol 106 pulmonary rheumatoid nodules may cavitate, occasionally leading to pneumothorax, pyopneumothorax, or fungal colonization. 107-1 14 caplan syndrome, also known as rheumatoid pneumoconiosis, occurs when rheumatoid nodules develop in patients with pneumoconiosis. originally described in coal workers with ra, the concept of caplan syndrome has been subsequently broadened to include ra patients with silica-associated or asbestos-associated lung disease. 115 -122 radiographically, multiple well-defined homogeneous rounded opacities are seen. 119 histologically, the nodules described by caplan are similar to rheumatoid pulmonary nodules, with the occasional addition of black coal dust in the centrally necrotic area of the nodules (see pleural effusion occurs in only 3% to 5% of patients with ra.1ii2. 153 it is usually associated with other manifestations of active disease such as arthritis, subcutaneous rheumatoid nodules, and high titers of rheumatoid factor. 42 pleural effusions in ra are typically exudative with a low ph, low glucose, and high concentrations of protein and lactate dehydrogenase. 102 . 147 .1 5 1.152,154.155 in most rheumatoid effusions, ra cells can be identified. rheumatoid arthritis cells are cytologically similar to lupus erythematosus (le) cells and are characterized by basophilic cytoplasmic inclusions that have been shown to represent phagocytosed igm rheumatoid factor in immune complex form. the ra cells are not specific for ra and can also be found in other connective tissue diseases, tuberculosis, various malignancies, and pleural effusion of unknown etiology.152 in contrast to adult-onset ra, there have been relatively few descriptions of pulmonary involvement in juvenile ra, and only rare cases with histologic documentation. 83 -85 ,90,156-158 however, over 50% of patients with juvenile ra have abnormal pulmonary function tests, indicating lung disease in juvenile ra may be more prevalent than the small number of reported cases would suggest. 157 pulmonary lesions in juvenile ra are identical to those seen in adult ra and include pleuritis, pleural adhesions, constrictive bronchiolitis, follicular bronchiolitis, lymphoid interstitial pneumonia, pulmonary vasculitis, pulmonary hypertension, and diffuse alveolar hemorrhage ( fig. 20.2) . 65, 84, 85, [158] [159] [160] [161] [162] [163] [164] systemic lupus erythematosus systemic lupus erythematosus (sle) is a multisystemic disease of autoimmune origin, characterized by a variety of autoantibodies, in particular antinuclear antibodies. acute or insidious in onset, sle is a chronic, often febrile disease characterized principally by injury to the skin, joints, kidney, and serosal membranes. however, virtually every organ can be affected, pleuropulmonary involvement is more common in sle than in any other connective tissue disease and is associated with an increased risk of mortality, 9, 12, 42, [165] [166] [167] [168] although the reported incidence ranges from as low as 9% to as high as 98%, the overall rate of pulmonary involvement in sle in most series is between 50% and 70%.9.12,42, 165-167.169,170 the number of patients with histologically documented significant lung lesions is around 20%. 11, 12, 167, [170] [171] [172] [173] [174] [175] [176] [177] [178] systemic lupus erythematosus is associated with an array of pulmonary manifestations, because of the common use of immunosuppressive therapy, sle patients are prone to infectious complications, and infection is the k.j. butnor and a. khoor most common cause of pulmonary infiltrates in these pa tients. 9 .1 2 ,165,172,175 other major complications include (1) diffuse alveolar hemorrhage, (2) acute lupus pneumonitis, (3) pulmonary hypertension, (4) antiphospholipid syndrome, (5) pleural disease, (6) shrinking lung syndrome, and (7) neoplasms. diffuse alveolar hemorrhage occurs in approximately 2 % of sle patients. 168 the typical presentation of acute dyspnea and extensive pulmonary infiltrates may mimic acute lupus pneumonitis, especially in the absence of hemoptysis,179,1811 both diseases are life-threatening and are treated empirically with corticosteroids and immunosuppression.l8l however, the differential diagnosis in such cases includes opportunistic infection. a misdiagnosis may lead to a potentially disastrous increase in immunosuppressive therapy. therefore, bal is recommended in sle patients with unexplained extensive pulmonary infiltrates to exclude opportunistic infection. in up to 20% of patients, diffuse alveolar hemorrhage is the presenting or dominant manifestation of sle. 179 ,180 in such cases, sle must be distinguished from other major causes of diffuse alveolar hemorrhage, such as wegener granulomatosis, goodpasture syndrome, microscopic polyangiitis, and idiopathic pulmonary hemorrhage. 65 histopathologic examination reveals capillaritis in most cases of sle-related diffuse alveolar hemorrhage, but in some patients, bland pulmonary hemorrhage without capillaritis has been reported. 65 ,179,182-184 capillaritis is manifested by an infiltrate of necrotic neutrophils within the alveolar septa and is often associated with destruction of the alveolar walls ( 185 although such inclusions are morphologically similar to nucleoprotein strands of myxoviruses, they are considered to be products of nonspecific cellular injury that can be induced by acid-labile a-interferon. acute lupus pneumonitis is characterized by the sudden development of severe dyspnea, tachypnea, fever, arterial hypoxemia, and diffuse pulmonary infiltrates. 186, 187 the clinical and radiographic findings of lupus pneumonitis are difficult to distinguish from those of infection and diffuse alveolar hemorrhage. 186 ,187 the mortality rate is around 50%. patients who survive often exhibit residual interstitial infiltrates and persistent pulmonary function test abnormalities. 186 histologic sections of the lungs reveal diffuse alveolar damage with hyaline membranes, interstitial edema, and arteriolar thrombosis. 186 pulmonary hypertension was once considered rare in sle, but now is being reported with increasing frequency. 181 abnormalities indicative of subclinical pulmonary hypertension are found on echocardiography in 10% of patients, usually in association with raynaud phenomenon. 18s the most common presenting complaints of sle patients with pulmonary hypertension are dyspnea on exertion, chest pain, nonproductive cough, edema, and fatigue or weakness.173 chest x-ray shows cardiomegaly, a prominent pulmonary artery, and clear lung fields. the diagnosis of pulmonary hypertension is established by demonstrating elevated pulmonary artery pressure at cardiac catheterization. symptoms may be mild with a protracted course. however, in some patients, the course is more rapid, with death resulting in only a few years. pathologic examination of the lungs in sle-related pulmonary hypertension reveals pulmonary arteriopathy with plexiform lesions or thromboembolic arteriopathy. [189] [190] [191] [192] [193] [194] rarely, pulmonary vasculitis is the cause of the pulmonary hypertension. 189 .1 95 anti-endothelial cell antibodies and immune complexes may stimulate endothelin-l release from endothelial cells. this mediator is thought to play an important role in the initiation and development of pulmonary hypertension in sle patients (see chapter 28) . 173, 196, 197 figure 20.10 . systemic lupus erythematosus. electron photomicrograph of tubuloreticular inclusion in alveolar endothelial cell of lupus lung. antiphospholipid antibodies are a family of autoantibodies that recognize various combinations of phospholipids, phospholipid-binding proteins, or both.19r the term antiphospholipid syndrome was first coined to denote the clinical association between antiphospholipid antibodies and a syndrome of hypercoagulability.199 currently, a patient with the antiphospholipid syndrome must meet at least one of two clinical criteria (vascular thrombosis or complications of pregnancy) and at least one of two laboratory criteria (anticardiolipin antibodies or lupus anticoagulant antibodies ).1 98 the antiphospholipid syndrome may be divided into two categories. 198 "primary" antiphospholipid syndrome occurs in patients without clinical evidence of another autoimmune disease, whereas "secondary" antiphospholipid syndrome occurs in association with autoimmune or other diseases. systemic lupus erythematosus is by far the most common disease with which the antiphospholipid syndrome occurs. pulmonary complications of the antiphospholipid syndrome include pulmonary embolism and infarction, both thromboembolic and perhaps nonthromboembolic pulmonary hypertension, pulmonary arterial thrombosis, pulmonary microthrombosis, adult respiratory distress syndrome, intraalveolar pulmonary hemorrhage, and postpartum syndrome. [s generally ascribed to diaphragmatic weakness?04 although neuropathy and myopathy can also occur in sle, the diaphragmatic dysfunction in shrinking lu~g syndrome appears to be unrelated to these disorders. 20 ).207.209 in one study, the characteristic restrictive defect could not be explained by a primary abnormality of the diaphragm and was attributed to an unspecified restriction in chest wall expansion.2io there are no specific histologic abnormalities in shrinking lung syndrome. 202 however, in rare cases, diaphragmatic fibrosis has been documented at autopsy. 204 neoplasms there seems to be an association between sle and non-hodgkin lymphoma, lung cancer, and kaposi sarcoma (see also chapter 34).211-214 bjornadal et a\.2j3 found a bimodal incidence of malignancy in sle patients, with an increased incidence of non-hodgkin lymphoma early in the patients' course and a later increase in the risk of lung cancer. however, another study of sle patients failed to confirm an overall association between sle and lung cancer and showed only a borderline statistically significant increased risk of lymphoma.215 pulmonary vasculitis has been reported rarely in sle, and chronic interstitial lung disease is much less prevalent in . systemic sclerosis (scleroderma) is a generalized connective tissue disorder characterized by thickening of the skin (scleroderma) and frequent involvement of internal organs, notably the heart, lungs, kidneys, and the gastrointestinal tract. traditional classification of systemic sclerosis is based on the extent of skin involvement. the diffuse form can involve any part of the body. the limited form, also known as the crest syndrome (calcinosis, raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectases), typically involves the face, sparing the trunk and the limbs proximal to the elbows and knees. pulmonary function abnormalities are common in both the limited and diffuse forms.219 the cumulative survival rate for patients with systemic sclerosis is less than 80% at 2 years, 50% at 8.5 years, and 30% at 12 years after diagnosis, with most patients dying of cardiopulmonary disease. 220 .221 renal, cardiac, pulmonary, and gastrointestinal involvement impacts negatively on surviva1. 220 major pulmonary complications of systemic sclerosis include (1) interstitial lung disease, and (2) pulmonary hypertension. in recent studies, nsip has surpassed uip as the most common histologic pattern of interstitial lung disease associated with systemic sclerosis. 15.26.7 1 bouros et a1. 15 reviewed surgical lung biopsies from 80 patients with systemic sclerosis and "fibrosing alveolitis." nonspecific interstitial pneumonia (n = 62, 77.5%), subcategorized as cellular nsip (n = 15) and fibrotic nsip (n = 47), was much more prevalent than uip (n = 6) , end-stage lung disease (n = 6), or other patterns (n = 6). in a similar study by kim et a1., 26 13 of 19 patients had nsip and five had uip. the ct appearance of systemic sclerosis-associated lung disease often resembles that of idiopathic nsip' 222 in the study by kim et ai., comparison of the clinical outcome of 12 patients followed for more than 12 months suggested a better prognosis for nsip than for uip. in contrast, the 5-year survival differed little between nsip (91 %) and uip (82%) in the similar study by bouros et a1. grossly, the lungs are firm in consistency (fig. 20.12 ). in general, honeycombing is relatively infrequent. the histologic patterns of systemic sclerosis-associated interstitial pneumonias are indistinguishable from those of their idiopathic counterparts (fig. 20.13 ). however, interstitial lung disease is likely to be accompanied by pleural fibrosis in systemic sclerosis.223 pulmonary hypertension develops in 10% to 33% of patients with diffuse systemic sclerosis and in 40% to 65 % of patients with the limited form of disease. 219 .224-229 in limited systemic sclerosis, pulmonary hypertension is usually not accompanied by interstitial fibrosis.219 increasing age (~60 years) at diagnosis is a risk factor for pulmonary arterial hypertension. 230 a decreasing carbon monoxide diffusing capacity (dlco) is a good predictor of subsequent development of isolated pulmonary hypertension in limited systemic sclerosis. 23 intimal thickening (fig. 20.14) .219,232-236 plexiform lesions are usually not seen, but there are occasional exceptions.235 rare cases of pulmonary occlusive venopathy (pulmonary veno-occlusive disease) have also been reported. 237 the cause of pulmonary hypertension in systemic sclerosis is unknown, but endothelial injury has been implicated as the inciting event, a concept that is supported by the detection of circulating endothelial cells in some patients. 238 dermatomyositis and polymyositis (dpm) are idiopathic inflammatory myopathies. patients with polymyositis manifest with proximal muscle weakness, elevated serum levels of enzymes derived from skeletal muscle, myopathic changes demonstrated by electromyography (emg), and evidence of inflammation on muscle biopsy. 255 the addition of skin rash suggests the diagnosis of dermatomyositis. 255 dermatomyositis-polymyositis is a relatively uncommon disease with an incidence of 5 to 5.5 cases per million in the united states.256257it occurs twice as frequently in women as in men in all age groups, with two peaks of onset, one in the first decade and the second in the fifth and sixth decades. 256 in recent studies, the prevalence of lung disease in dpm varies from 23% to 65%.258-260 lung disease precedes myositis or skin rash in at least one third of cases. 18 ,261-264 the first description of pulmonary involvement in dpm by mills and mathews 265 in 1956 was that of a 52year-old woman with "interstitial pneumonitis." interstitial lung disease remains the most frequent pulmonary problem in dpm. prior to the recognition of nsip, usual interstitial pneumonia (uip) was considered the most common pattern of interstitial lung disease in dpm. 18264 however, if current criteria are applied,nsip is the most common. clinically, interstitial lung disease in dpm usually presents in a manner similar to acute or subacute communityacquired pneumonia.27 chest radiographs and ct scans typically demonstrate bilateral irregular linear opacities involving the lung basesy jo-1 antibody is present in about 38% of patients. 27 survival of patients with dpmrelated interstitial lung disease is significantly better than that observed for patients with idiopathic uip, and is similar to idiopathic nsip' 27 there is no significant survival difference between jo-1 positive and jo-1 negative groups.27 histologically, dpm-related nsip (fig. 20.15 ), dad, organizing pneumonia ( fig. 20.16) , and vip are similar to their idiopathic counterparts. 22 .27,266 however, most cases of dpm-related interstitial lung disease are also associated with mild to moderate pleural fibrosis (fig.20.15b) . 27 aspiration pneumonia can be a problem in patients with dpm, particularly if there is upper airway or respiratory muscle weakness. pulmonary vasculitis and pulmonary hypertension have been reported rarely in patients with dpm. 264 ,267 as its name implies, mixed connective tissue disease (mctd) is characterized by overlapping features of other connective tissue diseases. the most common constituents of this disease, which some alternatively refer to as undifferentiated autoimmune rheumatic/connective tissue disease, are sle, dpm, and progressive systemic sclerosis.268.269 there is a striking female preponderance of 16: 1. 270 high titers of circulating antibodies to ribonucleoprotein (rnp) antigen are typically present. 268 antibodies to alveolar septal capillary endothelial cells have been found in some patients. 271 â�¢ 272 pulmonary involvement in mctd has been reported in 20% to 85% of cases. 12 with mctd show evidence of subclinical pulmonary involvement in the form of lymphocytic alveoli tis by bal. 283 the most commonly reported form of pulmonary involvement on biopsy is interstitial fibrosis in a pattern resembling uip'12.42.274.276-278.280-282 on hrct, interstitial fibrosis manifests as irregular linear and reticular opacities. 284 initially, such changes affect the lung bases, while in more advanced cases, honeycombing can observed. other forms of interstitial disease that have been reported include organizing pneumonia, particularly in patients with prominent lupus features. 2lo diffuse alveolar damage has also been reported. 274 pulmonary hypertension is relatively frequent, particularly in patients with prominent scleroderma features. 270 features identical to those observed in primary pulmonary hypertension, including plexiform lesions and fibrinoid necrosis, have been described. 273 .276.285-291 uncommon pulmonary vascular manifestations include vasculitis and recurrent thromboemboli. 276 â�¢ 282 massive and even fatal pulmonary hemorrhage has been reported, typically in association with glomerulonephritis. 279 .285.292.293 pulmonary amyloidosis has also been reported?94 pleural involvement in the form of effusions or pleuritis is seen occasionally, particularly in patients with other lupus-like manifestations.z74. 278.181.282.295 in patients with predominant clinical features of dpm or scleroderma, aspiration pneumonia can result from esophageal dysfunction. 296 .297 patients with dpm features are also prone to respiratory failure secondary to respiratory muscle dysfunction. 296.297 as might be expected from the heterogeneity of this disease, pulmonary involvement in mctd has a variable prognosis. 298 pulmonary disease in mctd patients with predominant scleroderma manifestations is particularly refractory to corticosteroids. 2 s 1 figure 20.17 . follicular bronchiolitis. bronchiole shows an adjacent lymphoid follicle. this finding may be seen in a number of collagen vascular diseases, including sjogren's syndrome. sjogren's syndrome (ss) is an autoimmune disease characterized by a triad of manifestations: keratoconjunctivitis sicca, xerostomia, and arthritis?99 two forms of the disease are recognized. in primary ss, this triad occurs in isolation. in secondary ss, which accounts for one half to two thirds of cases of ss, these manifestations occur in association with another collagen vascular disease, most typically rheumatoid arthritis.12,42 primary ss typically affects females in the fifth to seventh decades of life, while the patient population affected by secondary ss is somewhat more heterogeneous. patients with altered immunity, including bone marrow transplant recipients and individuals infected with human immunodeficiency virus (hiv), appear to have a higher rate of ss than the general population.3oo. 301 the occurrence of ss in families and the increased frequency of certain human leukocyte antigen (hla) subtypes in patients with ss suggests a genetic predisposition?02.303 however, the underlying etiology of ss has yet to be discovered. a number of autoantibodies are detectable in patients with ss, including rheumatoid factor, antinuclear antibody (ana), and the ribonucleoproteins ss-a(ro) and ss-b (la) . ss-b(la) is considered to be most specific for ss. [304] [305] [306] the most common clinical manifestations of ss, which include dry eyes, dry mouth, and salivary gland enlargement, reflect exocrine gland involvement by this disease. nine to 90% of patients, most commonly those with secondary ss, exhibit pulmonary manifestations. 42.304.305.107-31 7 pulmonary manifestations in ss typically develop late in the course of disease and only rarely are the presenting feature. 30r lesions affecting the small airways, such as follicular bronchiolitis, are among the most common histologic findings in patients with ss who have pulmonary involvement (fig. 20.17) . 22,42.304 large airway abnormalities include obstruction and desiccation of the tracheobronchial tree.30r jii.m the latter condition, which is referred to as xerotrachea, is characterized by lymphocytic inflammation and atrophy of the submucosal glands ( fig. 20.18 in series published prior to the recognition of nsip, lymphocytic interstitial pneumonia (lip) was the most common interstitial lung disease in ss (fig. 20.19 ). 304 however, in several more recent series, nsip has been the predominant pattern (fig. 20.20 ).23 uncommonly encountered patterns of interstitial lung disease in ss include uip and bronchiolitis obliterans-organizing pneumonia (boop). 1 2.42.299.304.3118.312.314.322-324 radiographically, interstitial disease in ss typically gives rise to a reticulonodular pattern. 325 follicular bronchiolitis and lip are but part of a spectrum of lymphoid lesions that may develop in ss, which also includes nodular lymphoid hyperplasia, lymphocytic alveoli tis, lymphomatoid granulomatosis, and malignant lymphoma. 12 343 through the use of modern clonality detection methods such as reverse-transcriptase polymerase chain reaction (rt-pcr), many cases previously designated as pseudolymphomas have been reclassified as malignant lymphomas (see chapter 32) . 344 on high-resolution computed tomography, pulmonary lymphoma in patients with ss may take the form of consolidation, solitary or multiple nodules, or be distributed along the pulmonary interstitium. 328 high-grade pulmonary lymphomas, such as diffuse large cell lymphoma, arising in the setting of ss are generally associated with a poor prognosis. in one series of patients with ss and lymphoma, four of 10 died within 4 years of receiving a malignant diagnosis.328 angioinvasion is a relatively frequent finding in pulmonary lymphomas associated with ss. however, this finding does not itself appear to directly impact the prognosis. 328 an array of other pulmonary abnormalities has been reported in ss. parenchymal nonnecrotizing granulomas may simulate hypersensitivity pneumonia, particularly in cases accompanied by peribronchiolar lymphocytic inflammation (fig. 20.21 ).305 rare cases of coexisting ss and sarcoidosis have been described, as have cases of coexisting ss and primary biliary cirrhosis, a condition that occasionally features pulmonary granulomas.334.345-347 perinuclear antineutrophil cytoplasmic antibody (p-anca) positivity has been demonstrated in a few patients with ss, most often in the setting of glomerulonephritis. [348] [349] [350] multiple bullae have been reported in some ss patients and are thought to be related to peribronchiolar lymphocytic inflammation and scarring. 35 1.352 thin-walled cysts accompanied by ground-glass opacities may be evident radiographically in ss patients with lip. 353 .354 histologically, such cysts correspond to dilated bronchioles with marked peri bronchiolar lymphocytic inflammation. pulmonary amyloidosis has also been reported in ss patients in association with lip, malt lymphoma, and bullous disease. 22 20.20 . nonspecific interstitial pneumonia (nsip) pattern in sjogren's syndrome. in this example of fibrotic nsip from a patient with sjogren's syndrome, the interstitium is diffusely and homogeneously expanded by mature fibrosis. and as part of a systemic vasculitis, has also been reported. 315 .318 pleural manifestations of ss include pleuritis and pleural effusions. 307.363 management of ss is largely influenced by the presence of other coexisting collagen vascular diseases. morbidity has been dramatically decreased by aggressive antimicrobial treatment of associated pulmonary infec-a k.j. butnor and a. khoor tions.312 small-airway disease in ss is usually steroidresponsive, while in patients with lip, corticosteroids and other immunosuppressive agents have been used with variable success. 314 . 364 ankylosing spondylitis ankylosing spondylitis (as), also known as marie-striimpell disease, is a seronegative spondyloarthropathy that predominantly involves the axial skeleton. in contrast to other collagen vascular disorders, as has a distinct male predominance, with a male-to-female ratio as high as 10:1. 299 ankylosing spondylitis is strongly associated with the hla-b27 histocompatibility antigen. while only 2% to 20% of hla-b27-positive individuals develop as, over 90% of patients with as are positive for hla_b27.36s-369 patients with as typically present in the third decade of life with symptoms referable to spinal arthritis and sacroiliitis. extraarticular manifestations include constitutional symptoms, such as fever and weight loss, as well as cardiovascular disorders, such as conduction defects, aortitis, and aortic insufficiency. pleuropulmonary involvement in as is rare, affecting only 1 % to 2% of patients. 37o involvement of the lungs and pleura typically occurs late in the course of disease and often is asymptomatic.344.371-380 the most common thoracic manifestations of as are upper lobe fibrobullous disease, which may be uni-or bilateral, and chest wall restriction (fig. 20.22 about 10% to 20% of patients with pleuropulmonary involvement suffer spontaneous pneumothorax, likely related to rupture of apical bullae. 379 the cystic changes in the lung apices of as patients are similar to those seen in patients with tuberculosis.379.392 while mycobacteria are not thought to playa role in the development of apical fibrocavitary lesions in as, such lesions may become infected secondarily by mycobacteria or aspergillus, resulting in either saprophytic mycetomas or invasive infection. 299 . 373 .382.388 interstitial fibrosis may occur in as, the pattern of which is nondescript. 376 ,381,383.385 rare cases of honeycomb fibrosis have been described. 3s1 accompanying chronic pleuritis, with or without pleural effusion, is not infrequent. 373 although the cause of pulmonary fibrosis in as is postulated to be immune-mediated, the exact pathogenesis is not yet known. 379 several cases of lung carcinoma have been reported in patients with as. some of these patients had previously received radiation therapy, a modality formerly used in the treatment of as. 393 . 394 survival data indicate that lung cancer mortality in patients previously irradiated for as has increased. 395 however, the frequency of lung cancer in as patients with no history of radiation therapy does not appear elevated? 44 various other pulmonary abnormalities have been reported in as, including bronchiectasis, organizing pneumonia, pulmonary hemorrhage, amyloidosis, and lip ' 373 ,389,396 in addition, the recently described pattern of acute lung injury designated as acute fibrinous and organizing pneumonia (afop) has been reported in a patient with underlying as (see chapter 4).397 many of these manifestations, such as interstitial fibrosis, apical bullae, and bronchiectasis, are readily detectable by hrct. 284 relapsing polychondritis (rp) is a systemic disease characterized by recurrent inflammation of cartilaginous tissue with subsequent degeneration and replacement by fibrosis. [398] [399] [400] [401] [402] [403] prior to the introduction of the term relapsing polychondritis in 1960, cases with features similar to rp were described under the designations "polychondropathia" and "chondromalacia.,,401,404-407 affected sites include the external ear, nose, ribs, larynx, trachea, and major bronchi. 401 .406,408,409 sites rich in glycosaminoglycans, such as the aorta, cornea, and sclera, may also be involved. 41 0--412 some series have shown a slight female predominance, while in others males and females are equally affected. 344 ,403 although rp can occur at any age, the peak reported incidence is in the fifth to seventh decades. 403 while the exact pathogenesis of rp is unknown, an autoimmune origin has been postulated. antibodies to type ii collagen are demonstrable in some patients. 413 granular deposits of immunoglobulin and the c3 component of complement at the chondrofibrous junction have also been reported, as have antibodies to type ii pneumocytes and clara cells.414.41 5 other coexisting collagen vascular diseases, such as rheumatoid arthritis, sle, or ss, are found in up to 25% of cases. 401 involvement of the large airways causes tracheobronchomalacia, which can lead to expiratory obstruction (fig. 20.23 ). 416 airway narrowing can sometimes be so severe as to be life threatening. 417 the pathologic findings in rp vary depending on the phase and extent of disease. in the acute phase, rp is characterized histologically by cartilage erosion, which is accompanied by a dense pericartilaginous inflammatory infiltrate comprised of neutrophils, lymphocytes, plasma cells, and, in some cases, giant cells (fig. 20.24 ). over time, active inflammation gives way to dissolution of the proteoglycan matrix and destruction of cartilage, which is followed in most cases by the formation of granulation tissue. in the resolving phase, damaged cartilage often appears metachromatic. 419 areas of complete destruction undergo fibrosis, which may be so exuberant as to result in stricture formation. 421 in addition to airway involvement, rare instances of parenchymal disease, manifesting radiographically as honeycombing, have been reported. 41s , 422 although laryngotracheal involvement is present in only about half of cases, it is associated with a poor progno-sis. 4111 ,402 management of such patients is aimed at maintaining patency of collapse-prone airways through ventilatory support or tracheostomy with stent placement. corticosteroids and immunosuppressive agents are typically employed in the treatment of rp ' 299 ,370,401,403,423-427 however, the spontaneous remitting and relapsing nature of rp makes it difficult to quantify the therapeutic effect of these agents. pleuropulmonary manifestations have been reported in 1 % to 8% of patients. 436 ,437 the predominant thoracic manifestations of beh<;et's syndrome are either directly related to or represent complications of necrotizing vasculitis (see chapter 29) . other pleuropulmonary disorders associated with beh<;et's syndrome include aneurysms, pulmonary thromboemboli, large airway stenosis and ulceration, pleuritis, pleural effusions, chyloptysis, and organizing pneumonia. 42 .430,432,438-448 acute interstitial pneumonia (alp) has also been reported, typically in the setting of underlying pulmonary hemorrhage. 439 hughes-stovin syndrome, which is characterized by pulmonary artery aneurysms and hemoptysis, most likely represents a limited from of beh<;et's. 443, 449 lung involvement in beh<;et's syndrome is often associated with a poor outcome. one third of beh<;et's patients with lung involvement die as a result of pulmonary complications, most commonly from intractable pulmonary hemorrhage. 429 eosinophilic fasciitis (ef), also known as schulman's syndrome, is a disorder of unknown etiology that predominantly affects patients in their third to sixth decades, which is characterized by painful sclerodermiform induration of the skin.450.451 a variety of extracutaneous manifestations have been reported, including synovitis, and esophageal dysmotility. 450.452.453 pleural effusions, often rich in eosinophils, have also been reported. 453 transbronchial biopsies may show infiltration of the bronchial mucosa by eosinophils. 453 pulmonary parenchymal disease, in the form of interlobular and peribronchial nodular interstitial thickening, has been noted radiographically in rare instances. 453 a case of bronchiolitis obliterans arising in a patient with ef while on penicillamine therapy has been also reported. 48 the features of ef show some overlap with those described in eosinophilic myalgia syndrome (see chapter 15) . 454.455 cutis laxa cutis laxa is an extremely rare systemic disorder of elastic tissue. both familial and acquired forms have been recognized. 456 the skin of patients with cutis laxa is devoid of elastic recoil, which manifests histopathologically as a decrease in the overall number of elastic fibers, as well as degenerative elastic fiber changes. 457 reported pulmonary complications of cutis laxa include emphysema and pulmonary artery stenosis. 45 8-461 ehlers-danlos syndrome ehlers-danlos syndrome is a heterogeneous group of inherited disorders of collagen synthesis and structure characterized by skin hyperextensibility and joint hyper-figure 20.25. ehlers-danlos syndrome. a,b. intraparenchymal fibrous pseudotumor characterized by a nodular proliferation of fibrous tissue. like fibrous scars, these lesions likely mobility. a variety of pulmonary manifestations have been described, including spontaneous or recurrent pneumothorax, bronchial dilation, parenchymal cysts, and arteriovenous anastomoses. 462 ,463 fibrous parenchymal scars, presumably a sequela of spontaneous tears, have been reported, as have fibrous nodules and pseudotumors, which are believed to be an exuberant fibroproliferative response to injury (fig. 20.25) . 464 the fragility of vessel walls in ehlers-danlos syndrome predisposes patients to pulmonary hemorrhage, which in rare cases has resulted in fatal hemoptysis. 465 both ehlers-danlos syndrome and cutis laxa have been associated with mounier-kuhn syndrome, a rare entity characterized by tracheobronchomegaly and trachiectasis (see chapter 5) . 466.467 pseudoxanthoma elasticum pseudoxanthoma elasticum, also known as gronblad-strandberg syndrome, is a heritable degenerative disease of elastic tissue. the etiology of pseudoxanthoma elasticum is not well understood, but mutations in the transmembrane protein abcc6, which is thought to play a role in extracellular matrix deposition and turnover of connective tissue, have been identified. 468 in addition to cutaneous manifestations, which usually become apparent in the second decade of life, other organs, such as the eyes, cardiovascular system, and lungs, may be affected. 469 histologically, the changes observed in the lungs in pseudoxanthoma elasticum are similar to those seen in other sites. 47o on routinely stained sections, elastic tissue within the lungs appears basophilic and disrupted (fig. 20.26 ). the basophilia of elastic fibers in pseudoxanthoma elasticum is due to dystrophic calcification and can be highlighted by von kossa or alizarin red stains.471 marfan's syndrome is a disorder caused by mutations in a gene for the elastin-binding protein fibrillin. while the majority of cases are inherited, about 15% are the result of acquired mutations. 299 ,472 skeletal changes, including thoracic cage deformities such as kyphoscoliosis, ocular lens dislocation, and cardiovascular abnormalities, characterize this disorder. well-recognized pulmonary complications of marfan's syndrome include emphysema, often with apical bulla formation, and spontaneous pneumothorax, which is frequently bilateral and recurrent.460.473-484 for a number of patients, pneumothorax is the initial manifestation of marfan's syndrome. 480 ,482 other pulmonary abnormalities in marfan's syndrome include congenital lobar and airway malformations, diffuse thin-walled cysts, airway hyperreactivity, bronchiectasis, saccular aneurysms of the pulmonary arteries, and respiratory infections, particularly mycobacterial infections.48s-49o a spectrum of interstitial changes, ranging from upper lobe fibrosis to honeycombing, have also been reported. 299 beals-hecht syndrome is another disorder that results from a defect in fibrillin; it is characterized by congenital contractures and external ear deformities. similar to marfan's syndrome, beals-hecht is associated with restrictive k.j. butnor a pattern of interstitial fibrosis similar to uip has been reported in several patients with pbc, most often in the setting of other coexisting autoimmune diseases. sou-s02 other pulmonary disorders that have been described include pulmonary hypertension, alp, lip, chronic interstitial pneumonia, boop, bronchiolitis, including constrictive bronchiolitis, and pulmonary hemorrhage accompanied by glomerulonephritis.so3-51o as noted previously, ss, which itself is associated with an array of pulmonary manifestations, may occur together with pbc. 34s .511 while ulcerative colitis (uc) and crohn's disease (cd) have long been recognized as system disorders, the pulmonary manifestations of idiopathic inflammatory bowel disease (ibd) have only recently been characterized. pulmonary involvement is more common in uc than cd.512 however, in both forms of ibd, the lungs are one of the least frequently affected extraintestinal sites. intestinal manifestations precede pulmonary abnormalities in 72 % to 84% of cases. 513 the interval between the onset of colitis and pulmonary disease is quite variable, ranging from weeks to decades. 514 active colitis is not a prerequisite for the development of lung disease. sis as many as 90% of patients have quiescent bowel disease at the onset of pulmonary symptoms. 513 roentgenographically, pulmonary disease in ibd most typically manifests as diffuse infiltrates, although bilateral germinal centers, which are typically prominent in usual bronchiectasis, are often absent in led-associated chronic bronchitis and bronchiectasis. 513 the overlying mucosa can exhibit squamous metaplasia, and mucous stasis is occasionally prominent. the second most common pattern of pulmonary involvement in led is interstitial lung disease. both nsip and boop patterns have been described. both may be accompanied by interstitial nonnecrotizing granulomas (fig. 20.28 ).528.538.539 a single case of dip in a patient with uc receiving sulfasalazine has also been reported. 540 subtle differences exist between the histologic pattern of boop seen in association with led and cryptogenic organizing pneumonia. 528 ,529,538,541 the former has been reported to show marked septal edema associated with eosinophils, rare cases of eosinophilic pneumonia have also been reported in association with ied.513 in such cases, drug toxicity due to sulfasalazine should be ruled out (see chapter 22) . five percent of led patients with pulmonary involvement exhibit necrobiotic parenchymal nodules. 513 to date, such lesions have only been reported in patients with uc. 513 in the early stages, necrobiotic nodules consist of neutrophils admixed with fibrin. as they mature, these nodules undergo necrosis and cavitation (fig. 20.29) . the histologic appearance of pulmonary necrobiotic nodules in uc is reminiscent of the cutaneous lesions seen in pyoderma gangrenosum. 513 as pulmonary necrobiotic nodules in uc may resemble infectious granulomas, infection should be excluded through histochemical stains and microbiologic cultures. ulcerative colitis-associated necrobiotic nodules have some similarities with lesions in wegener's granulomatosis. however, in contrast to wegener's granulomatosis, giant cells and vasculitis are notably lacking in necrobiotic nodules associated with uc. 513 in considering the differential diagnosis of pulmonary necrobiotic nodules, it is important to bear in mind that a subset of patients with led, in particular those with uc, demonstrate antineutrophil cytoplasmic (anca) antibodies.542.543 however, the antibodies observed in patients with led are perinuclear and directed against mpo antigen (p-anca) rather than the antiproteinase 3 cytoplasmic antibodies (c-anca) associated with wegener's gran uloma to sis. 544 along with infections, led-associated granulomatous inflammation must be distinguished from hypersensitivity pneumonia, sarcoidosis, and drug-induced granulomas. establishing a history of chronic active colitis is paramount. however, this may be difficult in cases where colitis has been inactive for a long period and essentially impossible in the rare instances in which pulmonary abnormalities precede other features of led. obtaining an exposure history is useful to search for an inciting antigen that might suggest hypersensitivity pneumonia. differentiating led-associated granulomatous inflammation from sarcoidosis on histology alone can also be difficult. the recognition of cases of coexisting sarcoidosis and led makes this distinction even more diagnostically challenging. 520 , [545] [546] [547] [548] [549] distinguishing pulmonary abnormalities related to led from drug-induced changes can be quite difficult, given that many patients with led are treated with immunosuppressive agents like sulfasalazine and 5-acetylsalicylic acid (asa). such drugs are known to induce a variety of pulmonary abnormalities, including eosinophilic pneumonia, nsip, boop, and vasculitis. [550] [551] [552] [553] [554] [555] [556] [557] [558] many other thoracic abnormalities have been described in led, including pleuritis, pleural effusions, amyloidosis, pulmonary thromboemboli, and apical fibrosis resembling the findings seen in ankylosing spondylitis. [559] [560] [561] the prognosis of led patients with pulmonary manifestations is good. most patients improve without any specific therapy. neither nonsteroidal agents typically used to control the intestinal manifestations of led nor proctocolectomy appears to ameliorate led-associated respiratory disease. 514 in fact, bowel surgery may exacer-. ' f ' fibd513517536ith bate the respiratory mam estatlons 0 .-" as been suggested that patients who develop pulmonary disease while being treated with sulfasalazine or 5-asa discontinue these medications to decrease the potential for confounding drug-induced abnormalities. 64 refractory cases of led-associated pulmonary disease may d . 'd 513541 respon to corilcosterol s. ' the collagen vascular diseases give rise to a panoply of pleuropulmonary manifestations. while all components of the lung can be affected, some diseases, like rheumatoid arthritis, preferentially involve the interstitium. others, such as sle, predominantly affect the vasculature, while still others, such as rp, principally target the airways. pleuropulmonary manifestations can be the first sign of collagen vascular disease in some cases. it is therefore important to consider collagen vascular disease in cases that show interstitial lung disease on biopsy, particularly when the pattern of involvement does not fit with one of the recognized idiopathic interstitial lung diseases or when other components of the lung are also affected. as pulmonary disease is a significant source of morbidity and mortality in patients with collagen vascular disease and is typically aggressively treated with immunosuppressant or cytotoxic agents, it is essential to correlate histopathologic findings with the clinical and radiographic impression to ensure an accurate diagnosis. pathologisch-anatomische und experimentell-pathologische tatsachen und ihre auswertung fur das anztliche rheumaproblem zur pathologischen anatomie des rhematismus diffuse collagen disease: acute disseminated lupus erythematosus and diffuse scleroderma the concept of collagen diseases is there a common denominator in scleroderma, dermatomyositis, disseminated lupus erythematosus, the libman-sack's syndrome and polyarteritis nodosa? hypersensitivity in disease, with special reference to periarteritis nodosa, rheumatic fever, disseminated lupus erythematosus and rheumatoid arthritis respiratory disease: task force report on problems, research approaches, needs. bethesda, md: national hearth and lung institute collagen vascular disease-related lung disease: high-resolution computed tomography findings based on the pathologic classification pulmonary manifestations of the rheumatic diseases pulmonary complications of collagen vascular disease histopathological findings of the lung in collagen diseases-especially on their differential diagnosis pulmonary manifestations of the collagen vascular diseases can interstitial pneumonia as the sole presentation of collagen vascular diseases be differentiated from idiopathic interstitial pneumonia? usual interstitial pneumonia: idiopathic pulmonary fibrosis versus collagen vascular diseases histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome fibroblastic foci in usual interstitial pneumonia: idiopathic versus collagen vascular disease interstitial pulmonary fibrosis with and without associated collagen vascular disease: results of a two year follow up interstitial lung disease in polymyositis and dermatomyositis. clinical features and prognosis as correlated with histologic findings open lung biopsy of patients with rheumatoid arthritis lung biopsy in rheumatoid arthritis structural features of interstitial lung disease in systemic sclerosis variations in histological patterns of interstitial pneumonia between connective tissue disorders and their relationship to prognosis pulmonary pathology in association with connective tissue disorders nonspecific interstitial pneumonia in collagen vascular diseases: comparison of the clinical characteristics and prognostic significance with usual interstitial pneumonia non-specific interstitial pneumonia as pulmonary involvement of systemic sclerosis the major histopathologic pattern of pulmonary fibrosis in scleroderma is nonspecific interstitial pneumonia polymyositis-dermatomyositis-associated interstitial lung disease idiopathic nonspecific interstitial pneumonia: prognostic significance of cellular and fibrosing patterns: survival comparison with usual interstitial pneumonia and desquamative interstitial pneumonia comparison of transbronchial and open biopsies in chronic infiltrative lung diseases step section preparation of trans bronchial lung biopsy. significance in the diagnosis of diffuse lung disease bilateral bronchoalveolar lavage in the diagnosis of opportunistic pulmonary infections the impact of bronchoalveolar lavage cell analysis on clinicians' diagnostic reasoning about interstitial lung disease this joint statement of the open biopsy for chronic diffuse infiltrative lung disease: clinical, roentgenographic, and physiological correlations in 502 patients the virtual disappearance of rheumatic fever in the united states: lessons in the rise and fall of disease. t. duckett jones memorial lecture pulmonary manifestations in the diffuse collagen diseases rheumatic pneumonitis pulmonary vascular changes associated with isolated mitral stenosis in india chronic rheumatic heart disease in india: a reappraisal of pathologic changes a treatise on gout and rheumatic gout the five manifestations of rheumatoid lung pulmonary involvement in the collagen vascular diseases interstitial pneumonia and its consequences in rheumatoid disease pulmonary involvement in rheumatoid arthritis rheumatoid arthritis in spain: occurrence of extra-articular bronchocentric granulomatosis associated with rheumatoid arthritis update on lymphoid interstitial pneumonitis connective tissue and inflammatory bowel diseases. in: non-neoplastic disorders of the lower respiratory tract. first series. washington, dc: american registry of pathology and the armed forces institute of pathology a clinicopathologic study of 34 cases of diffuse pulmonary hemorrhage with lung biopsy confirmation isolated pulmonary capillaritis and diffuse alveolar hemorrhage in rheumatoid arthritis and mixed connective tissue disease interstitial lung disease in recent onset rheumatoid arthritis pulmonary fibrosis in rheumatoid arthritis: a review of clinical features and therapy rheumatoid arthritisrelated lung diseases: ct findings nonspecific interstitial pneumonia/fibrosis. histologic features and clinical significance clinical features of non-specific interstitial pneumonia bronchiolitis obliterans organizing pneumonia and rheumatoid arthritis bronchiolitis obliterans organizing pneumonia in a patient with rheumatoid arthritis rheumatoid arthritis and cryptogenic organising pneumonitis lymphocytic interstitial pneumonitis. review of 13 cases interstitial lung disease in rheumatoid arthritis: assessment with high-resolution computed tomography high resolution computed tomography of the lungs in patients with rheumatoid arthritis and interstitial lung disease rapidly fatal pulmonary fibrosis: the accelerated variant of interstitial pneumonitis case records of the massachusetts general hospital. weekly clinicopathological exercises. case 32-2001. a 77-year-old man with rheumatoid arthritis and acute dyspnea and renal failure the value of serial bronchoalveolar lavages in assessing the clinical progress of patients with cryptogenic fibrosing alveolitis lower respiratory tract abnormalities in rheumatoid interstitial lung disease. potential role of neutrophils in lung injury pulmonary and pleural lesions in rheumatoid disease the triad of pneumonitis, pleuritis, and pericarditis in juvenile rheumatoid arthritis pulmonary manifestations of juvenile rheumatoid arthritis. a report of eight cases and review chronic diffuse lung diseases course and prognosis in rheumatoid arthritis. a further report the extra-articular features of rheumatoid arthritis. a systematic analysis of 127 cases subcutaneous nodules and the differentiation of rheumatoid arthritis from rheumatic fever pulmonary lesions and rheumatoid arthritis the lungs in rheumatoid arthritis prevalence of pulmonary involvement in rheumatoid arthritis and its relationship to some characteristics of the patients. a radiological and clinical study pulmonary necrobiotic nodules without rheumatoid arthritis pulmonary necrobiotic nodules as a presenting feature of rheumatoid arthritis multiple peripheral pulmonary nodules preceding rheumatoid arthritis pulmonary nodules in rheumatoid arthritis rheumatoid nodules. review of the spectrum of associated conditions and proposal of a new classification, with a report of four seronegative cases pulmonary nodules in rheumatoid disease: a report of two cases endobronchial necrobiotic nodule antedating rheumatoid arthritis nodular pulmonary opacities in patients with rheumatoid arthritis. a diagnostic dilemma pulmonary adenocarcinomas associated with rheumatoid nodules: a case report and review of the literature pleuropulmonary manifestations of rheumatoid arthritis widespread serous membrane involvement by rheumatoid nodules pleuropulmonary necrobiotic rheumatoid nodules. a review and clinicopathological study of six patients the rheumatoid nodule fine-needle aspiration cytology of pulmonary rheumatoid nodule: case report and review of the major cytologic features rheumatoid disease of the lung with cavitation darling ri pulmonary rheumatoid nodule with cavitation and chronic lipid effusion cavitation of a rheumatoid lung nodule apical fibrocavitary lesions of the lung in rheumatoid arthritis rheumatoid lung disease, pneumothorax, and eosinophilia necrobiotic pulmonary nodule leading to pyopneumothorax in a case of rheumatoid arthritis-a rare clinical presentation aspergillus colonization of pulmonary rheumatoid nodule fungus colonisation of pulmonary rheumatoid nodule certain unusual radiological appearances in the chest of coal-miners suffering from rheumatoid arthritis a broader concept of caplan's syndrome related to rheumatoid factors rheumatoid pneumoconiosis. documentation of onset and pathogenic considerations pulmonary connective tissue and environmental lung disease caplan's syndrome computed tomography findings of caplan syndrome pneumoconiosis" (caplan's syndrome) in an asbestos worker: a 17 years' follow-up rheumatoid pneumocomosls (caplan's syndrome) in an asbestos worker respiratory manifestations of rheumatoid arthritis systemic rheumatoid vasculitis: a clinical and laboratory study of 50 cases rheumatoid vasculitis: survival and associated risk factors immunofluorescent and immunologic studies of rheumatoid lung case records of the massachusetts general hospital. weekly clinicopathological exercises. case 3-1982. an elderly woman with mild rheumatoid arthritis and progressive pulmonary fibrosis clinical and prognostic significance of vasculitis as an early manifestation of connective tissue disease syndromes unexplained pulmonary hypertension with pulmonary arteritis in rheumatoid disease acute respiratory failure and pulmonary arteritis without parenchymal involvement: demonstration in a patient with rheumatoid arthritis igm rheumatoid factor and low molecular weight igm. an association with vasculitis rheumatoid factor and serum igg, igm and iga levels in rheumatoid arthritis with vasculitis incidence, target antigens, and clinical implications of antineutrophil cytoplasmic antibodies in rheumatoid arthritis pulmonary hypertension in rheumatoid arthritis pulmonary hypertension in a patient with rheumatoid arthritis the association of pulmonary hypertension with rheumatoid arthritis fatal pulmonary hypertension and rheumatoid vasculitis pulmonary hypertension in rheumatoid arthritis: report of a case with intimal sclerosis of the pulmonary and digital arteries report of a case in association with rheumatoid arthritis localised pulmonary arteritis in rheumatoid disease interstitial rheumatoid lung disease. a reassessment and review of the literature pulmonary alveolar proteinosis: report of two cases and comments on its pathogenesis pulmonary involvement in patients with rheumatoid arthritis risk of cancer in patients with scleroderma: a population based cohort study the lungs in rheumatoid arthritis-a clinical, radiographic and pulmonary function study pleuritis as a presenting manifestation of rheumatoid arthritis: diagnostic clues in pleural fluid cytology thoracoscopic, histological, and clinical findings in nine case of rheumatoid pleural effusion rheumatoid pleurisy and pericarditis characteristic and specific histological findings in rheumatoid pleurisy the pathognomonic cytologic picture of rheumatoid pleuritis. the 1989 maurice goldblatt cytology award lecture chemical and immunological features of pleural effusions: comparison between rheumatoid arthritis and other diseases decreased glucose in ra-cell-positive pleural effusion: correlation of pleural glucose, lactic dehydrogenase and protein concentration to the presence of racells pulmonary involvement in rheumatoid arthritis rheumatoid pleurisy. observations on the development of low pleural fluid ph and glucose level pulmonary and pleural lesions in rheumatoid disease pulmonary function in juvenile rheumatoid arthritis pulmonary lesions in the course of rheumatoid arthritis in children rheumatoid disease of the lung and cor pulmonale primary pulmonary hypertension in a patient with systemic-onset juvenile arthritis lymphocytic interstitial pneumonitis preceding polyarticular juvenile rheumatoid arthritis early onset of pulmonary parenchymal disease associated with juvenile rheumatoid arthritis fatal bronchiolitis obliterans in a patient with juvenile rheumatoid arthritis receiving chrysotherapy lymphoid interstitial pneumonia in juvenile rheumatoid arthritis respiratory manifestations of connective tissue disease acute pulmonary complications in systemic lupus erythematosus. immunofluorescence and light microscopic study pulmonary manifestations of systemic lupus erythematosus pulmonary alterations in systemic lupus erythematosus clinical manifestations of systemic lupus erythematosus diffuse interstitial lung disease in systemic lupus erythematosus the lung in systemic lupus erythematosus. analysis of the pathologic changes in 120 patients immunopathologic and clinical studies in pulmonary hypertension associated with systemic lupus erythematosus pleuro-pulmonary manifestations of systemic lupus erythematosus: clinical features of its subgroups. prognostic and therapeutic implications the pulmonary manifestations of systemic lupus erythematosus a long-term study of interstitial lung disease in systemic lupus erythematosus systemic lupus erythematosus: review of the literature and clinical analysis of 138 cases bronchiolitis in systemic lupus erythematosus diffuse alveolar hemorrhage and systemic lupus erythematosus. clinical presentation, histology, survival, and outcome pulmonary hemorrhage in systemic lupus erythematosus the lungs and connective tissue diseases acute massive pulmonary hemorrhage in systemic lupus erythematosus microangiitis in lupus-induced pulmonary hemorrhage pulmonary hemorrhage and immune-complex deposition in the lung. complications in a patient with systemic lupus erythematosus tubuloreticular inclusions in systemic lupus pneumonitis pulmonary manifestations of systemic lupus erythematosus: review of twelve cases of acute lupus pneumonitis severe, acute pulmonary disease in patients with systemic lupus erythematosus: ten years of experience at the national institutes of health pulmonary hypertension in systemic lupus erythematosus pulmonary vascular disease in systemic lupus erythematosus pulmonary hypertension and systemic lupus erythematosus an autopsied case of systemic lupus erythematosus with pulmonary hypertension-a case report plexogenic arteriopathy associated with pulmonary vasculitis in systemic lupus erythematosus vascular lesions in systemic lupus erythematosus (sle) with pulmonary hypertension pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions a fatal pulmonary complication of lupus in pregnancy endothelin-l release from cultured endothelial cells induced by sera from patients with systemic lupus erythematosus antiendothelial cell antibodies and their relation to pulmonary hypertension in systemic lupus erythematosus rauch 1. the antiphospholipid syndrome antiphospholipid antibodies and the antiphospholipid syndrome in systemic lupus erythematosus. a prospective analysis of 500 consecutive patients review: antiphospholipid antibodies and the lung the interstitial lung diseases associated with the collagen-vascular disorders unexplained" dyspnoea and shrinking lungs in systemic lupus erythematosus pulmonary dysfunction in systemic lupus erythematosus without pulmonary symptoms shrinking lung syndrome in sle-a clinical pathologic study respiratory muscle dysfunction in systemic lupus erythematosus inspiratory muscle dysfunction and unexplained dyspnea in systemic lupus erythematosus serial pulmonary function testing in patients with systemic lupus erythematosus presentation and prognosis of the shrinking lung syndrome in systemic lupus erythematosus bilateral phrenic paralysis in a patient with systemic lupus erythematosus diaphragm strength in the shrinking lung syndrome of systemic lupus erythematosus systemic lupus erythematosus and lymphoma non-hodgkin's lymphoma and other cancers among a cohort of patients with systemic lupus erythematosus increased cancer incidence in a swedish cohort of patients with systemic lupus erythematosus kaposi's sarcoma in a patient with sle risk of malignancy in an unselected cohort of icelandic patients with systemic lupus erythematosus bronchiolitis obliterans organizing pneumonia associated with systemic lupus erythematosus the association of bronchiolitis obliterans organizing pneumonia, systemic lupus erythematosus, and hunner's cystitis the association of lymphocytic interstitial pneumonia and systemic lupus erythematosus pulmonary involvement in systemic sclerosis (scleroderma) predictors of survival in systemic sclerosis (scleroderma) mortality and causes of death in a swedish series of systemic sclerosis patients ct features of lung disease in patients with systemic sclerosis: comparison with idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia pathologic observations in systemic sclerosis (scleroderma). a study of fiftyeight autopsy cases and fifty-eight matched controls pulmonary hypertension in the crest syndrome variant of progressive systemic sclerosis (scleroderma) prevalence and clinical correlates of pulmonary arterial hypertension in progressive systemic sclerosis pulmonary hypertension in the crest syndrome variant of systemic sclerosis pulmonary hypertension in systemic sclerosis pulmonary arterial hypertension in progressive systemic sclerosis prevalence and outcome in systemic sclerosis associated pulmonary arterial hypertension: application of a registry approach age and risk of pulmonary arterial hypertension in scleroderma predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited cutaneous involvement pulmonary vascular lesions in systemic scleroderma pulmonary scleroderma pulmonary vascular changes in scleroderma pathogenesis and evolution of plexiform lesions in pulmonary hypertension associated with scleroderma and human immunodeficiency virus infection the pulmonary pathologic manifestations of the crest syndrome pulmonary venoocclusive disease and the crest variant of scleroderma endothelial injury in scleroderma. a protease mechanism leroy ec endothelial injury in scleroderma circulating endothelial cells as a marker of ongoing vascular disease in systemic sclerosis a new era in the management of pulmonary arterial hypertension related to scleroderma: endothelin receptor antagonism progressive systemic sclerosis complicated by diffuse pulmonary haemorrhage endobronchial telangiectasias and hemoptysis in scleroderma bronchiolitis obliterans organizing pneumonia and scleroderma recurrent bronchiolitis obliterans organizing pneumonia in a patient with limited cutaneous systemic sclerosis bronchioloalveolar carcinoma and generalized amyloidosis complicating progressive systemic sclerosis recurrent spontaneous pneumothorax in diffuse scleroderma progressive generalized scleroderma: respiratory failure from primary chest wall involvement reversible hypercapnic respiratory insufficiency in scleroderma caused by respiratory muscle weakness carcinoma of the lung in progressive systemic sclerosis: a tabular review of the literature and a detailed report of the roentgenographic changes in two cases incidence of lung cancer in systemic sclerosis cancer and systemic sclerosis. an epidemiologic study bronchioloalveolar carcinoma in progressive systemic sclerosis incidence of cancer among patients with systemic sclerosis polymyositis and dermatomyositis (second of two parts) the epidemiology of polymyositis incidence of polymyositis-dermatomyositis: a 20-year study of hospital diagnosed cases in allegheny county, pa 1963-1982 interstitial lung disease in polymyositis and dermatomyositis interstitial lung disease in polymyositis and dermatomyositis: clinical course and response to treatment interstitial lung disease, a common manifestation of newly diagnosed polymyositis and dermatomyositis interstitial lung disease in polymyositis and dermatomyositis: analysis of six cases and review of the literature polymyositis and interstitial lung disease fibrosing alveoli tis in polymyositis. a review of histologically confirmed cases pulmonary disease in polymyositis/dermatomyositis: a clinicopathological analysis of 65 autopsy cases mathews who interstitial pneumonitis in dermatomyositis idiopathic inflammatory myopathy with diffuse alveolar damage pulmonary hypertension in polymyositis mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ena) symptomatic sjogren's syndrome in mixed connective tissue disease maddison pi overlap syndromes and mixed connective tissue disease direct and indirect immunofluorescence as a diagnostic adjunct in the interpretation of nonneoplastic medical lung disease anti-endothelial cell antibodies in the sera of patients with mixed connective tissue disease-the clinical significance mixed connective tissue disease with pulmonary hypertension: a clinical and pathological study a prospective evaluation emphasizing pulmonary involvement in patients with mixed connective tissue disease mixed connective tissue disease-clinical and radiological aspects of 20 cases severe pulmonary involvement in mixed connective tissue disease pulmonary involvement in mixed connective tissue disease intrathoracic manifestations in mixed connective tissue disease massive pulmonary hemorrhage in mixed connective tissue diseases mixed connective tissue disease: a clinicopathologic study of 20 cases respiratory complications in mixed connective tissue disease lungs in mixed connective tissue disease subclinical pulmonary involvement assessed by bronchoalveolar lavage in patients with early undifferentiated connective tissue disease interstitial lung diseases associated with collagen vascular diseases: radiologic and histopathologic findings fatal pulmonary hypertension and resolving immune-complex glomerulonephritis in mixed connective tissue disease. a case report and review of the literature mixed connective tissue disease with fatal pulmonary hypertension and a review of literature pulmonary vascular reactivity in severe pulmonary hypertension associated with mixed connective tissue disease increased serum angiotensin i-converting enzyme activity in patients with mixed connective tissue disease and pulmonary hypertension pulmonary hypertension in a child with mixed connective tissue disease pulmonary hypertension complicating mixed connective tissue disease mixed connective tissue disease with severe pulmonary hypertension and extensive subcutaneous calcification pulmonary hemorrhage and acute renal failure in a patient with mixed connective tissue disease fatal alveolar hemorrhage in a patient with mixed connective tissue disease presenting polymyositis features amyloidosis in mixed connective tissue disease bilateral exudative pleuritis, an unusual pulmonary onset of mixed connective tissue disease diaphragm dysfunction in mixed connective tissue disease. a case report pulmonary and neuromuscular complications of mixed connective tissue disease: a report and review of the literature pulmonary involvement in mixed connective tissue disease: comparison with other collagen vascular diseases using high resolution ct pulmonary involvement in collagen vascular disease: a review of the pulmonary manifestations of the marfan syndrome, ankylosing spondylitis, sjogren's syndrome, and relapsing polychondritis sjogren-type syndrome after allogeneic bone-marrow transplantation diffuse infiltrative lymphocytosis syndrome in children and adults infected with hiv-l: a model of rheumatic illness caused by acquired viral infection primary sjogren's syndrome and other autoimmune diseases in families. prevalence and immunogenetic studies in six kindreds sjogren's syndrome. a review with emphasis on immunological features pulmonary manifestations of sjogren's syndrome pulmonary disorders associated with sjogren's syndrome predictive value of ss-b precipitating antibodies in sjogren's syndrome pulmonary involvement in primary sjogren's syndrome pulmonary manifestations of sjogren's syndrome reappraisal of respiratory abnormalities in primary and secondary sjogren's syndrome. a controlled study respiratory manifestations in primary sjogren's syndrome. a clinical, functional, and histologic study sjogren's syndrome. a clinical, pathological, and serological study of sixty-two cases pulmonary and gastrointestinal manifestations of sjogren's syndrome alveolitis correlates with clinical pulmonary involvement in primary sjogren's syndrome pleuropulmonary abnormalities in primary sjogren's syndrome primary sjogren syndrome: clinical and immunopathologic features interstitial lung disease and primary sjogren's syndrome: clinical-pathological evaluation and response to treatment interstitial lung disease in primary sjogren's syndrome. clinicalpathological evaluation and response to treatment sjogren's syndrome: report of a fatal case with pulmonary and renal lesions xerotrachea and interstitial lung disease in primary sjogren's syndrome pulmonary involvement in sjogren's syndrome chronic obstructive airway disease in patients with sjogren's syndrome bronchiolitis obliterans organizing pneumonia and sjogren's syndrome usual interstitial pneumonia associated with primary sjogren's syndrome a case of bronchiolitis obliterans organizing pneumonia associated with primary sjogren's syndrome who died of superimposed diffuse alveolar damage primary sjogren's syndrome and associated lung disease: ct findings in 50 patients the spectrum of benign to malignant lymphoproliferation in sjogren's syndrome lymphomatoid granulomatosis of the lung, associated with a long history of benign lymphoepithelial lesions of the salivary glands and lymphoid interstitial pneumonitis pulmonary lymphoma in sjogren's syndrome sjogren's syndrome and fibrosing alveolitis complicated by pulmonary lymphoma lymphoid infiltration and amyloidosis of lung in sjogren's syndrome immunologically diagnosed malignancy in sjogren's pseudolymphoma primary sjogren's syndrome with diffuse cerebral vasculitis and lymphocytic interstitial pneumonitis lymphoid interstitial pneumonia: clinicopathological and immunopathological findings in 18 cases bronchopulmonary manifestations of sjogren's syndrome: the findings of the bal, tblb, and bronchoscopy pulmonary pseudolymphoma presented with a mass lesion in a patient with primary sjogren's syndrome: beneficial effect of intermittent intravenous cyclophosphamide low-grade lymphoma of immature t-cell phenotype in a case of lymphocytic interstitial pneumonia and sjogren's syndrome diffuse pulmonary lymphoreticular infiltrations associated with dysproteinemia lymphomatoid granulomatosis (liebow) pulmonary polymorphic centroblastic type malignant lymphoma in a patient with lymphomatoid granulomatosis, sjogren syndrome and other manifestations of a dysimmune state lymphomas in patients with sjogren's syndrome: review of the literature and physiopathologic hypothesis increased risk of lymphoma in sicca syndrome lymphomas in patients with sjogren's syndrome are marginal zone b-cell neoplasms, arise in diverse extranodal and nodal sites, and are not associated with viruses pathology and genetics of tumours of haematopoietic and lymphoid tissues ferrans vi the lung in connective tissue disorders pulmonary involvement in primary biliary cirrhosis association of sjogren's syndrome and sarcoidosis coexisting sjogren's syndrome and sarcoidosis in the lung ancaassociated pauci-immune crescentic glomerulonephritis complicating sjogren's syndrome sjogren's syndrome complicated by mpo-anca positive crescentic glomerulonephritis sjogren's syndrome, cavitating lung disease and high sustained levels of antibodies to serine proteinase 3 sjogren's syndrome with bronchial gland involvement and multiple bullae sjogren's syndrome with multiple bullae and pulmonary nodular amyloidosis lung cyst formation in lymphocytic interstitial pneumonia: ct features lymphocytic interstitial pneumonia: thin-section ct findings in 22 patients benign pulmonary lymphocytic infiltration and amyloidosis: computed tomographic and pathologic features in three cases ravin ceo nodular pulmonary infiltrate in a patient with sjogren's syndrome pulmonary nodular amyloidosis presenting as sjogren's syndrome nodular amyloidosis of the lungs associated with sjogren's syndrome pulmonary malt lymphoma with amyloid production in a patient with primary sjogren's syndrome pulmonary hypertension in primary sjogren's syndrome association of sjogren's syndrome with pulmonary hypertension: report of two cases and review of the literature the association of sjogren's syndrome and primary pulmonary hypertension sjogren's syndrome with pleural effusion pulmonary findings in patients with primary sjogren's syndrome clinical history as a screening test for ankylosing spondylitis the immunopathology of ankylosing spondylitis-a review the risk of developing ankylosing spondylitis in hla-b27 positive individuals. a comparison of relatives of spondylitis patients with the general population the risk of developing ankylosing spondylitis in hla-b27 positive individuals: a family and population study high association of an hl-a antigen, w27, with ankylosing spondylitis pulmonary manifestations of ankylosing spondylitis and relapsing polychondritis spondylarthritis ankylopoietica: a review and report of twenty cases ankylosing spondylitis lung disease-an under diagnosed entity? pleuropulmonary manifestations of ankylosing spondylitis upper lobe fibrosis associated with ankylosing spondylitis ankylosing spondylitis and upper lobe fibrosis and cavitation ankylosing spondylitis and lung fibrosis fibrocavitating pulmonary lesions in ankylosing spondylitis fibrobullous disease of the upper lobes: an extra skeletal manifestation of ankylosing spondylitis the pleuropulmonary manifestations of ankylosing spondylitis pulmonary involvement in ankylosing spondylitis fibrosing interstitial pneumonitis in ankylosing spondylitis pulmonary fibrosis, pulmonary tuberculosis and ankylosing spondylitis pulmonary manifestations of ankylosing spondylitis the chest radiograph in ankylosing spondylitis upper lobe fibrosis in ankylosing spondylitis pulmonary cystic disease in ankylosing spondylitis: two cases with unusual superinfection bronchoalveolar lavage and trans bronchial biopsy in spondyloarthropathies ankylosing spondylitis lung disease and mycobacterium scrofulaceum pulmonary diffuse amyloidosis and ankylosing spondylitis. a rare association johnston who apical pulmonary disease in ankylosing spondylitis simultaneous presentation of upper lobe fibrobullous disease and spinal pseudarthrosis in a patient with ankylosing spondylitis pulmonary fibrosis, mimicking tuberculosis, presenting before joint symptoms in a female with ankylosing spondylitis ankylosing spondylitis with fibrosis and carcinoma of the lung ankylosing spondylitis and adenocarcinoma of the lung cancer mortality following x-ray treatment for ankylosing spondylitis pulmonary hemorrhage associated with ankylosing spondylitis acute fibrinous and organizing pneumonia: a histological pattern of lung injury and possible variant of diffuse alveolar damage relapsing polychondritis respiratory complications of relapsing polychondritis relapsing polychondritis: prospective study of 23 patients and a review of the literature relapsing polychondritis. survival and predictive role of early disease manifestations airway complications in relapsing polychondritis uber einen fall von systematiseirter chondromalacie relapsing polychondritis. three cases with a clinico-pathological study and literature review relapsing polychondritis. clinical and pathologic features in fourteen cases report of two necropsied cases with histochemical investigation of the cartilage lesion relapsing polychondritis. analytical literature review and studies on pathogenesis relapsing polychondritis: review of current status and case report relapsing polychondritis: an autoimmune disease autoantibodies to cartilage and type ii collagen in relapsing polychondritis and other rheumatic diseases relapsing polychondritis. immunomicroscopic findings in cartilage of ear biopsy specimens relapsing poly(peri)chondritis associated with fibrosing alveolar disease and antibodies to pneumocytes type ii and clara cells pulmonary function in relapsing polychondritis bronchial disease in ulcerative colitis tracheal stenosis due to relapsing polychondritis in rheumatoid arthritis fatal bronchial stenosis due to isolated relapsing chondritis relapsing polychondritis. a clinical, pathologic-anatomic and histochemical study of 2 cases endobronchial stenting for severe airway obstruction in relapsing polychondritis pulmonary fibrosis with intractable pneumothorax: new pulmonary manifestation of relapsing polychondritis cyclosporin a treatment in a case of relapsing polychondritis progressive tracheobronchial polychondritis: need for early diagnosis relapsing polychondritis acute airway obstruction in relapsing polychondritis: treatment with pulse methylprednisolone relapsing polychondritis: new pulmonary manifestations pulmonary involvement in behcet's syndrome pulmonary manifestations in behcet's syndrome pulmonary disease in behcet's syndrome pulmonary involvement in behcet disease behcet's disease. report of 41 cases and a review of the literature pulmonary manifestations in behcet syndrome. case report and review of the literature hla-b5 and behcet's disease a study of familial occurrence of behcet's disease with and without ocular lesions pulmonary manifestations of behcet's disease pulmonary arterial aneurysms in behcet's syndrome: a report of 24 cases pulmonary vasculitis acute interstitial pneumonia in a patient with behcet's syndrome and common variable immunodeficiency behcet's syndrome with haemoptysis and pulmonary lesions pathology of the lung in behcet's disease. case report and review of the literature vascular involvement in behcet's disease: arterial and venous and vessels of all sizes possible behcet's disease revealed by pulmonary aneurysms large bilateral pulmonary artery aneurysms in behcet's disease: rupture of the contralateral lesion after aneurysmorrhaphy behcet's disease with pulmonary involvement, superior vena cava syndrome, chyloptysis and chylous ascites organizing pneumonia associated with pulmonary artery aneurysms in behcet's disease pulmonary complications of behcet's disease behcet's syndrome with pulmonary involvement-combined therapy for endobronchial stenosis using neodymium-yag laser, balloon dilation and immunosuppression pulmonary artery aneurysms with recurrent thrombophlebitis. the "hughes-stovin syndrome eosinophilic fasciitis report of six cases of a newly recognized scleroderma-like syndrome eosinophilic fasciitis: extracutaneous manifestations and associations eosinophilic fasciitis with pulmonary and pleural involvement fasciitis, and eosinophilia associated with the ingestion of tryptophan mixed connective tissue disease, overlap syndromes, and eosinophilic fasciitis genetic disorders of the elastic fiber system report of two cases and review of the literature cutis laxa (generalized elastolysis). a report of four cases with autopsy findings congenital cutis laxa with a dominant inheritance and early onset emphysema mottiaz 1. pulmonary elastic tissue in generalized elastolysis (cutis laxa) and marfan's syndrome: a light and electron microscopic study emphysema and cutis laxa abnormalities of the lungs and thoracic cage in the ehlers-danlos syndrome rare pulmonary manifestation of ehlers-danlos syndrome fibrous pseudotumours and cyst formation in the lungs in ehlers-danlos syndrome fatal hemoptysis in ehlers-danlos syndrome. old malady with a new curse tracheobronchiomegaly and acquired cutis lax a in a child. physiologic and immunologic studies pseudoxanthoma elasticum: a clinical, histopathological, and molecular update pseudoxanthoma elasticum: an update pulmonary calcification and elastic tissue damage in pseudoxanthoma elasticum clinical pathologic observations in pseudoxanthoma elasticum the marfan syndrome: diagnosis and management pulmonary emphysema in a neonate with the marfan syndrome pulmonary emphysema in a neonate with marfan syndrome pulmonary emphysema in a neonate with marfan syndrome elastic tissue in pulmonary emphysema in marfan syndrome pulmonary bullous disease in marfan syndrome fragile lung in the marfan syndrome familial multiple bilateral pneumothorax associated with marfan syndrome pneumothorax in the marfan syndrome: prevalence and therapy marfan's syndrome and spontaneous pneumothorax marfan's syndrome presenting as bilateral spontaneous pneumothorax report of a case and review of the pulmonary abnormalities in this disorder pulmonary disease in patients with marfan syndrome cystic lung in marfan's syndrome bronchial hyperreactivity in children with marfan syndrome bronchiectasis and marfan's syndrome pulmonary arterial aneurysms in marfan's syndrome pulmonary tuberculosis in marfan's syndrome bronchiectasis and spontaneous pneumothorax in marfan's syndrome beals-hecht syndrome antimitochondrial antibodies in primary biliary cirrhosis autoimmune associations in primary biliary cirrhosis primary biliary cirrhosis: lung involvement quantitation of hepatic granulomas and epithelioid cells in primary biliary cirrhosis sarcoidosis and primary biliary cirrhosis. associated disorders? sarcoidosis and primary biliary cirrhosis. literature review and illustrative case primary biliary cirrhosis. subclinical inflammatory alveolitis in patients with normal chest roentgenograms immunological features of lung lavage cells from patients with primary biliary cirrhosis may reflect those seen in pulmonary sarcoidosis pulmonary involvement in primary biliary cirrhosis multisystem involvement in chronic liver disease. studies on the incidence and pathogenesis primary biliary cirrhosis with fibrosing alveolitis acute interstitial pneumonia (fibrosing alveolitis) in a patient with primary biliary cirrhosis pulmonary hypertension associated with primary biliary cirrhosis in the absence of portal hypertension: a case report interstitial lung disease in primary biliary cirrhosis lymphocytic alveolitis associated with asymptomatic primary biliary cirrhosis pulmonary hemorrhage and glomerulonephritis in primary biliary cirrhosis bronchiolitis obliterans organising pneumonia and primary biliary cirrhosislike lung involvement in a patient with primary biliary cirrhosis case records of the massachusetts general hospital. weekly clinicopathological exercises. case 14-1998. a 49-year-old woman with primary biliary cirrhosis, pulmonary opacities, and a pleural effusion lymphocytic bronchitis/bronchiolitis in a patient with primary biliary cirrhosis sjogren's syndrome in patients with primary biliary cirrhosis pulmonary manifestations of gastrointestinal disease the lung in inflammatory bowel disease the lung in inflammatory bowel disease pulmonary complications in patients with inflammatory bowel disease inflammatory airways disease in ulcerative colitis: ct and high-resolution ct features chronic bronchial suppuration and inflammatory bowel disease airway involvement in ulcerative colitis pulmonary involvement in ulcerative colitis unexplained bronchopulmonary disease with inflammatory bowel disease severe inflammatory upper airway stenosis in ulcerative colitis ulcerative tracheobronchitis years after colectomy for ulcerative colitis crohn's disease with respiratory tract involvement bronchiectasis with ulcerative colitis and myelopathy noninfectious lung pathology in patients with crohn's disease constrictive bronchiolitis and ulcerative colitis diffuse panbronchiolit is preceding ulcerative colitis an unusual perilobular pattern of pulmonary interstitial fibrosis associated with crohn's disease are there characteristic alterations in lung tissue associated with crohn's disease? commentary: bronchiectasis and inflammatory bowel disease crohn's disease with pulmonary involvement in a 3 year old boy crohn's disease of the lung white hi inflammatory bowel disease associated with granulomatous lung disease: report of a case with endoscopic findings granulomatous bronchiolitis associated with crohn's disease severe pulmonary disease in association with crohn's disease in a 13-year-old girl bronchopulmonary involvement in ulcerative colitis early bronchopulmonary involvement in crohn disease: a case report nongranulomatous interstitial lung disease in crohn's disease cobden 1, et al. bronchiolitis obliterans organising pneumonia in a patient with ulcerative colitis desquamative interstitial pneumonitis complicating inflammatory bowel disease of childhood steroid-responsive alveolitis associated with ulcerative colitis autoantibodies to neutrophil cytoplasmic (anca) and endothelial cell surface antigens (aeca) in chronic inflammatory bowel disease pulmonary manifestations of ulcerative colitis mimicking wegener's granulomatosis are anti-neutrophil cytoplasmic antibodies (anca) clinically useful in inflammatory bowel disease (ibd)? sarcoidosis associated with regional enteritis (crohn's disease) familial occurrence of sarcoidosis and crohn's disease regional enteritis and sarcoidosis in one patient. a case report crohn's disease and sarcoidosis: systemic granulomatosis? association between inflammatory bowel disease and sarcoidosis. report of two cases and review of the literature pulmonary infiltrates and skin pigmentation associated with sulfasalazine pulmonary vasculitis-an uncommon complication of ulcerative colitis salazopyrininduced eosinophilic pneumonia fibrosing alveoli tis, bronchiolitis obliterans, and sulfasalazine therapy mesalamineinduced hypersensitivity pneumonitis. a case report and review of the literature mesalazineinduced eosinophilic pneumonia sulfasalazine pulmonary toxicity in ulcerative colitis mimicking clinical features of wegener's granulomatosis pulmonary vasculitis and ulcerative colitis bronchiolitis obliterans in a patient with ulcerative colitis receiving mesal amine pulmonary nodules due to reactive systemic amyloidosis (aa) in crohn's disease meadway 1. ulcerative colitis, coli tic spondylitis and associated apical pulmonary fibrosis arterial thromboembolic complications of inflammatory bowel disease key: cord-0023087-n4lffmo1 authors: khan, tanjila; jose, ricardo j.; renzoni, elisabetta a.; mouyis, maria title: a closer look at the role of anti-ccp antibodies in the pathogenesis of rheumatoid arthritis-associated interstitial lung disease and bronchiectasis date: 2021-08-27 journal: rheumatol ther doi: 10.1007/s40744-021-00362-4 sha: b69e1dd4bd968b696f8ec04942f468de1e008b2a doc_id: 23087 cord_uid: n4lffmo1 rheumatoid arthritis (ra) is an articular disease with extra-articular manifestations. pulmonary manifestations are not uncommon and can involve all compartments of the lungs with airway disease in the form of bronchiectasis or bronchiolitis, interstitial lung disease (ild), pleural effusions and parenchymal lung nodules. the pulmonary features may present synchronously or after the articular disease, but, importantly, it may be the first presentation in 10% of patients in the absence of articular symptoms. here we discuss the pathogenesis of ra lung involvement, particularly interstitial lung disease and bronchiectasis, focusing on the role anti-ccp antibodies (acpas). we highlight the complex interplay among genetic, environmental and immune factors. furthermore, we explore the relationship of citrullination and smoking as well as the concept of interstitial pneumonia with autoimmune features (ipaf), where patients do not have evidence of another known cause of interstitial pneumonia and have incomplete features of connective tissue disease (ctd). we surmise that the frequency and titers of rheumatoid factor (rf) and acpas are increased in bronchiectasis and ra-bronchiectasis compared to ra patients without lung disease. acpa is associated with more severe disease in both ra-ild and ra-bronchiectasis even in the absence of articular symptoms. there is no clear prediction of development of articular ra with high acpa levels in the context of positive acpa and ild; however, in ra-bronchiectasis, patients with positive antibodies can develop ra within a year after diagnosis of bronchiectasis. though the primary focus of this narrative is to highlight the role of acpa in pathogenesis and clinical practice, we also discuss the current treatment options and trials in ra-ild and ra-bronchiectasis. currently, there are no clear treatment guidelines. the treatments are now focusing on using a combination of immunosuppression and antifibrotic agents. combination treatment targets both the fibrotic and inflammatory components of the disease process. further studies are needed to identify the use of acpa as a biomarker to tailor the treatment in ra-ild and ra-bronchiectasis. lung disease and bronchiectasis, focusing on the role anti-ccp antibodies (acpas). we highlight the complex interplay among genetic, environmental and immune factors. furthermore, we explore the relationship of citrullination and smoking as well as the concept of interstitial pneumonia with autoimmune features (ipaf), where patients do not have evidence of another known cause of interstitial pneumonia and have incomplete features of connective tissue disease (ctd). we surmise that the frequency and titers of rheumatoid factor (rf) and acpas are increased in bronchiectasis and rabronchiectasis compared to ra patients without lung disease. acpa is associated with more severe disease in both ra-ild and rabronchiectasis even in the absence of articular symptoms. there is no clear prediction of development of articular ra with high acpa levels in the context of positive acpa and ild; however, in ra-bronchiectasis, patients with positive antibodies can develop ra within a year after diagnosis of bronchiectasis. though the primary focus of this narrative is to highlight the role of acpa in pathogenesis and clinical practice, we also discuss the current treatment options and trials in ra-ild and rabronchiectasis. currently, there are no clear treatment guidelines. the treatments are now focusing on using a combination of immunosuppression and antifibrotic agents. combination treatment targets both the fibrotic and inflammatory components of the disease introduction rheumatoid arthritis (ra) is a systemic autoimmune inflammatory disease with a global prevalence of approximately 0.46% that presents with mostly articular but also extraarticular manifestations [1] . pulmonary manifestations of rheumatoid arthritis include pulmonary nodules, pleural effusions, bronchiolitis, bronchiectasis and interstitial lung disease (ild) [2] . the latter two are the most common pulmonary manifestations and in 10% of cases can be the first presentation of ra [3] . in some cases, patients may present with respiratory symptoms and positive antibodies but without articular features of rheumatoid arthritis [4] , while in a minority of cases, the ild may precede the development of anticitrullinated peptide antibodies (acpa) by several years [4, 5] . in this review article, we provide an update on the pathogenesis of rheumatoid arthritis-associated bronchiectasis (ra-bronchiectasis) and ild, including the role of acpa [6] . this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. though the exact etiology of ra -associated ild (ra-ild) is not known, several risk factors have been identified. these include cigarette smoking, acpa, genetic variants, including hla alleles, male gender, [ 60 years of age, duration of ra 0-5 years and high ldh. no correlation has been observed between standard diseasemodifying antirheumatoid drugs (dmards) and ild [7, 8] . all the known histological interstitial pneumonia patterns are seen in ra, on high-resolution ct (hrct) scan or histology, often with evidence of multicompartment involvement including airways, pleura or vasculature. lung biopsies are rarely performed in ra-ild, as ct patterns provide prognostic separation. the pattern most frequently observed is usual interstitial pneumonia (uip) (on ct characterized by peripheral distribution of reticulation, with traction bronchiectasis with/without honeycombing, and on histology patchy fibrosis, microscopic honeycombing and fibroblastic foci), where fibrosis dominates over inflammation. non-specific interstitial pneumonitis (nsip) is the second most common pattern, characterized by a homogeneous distribution of fibrosis admixed with inflammation. more inflammatory patterns, including organizing pneumonia and lymphocytic interstitial pneumonia, are observed less frequently. a ct uip pattern has the worst prognosis, with an idiopathic pulmonary fibrosis (ipf)-like survival in patients with extensive disease on ct. features inconsistent with uip are associated with a more favorable prognosis [9] . features of small airway involvement are manifested on ct by the presence of a mosaic attenuation pattern and are often seen in ra-ild [9] . finally, concomitant emphysema is seen in a subset of ra-ild patients and is associated with a uip pattern and a worse outcome. jacob et al. report a prevalence of emphysema in 27% of never smokers with ra-ild, suggesting shared pathways, possibly autoimmune, between ild and emphysema in ra [9] . recently, there has been renewed interest in the role of acpas in disease pathogenesis. the presence of citrullinated peptides in the serum and synovial fluid in ra with high specificity and the facts that acpas can develop 3-5 years before the onset of arthritis [10] and that they are often related to more severe disease have suggested a key role in the pathogenesis of ra [11] . the lung is considered a potential site of initiation in acpa-positive ra [12] , possibly triggered by processes such as smoking and microbial dysbiosis [13] [14] [15] . furthermore, iga and igg ra-associated antibodies are found in the sputum of individuals at high risk of ra (e.g., with a first-degree relative with ra) even in the absence of serum acpa or rf. through loss of integrity of the mucosal barrier, these antibodies may subsequently leak into the systemic circulation [16, 17] . the citrullination process converts arginine to citrulline, which results in an immune response. the process relies on the extracellular release of peptidyl-arginine deiminase 4 (pad4) by neutrophils or macrophages. the full range of recognized citrullinated proteins is not known. acpa can be present in other diseases, including systemic lupus erythematosus (sle) (16.6%), ankylosing spondylitis (4%), psoriatic spondyloarthropathy (psa) (10.6%), unclassified rheumatism (33%) and tuberculosis [18, 19] . higher acpa titers are associated with an increased prevalence of ild, even when corrected for confounders such as ra and smoking [20] . approximately half of individuals with ra-ild have evidence of citrullinated proteins on examination of their lung tissue [21] . interestingly, the proportion with evidence of citrullination with idiopathic interstitial pneumonia is similar to that with ra-ild and double that seen in control populations [21] . some patients with ra have a genetic predisposition to developing ild. the presence of hla-b54, hladq1b*0601, hla-b40 and hla-dr4 is associated with increased risk of ild [22] . the muc5b gene encodes mucin 5b, a macromolecule glycoprotein secreted by airway cells in the airway mucus barrier. a promoter polymorphism of this gene, rs37505950, associated with increased muc5b expression, confers the greatest risk of developing ipf [23] . muc5b is overexpressed in the distal airways and honeycomb cysts of ipf lungs [24] . interestingly, the muc5b promoter variant is associated with the development of ra-ild, and specifically with a uip pattern, but is not a risk factor for the development of ra per se [25] . the mechanisms underlying the contribution of the muc5b polymorphism to the pathogenesis of ipf and ra-uip are not known, although aberrant mucociliary clearance in the small airways may be associated with a more detrimental impact of inhalational exposures such as smoking, known to be strongly associated with both ipf and ra-ild [26] . research is ongoing so that this genotype may guide better understanding of the pathogenesis and management of this condition. cigarette smoking is an independent risk factor in the development of ra and ild. it may play a role in inducing antibody formation and has been linked to higher titers of rheumatoid factor. smoking may also play a specific role in ra-ild by promoting citrullination of lung proteins by increasing pad 4 levels, thus leading to the development of acpa [27] [28] [29] . this seems to be especially the case for individuals who have the shared epitope hla-drb1. male sex has also been identified as a risk factor for ra-ild, although the exact reason is not understood [22] . a study has found that the proportion of smokers and non-smokers with acpa antibodies was not statistically different but smokers did have higher levels of acpa and more severe diseases with poorer response to treatments [30] . overall, there is a complex interplay among immune, genetic and environmental factors contributing to the pathogenesis of ra ild ( fig. 1 ) [22] . bronchiectasis is defined as the irreversible dilatation and damage of the bronchi [31] . it is associated with rheumatoid arthritis and can be either isolated or secondary to traction, as an expression of surrounding fibrosis, often seen in ild. although traction bronchiectasis is not considered to be associated with a productive cough and suppurative lung disease, in clinical practice this is seen, particularly when individuals are immunosuppressed and have bacterial colonization [32] . the prevalence of symptomatic free-standing bronchiectasis with ra is between 2 and 12% [33] but asymptomatic disease prevalent on a hrct is much higher, between 30 and 50% [34] . interestingly, bronchiectasis may precede articular manifestations of ra [35] but is most often seen as a delayed complication of ra, possibly related to immunosuppression predisposing to recurrent infection [36] . however, the fact that patients with other ctds such as systemic sclerosis or anti-synthetase syndrome-associated lung involvement only very rarely develop free of note, small airway involvement (bronchiolitis), even in the absence of free-standing bronchiectasis, is common in ra and is observed on chest high-resolution computed tomography (hrct) in almost two-thirds of patients, with or without associated ild [37] . the exact pathogenesis of ra-bronchiectasis remains poorly understood. in addition to the vicious cycle hypothesis of chronic infection leading to recurrent inflammation and airway damage (fig. 2) , risk factors such as positive autoantibodies (e.g., rf and acpa), cftr mutations (f508del) and hla associations (particularly hladqb1) have been identified [38, 39] . the role of chronic bacterial infections in inducing autoimmunity in bronchiectasis alone and ra-associated bronchiectasis has also been explored. the frequency and titers of rf and acpa are increased in bronchiectasis and rabronchiectasis compared to ra patients without lung disease, after adjusting for smoking history [40, 41] . furthermore, citrulline specificity of acpa was increased approximately two-fold in ra-bronchiectasis compared to bronchiectasis alone [40] . overall, this suggests that presence of these antibodies in bronchiectasis may potentiate the exacerbation of the autoimmune response in ra even before the articular manifestations develop. the bronchiectasis, asthma, control, rheumatoid arthritis (brac) study prospectively looked at the relationship between bronchiectasis and ra antibodies. comparison was made among three groups of patients with bronchiectasis, asthma and a group of controls [40] . rf positivity was 25%, 16% and 10.3%, respectively, in each group. in the rf-positive bronchiectasis group, 13% (4 patients) were also acpa positive compared to no acpa identified in the asthma/control groups. the acpa response in patients with bronchiectasis was not citrulline specific, suggesting epitope spreading to citrullinated peptides in patients who subsequently develop ra. importantly, half of the individuals with strongly positive acpa subsequently developed ra within 12 months. this supports the role of autoimmunity in some individuals with bronchiectasis and subsequent development of ra-bronchiectasis. a multicenter randomized control trial [42] has identified ra-bronchiectasis overlap to be an independent risk factor for mortality (28% in ra-bronchiectasis overlap vs. 18% in ra alone and 9% in bronchiectasis alone over a period of 48 months). these findings suggest that antibody positivity in ra-bronchiectasis may be associated with a poorer prognosis, but further randomized control trials are needed to compare mortality and prognosis with non-antibody-positive ra-bronchiectasis. genetic predisposition to ra-bronchiectasis has been investigated, and the role of cystic fibrosis transmembrane regulator (cftr) mutations has been identified. in a small french study, a heterozygous cftr mutation in f508del was identified in 15.4% of patients with ra-bronchiectasis versus none in the ra or bronchiectasis groups individually [43] . in a family-based study by the same group [44] , the frequency of general cftr mutations was higher in family members with rabronchiectasis or bronchiectasis only compared to unaffected relatives and to unrelated healthy controls but not to family members with ra only, suggesting an association between cftr fig. 2 cole's vicious cycle hypothesis mutations with bronchiectasis and ra. cftr mutations were five-fold more frequent in family members with ra-associated bronchiectasis than in those with ra only [44] . similarly to ra-ild, certain hla alleles have been linked with ra-associated bronchiectasis. these include dqb1*0601, dqb1*0301, dqb1*0201 and dqa1*0501 [45] . their clinical significance is not yet clear and requires further research. acpas have been shown to develop up to 14 years before the onset of clinical ra [10] . however, there are no clear predictors as to who will develop clinical manifestations of disease or when these features will be detected. in a group of patients with positive acpa and connective tissue disease, 6.1% developed ra over 8.9 years and titers were higher in patients with a diagnosis of ra compared to the non-ra/ctd group [46] . the presence of acpa in the context of ra is important as it can be used as a marker of prognosis for erosions and disease severity [47] . in clinical practice, when acpas are detected in individuals with only ild or bronchiectasis, it is unclear whether antibody levels correlate with pulmonary disease severity or if the treatment strategy needs to be altered. a research group first explored this in 2012 in a group of subjects with lung disease and autoimmune antibodies but no extrapulmonary manifestations of autoimmune disease. these included individuals with isolated airway disease, isolated ild, mixed airways disease and ild, and combined ild and emphysema. there was no correlation between acpa presence and hrct changes in this study. in the cohort of patients with high titer acpa, 9% of subjects developed articular rheumatoid arthritis over 1.2 years of median follow-up, while no patients in the medium or low acpa titer groups developed ra [4] . interestingly most patients in the total cohort had a smoking history, which as previously described is linked to acpa. the presence of high acpa titers is also associated with shared epitope alleles, higher inflammatory markers (il-6 and c-reactive protein) and presence of rheumatoid nodules [48] . a recent systematic review surmised that individuals with ra-ild have higher acpa titers than those with ra alone. higher acpa titers are associated with more extensive lung involvement. the quality of evidence however was rated as low or very low [48] [49] [50] . reticulation, honeycombing or traction bronchiectasis on ct scan seems to be two-fold more prominent in apca-positive patients compared to those who were negative, suggesting that usual interstitial pneumonitis (uip) is more common in those with higher acpa levels. furthermore, higher acpa levels are also associated with worse lung function [48] . interstitial pneumonia with autoimmune features (ipaf) has been suggested as a research entity to include those patients with ild without evidence of another known cause of interstitial pneumonia and incomplete features of ctd [51] . to meet criteria for ipaf at least one feature of at least two out of three domains must be reached (serological, clinical, morphological). all the idiopathic interstitial pneumonia patterns except for uip count as a feature in the morphologic domain, whereas in the presence of a uip pattern, at least one feature in the clinical and serologic domains needs to be present, unless there is an additional morphologic feature [51] . ipaf criteria are met in up to 25% of ild patients and will include patients presenting with ild and acpa but no articular manifestations of ra, provided there is an additional feature from the clinical or morphological domain if the pattern is uip. individuals with ctd-ild are more likely female and with higher incidence of joint pain, sicca symptoms and raynaud's phenomenon; patients with ipaf are more likely have high ro-52 titers and more honeycombing and pleural thickening on hrct and to present primarily with respiratory symptoms [52] . interestingly, a muc5b minor allele is associated with a worse survival in the context of ipaf, whereas it has been associated with a better survival in ipf in some, but not all, studies [53] . the presence of acpa in the context of lung disease may lead to intensification of the search for underlying ra and raises questions as to treatment choices, whether patients with ild and acpa should be treated as ra ild rather than idiopathic pulmonary fibrosis/ild. the treatment challenge lies in the fact that ild can occur at any point in a patient's journey with ra. treatment for the joints may not be effective for treating ild and vice versa [6] . acpa titers in the context of ra can be used to monitor disease activity. up to 25% reduction in levels has been noted in half of the patients on treatment. both rituximab and abatacept have been shown to reduce acpa levels in responders in ra patients [54, 55] . some reports have noted this with anti-tnf therapy but not consistently. the usefulness of assessing acpa levels in the context of ra-ild is less well understood, and further research is warranted. there are no treatment guidelines for the management of ra-ild. current evidence is based on retrospective data and small studies. corticosteroids have been shown to improve lung function, particularly in those with nsip, but their use increases the risk of infection at doses [ 10 mg [53, 55, 56] . in the context of a uip pattern, it is unclear whether immunosuppression may have similar adverse outcomes as seen in the panther trial for ipf [53] . on the other hand, song et al. [55] describe a small retrospective series where half of patients with ra-uip treated with immunosuppression either improved or had stable lung function. recent large case control studies have suggested that treatment with methotrexate may delay the onset of ild in patients with ra [57] . cyclophosphamide and mycophenolate have been investigated with rcts in systemic sclerosis-associated ild, with mmf having a better side effect profile compared to cyclophosphamide [55, 56, [58] [59] [60] . azathioprine is also an option [56, 61] . however, none of these immunosuppressants have been evaluated in controlled clinical trials in ra-ild. there are conflicting data regarding the safety of anti-tnf therapy, with concerns about its association with acute exacerbations of the ild. retrospective studies have suggested abatacept as a safe treatment for ra-ild, although controlled prospective trials are needed; one is currently ongoing (april trial) [6, 62, 63] ( table 1 ). rituximab has been used for the treatment of ra-ild, although rituximab-induced pulmonary toxicity is a concern and needs to be evaluated prospectively [64] . additionally, rituximab results in depletion of b cells, secondary immunodeficiency, recurrent infections and de novo bronchiectasis [65] [66] [67] . tofacitinib has been noted to be associated with a low incidence of ra-ild (0.18 per 100 patientyears) and is used to treat articular manifestations. it may therefore be a good treatment option in the future. trials are ongoing comparing tofacitinib versus methotrexate in the treatment of ild [55, 68] (table 1) . antifibrotic agents (e.g., nintedanib and pirfenidone) remain a treatment possibility in this cohort. as described, there is considerable overlap between ipf/ipaf and ra-ild with the muc5b gene being a potential link. both are more frequent in older male patients with a smoking history. treating both the fibrotic and inflammatory components may provide additional benefit; therefore, several trials are now underway looking at the safety and efficacy of these agents in ra-ild. efficacy data are available for nintedanib from the inbuild trial. a significant reduction in the rate of fvc decline was seen across ild entities, including ra-ild, which had progressed despite conventional treatment [6, 69] . the rct trail 1 using pirfenidone in treatment of ra-ild is still underway [53, 70] (table 1) . ra-ild has a 10-year mortality of 60% [71] promoting the need to ''treat to target'' as we do with patients with florid synovitis. it is unclear what the future holds, but it is likely that treatment regimens will include antifibrotic agents alongside immunosuppressive medication in selected subgroups. treatment will require an mdt approach and will need to be individualized based on the patient demographics and pattern of disease involvement as well as comorbidities. the challenge of treating both articular and pulmonary symptoms remains with few agents having an impact on both. research is needed in this area to improve management of ra-bronchiectasis. the identification of acpa is useful to phenotype individuals with the hope of future personalization of bronchiectasis treatments. it is plausible that systemic inflammation can drive bronchiectasis progression and severity and that suppressing ra-related systemic inflammation could attenuate this. in contrast, airway inflammation is thought to be a trigger for citrullination; optimal control of bronchiectasis-related airway inflammation may attenuate antibody formation. however, targeting both airway and/or systemic inflammation may be associated with bacterial colonization, overgrowth and infections that further drive bronchiectasis pathogens. bronchiectasis management in the context of associated ra involves interventions aimed at breaking the cycle in cole's vicious cycle hypothesis. infective exacerbations are treated with 10-14-day courses of antibiotics. azithromycin is used for its immunomodulatory properties in those with frequent exacerbations. in the presence of bacterial colonization and frequent exacerbations, prophylactic oral or nebulized antibiotics are often used. individuals with ra-bronchiectasis are often receiving dmards and biological therapies that suppress immune function, which results in frequent infections. the impact of biological therapies on respiratory infection risk has been reviewed elsewhere [50] . prophylactic antibiotics are useful but in some cases modification of immunosuppression therapy is needed. it is also important to evaluate individuals for the presence of secondary antibody deficiency as immunoglobulin replacement may be necessary. regular airway clearance with physiotherapy techniques, airway adjuncts and mucolytics is the mainstay of treatment in bronchiectasis. annual influenza vaccination is recommended, and consideration should be given for pneumococcal vaccination with pcv13 followed by ppv23 2 months later at least 2 weeks prior initiation of immunosuppressive medication in both ra-ild and ra-bronchiectasis [72] . as smoking is an important factor in the pathogenesis of both conditions, advising the current literature suggests that acpa levels are higher in patients with ra-ild and rabronchiectasis and associated with more severe lung disease. patients with ra-bronchiectasis and positive acpa may go on to develop articular manifestations of ra within a year of diagnosis. higher acpa titers do not predict the development of articular ra, but the presence of acpa alone is a predictor of articular ra over several years. although acpa levels drop with some treatments, it is still unclear whether they can be used as biomarkers of lung disease activity. there are no clear treatment pathways for patients with ra-ild, and on every occasion an mdt approach is advised. funding. no funding or sponsorship was received for this study or publication of this article. authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. author contributions. mm and rj contributed to the conception and design of the article as well as text and editing. tk helped to draft and edit the manuscript. er overviewed the drafts, reviewed the concept and provided great insight and expertise. disclosures. tanjila khan, ricardo j. jose and maria mouyis have nothing to disclose. elisabetta renzoni has received lecture fees paid to the institution by roche and boehringer and roche. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. open access. this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/bync/4.0/. the global prevalence of rheumatoid arthritis: a meta-analysis based on a systematic review pulmonary involvement in rheumatoid arthritis interstitial lung disease in rheumatoid arthritis: a review lung disease with anti-ccp antibodies but not rheumatoid arthritis or connective tissue disease the lung may play a role in the pathogenesis of rheumatoid arthritis treatment of rheumatoid arthritis-associated interstitial lung disease: lights and shadows a retrospective study on the predictive implications of clinical characteristics and therapeutic management in patients with rheumatoid arthritis-associated interstitial lung disease risk factors for interstitial lung disease: a 9-year nationwide population-based study predicting outcomes in rheumatoid arthritis related interstitial lung disease specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors maå�liå� ski w. the role of anti-citrullinated protein antibodies (acpa) in the pathogenesis of rheumatoid arthritis hutchinson d. the lung in acpa-positive rheumatoid arthritis: an initiating site of injury? environmental and genetic factors in the development of anticitrullinated protein antibodies (acpas) and acpa-positive rheumatoid arthritis: an epidemiological investigation in twins gene, environment, microbiome and mucosal immune tolerance in rheumatoid arthritis the lung microbiota in early rheumatoid arthritis and autoimmunity rheumatoid arthritis and the mucosal origins hypothesis: protection turns to destruction rheumatoid factor associated with a secretory component in rheumatoid arthritis anticyclic citrullinated peptide antibodies in rheumatoid and nonrheumatoid rheumatic disorders: experience with 1162 patients poncet's disease mimicking rheumatoid arthritis in a patient with suspected crohn's disease elevated anti-cyclic citrullinated peptide antibody titer is associated with increased risk for interstitial lung disease citrullination in extra-articular manifestations of rheumatoid arthritis. rheumatology (oxford) rheumatoid arthritis-associated lung disease regulation of muc5b expression in idiopathic pulmonary fibrosis mucins muc5b and muc5ac in distal airways and honeycomb spaces: comparison among idiopathic pulmonary fibrosis/ usual interstitial pneumonia, fibrotic nonspecific interstitial pneumonitis, and control lungs muc5b promoter variant and rheumatoid arthritis with interstitial lung disease genetic risk factors for idiopathic pulmonary fibrosis: insights into immunopathogenesis smoking, lung function, and rheumatoid factors smoking is a major preventable risk factor for rheumatoid arthritis: estimations of risks after various exposures to cigarette smoke smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in bal cells smoking habits and anti-ccp antibodies in patients with rheumatoid arthritis. gabriela chirea post covid-19 bronchiectasis: a potential epidemic within a pandemic prevalence of symptomatic bronchiectasis in patients with rheumatoid arthritis prospective study of the prevalence of bronchiectasis in rheumatoid arthritis using high-resolution computed tomography rheumatoid arthritis and bronchiectasis. a retrospective study of fourteen cases a late feature of severe rheumatoid arthritis bronchiectasis in rheumatoid arthritis: report of four cases and a review of the literature-implications for management with biologic response modifiers bronchiectasis in rheumatoid arthritis. a clinical appraisial bronchiectasis is a model for chronic bacterial infection inducing autoimmunity in rheumatoid arthritis increased disease activity, severity and autoantibody positivity in rheumatoid arthritis patients with co-existent bronchiectasis bronchiectasis rheumatoid overlap syndrome is an independent risk factor for mortality in patients with bronchiectasis: a multicenter cohort study increased frequency of cystic fibrosis deltaf508 mutation in bronchiectasis associated with rheumatoid arthritis mutations of the cystic fibrosis gene in patients with bronchiectasis associated with rheumatoid arthritis hla associations in subjects with rheumatoid arthritis and bronchiectasis but not with other pulmonary complications of rheumatoid disease autoantibodies to posttranslational modifications in rheumatoid arthritis anticitrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage association of fine specificity and repertoire expansion of anticitrullinated peptide antibodies with rheumatoid arthritis associated interstitial lung disease anti-cyclic citrullinated peptide antibodies and severity of interstitial lung disease in women with rheumatoid arthritis systematic review and meta-analysis of the risk of rheumatoid arthritisassociated interstitial lung disease related to anticyclic citrullinated peptide (ccp) antibody research statement: interstitial pneumonia with autoimmune features comparative analysis of connective tissue disease-associated interstitial lung disease and interstitial pneumonia with autoimmune features telomere length and genetic variant associations with interstitial lung disease progression and survival prednisone, azathioprine, and n-acetylcysteine for pulmonary fibrosis a systematic review of serum biomarkers anti-cyclic citrullinated peptide and rheumatoid factor as tests for rheumatoid arthritis clinical course and outcome of rheumatoid arthritis-related usual interstitial pneumonia risk of serious infection in patients with rheumatoid arthritis-associated interstitial lung disease methotrexate and rheumatoid arthritis associated interstitial lung disease randomized control multi-center clinical study of mycophenolate mofetil and cyclophosphamide in the treatment of connective tissue disease related interstitial lung disease cyclophosphamide versus placebo in scleroderma lung disease mycophenolate mofetil versus oral cyclophosphamide in scleroderma-related interstitial lung disease (sls ii): a randomised controlled, double-blind, parallel group trial 092 the effect of steroids, azathioprine and mycophenolate on the risk of death in rheumatoid lung disease abatacept in patients with rheumatoid arthritis and interstitial lung disease: a national multicenter study of 63 patients safety of abatacept in italian patients with rheumatoid arthritis and interstitial lung disease: a multicenter retrospective study effect of rituximab on pulmonary function in patients with rheumatoid arthritis de novo bronchiectasis in haematological malignancies: patient characteristics, risk factors and survival biological therapies in the treatment of inflammatory disease and cancer: impact on pulmonary infection development of bronchiectasis during long-term rituximab treatment for rheumatoid arthritis incidence rates of interstitial lung disease events in tofacitinib-treated rheumatoid arthritis patients: post hoc analysis from 21 clinical trials nintedanib in progressive fibrosing interstitial lung diseases the design and rationale of the trail1 trial: a randomized doubleblind phase 2 clinical trial of pirfenidone in rheumatoid arthritis-associated interstitial lung disease a population-based cohort study of rheumatoid arthritis-associated interstitial lung disease: comorbidity and mortality pneumococcal vaccination in immunocompromised hosts: an update thanks to william henderson of bedfordshire hospitals library and knowledge service with his help in the literature search. key: cord-0011129-z1f2d19o authors: anderson, martha; youngner, stuart; smith, regina dunne; nandyal, raja r.; orlowski, jeffrey p.; jessie hill, b.; barsman, sarah gutin title: neonatal organ and tissue donation for research: options following death by natural causes date: 2020-03-13 journal: cell tissue bank doi: 10.1007/s10561-020-09822-7 sha: 9eada110e2edb3036dafe9bfb49ab481bc2da9ac doc_id: 11129 cord_uid: z1f2d19o the donation of organs and tissues from neonates (birth to 28 days) for transplantation has been a relatively infrequent occurrence. less common has been the use of neonatal organs and tissues for research. specific ethical and legal questions beg for rational and transparent guidelines with which to evaluate referrals of potential donors. donation of organs and tissues from a neonate can play a key role in the care and support provided to families by health care professionals around the time of a neonate’s death. we report on the recovery of neonatal organs and tissues for research. a working group made up of bioethicists, neonatologists, lawyers, obstetric practioners as well as organ procurement and tissue banking professionals evaluated legal, ethical and medical issues. neonatal donor family members were also consulted. our primary goals were (a) to ensure that referrals were made in compliance with all applicable federal and state laws, regulations and institutional protocols, and (b) to follow acceptable ethical standards. algorithms and policies designed to assist in the evaluation of potential neonatal donors were developed. neonatal donation is proving increasingly valuable for research into areas including diabetes, pulmonary, gastrointestinal, genitourinary and neurological development, rheumatoid arthritis, autism, childhood psychiatric and neurologic disorders, treatment of mrsa infection and pediatric emergency resuscitation. the development of policies and procedures will assist medical professionals who wish to offer the option of donation to family members anticipating the death of a neonate. the developing practice of using neonatal organs and tissues for research raises specific ethical and legal questions that beg for rational and transparent guidelines with which to evaluate referrals. this is especially true because of the rapidly increasing use of neonatal organs for research. to this end, a working group of scholars representing disciplines including bioethics, neonatology, obstetrics, and law, as well as professionals from organ procurement organizations (opo) and tissue banks including iiam was formed. neonatal donor families were also consulted. its task was to consider the myriad of issues and to develop algorithms and policies for screening potential neonatal donors. our primary goals were: (a) to ensure that referrals were evaluated in compliance with all applicable federal and state laws, regulations, and institutional protocols; and (b) to follow acceptable ethical standards. the donation of organs and tissues from neonates (birth to 28 days) for transplantation has been a relatively infrequent occurrence for a number of reasons, including a relative scarcity of recipients who are size matched to these small donors (stiers et al. 2015; boucek et al. 2018) . director of research from the united network for organ sharing robert carrico shares that since 2010, annual totals of neonatal organ donors in the united states, both from donation after brain death (dbd) and donation after circulatory death (dcd), have ranged from 3 to 21 donors annually. this represents between 0.03 and 0.21% of organ donors recovered in any given year. the donation of neonatal organs and tissues for research is even less common than for transplantation, primarily because family members who receive a prenatal diagnosis of a lethal anomaly (la) or experience the death of a neonate are not routinely offered information about research donation. moreover, until recently researchers rarely sought neonatal organs and tissues. in the mid-2010s, some families who received a pre-natal diagnosis of a la such as anencephaly decided to carry their pregnancy to term regardless of the poor prognosis for long-term survival, often wishing to donate after the anticipated natural death of their neonate (gray 2016a, b, c, d, e, f, g; young 2017) . social media, blogs, and websites, including www.purposefulgift.org and www.anencephaly.info, have increasingly connected parents who receive a diagnosis of a la. some expectant parents began contacting the international institute for the advancement of medicine (iiam) with requests for assistance. iiam is among the largest 501c3 non-profit organizations in the u.s. that coordinates the placement of non-transplantable organs and tissues with organ procurement organizations (opos) and researchers. iiam is a division of mtf biologics, one of the largest non-profit providers of donated human tissues (e.g., musculoskeletal, dermal and placental). founded in 1986, iiam annually receives over 15,000 referrals of non-transplantable organs and tissues authorized for research from donors from u.s. opos. in the past 20 years, they have placed more than 14,000 research organs. matching donors and research projects is a challenging process, as researchers have very specific donor criteria and logistics requirements. research organs provided by iiam are used by academic researchers as well as by pharmaceutical and medical device companies in the u.s. and abroad. at the same time, academic researchers began requesting neonatal organs through iiam. prior to this time, researchers had limited access to such organs and relied either on organs from adult donors or fetal tissue. no national guidelines or standards existed for the coordination of neonatal donation for research so referrals of potential neonatal donors were sporadic and managed on an ad hoc basis. they typically depended on the interest/capabilities of the individual opo that would manage and coordinate the donation. as requests by expectant parents escalated, it became apparent that guidelines were necessary. the early interactions with expectant parents, opo staff and researchers prompted the work that has resulted in this paper. a more detailed taxonomy of acceptable and unacceptable donations appears later in this paper. after a brief discussion of ethical and legal issues as well as clinical ramifications of neonatal donation, we will explain how they helped shape our policy recommendations. lastly, we will present data about the outcomes of more than 224 neonatal referrals. although neonatal organs and tissues can be used for both transplantation and research, we will mainly focus on donation for research. neonatal organ and tissue donation represent a desire to make an altruistic gift and can play a key role in the care and support provided to families by hospital staff around the time of a neonate's death. indeed, many families who have donated the organs and tissues of their deceased neonate reported that the decision brought them solace and comfort (gray 2016a, b, c, d, e, f, g; rhodes 2014; purposeful gift) . moreover, neonatal donation is proving increasingly important for research into a variety of areas, including determining the causes of neural tube defects (ntd); treating vision impairment; diabetes; organ system development; rheumatoid arthritis; pancreatic cancer; treatment of mrsa infection; pediatric emergency resuscitation (gray 2016a, b, c, d, e, f, g; aguayo-mazzucato et al. 2017; gregg et al. 2012; ardini-poleske et al. 2017; cogger et al. 2017) . the primary goal of families with whom some of the authors (gds, jo, ma, sbg) have interacted was to carry a fetus diagnosed with la to term (or as close to term as possible) in order to achieve a live birth, knowing that aggressive treatment after birth would be futile. the possibility of organ and tissue donation was a secondary goal and provided an opportunity to find something positive in the midst of a tragic situation. legal and ethical questions that must be addressed require adherence to a variety of laws, regulations, and ethical principles. various state and federal laws regulate the donation of organs and tissues regardless of the age of a potential donor. federal law does not contain any restrictions that are uniquely relevant to neonatal organ and tissue donation. the federal national organ transplant act (nota) and related regulations prohibit the sale of organs or tissues and creates a framework to facilitate and standardize the donation and recovery process (42 u.s.c. § 273 et seq). at the state level, there are laws that may affect tissue and organ donation for research. for example, every state has an anatomical gift act that adopts some version of the uniform anatomical gift act (uaga) (uaga 2019). all states have adopted the uniform determination of death act (dda), which describes the events that trigger the possibility of organ and tissue donation (dda 1980) . organ and tissue donation must conform to the so-called dead donor rule, which states that no patient's death may be caused by organ recovery (robertson 1999) . under the policies stipulated by the organ procurement and transplant network, it is mandatory to separate the medical care of a patient and the determination of death from the recovery of organs or tissues in all cases of donation (organ procurement and transplant network policies (pp. 28-29) available at: https://optn.transplant.hrsa.gov/media/1200/ optn_policies.pdf). if life-sustaining treatment is withdrawn, donation may occur if the patient dies and organ and tissue recovery can occur within a time period specified by the intended researcher. obtaining authorization for donation from parents of neonatal patients can be complex, and has received significant attention in previous studies (martin et al. 2015) . typically, opo staff conduct the authorization process, but they are not involved in the routine care of the neonate, or in determining or pronouncing the death of the neonate. family members are given the latitude to determine which organs or tissues they wish to donate, and whether they wish to donate solely for transplantation or transplantation and research. some families may wish to donate organs in the context of actively terminating a pregnancy with an ultimately lethal outcome. the american college of obstetricians and gynecologists (acog) has established guidelines regarding medically acceptable indications for pre-term delivery for maternal or newborn benefit, but such a choice raises the legally and ethically fraught issue of abortion (nicholson 2015; committee opinion no. 560 2013) . many state laws have unique implications for the legality of neonatal organ donation in certain circumstances. in particular, laws forbidding donation of, or research upon, tissue resulting from an abortion may be relevant in cases in which a pre-term delivery technically meets the statelaw definition of abortion. recently, several states and the federal government have passed or considered such laws (ark. code § 20-17-802, ind. code § § 16-34-3-4(a); 16-41-16-4(d); 16-41-16-5; 16-41-16-7.6.; 15a n.c. admin. code 13b.1301.; 41 tex. reg. 9709-41). although abortion is a legal medical procedure in the united states, for purposes of this project we rejected the possibility of accepting donations that resulted from abortions. our guidelines do, however, allow acceptance of donations from some neonates who are delivered pre-term-but only if the delivery would not qualify as an abortion under applicable law. finally, we considered federal and state laws governing the withholding or withdrawing of lifesustaining care from disabled neonates. these laws, most specifically the baby doe regulations (bdr) and the born alive infant protection act (baipa), apply primarily to clinicians and hospitals that are directly involved in the pre-term delivery and subsequent care of a neonate, and do not constrain organ donation. bdr and baippa are federal laws that apply to pre-term delivery and resuscitation. bdr are federal statutory provisions that impose certain requirements on states as a condition of accepting federal child abuse prevention and treatment act (capta) funds (pub.l. 98-457, 98 stat. 1749 (codified as amended at 42 u.s.c. § § 5101-5106i (2006)). states are required to enact procedures for reporting and responding to ''medical neglect,'' which is defined to include withholding treatment (including appropriate nutrition, hydration, and medication) from neonates with disabilities and life-threatening conditions (42 u.s.c. § 5106a(b)(2)(c)). this requirement imposes a fairly sweeping mandate to provide care for neonates born with potentially lethal conditions. there are, however, three exceptions to the requirement of providing medically indicated treatment. treatment is not required if, ''in the treating physician's or physicians' reasonable medical judgment: • the neonate is chronically and irreversibly comatose''; • providing treatment would only prolong the death of the neonate, would not be effective in correcting or ameliorating the conditions, or would ''otherwise be futile in terms of the survival of the neonate'' and, • the treatment ''would be virtually futile in terms of the survival of the neonate'' and ''the treatment itself under such circumstances would be inhumane'' (42 u.s.c. § 5106 g(a) (5)). the american academy of pediatrics and the ethics committee of the american college of critical care medicine have both affirmed that it is ethically and legally justifiable to withhold or withdraw aggressive life-sustaining treatment in neonates when the burdens of such treatment far outweigh its benefits (ethics committee 2001; committee on bioethics 2013; sarnaik 2015; aap policy statement 2010; kon et al. 2016; gries et al. 2013) . baipa is a second federal law that has an intent similar to that of bdr. baipa states that any infant who is ''born alive,'' at any stage of development, must be treated as a person for purposes of the protections of federal law (1 u.s.c. § 8). this law has long been viewed as symbolic legislation with an antiabortion message, since it is unclear that it has any actual impact on the application of any federal law. it might, however, mean that the emergency medical treatment and active labor act's (emtala) requirement of stabilizing patients who arrive at the hospital in an emergency condition would apply to very premature infants as well (42 u.s.c.a. § 1395dd) emtala, combined with the baipa, may therefore be understood to require some treatment of extremely premature infants who are born with a heartbeat or other signs of life. futile or non-medically-indicated treatment would not likely be required for a very premature infant, since such treatment would not, within reasonable medical probability, prevent the neonate's condition from deteriorating. thus, as long as it is appropriate to characterize any care withheld from very premature neonates as being futile (in reasonable medical judgment), there is little danger of running afoul of bdr or baipa, as applied to emtala. in 2015, there were 15,652 total u.s. neonatal deaths (3.93 per 1000 live births) (murphy et al. 2017) . congenital malformations, deformations, and chromosomal abnormalities were noted to be primary causes of neonatal death. one such congenital malformation is anencephaly, one of the most common central nervous system disorders. anencephaly is a neural tube defect that occurs when the cephalic (head) end of the neural tube fails to close, usually between the 23rd and 26th days of pregnancy; this results in the absence of the major portion of the brain, skull, and scalp. some rudimentary forebrain, a part of the brain consisting mainly of the cerebrum, may exist. a functioning brainstem is usually present. prenatal diagnosis of anencephalic neonates typically occurs at 12-14 weeks. the majority of anencephalic pregnancies are terminated early in the pregnancy (brierley 2010; stiers et al. 2015) . the cdc estimates that anencephaly annually affects approximately 3 pregnancies in every 10,000, or 1206 pregnancies (cdc 2015) . most anencephalic neonates die within days or weeks without life-supporting interventions (shewmon 1989) . the care of a mother carrying a baby with a la can be complex, requiring close coordination between the obstetrician, neonatologist, and other healthcare professionals. in some instances, mothers experience a typical pregnancy except for the fact that they have received a diagnosis of a la; those who receive minimal or no prenatal care are unaware of a la until the baby is born. in many cases, the syndrome or condition afflicting the fetus may cause concern for maternal, fetal, and/or both maternal and fetal health and may lead to a decision to induce pre-term (iatrogenic) labor. alternatively, pre-term delivery may occur spontaneously without intervention. the leading causes for pre-term delivery are: 1. spontaneous labor with intact membranes 2. pre-term premature rupture of membranes (pprom) 3. delivery for maternal or fetal indications (thakor et al. 2008 ). tucker reports that 15-25% of pre-term infants are iatrogenically delivered early because of maternal or fetal pregnancy complications (tucker and mcguire 2004) . complications may include hypertension, preeclampsia or eclampsia, polyhydramnios, oligohydramnios, gestational diabetes, intrauterine growth restrictions, infection, and twin-to-twin transfusion syndrome. despite the recent widespread use of hypothermia therapy, hypoxic ischemic encephalopathy (hie) is a major cause of neurologic disabilities in term neonates. the incidence of hie ranges from 1 to 8 per 1000 live births in developed countries and is as high as 26 per 1000 live births in underdeveloped countries (douglas-escobar and weiss 2015). hie neonates frequently receive ventilation, hypothermia, or other therapy, until brain death is confirmed or until a decision is made to withdraw treatment and to allow for a natural death, also known as ''and''. medical literature is scant regarding discussion about neonates with hie who became organ donors for transplantation or research. there are anecdotal reports of neonatal organ donation from hie donors, including one author's center. jadcherla, et al., reported a case of neonatal organ donation in a fullterm neonate with severe hie complicated by multiorgan dysfunction who underwent therapeutic hypothermia (bokisa et al. 2015) . in non-ventilated cases, palliative care is provided to the neonate (hydration, comfort, etc.) allowing for a natural death (and). it is at that point that criteria for dcd may be applied. medical challenges include working with hospital staff who may have inadequate information or awareness about the potential of neonates to become donors. because transplant surgeons normally perform organ recovery only for transplant, opo staff need to be trained in organ recovery for research. specific details of the delivery of neonates can have a significant effect on the donation process-i.e., whether delivery occurred at term or prior to term; whether the neonate was delivered by natural delivery or whether labor was induced; the indications for medical intervention if labor was induced. the criteria used for the acceptance of organ donors for research may vary, based on the type of research and individual research protocols. a neonate with la may be considered as a potential donor for liver, lung, heart, kidney, pancreas, intestine, thymus, or tissue (skin, eyes, bone marrow, musculoskeletal and reproductive tissues). multiple recipients or research studies can often benefit from a single donation. the researcher (or the protocol) needs to establish requirements such as minimum gestational age of the neonate. exact time of death and a clear plan for organ recovery surgery have to be determined and documented. donation must occur in a timely manner because of concern regarding the effect of warm ischemic time (wit) on organ viability; this concern should be balanced with the family's need for time with their neonate after the neonate has passed away. neonatal donors, especially anencephalic donors, provide a unique set of challenges to an opo. whether the donation will ultimately result in transplanted organs or tissues, research organs or tissues, or some combination of these outcomes is often unclear until the surgical recovery occurs. the lack of formation of cerebral cortex and exposed brain structure make it impossible to determine brain death in anencephalic neonates. in other circumstances, such as hie, it may be possible because the cerebral cortex is formed, and the skull structure is intact. in ventilated neonatal donors, including neonates with hie, the process will often unfold in the same way as in older donors. thus, coordinating neonatal anencephalic donation requires flexibility and establishment of contingency plans to maximize successful donation. the challenges faced by opos in coordinating anencephalic donors fall largely into one of three categories: donor identification and referral; family and staff counseling; donation logistics. a brief discussion of each follows. in the united states, opos are to receive referrals on all in-hospital and imminent deaths. most opos have established ''clinical triggers'' for referral, which include variables such as glasgow coma scale 5 or below, discussion of withdrawal of care, initiation of brain death testing, or cardiac arrest. in the context of ventilated neonatal donors, these clinical triggers may apply; in anencephalic donors, who are typically nonventilated, they will not. the challenge, therefore, is to establish referral processes for practitioners working with the families of anencephalic neonates, to educate both opo staff and donor hospital professionals about neonatal donation, and to implement protocols for both opo and hospital staff to manage these cases. sample protocols are available upon request to iiam (www.iiam.org). the standard approach for opos in terms of counseling donor family and hospital staff regarding donation begins with the initial referral and after an evaluation of the clinical suitability for donation. because the timing of a referral is quite different in the anencephalic donor, two variables come into play. first, the donor family may be several months short of the actual delivery and will have much more time to consider their options for donation. this is unusual and requires multiple follow-ups with a family over an extended period as their decision-making evolves. second, the context of the discussions with perinatal and neonatal practitioners regarding the pregnancy and other related decisions is quite different from traditional organ donor situations, in which the discussion centers around brain death, withdrawal of care in a patient who is not brain dead and helping the family come to grips with the finality of the patient's brain injury. with anencephalic neonates, the family is addressing a far different set of issues, such as whether to carry to term and development of an appropriate birth plan. the process typically proceeds as with any organ/tissue donor referral in scenarios with hie or la. in standard dbd or dcd scenarios, regardless of the age of the potential donor, opos obtain authorization for donation, evaluate organ function, attempt to maximize organ function, match organs to recipients, and then coordinate the surgical recovery, which may include allowing teams from distant centers to fly to the hospital for the surgical recovery. surgical recovery is scheduled after organ evaluation and preliminary acceptance by a transplant center or, in the case of research donation, by a researcher. donor blood type, height, weight, and many other data factor into this process. a ventilated neonatal donor will follow much the same path. anencephalic donors are not typically intubated. as a result, the opo will have limited clinical information about the donor. it is vital to underscore that no donation can take place until death has occurred, through the pronouncement of death either by neurological criteria (i.e., brain death) or by circulatory death. opos can implement a few key strategies to maximize the benefit of the donation for the neonatal donor family, transplant recipients, and researchers. they include: 1. proactively identify centers where these potentially complex cases may be referred and transplant centers willing to consider neonatal organs for transplant. 2. have research outlets for all possible organs and tissues since transplantation rarely occurs with these potential donors. 3. closely collaborate with the specialists caring for the mother and neonate. it is not uncommon for these cases to unfold over months. 4. have at least two opo staff supporting and counseling the family. the prolonged process of neonatal donation and the importance of bonding with the family require a team approach in order to avoid compassion fatigue (nicely and delario 2011; maloney and wolfelt 2011; larowe 2005) . in 2012, bethany c., who was pregnant with an anencephalic baby, contacted iiam. she wanted to donate her son's organs and tissues following his death. despite contacting her obstetrician, local medical schools and hospitals, and the opo that serves her hometown, she had been unsuccessful. she found iiam's website and contacted iiam just days before her scheduled c-section. within 2 h, iiam found researchers who were willing to accept her baby's liver and pancreas, and another researcher who was interested in receiving his entire body after organ donation. their son, amalya nathaniel, lived for 80 min, surrounded by his parents, maternal and paternal grandparents, and other extended family members. the opo performed the organ recovery after his death. as a result of his donation, researchers were able to evaluate pancreatic beta cells in their earliest stages of development, leading them to understand why some people develop type 1 diabetes; other researchers studied hepatocytes, which is critical to understanding cell generation. images generated by his body led to fda clearance for a medical device used for rapid pediatric resuscitation (ardini-poleske et al. 2017; gray 2016a, b, c, d, e, f, g) . between 2012 and 2017, iiam coordinated the recovery of organs and tissues from 86 donors, placing 281 organs and tissues. most neonates who became donors were anencephalic (60%), followed by neonates who died because of anoxia or hie (23%). other causes of death included trisomy 18, fatal cardiac and renal anomalies, trauma, and other genetic and neurological conditions. gestational age (ga) varied, with the majority being delivered at term (which includes early term, full term and late term; 37 0/7 weeks-41 6/7 weeks) (see fig. 1 ) (taylor et al. 2019 ). pre-term deliveries (20 0/7 weeks-36 6/7 weeks) often occurred for medically indicated reasons (either maternal or fetal health or both); the majority of these deliveries were iatrogenic. pre-term deliveries for non-medical reasons were not accepted as referrals. one was referred following miscarriage at 16 weeks and was able to donate skin. all of the families who pursued donation did so with the intent of having a live birth, spending as much time as possible with the neonate, and donating, if possible, after the neonate's death. the time elapsed from birth to death ranged from minutes to days (see fig. 2) . in most cases, the neonate remained in the labor and delivery area; on one occasion, the neonate was taken home, given hospice care, and then her body was returned to the hospital following her death at 5 days. considering the legal, medical, ethical and practical issues surrounding these donation opportunities, the working group developed a set of algorithms designed to guide staff from opos and iiam in evaluating potential donors. three separate algorithms were developed: one for neonates being delivered at term with a la (see fig. 3) ; one for neonates being born pre-term with a la (see fig. 4) ; one for neonates who die as a result of other conditions, such as hie or trauma (see fig. 5 ). referrals are now evaluated according to these algorithms. reasons for declining referrals of potential neonatal donors include: • iatrogenic labor planned to terminate a pregnancy, not for maternal and/or fetal health (1%) • ga less than 24 weeks (3%) researchers who accepted neonatal organs and tissues through this program study such diverse areas as regenerative medicine, diabetes, chronic kidney disease, cancer, congenital organ malformation, abnormal lung development, and fertility (see fig. 6 ). pulmonary research in particular, has been advanced using donated neonatal lungs. over 75 neonatal donors have been provided to the biorepository for the investigation for diseases of the lung (brindl)/university of rochester medical center (urmc) human tissue core and distributed to more than 10 other academic laboratories. brindl and the urmc human tissue core are responsible for all the human data that currently appears on lungmap.net. papers and presentations arising from the lungmap project primarily focus on characterization of neonatal lung cells, and clinical and translational studies in rare lung diseases. this project has also explored why neonates and infants frequently progress to severe lung failure when exposed to respiratory syncytial virus (rsv) versus adults whose systems respond to rsv in a less severe manner. this information is expected to provide new options for treatment and prevention (taylor et al. 2019; jiang et al. 2019; wang et al. 2019; lal et al. 2018; whitsett 2018; kyle et al. 2018; bandyopadhyay et al. 2018; luo et al. 2018; zhu et al. 2018; zhou et al. 2018; ardini-poleske et al. 2017; warburton 2017; du et al. 2017 ). research findings from other projects using neonatal organs have been presented at scientific conferences and published in scientific journals (gray 2016a, b, c, d, e, f, g; ardini-poleske et al. 2017; gittinger 2015) . placements of research organs and tissues range from one organ per donor to over ten organs and tissues per donor. donation of organs and tissues for research from neonates who die following a live birth has recently become an option some families wish to consider. pursuing this donation option requires careful attention to legal, ethical, medical, and procedural issues. it is vital to ensure that the integrity of the donation process be safeguarded, and that the desire to support neonatal donor families does not compromise ethical and legal standards. the comfort and solace that donation provides following the death of a neonate can be vital to families' healing processes and should be post-natal fetal defect supported whenever possible. given the potential legal and ethical barriers to donation and the fact that not all opos are currently equipped to provide this service even if the request falls within acceptable standards, it is important that families have realistic expectations with respect to donation opportunities. coordinating neonatal referrals requires careful collaboration between hospital administration, health care professionals, opo staff, staff of the agency interfacing with researchers and opos, and the researchers themselves. the uses of neonatal organs and tissues have led to remarkable breakthroughs in science. family donation of organs and tissues from a neonate represents a desire to make an altruistic gift and can play a key role in the care and support provided to families by hospital staff at the time of a neonate's pending death. family members who suffer the tragic loss of a newborn through either congenital abnormalities such as anencephaly, or situations arising from a traumatic birth or hie, have found ''unexplainable peace, joy and healing'' in their ability to donate, thereby allowing their child to make an ''impact on the world'' (rhodes 2014 ). author's contribution ma: conceptualization, research, writing, rds: conceptualization, research, writing, sgb: research, writing, bjh: conceptualization, research, writing, rrn: research, writing, jpoi: research, writing, sy: conceptualization, research, writing. funding mtf biologics provided funding for the development of the project.the authors wish to thank wee siang tay for his assistance with references. ) b cell aging markers have heterogeneous distribution and are induced by insulin resistance lungmap consortium. lungmap: the molecular atlas of lung development program dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues death by neurologic criteria in a neonate: implications for organ donation pediatric heart transplantation after declaration of cardiocirculatory death neonatal organ donation: has the time come? updated estimates of neural tube defects prevented by mandatory folic acid fortification -united states glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors ethical controversies in organ donation after circulatory death hypoxic-ischemic encephalopathy: a review for the clinician lung gene expression analysis (lgea): an integrative web portal for comprehensive gene expression data analysis in lung development american college of critical care medicine, society of critical care medicine: recommendations for non-heartbeating organ donation normal delivery of the infant: overview, indications amalya's story amalya's story amalya's story eli's story sophie's choice: backward at 100 mph the dance. in: a life everlasting thomas's ride formation of a human b-cell population within pancreatic islets is set early in life official american thoracic society/international society for heart and lung transplantation/society of critical care medicine/association of organ and procurement organizations/united network of organ sharing statement: ethical and policy considerations in organ donation after circulatory determination of death alteration of cystic airway mesenchyme in congenital pulmonary airway malformation defining futile and potentially inappropriate interventions cell type-resolved human lung lipidome reveals cellular cooperation in lung function genomics, microbiomics, proteomics, and metabolomics in bronchopulmonary dysplasia a comparison of adult and neonatal human hepatocytes in drug metabolizing enzyme activities spatial and temporal changes in extracellular elastin and laminin distribution during lung alveolar development caring for donor families pediatric deceased donation-a report of the transplantation society meeting in geneva deaths: final data for 2015 virginia henderson's principles and practice of nursing applied to organ donation after brain death the 39-week rule and term stillbirth: beneficence, autonomy, and the ethics of the current restrictions on early-term labor induction in the us organ procurement and transplant network. policies the littlest donors: neonatal organ donation offers hope in tragedy. nbcnews.com the dead donor rule neonatal and pediatric organ donation: ethical perspectives and implications for policy. front pediatr 3:100 the use of anencephalic infants as organ sources potential and actual neonatal organ and tissue donation after circulatory determination of death the pediatric cell atlas: defining the growth phase of human development at single-cell resolution anaemia, weight loss, and round shadows in the lungs epidemiology of preterm birth a novel in vitro model of primary human pediatric lung epithelial cells overview of lung development in the newborn human airway epithelial differentiation and mucociliary clearance young r we spent months bracing and preparing for the death of our daughter extracellular matrix in lung development, homeostasis and disease proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanolc-ms publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-0015130-hij65gru authors: mosor, erika; studenic, paul; alunno, alessia; padjen, ivan; olsder, wendy; ramiro, sofia; bini, ilaria; caeyers, nele; gossec, laure; kouloumas, marios; nikiphorou, elena; stones, simon; wilhelmer, tanita-christina; stamm, tanja a title: young people’s perspectives on patient-reported outcome measures in inflammatory arthritis: results of a multicentre european qualitative study from a eular task force date: 2021-01-29 journal: rmd open doi: 10.1136/rmdopen-2020-001517 sha: 73ec0e636e56f83f216fcf088d423cc666d62f27 doc_id: 15130 cord_uid: hij65gru introduction: although patient-reported outcome measures (proms) are increasingly used in clinical practice and research, it is unclear whether these instruments cover the perspective of young people with inflammatory arthritis (ia). the aims of this study were to explore whether proms commonly used in ia adequately cover the perspective of young people from different european countries. methods: a multinational qualitative study was conducted in austria, croatia, italy and the netherlands. young people with either rheumatoid arthritis (ra), juvenile idiopathic arthritis (jia), still’s disease, psoriatic arthritis (psa) or spondyloarthritis (spa), aged 18–35 years, participated in semistructured focus group interviews. thematic analysis was used and data saturation was defined as no new emergent concepts in at least three subsequent focus groups. results: fifty-three patients (21 with ra/jia/still’s, 17 with psa, 15 with spa; 72% women) participated in 12 focus groups. participants expressed a general positive attitude towards proms and emphasised their importance in clinical practice. in addition, 48 lower level concepts were extracted and summarised into 6 higher level concepts describing potential issues for improvement. these included: need for lay-term information regarding the purpose of using proms; updates of certain outdated items and using digital technology for data acquisition. some participants admitted their tendency to rate pain, fatigue or disease activity differently from what they actually felt for various reasons. conclusions: despite their general positive attitude, young people with ia suggested areas for prom development to ensure that important concepts are included, making proms relevant over the entire course of a chronic disease. patient-reported outcomes (pros) constitute an essential part of health outcomes. 1 on an individual level, the measurement of pros is a crucial component of patientcentred care, building the basis for shared what is already known about this subject? ► patient-reported outcome measures (proms) have been used for different purposes in routine clinical practice, research and health services management in recent decades, particularly in the field of chronic diseases. ► this is the first study that explores perspectives and views of young people with inflammatory arthritis (ia) on widely used proms. ► young people in all countries and disease areas did not feel sufficiently informed about the value of proms and the reasons for collecting patient-reported outcomes in addition to clinical outcomes. ► from the perspective of young people, commonly used functional assessments seem to be outdated and overlook current relevant issues. these include career planning, caring for others such as children, losing friends, participating in social life (like going out in the evening), being excluded from physical activities at school and university, and using technological devices including smartphones and computers. ► young people considered a large number of functional items too 'easy', for example, walking and eating, while other more complex activities in daily life, such as using public transport or preparing meals were missing at all. how might this impact on clinical practice? ► the results of our study will change the use of proms in young people with ia in clinical practice and research. in the future, young people with ia should be involved in the adaptation of existing proms and the development of new instruments to ensure that important concepts are included, making proms relevant over the entire course of a chronic disease. decision-making, patient empowerment, engagement and self-management. 2 when used in routine clinical practice, pros can positively influence the relationship between patients and their healthcare providers. 3 4 pros allow a structured assessment of the type and severity of symptoms that patients experience, as well as the impact of their disease and subsequent treatment on their life. furthermore, aggregated pro data can also be used to drive healthcare quality improvement initiatives on an institutional level; and for population health monitoring and reimbursement decision-making on a macro-level. 2 several outcome domains can only be measured in a self-reported manner. examples are pain, fatigue, functioning in real-life situations and health-related quality of life. 2 5 to accurately quantify patients' experiences, patient-reported outcome measures (proms) are used. proms are defined as assessments of subjective health outcomes, based on responses provided directly by patients themselves without subsequent interpretation or alteration of the responses by health professionals (hps) or anyone else. 6 in the development and the selection of suitable proms, various methodological issues and measurement properties must be considered. these include the reliability, validity, responsiveness and interpretability of the respective instruments. 7 8 proms have been increasingly applied in routine clinical practice and research in recent decades, particularly in the field of chronic diseases. 9 10 in their work on pros in rheumatoid arthritis (ra), van tuyl and michaud 11 provided key examples of valid and reliable, commonly used proms in rheumatology. however, in order to address the impact of chronic diseases over their entire course, proms should be equally applicable and valid across a patient's lifespan. otherwise, adaptations for certain age groups may be required. 3 in addition to the validity, reliability, responsiveness and acceptability of the measurements, proms need to cover what matters to patients. 12 while a few studies have been conducted in rheumatology to explore whether proms cover the issues important to patients with different chronic autoimmune diseases, none of them focused specifically on young people. [13] [14] [15] inflammatory arthritis (ia) affects people of all age groups and proms play an important role to determine if a treatment is successful or not. however, to date, it has not been investigated whether proms commonly used in ia adequately include the perspectives of young people. this need was recognised by the eular and an international task force on incorporating the perspective of young people with ia into outcomes assessment was established. a qualitative approach was adopted to explore the perspectives of young people with ia on the content and practical use of the most commonly used proms in a broader european context. on this basis, the aims of our study were to explore whether commonly used proms in ia adequately cover the perspective of young people with ia from different european countries. a multinational qualitative study was conducted in austria (at), croatia (hr), italy (it) and the netherlands (nl). young people with ia aged between 18 and 35 years, treated in rheumatology centres, with a disease duration of at least 1 year, and a formal diagnosis of one of three ia disease areas: (1) ra, juvenile idiopathic arthritis (jia) and still's disease; (2) spondyloarthritis (spa); (3) psoriatic arthritis (psa), were included in the present study. all participants were contacted by telephone either by local investigators or patient organisations and appointments for participation in focus groups at the local centres or the location of the patient association (in the netherlands) were made. qualitative research typically uses small sample sizes with a diverse range of participants to explore the personal experiences and views of people on a specific topic. based on earlier studies, 12 15 16 disease-specific focus groups were conducted in each country. data saturation was defined as no new emergent concepts in at least three subsequent focus groups. 17 18 in order to determine the number of emergent concepts in each focus group session, data analysis commenced when first transcripts were available and proceeded in parallel to data collection. 19 data collection hps and patient research partners (prps) co-developed, piloted and finalised the semistructured interview guide. 21 it included questions on the perspectives and views of the participants on currently used proms which were selected based on a literature review and an online voting process. from 16 proms for ra, 19 proms for psa and 15 proms for spa, which were identified in the literature review, the study team in each country ranked the top five most commonly used proms based on their own experience. subsequently, an agreement was made regarding five often used proms for each of the disease areas to be provided to the focus group members in printed form as a basis for discussion ( a focus group facilitation guide, including transcription instructions, was provided to support local centres while ensuring data collection was harmonised. the interviews were conducted by trained local investigators with experience in qualitative research data acquisition between march and august 2018, audio-recorded, transcribed verbatim and translated into english. data coding and initial analyses were primarily undertaken by the first author (em). during a face-to-face meeting of the eular task force members, the concepts were rephrased and organised into a scheme of higher and lower level concepts, with input from the local investigators, prps and hps. descriptive statistics were used to summarise the characteristics of participants. 22 thematic analysis of qualitative data followed a modified form of 'meaning condensation', 23 facilitated by using atlas. ti software 24 to manage and organise the data. thematic analysis comprised the following steps (figure 1): all transcripts were screened and read. queries concerning content were sent to the country teams. transcripts were then divided into meaning units (defined as specific parts of text, either a few words or a few sentences with a common meaning). subsequently, initial codes were assigned to the meaning units. codes could refer to the main topic of a meaning unit, but one meaning unit could also contain more than one code. associated codes were then grouped into lower level concepts. in a final step, the lower level concepts were summarised into higher level concepts. rigour and accuracy of the analysis several strategies were used to enhance the trustworthiness of the qualitative data. 25 debriefing notes were recorded after each focus group interview. all local investigators who conducted focus groups checked the transcripts against the audio-recordings for accuracy. after analysing all focus group interviews, the results were discussed with researchers of all centres and reviewed by other task force members (prps and hps who were not involved in the analysis of the transcripts). finally, the consolidated criteria for reporting qualitative research checklist 26 was used to ensure the high quality of reporting the study results (online supplemental table 2). prps (wo, ib, nc, mk, ss and t-cw) were part of the task force and included in all stages of the study. furthermore, they will disseminate the results in lay language after publication. saturation was reached after conducting 12 focus groups (online supplemental table 3 ; see table 2 ). in total, 18 hours and 22 min of discussion were recorded, resulting in 269 pages of transcript. a general positive attitude towards proms all participants expressed a general positive attitude towards proms and acknowledged their importance in clinical practice. participants of all focus groups across all diseases described that proms had made a meaningful difference in their treatment, in that hps addressed important issues which impacted on daily life, based on proms. furthermore, proms were perceived as useful suggestions for adapting proms to young people forty-eight lower level concepts pertaining to suggested improvement of proms from the perspective of young people with ia were organised into six higher level concepts (table 3) . higher level concept one addressed the need for additional information regarding the purpose of using proms. young people in all countries and disease areas did not feel sufficiently informed about the value of proms and the reasons for collecting pros in addition to clinical outcomes. they thought that they were asked to fill in proms because it was commonly done, for study reasons only or to keep them busy while waiting at the outpatient clinic. participants in all 12 focus groups (100%) described uncertainties regarding the terminology used in the proms. they suggested simpler wording or clearer definitions, as young people were often confused, ashamed or even scared in case of difficulties to understand and would not ask for clarification. some participants also pointed out that the value of proms seems limited, if the hps have no time to discuss the prom results with them and if the results are not available for all members of the healthcare team. furthermore, some participants mentioned that in order to be suitable to young people, some of the items within proms would need to be updated to ensure they are relevant for young people in current times (higher level concept two). in particular, this related to the proms which address functioning in daily life. a young man expressed it as follows: i think it's just an old people's questionnaires, indeed. maybe we have kind of an old people's body, but with this you are really confronted with that. (male, 22, spa, the netherlands) moreover, young people considered a large number of functional items too 'easy', for example, walking, while other issues related to mobility and physical activity, such as participating in physical activities similar to people without chronic diseases of the same age, or sitting in front of a computer for the entire day, are not covered at all. likewise, eating was considered too easy, while preparing meals was missing. some participants explained that reference to 'easy' items had incited fear, implying that someone might not be able to walk in later stages of ia. interestingly, participants in one focus group (ra/ jia/still's disease, the netherlands) discussed the need for developing different proms for people of different ages. in comparison, all other participants suggested to extend the questionnaires to use the same proms for younger and older adults. issues important to young people should be added and regularly assessed (higher level concept three). these include problems with using technological devices, like smartphones and computers, difficulties with career plans, caring for others (for example, children), loss of friends, social life participation (such as going out in the evening), being excluded from physical activities at school and university, and challenges with regard to sexuality. the fourth higher level concept referred to planned, erroneous reporting from young people. some participants mentioned that they had purposely rated their pain, fatigue, disease activity or other symptoms differently from what they actually felt. some of them had rated better, others worse than the situation had been experienced. the reasons behind this were diverse and included intentions to trigger changes in their disease management, often as an attempt to more accurately demonstrate how they had been feeling since their last visit, or even mirrored undisclosed fears. however, these reports may have a severe impact on an individual's treatment, independent of the reason. a participant described the following example: well, i answer those questions more positively than how it goes, because i'm afraid they might think i'm depressed or something and send me to a psychiatrist. (female, 25, spa, the netherlands) higher level concept five focused on individualising proms. participants in the majority of the focus groups expressed that they would like to talk about their personal experience with ia and wished proms to be tailored to their individual needs and goals. in addition, length and comprehensiveness of proms were included in this higher level concept. some participants requested that several issues important and meaningful for young people with ia should be adequately addressed in the proms and taken up and discussed in the subsequent interactions with interdisciplinary healthcare teams. some of these participants even questioned the comprehensiveness of proms at all, since these tools can never encompass the entire spectrum and the full impact of ia on daily life. this theme was somewhat ambiguous, as other participants criticised the length of some proms, contradicting the suggestion of comprehensiveness. nonetheless, single item scales were seen as insufficient and too narrow in focus, compared with tools which comprehensively assess the impact of a disease in daily life by young people in all countries and disease areas. the sixth higher level concept covered the desire to use digital technology for data acquisition and access to prom data. participants were of the opinion that continuous monitoring could support young people in self-managing their health and well-being. moreover, participants felt that by using technology, hps would be more able to share the information about pros with other hps involved in the care of young people with ia. to our knowledge, this is the first study investigating the perspective of young people with ia on proms in a wider european context. although the use of proms was highly valued by young people, participants across all countries and disease groups expressed that proms often fail to sufficiently encompass the daily challenges of young people with ia and are often experienced as 'too easy', for example, the health assessment questionnaire 27 and the bath ankylosing spondylitis functional index. 28 these proms lack more 'difficult' items referring to the instrumental activities of daily living 29 which are essential for an independent life, especially at a younger age. in this context, item response theory (irt) and computerised adaptive testing are used for the development of innovative patient-reported instruments, such as the patient-reported outcomes measurement information system. 30 in their study, fries et al could show that physical function scales using a common metric or irt-based items can result in greater responsiveness and precision across a broader range of functioning. 31 some issues were seen controversially, for example, the length/shortness versus comprehensiveness of proms or developing age-appropriate proms versus using the same proms across the life course. although not all participants shared the same opinion, they agreed to complete proms no matter how long it would take, if they were adequately informed about the purpose of using these questionnaires and rating scales. the wish for more information, transparency and clarity regarding the reasons for using proms and their advantages in healthcare and research were also reported in previous studies with people of an older age. [32] [33] [34] [35] [36] therefore, simple and clear explanations for the use of proms can be seen as prerequisites in clinical practice and research to ensure that patients feel confident and provide accurate information. it is known that chronic diseases influence major lifechanging decisions related to social life, education, job, career choice and family planning. 37 participants in our study suggested that topics related to these areas should be discussed on a regular basis at the time of diagnosis and during subsequent follow-up visits with the healthcare team, for which proms could also be used. interestingly, topics like sexuality, intimate relationships, family planning and work were not mentioned in all focus groups, potential reasons being the different cultural backgrounds or the assumption that these topics were not as relevant as others to be raised during consultations. although it is advisable that private life aspects are increasingly explored among young people with rheumatic and musculoskeletal diseases (rmds) including ia, sensitivity is required to prevent unnecessary pressure on young people. discussing the responses of proms together with patients, analysing these responses over time, and including them in shared decisions were highly valued by those participants who had experienced these processes before and were also described in the literature by fautrel et al and chewning et al. 38 39 however, young people in all focus groups sometimes missed this valuable exchange of information due to time restrictions during consultations. therefore, some participants in our study suggested using new technology to capture patient-reported data to a larger extent, in order to eventually meet this challenge. for example, patients could complete proms ahead of their consultation via a smartphone, tablet or computer, so that the results could be viewed and discussed in clinic. collecting pros electronically, as well as sharing and discussing them with healthcare providers in a remote way, might be of great importance during times of a pandemic, such as experienced due to covid-19. many young people participating in our study also wished to talk about their personal experiences with their disease and expected proms to be tailored to their individual needs and goals. one outcome measure that is used both in clinical work and research to assess the effectiveness of treatment based on personally relevant goals is the goal attainment scale. 40 41 it is often used to define and evaluate personalised patients' treatment goals that are meaningful to patients in a standardised way. 42 our study enabled us to gain deep insights into the perspectives of young people with ia by means of a qualitative study using focus group interviews in four european countries. we included on purpose researchers and patients from one country from western (nl), central (at), southern (it) and southeastern (hr) europe in our study to ensure cultural diversity, but also include countries with different healthcare systems. interestingly, concepts that were important from the participants' point of view were the same, independent from the country data collected. our results might thus be transferable to some other countries as well. however, there might still be differences between different countries, especially outside europe, as only a limited number of countries were involved in our study. future studies could include other countries and/or other disease areas using a quantitative survey that builds on our results. to conclude, the results of our study will change the use of proms in young people with ia in clinical practice and research. young people with ia described a substantial potential for improving proms. first, optimising the current use of proms in their present form; and second, the potential for adapting proms so that they meet the current needs of young people. our study provides the basis for further research in the field of outcomes research, since the assessment of young people's perspectives should reach beyond the issues covered in proms used within rheumatology. accordingly, young people with ia and other rmd-related conditions should be involved in the development of new proms and the inflammatory arthritis inflammatory arthritis inflammatory arthritis potential adaptation of existing proms, to ensure that important concepts are included and address the entire course of a chronic disease. in broader terms, our findings may also be relevant to the use of proms in the context of other chronic diseases where individual needs, the perception of health and experience of symptoms vary during the course of life. patient-reported outcome measures: literature review patient reported outcome measures in practice the impact of patientreported outcome measures in clinical practice for pain: a systematic review health-related quality-of-life assessments and patient-physician communication: a randomized controlled trial impact of patient-reported outcome measures on routine practice: a structured review fda guidance on patient reported outcomes the cosmin study reached international consensus on taxonomy, terminology, and definitions of measurement properties for health-related patientreported outcomes patient-reported outcomes (pros) and patient-reported outcome measures (proms) the history of patient-reported outcomes in rheumatology incorporating the patient's perspective in outcomes research patient-reported outcomes in rheumatoid arthritis concepts of functioning and health important to people with systemic sclerosis: a qualitative study in four european countries do patient-reported outcome measures cover personal factors important to people with rheumatoid arthritis? a mixed methods design using the international classification of functioning, disability and health as frame of reference development and psychometric validation of a patient-reported outcome measure to assess fears in rheumatoid arthritis and axial spondyloarthritis: the fear assessment in inflammatory rheumatic diseases (fair) questionnaire concepts important to patients with psoriatic arthritis are not adequately covered by standard measures of functioning concepts important to persons with systemic lupus erythematosus and their coverage by standard measures of disease activity and health status qualitative research interviewing: biographic narrative and semi-structured methods descriptions of sampling practices within five approaches to qualitative research in education and the health sciences qualitative research in health care. analysing qualitative data world medical association declaration of helsinki: ethical principles for medical research involving human subjects developing questions for focus groups ibm spss statistics for windows interviews: an introduction to qualitative research interviewing atlas. ti scientific software development gmbh. atlas. ti [8.0] [program. berlin: atlas. ti scientific software development gmbh qualitative research in health care. assessing quality in qualitative research consolidated criteria for reporting qualitative research (coreq): a 32-item checklist for interviews and focus groups measurement of patient outcome in arthritis a new approach to defining functional ability in ankylosing spondylitis: the development of the bath ankylosing spondylitis functional index the hierarchical relationship between activities of daily living and instrumental activities of daily living advancing promis's methodology: results of the third patient-reported outcomes measurement information system (promis(®)) psychometric summit improved responsiveness and reduced sample size requirements of promis physical function scales with item response theory patients' views on routine collection of patient-reported outcomes in rheumatology outpatient care-a multicenter focus group study. arthritis care res2019 patient global assessment in measuring disease activity in rheumatoid arthritis: a review of the literature discrepancies between patients and physicians in their perceptions of rheumatoid arthritis disease activity i suppose that depends on how i was feeling at the time': perspectives on questionnaires measuring quality of life and musculoskeletal pain systematic review of patient-reported outcome measures (proms) for assessing disease activity in rheumatoid arthritis chronic diseases influence major life changing decisions: a new domain in quality of life research call for action: how to improve use of patient-reported outcomes to guide clinical decision making in rheumatoid arthritis patient preferences for shared decisions: a systematic review goal attainment scaling: a general method for evaluating comprehensive community mental health programs goal attainment scaling: applications, theory, and measurement goal attainment scaling in rehabilitation: a literature-based update psychometric properties of three single-item pain scales in patients with rheumatoid arthritis seen during routine clinical care: a comparative perspective on construct validity, reproducibility and internal responsiveness measurement of fatigue in cancer, stroke, and hiv using the functional assessment of chronic illness therapy -fatigue (facit-f) scale the mos 36-item short-form health survey (sf-36). i. conceptual framework and item selection acknowledgements we would like to thank all young people who took part in this study for sharing their valuable perspectives. we also thank dr elena picchiassi, ms linda van nieuwkoop, dr mirna reihl and ms gordana maligec for their support in data collection. personal fees from ciscrp, personal fees from janssen, personal fees from parexel, outside the submitted work; tas reports grants from abbvie, grants and personal fees from roche, personal fees from sanofi genzyme, personal fees from takeda, outside the submitted work. ethics approval the study was approved by the local ethics committees including the ethics committee of the medical university of vienna, austria ek number 2117/2017. all participants were informed about the purpose and procedures of the study and gave their written informed consent in accordance with the declaration of helsinki.provenance and peer review not commissioned; externally peer reviewed.data availability statement no data are available. this is a qualitative study and therefore the data generated are not suitable for sharing beyond that contained within the report. further information can be obtained from the corresponding author.open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ 4. 0/. key: cord-0016807-apofatya authors: wang, juan; yang, zhe; zheng, yan; peng, yaling; wang, qing; xia, hongli; wang, yan; ding, jin; zhu, ping; shang, lei; zheng, zhaohui title: effects of illness perceptions on health-related quality of life in patients with rheumatoid arthritis in china date: 2021-04-20 journal: health qual life outcomes doi: 10.1186/s12955-021-01770-4 sha: 52c201ea6b40a65eb2bf02bb8e013480f9b33a87 doc_id: 16807 cord_uid: apofatya objectives: for patients with rheumatoid arthritis (ra) in china, little is known of how their illness perceptions affect their health-related quality of life (hrqol). the present study investigated associations between specific illness perceptions due to ra and hrqol features. methods: for 191 patients with ra, illness perceptions were measured using the brief illness perceptions questionnaire (bipq) comprising 8 domains. hrqol was determined with the medical outcomes study 36-item short-form health survey (sf-36). multivariate linear regression analyses were performed. results: the overall bipq of patients with ra was 49.09 ± 11.06. the highest and lowest scores were for concern (9.15 ± 1.81) and personal control (4.30 ± 2.52), respectively. multivariate stepwise regression analyses showed that the overall bipq was significantly negatively associated with each hrqol feature, and hrqol total score (β = − 0.343, p < 0.001, 95% ci − 7.080 to − 4.077). positive associations between bipq features and hrqol included personal control (β = 0.119, p = 0.004, 95% ci 2.857–14.194) and treatment control (β = 0.084, p = 0.029, 95% ci 0.640–12.391). negative associations with hrqol were identity (β = − 0.105, p = 0.034, 95% ci − 13.159 to − 0.430) and emotional response (β = − 0.207, p < 0.001, 95% ci − 18.334 to − 6.811). conclusions: patients with ra in china perceive their illness in ways that affect their hrqol. these results suggest that strategies that target these perceptions may improve the quality of life of these patients. rheumatoid arthritis (ra) is a chronic disease characterized by symmetric polyarticular arthritis. it is the most common autoimmune disease. clinical manifestations of ra include joint swelling, pain, and limited function [1, 2] . progression of ra may eventually lead to joint deformities which seriously affect patients' quality of life [3, 4] . in ra, each patient's presentation and course of disease is unique. to understand the individualized course of chronic diseases, there has been growing interest in common sense models. these tested models suggest that the patient's perception of illness, that is, their own cognitive and emotional responses, direct their response to that illness [5] [6] [7] [8] [9] . individuals actively try to make sense of their symptoms and form personal beliefs about their illness. these beliefs, in turn, determine their subsequent coping behavior and quality of life [10] . health-related quality of life (hrqol) may be measured to reveal the physiological, psychological, and social functions of patients with ra [3, 11, 12] . illness perception is associated with quality of life, social function, and disease prognosis [13] [14] [15] [16] [17] [18] . such knowledge is applied to guide clinicians in the management of ra, to improve the hrqol and the prognosis of their patients. in rheumatology, the effect of illness perception has been studied extensively in western populations, specifically for ra [19, 20] , systemic sclerosis [21] , lupus nephritis [22, 23] , psoriatic arthritis [16] , multiple sclerosis [24] , and systemic lupus erythematosus [25, 26] . research has highlighted the importance of the beliefs of patients with ra about their illness and symptoms as they affect their hrqol. the identification of these patients' perceptions could positively influence quality of life, as illness perception is amenable to intervention [27] . however, to our knowledge, there have been no studies on illness perception and its association with hrqol for patients with ra in china. the purpose of the present study was to identify those perceptions of illness that influence patients' quality of life, to better guide clinicians in the management of ra, and improve the ability of patients with ra to self-manage and improve their hrqol. this cross-sectional study was conducted in an outpatient clinic at xijing hospital, xi'an, shaanxi, china from march 2017 to december 2017. the ethics committee of xijing hospital approved the study (ky20140902-5), and all subjects provided written informed consent prior to their participation. the study included patients with ra diagnosed according to the acr (american college of rheumatology)/ eular (european league against rheumatism) 2010 classification criteria [28] . all the patients were at least 18 years of age; able to understand and communicate in chinese; and willing to participate. patients with any of the following were excluded: suffering from other chronic diseases; recent major surgery; unstable condition; or intellectual or cognitive impairments. this study is based on the infinite population sampling formula:n = u α/2 σ /δ 2 , where u α/2 = 1.96 , σ = 5 , and δ = 0.7 . the values σ and δ refer to the literature related to illness perceptions [27] ; the sample size was calculated as 196. sociodemographic and clinical data were collected through face-to-face interviews with the patients. disease activity estimates were based on the disease activity score 28 (das28): an index of physician-rated tenderness and swelling scores for 28 joints and an inflammatory biomarker (erythrocyte sedimentation rate or c-reactive protein) crp as the inflammatory index [29] . illness perceptions and hrqol were assessed via patient-reported outcome measures. the brief illness perceptions questionnaire (bipq) assesses an individual's perceptions and cognitions regarding their disease [7, 30] . the chinese bipq has been tested and validated previously [31, 32] . the questionnaire measures the following 8 domains of illness perception: consequences; timeline; personal control; treatment control; identity; concern; coherence; and emotional response. the 8 bipq domains that together reflect an individual's perception of their disease. the score of each domain may range from 0 to 10. the overall bipq score ranges from 0 to 80, a higher score reflects a more negative view of the illness. hrqol was measured with the chinese version of the medical outcomes study 36-item short-form health survey (sf-36). the sf-36 consists of 36 items that measure the following 8 dimensions: physical function (pf); role limitations related to physical problems (rp); bodily pain (bp); general health perception (gh); vitality (vt); social functioning (sf); role limitations due to emotional problems (re), and mental health (mh). the score of each dimension is converted to a standard score ranging from 0 to 100, with a highest score indicating the best hrqol [33] . the sf-36 has shown good reliability and validity among various chinese patient populations [34, 35] . statistical analyses were performed using statistical package for social science 18.0 (spss, chicago, il) software. the descriptive statistics are presented as the mean and standard deviation for quantitative data, and percentage for count data. the independent samples t-test and oneway analysis of variance were used to analyze inter-group differences with normal distribution. linear regression analyses were used to test the univariate correlations between the domains of illness perception and hrqol, and to screen the significant independent variables (p < 0.1) for subsequent multivariate regression analyses. multivariate stepwise regression analysis was used to explore the effect of illness perceptions on hrqol. after controlling for demographics and disease characteristics, independent variables were entered stepwise into the model. p values < 0.1 were added into the regression, and only p values < 0.05 were considered statistically significant. the total score for quality of life was normally distributed (p = 0.066); the scores of each dimension did not conform to a normal distribution and were analyzed after normalization conversion (p < 0.001). the questionnaires were distributed to 200 eligible patients. because 9 questionnaires were missing data, there were finally 191 study participants. the average age of the participants was 45.06 ± 13.32 years, 140 (73.30%) were women (table 1 ), 37.7% had a disease duration of more than 5 years, 26.18% were in remission, and 16.75% had severe disease. the overall bipq score of the participants was 49.09 ± 11.06. the scores of each dimension of the bipq were as follows: concern (9.15 ± 1.81); timeline (7.68 ± 2.78); treatment control (7.83 ± 2.3); emotions (7.15 ± 2.9); consequences (6.72 ± 3.02); identity (6.61 ± 2.74); coherence (6.08 ± 2.60); and personal control (4.30 ± 2.52). concern and timeline received the highest, while personal control was the lowest. for the unstratified (overall) population, the sf-36 scores of the 8 dimensions, from highest to lowest, were social functioning, mental health, physical function, vitality, bodily pain, general health perception, role limitations due to emotional problems, and role limitations related to physical problems. compared with the younger subgroup (age < 45 y), the older subgroup had significantly lower scores for physical function, vitality, and social functioning (p < 0.05, = 0.018, and = 0.039, respectively). when stratified by occupation (unemployed, blue, and white collar) and 3 levels of education, each dimension differed significantly among the subgroups except for role limitations due to emotional problems. significant differences in sf-36 scores based on disease duration were found in all dimensions except general health perception, role limitations due to emotional problems, and mental health. when the population was stratified by das28 (remission, low, moderate, or high), each dimension differed significantly among these subgroups (p < 0.05, table 2 ). based on the linear regression analysis of the sf-36 dimensions, age and disease duration were each significantly associated with every sf-36 dimension, except mental health; and das28 was significantly associated with every sf-36 dimension ( table 3) . the overall bipq score was significantly associated with every sf-36 dimension, as was the following individual bipq domains: consequences, personal control, identity, and emotional response. timeline was significantly associated with every sf-36 dimension, except mental health. treatment control was associated with general health perception and role limitations due to emotional problems. illness concern was associated with bodily pain, general health perception, and vitality. coherence was associated only with physical function. only the bipq domains treatment control and coherence were not significantly associated with the total sf-36 score (table 3) . to evaluate the effects of certain demographics and illness perceptions on various dimensions of hrqol in chinese patients with ra, each hrqol dimension was taken as a dependent variable, while the general characteristics and all bipq dimensions were considered independent variables in the multivariate stepwise regression analysis of hrqol. according to the results (tables 2 and 3) , only the variables with a significant association (p < 0.1) were selected as the independent variables for multivariate stepwise regression (table 4) . four models were analyzed, with different variables. model 1 included demographic variables (gender, age, education, type of residence, family income, and employment). age, education, and occupation could account for 17.1% of the variance of the total sf-36 score (model 1, r 2 = 0.171, f = 12.855, p < 0.001). the selection of independent variables for multiple regression analyses was based on these analyses (tables 2 and 3 in addition, the bipq domains personal control, treatment control, identity, and emotional response were associated with hrqol when the demographic factors and disease characteristics were controlled. the bipq domains explained 9.0% of the variance of the total sf-36. identity and emotional response were negatively associated with hrqol, however personal control and treatment control were positively associated with hrqol. furthermore, consequences were associated with vitality, and coherence with mental health (model 4, p < 0.05). the total bipq had a significant negative association with each component of the sf-36, and also the total sf-36 (model 3). this study investigated the illness perception and hrqol of patients with ra in china, and associations between illness perception and each domain of hrqol. it was found that the total bipq had a significant negative association with sf-36: as scores of illness perception increased, the quality of life worsened. thus, illness perceptions were identified as likely targets for strategies to improve the quality of life of patients with ra. this is the first report of the illness perceptions of patients with ra in china. the overall bipq score was 49.09 ± 11.06, which was higher than reported by a study from greece (40.08 ± 1.06) [18] . this showed that the patients with ra in the present cohort had a more negative view of the disease than did patients in greece. in western countries, the domain with the highest bipq score was timeline, and the lowest was identity [16] [17] [18] . the present study found that the highest bipq score was for illness concern and timeline. our results showed that the timeline score was higher, in accord with previous studies on ra [17, 18] . although the survey populations differed, these study comparisons show that patients with ra in china and in western countries generally recognize that ra is a chronic disease with a long course. the present study also found that patients older than 45 years had significantly lower scores for physical function, vitality, and social functioning compared with younger patients. patients with a disease duration more than 5 years, and more serious disease activity, had the worst hrqol. patients with higher education and engaged in white collar occupations had better hrqol compared with patients with less education or poorer employment. a literature review reported that increased age was associated with reduced physical function and physical component summary scores for hrqol [11] . the present analysis also showed that age was negatively associated with physical function, bodily pain, general health perception, vitality, and social functioning. in addition, the multivariate analyses showed that disease activity was negatively associated with each of the sf-36 components. education and das28 were associated with total hrqol (model 2, r 2 = 0.650, f = 174.913, p < 0.001). it must be noted that clinical characteristics accounted for 47.9% of the variance in total hrqol, in addition to the demographic variables (table 4 ). these findings revealed that disease activity had a dramatic effect on the hrqol of patients with ra. the data in the present study support that increased disease activity is associated with reduced hrqol in ra [36, 37] . ra has also been noted to affect the hrqol of patients, by the clinical manifestations of the disease, and by socioeconomic, personal, and environmental factors [4] . berner et al. [27] reported that illness perception accounted for 51% and 45% of variance in physical and mental hrqol, respectively. however, we found that total bipq was negatively associated with each of the sf-36 components (model 3). in addition, the domains of the bipq explained 9.0% of the variance in total sf-36, besides clinical characteristics and demographic variables (model 4, r 2 = 0.740, f = 87.37, p < 0.001). although this association is not strong, it obviously has importance. the reason for such discrepancy may be the different study populations and recruitment methods. it has also been reported that sleep impairment is a common clinical condition in patients with ra [38, 39] , and has been confirmed to affect quality of life [40] , the majority of chinese patients with ra suffer from poor sleep and impaired quality of life [41] . fatigue is highly prevalent in individuals with ra, and is perceived to have a significant detrimental effect on health status, and physical and social functioning [44] , these are significant factors that affect the quality of life in patients with ra [12, 42] , and further study is warranted. hyphantis et al. [17] reported that perceived consequences of the disease were independent correlate of physical hrqol. the present study found that perceived consequences were associated specifically to vitality in hrqol. the high scores indicated that patients with strongly held beliefs regarding the serious consequences of ra had worse vitality. kotsis et al. [16] reported that anxiety, depression, and identity were associated with hrqol in patients with ra, but not personal control or treatment control. however, the present multivariate analysis showed that personal control and treatment control were positively associated with hrqol. effective treatment and the ability of the patient toward selfmanagement can control the disease, and improve the hrqol. the difference in results may be related to differences in the survey population and the survey scales. a review regarding illness perceptions concluded that negative emotions can affect the treatment of diseases, limit the physiological functions of patients, and seriously affect the prognosis of diseases. illness perceptions, depression, anxiety, and quality of life were closely related [43] . illness perception is associated with depression in patients with chronic illness [44] . lu et al. [45] reported that components of illness perception were associated with negative emotions in depressed patients. during the covid-19 pandemic, patients with ra were reported to experience severe anxiety and depression [46] . we propose that during this time patients with ra should be encouraged to enhance their illness perception and selfmanagement. in so doing, their personal control of ra may increase, with fewer negative emotions, and hrqol improve despite the pandemic. this deserves further study. ho et al. [44] reported that up to 52% of patients with ra experienced symptoms of anxiety and/or depression [44] . liu et al. [47] found that the il-17 level positively correlated with the severity of anxiety in patients with ra. a meta-analysis showed that patients with ra with depression tended to have lower quality of life than patients without depression [48] . depressed patients with ra have more pain [49] , high disease activity [50] , and reduced hrqol [2] . the present study also found that emotional response in patients with ra was negatively associated with hrqol (physical function, general health perception, vitality, social functioning, role limitations due to emotional problems, and mental health). in addition, identity was negatively associated with hrqol (role limitations related to physical problems, bodily pain). our finding emphasizes that patients with ra who experience serious negative emotions caused by their disease usually have a heavier mental burden and worse quality of life. this study specifically showed that illness perceptions influenced different dimensions of hrqol in patients with ra. patients with the worst illness perceptions had the worst hrqol. previous research in ra showed that illness perceptions had significant implications for adaptation to illness and notably affected medical disease status, even more so than depression, physical function, or pain [51] . berner et al. [27] highlights the importance of patients' beliefs about their illness and symptoms in relation to hrqol. identification of patients' perception of ra may be a way to influence quality of life for the better. health interventions based on understanding and modifying perceptions of illness proved useful in facilitating patient's hrqol [51, 52] . illness perceptions can change over time, and these changes affect patients' outcomes [53] . patients with ra may benefit from illness perception modification. future evidence-based interventions that focus on illness perception are required to enhance the hrqol of patients with ra [54, 55] . there are some limitations to this study. the cases were from a single hospital and the sample size was relatively small. longitudinal studies are needed before further conclusions can be drawn. future studies should add variables such as sleep disturbance, depression, anxiety, and fatigue, and explore how these factors and illness perceptions can affect hrqol. illness perceptions were associated with the hrqol in this study of a patient population with ra in china. patients with the worst illness perceptions had the worst hrqol. illness perceptions are important potential targets to improve the quality of life of patients with ra. illness perceptions may therefore be a useful basis for future quality of life interventions. concurrent psychiatric disorders are associated with significantly poorer quality of life in patients with rheumatoid arthritis disease activity, resilience and health-related quality of life in chinese patients with rheumatoid arthritis: a multicenter, cross-sectional study functional disability associated with disease and quality-of-life parameters in chinese patients with rheumatoid arthritis illness perceptions among cancer survivors illness perceptions, adjustment to illness, and depression in a palliative care population the brief illness perception questionnaire the common-sense model of self-regulation of health and illness: how can we use it to understand and respond to our patients' needs? why illness perceptions matter illness perception in association with psychological functioning in patients with discoid lupus erythematosus the impact of rheumatoid arthritis on quality-of-life assessed using the sf-36: a systematic review and meta-analysis health-related quality of life and its predictors among patients with rheumatoid arthritis illness perceptions and coping determine quality of life in copd patients illness perception in patients with coronary artery disease: a systematic review illness perceptions and quality of life in japanese and dutch patients with non-small-cell lung cancer anxiety and depressive symptoms and illness perceptions in psoriatic arthritis and associations with physical health-related quality of life the relationship between depressive symptoms, illness perceptions and quality of life in ankylosing spondylitis in comparison to rheumatoid arthritis illness perceptions and psychological distress associated with physical health-related quality of life in primary sjogren's syndrome compared to systemic lupus erythematosus and rheumatoid arthritis mood and anxiety disorders in systemic sclerosis patients the course of depression in recent onset rheumatoid arthritis: the predictive role of disability, illness perceptions, pain and coping systemic sclerosis: patients' perceptions of their condition relationship of illness perceptions with depression among individuals diagnosed with lupus illness perceptions in patients with systemic lupus erythematosus and proliferative lupus nephritis illness perception, treatment beliefs, self-esteem, and self-efficacy as correlates of self-management in multiple sclerosis the impact of illness perceptions on sexual functioning in patients with systemic lupus erythematosus illness perception is significantly determined by depression and anxiety in systemic lupus erythematosus a cross-sectional study on self-reported physical and mental health-related quality of life in rheumatoid arthritis and the role of illness perception rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative modified disease activity scores that include twenty-eightjoint counts. development and validation in a prospective longitudinal study of patients with rheumatoid arthritis the brief illness perception questionnaire psychometric assessment of the chinese version of the brief illness perception questionnaire in breast cancer survivors negative illness perceptions associated with low mental and physical health status in general hospital outpatients in china the mos 36-item short-form health survey (sf-36). i. conceptual framework and item selection effects of social support, hope and resilience on quality of life among chinese bladder cancer patients: a cross-sectional study health-related quality of life and utility: comparison of ankylosing spondylitis, rheumatoid arthritis, and systemic lupus erythematosus patients in taiwan association between helplessness, disability, and disease activity with health-related quality of life among rheumatoid arthritis patients in a multiethnic asian population the association between anxiety and disease activity and quality of life in rheumatoid arthritis: a systematic review and meta-analysis sleep quality and correlates of poor sleep in patients with rheumatoid arthritis sleep loss exacerbates fatigue, depression, and pain in rheumatoid arthritis role of sleep disturbance, depression, obesity, and physical inactivity in fatigue in rheumatoid arthritis sleep quality in chinese patients with rheumatoid arthritis: contributing factors • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year submit your research ? choose bmc and benefit from: ? choose bmc and benefit from: and effects on health-related quality of life sleep impairment: an obstacle to achieve optimal quality of life in rheumatoid arthritis a systematic review and meta-analysis of the brief illness perception questionnaire clinical and psychosocial factors associated with depression and anxiety in singaporean patients with rheumatoid arthritis a regressional analysis of maladaptive rumination, illness perception and negative emotional outcomes in asian patients suffering from depressive disorder psychological state and associated factors during the 2019 coronavirus disease (covid-19) pandemic among filipinos with rheumatoid arthritis or systemic lupus erythematosus the role of interleukin (il)-17 in anxiety and depression of patients with rheumatoid arthritis depression has an impact on disease activity and health-related quality of life in rheumatoid arthritis: a systematic review and meta-analysis types of pain and their psychosocial impact in women with rheumatoid arthritis. women's midlife health the correlations of socioeconomic status, disease activity, quality of life, and depression/ anxiety in chinese patients with rheumatoid arthritis the role of perceived and actual disease status in adjustment to rheumatoid arthritis illness perceptions and coping in physical health conditions: a meta-analysis illness perceptions in patients with osteoarthritis: change over time and association with disability 2016 update of the eular recommendations for the management of early arthritis eular recommendations for the management of early arthritis: report of a task force of the european standing committee for international clinical studies including therapeutics (escisit) authors' contributions jw designed the study and wrote the paper, and zy analyzed the data. jw and zy contributed equally to this work. zhz, ls, and pz corrected the manuscript, and share corresponding authorship. all authors participated in the questionnaire collection, data input, and read and approved the final version of this manuscript for submission for publication. all authors contributed to the study conception and design. the authors ls, jd, and pz received funds for this study from the national natural science foundation of china, nos. 81773540; 81701617; and the national science and technology major projects for new drugs, no. 2017zx10201302-003. the data used are under license for the current study. they are not publicly available. ethical approval was obtained from the xijing hospital ethics committee. ethics approval code: (ky20140902-5). informed consent was obtained from all individual participants included in the study. the authors declare that they have no competing interests.received: 3 september 2020 accepted: 14 april 2021 key: cord-0025437-0tntvkwn authors: brkic, alen; diamantopoulos, andreas p.; haavardsholm, espen andre; fevang, bjørg tilde svanes; brekke, lene kristin; loli, liz; zettel, camilla; rødevand, erik; bakland, gunnstein; mielnik, pawel; haugeberg, glenn title: exploring drug cost and disease outcome in rheumatoid arthritis patients treated with biologic and targeted synthetic dmards in norway in 2010–2019 – a country with a national tender system for prescription of costly drugs date: 2022-01-10 journal: bmc health serv res doi: 10.1186/s12913-021-07425-w sha: ba41f8e8d2f7f2475997a5044e2cdc163e586124 doc_id: 25437 cord_uid: 0tntvkwn background: in norway, an annual tender system for the prescription of biologic and targeted synthetic disease-modifying antirheumatic drugs (b/tsdmards) has been used since 2007. this study aimed to explore annual b/tsdmards costs and disease outcomes in norwegian rheumatoid arthritis (ra) patients between 2010 and 2019 under the influence of the tender system. methods: ra patients monitored in ordinary clinical practice were recruited from 10 norwegian centers. data files from each center for each year were collected to explore demographics, disease outcomes, and the prescribed treatment. the cost of b/tsdmards was calculated based on the drug price given in the annual tender process. results: the number of registered ra patients increased from 4909 in 2010 to 9335 in 2019. the percentage of patients receiving a b/tsdmard was 39% in 2010 and 45% in 2019. the proportion of b/tsdmards treated patients achieving das28 remission increased from 42 to 67%. the estimated mean annual cost to treat a patient on b/tsdmards fell by 47%, from 13.1 thousand euros (eur) in 2010 to 6.9 thousand eur in 2019. the mean annual cost to treat b/tsdmards naïve patients was reduced by 75% (13.0 thousand eur in 2010 and 3.2 thousand eur in 2019). conclusions: in the period 2010–2019, b/tsdmard treatment costs for norwegian ra patients were significantly reduced, whereas das28 remission rates increased. our data may indicate that the health authorities’ intention to reduce treatment costs by implementing a tender system has been successful. supplementary information: the online version contains supplementary material available at 10.1186/s12913-021-07425-w. inflammatory joint disorders, e.g., rheumatoid arthritis (ra), where remission is now an attainable treatment goal [1] [2] [3] [4] . however, the high cost of b/tsdmards has caused restrictions on the usage of these drugs, contributing to inequality of care worldwide [5] [6] [7] . in some countries (e.g., norway and denmark) with a public tax-funded healthcare system, tender systems, and the possibility of a mandatory switch to potentially cheaper biosimilar drugs have been implemented to reduce the drug expenditure (particularly for costly drugs). to our knowledge, this is the first study to explore changes in b/tsdmard treatment costs set against changes in disease outcomes in ra following the implementation of a tender system. this study aimed to explore treatment cost and disease outcomes in ra patients treated with b/tsdmards in norway during a 10-year period (2010 to 2019) with a tender system in effect. data were obtained from the biorheuma project (biologic treatment of patients suffering from inflammatory rheumatic disorders in norway) that started in 2010. the objective of the biorheuma project was to facilitate the use of recommended and validated outcome measures to monitor patients with inflammatory joint disorders as part of ordinary care in norwegian outpatient clinics. patient monitoring at the participating centers was standardized using the computer tool gotreatit ® rheuma (www. diagr aphit. com). the clinical expectations of the project were to reveal annual changes in the usage of conventional synthetic dmards (csdmards) and b/ tsdmards, viewed against changes in demographics, disease activity, and patient-reported outcome measures (proms) during follow-up. the 10 biorheuma centers providing data for this study were located across the country (bergen, baerum, førde, haugesund, kristiansand, lillehammer, oslo, skien, tromsø, and trondheim). we estimated the completeness of included patients from each center by comparing with published prevalence figures for ra in norway [8, 9] . biorheuma prevalence figures were calculated using the number of included ra patients at each center divided by the background population the various centers were covering. for each of the 10 years, data was extracted from each participating center's database using predefined queries. one query retrieved ra patients registered with at least one visit in the examined year. data from the latest visit was used if multiple visits occurred in that year. another query retrieved all patients starting on either bdmard or tsdmard for the different years. anonymized data files from the 10 participating centers were merged and analyzed using excel and the statistical package for social sciences (spss). data collection for each year included demographic variables, diagnosis-related variables, disease activity measures, proms, and ra treatment medications. demographic variables include patient age, sex, body mass index (bmi, kg/m2), current smoking status, years of education, disease duration, and occupational status. the occupational status of participants younger than 65 years was categorized as enabled workers or disabled workers. patients who reported their occupational status as a full-time job, part-time job, student, maternity leave, paternity leave, sick leave, unemployed, early retirement, part-time job/sick leave, part-time job/unemployed were defined as "enabled workers. " in contrast, patients who reported part-time job/disabled pensioner, disabled pensioner, disabled pensioner due to ra, medical rehabilitation, and occupational rehabilitation were defined as "disabled workers. " participants ≥ 65 years were omitted and defined as pensioners. disease duration was calculated from the date of diagnosis until the latest visit at the outpatient clinic for the examined year. diagnosis-related variables include rheumatoid factor (rf) and anti-cyclic citrullinated peptide (anti-ccp). measures reflecting disease activity encompass laboratory measures (erythrocyte sedimentation rate (esr), c-reactive protein (crp), the clinical measures 28 swollen and tender joint count (28sjc/28tjc), investigator global assessment (iga) scored on a visual analog scale (vas; 0-100 mm), and composite 28 joint count disease activity score using crp (das28) [10] . the proms included were pain, patient global assessment (pga), and fatigue scored on a vas-scale (0-100 mm), as well as morning stiffness (reported in 15-min units) and modified health assessment questionnaire (mhaq) [11] to evaluate the physical function of the ra patients. among available composite scores, das28 was used to define the disease activity status with the following cutoff values; remission ≤2.6, low disease activity between > 2.6 and ≤ 3.2, moderate disease between > 3.2 and ≤ 5.1, and high disease activity for those > 5.1 [10] . for each of the 10 years, the annual total cost for b/tsd-mards as well as mean b/tsdmard cost per patient was calculated for all patients receiving ongoing b/tsd-mards (current b/tsdmard users), for those who started on their first b/tsdmard (naïve b/tsdmard users) and for those who started on a new b/tsdmards but were previous users of b/tsdmards. the cost was calculated based on price offers given for the separate drugs at the annual tender process for the given year. adjusted cost was also calculated using the norwegian consumer price index (cpi) for pharmaceuticals from 2010 norwegian kroners (nok) [12] . only average prices (no drug-specific prices) are presented due to an agreement between the pharmaceutical companies and the norwegian authorities to keep the costs for individual drugs confidential and exempt from the public. due to the challenging covid-19 pandemic situation, clinical data for 2020 was not collected, but the cost for 2020 was calculated using 2019 population data. all costs were converted to euros (eur) based on the average nok-to-eur conversion rate between 2010 and 2020 (1 nok = 8.839 eur). the b/tsdmards included were tumor necrosis factor inhibitors (tnfi) (etanercept reference, etanercept sb4, infliximab reference, infliximab ct-p13, adalimumab, golimumab, certolizumab pegol), non-tnfi (rituximab reference, rituximab gp2013, abatacept, and tocilizumab), and tsdmards (baricitinib and tofacitinib). for 2020 the biosimilars infliximab gp1111 and adalimumab gp2017 won the tender and were used in the cost analysis for 2020. data collection also included the use of csdmards and prednisolone. categorical variables are reported as numbers and percentages and continuous variables as mean with standard deviation (sd), or mean with range. change and association between variables over the 10-year period were analyzed with spss using one-way analysis of variance (anova) for continuous variables and the chi-square test for categorical variables. only available data were used without imputation of missing data. a p-value of < 0.05 was considered statistically significant. the study was approved by the regional ethical committee (rec) (regional etisk komite midt-norge 2010/3078) and follows the declaration of helsinki ethical principles of medical research involving human subjects. no consent from patients was required by the rec, as all data were anonymized and collected as part of routine clinical care. the number of ra patients registered in the biorheuma project in the 10-year period ranged from 4909 patients in 2010 to a maximum of 9335 in 2019, and the percentage of patients registered as b/tsdmard users increased from 40% (n = 1959) to 45% (n = 4209), respectively. in table 1 , annual results are shown for demographics, biomarkers, disease activity, and prom variables for current users of b/tsdmards. the percentage of patients currently treated with b/tsdmards increased from 39% in 2010 to 45% in 2019. an improvement was seen for disease activity measures, mhaq, and fatigue, but not for pga, pain, and morning stiffness. the proportion of patients in das28 remission who received a b/tsdmard increased from 42% in 2010 to 67% in 2019. the percentage of enabled workers did not change significantly, ranging from 63% in 2010 to 59% in 2019. a supplementary table (see additional file 1) compares mean values and range for the 10 years between b/ tsdmard-treated patients and non-b/tsdmards ra patients. in general, no relevant differences for disease activity measures and proms were seen between b/ tsdmards and non-b/tsdmards treated ra patients. however, more b/tsdmards treated patients were rf and ccp positive. numerically only minor, yet statistically significant differences were found for most demographic variables. however, disease duration was markedly longer for b/tsdmards than non-b/tsd-mards treated patients (14.0 vs. 8.9 years, p = < 0.001). baseline values for demographics, disease activity, and proms are shown in table 2 for naïve b/tsdmards users and in table 3 for patients starting subsequent b/ tsdmard. for patients naïve to b/tsdmards, disease duration was the only demographic variable with a significant change during the 10 years. in contrast, significant changes were found for all demographic variables apart from work status in the non-naïve group. both in naïve and non-naïve treatment groups, the disease activity level at the start of a new b/tsdmard treatment decreased from 2010 to 2019. for naïve users, the mean das28 was 5.0 in 2010 and 3.8 in 2019, whereas das28 fell from 5.3 in 2010 to 3.8 in 2019 in the nonnaïve group. a statistically significant difference was found for all prom variables for non-naïve patients. however, in ra patients naïve to b/tsdmards, there were non-significant changes in vas for pain and fatigue. the total treatment expenditure for b/tsdmards was lowest in 2010 (treating 1959 ra patients) with 25.6 million eur, highest in 2014 (39.6 million eur for treating 3448 patients), and second lowest in 2019 (28.9 million eur for treating 4209 patients). detailed information is shown in table 4 for current users of b/tsdmards and the subgroups tnfi, non-tnfi, and tsdmards for the different 10 years. table 4 also shows the numbers treated, the cost of b/tsdmards drugs started in the different years (for all and those naïve to b/tsdmards), and the subgroup tnfi non-tnfi and tsdmards. age (years) 60 (13) 60 (14) 59 (14) 59 (14) 59 (14) 59 (14) 59 (14) 59 (14) 60 (14) 60 (14) 59 (10) 13 (11) 14 (11) 14 (11) 14 (11) 14 (11) 15 (11) 14 (11) 15 (11) 15 ( 1.4 15%, 9-19% < 0.001 iga (vas, 0-100 mm) 18 (16) 18 (16) 17 (16) 16 (15) 16 (15) 15 (15) 14 (15) 14 (15) 13 (15) 12 (14) 15 pga (vas, 0-100 mm) 33 (24) 33 (25) 33 (25) 32 (25) 32 (25) 33 (25) 33 (25) 32 (26) 32 (26) 32 (26) 33 pain (vas, 0-100 mm) 32 (25) 34 (25) 33 (24) 32 (25) 32 (25) 33 (25) 32 (25) 32 (26) 32 (25) 32 ( 38 (29) 37 (29) 38 (29) 37 (29) 38 (30) 39 (30) 39 (30) 39 (30) 40 ( age (years) 57 (15) 56 (15) 57 (14) 55 (15) 55 (15) 55 (14) 56 (15) 54 (16) 55 (15) 55 (15) 39.1 (19) 38 (18) 38 (18) 37 (18) 38 (20) 36 (18) 33 (19) 32 ( (25) 47 (25) 48 (25) 47 (24) 47 (27) 47 (25) 45 (26) 45 (27) 57 (14) 58 (14) 57 (15) 55 (14) 578 (15) 59 (14) 58 (14) 58 (15) 59 (14) 58 0.3%, 0-3% 13 (10) 14 (11) 13 (10) 13 (11) 13 ( 474 [24] 748 [26] 840 [27] 826 [27] 979 [28] 1189 1183 [30] 1084 [26] 28% ( 218 [17] 232 [25] 133 [20] 321 [30] 35% ( 190 [29] 227 [21] 19% (6.0-29%) the mean cost to treat a current ra user with b/tsd-mards decreased by approximately 47% from 13.1 thousand eur in 2010 to 6.9 thousand eur in 2019 (table 4) . for both naïve and non-naïve b/tsdmard users, the annual mean cost was markedly reduced from 2010 to 2019 by approximately 75 and 64% (13,0 thousand to 3.2 thousand and from 12.9 thousand to 4.6 thousand, respectively). adjusted for cpi as displayed in table 4 , the reduction from 2010 to 2019 was even higher: for mean current users 56%, naïve users 80%, and non-naïve users 70%. when applying the tender results from 2020 on the 2019 population, the reduction was even higher with the estimated annual mean cost for current b/tsd-mards users 5.8 thousand eur and for naïve users 2.4 thousand eur, which yields a cost reduction from 2010 of 56 and 82% and adjusted for cpi 64 and 85%, respectively. figure 1a visualizes the change in total costs for treating ra patients with b/tsdmards for current users and for naïve and non-naïve starters of b/tsdmards and numbers of treated patients. figure 1b shows the mean cost to treat one patient in the three groups. the estimated ra-prevalence based on biorheuma data for each year and center is shown in a supplementary table (see additional file 2). in 2019 the estimated overall prevalence (≥20 years old) was 0.3%, ranging at the single centers from 0.2 to 0.5%. the main finding in this study is an estimated 47% reduction (56% cpi-adjusted) in the annual per-patient cost of b/tsdmard from 2010 to 2019 in norway. during this period, a national tender system for the prescription of b/ tsdmards was implemented. the estimated annual cost reduction for naïve b/tsdmard users was 75% (79.5% cpi-adjusted). cost simulation using 2020 tender results on the 2019 population treatment data found that reduction increased further to 82% (85% cpi-adjusted) from 2010 for naïve patients. the findings in our study suggest that the implemented tender system for b/tsdmard procurements in norway for the last 10 years may have facilitated positive competition between pharmaceutical companies and thus served as a market mechanism to reduce prices. the norwegian pharmaceutical procurement cooperation, a subdivision of the norwegian hospital procurement trust, has annually released lists of their recommendation for b/tsdmards use based on the results of the tender. the prescribing physicians are not obliged by law to follow the annual recommendations and may therefore choose another drug in case of individual reasons. however, the regional health trusts strongly advise and monitor the adherence to the annual (tender-based) recommendations. since the original cost on specific b/tsdmard is confidential, we can only report the total average cost of the assessed b/tsdmards. however, among the current b/tsdmards users, many patients are also using more expensive b/tsdmards on the tender list, which is reflected in the slower drop in prices shown in table 4 and fig. 1b . the expiration of patents for reference bdmards has enabled the development and production of biosimilar bdmards, reaching the market at lower costs. in 2014 infliximab ct-p13 was the first biosimilar to reach the norwegian market, followed by etanercept sb4 in 2016 [13, 14] . in 2016, a high increase was observed in prescription among ra patients who started on a b/tsd-mards not being naïve to b/tsdmards compared to the steady rate years before. this is explained by the mandatory switching from reference agent to etanercept sb4, which in this study is defined as non-naïve starters on b/ tsdmards. in the 2019 norwegian tender process, several companies manufacturing biosimilar adalimumab drugs gave price offers. however, the reference adalimumab won the tender by offering a lower price than what was offered for the biosimilars. the same was seen for etanercept in 2020, where the reference and not a biosimilar drug won. this shows that biosimilars influence the competition between pharmaceutical companies by influencing producers of reference bdmards to reduce their prices in order to win the tender. in 2020 however, the biosimilar gp2017 adalimumab won the tender process. in denmark, estimated accumulated price and quantitative data have been published for infliximab, etanercept, and adalimumab after the expiration of a patent [15, 16] . when the adalimumab biosimilar reached denmark's market in october 2018, the price for adalimumab dropped by 83% within 3 months. whereas between september 2018 to september 2019, the use of adalimumab increased by approximately 35% [15] . the third mechanism used in norway and denmark to promote rapid cost reduction for bdmards is the recommended switch to the cheapest available substance when generics or biosimilars are available. in norway, this switch has to be done by the treating rheumatologist and cannot be performed by the pharmacist, e.g., at the pharmacy. as shown in our study, the impact of a tender system to reduce drug cost is a mechanism that may increase the availability of b/tsdmards to treat inflammatory arthritis, e.g., ra. this may be particularly important for low-income countries where ra patients have been shown to have higher disease activity than higherincome countries [5-7, 17, 18] . the previously documented improvement in clinical outcomes for ra patients in the new millennium in norway [2, 3] and other countries [19] [20] [21] [22] [23] [24] was also found in our study. aga et al., in the nor-dmard multicenter study, found that remission rates in ra patients after 6 months of tnfi (and methotrexate) treatment had increased from 17% in the period 2000-2002 to 46% in the period 2009-2010 [3] . disease duration before starting a tnfi had decreased from a median of 8.0 years (2000-2002) to 3.8 years (2009-2010) [3] . in comparison, in our study, the percentage of patients in das28 remission increased from 42% in 2010 to 67% in 2019, whereas disease duration in ra patients who started naïve on b/tsdmards did not change substantially. treatment with b/tsdmards in randomized clinical trials has been shown to improve occupational outcomes [25] [26] [27] . from the swedish bdmard registry, 35% of work-disabled ra patients with a disease duration of fewer than 5 years were found to regain their work ability within 3 years after starting a tnfi. with a disease duration of 5 years or more, the work recovery proportion was only 14% [28] . in our study, we did not see a significant change in the proportion of enabled workers across the 10 years. however, we saw a significant difference of roughly 10% (59% vs. 70%) among enabled workers when comparing those who were b/tsdmard users vs. non-b/ tsdmard users (supplementary table 1 ). respectively, their average disease duration was 14 years vs. 9 years. when comparing the mean of naïve b/tsdmards users ( table 2) with non-naïve b/tsdmards users (table 3) in the same manner, we observed 72% enabled workers with a six-year disease duration vs. 57% enabled workers with 12 years disease duration. in the quest-ra study with data collected between 2005 and 2009 from 32 countries, 37% of previously work-enabled ra patients aged 65 years and younger reported occupational disability at the onset of ra symptoms (median observation period of 9 years) [29] . despite the major differences in disease activity in their study, there was no significant difference in the proportion of work-enabled ra patients between countries with high and low gross domestic product (gdp). ra patients in low-gdp countries remained working despite high levels of disability and disease activity, suggesting that cultural and economic differences between societies also impact work disability rates in ra patients [29] . our study's major strength is that the data collected is standardized for all ra outpatients independent of treatment using the same hospital computer system. this is in contrast to some registry-based studies that either only included selected patient groups using b/tsdmards or patients who initiated treatment with csdmards and/or b/tsdmards (e.g., the norwegian nor-dmard registry) [30] . another strength is that the included patients come from 10 centers spread across norway. selection bias, if present, would most likely affect the first years of the 10-year period as the number of registered patients was lower than at the end of the period. however, no significant changes were seen between the ra patients for age, sex, ccp, and rf status. furthermore, comparing the estimated mean prevalence for ra of 0.3% in 2019 (single centers range 0.2 to 0.5%) in our study with a population-based prevalence of 0.4% in oslo (1994) for the age group 20-80 years and 0.5% in tromsø (1994) for the age group 20 years and older indicate a low grade of selection bias, at least in some centers [8, 9] . ra patients followed by privately practicing rheumatologists have not been included in the analysis and may partly explain lower prevalence estimates in some centers. however, we have reason to believe that both internal validity for each center and external validity for norway are satisfactory. the relatively high rate of missing data for disease activity measures is a limitation. nevertheless, as argued above, we find this less likely to be caused by a systematic bias and is most likely based on random. another limitation is the reduced effort of including patients in the biorheuma projects during the early phase of the 10-year period. therefore, the increasing percentage of included patients may be strongly affected by the examining physician's interest in including the patient into the gotreatit rheuma database. also, it cannot be excluded that the improved disease outcome across the 10 years may have improved due to other factors such as earlier diagnosis, starting b/tsdmards at a lower disease activity, improved self-management, fewer comorbidities, and other aspects that may have reduced the patient global assessment (a key component of das28) besides the effect of b/tsdmards. in conclusion, our data shows that the average annual costs of treating a norwegian ra patient with b/tsd-mard over the 10 year period 2010-19 were reduced by 47% for any user, and by 75% for naïve b/tsdmard users. when adjusting for cpi, the percentage reduction was even higher. in norway, with a tax-based healthcare system, we show that treatment with b/tsdmards has become more available at a lower cost, and the threshold for starting b/tsdmards has decreased significantly. although not confirming causality, there is strong reason to believe that the national tender system has contributed the annual total cost of b/tsdmards in million euro eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update ten years of change in clinical disease status and treatment in rheumatoid arthritis: results based on standardized monitoring of patients in an ordinary outpatient clinic in southern norway time trends in disease activity, response and remission rates in rheumatoid arthritis during the past decade: results from the nor-dmard study rheumatoid arthritis: strategy more important than agent inequities in access to biologic and synthetic dmards across 46 european countries variations in criteria regulating treatment with reimbursed biologic dmards across european countries. are differences related to country's wealth? inequity in access to bdmard care and how it influences disease outcomes across countries worldwide: results from the meteor-registry the prevalence and severity of rheumatoid arthritis in oslo. results from a county register and a population survey incidence and prevalence of rheumatoid arthritis in the county of troms, northern norway validation of rheumatoid arthritis improvement criteria that include simplified joint counts assessment of patient satisfaction in activities of daily living using a modified stanford health assessment questionnaire consumer price index • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year your research ? choose bmc shift from adalimumab originator to biosimilars in denmark the danish model for the quick and safe implementation of infliximab and etanercept biosimilars disparities in rheumatoid arthritis disease activity according to gross domestic product in 25 countries in the quest-ra database lower education and living in countries with lower wealth are associated with higher disease activity in rheumatoid arthritis: results from the multinational comora study comparative effectiveness of rituximab, abatacept, and tocilizumab in adults with rheumatoid arthritis and inadequate response to tnf inhibitors: prospective cohort study efficacy and safety of rituximab with and without methotrexate in the treatment of rheumatoid arthritis patients: results from the gisea register abatacept and its use in the treatment of rheumatoid arthritis (ra) in the czech republic-data from the attra registry comparative effectiveness of antitumour necrosis factor agents, biologics with an alternative mode of action and tofacitinib in an observational cohort of patients with rheumatoid arthritis in switzerland persistence rates of abatacept and tnf inhibitors used as first or second biologic dmards in the treatment of rheumatoid arthritis: 9 years of experience from the rhumadata ® clinical database and registry effectiveness and survival-on-drug of certolizumab pegol in rheumatoid arthritis in clinical practice: results from the national swedish register infliximab treatment maintains employability in patients with early rheumatoid arthritis effect of certolizumab pegol with methotrexate on home and work place productivity and social activities in patients with active rheumatoid arthritis association between etanercept use and employment outcomes among patients with rheumatoid arthritis. arthritis rheum predictors of work disability after start of anti-tnf therapy in a national cohort of swedish patients with rheumatoid arthritis: does early anti-tnf therapy bring patients back to work? work disability remains a major problem in rheumatoid arthritis in the 2000s: data from 32 countries in the quest-ra study european biologicals registers: methodology, selected results and perspectives publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations to the statistician are hugo pripp ph.d. (faculty of health sciences, oslo and akershus university college of applied sciences, oslo, norway) for statistical support. we are also grateful to all physicians, nurses, clerks, and patients at the participating outpatient clinics who have contributed in daily clinical practice to make this real-life study possible. hospital for rheumatic diseases, haugesund, norway. 7 significantly to this favorable price reduction for b/tsd-mards in norway. the online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12913-021-07425-w. all authors have contributed with critical components to enable the delivery of the study and manuscript. these include: patient recruitment and/or data generation and/or analysis, as well as writing or critically revising the present manuscript and/or raising infrastructure to support the study. all authors read and approved the final manuscript. no financial support or other benefits from commercial sources for this work have been provided. data are available on reasonable request and must be approved by all participating centers. please contact the corresponding author by email to request the data from this study. the study was approved by the regional ethical committee (rec 2010/3078). no consent from patients was required by the rec as we only use anonymized data. not applicable. the authors declare no competing interests. the authors have no financial interests that could create a potential conflict of interest or the appearance of a conflict of interest concerning the submitted work. key: cord-0022896-cfh54kf5 authors: li, jing; zhang, ying; kang, ya-juan; ma, nan title: effect of family caregiver nursing education on patients with rheumatoid arthritis and its impact factors: a randomized controlled trial date: 2021-10-06 journal: world j clin cases doi: 10.12998/wjcc.v9.i28.8413 sha: 7395ee3973f802098dad67d6066b760f477ea6f5 doc_id: 22896 cord_uid: cfh54kf5 background: rheumatoid arthritis (ra) is a common autoimmune disease. nursing education for family caregivers is considered a workable and effective intervention, but the validity of this intervention in ra has not been reported. aim: to explore whether family caregiver nursing education (fcne) works on patients with ra and the factors that influence fcne. methods: in this randomized controlled study, a sample of 158 pairs was included in the study with 80 in the intervention group and 78 in the control group. baseline data of patients and caregivers was collected. the fcne intervention was administered to caregivers, and inflammation level indicators, disease activity indicators and mood disorder indicators of patients were followed up and analyzed. results: baseline characteristics of the intervention and the control groups had no significant difference. indicators were significantly reduced in the intervention group compared to the control group. the intervention group showed significant differences in stratification of relationship, education duration and age. conclusion: the effect of fcne on ra is multifaceted, weakening inflammation level, alleviating disease activity and relieving mood disorder. relationship between caregiver and patient, caregiver’s education level and patient’s age may act as impact factors of fcne. rheumatoid arthritis (ra) is a common autoimmune disease characterized by chronic inflammation [1] . a recent survey has been reported that the prevalence of ra in the united states population ranges from 0.5% to 0.8%, with rates as high as 1.7% for specific groups of older adults [2] . in china, ra is one of the top ten chronic diseases, and its prevalence has been recorded at 1.02% [3] . patients not only suffer from reduced physical function but also frequently experience increased mental stress accompanied by depression and anxiety [4] . as ra patients are more likely to be diagnosed between the ages of 35 and 60 [5] and the disease is persistent and difficult to eradicate, long-term care is a necessity. family nursing can no longer be ignored in the care of patients, and family caregivers have become the mainstay of caregivers [6] . family nursing is gradually emerging, and support for family caregivers is increasingly valued [7] . nursing education for family caregivers is considered as a workable and effective intervention that directly improves their disease knowledge, physiological management abilities and psychological support skills to provide better care to patients [8] . studies have shown this intervention plays an active role in the course of specific diseases including stroke [9] , asthma [10] and kidney injury [11] . however, the effectiveness of care education for family caregivers of patients with ra has not been reported. in this study, we designed a health education program called family caregiver nursing education (fcne), a series of professional training courses for family caregivers that focused on care techniques of ra patients and main points of ra-related knowledge. indicators of inflammation level, disease activity and mood disorder were also collected and followed up to explore the effect of fcne on patients with ra and its impact factors. participants included ra patients and their corresponding family caregivers, and the effect on patients was observed by implementing the intervention on caregivers. patients were selected from those who were hospitalized in the immune-rheumatology department of a governmental and university-affiliated hospital from june 2017 to december 2018, on the basis of the 2010 revised ra classification criteria of the american rheumatism association, the european league against rheumatism and the 1987 american college of rheumatology classification [12] . each patient was by using the computer assignment procedure in spss 21.0, sequential numbers were generated and placed in a sealed opaque box, and a separate researcher was arranged to randomly assign the selected participants to the intervention group or the control group. until all the baseline questionnaires were completed, neither the researchers nor the participants were aware of the group assignment [13] . all patients in both groups received rheumatoid routine primary care and were treated with a uniform regimen of dmards represented by methotrexate plus hormonal medication represented by prednisone acetate for 6 mo. in addition, the family caregivers of the intervention group received the fcne for 6 mo. all interventions were unchanged during the trial. the original content of fcne came from literature reviews and consensus guidelines in national guideline clearinghouse [14] . a total of eight experienced rheumatologists and nurses then worked together to add, delete, adapt and revise the teaching content in conjunction with expert advice and to develop an appropriate teaching scheme based on the predetermined study period. the final items covered seven primary areas: psychological guidance, medication guidance, functional exercise, diet, clean skin care, care during the active phase of the lesion and care during the stable phase of the lesion. in addition, a brief supplementary course on the epidemiology, pathogenesis and clinical symptoms of ra was interspersed between the main items. fcne was carried out around five major approaches: group education, individual training, distribution of written materials, web-based information dissemination and appraisal system. a 45 min one-to-one training and a 1.5 h group training were conducted at regular intervals each month, for a total of six one-to-one training sessions and six group training sessions. each group training was followed by a workshop on the content of the course and the distribution of the corresponding paper material. electronic data were released through the network at irregular intervals. a week after each session, participants were followed up by telephone calls of 15 min each, through which researchers checked acceptance and implementation of the last session and arranged additional courses if required [15] . every 2 wk after the training was completed, an examination was used to test and evaluate the effectiveness of the teaching. for subjects who failed the test, retraining and make-up examinations were conducted. those who still failed the make-up examination were removed from the intervention group. those with an attendance rate of less than 80% were also removed from the intervention group. all of the above assessments were randomly assigned to five independent researchers and completed using a double-blind method. general information of patients and caregivers was collected from questionnaire or medical chart at baseline. indicators of patients including c-reactive protein (crp), erythrocyte sedimentation rate (esr), tumor necrosis factor (tnf-α), tender joint counts out of 28 joints (tjc28), swollen joint counts out of 28 joints (sjc28), pain on visual analogue scale, provider global assessment by visual analogue scale (pga), patient global assessment of disease activity by visual analogue scale (ptga), health assessment questionnaire (haq), self-rating depression scale (sds) and self-rating anxiety scale (sas) were followed up at baseline, 1 st month, 3 rd month and 6 th month when patients came for their routine visits. ∆crp, ∆esr, ∆tnf-α, clinical disease activity index (cdai), simplified disease activity index (sdai) and disease activity october 6, 2021 volume 9 issue 28 general information: general information included the patient's age, gender, presence of comorbidity (hypertension, coronary heart disease and diabetes), drug therapy and disease duration and the caregiver's age, gender, work status, relationship with the patient and education duration (representing the education level). indicators of inflammation level: crp, esr and tnf-α were used to assess the biochemical level of inflammation; ∆crp, ∆esr and ∆tnf-α were used to assess the degree of decline in inflammatory indicators. crp, esr and tnf-α are considered to be the main pathophysiological factors in ra. biomarkers in the blood become higher when inflammation is severe, while ∆crp, ∆esr and ∆tnf-α rise accordingly when inflammation subsides [16] . indicators of disease activity: cdai, sdai, das28 and haq were used to evaluate the level of disease activity in ra. the specific formulas for cdai, sdai and das28 have been listed previously with tjc28, sjc28, pga, ptga, crp and esr. haq covers daily activities such as dressing, standing, eating, walking and hygiene. high values of these scores indicate deterioration in physical function [15] . indicators of mood disorder: sds and sas were used to appraise the level of mental health and mood disorder. the sds and sas assess 20 symptoms of depression and anxiety, respectively, rated numerically on a scale for each item, with higher scores indicating a higher intensity of the symptom in question. sds ≥ 50 is defined as depression, 50-59 as mild depression, 60-69 as moderate depression and 70 or more as severe depression. sas ≥ 50 is defined as anxiety, 50-59 as mild anxiety, 60-69 as moderate anxiety and 70 or more as severe anxiety [17] . sds and sas have been used to test the psychological level of ra patients [18] . october 6, 2021 volume 9 issue 28 this study utilized spss 21.0 software to process the data. a total of 158 cases were ultimately included in the statistical analysis, including 80 cases in the intervention group and 78 cases in the control group. if the quantitative data were normally distributed, the mean ± sd were used to describe. if the data showed a skewed distribution, the median and interquartile range were applied. frequency and percentage reports were used to describe the categorical data. depen-ding on the type of data analyzed, baseline data was analyzed using the t-test, the mann-whitney u test and the χ 2 test. for follow-up data, repeated measures analysis of variance (anova) was used to analyze the difference, and pearson correlation analysis was used to analyze the correlation. all the statistical analyses were considered significant at p < 0.05. in total, 80 pairs were included in the statistical analysis in the intervention group and 78 pairs in the control group. for family caregivers, the majority were women with full-time jobs. the mean age was 47.4-years-old, ranging from 28-years-old to 65-yearsold. the median education duration was 9 years, which meant they had senior high school education or near university education. for patients, most were also female, and the mean age was 59.2-years-old distributed between 34-years-old and 86-yearsold. patients with the median disease duration of 5.5 years were mainly treated with dmards + glucocorticoid and had no comorbidity. in addition, indicators were counted to assess the patient's initial condition. there was no significant difference in all general information and indicators between the intervention group and the control group at baseline. specific values and statistical results of the characteristics were shown in table 1 . all follow-up indicators of the intervention group and the control group were shown in table 2 . repeated measures anova was performed. main effect of time and interaction effect of time × group were significant in all inflammation indicators (p < 0.001), meaning that they had a downward trend over time while time interacted with fcne. effect of group was also significant in crp, esr and tnf-α (p < 0.001, p = 0.001, p = 0.019, respectively), implying that fcne promoted containment of inflammation and reduced indicators of inflammation level in ra. except that the repeated measures anova result of haq did not show significant difference, effect of time and time × group was significant in cdai, sdai and das28 (p < 0.001), and effect of group was significant in cdai, sdai and das28 (p < 0.001, p < 0.001, p = 0.013, respectively), indicating that fcne helped to curb disease progression and reduced indicators of disease activity in ra. according to the scoring criteria, at baseline, 77 people were depressed in the intervention group (56 mildly depressed and 21 moderately depressed) and 71 in the control group (41 mildly depressed, 26 moderately depressed and 4 severely depressed), with no significant difference; after 6 mo of follow-up, 10 people were depressed in the intervention group significantly lower than 39 in the control group (p < 0.001). similarly, 54 people in the intervention group suffered from anxiety at baseline (32 with mild anxiety, 19 with moderate anxiety and 3 with severe anxiety) and 48 in the control group (26 with mild anxiety, 14 with moderate anxiety and 8 with severe anxiety), with no significant difference; after 6 mo of follow-up, 6 people in the intervention group suffered from anxiety significantly lower than 23 in the control group (p < 0.001). in addition, the repeated measures anova result also showed that time, time × group and group effect of sds and sas was significant (p < 0.001). all these suggested that fcne contributed to mental health and reduced indicators of mood disorder in ra. relationship between caregiver and patient: the intervention group was reclassified based on the relationship between caregiver and patient: 23 cases in son or daughter group, 30 cases in spouse group and 27 cases in other relationships group. inflammation indicators of these three groups were shown in table 3 . for crp, effect of relationship-group was significant (p = 0.033), and further pairwise comparisons revealed that spouse group had a significantly lower reduction in crp than other relationships group (p = 0.012). for esr, relationship-group effect was also significant (p = 0.041), and pairwise comparisons showed that esr reduction of spouse group was significantly lower than that of other relationships group (p = 0.024), while son or daughter group had a significantly lower esr reduction than other relationships group (p = 0.035). however, tnf-α did not show significant stratification. both crp and esr results suggested a more efficient effect of fcne for spousal relationship, resulting in a more pronounced reduction in inflammatory indicators. relationship between caregiver and patient was an impact factor of fcne. education duration of caregiver: the means of ∆crp, ∆esr and ∆tnf-α were 84.74% (sd = 14.32%), 31.01% (sd = 14.89%) and 32.03% (sd = 9.75%), respectively. the results of pearson correlation analysis showed that ∆crp (r = 0.516, p < 0.001), ∆esr (r = 0.507, p < 0.001) and ∆tnf-α (r = 0.734, p < 0.001) were significantly and positively correlated with education duration. the longer the caregiver's education duration, the higher the patient's inflammation decline, and the better the effect of fcne, which meant that caregiver's education duration was an impact factor of fcne. the intervention group was reclassified by patient age: 42 cases in middle-aged group and 38 cases in elderly group (the world health organization defines 45 years to 59 years as middle-aged people and 60 years and above as elderly people). disease activity and mood disorder indicators of these two groups were shown in table 4 . for disease activity indicators, except for no difference in haq stratification, effect of age-group in cdai, sdai and das28 was significant (p < 0.001). cdai, sdai and das28 were higher in the elderly group than in the middleaged group at baseline, but the level of the elderly group was approaching that of the middle-aged group by the 6 th month of follow-up. the degree of disease activity decline was more evident in the elderly group. for mood disorder indicators, age-group effect for both sds (p = 0.014) and sas (p < 0.001) was significant, meaning that the middle-aged group with higher mood disorder scores before the intervention was close to or even lower than the elderly group after 6 mo of fcne intervention. fcne had a significant psychological improvement effect on the middle-aged group and a significant disease mitigation effect on the elderly group, showing that patient's age was another impact factor of fcne. traditional nursing education for ra is aimed at patients. in addition to the patients themselves, to a certain extent, the quality of life for patients also depends on the support of their families [19] . a study has proven that the mood of caregivers also affected the disease progression of ra patients [20] . more studies on family interventions have been published in recent years, and the majority of these showed benefit to the identified patient [21] . but there is little research on nursing education or family nursing for ra patients. therefore, we designed the fcne. for the selection of outcome measures, we chose a total of nine characteristic indicators in terms of inflammation level, disease activity and mood disorder for a 6 mo intervention and followup. the aim was to assess the effect of fcne on ra and its influencing factors in a holistic manner. initially, we selected biochemical indicators of inflammation for evaluation due to their importance in the pathogenesis of ra [22] . in addition to the two traditional indicators of crp and esr[23], we also included tnf-α, an emerging marker of ra [16] . the results found that the intervention group showed a significantly better reduction in all three indicators than the control group, which corroborated the reliability of tnf-α. afterwards, we calculated and appraised the disease indexes for ra and found that fcne had a distinct advantage for the reduction of cdai, sdai and das28 but did not show the same effect for haq, probably due to errors caused by small values with insignificant changes. more and more care models were proven to work for ra, and a nurse-led study found that nursing education by telephone was effective in improving medication adherence in ra patients [14] . as a rising approach, fcne plays a positive role in the prognosis of diseases including lung cancer [24] and stroke [25] . fcne enhances caregivers' knowledge of the disease and improves nursing skills, which is conducive to providing better care to patients while identifying risk factors and complications in time to reduce injuries. it also provides a communication platform, bringing participants together for exchange and discussion, which not only allows them to obtain more practical experience but also benefits the release of october 6, 2021 volume 9 issue 28 negative emotions [25] . according to surveys, the prevalence of depression in ra patients is between 14.8% and 48.0%, which is twice that of the general population[26] and increases the mortality rate of ra patients to a certain extent [27] . relevant studies have shown that fcne can reduce depression, anxiety and self-harm in certain patient populations, such as ischemic stroke patients [28] , older patients[29] and suicidal patients [30] . in this study, the effect of fcne on alleviating mood disorder and promoting mental health in ra patients was similarly confirmed. this role of fcne may be achieved by facilitating family communication, relieving misunderstandings and conflicts and supporting the maintenance of an enabling environment characterized by understanding and cooperation [31, 32] . the effectiveness of mindfulness interventions for ra[33] also supported this speculation. after confirming the effect of fcne on ra, we had a stratified study of the intervention group according to different factors to explore the possible influencing fac -tors of fcne. the results showed that inflammation reduction was further enhanced when the caregiver was a spouse and had an advanced education level. a crosssectional study indicated that spouses took on a vital part as family caregivers but also carried more of the role load [34] . besides, we found that the initial disease activity was higher but declined faster in elderly people compared to middle-aged people. interestingly, the initial mood disorder was more severe but resolved more rapidly in middle-aged people compared to elderly people. a survey revealed that younger caregivers were more likely to report adverse psychological symptoms [35] . thus, we considered that relationship between caregiver and patient, caregiver's education level and patient's age operated as influencing factors affecting the efficacy of fcne, which also suggests priorities for fcne participants, such as giving preference to spouses or caregivers with high education level as they are likely to have better intervention outcomes. in this study we were surprised to find that fcne had a significant improvement in a number of indicators, particularly inflammatory indicators including tnf-α, which we hypothesize is related to fcne improving adherence to drug treatment. patients with positive adherence to medication may be better able to contain the disease and slow its progression [36] . several previous studies have confirmed the effectiveness of educational interventions tailored to ra, which are achieved by improving and maintaining patients' medication adherence [37, 38] . nurses are increasingly prominent in this process, assisting patients to improve adherence and self-management [39] . studies have shown that education by experienced rheumatology nurses can help promote patient behavior, including maintaining medication adherence [40] and that fcne, as a nurse-led intervention that takes into account patient needs and disease characteristics, can help increase patients' confidence, motivation and skills to take their medication in the long term [41] . further research is needed on the specific mechanisms that improve indicators including biochemical levels of inflammation and more evidence related to improving adherence. this study has some limitations. primarily, the sample was small, and it was a single center study. the conclusions have yet to be validated in a large sample and multicenter experiment. furthermore, there was a slight improvement in some indicators in the control group, which we speculate may be related to the conventional treatment they received and remains to be demonstrated. finally, based on follow-up data for all indicators, the effect of fcne is most pronounced after 1 mo and especially between 3 and 6 mo, demonstrating its short-term impact. however, the lack of longterm follow-up has demonstrated its role in relation to the chronic effects. the effect of fcne on ra is multifaceted, weakening inflammation level, alleviating disease activity and relieving mood disorder. relationship between caregiver and patient, caregiver's education level and patient's age may act as impact factors of fcne. one year in review 2020: pathogenesis of rheumatoid arthritis a systematic review of the incidence, prevalence, costs, and activity and work limitations of amputation, osteoarthritis, rheumatoid arthritis, back pain, multiple sclerosis, spinal cord injury, stroke, and traumatic brain injury in the united states: a 2019 update burden of rheumatoid arthritis from a societal perspective: a prevalence-based study on cost of this illness for patients in china the evidence base for psychological interventions for rheumatoid arthritis: a systematic review of reviews rheumatoid arthritis: a brief overview of the treatment family nursing: the family as the unit of research and care interventions to support family caregivers in pain management: a systematic review evaluation of a therapeutic education program for french family caregivers of elderly people suffering from major neurocognitive disorders: preliminary study evaluation of family stroke education caregiver education to promote appropriate use of preventive asthma medications: what is happening in primary care? effects on caregiver burden of education related to home care in patients undergoing hemodialysis classification of rheumatoid arthritis: comparison of the 1987 american college of rheumatology criteria and the 2010 american college of rheumatology/european league against rheumatism criteria effectiveness of a self-management program for joint protection and physical activity in patients with rheumatoid arthritis: a randomized controlled trial a randomized controlled trial of the effects of a telehealth educational intervention on medication adherence and disease activity in rheumatoid arthritis patients effectiveness of health education by telephone follow-up on self-efficacy among discharged patients with rheumatoid arthritis: a randomised control trial can cytokines be used as an activation marker in rheumatoid arthritis? the depression status inventory: an adjunct to the self-rating depression scale anxiety and depression in primary care patients suffering of rheumatoid diseases the role of demographic and clinical variables in assessing the quality of life of outpatients with rheumatoid arthritis association of the commitments and responsibilities of the caregiver within the family to the disease activity in patients with rheumatoid arthritis: a family involvement in adult chronic disease care: reviewing the systematic reviews elevated serum granzyme b levels are associated with disease activity and joint damage in patients with rheumatoid arthritis identifying reliable diagnostic/predictive biomarkers for rheumatoid arthritis effect of caregiver education on pulmonary rehabilitation, respiratory muscle strength and dyspnea in lung cancer patients effect of family education program on cognitive impairment, anxiety, and depression in persons who have had a stroke: a randomized, controlled study the prevalence of depression in rheumatoid arthritis: a systematic review and meta-analysis: reply comorbid depression is an independent risk factor for mortality in patients with rheumatoid arthritis a newly designed intensive caregiver education program reduces cognitive impairment, anxiety, and depression in patients with acute ischemic stroke targeted education for general practitioners reduces risk of depression or suicide ideation or attempts in older primary care patients short-term effects of a suicide education intervention for family caregivers of people who are suicidal clinical practice. rehabilitation after stroke education and depression mindfulness interventions for rheumatoid arthritis: a systematic review and meta-analysis stressors and resources related to medication management: associations with spousal caregivers' role overload mental health status of health-care professionals working in quarantine and non-quarantine egyptian hospitals during the covid-19 pandemic what are the effects of medication adherence interventions in rheumatic diseases: a systematic review nursing sensitive outcomes in patients with rheumatoid arthritis: a systematic literature review what do we know about rheumatoid arthritis patients' support needs for self-management? patient preferences for psychological support in inflammatory arthritis: a multicentre survey nurse telephone education for promoting a treat-to-target approach in recently diagnosed rheumatoid arthritis patients: a pilot project patients' perceived health information needs in inflammatory arthritis: a systematic review the authors would like to thank all participants for their contribution to the study. rheumatoid arthritis (ra) is a common disease that requires long-term care, and nursing education for family caregivers is considered as a workable and effective intervention. the effectiveness of care education for family caregivers of patients with ra has not been reported. this study aimed to explore whether family caregiver nursing education (fcne) works on patients with ra and the factors that influence fcne. in this study, we designed a health education program called fcne, a series of professional training courses for family caregivers that focused on care techniques of ra patients and main points of ra-related knowledge. the fcne intervention was administered to caregivers, and inflammation level indicators, disease activity indicators and mood disorder indicators of patients were followed up and analyzed. indicators were significantly reduced in the intervention group compared to the control group. the intervention group showed significant differences in stratification of relationship, education duration and age. the effect of fcne on ra is multifaceted, weakening inflammation level, alleviating disease activity and relieving mood disorder. relationship between caregiver and patient, caregiver's education level and patient's age may act as impact factors of fcne. this study indicates that fcne is feasible and efficient for patients with ra. it also suggests priorities for fcne participants, such as giving preference to spouses or caregivers with high education level as they are likely to have better intervention outcomes. key: cord-0030919-jy3cyl42 authors: ma, yuanchen; chen, jieying; wang, tao; zhang, liting; xu, xinhao; qiu, yuxuan; xiang, andy peng; huang, weijun title: accurate machine learning model to diagnose chronic autoimmune diseases utilizing information from b cells and monocytes date: 2022-04-20 journal: front immunol doi: 10.3389/fimmu.2022.870531 sha: 8c5380d907492674f308cffc3043fce24a5177fd doc_id: 30919 cord_uid: jy3cyl42 heterogeneity and limited comprehension of chronic autoimmune disease pathophysiology cause accurate diagnosis a challenging process. with the increasing resources of single-cell sequencing data, a reasonable way could be found to address this issue. in our study, with the use of large-scale public single-cell rna sequencing (scrna-seq) data, analysis of dataset integration (3.1 × 10(5) pbmcs from fifteen sle patients and eight healthy donors) and cellular cross talking (3.8 × 10(5) pbmcs from twenty-eight sle patients and eight healthy donors) were performed to identify the most crucial information characterizing sle. our findings revealed that the interactions among the pbmc subpopulations of sle patients may be weakened under the inflammatory microenvironment, which could result in abnormal emergences or variations in signaling patterns within pbmcs. in particular, the alterations of b cells and monocytes may be the most significant findings. utilizing this powerful information, an efficient mathematical model of unbiased random forest machine learning was established to distinguish sle patients from healthy donors via not only scrna-seq data but also bulk rna-seq data. surprisingly, our mathematical model could also accurately identify patients with rheumatoid arthritis and multiple sclerosis, not just sle, via bulk rna-seq data (derived from 688 samples). since the variations in pbmcs should predate the clinical manifestations of these diseases, our machine learning model may be feasible to develop into an efficient tool for accurate diagnosis of chronic autoimmune diseases. systemic lupus erythematosus (sle), multiple sclerosis (ms), and rheumatoid arthritis (ra) are all chronic autoimmune diseases associated with progressive widespread organ damage (1) (2) (3) . the course of these three diseases is typically progressive with intermittent remission (4, 5) . it is generally accepted that early treatment could increase the remission probability of these diseases and improve their prognosis (6, 7) . if appropriate treatment is not given in a timely manner, these diseases may progress, causing work disability and life quality reduction for patients. furthermore, such progression would lead to enormous financial burdens to the patients, their families, and society (8) (9) (10) . hence, it is crucial to develop an efficient method of accurate diagnosis to enable early intervention for these diseases. unfortunately, it seems that diagnosing sle, ms, and ra may still be a challenging process that relies on a set of criteria (11) (12) (13) , including clinical manifestations, functional outcomes, and serological and radiological evidence, that have to be met to make an accurate diagnosis (14, 15) . under non-specific and insensitive criteria, the misdiagnosis and underdiagnosis of these diseases are relatively common (16) . the average time from symptom onset to diagnosis confirmation was approximately two years (17) . this may cause patients to miss the optimal time for treatment. to break the bottleneck of early diagnosis, many studies have focused on biomarker detection to develop an accurate diagnostic criterion (18) (19) (20) (21) . however, the results were unsatisfying, owing to the tremendous heterogeneity of these diseases and limited comprehension of the disease pathophysiology (22) . in detail, although it is well known that the loss of immune tolerance and persistent release of autoantibodies are the two important bases for the pathophysiology of chronic autoimmune disease (23, 24) , most studies have focused on investigating the contribution of certain cellular or molecular mechanisms rather than comprehensively and systematically illustrating the pathogenesis. this might be due to the limitation of methods or means. with the development of single-cell sequencing technology, the increased resources of data, and the improvement of bioinformatic tools (e.g., seurat, sharp, cellchat, etc.) (25) (26) (27) , these would together help us to comprehend the pathophysiology of these diseases, thus their crucial features would be efficient for being mined. for example, nehar-belaid et al. thoroughly analyzed the major cell types among peripheral blood mononuclear cells and revealed an expanded subpopulation that has a specific interferonstimulated gene (isg) expression pattern in sle patients (28) . meena subramaniam et al. also found that monocytes from sle patients highly expressed isgs (29) . both of these studies comprehensively illuminated the cytological changes of sles. according to these public single-cell rna sequencing (scrna-seq) data of sle, we seek for a feasible way for sle accurate diagnosis. firstly, integration and cellular cross-talking analysis were performed to obtain the powerful information labeling the disease. this information was then combined with an unbiased random forestry machine learning algorithm which rendered an efficient mathematical model for sle diagnosis. the accuracy of the mathematical model to identify patients with ra and ms was also validated. furthermore, the diagnostic precision of our model was evaluated using an independent sle cohort ( figure 1 ). the single-cell rna sequencing data were deposited in the gene expression omnibus (geo), and the accession numbers were gse137029 and gse135779 for sle patients and gse164378 for healthy donors. bulk rna-sequencing data were deposited to gse72509 and gse164457 for peripheral blood mononuclear cells (pbmcs) of sle patients, gse90081 for pbmcs of ra patients, gse89408 for synovial tissues of ra patients, gse159225 for pbmcs of ms patients, and gse89408 for cd14-positive cells of ms patients, and gse183204 and gse169687 for pbmcs of healthy donors. reciprocal principal component analysis (rpca)-based integration could effectively detect a state-specific cell cluster and run significantly faster on large datasets. compared with other integration tools (e.g., bbknn and liger), rpca could conserve more distinct cell identities when removing batch effect, particularly for the data of immune cells (30) . considering its balancing capability on batch effect removal and biological variance preserving, rpca would be used for our dataset integration. before the integration, two lists were created: one containing merged sle data and the other containing merged healthy data. these two lists were then combined and integrated through seurat (version 4.0.5) following the guidelines at https:// satijalab.org/seurat/articles/integration_rpca.html. to discover sle-dominant cell clusters, pbmcs and their subpopulations were clustered through seurat (version 4.0.5), respectively. cell proportions of each cluster were calculated subsequently. for pbmc cell clustering, each cell subcluster was annotated based on a canonical marker. any cluster that has sle cells containing more than 75% would be considered as sle dominant. within those pbmc subpopulations (e.g., b cells and monocytes) which contain the sle-dominated cluster, differential expression gene (deg) analysis would be applied on all of their cell clusters with function findallmarkers embedded in seurat (version 4.0.5) to find out useful information that mark the sle state. top five genes based on their log2 fold change value were selected as the first part of feature input for machine learning. meanwhile, these deg functions were annotated through literature search. the machine learning model can be optimized with powerful sources of information. thus, cellchat (version 1.1.3) analysis was performed following the guidelines at https://github.com/ sqjin/cellchat. in details, overall interaction, overall signaling pattern, outgoing/incoming signaling pattern, and ligandreceptor pair were checked step by step. samples were analyzed independently. datasets of patients and health donors were analyzed separately and merged to make a comparison analysis. ligand-receptor pairs which disappeared at sle were selected as a second part of feature input for machine learning. 1 | workflow for establishment of an accurate machine learning model to diagnose chronic autoimmune diseases. step i, to figure out the most crucial information that characterizes diseases using public scrna-seq datasets. from analysis of integration and clustering, 67 top five cluster-specific genes basing on the differential expression gene identification within sle dominant pbmc subpopulations were derived. from cellular cross-talking analysis, 21 genes constituting ligandreceptor pairs disappeared in sle patients and showed that more than two kinds of pbmc subpopulation were derived. a union of these two gene sets would be used in the next step. step ii, to establish the machine learning model diagnosing diseases. a random forest machine learning model was implemented, and genes derived from step i were combined as feature input. 56 and 527 samples were used as sample input for scrna-seq and bulk rna-seq data, respectively. step iii, to validate the accuracy of our machine learning model. receiver operating characteristic (roc) analysis was used to test the accuracy, and multiple times of ten-fold cross-validation tests were adopted to avoid bias. the diagnostic accuracy of our model was also validated using an independent bulk rna-seq cohort containing 120 sle patients and 41 health donors. the random forest machine learning model was implemented with sklearn (version 0.23.2). the gene set which derived from integration and cellchat analysis were combined as feature input, aiming at selecting information within the sequencing datasets, thus improving the performance of the machine learning model. 56 and 527 samples were used as sample input for scrna-seq and bulk rna-seq data, respectively. samples from patients and healthy donors were labeled with 1 and 0, respectively. with the function train_test_split within sklearn.model_selection, the data were split into two parts, 70% for training and 30% for testing, according to previous study (31) . data balancing was performed when the cell/ sample ratio between patients and healthy donors was above 1:2, at random forest model initialization. receiver operating characteristic (roc) analysis was used to test models' accuracy. the models for each disease were independent. to avoid bias of data composition, the sklearn module stratifiedkfold was used to split data into ten parts preserving the ratio of samples and perform a ten-fold cross-validation with a loop of one hundred. the average and standard deviation of area under curve (auc) were documented. an independent bulk rna-seq cohort containing 120 sle patients and 41 health donors was enrolled into the diagnostic accuracy validation of our machine learning model. basic information of this cohort including sle severity, age, and gender was documented. genes which were used as feature input for the machine learning model were confirmed to be expressed in each sample. the diagnostic accuracy of our machine learning model for sle and healthy donors was tested separately. the statistical significance of differential gene expression was analyzed with the wilcoxon test, a default parameter in function findallmarkers of seurat packages. all the software mentioned above were based on r (version 4.1.1) and python (3.7). integration analysis and cell clustering were based on seurat (version 4.0.5), and cellular cross-talking analysis was based on cellchat (1.1.3). machine learning was based on sklearn (version 0.23.2). to discover the sle-dominated alterations of pbmc composition in sle patients, two single-cell transcriptomic datasets with more than 3.15 × 10 5 cells from 15 sle patient (gse137029) and 8 healthy donor (gse164378) samples were enrolled in our study. the uniform manifold approximation and projection (umap) and louvain algorithm were applied for unsupervised dimension reduction and clustering, respectively (32, 33) . as shown in figures 2a, b , the pbmcs of these two datasets could be grouped into sixteen molecularly distinct clusters. the clusters were annotated based on the gene expression values compared to all other cells. the results illustrated two clusters of t cells, b cells, natural killer cells, and erythroid cells, three clusters of monocytes and dendritic cells, and one platelet cluster (figures 2a, d) . unfortunately, sle-dominated (clusters 13 and 15) clusters were tiny and might come from erythrocytes (hbb specifically expressed). the rest of the cell cluster proportions of sle patients and healthy donors were evenly balanced or healthy donor dominant ( figure 2c ). this is partly because the difference between sle patients and healthy donors might be attenuated under the overall pbmc perspective. hence, to strengthen the power of detecting sle-dominant information, further analyses were performed in the subpopulations of pbmcs according to the cluster annotation above. increasing evidence indicates that specialized immune cell subsets are involved in the pathophysiological process of autoimmune diseases through multiplex pathways and signals (34) (35) (36) . thus, we re-clustered the subpopulations of pbmcs to identify the sledominated clusters in which the cell proportion of sle exceeds 75%. interestingly, the sle-dominated clusters were identified only in b cells (clusters 2, 6, and 7, figures 3a, b) and monocytes (clusters 1 and 7, figures 3e, f) ; the rest of the pbmc subpopulation is shown in figure s2 . table s1 . interferon inflammatory signatures are closely related to the sle (37) . consistently, we found that cluster 7 of b cells has interferon-stimulated gene (isg) expression patterns (ifi27, mx1, isg15, and ifi44l). moreover, we identified that this cluster simultaneously possess the typical expression patterns of naïve and autoactive b lymphocytes (naïve: igd+, cd27-, cd38 low, cd24 low; autoactive: tbx21, itgax, cxcr5, traf5, cr2, figure 3d ) (38, 39) . in addition, we also found that cluster 1 of monocyte highly expressed isgs (ifi27, mx1, isg15, ifi44l), and cluster 7 of monocyte had a proinflammatory character (fkbp5, figure 3h ) (40) . taken together, these findings revealed that there were enhanced signals of an autoreactive/inflammatory state in b cells and monocytes of sle patients, which suggested the essential roles in the pathophysiological process of sle. to systematically explore the alterations of pbmcs in sle patients and obtain a powerful source of information for the training of the machine learning model, we employed cellchat to analyze cellular cross talking from scrna-seq data. three scrna-seq datasets (gse137029, 15 adult patients with sle; gse135779, 13 child patients with sle; gse164378, 8 healthy donors) with more than 3.80 × 10 5 cells were included in this analysis. the total number and strength of ligand-receptor pairs were significantly reduced in both adult and child sle patients compared with healthy donors ( figure 4a) . remarkably, the interactions of pbmc subpopulations in sle patients were weakened ( figure 4b ). comparing overall and detailed outgoing/incoming signaling pattern variations among sle and healthy donors, we identified that abundant signal patterns could be observed for the healthy donors, but in the sle groups, the number of involved pathways was reduced ( figures 4c, d) . in detail, there were several signal patterns that specifically disappeared under the disease state. among them, flt3, cd48, and tgf-beta signal patterns have been reported to have a negative correlation with sle development (41) (42) (43) (44) . taken together, the disappearance of multiple signal patterns might be a potential feature during sle development. as the above results indicated that numerous signal patterns disappeared in sle compared with healthy states, to find detailed information, we further explore the discrepancy of ligand-receptor pairs from all pbmc subpopulations (b cells, monocytes, t cells, natural killer cells, and dendritic cells) among healthy donor, adult sle (asle), and child sle (csle) groups ( figures 5a-e) . we identified that eighty-seven ligandreceptor pairs disappeared in sle patients, which were composed of sixty-one genes. the frequency of each gene appeared at each pbmc subpopulation, as listed in table s2 . the genes which showed more than two kinds of pbmc subpopulation were recognized as significant ones to be selected as a second part of feature input for machine learning. among them, tgfbr1, tgfbr2, ccl5, cd48, cd244a, and cd72 have been reported to be closely related to the pathophysiologic processes of autoimmune diseases (41, 43, (45) (46) (47) . for example, tgfbr1, tgfbr2, and ccl5 levels are negatively correlated with sle development (43, 45) . cd48, also known as slamf2, which could regulate both natural killer cells and cytotoxic cd8+ t cells (48) , could protect mice from autoimmune nephritis (41), cd244a and cd72 were specifically decreased in monocytes and b cells during sle development (47, 49) . interestingly, all these selected pairs are all in b cells or monocytes, suggesting the key roles of monocytes and b cells on the pathophysiologic processes of autoimmune diseases. all these findings were consistent with our results of integration analysis. to establish a mathematical model of unbiased random forest machine learning for sle accurate diagnosis, sixty-seven top five cluster-specific genes derived from integration analysis and twenty-one significant genes identified via cellular cross-talking analysis were combined as feature input. the dataset gse135779, containing 3.60 × 10 5 pbmcs (derived from 33 csle, 7 asle, and 11 healthy children, 5 healthy adults), was included to evaluate the diagnosis efficiency of our mathematical model. the results indicated that our machine learning model could separate sle and healthy status with acceptable accuracy (auc = 0.776 ± 0.097, figure 6a ). the feature importance of our gene set for sle is shown in figure 6c . considering the signal intensity of our gene sets and the denoising ability of machine learning, a further investigation was conducted to evaluate the disease distinguishing the efficiency of our mathematical model using bulk rna-seq data. the bulk rna-seq datasets (gse72509, gse183204), which include 99 sle patients and 30 healthy donors were used in this investigation. the results indicated that our mathematical model has great adaptability (auc = 0.998 ± 0.004, figure 6b ). the corresponding feature importance was also calculated ( figure 6d ). this revealed that combined with the unbiased random forestry machine learning model, our gene sets rendered a powerful mathematical tool for distinguishing sle. it is reported that chronic autoimmune diseases including sle and ra might share some similar cellular pathogeneses with ms (50) . thus, we investigated whether our machine learning model could efficiently distinguish ra and ms based on bulk rna-seq data. three datasets were included in this study, including a set of pbmc datasets (gse90081, gse183204) with 12 ra patients and 24 healthy donors, a synovial tissue dataset (gse89408) with 152 ra patients and 28 healthy donors, and a pbmc dataset (gse159225) with 20 relapse-andremission ms patients, 10 secondary progressive ms patients, and 20 healthy donors. surprisingly, our machine learning model could separate patients with ra/ms and healthy donors with excellent accuracy in ra patients (auc = 0.967 ± 0.099 in ra pbmc datasets, figure 7a ; auc = 0.997 ± 0.006 in the ra synovial dataset, figure 7c ). for ms patients, our figure rendered an acceptable accuracy (auc = 0.775 ± 0.236 in ms pbmc datasets, figure 7e ). the corresponding feature importance shown in figures 7b, d, f illustrated that although our gene sets have extensive applicability and great accuracy for these diseases, each gene has different importance across each of these diseases. it suggested that our machine learning model requires a fine adjustment when applied to these diseases. to determine the contribution of positive signals to the accuracy of our machine learning model, we obtain a public bulk rna-seq dataset (gse137143, 122 ms patients and 22 healthy donors), which consists of only cd14-positive monocytes. unfortunately, the auc value dropped to 0.673 ± 0.136, indicating that the accuracy sharply decreased ( figure s4 ). this result suggested that the distinguishing power of our model was reduced on account of a loss of positive signals, for example, the signals from b cells. to evaluate the diagnosis accuracy of our machine learning model, an independent cohort containing 120 sle patients (gse164457) and 41 healthy donors (derived from gse169687) were enrolled into the study. the basic information and the gene expression pattern of objects within this cohort are shown in figures 8a, c. notably, the precision rate of our machine learning model diagnosis was 100% (120/120) and 92.7% (38/41) for sle patients and healthy donors, respectively ( figure 8b ). this result confirmed the diagnostic accuracy of our machine learning model, which suggested that it may be feasible to develop into an efficient tool for accurate disease diagnosis in the future. we aimed to develop a feasible strategy for distinguishing patients with sle and other major chronic autoimmune diseases in the early stage from healthy people. to achieve our purpose, the most crucial information that characterizes diseases should be filtered out first. from public single-cell rna sequencing datasets, we found that b cells and monocytes were the only two subpopulations containing sle-dominated clusters in the pbmcs of patients, which suggested that they might carry much stronger signals that indicate sle than other pbmc subpopulations. to date, conclusions about the contribution of pbmc subpopulations to the development of sle and other autoimmune diseases are not consistent, even when based on single-cell rna sequencing data (51) (52) (53) (54) (55) . most studies mainly focus on specific disease aspects, which might result in imbalanced data selection, background noise interference, and biased conclusions. hence, we selected the single-cell rna sequencing data from over 1.50 × 10 5 cells for each category with a balanced ratio between patients and controls (approximately 1:1) to avoid rushing into any prejudicial conclusions. further investigation of differentially expressed genes revealed the details of the most significant information that marks a disease within b cells and monocytes. a few interferon-stimulated genes were active in the sle-dominated b cells and monocytes, indicating that these cells might be a consequence of the inflammatory microenvironment. it is well known that the inflammatory microenvironment may be crucial to the progression of sle and other chronic autoimmune diseases. tsokos et al. reported that the production of autoantibodies triggered by both the innate and adaptive immune responses against self-antigens in sle patients resulted in the accumulation of monocytes and activation of lymphocytes (56) . our results confirmed this suggestion. interestingly, we found an activated naïve cluster of b cells in all of the pbmc subpopulations were influenced mutually in the progression of chronic autoimmune diseases, and analyses based on individual subpopulations may lose important information of reciprocities that accounts for disease progression. most current scrna-seq data analysis tools focus on detailed categorizations and trajectories of cells (28, (57) (58) (59) . recently, bioinformatic tools (e.g., cellchat, cellphonedb, italk) were developed to infer cellular cross talking from scrna-seq data, which make it possible to decipher reciprocities among cells under a single-cell level (57, (60) (61) (62) . therefore, we carried out cellular cross-talking analyses to reveal dynamic interactions across pbmc subpopulations and systematically decipher the etiology of diseases. surprisingly, we found that the interactions among the pbmc subpopulations of sle patients were weakened. it was reported that monocytes might contribute to the hyperactivity of b cells in sle patients (63) . a study also revealed that monocytes may function as a bridge during ra pathogenesis, and colocalization of cd14+ cells with cd4+ t effectors was found at sites of the inflamed rheumatoid synovium (64) . together, these reports illustrate that immune cells weave a network and that their interaction would provide significant information for autoimmune disease pathogenesis. further detailed analysis revealed that the major changes occurred in b cells or monocytes, including flt3, cd48, tnf, and tgf-beta signal patterns that have been reported to have a negative correlation with sle development (41) (42) (43) (44) . our results were consistent with previous studies on the variations in b cells (65) (66) (67) and monocytes (68) (69) (70) in sle. considering the repeatable results gained from our study, it should be convincing that the interactions among the pbmc subpopulations of sle patients may be weakened, which could result in abnormal emergences or variations in signaling patterns within pbmcs. based on our finding of powerful information that characterizes diseases, we tried to establish a machine learning model to distinguish chronic autoimmune diseases. several reports have proven that the random forest (rf) machine learning method would give a high accuracy in disease classification when abundant features were included (71, 72) , and another reason for the random forest model was its interpretability-each gene contribution in the rf machine learning model was visible. our area under curve (auc) score for sle indicates that our machine learning model has the potential to become an efficient tool for accurate diagnosis of sle at the single-cell rna level. considering that the information we identified was not specific to the early stage of the disease, further optimization should be performed to identify the sensitive information in the early stage of the disease to strengthen the diagnostic power of our machine learning model. further investigation is also needed to evaluate the efficiency of our machine learning model using bulk rna-sequencing data. our auc score illustrates that although other immune cell background noise might be introduced into rna-seq data, the gene set still has high accuracy in distinguishing patients with the disease from healthy donors. this might be attributed to the low correlation between each gene since they were derived from the two different analysis frameworks, and this low gene correlation in turn increased the random forest model accuracy (73) . given the cost and convenience of bulk rna sequencing, our results suggested that this machine learning model should be highly applicable going forward. in addition, our classification results for bulk rna sequencing data of pbmcs and synovial tissues derived from ra and ms patients indicated that this machine learning model also showed high accuracy in distinguishing these diseases. numerous studies have reported that chronic autoimmune diseases, such as sle, ra, and ms, might share some similar cellular pathogeneses (46, 50, 74) . our findings further confirmed this viewpoint and suggested that this machine learning model with the information we filtered out might be powerful enough to discriminate patients with common chronic autoimmune diseases from healthy donors, not just sle patients. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. author contributions are shown as follows. conception and design: ax, ym, and wh. acquisition of data: xx, yq. analysis and interpretation of data: tw, lz, jc and ym. writing, review, and/or revision of the manuscript: all authors. all authors contributed to the article and approved the submitted version. quality of life and unmet needs in patients with inflammatory arthropathies: results from the multicentre, observational rapsodia study lupus activity, and permanent organ damage axonal transection in the lesions of multiple sclerosis risk of relapse after antidepressant discontinuation in anxiety disorders, obsessive-compulsive disorder, and post-traumatic stress disorder: systematic review and meta-analysis of relapse prevention trials multiple sclerosis relapses: epidemiology, outcomes and management long-term outcomes in systemic lupus erythematosus: trends over time and major contributors sle diagnosis and treatment: when early is early work disability remains a major problem in rheumatoid arthritis in the 2000s: data from 32 countries in the quest-ra study the global burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study cardiovascular disease in rheumatoid arthritis: state of the art and future perspectives systemic lupus erythematosus: state of the art on the tension between early diagnosis and misdiagnosis of multiple sclerosis two-year clinical and radiologic follow-up of early ra patients treated with initial step up monotherapy or initial step down therapy with glucocorticoids, followed by a tight control approach: lessons from a cohort study in daily practice how do patients with newly diagnosed systemic lupus erythematosus present? a multicenter cohort of early systemic lupus erythematosus to inform the development of new classification criteria clinically isolated syndromes and the relationship to multiple sclerosis misdiagnosis of multiple sclerosis: impact of the 2017 mcdonald criteria on clinical practice the main challenges in systemic lupus erythematosus: where do we stand? new biomarkers in sle: from bench to bedside interferon pathway in sle: one key to unlocking the mystery of the disease serum biomarker panel for the diagnosis of rheumatoid arthritis molecular biomarkers in multiple sclerosis current and future therapies for sle: obstacles and recommendations for the development of novel treatments breaking immunological tolerance in systemic lupus erythematosus autoantibodies in systemic autoimmune diseases: specificity and pathogenicity comprehensive integration of single-cell data sharp: hyperfast and accurate processing of single-cell rna-seq data via ensemble random projection inference and analysis of cell-cell communication using cellchat mapping systemic lupus erythematosus heterogeneity at the single-cell level predicting patient phenotypes from single-cell rna-seq benchmarking atlas-level data integration in single-cell genomics why 70/30 or 80/20 relation between training and testing sets: a pedagogical explanation dimensionality reduction for visualizing single-cell data using umap fast unfolding of communities in large networks relative contributions of lean and fat mass to bone strength in young hispanic and non-hispanic girls enhanced inflammasome activity in systemic lupus erythematosus is mediated via type i interferon-induced up-regulation of interferon regulatory factor 1. arthritis rheumatol b-cell targeted therapeutics in systemic lupus erythematosus: from paradox to synergy? modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type i and type ii interferon signatures challenges and opportunities for consistent classification of human b cell and plasma cell populations distinct effector b cells induced by unregulated toll-like receptor 7 contribute to pathogenic responses in systemic lupus erythematosus epigenetic upregulation of fkbp5 by aging and stress contributes to nf-kb-driven inflammation and cardiovascular risk the slam family member cd48 (slamf2) protects lupus-prone mice from autoimmune nephritis the role of tumor necrosis factor-alpha in systemic lupus erythematosus impaired tgf-b signaling in patients with active systemic lupus erythematosus is associated with an overexpression of il-22 mesenchymal stem cell therapy induces flt3l and cd1c+ dendritic cells in systemic lupus erythematosus patients whole-genome transcription and dna methylation analysis of peripheral blood mononuclear cells identified aberrant gene regulation pathways in systemic lupus erythematosus the role of monocytes and macrophages in autoimmune diseases: a comprehensive review cd72 is a negative regulator of b cell responses to nuclear lupus self-antigens and development of systemic lupus erythematosus selective loss of signaling lymphocytic activation molecule family member 4-positive cd8+ t cells contributes to the decreased cytotoxic cell activity in systemic lupus erythematosus brief report: decreased expression of cd244 (slamf4) on monocytes and platelets in patients with systemic lupus erythematosus b cell depletion therapies in autoimmune disease: advances and mechanistic insights single-cell multi-omics analysis reveals ifn-driven alterations in t lymphocytes and natural killer cells in systemic lupus erythematosus single-cell analysis of human mononuclear phagocytes reveals subset-defining markers and identifies circulating inflammatory dendritic cells newly defined pro-inflammatory dc subset expanded in sle transcriptomic studies of systemic lupus erythematosus review: transcriptional regulation of cd4+ t cell differentiation in experimentally induced arthritis and rheumatoid arthritis new insights into the immunopathogenesis of systemic lupus erythematosus single-cell rna-seq reveals transcriptional heterogeneity and immune subtypes associated with disease activity in human myasthenia gravis comprehensive analysis of a mouse model of spontaneous uveoretinitis using single-cell rna sequencing single-cell rna sequencing of murine islets shows high cellular complexity at all stages of autoimmune diabetes prednisone reprograms the transcriptional immune cell landscape in cns autoimmune disease glomerular endothelial cells are the coordinator in the development of diabetic nephropathy single-cell multi-omics analysis of the immune response in covid-19 induction of dendritic cell differentiation by ifn-alpha in systemic lupus erythematosus macrophage subpopulations in rheumatoid synovium: reduced cd163 expression in cd4+ t lymphocyte-rich microenvironments cd11b activation suppresses tlr-dependent inflammation and autoimmunity in systemic lupus erythematosus relationship between cd4+/cd8+ t cell ratio and t cell activation in systemic lupus erythematosus cd47 potentiates inflammatory response in systemic lupus erythematosus. cells (2021) intercellular adhesion molecules in systemic lupus erythematosus patients with lupus nephritis recent insights into the genetic basis of systemic lupus erythematosus fecal mycobiota combined with host immune factors distinguish clostridioides difficile infection from asymptomatic carriage machine learning-based scoring models to predict hematopoietic stem cell mobilization in allogeneic donors random forests: from early developments to recent advancements monocyte subsets involved in the development of systemic lupus erythematosus and rheumatoid arthritis the authors thanks prof. min zhang of the first affiliated hospital, sun yat-sen university for manuscript revision. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.870531/ full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-0025521-5zqdj2cv authors: rahman, md. mominur; rahaman, md. saidur; islam, md. rezaul; rahman, firoza; mithi, faria mannan; alqahtani, taha; almikhlafi, mohannad a.; alghamdi, samia qasem; alruwaili, abdullah s; hossain, md. sohel; ahmed, muniruddin; das, rajib; emran, talha bin; uddin, md. sahab title: role of phenolic compounds in human disease: current knowledge and future prospects date: 2021-12-30 journal: molecules doi: 10.3390/molecules27010233 sha: ebed223cfcf09e54e34136e9dde592d1b278e7ba doc_id: 25521 cord_uid: 5zqdj2cv inflammation is a natural protective mechanism that occurs when the body’s tissue homeostatic mechanisms are disrupted by biotic, physical, or chemical agents. the immune response generates pro-inflammatory mediators, but excessive output, such as chronic inflammation, contributes to many persistent diseases. some phenolic compounds work in tandem with nonsteroidal anti-inflammatory drugs (nsaids) to inhibit pro-inflammatory mediators’ activity or gene expression, including cyclooxygenase (cox). various phenolic compounds can also act on transcription factors, such as nuclear factor-κb (nf-κb) or nuclear factor-erythroid factor 2-related factor 2 (nrf-2), to up-or downregulate elements within the antioxidant response pathways. phenolic compounds can inhibit enzymes associated with the development of human diseases and have been used to treat various common human ailments, including hypertension, metabolic problems, incendiary infections, and neurodegenerative diseases. the inhibition of the angiotensin-converting enzyme (ace) by phenolic compounds has been used to treat hypertension. the inhibition of carbohydrate hydrolyzing enzyme represents a type 2 diabetes mellitus therapy, and cholinesterase inhibition has been applied to treat alzheimer’s disease (ad). phenolic compounds have also demonstrated anti-inflammatory properties to treat skin diseases, rheumatoid arthritis, and inflammatory bowel disease. plant extracts and phenolic compounds exert protective effects against oxidative stress and inflammation caused by airborne particulate matter, in addition to a range of anti-inflammatory, anticancer, anti-aging, antibacterial, and antiviral activities. dietary polyphenols have been used to prevent and treat allergy-related diseases. the chemical and biological contributions of phenolic compounds to cardiovascular disease have also been described. this review summarizes the recent progress delineating the multifunctional roles of phenolic compounds, including their anti-inflammatory properties and the molecular pathways through which they exert anti-inflammatory effects on metabolic disorders. this study also discusses current issues and potential prospects for the therapeutic application of phenolic compounds to various human diseases. polyphenols are secondary plant metabolites that play a vital role in protecting plants from uv radiation and disease attacks [1] . they are naturally occurring compounds present in many foods, including fruits, vegetables, cereals, and beverages. polyphenols can be found in up to 200-300 mg per 100 g of fresh weight in grapes, apples, pear, cherries, and berries. their presence is also high in the products manufactured from these fruits. about 100 mg polyphenols are found in a glass of red wine, a cup of tea, or coffee. polyphenols can also be found in cereals, dried legumes, and chocolate [2, 3] . there has been a lot of focus on the potential health benefits of dietary plant polyphenols as antioxidants in the last decade. according to epidemiological research and related meta-analysis, long-term consumption of diets high in plant polyphenols protects against cancer, cardiovascular disease, diabetes, osteoporosis, and neurological diseases [4, 5] . bitterness, astringency, color, flavor, odor, and oxidative stability are all things that polyphenols can help with in food. plant polyphenols in the diet have a plethora of health benefits [6] . these compounds can prevent the negative reactivity of undesired reactive oxygen/nitrogen species produced by metabolic activities in the body. because of their potential health benefits, polyphenols and other dietary phenolics are generating much attention in the scientific world. inflammation is a biological response when tissue homeostasis is disrupted by a natural, synthetic, or foreign agent [7] . pathogens (parasites, fungi, and microorganisms), stress (shock or burns), and toxic compounds can all trigger an immune system response [8] . during infections, microorganisms and macrophages can stimulate the generation of proinflammatory mediators, such as interleukin il-1, il-6, and il-8, reactive oxygen species (ros), nitric oxide, and prostaglandins. chronic infection, resulting in the excessive release of pro-inflammatory factors, has been associated with the development of degenerative conditions, including arthritis, atherosclerosis, asthma, alzheimer's disease (ad), and malignancies [9] . ad is the most prevalent neurodegenerative disease, accounting for 60-70% of dementia cases [10, 11] . approximately 44 million people are estimated to have been diagnosed with ad or associated dementia to date, and this population is expected to grow to over 135 million by 2050 [12, 13] . the primary neuropathological hallmarks of ad are proteinaceous aggregates, such as intracellular neurofibrillary tangles containing hyperphosphorylated tau and extracellular senile plaques containing amyloid-beta deposits of varying lengths [14] [15] [16] [17] , resulting in neuronal cell death and degeneration associated with memory loss and severe cognitive impairment, disrupting activities of daily living [18] [19] [20] [21] . in addition to protein aggregation, ad is associated with a neuropathological level of glial activation and neuronal cell death [22] [23] [24] [25] [26] . environmental, lifestyle, and genetic risk factors can contribute to ad development [27] [28] [29] . hypertension is a common and often progressive condition associated with a high risk of cardiovascular disease and related complications [30] . hypertension is a critical health problem, and the incidence of hypertension is increasing globally, with some estimates suggesting that hypertension affects one-fourth of the total adult population worldwide [31] . the inhibition of the angiotensin-converting enzyme (ace), which converts angiotensin i to angiotensin ii, has been used to treat inflammatory disease (id) and has demonstrated powerful antihypertensive effects [32] . sugar uptake issues can lead to diabetes mellitus (dm), obesity, and oral diseases [33] . one of the most critical ongoing issues is the increased incidence of dm, caused by hyperglycemia, which describes an increase in the circulating blood glucose concentration. two types of dm have been identified: type i, which is caused by insufficient insulin production, and type ii, which is the result of insulin inefficiency or also insufficient insulin production [34] . mammalian skin tone is prevalently determined by the level of melanin, which is a pigment responsible for protecting the skin from ultraviolet (uv) damage. the melanin content is related to glycemic control of diabetes and obesity. the lower the melanin content, the higher possibility of microangiopathy [35] . in recent years, epidemiological examinations have revealed a critical increase in melanoma, especially among the white populace [36] [37] [38] . polyphenols can be helpful to delay detrimental effects in neuronal, behavioral, age-related diseases due to their high antioxidant activities. one study examined the antioxidant capacity of grape seed extract (gse) in different regions of the central nervous system (cns) in young and old rats [39] . reducing the inflammatory response is often critical for many disease states, and non-steroid anti-inflammatory drugs (nsaids) are often used for this purpose. however, nsaids are often associated with adverse side effects, including gastrointestinal complications, water retention, kidney deficiency, bronchospasm, and hypersensitive responses [40, 41] . phenolic and polyphenolic products, either alone or in combination with vitamins, such as carotenoids, vitamin e, and vitamin c, act as antioxidants that protect the tissues in the human body from the damaging effects of oxidative stress. polyphenols are the most common antioxidants found in fruit and vegetable-based diets. gallic, ellagic, protocatechuic, and 4-hydroxybenzoic acids are the most common benzoic acids consumed by humans, whereas caffeic, ferulic, sinapic, and p-coumaric acids are the most common cinnamic acids. plant-based diets are high in polyphenols, which provide nutritional advantages and contribute to preventing chronic diseases [42] . the current level of knowledge about the biochemical effects of dietary polyphenols and their involvement in human health and disease is discussed in this study. this study aims to describe the multifunctional roles of phenolic compounds in the treatment of human diseases. phenolic compounds have been used for many therapeutic purposes due to their effects on inflammation and other characteristics of human diseases, which may guide future research. phenolic compounds can be found in various fruits and vegetables, especially grapes, berries, and tomatoes. phenolic compounds can benefit one's health by reducing the risks of developing metabolic disorders, such as type 2 dm [33] . the biological properties of phenolic compounds are diverse, although the specific mechanisms they exert their disease preventive effects remain unknown. antioxidants contribute to the removal of these oxidative products. under normal cellular circumstances, ros and reactive nitrogen species (rns) are incredibly reactive molecules; for example, ros and rns can disrupt mitochondrial respiration, damaging critical biological macromolecules, including proteins and dna [43] [44] [45] . the anti-inflammatory, anti-aging, antiproliferative, and antioxidant properties of phenolic compounds have been described in several studies. in totaling the upstairs modifications, antioxidant enzymes are crucial for preventing oxidative damage [46] . reactive oxygen (ros) and reactive nitrogen (rns) species are highly reactive oxidized molecules, including superoxide, peroxide, singlet oxygen, hydroxyl radical, nitric oxide (no), and peroxynitrite (oono − ), that are constantly produced under normal cellular conditions, such as during homeostasis, impaired antioxidant functions can lead to cellular damage, resulting in aging, disease, and cell death ( figure 1 ) [47] . in intact cell systems, photo-activated reactive oxygen species (ros) may activate the mitochondrial permeability transition pore (mptp) within individual mitochondria. after the occurrences of ros-triggering of the mptp followed by additional stimulation of ros production, the phenomenon of ros-induced ros release (rirr) [49] was named. mptp opening is a mitochondrial response to oxidative stress that causes an increase in ros signal, which can have various consequences depending on ros levels. in addition to the effects of ros in those mitochondria, ros released into the cytosol could stimulate a complicated cellular signaling response and rirr in neighboring mitochondria (where the rirr originated). in the latter case, ros trafficking between mitochondria could serve as a positive feedback mechanism, increasing ros production that spreads throughout the cell and causes visible mitochondrial and cellular damage. although photo-induced ros formation in the laboratory could be used to trigger more massive, avalanche-like ros release, this phenomenon represents a more fundamental mechanism, such as lightindependent spontaneous redox transitions associated with the induction of mptp or another mitochondrial channel(s) that may occur under various physiological or pathological conditions. this review will cover a broad spectrum of physiological and pathological rirr-related processes, such as mitochondrial ros production and scavenging. finally, an imbalance in the intake, neutralization, and outflow of ros and associated triggers in particular cell signaling pathways can lead to oxidative and reductive stressors, which can cause a range of illnesses or even cell organismal death [50] . the primary sources of phenolic compounds are fruits [51] , vegetables, and beverages, such as coffee, tea, wine, and fresh fruit juices. although coffee is known for its stimulating properties attributed mainly to caffeine, it also contains other biologically active compounds, including phenolic compounds, with chlorogenic acids being the most abundant. the critical factor that affects these compounds in green coffee is the roasting time-temperature profile [52] . polyphenols found in green tea include, but are not limited to, epigallocatechin gallate (egcg), epigallocatechin, epicatechin gallate, and epicatechin; flavanols such as kaempferol, quercetin, and myricetin are also found in green tea [53] . in addition to exploring the potential protective effects, these compounds provide health benefits against chronic diseases, understanding the modifications during food processing techniques and, therefore, overall bioavailability is essential. the bioavailability of bioactive compounds is the absorptive process of these molecules across the intestine into the circulatory system, after food ingestion. several polyphenols can be ingested as either purified, isolated substances or in foods. following the intake of polyphenols ranging from 6.4 to 1000 mg/day, detectable plasma levels ranged from 0.072 to 5 µm [54] . the polyphenol intake measured for an older japanese population was reported to range from 183 to 4854 mg/day, with average information of 665-1492 mg/day. coffee and green tea were the most common sources of these compounds [55] . phenolic acids typically constitute approximately one-third of the total phenolics consumed, whereas flavonoids comprise the remaining two-thirds. phenolic and polyphenolic products, either alone or in combination with vitamins, such as carotenoids, vitamin e, and vitamin c, can serve as antioxidants to protect various tissues in the human body from oxidative stress. polyphenols are the most common antioxidants found in fruit and vegetable-based diets. gallic, ellagic, protocatechuic, and 4-hydroxybenzoic acids are the most common benzoic acids consumed by humans, whereas caffeic, ferulic, sinapic, and p-coumaric acids are the most common cinnamic acids. plant-based diets are commonly high in polyphenols, providing nutritional advantages and protecting against the emergence of chronic diseases. however, food processing techniques, including blanching and thermal treatments, can alter polyphenol levels or induce conversion into secondary compounds. enzymatic and nonenzymatic reactions can activate the absorption and metabolism of phenolics, in addition to molecular changes that might occur during food production ( figure 2 ). conjugation reactions may also increase or decrease the bioavailability of these molecules [42] . during the absorption process, gastric acid from the stomach can cause initial modifications to oligomeric polyphenols. following ingestion, glycosidic polyphenols are cleaved in the small intestine, releasing the glycoside radical. lactase phlorizin hydrolase and cytosolic glucosidase are enzymes with an affinity for glucose, xylose, and galactose [56] . however, polyphenols that are not cleaved by these enzymes are not absorbed by the small intestine and can be cleaved into small molecules known as phenolic acids produced by intestinal bacteria. polyphenol structures can also be involved in conjugation reactions, resulting in methyl, glucuronide, or sulfate groups. the remaining polyphenols, especially those attached to rhamnose, can be processed by rhamnosidase released by the colonic microbiota. following these absorptive processes, phenolics will typically follow one of four paths: (1) excretion in the feces; (2) absorption by the mucosa of the intestines or the colon, followed by entry into the portal vein for delivery to the liver; (3) further conjugation in the liver can result in the addition of with methyl, glucuronide, or sulfate groups, followed by release into the bloodstream for tissue absorption; and (4) excretion in the urine [42] . bioavailability refers to the percentage of a nutrient that is digested, absorbed, and metabolized normally. the bioavailability of each polyphenol varies, but there is no link between the number of polyphenols consumed and their bioavailability in the human body. although aglycones can be absorbed through the small intestine, most polyphenols found in food are present as esters, glycosides, or polymers, which cannot be absorbed in their natural state [57] . before these compounds may be taken, they must be processed by intestinal enzymes or colonic microbes. polyphenols undergo significant changes during absorption; they are conjugated in intestinal cells and later in the liver by methylation, sulfation, and glucuronidation [58] . as a result, the forms that reach the bloodstream and tissues differ from those found in food, making it difficult to identify and quantify all of the metabolites' biological activity [59] . the chemical structure of polyphenols, rather than their content, determines the rate and amount of absorption and the type of metabolites circulating in the plasma. because the most common polyphenols in our diet do not necessarily have the highest amounts of active metabolites in target tissues, polyphenol biological activities vary greatly. the increased plasma antioxidant capacity after consuming polyphenol-rich meals suggests that they are absorbed via the gastrointestinal barrier [60, 61] . polyphenols have distinct absorption locations in humans. some polyphenols are absorbed well in the stomach, whereas others are absorbed more efficiently in the intestine or other regions of the digestive tract. all flavonoids in foods are glycosylated, except for flavanols. the fate of glycosides in the stomach is largely unknown. most glycosides are unlikely to be hydrolyzed by stomach acid and hence reach intact in the intestine [62] , where only aglycones and a few glucosides are absorbed. some flavonoids, such as quercetin, can be absorbed at the stomach level, but not their glycosides, according to rat study [63] . furthermore, anthocyanins are recently shown to be absorbed from the stomach in rats and mice [57, 64] . according to one theory, glucosides are transported into enterocytes by the sodium-dependent glucose transporter sglt1 and then destroyed by a cytosolic -glucosidase. isoflavones, on the other hand, appear to be less affected by glucosylation than quercetin in terms of absorption [65] . proanthocyanidins differ from most other plant polyphenols because of their polymeric shape and huge molecular weight. this feature should limit their absorption through the gut barrier, and oligomers larger than trimers in their native forms are unlikely to be absorbed in the small intestine [57, 66] . when eaten in their free form, hydroxycinnamic acids are swiftly absorbed by the small intestine and conjugated into flavonoids [67] . because the intestinal mucosa, liver, and plasma lack esterase capable of hydrolyzing chlorogenic acid to liberate caffeic acid, and hydrolysis can only be conducted by colonic microbiota. these chemicals are naturally esterified in plant products, and esterification hinders their absorption [68] . even though the majority of polyphenols are absorbed in the gastrointestinal tract and intestine, some polyphenols are not. these polyphenols travel to the colon, where bacteria hydrolyze glycosides into aglycones, which are then converted into a variety of aromatic acids [69] . aglycones are split at different points along the heterocycle depending on their chemical structure, resulting in different acids that are then metabolized to benzoic acid derivatives. following absorption, polyphenols go through a variety of conjugation pathways. these activities generally consist of methylation, sulfation, and glucuronidation, which are metabolic detoxication processes that increase the hydrophilicity of xenobiotics and so assist biliary and urine excretion. polyphenol methylation is also very specific; it normally occurs in the c 3 position of the polyphenol, but it can also occur in the c 4 position: in fact, a large amount of 4 -methylepigallocatechin has been found in human plasma after tea drinking [70] . during the sulfonation process, sulfo-transferases catalyze the transfer of a sulfate moiety. sulfation occurs largely in the liver, but the precise location of sulfation for polyphenols is unknown [71] . glucuronidation occurs in the stomach and liver, with the largest rate of conjugation occurring in the c 3 position [72] . green tea catechins, whose aglycones can account for a significant portion of the overall amount in plasma [73] , have highly efficient conjugation processes, and free aglycones are frequently absent or present in low amounts in plasma following dietary dosages. because the nature and placements of the conjugating groups on the polyphenol structure can affect the biological properties of the conjugates, it is vital to identify the circulating metabolites, including their type and placements on the polyphenol structure. polyphenol metabolites attach to proteins and circulate in the bloodstream; albumin is the most important protein involved in this process. in order for polyphenols to be bioavailable, albumin is required. the affinity of polyphenols for albumin varies according to their chemical structure [74] . the rate of metabolite elimination, as well as their distribution to cells and tissues, may be affected by albumin binding. it is possible that metabolite absorption is related to the concentration of unbound metabolites in the cell. finally, whether polyphenols must be in their free state to have biological effect or if albumin-bound polyphenols might have biological activity is unknown [57] . because this is the concentration at which polyphenols are biologically active for exerting their effects, the accumulation of polyphenols in tissues is the most important phase of polyphenol metabolism. polyphenols have been shown in tests to permeate tissues, especially those that process them, such as the intestine and liver. in the urine and bile, polyphenols and their derivatives are eliminated. highly conjugated metabolites are more likely to be excreted in bile, whereas small conjugates, such as monosulfates, are eliminated preferentially in urine, according to new research. the extent of metabolites removed in urine is roughly proportional to maximum plasma concentrations. the urine excretion % of citrus flavanones is high, but it decreases as you proceed from isoflavones to flavonols. as a result, polyphenols' health advantages are reliant on their ingestion as well as their bioavailability [60] . phenolic compounds are a heterogeneous group of secondary metabolites generated during plant metabolism. due to their beneficial health effects, phenolic compounds have generated the interest of various experts, particularly their presence in foods. phenolic compounds contain at least one aromatic ring to which one or more hydroxyl group is attached, and they may be aromatic or aliphatic. flavonoids and non-flavonoids are two types of phenolic compounds [75] . these are heterocyclic compounds consisting of two aromatic rings linked by an oxygen. flavonoids can be divided into flavones, flavonols, anthocyanins, and isoflavones, depending on the hydrogenation status and the identities of heterocyclic substitution. flavonoids are typically represented by glycosides ( figure 3 ). two of the most representative non-flavonoid compounds are benzoic and cinnamic acids, also identified as phenolic acids ( figure 4) . stilbenes, tannins, and lignins are derived from phenolic acids. the absorption of phenolic fats (catechol, resorcinol, and hydroquinone) [77] from food products has been linked to the reduced occurrence of chronic illnesses, such as dm, cardiovascular disease, ad, parkinson's disease (pd), and infection, according to epidemiological data [45, 47] . phenolic compounds are considered to be responsible for these beneficial effects. prolonged and severe illnesses have been associated with the development of chronic conditions, as described. as a result, invasive events that alter the inflammatory cascade associate with disease development may represent potential targets for disease prevention [78] [79] [80] . some phenolic compounds have been shown to have anti-inflammatory properties. although the specific mechanisms that underlie these anti-inflammatory activities are not yet understood, there is an element between a high absorption of these intensifies and a devaluation in intemperate response [81] . the relationship between phenolic complexes and anti-inflammatory activities has been examined, and the following criteria have been established based on the observed reactions with various inflammatory targets [53, 54] . (i) for flavonoid molecules to be active, they must have a planar ring structure. (ii) the c ring must be unsaturated, due to the presence of a ketone carbonyl at c 4 or a double bond between c 2 and c 3 , for example. (iii) oh groups must be conjugated to the b ring and at c 5 and c 7 of the a ring. (iv) flavones and flavones featuring an oh group at the 4 position of the b ring had more significant activity than those without an oh group. (v) the activity improved following the methylation of the oh groups at positions 3, 5, or 4 . the methylation of the 3-oh group reduced (vi) cytotoxicity. (vii) flavones had more incredible activities than isoflavone, and flavonoids, although flavones are a type of flavonoid. (viii) because aglycones are not glycosylated, they have more potent effects than glycosides. the role of glycosides remains under debate because they have occasionally been shown to reduce anti-inflammatory activity [82] but facilitate absorption [83] . the relationship between the phenolic system and pro-inflammatory intermediaries has been demonstrated in further specialized applications. flavonoids can inhibit no due to three structural characteristics: (a) the c 2 =c 3 double bond; (b) an unwieldy substituent group can either decrease inhibition by several-fold (aglycones have a stronger inhibitory effect than glycosides); and (c) 7 and 4 oh groups can also affect inhibition, although to a lesser extent than the other characteristics [84] . phenolic compounds tend to act in a complementary manner with nsaids, but some phenolic compounds can also inhibit pro-inflammatory mediators' activity or gene expression, such as cyclooxygenase (cox). phenolic compounds can also up-or downregulate transcriptional elements involved in antioxidant pathways, such as nuclear factor-κb (nf-κb) or nuclear factor-erythroid factor 2-related factor 2 (nrf-2) [59, 85] . the structures of phenolic compounds can significantly impact their anti-inflammatory mechanisms. by resonance, unsaturation in the c ring, for example, appears to affect the strength of binding interactions. furthermore, the formation of a double bond between c 2 and c 3 causes coplanarity between the a and c rings, which increases the interaction between flavonoids and synthetic active sites [86] . enzymatic activity inhibited by catechols improves depending on the structure of the b ring, which relies on the development of electrophilic domains and requires nucleophilic additions. phenolic ligands can enhance the formation of covalent bonds between flavonoids and macromolecules [81] . phenolic compounds are thought to suppress the binding of pro-inflammatory mediators, regulate eicosanoid synthesis, inhibit stimulated resistant units, or inhibit the activity of no synthase and cox-2 through inhibitory effects on nf-κb [52, 60, 62] . inflammatory mediators, such as il-6, are affected by dietary flavonoids, such as flavones found in cocoa and tea, which have a dose-response effect on il-6 levels in the blood [87] . some subjects demonstrated a positive impact on the reduction in inflammatory markers. however, the mechanism linking the absorption of phenolic compounds from cocoa and the resulting levels of inflammatory markers (il-1, 1l-6, tumor necrosis factor [tnf]-α), but a reduction in low-density lipoprotein (ldl) was reported, which could result in reduced vascular inflammation, oxidative stress, and no levels and the prevention of platelet aggregation, reducing the risk of heart disorders [49, 64] . extensive reviews describing the anti-infection properties of phenolic compounds have again been directed toward us and red flame consumption. grape phenolic extracts have been used for both in vitro and in vivo experiments. procyanidins have been shown to inhibit inflammatory mediators, resulting in reduced concentrations of no, prostaglandin e 2 , and ros. the antioxidant properties of phenolic compounds were primarily responsible for these effects [88] [89] [90] [91] [92] . the inhibitory activities of pro-inflammatory intermediaries or changes in gene expression are involved in the impact exerted by phenolic compounds ( figure 5 ). an increase in the level of phenolic compounds or multiple phenolics may have anti-inflammatory effects through various pathways, whereas synthetic molecules can typically only affect one component (table 1 ). red circles indicate inhibition, and the numbers refer to the following phenolic compounds. (1) genistein [84, 93] , (2) daidzein [84] , (3) isorhamnetin [89] , (4) pelargonidin [91] , (5) kaempferol [92] , (6) apigenin [90] , and (7) epicatechin [93] . table 1 . mechanisms of anti-inflammatory activity mediated by dietary phenolic compounds [76] . genistein daidzein the inhibitor of nf-kb is one of the critical molecular targets of genistein. the inhibitory effect of genistein and daidzein was moderate (57-72%). inhibiting stat-1 activation also was genistein and daidzein expression and no output in vitro [84, 93] isorhamnetin pelargonidin isorhamnetin and pelargonidin both suppressed nf-b activation, but not stat-1 in vitro [84] kaempferol flavonol the mechanisms through which kaempferol inhibits stat-1 activation are unknown. however, they may be linked to stat-1 or its upstream kinase jak2 phosphorylation in vitro [94] apigenin flavone apigenin inhibits the nf-b pathway, which has anti-proliferative, anti-inflammatory, and anti-carcinogenic properties. apigenin inhibits stat1-induced cd40 expression, which modulates microglial activation in vitro [95, 96] epicatechin flavan-3-ol the suppression of the nf-b pathway by epicatechin protects against ulcerative colitis. the suppression of transcription factors stat1 and nf-b in intestinal cells is thought to be the primary cause of this impact in vitro [97, 98] inflammation is a biochemical reaction to tissue injury that is essential for survival. the immune system responds to stimuli such as infection, injury, or irritation through the release of pro-inflammatory cytokine [99] . the overproduction of pro-inflammatory cytokines, including il-1b, il-6, and tnf-α, leads to severe illnesses among adults, including asthma, atherosclerosis, allergies, and cancer ( figure 6 ) [100] . preventing inflammation-associated diseases requires the inhibition of pro-inflammatory cytokine overproduction. since ancient times, phytochemicals derived from plant-based formulations have been widely used to treat inflammation and associated disorders. among identified phytochemicals, phenolics are essential for suppressing inflammation, and recent research has revealed their potent anti-inflammatory properties. pragasam et al. [101] investigated the anti-inflammatory efficacy of p-coumaric acid by measuring the expression of the inflammatory mediator tnf-α in the synovial tissue of adjuvant-induced arthritic rats. they discovered that p-coumaric acid has a potent anti-inflammatory function through the reduction of tnf-α expression. prescription medications are often designed to act as inhibitors of compounds involved in disease mechanisms. concerns regarding harmful adverse effects caused by engineered catalyst inhibitors have prompted the search for new effective and protective inhibitors derived from natural sources. plant phenolic compounds are among the most commonly explored groups due to their enormous scope of biological effects. this section provides an overview of known phenolic compounds with protein inhibitory activity. to examine the chemical inhibitory capacities of phenolic compounds against various proteins known to be involved in severe human conditions, wide-reaching research has been conducted [103] . catalysts are perhaps the best focal point for identifying drug-based inhibitors for treating human diseases because they are significantly involved in several physiological cycles [104] . the utilization of compound inhibitors found in specific fundamental human food sources has been examined, which have reported effects on hypertension, metabolic issues, incendiary infections, and neurodegenerative diseases. several food sources containing identified inhibitors have been reported in treating a variety of symptoms, including those associated with hepatotoxicity, gastrointestinal problems, and diarrhea [76, 77] . hypertension is a common and often progressive condition associated with a high risk of cardiovascular disease and related complications [30] . hypertension is a critical and increasingly common health problem worldwide, and estimates suggest that up to one-quarter of the global adult population suffers from hypertension [31] . ace inhibition is a common goal for treating id and has been shown to have antihypertensive effects [32] . ace catalyzes the conversion of angiotensin i into angiotensin ii, a vasoconstrictive peptide, and degrades bradykinin, a potent vasodilator [105] . common ace inhibitors, including captopril, benazepril, and enalapril, among others [106] , have been associated with adverse effects, including an overall reduction in proteolytic activity. consequently, attempts have been made to identify novel ace inhibitors from natural sources, mainly plant sources. many studies have shown that food sources rich in polyphenols are powerful for preventing and treating hypertension, specifically through ace inhibition (figure 7 ) [107] . in a new report, 74 plant families with significant ace inhibitory action were distinguished by patten et al. [108] . field and newton [109] have similarly demonstrated that cocoa polyphenols (catechins, flavonol glycosides, anthocyanins, procyanidins) are bioavailable particles with antihypertensive activity through ace inhibition [110] . using an in vitro model, bhandari et al. studied the antidiabetic efficacy of bergenia ciliata. the active chemicals (−)-3-o-galloylepicatechin and (−)-3-o-galloylcatechin, found in the ethyl acetate soluble b. ciliata extract, were found to be responsible for the substantial inhibition of porcine pancreatic-amylase and rat intestinal maltase activity in a dosedependent manner [111] . the half-maximal inhibitory concentration (ic 50 ) value, often used to quantify inhibitor potency, is defined as the concentration of an inhibitor necessary to inhibit enzyme activity by 50%. the ic 50 values for (−)-3-o-galloylepicatechin were 334 and 739 m for rat intestinal maltase and porcine pancreatic-amylase, respectively, and those for (−)-3-o-galloylcatechin were 150 and 401 m, respectively [111] . the inhibitory activities of these compounds against α-glucosidase and α-amylase in vivo and in vitro demonstrated that they have an excellent potential for development as a treatment for type 2 dm [112] . delaying glucose absorption through inhibiting -glucosidase activity is one potential treatment option for type 2 dm prevention. terminalia chebula fruits contain phenolics that present significant enzymatic inhibitory activity against mammalian-glucosidases, which could contribute to the management of blood glucose levels in patients with type 2 dm without causing severe adverse effects quercetin, kaempferol, luteolin, quercetagetin, and scutellarein are naturally occurring flavonoids that function as inhibitors of human α-amylase, making them intriguing candidates for limiting starch digestion (figure 7 ) [34] . natural health substances including non-flavonoid polyphenols (e.g., resveratrol, curcumin, tannins, and lignans), flavonoids (e.g., anthocyanins, epigallocatechin gallate, quercetin, naringin, rutin, and kaempferol), plant fruits, vegetables, and other products (e.g., garlic, green tea, blackcurrant, rowanberry, bilberry, strawberry, cornelian cherry, olive oil, sesame oil, and carrot) may be a safer alternative to primary pharmacological therapy. flavonoids are essential components of the human diet, and they may contribute to dm management by delaying the deterioration of pancreatic beta-cell function caused by oxidative stress. they are recommended as food supplements to prevent and ameliorate t2dm-related complications [113, 114] . mammalian skin tone is prevalently dictated by the levels and locations of the pigmentation melanin. melanin protects the skin from the harmful effects of uv light; however, melanin overproduction can result in the development of various dermatological problems. for example, melasma and age spots are caused by the abnormal accumulation of epidermal pigmentation [115] . three reactions in the biosynthetic cycle of melanin in melanocytes are catalyzed by tyrosinase. tyrosine is first hydroxylated to l-dopa and then oxidized to dopa quinone. following a series of oxidoreduction reactions, the moderate dihydroxy indole (dhi) and dihydroxy indole carboxylic corrosive are generated and polymerize to frame melanin [116] . tyrosinase inhibition is one of the key strategies used to treat hyperpigmentation; however, concerns regarding the toxicity and adverse effects of synthetic inhibitors have prompted the search for new protective and effective tyrosinase inhibitors derived from natural products. tyrosinase is often used as a soil-conditioning agent, and inhibitors of this compound are often added to plant-based nourishments. for the development of skin-brightening agents, similar to pest control substances, the identification of tyrosinase inhibitors is fundamental. various scientists have attempted to identify inhibitors from natural sources, such as plants. phenolics and polyphenols represent the largest group of phytochemical substances with dynamic tyrosinase inhibitory activity (figure 7 ) [117] . the largest group of newly identified natural tyrosinase inhibitors are flavonoids, and their structures contain elements similar to those of tyrosinase substrates and known tyrosinase inhibitors [117, 118] . steppogenin, for example, is a flavanone derivative isolated from cudrania tricuspidata, which showed significantly more decisive inhibitory action against tyrosinase than kojic acid, a known tyrosinase inhibitor [119] . some stilbenes have also been recognized as having tyrosinase inhibitory and degradation properties, including resveratrol, oxyresveratrol, and chlorophorin [118, 120, 121] . numerous ailments are associated with chronic aggravation, including dm, obesity, malignancy, osteoarthritis, atherosclerosis, and crohn's disease. the aggravation systems remember a grouping of occasions for which arachidonic acid digestion plays a significant role in catalyzing the conversion of arachidonic acid to prostanoids by cox-1 and cox-2. 5-lipo oxygenase (lox) is involved in a second pharmacologically practical metabolic pathway for arachidonic acid, resulting in the biosynthesis of leukotrienes, a class of inflammatory mediators. pro-inflammatory compounds include coxs, which affect platelet aggregation, vasoconstriction, vasodilatation, and loxs, affecting atherosclerosis development [122, 123] . much attention has been paid to the two cox proteins and 5-lox because they have been identified as putative malignancy prevention targets. nonsteroidal and steroidal anti-inflammatory drugs exert their activity by inhibiting these pro-inflammatory mediators through various mechanisms [124] . although current anti-inflammatory agents can inhibit intense inflammatory responses, unfavorable effects are associated with the continued use of these medications to combat chronic inflammatory states. these adverse effects legitimize the search for new and safe inflammatory mitigating agents derived from plant compounds. recently, interest in flavonoids' inhibitory and immunomodulatory capability has increased, including their capacity to inhibit pro-inflammatory secretion [125] [126] [127] . polyphenolic compounds inhibit phosphatidylinositide 3-kinases/protein kinase b (pi3k/akt), inhibitor of kappa kinase/c-jun amino-terminal kinases (ikk/jnk), mammalian target of rapamycin complex 1 (mtorc1) which is a protein complex that controls protein synthesis, and jak/stat. they can suppress toll-like receptor (tlr) and pro-inflammatory genes' expression. their antioxidant activity and ability to inhibit enzymes involved in the production of eicosanoids contribute as well to their anti-inflammation properties. a series of in vitro studies found that polyphenols like oleanolic acid, curcumin, kaempferol-3-o-sophoroside, egcg and lycopene inhibit high mobility group box1 protein, an important chromatin protein that interacts with nucleosomes, transcription factors, and histones regulating transcription and playing a key role in inflammation. all of these examples support the anti-inflammatory effects of polyphenols [128] . ad is a progressive, age-related neurodegenerative disease that represents the most predominant form of dementia. the rapidly aging human population has increased the incidence of ad worldwide, and global ad rates are projected to improve immensely, particularly in developing regions. although the exact pathogenesis of ad has yet to be clarified, it is currently believed to be a multifactorial disease. postmortem research performed during the mid-1970s showed that choline uptake and acetylcholine levels were diminished in the cerebrums of ad patients, which was associated with severe presynaptic cholinergic deficits [129] . this finding prompted the cholinergic-deficiency hypothesis, which suggests that a disruption in the cholinergic capacity is an underlying factor in ad development, associated with deficits in learning, memory, behavior, and excitatory reactions in various cerebral regions, including the neocortex and the hippocampus. acetylcholine is rapidly hydrolyzed by acetylcholinesterase (ache) [96, 97] , and acetylcholine levels at the synapse are responsible for the conduction of electrical impulses that transmit from one neuron to the next, which becomes diminished under conditions of acetylcholine deficiency. butyryl cholinesterase (bche) is a catalyst firmly identified with ache and serves as a co-regulator of acetylcholine hydrolysis and cholinergic neurotransmission [130] . during ad progression, some studies have shown expanded activity of bche in the most affected brain regions. the inhibition of both ache and bche increases acetylcholine availability and diminishes amyloid-beta accumulation, significant ad features. bche expression is primarily restricted to the fringe tissues, with only small quantities of bche found in the primary cerebral cortex. the potential advantages of the specific inhibition of ache without inhibiting bche could result in reduced responses due to the remaining cholinesterase activity in the fringe regions [131] . however, the mechanisms through which polyphenols act on important cellular events have not been completely elucidated to date. phenolic compounds interact with the amino acid residues that define the active site of ache via the formation of hydrogen bonds and hydrophobic and π-π interactions [132] . multiple hydroxyl groups in the phenolic compound are thought to enhance the inhibition of ache due to the enhanced binding capacity [133] . these inhibitory activities explain the functional potential associated with most phenolic compounds, but not all act through the exact mechanism [134] . the role of different polyphenols and their modes of action are shown in table 2 . the skin is an essential organ of the human body, representing the largest surface area and directly contacting the environment. various skin diseases can undermine our lives, such as malignancy (table 3 ) [142] . table 3 . protective effects of plant extracts and phenolic compounds against oxidative stress and inflammation induced by airborne particulate matter [143] . keratinocytes from the hacat strain membrane irritation eupafolin from phyla nodiflora [144] keratinocytes from the hacat strain -eupafolin nanoparticles [145] keratinocytes from the hacat strain membrane irritation nanoparticles comprising 7,3 ,4 -trihydroxy isoflavone [146] fibroblast-like synoviocyte membrane irritation resveratrol [147] keratinocytes from the hacat strain 3d-skin models painful joints resveratrol, resveratryl triacetate [148] ea.hy926 endothelial cubicles, monocytic thp-1 cells membrane irritation ellagic acid, punicalagin, punica granatum abstract [149] keratinocytes from the epidermis of humans irritation punicalagin, (−)-epigallocatechin gallate [150] dermal fibroblasts from humans membrane irritation (−)-epigallocatechin gallate [151] keratinocytes from the hacat strain3d-skin models membrane irritation e. cava extract, dieckol [152] keratinocytes from the hacat strain membrane irritation afzelin from thesium chinense [153] keratinocytes from the hacat strain 3d-skin models membrane irritation formononetin from astragalus mongholicus [154] in vivo the most dangerous skin diseases are skin tumors, including squamous cell carcinoma, basal cell carcinoma, and threatening melanoma. although threatening melanoma is less common than the other two types, it is often the most serious. melanoma can be treated adequately through a simple medical procedure during the early phases; however, melanoma is often associated with a high death rate due to high levels of metastasis and poor response to chemotherapy. in recent years, epidemiological examinations have shown a critical expansion in the occurrence of melanoma, especially among the white populace [36] [37] [38] . high death rates among melanoma patients have prompted numerous scientists to search for effective feature-based treatments. spices and medications derived from plants have long been used to treat tumors and are increasingly used in modern society [157, 158] . among the currently available anticancer drugs, 60% depend on natural compounds and their metabolites. it has increased interest and trust in biological agents, which represent significant hotspots for developing viable therapeutic agents for various diseases [159] . phenolic compounds that can affect the cell cycle represent promising natural compounds that inhibit malignant growth, such as skin tumors. curcumin, which is a wellknown apoptotic compound, is one possible compound. studies have shown that p53 is not activated by curcumin, which is significant for treating p53-transformed melanomas that are impervious to regular chemotherapy. curcumin activates caspase-3 and caspase-8 but not caspase-9, and, through a layer intervened system, apoptosis happens [160] [161] [162] . the chemopreventive effects of polyphenols as anthocyanins, ellagitanins, egcg, oleuropeindihydroxy phenyl, punicalagin, quercetin, resveratrol and theaflavin, were mainly examined in treatment of melanoma as the highly metastatic form of the cutaneous cancer. these polyphenols are mediated by several signaling pathways against skin carcinogenesis and metastasis, implying the importance of polyphenols to open up new horizons in development of anti-skin cancer therapeutic strategies [163] . psoriasis is a hereditary disease characterized by cutaneous irritation, expanded epidermal growth, hyperkeratosis, angiogenesis, and strange keratinization, with a t cell infiltration into the inflamed skin tissue [162, 164] . psoriasis is often characterized by dry or red patches of skin, coated with gleaming scales and red lines, and can affect various regions of the body, and all areas of the skin can be affected. psoriasis often presents in the fingernails and toenails, on the skin of the trunk, elbows, knees, and scalp. skin injuries, breaks in the skin, aggravation, scratching, joint pain, increased irritation of the eyes, and small flaky skin spots on the skin, particularly among infants, are additional symptoms. irritation and angiogenesis correspond with the pathophysiology of psoriasis, promoting uncontrolled keratinocyte outgrowth. to date, psoriasis, alongside natural elements, is solidly distinguished as a solid, unique hereditary background [165, 166] . phototherapies and medicines with antiproliferative effects that inhibit keratinocyte growth are the primary treatment options for psoriasis [125] . however, existing treatments can aggravate symptoms and induce phototoxicity, excessive sensitivity, organ damage, malignant growth, and systemic immunosuppression, and the identification of natural treatment options represents a vital alternative [166] . immunosuppressive and growth mitigation effects against psoriasis have been demonstrated by various natural compounds, including polyphenolic compounds [167] . psoriasis is a stable, recurring skin disease that affects up to 2% of the global population, characterized by well-defined macroscopic skin changes [168] . the development of psoriatic lesions involves two diverse cell types, mononuclear leukocytes, and epidermal keratinocytes. keratinocytes produce factors that enable t cells and antigen-presenting cells to communicate directly [169] . dietary activities have been linked to reductions in the clinical course and incidence of psoriasis in evidence-based clinical studies [170] . dietary alterations can also diminish the incidence of side effects associated with the use of immunosuppressive drugs; from a pharmacoeconomic perspective, a wellbalanced diet can lessen the costs of chronic disease care while also lowering the risks of complications [171] . it has been demonstrated that phytomedicine, which is used for psoriasis patients, provides some advantages, including natural sources, a lower risk of adverse effects, and the avoidance of dissatisfaction with conventional therapy. the herbal products' structural diversity and multiple mechanisms of action have enabled the synergistic activity to mitigate psoriasis through the inhibition of keratinocyte-proliferation [172] . there are several modern drugs available for the treatment of psoriasis from polyphenol-rich dietary active compounds (table 4 ) [173] . table 4 . phenolic compounds in psoriasis treatment [173] . quercetin smilax china leucocyte migration and epidermal thickness are reduced [174] capsaicin because of the release of substance-p, it's helpful in neurogenic inflammation [175] wrightia dione wrightia tinctoria anti-inflammatory [175] thespesin thespesia populnea retention of the stratum granulosum and significant reduction in the total epidermal thickness [176] chamazulene/matricin matricaria recutita by reducing the function of lipoxygenase, has an anti-inflammatory effect [177] silymarin silybum marianum it decreases liver damage by inhibiting leukotriene production and camp phosphodiesterase action [178] 6.9. acne vulgaris the anaerobic bacterium propionibacterium acnes plays a significant role in the pathogenesis of skin inflammation. antimicrobial treatments applied to the treatment of skin rashes, rosacea, and other non-resistant infections may also prevent propionibacterium acnes colonization. a minimal skin inflammatory response following the application of antimicrobial agents, such as erythromycin and antibiotics, has been reported, leading to a lack of satisfaction with treatment. benzoyl peroxide (bpo) is an exceptionally viable antibacterial agent against propionibacterium acnes, which has no known targeted treatment option to date. in a combined treatment for skin rashes, including antimicrobials, retinoids represent a fundamental component and may represent an alternative treatment option [179] . flavonoids identified in eucalyptus maculata extract [180] or terminalia arjuna [181] include alpha-mangostin, the principal compound in mangosteen natural skin products, which demonstrated significant antimicrobial activity against propionibacterium acnes strains [182] . honokiol and magnolol (isolated from magnolia sp.), in addition to gallic, caffeic, chlorogenic, ferulic, myricetin, and cinnamic acids, quercetin, apigenin, luteolin, and thymol, derived from wild watermelon leaves, are other phenolic compounds that have demonstrated antibacterial effects against propionibacterium acnes [183, 184] . this makes the invulnerable framework incapable of adapting to allergens around us, and an ever-increasing number of people experience the adverse effects of skin hypersensitivities and atopic dermatitis. a polluted environment can disrupt nourishment due to an increase in synthetics and stress. the incidence of skin hypersensitivity has increased globally over the past 20 years. changes in dietary patterns are thought to represent an ecological factor contributing to the unfavorable changes in the presentation of detrimental symptoms and increased sensitivity to various factors. changes in diet have been demonstrated to prevent hypersensitive reactions and improve the manifestation of symptoms. devereux et al. have performed a longitudinal study showing that the dietary intake of cell reinforcements and lipids during pregnancy and youth may be associated with a lower risk of hypersensitive skin diseases onset [185] . in another investigation, shaheen et al. showed that asthma incidence and severity was negatively associated with apple and red wine consumption in a population-based case-control study in london, likely due to the protective impacts of flavonoids [186] . the clinical implications of specific flavonoid-rich vegan diets that feature fewer calories in adult patients with atopic dermatitis were examined in a different examination, which showed a reduction in disease severity and improved serological parameters [187] . some plant species produce a wide range of phenolic compounds. camellia assumica and camellia sinensis are familiar catechin sources. green tea primarily consists of water and phenolic substances (flavandiol, flavanols, phenolic acid, and flavonoids), and catechins represent greater than 75% of the polyphenols found in tea leaves [188] . c 60 (oh) 44 is active catechin, often used as a regulator in studies of fullerenes, and its hydroxylated compounds feature antimicrobial activity [189] . alvarez-suarez et al. investigated the presence of carotenoids, flavonoids, amino and ascorbic acids, proteins, and total phenolic compounds and explored their antimicrobial properties in a variety of cuban honey. honey displays antibacterial effects, which moderately effectively against bacillus subtilis and escherichia coli (figure 7) [190] . pinosylvin, piceatannol, and pinosylvin monomethyl ether, all stilbenes, have been proposed to feature antibacterial activities. pinosylvin susceptibility was exceptionally high for listeria monocytogenes [191] . when assessing the antibacterial activities of phenolic compounds isolated from tobacco leaf, the ability to inhibit proliferation in staphylococcus aureus, escherichia coli and bacillus subtilis was measured [192] . the in vitro activities of seven antimicrobial agents, including ceftazidime, ciprofloxacin, tetracycline, sulfamethoxazole, trimethoprim, piperacillin, and polymyxin b, and six polyphenols (gallic acid, ellagic acid, protocatechuic acid, rutin, myricetin, and berberine) against five pseudomonas aerugi varieties were tested, both individually and in combination [193] . although their bactericide and fungicide properties have been extensively studied, few studies have examined how biopolymers may behave as carriers of antiviral compounds or how they interact with the constituents of edible films or coatings. furthermore, edible antiviral coatings can be engineered to inactivate viruses, usually more resistant to treatments than bacteria [194] . for example, gse and green tea extract are two plant substances with known antiviral activities that can be useful in the food industry [195] [196] [197] [198] . with increasing age, aging is defined as the accumulation of various harmful changes in cells and tissues, increasing the risk of disease and mortality. one of the most widely recognized hypotheses for understanding the mechanism of aging is the free radical/oxidative stress theory [199] . even under normal circumstances, some oxidative damage occurs; however, as the efficiency of antioxidative and repair mechanisms declines with age, the rate of this damage increases [200, 201] . the antioxidant capacity of plasma is connected to antioxidant food intake; it has been discovered that eating an antioxidant-rich diet can help to reduce the adverse effects of aging and behavior. several studies have suggested that a combination of antioxidant/anti-inflammatory polyphenolic chemicals in fruits and vegetables could be effective anti-aging agents [202] . brightly colored fruits, such as berry fruits, concord grapes, and grape seeds, are exceptionally high in anthocyanins, a subset of flavonoids. fruit pigments called anthocyanins have been proven to have powerful antioxidant and anti-inflammatory properties and suppress lipid peroxidation and the inflammatory mediator's cyclo-oxygenase (cox)-1 and -2. fruit and vegetable extracts with high quantities of flavonoids, such as spinach, strawberries, and blueberries, have high total antioxidant activity. dietary supplementation with spinach, strawberry, or blueberry extracts in a control diet was likewise helpful in restoring age-related deficiencies in the brain and behavioral function in elderly rats, according to the findings [46] . according to a new study, tea catechins have potent anti-aging properties, and drinking green tea rich in these catechins may help delay the onset of aging [203] . polyphenols can also help reduce the adverse effects of aging on the neurological system and brain. the ability of dietary polyphenols to cross the blood-brain barrier (bbb), which tightly limits the entry of metabolites, nutrients, and medications into the brain, is critical for their relevance in protecting the aging brain. resveratrol has been demonstrated to extend life expectancy continuously; its activity is linked to a condition known as calorie restriction or partial food deprivation. resveratrol, a grape polyphenol, is a relatively new antiaging agent. the sirtuin class of nicotinamide adenine dinucleotide (nad)-dependent deacetylases has been demonstrated to be an early target of resveratrol. in mammals, seven sirtuins have been discovered, with sirt-1 thought to mediate the health and longevity benefits of calorie restriction and resveratrol [204] . resveratrol improved insulin sensitivity, reduced igf-1 expression, and raised the activity of amp-activated protein kinase (ampk) and peroxisome proliferator-activated receptor-c coactivator 1a (pgc-1a). when the mechanism was investigated, it activated forkhead box o (foxo), which regulates the expression of genes involved in lifespan and stress resistance, as well as insulin-like growth factor binding protein 1 (igfbp1) (igfbp-1) [205] . experiments have shown that resveratrol can lengthen the lifetime of yeast. fruit fly saccharomyces cerevisiae drosophila melanogaster, c. elegans (nematode worm), and nothobranchius furzeri (seasonal fish). recently, quercetin has been linked to an anti-aging effect [206] . epidemiological evidence has demonstrated the remarkable health-promoting effects of phenolics against chronic ailments, including anticarcinogenic, anti-inflammatory, and antioxidant activities (table 5 ). flavonoids are the most common phenolic compounds, comprised of chalcones that contain three aromatic rings and fifteen carbons [207] [208] [209] . other phenolic acids, such as ferulic, feruloyl-l-arabinose, and coumaric, have been studied in numerous cell lines for their anticarcinogenic potential [210] . ferulic acid has an anticancer impact on mia paca-2 cells through effects on the cell cycle, invasion, and apoptotic behavior (human pancreatic cells) [211] . researchers examined the synergistic anticancer potential of ferulic acid and -tocotrienol against the proliferation of various cancer cells and discovered that, when used together, this combination inhibits the proliferation of du-145 (prostate cancer), mcf-7 (breast cancer), and panc-1 (pancreatic cancer) cells better than when used separately [212] . according to choi and park, ferulic acid suppresses homologous recombination during dna repair and rad51 (eukaryotic gene) production in breast cancer cells. furthermore, when combined with veliparib therapy, ferulic acid showed significant chemotherapeutic efficacy [213] . by slowing the cell cycle progression of caco-2 colon cancer cells, p-coumaric acid was able to protect against the development of colon cancer. in lung cancer cells, feruloyl-l-arabinose inhibited cell penetration, motility, and ros generation. furthermore, flavonoids have demonstrated excellent anticarcinogenic capacities, such as troxerutin, apigenin, kaempferol, and myricetin [214] . despite a lack of research regarding its pharmacological activities and chemical constituents, urtica atrichocaulis, a plant indigenous to china, is widely used to treat rheumatoid arthritis. the chemical compositions of the phenolic compound-rich fraction of u. atrichocaulis (tfua) and their anti-rheumatoid arthritis activities were examined. tfua treatment significantly reduced the effects of adjuvant-induced arthritis and carrageenaninduced paw edema in rats and cotton pellet-induced granuloma and acetic acid-induced writhing reactions in mice [216] . inflammatory bowel disease (ibd) is a common condition with an unknown cause in western and eastern countries. although recognized therapies exist for the treatment of ibd, their clinical efficacy remains lacking. plant-derived substances have shown great promise in pharmacological models of inflammation, and a few have been tested in preliminary clinical trials. isoprenoids, stilbenes, flavonoids, and alkaloids, in addition to structurally related chemicals, are examples of secondary metabolites with anti-inflammatory activities. most of these chemicals suppress cytokine release by inhibiting nf-κb activity, modulating enzymes and transcription factors [217] . body, food, and respiratory allergies comprise the majority of allergy problems. when an insusceptible system becomes sensitive to a typically innocuous allergen, the immune response becomes overwhelmingly oriented to a t-helper type 2 reaction. when the allergen is again introduced, the body releases a critical number of allergy-related intermediaries, resulting in the presentation of symptoms. our insight into these conditions has improved treatment techniques to balance the acute response or regulate allergen intermediaries, reducing susceptibility to adverse side effects. polyphenols have been investigated for their antiallergic properties in animal models and human clinical trials. their mitigating properties have been associated with the recruitment of immune cells to the skin and the anticipation of potential infections following skin breaks. polyphenols may regulate hypersensitivity through interactions with proteins, and their immediate effects on unfavorable effector cells like pole cells repress the secretion of inflammatory mediators, bringing about sign alleviation. moreover, the level of cellular damage caused by free radicals during the hypersensitive insult is restricted by polyphenols' endogenous cancer prevention activities. generally, polyphenols show promise as antiallergenic agents, potentially influencing various natural pathways and protecting cells during the hypersensitivity reactions, and warrant further consideration [218] . according to the literature review, flavonoid and phenolic compounds are found in medicinal plants and shown cardioprotective effects, [219] [220] [221] [222] according to an extensive review by razavi-azarkhiavi [223] a cardioprotective function was identified for various phenolic compounds. although doxorubicin (dox) is among the most widely used anticancer agents, its clinical application is hampered owing to its cardiotoxicity. adjuvant therapy with an antioxidant has been suggested as a promising strategy to reduce dox-induced adverse effects. in this context, many phenolic compounds have been reported to protect against dox-induced cardiotoxicity [223, 224] . centaurea transcaucasica sosn. ex grossh was studied for their protective effects on doxorubicin-treated cardiomyocytes [225] . the cardioprotective effects of phenolic compounds are exerted via multiple mechanisms including inhibition of reactive oxygen species generation, apoptosis, nf-κb, p53, mitochondrial dysfunction, and dna damage [223] . nanotechnology plays an increasingly important role in reducing oxidative stress, which has been linked to various diseases, such as cancer, ad, and pd [226] ; however, the role of this technology in other conditions has yet to be determined. depending on their antioxidant functionality, nanoparticles can play a critical role in preventing or treating disease by reducing oxidative stress levels, which is a standard function of nano bioactive compounds [227] . the most popular method for delivering antioxidant nanoparticles involves shrinking a natural bioactive molecule to a nanoscale that can rapidly target a site with minimal activity loss [220] . nanosized bioactive compounds can range in size from 10 to 1000 nanometers, which can enhance both bioactivity and target specificity while simultaneously reducing toxicity and improving safety [228] . the size of the surface activity, carrier toxicity, and the type of bioactive compound are the most important characteristics to determine when designing a nanoparticle for the treatment of pd [229] . bioactive nanoparticles with smaller sizes can reach a target in the brain target faster than larger nanoparticles. the anti-phagocytosis properties of hydrophilic coatings applied to nano bioactive compounds can prevent their premature degradation, and the nanoparticle carrier should be nontoxic [230] . the process used to prepare nano bioactive compounds determines whether the nano bioactive compounds are packaged in the core or on the surface of the nanoparticles. curcumin oxidation or hydroxylation can be prevented by packaging curcumin within the core of a nanoparticle [231] . thiamine-coated nanoparticles have been prearranged on the particle surface to boost antioxidant transport to the brain [232] . the most common neurodegenerative disorder, ad, currently lacks any accepted treatment options. ad is characterized by the loss of cognitive, learning, memory, and language skills. ad is associated with extracellular amyloid-beta plaques and intracellular neurofibrillary tangles containing hyperphosphorylated tau. approximately 10% of all individuals older than 65 suffer from ad [233] . as a result, current and future patients are likely to face various financial and social issues. effective interventions for treatment and prevention must be developed rapidly to confront these challenges. researchers have identified several factors contributing to ad, but the underlying mechanisms that increase neuronal susceptibility to ad with age have not yet been discovered [234] . the study of phenolic compounds in alzheimer's disease are shown in table 6 . table 6 . some proposed mechanisms for the beneficial effects of polyphenols in alzheimer's disease. resveratrol encourages deprivation of ab via proteasome in vitro [235] protects against ab-mediated cell death via pkc phosphorylation in vitro [236] egcg hinders creation, delay and steadiness of ab fibrils in vitro in vitro [237] keeps since ab-induced apoptosis in vitro [238] encourages non-amyloidogenic way in animal and cell models in vitro [239] curcumin hinders construction of ab fibrils in vitro in vitro [239] diminishes oxidative stress and plaques construction in appsw transgenic mice in vitro [240] shields cells since oxidative ab insult in vitro [241] as indicated by numerous investigations, the risks of developing certain diseases can be increased or decreased depending on the components of our diets. diet-associated shifts in trouble have been identified for neurodegenerative diseases [242] and cardiovascular disease. epidemiological data suggest that particular enhancements (such as malignant growth anticipation agents, vitamins e and b supplements, and polyunsaturated unsaturated fats) [243] and food assortments (such as wine, fish, and vegetables) [244] can prevent or delay cognitive impairments, particularly for ad [244] . reductions in ros and cancer prevention and protective agents are critical in neurodegenerative disease. cellular support, for example, phenolic compounds from dietary plants, might play a role in preventing and treating ad, given the evidence supporting the effects of oxidative stress during ad development. the natural sources of these phenolic compounds and their multitarget reactivity make them potentially valuable tools for managing multifactorial diseases [245] . another factor that must be considered when examining the effects of dietary phenolic compounds is their capacity to alter the permeability of the blood-brain barrier through impacts on lipid digestion, which might also decrease the risk of stroke [246] [247] [248] [249] . increased dietary antioxidant intake has freshly piqued public, media, and science interest in the possibility dietary antioxidants may protect against certain chronic diseases. according to epidemiological evidence, the information of fruits and vegetables may minimize the risks of certain forms of cancer and cardiovascular disease, which is thought to be due to their antioxidant contents. these antioxidants include the well-studied vitamin e and β-carotene, in addition to a wide range of polyphenolic compounds. the presence of flavonoids and phenolic acids in fruit and vegetable-based beverages, such as red wine and green and black teas, has also piqued interest [250] . the oxidized ldl (ldlox) hypothesis suggests ldlox contributes to all stages of atherosclerosis, including inflammatory activation, endothelial degradation, and macrophage enrollment, and the absorption of ldlox by these cells, resulting in the generation of foam cells a hallmark of early atherosclerotic lesions. although the role of ldlox in atherogenesis is currently well understood, determining the pathways that promote ldlox generation in vivo has proven difficult. oxidation is thought to occur in the subendothelial space of the arterial wall. a recent study of stable compounds formed via specific pathways has suggested that both ros and rns, in addition to other enzymes, such as myeloperoxidase and lipoxygenase, could be involved [145] . plasma ldl only becomes atherogenic after oxidation. research suggests that oxidative stress causes atherosclerosis by inducing lipid peroxidation [251] . extra-virgin olive oil contains polyphenolic compounds that are critical to the prevention of atherosclerotic harm. inhibitors of 3-hydroxy-3-methylglutaryl-coa (hmg-coa) reductase (statins) actively reduce the levels of saturated fatty acids in the plasma (cholesterol) [251] . the effects of polyphenolic composites derived from extra-virgin olive oil on lipid absorption have been the subject of much research [252] . the biological properties of olive oil phenolics are shown in table 7 . table 7 . biological properties of olive oil phenolics [253] . hydroxytyrosol, protocatechuic acid, phenyl ethanol-elenolic acid, caffeic acid and are some of the compounds checked in oleuropein. the shyness of viral and bacterial evolution and motion in vitro antimicrobial and antiviral motion ancel keys coined the term mediterranean diet (md) during the 1960s to describe the epidemiological observation that italian and greek populations had reduced mortality rates and the lower recurrence of harmful neurodegenerative and cardiovascular diseases than other populations [254] . more than 12,000 people from america, europe, and asia were included in the seven countries study. since then, various clinical and epidemiological studies have been published, confirming these discoveries, as demonstrated by the dramatic increase in unique publications regarding the md since 1999 [255] . the md involves the high consumption of oats, grains, vegetables, and unsaturated fats (typically from olive oil), the low consumption of red meats, poultry, and submerged unsaturated fats, and the moderate consumption of fish, milk, and dairy items, and moderate ethanol intake (such as drinking wine with meals) [256] [257] [258] . some studies of the md have suggested that adherence to this eating regimen can reduce the risks of developing an assortment of physiological issues, including cardiovascular and cerebrovascular infections, dm, metabolic disorder, malignant growths, and neurodegenerative disease [259] . the mediterranean dietary pattern includes as distinctive features the moderate intake of red wine and extra virgin olive oil, both of them rich in polyphenolic compounds, such as resveratrol, oleuropein and hydroxytyrosol and their derivatives, which have demonstrated the therapeutic effects [260] . osteoporosis is a bone disease that causes increased skeletal fragility and fractures due to decreased bone mass and microstructural deterioration [173] . the loss of bone mineral density (bmd) is the most common symptom of osteoporosis [261, 262] . the most common symptom of osteoporosis is the appearance of fragility bone fractures, which are most commonly found in the vertebrae, wrists, and hip. such fractures are associated with significant morbidity and death, and despite the availability of various therapies for osteoporosis, the global burden of osteoporotic fractures is growing [263] . natural medicines offer fewer adverse effects and are better for long-term usage than synthetic drugs. in addition, plant medicines with different chemical ingredients usually have several therapeutic pathways and targets, which is similar to the multiple variables that contribute to osteoporosis pathogenesis [262] . natural compounds including phytoestrogens with estrogenic effects (e.g., genistein, daidzein, icariin, dioscin, ginkgo biloba), antioxidant and anti-inflammatory agents (e.g., acteoside, curcumin, resveratrol, camellia sinensis), treatments that exert their effects by multiple actions (e.g., kinsenoside, berberine, olea europaea, prunus domestica, allium cepa) could provide a safer alternative to primary pharmacological strategies for osteoporosis [264] . phenolic compounds commonly found in many plants may represent promising candidates for future medical and pharmaceutical product development. future studies should consider comparisons of raw plant materials obtained from various geographical areas to determine whether differences exist in the composition of the extracts. future research should focus on local medicinal plant species and wild or endangered species to explore new phytochemical compounds and expand the number of alternative raw materials for medical and pharmaceutical purposes. the underlying mechanisms associated with some wellknown phenols, including the signaling pathways and molecular processes through which they exert their effects, require continued investigation, as this information can be utilized during drug development and targeting. various medicinal plant cultivars can provide different phenolic compounds with a wide range of biological activities. therefore, future research should continue to explore different cultivars to identify new compounds [265] . scientists are constantly searching for medications to combat the coronavirus pandemic (sars-cov-2) that has been going on for over a year. compounds of natural origin, such as phenolic acids and flavonoids, have shown promising antiviral potential in silico study and chosen experimental data. attachment (disturbance of the interaction between cellular and viral receptors), penetration (inhibition of viral pseudo-particle fusion to the host membrane), replication (inhibition of integrase and 3c-like protease), assembly, and maturation (inhibition of microsomal triglyceride transfer protease) are all stages of the viral life cycle where phenolic compounds inhibit virus multiplication [266] . the black carrot has been shown to help those with type 2 diabetes. this feature is due to the phenolic chemicals found in black carrots. still, little information regarding the mechanism of action and target enzymes were known phenols and related compounds because they are stable for heating and drying. moreover, they are unaffected by organic compounds [267] . when used in its pure form, phenol is harmful to tissues. it also has a disagreeable odor. phenolics are a heterogeneous collection of compounds generated as secondary metabolites in plants. phenolic compounds are aromatic or aliphatic compounds with at least one aromatic ring to which one or more oh groups are connected. hypertension is a common and sometimes progressive disorder that significantly increases the risk of de-veloping cardiovascular disease and associated complications. some commonly used ace inhibitors, such as captopril, benazepril, and enalapril, are associated with side effects, such as the inability to counteract the effects of proteolytic degradation. the inhibition of carbohydrate hydrolyzing enzyme for type 2 dm and cholinesterase inhibition in ad are goals that phenolic compounds can accomplish. phenolic compounds have anti-inflammatory properties that can be used to treat skin diseases. the most common forms of natural antioxidants are phenols, which feature both antioxidant and anti-inflammatory properties. despite significant improvements in medicine and pharmacology, researchers still face difficulties understanding the immune system and diseases associated with inflammation. this review described the action and bioavailability of phenolic compounds, including currently known information regarding their biological activities in cancer, rheumatoid arthritis, allergic diseases, cardiovascular disease, and neurogenerative disease. by collecting this information, we can conclude that using phenolic compounds encapsulated in synergistic nanoparticles is likely to enhance our ability to prevent and treat a variety of anti-inflammatory diseases. through these mechanisms, science can contribute to humanity's advancement. the disadvantages associated with some phenolic compounds might be mitigated by using a suitable delivery method, such as a nano-based drug delivery system. several polyphenols that have been examined have performed better when delivered in the core or surface of a nanoparticle than when given as a free soluble compound. the authors declare no conflict of interest. dietary polyphenols and the prevention of diseases biomarkers of the intake of dietary polyphenols: strengths, limitations and application in nutrition research phenolic-storing cells: keys to programmed cell death and periderm formation in wilt disease resistance and in general defence responses in plants? flavones, flavanones, and human health: epidemiological evidence polyphenols and disease risk in epidemiologic studies plant polyphenols as dietary antioxidants in human health and disease origin and physiological roles of inflammation inflammation: mechanisms, costs, and natural variation evaluation of the contribution of multiple damps and damp receptors in cell death-induced sterile inflammatory responses emerging signal regulating potential of genistein against alzheimer's disease: a promising molecule of interest. front guideline: strengthening the reporting of cohort studies in surgery stability of perovskite solar cells effect of brick kiln on arable land degradation, environmental pollution and consequences on livelihood of bangladesh alzheimer's disease: the challenge of the second century insulindegrading enzyme regulates extracellular levels of amyloid β-protein by degradation prevalence and distribution of e-cigarette use among u.s. adults: behavioral risk factor surveillance system exploring the multimodal role of phytochemicals in the modulation of cellular signaling pathways to combat age-related neurodegeneration autophagic dysfunction in alzheimer's disease: cellular and molecular mechanistic approaches to halt alzheimer's pathogenesis a potent highly selective and reversible mao-b inhibitor for alzheimer's disease analyzing the chance of developing dementia among geriatric people: a cross-sectional pilot study in bangladesh melatonin in alzheimer's disease: a latent endogenous regulator of neurogenesis to mitigate alzheimer's neuropathology emerging potential of naturally occurring autophagy modulators against neurodegeneration neuropathological alterations in alzheimer disease nmda receptor antagonists: repositioning of memantine as a multitargeting agent for alzheimer's therapy nootropic and anti-alzheimer's actions of medicinal plants: molecular insight into therapeutic potential to alleviate alzheimer's neuropathology novel anti-alzheimer's therapeutic molecules targeting amyloid precursor protein processing molecular insight into the therapeutic promise of targeting apoe4 for alzheimer's disease emerging signal regulating potential of small molecule biflavonoids to combat neuropathological insults of alzheimer's disease emerging promise of sulforaphane-mediated nrf2 signaling cascade against neurological disorders world health organization-international society of hypertension guidelines for the management of hypertension. guidelines sub-committee of the world health organization angiotensin-converting enzyme inhibitory effects by plant phenolic compounds: a study of structure activity relationships protective effects of blocking renin-angiotensin system on the progression of renal injury in glomerulosclerosis inhibitors: a review of raw material and isolated compounds from plant source anti-diabetic potential of phenolic compounds: a review lower melanin content in the skin of type 1 diabetic patients and the risk of microangiopathy international trends in the incidence of malignant melanoma 1953-2008-are recent generations at higher or lower risk? malignant melanoma in elderly patients: biological, surgical and medical issues rejuvenation of antioxidant system in central nervous system of aged rats by grape seed extract non-steroidal anti-inflammatory drugs: overall risks and management. complementary roles for cox-2 inhibitors and proton pump inhibitors defining inappropriate practices in prescribing for elderly people: a national consensus panel phenolic compounds: functional properties, impact of processing and bioavailability effect of diet on cancer development: is oxidative dna damage a biomarker? free radic oxidative stress, exercise, and antioxidant supplementation efficient binomial mixed model for identifying differential dna methylation in bisulfite sequencing data berry fruit supplementation and the aging brain an overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes a model for the role of the proline-linked pentose-phosphate pathway in phenolic phytochemical bio-synthesis and mechanism of action for human health and environmental applications reactive oxygen species (ros-induced) ros release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes mitochondrial reactive oxygen species (ros) and ros-induced ros release phenolic compounds in fruits-an overview phenolic compounds in coffee compared to other beverages. coffee heal tea and health: studies in humans dietary intake and bioavailability of polyphenols estimated dietary polyphenol intake and major food and beverage sources among elderly japanese comprehensive invited review dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases polyphenols, dietary sources and bioavailability biomarkers for exposure to dietary flavonoids: a review of the current evidence for identification of quercetin glycosides in plasma comparing the pharmacokinetics of daidzein and genistein with the use of 13c-labeled tracers in premenopausal women effect of fruit juice intake on urinary quercetin excretion and biomarkers of antioxidative status the effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers quercetin glucosides interact with the intestinal glucose transport pathway. free radic quercetin, but not its glycosides, is absorbed from the rat stomach fast access of some grape pigments to the brain bioavailability and bioefficacy of polyphenols in humans. i. review of 97 bioavailability studies the gastrointestinal tract: a major site of antioxidant action? chlorogenic acids and other cinnamates-nature, occurrence and dietary burden chlorogenic acid and caffeic acid are absorbed in humans a class of semi-essential food components: their role in human nutrition pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability enzymology of human cytosolic sulfotransferases the small intestine can both absorb and glucuronidate luminal flavonoids bioavailability of flavonoids from tea binding of flavonoids to plasma proteins polyphenols: chemistry, dietary sources, metabolism, and nutritional significance phenolic compounds: natural alternative in inflammation treatment. a review. cogent food agric amdo tibetans in transition: society and culture in the post-mao era chocolate at heart: the anti-inflammatory impact of cocoa flavanols dark chocolate/cocoa polyphenols and oxidative stress resveratrol as an anti-inflammatory and anti-aging agent: mechanisms and clinical implications online brca1 and brca2 germline mutations in korean patients with sporadic breast cancer absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers anti-inflammatory effects of flavonoids: genistein, kaempferol, quercetin, and daidzein inhibit stat-1 and nf-κb activations, whereas flavone, isorhamnetin, naringenin, and pelargonidin inhibit only nf-κb activation along with chemical profiles, pharmacological properties, and in silico studies provide new insights on cycas pectinata mechanism of inhibition of human secretory phospholipase a2 by flavonoids: rationale for lead design effect of cocoa and green tea on biomarkers of glucose regulation, oxidative stress, inflammation and hemostasis in obese adults at risk for insulin resistance modulation of endogenous antioxidant system by wine polyphenols in human disease factor analysis of the big five questionnaire using polychoric correlations in children effectiveness of training programs: a meta-analysis view project use of online therapy among spanish psychologists view project grape-seed procyanidins prevent low-grade inflammation by modulating cytokine expression in rats fed a high-fat diet metabolic syndrome as a prognostic factor for breast cancer recurrences resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells molecular mechanisms of action of genistein in cancer: recent advances genistein, a specific inhibitor of tyrosine-specific protein kinases suppression of nf-?b and nf-?b-regulated gene expression by apigenin through i?bα and ikk pathway in tramp mice apigenin and luteolin modulate microglial activation via inhibition of stat1-induced cd40 expression the protective effect of epicatechin on experimental ulcerative colitis in mice is mediated by increasing antioxidation and by the inhibition of nf-κb pathway inhibition of ulcerative colitis in mice after oral administration of a polyphenol-enriched cocoa extract is mediated by the inhibition of stat1 and stat3 phosphorylation in colon cells increased anti-inflammatory activity and enhanced phytochemical concentrations in superfine powders obtained by controlled differential sieving process from four medicinal plants kaempferol and inflammation: from chemistry to medicine immunomodulatory and anti-inflammatory effect of p-coumaric acid, a common dietary polyphenol on experimental inflammation in rats bioactivities of phenolics by focusing on suppression of chronic diseases: a review the interactions between polyphenols and microorganisms, especially gut microbiota evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists ace inhibition and bradykinin-mediated renal vascular responses: edhf involvement angiotensin-converting enzyme inhibitors: do we utilize our knowledge in heart failure patients? polyphenol protection and treatment of hypertension inhibition of angiotensin converting enzyme, angiotensin ii receptor blocking, and blood pressure lowering bioactivity across plant families a comprehensive review on processing, therapeutic benefits, challenges, and economic scenario of unconventional oils polyphenols from cocoa and vascular health-a critical review glucosidase and α-amylase inhibitory activities of nepalese medicinal herb pakhanbhed (bergenia ciliata, haw.). food chem neurodegenerative diseases and oxidative stress pharmaceutical drugs and natural therapeutic products for the treatment of type 2 diabetes mellitus natural products for the treatment of type 2 diabetes mellitus tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future mecanismos reguladores da melanogênese flavonoid derivatives as potent tyrosinase inhibitors-a survey of recent findings between natural, semisynthetic and synthetic tyrosinase inhibitors characterization of tyrosinase inhibitors in the twigs of cudrania tricuspidata and their structure-activity relationship study effects of resveratrol, oxyresveratrol, and their acetylated derivatives on cellular melanogenesis oxyresveratrol and hydroxystilbene compounds: inhibitory effect on tyrosinase and mechanism of action effect of dietary stilbenes on 5-lipoxygenase and cyclooxygenases activities in vitro impact of wines and wine constituents on cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase catalytic activity anti-inflammatory drugs and their mechanism of action macrophage-mediated innate host defense against protozoan parasites the flavonoid baicalin exhibits anti-inflammatory activity by binding to chemokines the immunomodulatory and anti-inflammatory role of polyphenols treatment recommendations for interferon-β in multiple sclerosis widely spread butyrylcholinesterase can hydrolyze acetylcholine in the normal and alzheimer brain a review on cholinesterase inhibitors for alzheimer's disease structural aspects of flavonoids as inhibitors of human butyrylcholinesterase interaction between plectranthus barbatus herbal tea components and acetylcholinesterase: binding and activity studies screening flavonoids for inhibition of acetylcholinesterase identified baicalein as the most potent inhibitor cholinesterase targeting by polyphenols: a therapeutic approach for the treatment of alzheimer's disease antioxidant, tyrosinase inhibitory, and acetylcholinesterase inhibitory activities of green tea (camellia sinensis l.) seed and its pericarp journal of enzyme inhibition and medicinal chemistry design, synthesis and evaluation of novel cinnamic acid derivatives bearing n-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for alzheimer's disease. taylor fr pyridoxine-resveratrol hybrids mannich base derivatives as novel dual inhibitors of ache and mao-b with antioxidant and metal-chelating properties for the treatment of alzheimer's disease inhibition of cholinesterase and amyloid-β aggregation by resveratrol oligomers from vitis amurensis resveratrol prevents memory deficits and the increase in acetylcholinesterase activity in streptozotocin-induced diabetic rats curcumin and its derivatives: moderate inhibitors of acetylcholinesterase, butyrylcholinesterase and trypsin the potential of plant phenolics in prevention and therapy of skin disorders can plant phenolic compounds protect the skin from airborne particulate matter? antioxidants eupafolin nanoparticles protect hacat keratinocytes from particulate matter-induced inflammation and oxidative stress mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis preparation, characterizations and anti-pollutant activity of 7,3 ,4 -trihydroxyisoflavone nanoparticles in particulate matter-induced hacat keratinocytes diphlorethohydroxycarmalol attenuates fine particulate matter-induced subcellular skin dysfunction resveratrol inhibits urban particulate matter-induced cox-2/pge2 release in human fibroblast-like synoviocytes via the inhibition of activation of nadph oxidase/ros/nf-κb effects of resveratrol and resveratril triacetate (rta) on the inflammatory response of human epidermal keratinocytes exposed to atmospheric particulate matter pm10 epigallocatechin-3-gallate rescue cell viability and attenuate inflammatory responses of human epidermal keratinocytes exposed to airborne particulate matter pm10 the effects of eucheuma cottonii on alveolar macrophages and malondialdehyde levels in bronchoalveolar lavage fluid in chronically particulate matter 10 coal dust-exposed rats potentials and prospects for renewable energies at global scale ecklonia cava extract and dieckol attenuate cellular lipid peroxidation in keratinocytes exposed to pm10. evid.-based complement eckol inhibits particulate matter 2.5-induced skin keratinocyte damage via mapk signaling pathway afzelin suppresses proinflammatory responses in particulate matter-exposed human keratinocytes astragali radix and its compound formononetin ameliorate diesel particulate matter-induced skin barrier disruption by regulation of keratinocyte proliferation and apoptosis plant natural products: from traditional compounds to new emerging drugs in cancer therapy anti-cancer agents from medicinal plants plants as a source of anti-cancer agents curcumin induces apoptosis in human melanoma cells through a fas receptor/caspase-8 pathway independent of p53 inhibition of nuclear factor-κb and nitric oxide by curcumin induces g2/m cell cycle arrest and apoptosis in human melanoma cells anti-proliferative effect of curcumin on melanoma cells is mediated by pde1a inhibition that regulates the epigenetic integrator uhrf1 advances on natural polyphenols as anticancer agents for skin cancer psoriasis: epidemiology, clinical features, and quality of life assessment of anti-psoriatic activity of cassia fistula l. extract incorporated cream classical to current approach for treatment of psoriasis: a review using old solutions to new problems: natural drug discovery in the 21st century pathogenesis and therapy of psoriasis dietary compounds as potential modulators of microrna expression in psoriasis dietary recommendations for adults with psoriasis or psoriatic arthritis from the medical board of the national psoriasis foundation: a systematic review antioxidants and atherosclerotic heart disease apoptotic or antiproliferative activity of natural products against keratinocytes for the treatment of psoriasis recent advances in psoriasis therapy: trends and future prospects tremendous health benefits and clinical aspects of smilax china. afr a review of the effects of capsicum annuum l. and its constituent, capsaicin, in metabolic syndrome study of anti-psoriatic activity and evaluation of e2a gene expression for psoriasis levels by rt-pcr of thespesia populnea l. methanolic leaf extract evaluation of the efficacy of a topical chamomile-pumpkin oleogel for the treatment of plaque psoriasis: an intra-patient, double-blind, randomized clinical trial an insight into medicinal and therapeutic potential of silybum marianum (l.) gaertn an insight into medicinal and therapeutic potential of silybum marianum (l.) gaertn international journal of biosciences clinical considerations in the treatment of acne vulgaris and other inflammatory skin disorders: focus on antibiotic resistance antimicrobial activities of eucalyptus leaf extracts and flavonoids from eucalyptus maculata development and evaluation of anti-acne products from terminalia arjuna bark pharmaceutical biology free radical scavenging and anti-acne activities of mangosteen fruit rind extracts prepared by different extraction methods free radical scavenging and anti-acne activities of mangosteen fruit rind extracts prepared by different ext multifunctional therapeutic potential of phytocomplexes and natural extracts for antimicrobial properties inhibitory effects of wild bitter melon leaf extract on propionibacterium acnes-induced skin inflammation in mice and cytokine production in vitro diet as a risk factor for atopy and asthma dietary antioxidants and asthma in adults: population-based case-control study vegetarian diet ameliorates symptoms of atopic dermatitis through reduction of the number of peripheral eosinophils and of pge2 synthesis by monocytes polyphenols as active ingredients for cosmetic products antimicrobial activity of fullerenes and their hydroxylated derivatives antioxidant and antimicrobial capacity of several monofloral cuban honeys and their correlation with color, polyphenol content and other chemical compounds the antimicrobial effects of wood-associated polyphenols on food pathogens and spoilage organisms identification of polyphenols in tobacco leaf and their antioxidant and antimicrobial activities activity and interactions of antibiotic and phytochemical combinations against pseudomonas aeruginosa in vitro evaluation of silver-infused polylactide films for inactivation of salmonella and feline calicivirus in vitro and on fresh-cut vegetables fostering the antiviral activity of green tea extract for sanitizing purposes through controlled storage conditions effect of green tea extract on enteric viruses and its application as natural sanitizer effect of grape seed extract on human norovirus gii.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water antiviral effects of grape seed extract against feline calicivirus, murine norovirus, and hepatitis a virus in model food systems and under gastric conditions free radical theory of aging: an update alterations in antioxidant enzymes during aging in humans markers of oxidative stress in erythrocytes during aging in humans increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables protective role of tea catechins on erythrocytes subjected to oxidative stress during human aging resveratrol in prevention and treatment of common clinical conditions of aging correction: a low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice quercetin increases oxidative stress resistance and longevity in saccharomyces cerevisiae dietary flavonols: chemistry, food content, and metabolism. nutrition application of nano/microencapsulated phenolic compounds against cancer the biochemistry and medical significance of the flavonoids ferulic acid decreases cell viability and colony formation while inhibiting migration of mia paca-2 human pancreatic cancer cells in vitro synergistic inhibition of cancer cell proliferation with a combination of δ-tocotrienol and ferulic acid ferulic acid in combination with parp inhibitor sensitizes breast cancer cells as chemotherapeutic strategy the antiproliferative effect of dietary fiber phenolic compounds ferulic acid and p-coumaric acid on the cell cycle of caco-2 cells feruloyl-l-arabinose attenuates migration, invasion and production of reactive oxygen species in h1299 lung cancer cells polyphenols: food sources and bioavailability antirheumatoid arthritis activities and chemical compositions of phenolic compounds-rich fraction from urtica atrichocaulis, an endemic plant to china. evid.-based complement glochidion ellipticum wight extracts ameliorate dextran sulfate sodium-induced colitis in mice by modulating nuclear factor kappa-light-chain-enhancer of activated b cells signalling pathway dietary polyphenols in the prevention and treatment of allergic diseases health-promoting properties of common herbs enhanced neuroprotective effects of resveratrol delivered by nanoparticles on hydrogen peroxide-induced oxidative stress in rat cortical cell culture highly stabilized curcumin nanoparticles tested in an in vitro blood-brain barrier model and in alzheimer's disease tg2576 mice neuroprotective ferulic acid (fa)-glycol chitosan (gc) nanoparticles for functional restoration of traumatically injured spinal cord the protective role of phenolic compounds against doxorubicin-induced cardiotoxicity: a comprehensive review evaluation of the phytochemical composition and protective activities of methanolic extracts of centaurea borysthenica and centaurea daghestanica (lipsky) wagenitz on cardiomyocytes treated with doxorubicin flavonoids and other phenolic compounds in andean indigenous grains: quinoa (chenopodium quinoa), kañiwa (chenopodium pallidicaule) and kiwicha (amaranthus caudatus) solid lipid nanoparticles as delivery systems for bioactive food components protective effects of nanoparticulate coenzyme q 10 and curcumin on inflammatory markers and lipid metabolism in streptozotocin-induced diabetic rats: a possible remedy to diabetic complications nanoencapsulation of food ingredients using lipid based delivery systems design of nano-laminated coatings to control bioavailability of lipophilic food components optimised nano-formulation on the bioavailability of hydrophobic polyphenol, curcumin, in freely-moving rats innovative pharmaceutical development based on unique properties of nanoscale delivery formulation leukocyte extravasation as a target for anti-inflammatory therapy-which molecule to choose? genetic variation in pcdh11x is associated with susceptibility to late-onset alzheimer's disease oxidative damage in mild cognitive impairment and early alzheimer's disease resveratrol promotes clearance of alzheimer's disease amyloid-β peptides neuroprotective effects of resveratrol against β-amyloid-induced neurotoxicity in rat hippocampal neurons: involvement of protein kinase c potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of alzheimer's disease il the green tea polyphenol (−)-epigallocatechin gallate attenuates β-amyloid-induced neurotoxicity in cultured hippocampal neurons adam10 activation is required for green tea (-)-epigallocatechin-3-gallate-induced α-secretase cleavage of amyloid precursor protein curcumin has potent anti-amyloidogenic effects for alzheimer's β-amyloid fibrils in vitro curcuminoids from curcuma longa l. (zingiberaceae) that protect pc12 rat pheochromocytoma and normal human umbilical vein endothelial cells from βa(1-42) insult flavonoids, cognition, and dementia: actions, mechanisms, and potential therapeutic utility for alzheimer disease. free radic polyunsaturated fatty acids and reduced odds of mci: the mayo clinic study of aging hiigh fruit and vegetable intake is positively correlated with antioxidant status and cognitive performance in healthy subjects emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets dietary habits in patients with ischemic stroke: a case-control study flavonol intake and stroke risk: a meta-analysis of cohort studies potential role of olive oil phenolic compounds in the prevention of neurodegenerative diseases coffee, tea, and cocoa and risk of stroke the role of oxidized lipoproteins in atherogenesis. free radic lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis effects of feeding virgin olive oil or their polyphenols on lipid of rat liver the phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health the diet and 15-year death rate in the seven countries study scientific evidence of interventions using the mediterranean diet: a systematic review traditional mediterranean diet and longevity in the elderly: a review. public health nutr adherence to a mediterranean diet and survival in a greek population adherence to a mediterranean diet, cognitive decline, and risk of dementia evidencia actual sobre los beneficios de la dieta mediterránea en salud chemistry and biochemistry of winemaking, wine stabilization and aging the prevention and therapy of osteoporosis: a review on emerging trends from hormonal therapy to synthetic drugs to plant-based bioactives potential antiosteoporotic agents from plants: a comprehensive review. evid.-based complement burden of high fracture probability worldwide: secular increases 2010-2040 pharmacological agents and natural compounds: available treatments for osteoporosis flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview drug design strategies for the treatment of viral disease. plant phenolic compounds and their derivatives key: cord-0027574-4yr8n5qx authors: deloch, lisa; hehlgans, stephanie; rückert, michael; maier, andreas; hinrichs, annika; flohr, ann-sophie; eckert, denise; weissmann, thomas; seeling, michaela; nimmerjahn, falk; fietkau, rainer; rödel, franz; fournier, claudia; frey, benjamin; gaipl, udo s. title: radon improves clinical response in an animal model of rheumatoid arthritis accompanied by increased numbers of peripheral blood b cells and interleukin-5 concentration date: 2022-02-16 journal: cells doi: 10.3390/cells11040689 sha: cbc517a7bd12364c3ebf347fdbb389e23cc833be doc_id: 27574 cord_uid: 4yr8n5qx radon treatment is used as an established therapy option in chronic painful inflammatory diseases. while analgesic effects are well described, little is known about the underlying molecular effects. among the suspected mechanisms are modulations of the anti-oxidative and the immune system. therefore, we aimed for the first time to examine the beneficial effects of radon exposure on clinical outcome as well as the underlying mechanisms by utilizing a holistic approach in a controlled environment of a radon chamber with an animal model: k/bxn serum-induced arthritic mice as well as isolated cells were exposed to sham or radon irradiation. the effects on the anti-oxidative and the immune system were analyzed by flow-cytometry, qpcr or elisa. we found a significantly improved clinical disease progression score in the mice, alongside significant increase of peripheral blood b cells and il-5. no significant alterations were visible in the anti-oxidative system or regarding cell death. we conclude that neither cell death nor anti-oxidative systems are responsible for the beneficial effects of radon exposure in our preclinical model. rather, radon slightly affects the immune system. however, more research is still needed in order to fully understand radon-mediated effects and to carry out reasonable risk-benefit considerations. the noble gas radon is a naturally occurring radioactive substance. while it greatly contributes to the exposure from naturally occurring sources of ionizing radiation (ir) it is also widely used in the treatment of benign inflammatory and non-inflammatory diseases, in many countries. today, it is widely accepted that the emitting alpha-particles from radon in these springs are responsible for the main dose and thus for several beneficial effects [1] [2] [3] . at present, the analgesic effects of radon therapies are exploited in the form of bathing in water containing radon, drinking radon-infused water or via inhalation therapy in galleries or caves with enhanced radon concentrations [2] [3] [4] [5] . lead to relatively small measurable effects that are often masked by other lifestyle factors (e.g., smoking) [27] . in that matter, paz et al. [4] performed analyses in patients that were exposed to radon treatment and compared them to healthy donors. they found that radon treatment had no significant impact on aberration frequency or the fraction of complex aberrations in human peripheral blood lymphocytes. they thus conclude that in their cohort and based on biological dosimetry utilizing aberration analyses, no increased health risk was detected [4] . nevertheless, a careful risk-benefit assessment should always be carried out prior to treatment with ir. therefore, we aimed to take a closer look on the molecular mechanisms under controlled conditions in an in vivo and ex vivo setting taking multiple parameters such as clinical progression, the involvement if the immune system alongside systemic cytokine alterations as well as examinations of the anti-oxidative system into account. one disease that is treated with radon and that also has been linked to oxidative stress [28] is ra. we therefore chose the k/bxn serum-transfer arthritis model as our model system for this study, as it closely resembles antibody-mediated ra in patients. as it is an antibody-mediated model, immune cells, as well as cytokines and chemokines and factors involved in pain perception are all involved in this model. this leads to a progression of the disease also including bone erosions and pannus formation. further, as serum of k/bxn mice can be collected and then injected in other strains [29] , obtaining larger groups of sex-and age-matched animals alongside similar onset and progression of ra is another advantage, especially in radon research, as doses are low and effects are expected to be small. as mentioned above, further challenges when studying radon-induced effects include the uniformity of applied doses over all treatment groups, the measurements of actual radon uptake and the feasibility of placebo controlled, randomized studies. similarly, most other in vitro studies mainly use α-sources to carry out their experiments in order to have a better controlled environment, but which neglect the effects of mixed radiation qualities of radon decay. therefore, we decided to opt for a radon chamber that was built at the gsi helmholtzzentrum für schwerionenforschung (gsi), darmstadt [27] where parameters are easier to control than in actual radon galleries. here, we were also able to focus on radon-induced effects, as other parameters such as elevated temperature or humidity are able to be set, controlled, or switched off, as needed. this allowed us to carry out in vivo and ex vivo analysis using radon in a controlled environment. 10 weeks old female c57bl/6 mice were ordered from janvier labs (le genest-saint-isle, france) and maintained in the animal facility at gsi. all animal procedures have been approved by the "regierung von unterfranken" as well as the "regierung von hessen" (approval numbers: 55.2-dms-2532-2-114 from 13 november 2015 and 10 december 2015; ts-3/14 from 21 february 2014) and were conducted in accordance with the guidelines of federation of european laboratory animal science associations (felasa). pooled serum from transgenic k/bxn mice was provided by the group of falk nimmerjahn. 200 µl or 300 µl serum, depending on the determined efficacy of harvested sera, was injected intraperitoneally into the mice, figure 1a ,b. this model was first reported by kouskoff et al. [30] and was discovered by crossing t cell receptor (tcr) transgenic krn mice with autoimmune-prone, non-obese diabetic (nod) mice. the f1 generation of these mice was called k/bxn and developed severe arthritis within the first couple of weeks. serum of these mice resulted in a reproducible arthritis in many mouse strains when being transferred to them [29] . serum of these mice resulted in a reproducible arthritis in many mouse strains when being transferred to them [29] . figure 1. exposure to radon gas for one hour results in an improved clinical score in k/bxn seruminduced c57bl76 mice in comparison to mock-treated controls. female c57bl/6 mice were injected i.p. with 200 µl or 300 µl, depending on the respective serum batch, of pooled k/bxn serum. within a few days, mice develop a visible swelling of the paws (a,b). on d3 after the injection, mice were scored and randomly distributed into two groups (e) and placed into the radon chamber (c) for treatment. while one group received mock treatment, the other group was exposed to radon gas. both groups were scored again on d7 and d10. on d10 mice were sacrificed and samples (whole blood, serum, bone marrow and hind feet) were collected for further analysis ((d), timeline). clinical score of serum-induced mice was determined according to swelling (0-no swelling, 5-complete swelling of paws and toes), whereas the score of both hindlegs/mouse was added up for final evaluation. 6 out of 9 animals in the radon group (f) showed an improvement in clinical score whereas only one in 8 control animals (g) showed improvement during the time of observation. these effects are also visualized in (h), where the δ clinical score [d7-d10] of clinical progression is significantly better in radon treated animals when compared to mock controls. figure shows data of two independent experiments with in total 10 mice/group and is shown as median + iqr. animals that did not show swelling on d3 were removed from the experiment; ncontrol = 7; nradon = 9. statistics were calculated using mann-whitney-u test; (* p < 0.05). arthritis score evaluation was carried out in a blinded manner with a score system ranging from 0 (no swelling) to 3 (massive swelling) as described previously [31] . the experimental set-up for in vivo radon exposure can be seen in figure 1c ,d. bone marrow (bm) from 6-week-old, female c57bl/6 mice (janvier labs) was isolated from the long bones of the hind legs (femur, tibia), followed by lysis of erythrocytes for 5 min at room temperature. next, cells were frozen in 10%dmso/fcs at −80°c and transported to the gsi on dry ice. prior to experiments, cells were defrosted and seeded figure 1 . exposure to radon gas for one hour results in an improved clinical score in k/bxn seruminduced c57bl76 mice in comparison to mock-treated controls. female c57bl/6 mice were injected i.p. with 200 µl or 300 µl, depending on the respective serum batch, of pooled k/bxn serum. within a few days, mice develop a visible swelling of the paws (a,b). on d3 after the injection, mice were scored and randomly distributed into two groups (e) and placed into the radon chamber (c) for treatment. while one group received mock treatment, the other group was exposed to radon gas. both groups were scored again on d7 and d10. on d10 mice were sacrificed and samples (whole blood, serum, bone marrow and hind feet) were collected for further analysis ((d), timeline). clinical score of serum-induced mice was determined according to swelling (0-no swelling, 5-complete swelling of paws and toes), whereas the score of both hindlegs/mouse was added up for final evaluation. 6 out of 9 animals in the radon group (f) showed an improvement in clinical score whereas only one in 8 control animals (g) showed improvement during the time of observation. these effects are also visualized in (h), where the ∆ clinical score [d7-d10] of clinical progression is significantly better in radon treated animals when compared to mock controls. figure shows data of two independent experiments with in total 10 mice/group and is shown as median + iqr. animals that did not show swelling on d3 were removed from the experiment; n control = 7; n radon = 9. statistics were calculated using mann-whitney-u test; (* p < 0.05). arthritis score evaluation was carried out in a blinded manner with a score system ranging from 0 (no swelling) to 3 (massive swelling) as described previously [31] . the experimental set-up for in vivo radon exposure can be seen in figure 1c ,d. bone marrow (bm) from 6-week-old, female c57bl/6 mice (janvier labs) was isolated from the long bones of the hind legs (femur, tibia), followed by lysis of erythrocytes for 5 min at room temperature. next, cells were frozen in 10%dmso/fcs at −80 • c and transported to the gsi on dry ice. prior to experiments, cells were defrosted and seeded into 6 well plates in dmem (gibco life technologies, carlsbad, ca, usa) supplemented with 10%fcs (sigma aldrich, st. louis, mo, usa) and 1% penicillin streptomycin (gibco life technologies). monocyte and macrophage medium were further supplemented with 5 ng ml m-csf (peprotech, rocky hill, nj, usa). bm was seeded 1 h prior to treatment. for monocytes, bm cells were seeded in 10 cm dishes 6 h prior to mock or radon treatment and the non-adhering fraction was transferred into 6 well plates 1 h before irradiation. for macrophage differentiation, bm cells were also seeded in 10 cm dishes for 6 h, the nonadhering fraction was then differentiated into m0 macrophages for 7 days in the presence of 5 ng/ml m-csf. all cells were kept under standard cultivation conditions (37 • c, 5% co 2 , 90% humidity). animals and cells were exposed to radon gas in a radon chamber in a controlled environment at gsi, darmstadt, germany for one hour as described in [27, 32] . mice were placed in a cage within the chamber, see figure 1c , and exposed to radon gas for one hour, followed by fresh air for 30 min. 7 days after mock or radon exposure, animals were sacrificed and bone marrow, full blood, serum and organs were harvested for further analysis. exposition conditions for in vivo experiments can be found in table 1 . cells were exposed for 1 h and chamber was flushed with fresh air for 30 min afterwards. the optimum irradiation practice was tested out beforehand. covering cells with 750 µl medium during treatment ensured that cells did not fall dry during this period, while radon treatment was carried out as effective as possible with the smallest possible liquid barrier. 24 h after the mock or radon treatment cells and supernatants (sn) were harvested and used for further experiments. mean exposition parameters for ex vivo exposure are displayed in table 2 . for flow cytometry analysis whole blood and bone marrow (bm) of animals was collected. erythrocytes in 100 µl whole blood were lysed by 0.12% formic acid for 10 s, followed by a neutralization step and subsequent fixation in 4% paraformaldehyde. for analysis of bm, 1 × 10 5 cells/stain and for blood samples 35 µl of whole blood/stain was used. for ex vivo experiments, cells were collected 24 h after radon or mock exposure. cells (lysed blood cells, bm as well as monocytes and macrophages) were resuspended in 50 µl fc-block solution (ebioscience, san diego, ca, usa) and incubated for 10 min at cells 2022, 11, 689 6 of 23 room temperature. staining for antibody panels was then carried out at 4 • c for 30 min as described previously [33] and as it can be seen in table a1 in appendix a. for 2 ,7 -dichlordihydrofluorescein-diacetat (dcf) assay, cells were washed and treated with either 2 µm dcf or 0µm dcf (invitrogen, waltham, ma, usa) for 90 min at standard culture conditions in the dark. subsequently, cells were detached and analyzed in a flow cytometer. for evaluation, ∆mean fluorescence intensity (mfi) of (2 µm dcf)-(0 µm dcf) was calculated. cells were analyzed using a cytoflex s flow cytometer and data was analyzed with the help of the kaluza analysis software (both: beckman coulter, brea, ca, usa). gating strategies are depicted in appendix a figures a1 and a2. multiplex elisa was carried out using the meso scale discovery ® (msd ® ; rockville, md, usa) system that was used according to the manufacturer's recommendation. for in vivo experiments, the proinflammatory panel 1 (mouse) kit v-plex ® assay as well as the th17 panel 1 (mouse) kit v-plex ® assay were used. samples were diluted according to the manufacturer's recommendations, 1:2 for the proinflammatory and 1:4 for the th17 panel, respectively. subsequent analysis was carried out using msd ® discovery workbench ® . cells were harvested 24 h after radon exposure by centrifugation, washed once with pbs and the cell pellets were lysed in 300 µl lysis buffer ml (macherey-nagel, düren, germany) and stored at −80 • c. rna was subsequently prepared with the nucleospin mirna kit (macherey-nagel, düren, germany) and 500 µg rna was reverse transcribed using 200 units m-mlv reverse transcriptase (promega, walldorf, germany), dntps (500 µm; carl roth, karlsruhe, germany) and random hexamer primers (5 µm; thermo fisher scientific, darmstadt, germany) for 10 min at 25 • c, 30 min at 37 • c and 30 min at 42 • c. expression of murine anti-oxidative enzymes superoxide dismutase 1 (sod1), glutathione peroxidase 1 (gpx1), catalase and the transcription factor nuclear factor-erythroid-2-related factor 2 (nrf2; gene name: nuclear factor, erythroid derived 2, like 2, nfe2l2) was measured by quantitative real-time pcr (qpcr). analyses were performed using a quantstudio 5 real-time pcr system (thermo fisher scientific), specific primers (0.5 µm), internal fluorescent taqman probes (0.25 µm; eurofins genomics, ebersberg, germany) and the gotaq probe qpcr master mix (promega). the sequences of primers and probes are listed in table a2 . the following conditions were applied: 95 • c for 2 min and 40 cycles of 95 • c for 15 s and 60 • c for 1 min. amplifications were performed using the standard curve methodology with serial dilutions of a pcr2.1 plasmid containing the specific fragment of the gene of interest, amplified with the individual forward and reverse primers (table a2) and cloned using the original ta cloning kit (thermo fisher scientific). plasmid dna was purified with the nucleobond xtra midi plus ef kit (macherey-nagel) according to the manufacturer's recommendations and the correct insertion of the specific fragment was confirmed by sequencing (eurofins genomics). all samples were run in duplicate and normalized to the expression of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (hprt). data is presented as median + interquartile range (iqr). samples were tested for normal distribution and variance equality and subsequently analyzed using two-tailed mann-whitney-u test in comparison to untreated controls. generation of graphs as well as statistical analysis was done using graphpad prism software (version 8.3.0; graphpad software, inc., san diego, ca, usa). p-values ≤ 0.05 were considered to be statistically significant. as there is a lack in placebo-controlled studies for the treatment of musculoskeletal diseases with a radon inhalation therapy as it takes place in radon spa galleries, we exposed k/bxn serum-induced c57bl76 mice to a radon inhalation therapy or mock treatment inside the radon chamber built at gsi, darmstadt [27] . the mice remained in the cage within the radon chamber for one hour at room temperature (23 • c) and either received mock treatment or where exposed to a radon activity concentration of~470 kbq/m 3 . in one of the most well-known radon spa galleries in bad gastein, austria, patients receive approximately 44 kbq/m 3 radon/therapy session in the course of 10 treatment sessions, each lasting for the duration of one hour. unlike in our experiment where we wanted to focus on only radon-induced effects, therapy includes enhanced temperature (37-41.5 • c) and humidity (70-100%) [1, 8] . clinical score of mice was assessed as described in [31] at multiple time points throughout the experiments (see figure 1d ) which was also used to randomly distribute them into two groups at the beginning of the experiments ( figure 1e ).when comparing clinical scores of mock treated animals with those that received radon therapy that was set in relation to the clinical score at the time of mock or radon treatment, we found that one in 7 mice from the mock group ( figure 1f ) showed an improvement in clinical parameters while 7 in 9 mice from the radon group ( figure 1g ) showed an improvement or remained stable in clinical score after radon treatment. when comparing d7 with d10 of the experiment, we found that radon had a significant impact on clinical progression of serum-induced mice (p = 0.0360, figure 1h ). as mentioned above, ros can be induced by extrinsic stimuli such as ionizing radiation. therefore, we first evaluated a putative effect of radon exposure on the expression of anti-oxidative enzymes superoxide dismutase 1 (sod1), glutathione peroxidase 1 (gpx1) and catalase and their redox-sensitive transcription factor nuclear factor-erythroid-2-related factor 2 (nrf2) in the peripheral blood of serum-induced mice. while the expression of sod1 and nrf2 was not modulated by radon gas inhalation, gpx1 and catalase expression was slightly increased in the radon group when compared to mock-treated animals (figure 2a -d). as suggested by rühle et al. [3] , we checked for modulations of immune cell subsets in the peripheral blood, as well as in the bone marrow, where, as described by maier et al. [2] , an elevated dose can be expected. in the bone marrow of exposed mice, we found only mild alterations in immune cell subsets, such as a reduction in monocytes/macrophages, see figure 3 . in the peripheral blood, a significant increase of b cells in the radon group ( figure 4b , p = 0.0286) as well as a very slight increase in t cell subsets was evident. likewise, pd1 was also slightly increased on b cells and cd8+ t cells after radon exposure (figure 4d ,e,g,i). as alterations in the peripheral blood but not in the bone marrow were observed, we checked for cytokine expression using a multiplex elisa system in the serum of radon and mock treated mice. while slight modulations were detected in some of the investigated cytokines (slight reduction of il-16, il-17a, and il-22; minor increase in il-4 ( figure 5c ,g-i)), we found a significant increase of il-5 within the radon group ( figure 5d , p = 0.0229). all other cytokines revealed no alterations after the radon treatment when being compared to the control group. exposure to radon gas for one hour results in a slightly increased expression of antioxidative enzymes glutathione peroxidase (gpx1) and catalase in the peripheral blood of k/bxn serum-induced c57bl76 mice in comparison to mock-treated controls. serum-induced mice were either exposed to radon or were mock-treated (w/o) on day 3. after 7 days (day 10), mice were sacrificed and peripheral blood was collected and subjected to rna isolation and quantitative realtime pcr to measure the expression of superoxide dismutase 1 (sod1) (a), glutathione peroxidase 1 (gpx1) (b), catalase (c) and nuclear factor-erythroid-2-related factor 2 (nrf2) (d). data are presented as median + iqr and were derived from 2 independent experiments ncontrol = 7; nradon = 9. statistics were calculated using mann-whitney-u test. as suggested by rühle et al. [3] , we checked for modulations of immune cell subsets in the peripheral blood, as well as in the bone marrow, where, as described by maier et al. [2] , an elevated dose can be expected. in the bone marrow of exposed mice, we found only mild alterations in immune cell subsets, such as a reduction in monocytes/macrophages, see figure 3 . exposure to radon gas for one hour results in a slightly increased expression of antioxidative enzymes glutathione peroxidase (gpx1) and catalase in the peripheral blood of k/bxn serum-induced c57bl76 mice in comparison to mock-treated controls. serum-induced mice were either exposed to radon or were mock-treated (w/o) on day 3. after 7 days (day 10), mice were sacrificed and peripheral blood was collected and subjected to rna isolation and quantitative realtime pcr to measure the expression of superoxide dismutase 1 (sod1) (a), glutathione peroxidase 1 (gpx1) (b), catalase (c) and nuclear factor-erythroid-2-related factor 2 (nrf2) (d). data are presented as median + iqr and were derived from 2 independent experiments n control = 7; n radon = 9. statistics were calculated using mann-whitney-u test. as it is often suggested that ionizing radiation (ir) exerts some of its properties through the induction of cell death, we performed annexinv/propidiumiodide (axv/pi) staining in isolated bone marrow cells as well as in bone marrow-derived ex vivo differentiated monocytes and macrophages. when analyising cell death utilizing axv/pi staining we found no significant influence of radon treatment on cell death in all ex vivo experiments we have performed ( figure 6 ). as monocytes and macrophages are considered to be key players in ros-mediated effects, and as slight increase in gpx1 and catalase expression were found in the in vivo experiments (figure 2 ), we checked for ros release and anti-oxidative enzymes in an ex vivo setting. as it is also known, that inflammatory processes are often vital for low doses of ir [34, 35] , as it is also true in radon treatment, we added tumor necrosis factor (tnf)α to the cells in order to mimic inflammation. intracellular ros levels, as measured via dcf assay, in radon treated monocytes ( figure 7c ) showed no significant alterations compared to mock treated ones. however, we observed significantly increased ros levels ( figure 7i , p = 0.0286) in radon −tnfα treated samples compared to those with radon +tnfα treated ones as well as in mock treated ones compared to radon +tnfα treated ones ( figure 7i , p = 0.0286). this suggests a stronger effect of inflammatory stimulus than radon induced effects. we also found no differences in the distribution of classical or non-classical monocytes ( figure 7a ,b) or macrophage numbers ( figure 7h ). in order to examine the impact of pro-inflammatory stimulation with tnfα and radon exposure on the anti-oxidative system of immune cells ex vivo, we measured the mrna expression of anti-oxidative enzymes superoxide dismutase 1 (sod1), glutathione peroxidase 1 (gpx1) and catalase, as well as their redox-sensitive transcription factor nuclear factorerythroid-2-related factor 2 (nrf2) in tnfα-stimulated vs. non-stimulated, mock-treated or radon-exposed monocytes and macrophages and mock vs. radon-exposed murine bone marrow. in monocytes, the expression of sod1, gpx1, catalase or nrf2 was not modulated by tnfα or radon treatment ( figure 7d-g) . cells 2022, 11, x for peer review 9 of 25 figure 3 . inhalation of radon gas results only in minor immune cell alterations of the bone marrow in exposed mice. bone marrow of the long bones of k/bxn serum-induced age and sex matched c57bl/6 mice was collected, followed by lysis of erythrocytes and multicolor flow cytometry analysis for immune cell subtypes was carried out (a-i). depicted are various immune cell subsets of radon and mock treated mice. data shows two independent experiments with in total ncontrol = 7; nradon = 9 mice/group and is presented as median + iqr. statistics were calculated using mann-whitney-u test. in the peripheral blood, a significant increase of b cells in the radon group ( figure 4b , p = 0.0286) as well as a very slight increase in t cell subsets was evident. likewise, pd1 was also slightly increased on b cells and cd8+ t cells after radon exposure ( figure 4d ,e,g,i). figure 3 . inhalation of radon gas results only in minor immune cell alterations of the bone marrow in exposed mice. bone marrow of the long bones of k/bxn serum-induced age and sex matched c57bl/6 mice was collected, followed by lysis of erythrocytes and multicolor flow cytometry analysis for immune cell subtypes was carried out (a-i). depicted are various immune cell subsets of radon and mock treated mice. data shows two independent experiments with in total n control = 7; n radon = 9 mice/group and is presented as median + iqr. statistics were calculated using mann-whitney-u test. by contrast, tnfα stimulation significantly increased the expression of gpx1 (p = 0.0286), catalase (p = 0.0286) and nrf2 (p = 0.0286) in macrophages, however independent of radon treatment, but dependent on inflammatory stimuli, while sod1 expression was not significantly modulated ( figure 7j-m) . concurrently, ros was decreased upon tnfα treatment of macrophages ( figure 7i ). as alterations in the peripheral blood but not in the bone marrow were observed, we checked for cytokine expression using a multiplex elisa system in the serum of radon and mock treated mice. while slight modulations were detected in some of the investigated cytokines (slight reduction of il-16, il-17a, and il-22; minor increase in il-4 ( figure 5c ,g-i)), we found a significant increase of il-5 within the radon group ( figure 5d , p = 0.0229). all other cytokines revealed no alterations after the radon treatment when being compared to the control group. as it is often suggested that ionizing radiation (ir) exerts some of its properties figure 5 . exposure to radon gas for one hour results in altered cytokine expression levels in the serum of treated animals in comparison to mock treated animals. serum of k/bxn serum-induced age and sex matched c57bl/6 mice was collected and analyzed via msd multiplex assay (a-j). data shows cytokine levels of two independent experiments with in total n control = 7; n radon = 9 mice/group and is presented as median + iqr. statistics were calculated using mann-whitney-u test (* p < 0.05). in vitro exposure of c57bl/6-derived bone marrow to radon slightly decreased expression of gpx1 and catalase ( figure 8b,c) , however, these findings were not significant. cells 2022, 11, x for peer review 12 of 25 figure 6 . ex vivo treatment of bone marrow cells as well as monocytes and macrophages with radon does not enhance cell death. isolated bone marrow cells (168 kbq/m³) from c57bl/6 mice as well as bone marrow-derived monocytes and macrophages (177 kbq/m³) were exposed to radon or mock treatment for one hour under standard cell culture conditions. 24 h after treatment, cells were collected, stained for axv/pi and subsequently analyzed (a-c). for bone marrow cells (d-f), 4 cell pools, each derived from 10 c57bl/6 mice, were exposed in two independently performed experiments. for monocytes (g-i) and macrophages (j-l), 3 and 4 cell pools were exposed in 3 figure 6 . ex vivo treatment of bone marrow cells as well as monocytes and macrophages with radon does not enhance cell death. isolated bone marrow cells (168 kbq/m 3 ) from c57bl/6 mice as well as bone marrow-derived monocytes and macrophages (177 kbq/m 3 ) were exposed to radon or mock treatment for one hour under standard cell culture conditions. 24 h after treatment, cells were collected, stained for axv/pi and subsequently analyzed (a-c). for bone marrow cells (d-f), 4 cell pools, each derived from 10 c57bl/6 mice, were exposed in two independently performed experiments. for monocytes (g-i) and macrophages (j-l), 3 and 4 cell pools were exposed in 3 independently carried out experiments. data is presented as median + iqr. statistics were calculated using mann-whitney-u test. by contrast, tnfα stimulation significantly increased the expression of gpx1 (p = 0.0286), catalase (p = 0.0286) and nrf2 (p = 0.0286) in macrophages, however independent of radon treatment, but dependent on inflammatory stimuli, while sod1 expression was . ex vivo radon treatment of bone-marrow derived monocytes and macrophages has no effects on the anti-oxidative system while stimulation with tnf-α induces significant effects in macrophages. c57bl/6 bone marrow-derived monocytes and macrophages were exposed to radon (177 kbq/m 3 ) or mock treatment for one hour under standard cell culture conditions. 24 h after radon not significantly modulated ( figure 7j-m) . concurrently, ros was decreased upon tnfα treatment of macrophages ( figure 7i ). in vitro exposure of c57bl/6-derived bone marrow to radon slightly decreased expression of gpx1 and catalase ( figure 8b,c) , however, these findings were not significant. figure 8 . ex vivo radon treatment of bone-marrow derived from c57bl/6 mice has no effects on the anti-oxidative system. c57bl/6-derived bone marrow was exposed to radon for 1 h at standard cell culture conditions. the expression of sod1 (a), gpx1 (b), catalase (c) and nrf2 (d) was measured by qpcr 24 h after radon exposure of bone marrow. 4 cell pools were exposed in 2 independent experiments. data is presented as median + iqr. statistics were calculated using mann-whitney-u test. similar to the peripheral blood analysis in in vivo experiments of k/bxn seruminduced mice, we found a significant increase of b cells in radon exposed bone marrow cells ( figure 9c , p = 0.0286). significant alterations were also present in cd34+ hematopoietic stem cells within the radon group ( figure 9j , p = 0.0286), whereas only mild alterations were found in t cells as well as cd115+ monocytic precursors ( figure 9e ,f,h,k). figure 8 . ex vivo radon treatment of bone-marrow derived from c57bl/6 mice has no effects on the anti-oxidative system. c57bl/6-derived bone marrow was exposed to radon for 1 h at standard cell culture conditions. the expression of sod1 (a), gpx1 (b), catalase (c) and nrf2 (d) was measured by qpcr 24 h after radon exposure of bone marrow. 4 cell pools were exposed in 2 independent experiments. data is presented as median + iqr. statistics were calculated using mann-whitney-u test. similar to the peripheral blood analysis in in vivo experiments of k/bxn seruminduced mice, we found a significant increase of b cells in radon exposed bone marrow cells ( figure 9c , p = 0.0286). significant alterations were also present in cd34+ hematopoietic stem cells within the radon group ( figure 9j , p = 0.0286), whereas only mild alterations were found in t cells as well as cd115+ monocytic precursors ( figure 9e,f,h,k) . figure 9 . ex vivo treatment of bone marrow cells with radon significantly increases b cell numbers and cd34+ hematopoietic precursor cells. isolated bone marrow cells derived from c57bl/6 mice were exposed to radon (168 kbq/m³) or mock treatment for one hour under standard cell culture conditions. 24 h after treatment cells were collected, stained for multicolor flow-cytometry analysis and subsequently analyzed (a-k). four cell pools, each derived from 10 c57bl/6 mice, were exposed in two independently performed experiments. data is presented as median + iqr. statistics were calculated using mann-whitney-u test (* p < 0.05). similar to the observations made in patient studies, we found a significant improvement in the clinical course of ra in radon treated animals in comparison to sham irradiated control animals ( figure 1h ). as radon exposure was carried out at room temperature and humidity was also not enhanced, we suggest that the observed effects regarding the clinical score ( figure 1f-h) , as well as modulation of immune cells in the bone marrow (figure 3) , the peripheral blood ( figure 4 ) and the cytokine milieu in the serum ( figure 5 ) in the treatment group are radon-mediated effects. this is in line with observations made in patient studies: while probably due to the spa effects with elevated temperatures and humidity, initial improvement in treatment and control groups were observed, long lasting analgesic effects were only obvious in radon treatment groups figure 9 . ex vivo treatment of bone marrow cells with radon significantly increases b cell numbers and cd34+ hematopoietic precursor cells. isolated bone marrow cells derived from c57bl/6 mice were exposed to radon (168 kbq/m 3 ) or mock treatment for one hour under standard cell culture conditions. 24 h after treatment cells were collected, stained for multicolor flow-cytometry analysis and subsequently analyzed (a-k). four cell pools, each derived from 10 c57bl/6 mice, were exposed in two independently performed experiments. data is presented as median + iqr. statistics were calculated using mann-whitney-u test (* p < 0.05). similar to the observations made in patient studies, we found a significant improvement in the clinical course of ra in radon treated animals in comparison to sham irradiated control animals ( figure 1h ). as radon exposure was carried out at room temperature and humidity was also not enhanced, we suggest that the observed effects regarding the clinical score ( figure 1f-h) , as well as modulation of immune cells in the bone marrow (figure 3) , the peripheral blood ( figure 4 ) and the cytokine milieu in the serum ( figure 5 ) in the treatment group are radon-mediated effects. this is in line with observations made in patient studies: while probably due to the spa effects with elevated temperatures and humidity, initial improvement in treatment and control groups were observed, long lasting analgesic effects were only obvious in radon treatment groups [6, 10] . this suggests radon to be responsible for inducing long-lasting effects in improvement of symptoms. as mentioned above, ros and the anti-oxidative system are suspected to be one of the modes of action through which radon exerts some of its molecular effects. therefore, we examined key factors of the anti-oxidative system in the in vivo exposed animals using qpcr. however, unlike other reports [14] [15] [16] , that indicated a significant influence of radon exposure especially on sod1, we found no significant radon induced alterations of the antioxidative enzymes sod1, gpx1, catalase, and nrf2 in in vivo exposed animals (figure 2 ). this discrepancy is most likely due to the differences in experimental set-up (dose and duration of radon treatment), time of sampling (7d vs right after exposure), differences in utilized methods, the organs that have been looked into, and the mouse model used. all the aforementioned experiments were carried out using balb/c mice, whereas we used k/bxn serum induced c57bl/6 mice. further, we have shown before, that an inflammatory background is essential in the induction of anti-inflammatory effects [34, 35] , therefore the observed differences could also be due to the different inflammatory background. these important facts have to be well-thought-out when a radon treatment for patients is being considered. this indicates that personalized treatment approaches should be taken into mind, in dependence of the mentioned factors. further, immune alterations might play a more important role on such individual settings. we performed immunephenotyping in the bone marrow ( figure 3 ) as well as in the peripheral blood ( figure 4 ) of sham or radon irradiated mice. while we expected the accumulated dose to be higher in the bone marrow [2, 36] , no significant alterations in immune cells besides a minor reduction in monocytes/macrophages ( figure 3a ) was found. interestingly however, in the peripheral blood, a significant increase of b cells ( figure 4b ) alongside minor, non-significant alterations in t cells ( figure 4d -f,h,i) was observed. in general, b cells are considered to be positive regulators of the immune system as they produce a number of cytokines, including il-4, -6, 10 as well as interferon (ifn)γ and tgfβ. further, they are involved in cd4+ t cell expansion, but may also positively regulate cd8+ t cells, as it has been shown in mouse models of autoimmune diseases. further, they can be a source of autoreactive antibodies. however, they can also be negative regulators of the immune system, especially during inflammation and autoimmunity and specific b cell subsets, so called regulatory b cells, have been reported [37] [38] [39] [40] [41] . these regulatory b cells have been identified regularly in mouse models for inflammation and autoimmunity, where several subsets of these cells have been acknowledged [39] . as we have identified b cells to be cd19+ only [33] , we cannot further determine b cell subsets in our set-up, as it would be necessary to identify regulatory b cells [41, 42] . therefore, further examinations are needed in order to deeper understand the radon-mediated increase of b cell numbers and the subsequent effects. nevertheless, one has to stress, that in the rad-on01 patient study, a slight temporary increase of b cells in the peripheral blood of the radon patients was observed 6 weeks after therapy [3] . further, while we found only minor alterations in t cell numbers ( figure 4d -f), rühle et al. suggested that radon balneology only induced minor alterations in total t cell numbers, but significant and long-lasting alterations were found in their activation status [3] . this is in line with our findings in the cytokine milieu in the peripheral blood where il-16 ( figure 5g ), il-17a ( figure 5h ) and il-22 ( figure 5i ) were slightly reduced in radon treated animals. all of these cytokines are involved in t h 17 response [43] and could therefore point to an alteration in t cell subsets and/or activation status. next to the slight alterations in cytokines involved in t h 17 response, we detected a significant increase of il-5 in the radon group ( figure 5d ). il-5 can be produced by e.g., t h 2 cells, and there are indications that t h 2-produced il-5 is able to promote and expand antigen-specific tregs that have been activated by il-4 and autoantigens and that are involved in the suppression of autoimmunity, suggesting a beneficial effect of radon treatment in autoimmune diseases such as ra [44] . however, as our animals have been exposed to radon inhalation therapy, it is also of note that il-5, alongside with il-4, is also known to be involved in airway inflammation and hyperreactivity especially in asthma [45] [46] [47] . next to its role in t cell biology, il-5 is also involved in the activation of b cells as well as eosinophils, which we have found to be significantly ( figure 4b ) and slightly ( figure 4c ) elevated in the radon group [48, 49] . when comparing the preclinical results with patient outcome after a radon spa treatment in bad steben (series of 9 baths/20 min each over a course of three weeks, natural radon spring water with 600-1200 bq/l radon), similar alterations in the immune system were found. in that matter rühle et al., as mentioned above, observed that t cells, monocytes and eosinophils in peripheral blood were significantly increased at the first time point after therapy alongside a tendency for increased b cell numbers [3] . similarly, in our mouse model, b cells were significantly increased in the peripheral blood while t cells, monocytes and eosinophils showed a tendency for it ( figure 4 ). future research should additionally include analyses of the activation state of immune cell subsets. again, similar to our results ( figure 5 ), kullmann et al. found no significant alterations in serum levels of tnf-α, il-10 and il-1β [21] . with regard to the antioxidative system, kuciel-lewandowska et al. found, in a pilot study examining the integrated antioxidant system of the body in the course of radon therapy that the total antioxidant status of patients increased after radon therapy. however, as the applied methods differ from those we have used in our preclinical study and as patient numbers are relatively low, further research on the role of the anti-oxidative system after radon therapy is needed [17, 50] . nevertheless, of course, mice and humans cannot be compared one-to-one, and preclinical examinations can only give hints as to which parameters should be looked at in patient studies in more detail. furthermore, while we chose similar exposure parameters for our animal experiments, there are, of course, other factors that need to be taken into consideration, as e.g., different anatomical composition and a higher respiratory rate in mice compared to humans. sakoda et al. found the calculated whole lung dose to be 3-times higher in mice compared to humans [51] . however, the equilibrium factor that was used in these calculations was 0.4, suggesting comparatively high amounts of decay products. the decay products, according to the international commission on radiological protection (icrp), are responsible for most of the lung dose [52] , as the radon progeny is either inhaled in a higher rate with increased respiratory rate, resulting at a higher lung dose. however, this is different in our radon chamber. within this chamber, we have a very low amount of radon progeny (equilibrium factor~0.01) and thus a homogenous distribution of radon in the air and mouse lungs, independent of respiratory frequency. furthermore, radon has a half-life period of 3.8 days, which is a lot longer than the respiratory cycle of mice, therefore it is unlikely that radon is decaying within a breathing cycle. thus, for pure radon, respiratory frequency is most likely not an important factor in this setting. for the examinations in peripheral blood, we assume, according to the biokinetic models of icrp, radon to merge directly into the blood from the lungs. blood is able to take up radon until a saturation is reached: the blood is then transporting the dissolved radon to the organs. as mice have a higher metabolic rate than humans, this likely happens faster in mice than in humans; however, as both, mice and humans can only take up a certain amount of radon, this is independent of the amount of available radon and also independently of metabolic rates or size. therefore, we suggest that in terms of radon uptake in the blood, our preclinical approach is comparable to the human situation. further, when applying ir it is often suggested that the main effects that can be observed following exposure to ir are mainly mediated via the direct induction of cell death or ros-induced dna damage. however, we found no significant radon mediated alterations regarding cell death in the examined bone marrow, monocytes or macrophages ( figure 6) . we then took a closer look into the induction of ros as well as the antioxidative system in cells that are known to produce large amounts of ros. therefore, we exposed ex vivo differentiated monocytes and macrophages to radon and performed dcf-analysis as well as qpcr analysis of key factors of the anti-oxidative response system (figure 7) . no significant alterations in the induction of ros were found, however, the dcf signal was slightly increased in monocytes after radon exposure ( figure 7c ). next to its putative effects in radon treatment, ros is also involved in the initiation, progression, and resolution of inflammatory processes thus making it a central key player in the progression of inflammatory processes in general [53, 54] . we found significant effects in healthy monocytes and macrophages only after inducing an inflammatory environment by adding tnfα (figure 7d -g,j-m) that exceeded those of radon induced effects. we therefore suggest that the inflammatory environment causes greater alterations within the antioxidative system than radon. similar to our results in in vivo exposed mice, we also found no significant alterations of anti-oxidative enzymes sod1, gpx1, catalase, and nrf2 in ex vivo exposed bone marrow (figure 8 ). however, in line with our findings in the peripheral blood of in vivo exposed k/bxn serum-induced, we found a significant increase of b cells ( figure 9b ) and here additionally a significant increase of hematopoietic precursor cells ( figure 9j ) alongside slight alterations in t cells ( figure 9e ,f,h,i) and a minor induction of the monocytic marker cd115 ( figure 9k) . a possible explanation for the in part discrepant effects in in vivo versus ex vivo exposed bone marrow could be on the one hand due to the differences in solubility or radon. sanjon et al. described that radon solubility is reduced in water (and therefore probably also cell culture medium) in comparison to fat [32] . as described by maier et al. [2] , in vivo the bone marrow holds a higher fat content as it is composed of both, bone marrow cells and fat cells. however, this was only given in our in vivo experiments as ex vivo bone marrow was flushed out and only bone marrow cells were seeded in cell culture medium. on the other hand, radon uptake differs in both models, while in in vivo radon is actively distributed in the body, in ex vivo settings radon is dissolved passively in the medium. meaning, in vivo exposed mice inhaled the radon, which was then dissolved in blood. this way it is actively transported and thus distributed where it can reach and accumulate in the cells. on the contrary, in ex vivo treatment, radon is passively dissolved in the medium where it can only diffuse. this way, in order for α-particles deposit its energy within a cell, cells and α-particles have to be in close vicinity by chance. taking all these results into consideration, we consequently suggest that the clinical response in arthritic mice to radon as well as the observed modulations in immune cell subsets and cytokines in the described experimental set-ups are likely not cell death-or ros-related, but rather immune-mediated. additionally, as in vivo radon exposed mice showed significant improvement of clinical parameters in comparison to sham irradiated animals without further co-factors such as temperature, minerals or humidity [55] , a pure placebo effect can be excluded. further, additional research is needed in order to better understand the radon-mediated effects on b cell and t cell biology. with regard to radon induced risk factors, while to date no clear answer can be given, our results also suggest that further research is needed, but that the radon-induced immune alterations are minor and therefore not actively health-threatening in this low dose and not chronic exposure scenario. figure a1 . exemplary gating strategy for 2′,7′-dichlordihydrofluorescein-diacetat (dcf) measurements. for each condition, two wells were prepared, treated and washed. one well was subsequently treated with 2 µm dcf (+dcf), while the other one received 0 µm dcf (−dcf), followed by a 90 min incubation period at standard culture conditions in the dark. afterward, cells were detached and dcf activity was measured. for analysis, ∆mfi was calculated from mfi(+dcf)-mfi(−dcf). figure a2 . exemplary gating strategy for ex vivo differentiated monocytes and macrophages. in both cases, singlets were pre-chosen, followed by gating of viable cells and a live/dead control using zombie nir. afterward, cells were gated for f4/80+ or f4/80−. in case of monocytes, f4/80− cells were further classified into "classical" (f4/80-cd11b+ly6c+) or "non-classical" (f4/80figure a1 . exemplary gating strategy for 2 ,7 -dichlordihydrofluorescein-diacetat (dcf) measurements. for each condition, two wells were prepared, treated and washed. one well was subsequently treated with 2 µm dcf (+dcf), while the other one received 0 µm dcf (−dcf), followed by a 90 min incubation period at standard culture conditions in the dark. afterward, cells were detached and dcf activity was measured. for analysis, ∆mfi was calculated from mfi(+dcf)-mfi(−dcf). figure a1 . exemplary gating strategy for 2′,7′-dichlordihydrofluorescein-diacetat (dcf) measurements. for each condition, two wells were prepared, treated and washed. one well was subsequently treated with 2 µm dcf (+dcf), while the other one received 0 µm dcf (−dcf), followed by a 90 min incubation period at standard culture conditions in the dark. afterward, cells were detached and dcf activity was measured. for analysis, ∆mfi was calculated from mfi(+dcf)-mfi(−dcf). figure a2 . exemplary gating strategy for ex vivo differentiated monocytes and macrophages. in both cases, singlets were pre-chosen, followed by gating of viable cells and a live/dead control using zombie nir. afterward, cells were gated for f4/80+ or f4/80−. in case of monocytes, f4/80− cells were further classified into "classical" (f4/80-cd11b+ly6c+) or "non-classical" (f4/80figure a2 . exemplary gating strategy for ex vivo differentiated monocytes and macrophages. in both cases, singlets were pre-chosen, followed by gating of viable cells and a live/dead control using zombie nir. afterward, cells were gated for f4/80+ or f4/80−. in case of monocytes, f4/80− cells were further classified into "classical" (f4/80-cd11b+ly6c+) or "non-classical" (f4/80-cd11b+ly6c−) monocytes. in case of ex vivo differentiated macrophages, f4/80+cd11b+ cells were considered to be macrophages. endogenous anandamide and self-reported pain are significantly reduced after a 2-week multimodal treatment with and without radon therapy in patients with knee osteoarthritis: a pilot study radon exposure-therapeutic effect and cancer risk modulation of the peripheral immune system after low-dose radon spa therapy: detailed longitudinal immune monitoring of patients within the rad-on01 study chromosome aberrations in lymphocytes of patients undergoing radon spa therapy: an explorative mfish study impact of radon and combinatory radon/carbon dioxide spa on pain and hypertension: results from the explorative rad-on01 study long-term efficacy of radon spa therapy in rheumatoid arthritis-a randomized, sham-controlled study and follow-up long-term benefit of radon spa therapy in the rehabilitation of rheumatoid arthritis: a randomised, double-blinded trial radon within therapeutic strategies of ankylosing spondylitis low-dose radiotherapy for painful osteoarthritis of the elderly: a multicenter analysis of 970 patients with 1185 treated sites radon therapy for the treatment of rheumatic diseases-review and meta-analysis of controlled clinical trials cost effectiveness of combined spa-exercise therapy in ankylosing spondylitis: a randomized controlled trial combined spa-exercise therapy is effective in patients with ankylosing spondylitis: a randomized controlled trial study of antioxidative effects and anti-inflammatory effects in mice due to low-dose x-irradiation or radon inhalation evaluation of the redox state in mouse organs following radon inhalation radon inhalation decreases dna damage induced by oxidative stress in mouse organs via the activation of antioxidative functions comparison of antioxidative effects between radon and thoron inhalation in mouse organs the assessment of the integrated antioxidant system of the body in the course of radon therapy: a pilot study oxidative damage in various tissues of rats exposed to radon effect of radon on the immune system: alterations in the cellularity and functions of t cells in lymphoid organs of mouse decrease of markers related to bone erosion in serum of patients with musculoskeletal disorders after serial low-dose radon spa therapy. front temporarily increased tgfβ following radon spa correlates with reduced pain while serum il-18 is a general predictive marker for pain sensitivity could pulmonary low-dose radiation therapy be an alternative treatment for patients with covid-19 pneumonia? preliminary results of a multicenter seor-gicor nonrandomized prospective trial (ipacovid trial) free radicals in the physiological control of cell function reactive oxygen species (ros) homeostasis and redox regulation in cellular signaling nrf2:inrf2 (keap1) signaling in oxidative stress. free radic reactive oxygen species in endothelial function-from disease to adaptation experimental setup for radon exposure and first diffusion studies using gamma spectroscopy inflammatory and oxidative stress markers-mirror tools in rheumatoid arthritis k/bxn serum-transfer arthritis as a model for human inflammatory arthritis organ-specific disease provoked by systemic autoimmunity siglec-15 on osteoclasts is crucial for bone erosion in serum-transfer arthritis a combined experimental and theoretical study of radon solubility in fat and water combinations of radiotherapy with vaccination and immune checkpoint inhibition differently affect primary and abscopal tumor growth and the tumor microenvironment dose radiotherapy has no harmful effects on key cells of healthy non-inflamed joints low-dose radiotherapy ameliorates advanced arthritis in htnf-α tg mice by particularly positively impacting on radon as a causative factor in induction of myeloid leukaemia and other cancers regulatory b cells as inhibitors of immune responses and inflammation skin-associated b cells in the pathogenesis of cutaneous autoimmune diseases-implications for therapeutic approaches regulatory b cells in human inflammatory and autoimmune diseases: from mouse models to clinical research regulation of immunity and autoimmunity by b cells a case for regulatory b cells b10 cells and regulatory b cells balance immune responses during inflammation, autoimmunity, and cancer development, cytokine profile and function of human interleukin 17-producing helper t cells il-5 promotes induction of antigen-specific cd4+cd25+ t regulatory cells that suppress autoimmunity interleukin-5-producing cd4+ t cells play a pivotal role in aeroallergen-induced eosinophilia, bronchial hyperreactivity, and lung damage in mice a novel t cell-regulated mechanism modulating allergen-induced airways hyperreactivity in balb/c mice independently of il-4 and il-5 aeroallergen-induced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of il-4 and allergen-specific immunoglobulins role of il-5 in the innate immune system and disease control the role of il-5 in iga b cell differentiation the assessment of the integrated antioxidant system of the body and the phenomenon of spa reaction in the course of radon therapy: a pilot study lung dosimetry of inhaled radon progeny in mice occupational intakes of radionuclides: part 3. icrp publication 137 role of reactive oxygen species in inflammation: a minireview reactive oxygen species in inflammation and tissue injury effectiveness of balneotherapy and spa therapy for the treatment of chronic low back pain: a review on latest evidence lack of the antioxidant enzyme glutathione peroxidase-1 accelerates atherosclerosis in diabetic apolipoprotein e-deficient mice lack of the antioxidant glutathione peroxidase-1 does not increase atherosclerosis in c57bl/j6 mice fed a high-fat diet nrf2 is a key target for prevention of noise-induced hearing loss by reducing oxidative damage of cochlea transcription factor nrf1 negatively regulates the cystine/glutamate transporter and lipid-metabolizing enzymes control of regulatory t cell development by the transcription factor foxp3 key: cord-0019844-cb6djwx0 authors: yan, qingran; du, fang; kang, yuening; ye, ping; wang, xiaodong; xu, jianhua; tang, jianping; wang, niansong; jiang, gengru; li, zhijun; wang, xuan; xue, qin; huang, xinfang; zhang, xiaoyan; zhou, ying; dai, min; bao, chunde title: comparison of iguratimod and conventional cyclophosphamide with sequential azathioprine as treatment of active lupus nephritis: study protocol for a multi-center, randomized, controlled clinical trial (igelu study) date: 2021-08-11 journal: trials doi: 10.1186/s13063-021-05475-3 sha: 4b3ea9830988d8548358dc34c6f56c186773a528 doc_id: 19844 cord_uid: cb6djwx0 background: systemic lupus erythematosus (sle) is an autoimmune disease that can involve multiple organs or systems. lupus nephritis (ln) is associated with high mortality and morbidity. however, plenty of patients do not respond to present treatment or relapse. iguratimod (igu) is a new small molecular, anti-rheumatic drug and has shown the potential for drug repurposing from rheumatoid arthritis (ra) to ln treatment. it has been approved for treating ra in northeast asia. beyond expectation in a recent observational study, over 90% of thirteen refractory ln patients responded to iguratimod monotherapy in 24 weeks, with no steroids dose increasing or any other medication add-on during the entire follow-up. methods/design: this study is a multi-center, randomized, 52-week parallel positive drug-controlled study. the study was designed as a head-to-head comparison between the iguratimod and present first-line therapy on ln patients. a total of 120 patients (60 patients each group) is in the enrolling plan. all enrolled patients are assigned randomly into trial and control groups. the patients will be selected from six study sites in china and will all have biopsy-proven active lupus nephritis. in the first 24 weeks of the trial, igu is compared with cyclophosphamide as an induction therapy, and in the second 24 weeks, igu is compared with azathioprine as a maintenance therapy. the primary outcome is renal remission rate including both complete remission and partial remission at week 52, which will be analyzed using a non-inferiority hypothesis test. discussion: most patients diagnosed with sle will develop ln within 5 years and ln remains a major cause of morbidity and death for sle patients. although some medications are proven effective for the treatment of this condition, at least 20–35% ln patients have to suffer from relapse or ineffective treatment and medication intolerance is also frequent. this trial is designed to demonstrate whether iguratimod can be used as an alternative induction or maintenance therapy in subjects who have lupus nephritis. data from this study will provide an evidence on whether or not iguratimod should be recommended to active ln patients. trial registration: clinicaltrials.govnct 02936375. registered on october 18, 2016. supplementary information: the online version contains supplementary material available at 10.1186/s13063-021-05475-3. systemic lupus erythematosus (sle) is an autoimmune disease that can involve multiple organs or systems [1] [2] [3] . lupus nephritis (ln) is associated with high mortality and morbidity rates. over recent decades, substantial progress has been made in developing immunosuppressant agents and biologic therapies [4] . however, a significant proportion of patients either do not respond to first-line immunosuppressive drugs or quickly relapse after initial remission. approximately 5-20% of patients especially in class iv ln (44% of patients) will experience continued worsening of renal function and go on to develop end-stage renal disease in developing vs developed countries [5, 6] . to treat ln, a high dose of steroids plus traditional immunosuppressants, especially cyclophosphamide (cyc) or mycophenolate mofetil (mmf), is still the first-line option in most clinical recommendations [4, 7] . in addition, b cell depletion therapy with rituximab, multiple target therapy with a combination of mmf, and calcineurin inhibitor have emerged as the second-line choice for ln treatment [4] . of note, these newly recommended regimes, as well as other promising agents that succeeded in recent phase iii ln trials, such as belimumab and voclosprorin, are supposed to be applied in combination, for most successful studies on ln adopted add-on strategy. for example, rituximab has been added to another immunosuppressant, typically cyc [8] [9] [10] , and belimumab and voclosprorin were both added to mmf in phase iii trials [11, 12] . the add-on strategy may help to ensure efficacy, but could raise extra safety concerns and may make drug adjustment more difficult when a patient does not respond or tolerate the combinational regime. with this circumstance, a new instead-of medicine for ln would well meet the clinical request. iguratimod is a new small molecular, anti-rheumatic drug and has shown the potential for drug repurposing from rheumatoid arthritis (ra) to ln treatment. it has been approved for treating ra in northeast asia. according to data from ra clinical trials in japan and china, iguratimod is superior to a placebo and noninferior to methotrexate and sulfasalazine [13] [14] [15] [16] . in a preclinical study on lupus, iguratimod prevented autoimmune nephritis in mrl/lpr mice, decreased the amount of proteinuria, and reduced immune complex deposition [17] . beyond expectation in a recent observational study, over 90% of thirteen refractory ln patients responded to iguratimod monotherapy in 24 weeks, with no steroids dose increasing or any other medication add-on during the entire follow-up [18] . previous studies on possible mechanisms have provided the-other-side evidence supporting the rationale for using iguratimod to treat lupus, especially the interference with b cell differentiation. it was found to suppress b cell production of immunoglobulins over a decade ago [19] . in a phase iii clinical trial on ra, iguratimod reduced serum immunoglobulin concentrations [14, 16] . in ra and lupus animal models, iguratimod has decreased autoantibody titers, including anti-collagen antibody [20, 21] and anti-double strand (dsdna) antibody [17] . interestingly, iguratimod reportedly decreases peripheral plasma cell counts without affecting the total b cell population in mrl/lpr mice [17] and patients with ra who are receiving iguratimod monotherapy [22] . further investigation has shown that iguratimod regulates the key transcription factors affecting plasma cell differentiation, especially blimp-1, through the pkc/ egr1 axis [22] . given the strong evidence of implicating immunomodulatory of iguratimod, we designed and implanted a multi-center randomized, open-labeled, parallel positive drug control clinical trial with non-inferiority hypothesis. this trial focused on patients with active lupus nephritis. and our research is the first randomized controlled trial of iguratimod designed to treat lupus nephritis as well as sle in the world. we hypothesize that iguratimod monotherapy has non-inferior efficacy to traditional cyc-azathioprine (aza) sequential treatment on renal remission rate of at week 52 of treatment. objective this study is aimed to explore the efficacy and safety of iguratimod for treating biopsy-proven active ln patients, using an instead-of designing. we hypothesize that iguratimod monotherapy has non-inferior efficacy to traditional cyc-azathioprine (aza) sequential treatment on renal remission rate of at week 52 of treatment. the secondary objectives include exploring the effect, safety, and tolerance of iguratimod in active ln patients, and recording indicators of immunological markers, laboratory biomarkers, and behavioral factors during the trial. this study is a multi-center randomized, open-labeled, parallel positive drug control, clinical trial with noninferiority hypothesis. the patients will be selected from the rheumatology or nephrology department of six study sites after being diagnosed with active ln by biopsyproved in recent three months and satisfying the inclusion criteria. the flow chart of the study is shown in figs all candidates will receive clinical study information about the trial. written consent will be obtained from each participant. the purpose, procedures, and potential risks and benefits of the study will also be explained thoroughly to the participants. the participants will be able to withdraw from the study at any time without consequence. appointments are free of cost to patients. a copy of the signed consent form will be given to the participant and a further copy will remain in the patient's records at the recruitment site. prescreened patients, who are in the specified age range, have active lupus nephritis, will be scheduled for a screening visit (figs. 2 and 3 ). after obtaining written informed consent, the subject will complete medical history, family history, and physical examination. concomitant medications will be recorded. laboratory testing will be done, including complete blood count with differential, blood chemistry, urinalysis, urine routine, 24-h urine protein, pregnancy test, c reactive protein, erythrocyte sedimentation rate, anti-dsdna body, antinuclear antibody (ana), immunoglobulin, and complements 3 and 4. individuals who pass the screening laboratory testing and the physical examination will be scheduled for a baseline visit, to take place within 4 weeks of the screening visit. at the baseline visit (randomization visit), the patient will first complete a physical examination and review of concomitant medications and events will be carried out by the investigator. the following items will be completed: health assessment questionnaire (haq), evaluation for lupus, the modified sledai-2 k disease activity instrument for sle [23] , british isles lupus assessment group score (bilag score); the slicc/acr damage index [24] and the provider's global assessment (pga) and sle responder index (sri) face to face with clinicians. a random number table generated using statistical analysis software (sas), version 9.2, using the randomization method, will be used to assign the participants in a ratio of 1:1, with 60 patients each in the trial group and control group. participants in the iguratimod treatment group will additionally receive 50 mg oral iguratimod. and participants in the control group were treated with pulse cyclophosphamide for 24 weeks and sequential oral azathioprine. the study participants will be followed for a total of 52 weeks, including the enrolment and follow-up periods (figs. 1 and 2), after randomization at visit 1, visit 2 will fig. 1 trial design occur at 2-week, visits 3-10 will occur at 4-week intervals, and visits 11-12 will occur at 8-week intervals following the schedule. each study visit will include medication and adverse event review, physical examination, laboratory examination, completion of the sle-dai, slicc criteria, and acr criteria. medication compliance is determined by pill count and a new supply of pills dispensed. participants who achieve renal remission at the end of 52-week visit will undergo extended follow-up visits. any potential adverse events or significant changes in the subject's medical condition will prompt an unscheduled study visit. unscheduled visits may be performed to document adverse events, worsening of the subject's medical condition, or withdrawal from the study. within 4 weeks of completion of the treatment period, participants will undergo another visit to make sure no new adverse events happen. standard protocol items on the list of recommendations for interventional trials (spirit) are provided as a completed checklist (additional file 1). inclusion criteria are shown in table 1 . exclusion criteria are shown in table 2 . any participant unwilling or unable to continue the clinical trial for any reason could withdraw informed consent and withdraw the trial. and any subjects who have not reached remission or partial remission by 24 weeks will be terminated. termination of study may occur by investigators for the following reasons (table 3) : interventions experimental arm simcere pharmaceutical). participants in the b proteinuria of no less than 1.0 g/24 h within the 4 weeks before enrollment, with or without microscopic hematuria. c active lupus nephritis confirmed by biopsy and classified as class iii/iv/v type or compound type (iii+v or iv+v) according to the classification of lupus nephritis within 3 months prior to screening. 3 systemic lupus erythematosus disease activity index 2000 (sledai-2000) scores ≥ 8 points within 2 weeks before enrollment. 4 weight no less than 40 kg. 5 the participant agrees to take measures for birth control contraception 6 the patient agrees to participate and provides informed consent table 2 exclusion criteria of igelu study 1 severe manifestations of sle, including: a. neuropsychiatric lupus within 1 month before screening. b. extensive crescentic glomerulonephritis confirmed by biopsy with a ratio of crescents higher 50%. c. estimated glomerular filtration rate (egfr) < 60 ml/min/1.73m 2 (calculated by the epi formula) at least two tests within 30 days before screening and at least 14 days between the two tests. d. evidence of significant abnormal laboratory value in peripheral blood not related to lupus (white blood cells (wbc) < 3 × 10 9 /l, platelets (plt) < 50 × 10 9 /l) within 1 week before screening. e. moderate to severe anemia within one week before screening. f. ast and alt values more than two times the upper limit of normal within one week before screening. 2 participant has been diagnosed with another autoimmune disorder, including but not limited to: rheumatoid arthritis, sjogren's syndrome, inflammatory myositis, systemic sclerosis, autoimmune liver disease. 3 prior use of the following agents within 3 months of screening. a. mmf or cnis for more than 14 days in accumulation. b. anti-tnf-α, anti-interleukin-6 (il-6), anti-il1 or jak inhibitor. c. b cell depletion therapy (e.g., anti-cd20 monoclonal antibody) or anti-b lymphocyte stimulator therapy (e.g., belimumab) d. high dose of glucocorticoid therapy (> 100 mg/d calculated by prednisone). e. plasma exchange, blood adsorption, hemodialysis, or mesenchymal stem cell translation. 4 gene test for aza demonstrates a high risk of side effects. 5 active bacterial, viral, fungal, or opportunistic infection during the screening period. 6 active viral hepatitis. 7 active tuberculosis. 10 suspected or confirmed history of alcohol or drug abuse within two years 11 history of malignant neoplasm except carcinoma in situ, basal cell carcinoma and cured carcinoma. 12 severe, progressive, or uncontrolled pulmonary, cardiac, or hypertension medical condition. 13 participant suspected of mental disability. 14 participant with epilepsy or nervous system dysfunction. 15 pregnant, breastfeeding, or unwilling to practice birth control during participation in the study. iguratimod group will be prescribed oral iguratimod at a dose of 25 mg twice daily until the end of trial. 1. cyclophosphamide (cyc, 0.2 g per potion). participants in the control group will be prescribed intravenous cyc as induction immunosuppression at a dosage of 0.5-1.0 g/m 2 once for 4 weeks. 2. azathioprine (aza, 50 mg per tablet). participants in the control group who has achieved remission (pr/cr) at week 24 will be prescribed oral azathioprine at an initial dose of 50 mg/d. if there is no acute leukocytopenia in weeks 25 to 28, the dosage would be increased to 2 mg/kg per day in the next 1 month. 1. glucocorticoid: all participants will be treated with glucocorticoid (calculated by prednisone) following the routine: 1.1 oral prednisone (1 mg/kg/d) for first 4 weeks. 1.2 the dosage of prednisone will be reduced by 5-10 mg every 2 weeks from the 5th week until 30 mg/d, followed by reduction of 2.5-5 mg for every 2 weeks, followed by a taper to no more than 10 mg/d until the end week 24, followed by maintenance therapy with daily oral prednisone no more than 10 mg/d. the primary outcome is reported as the proportion of participants with renal remission (pr or cr) at 52 weeks. the key secondary outcomes include renal remission rate by week 24, renal flare rate by week 52, number of treatment-related adverse events, and changes in sledai-2 k score, bilag score, and pga. the detail of key secondary outcomes is displayed in table 4 . exploratory secondary outcomes are also planned as follows ( 12 serum immunoglobulin levels. eligible patients who sign written informed consent will be randomly assigned across study centers. the study will be conducted in accordance with the currently approved protocol, international conference on harmonisation good clinical practice (ich gcp), and relevant regulations. to maintain confidentiality, all laboratory specimens, evaluation forms, reports, and other records will be identified by a coded number and initials only. case report forms (crfs), medical reports, and laboratory test reports will make up the main data source file for the subjects in this clinical trial. investigators must fill out the case report form truthfully. crf-related data are provided by the research team to a professional institution for clinical data management and study data statistics. data will be modified by investigators strictly following inductions of case report form with signature and date every time. the collected data in crf will be audited and verified by referring to the source document by the clinical researchers of the project during a scheduled visit to ensure data integrity with signature and date for every visit. the medical record data will be entered into the database and consistent with crfs. after data are input into electronic care report tables, quality control and data audit procedures will be performed by individual investigator to ensure the accuracy and reliability of these data. during the study, an independent data monitoring committee (dmc) will be set up to carry out periodic interim evaluation and optimize the study when appropriate based on the results of the interim evaluation. the dmc is authorized to discontinue the clinical study in case of unexpected adverse reactions. during implementation of the project, staff of dmc will check each electronic case report form (ecrf) for validity and consistency periodically or irregularly, and study compliance will be checked, so that data integrity and accuracy will be fully guaranteed and authenticity and reliability of the study results are ensured. the effect of iguratimod was carried out as one of the main efficacy indexes, the primary hypothesis is that iguratimod therapy is not inferior to cyz+aza therapy. thus, the required sample size is: where p a is the remission rate on the basis of previous investigational study estimated as 0.8, p b is estimated as 0.7 based on related data. the δ value between the two groups is expected to be − 0.1 with a significance level of α = 0.05 and an assurance of 1 -β = 0.8. thus, the total sample size was calculated as 53 cases if the iguratimod group and the control group were included at a ratio of 1:1. we will increase the sample size by approximately 10% to reach 60 cases in consideration of the possibility that some patients could miss visits due to the long illness course and follow-up time. the data obtained from the participants of this clinical trial will mostly be divided into a fas (full analysis set) group, a pps (per protocol set) group, and a ss (safety set) group. full analysis set (fas) based on the principle of intentionality treatment (itt principle), the fas group will include all randomized subjects who met entry criteria. the primary efficacy analyses will be based on the fas group, as well as key secondary efficacy and exploratory secondary outcomes. for the fas group, if a defect value happens at any point or a dropout occurs before the clinical trial ends, the most recent data will be analyzed as if it were obtained at that point in time locf (last observation carried forward analysis). in a locf analysis, a missing follow-up visit value is replaced by (imputed as) that subject's previously observed value, that is, the last observation is carried forward. and patients who did not reach remission would be identified as ineffective subjects. per protocol set modify (pps/ppsm) the pps group will include participants from the fas group who successfully completed this clinical trial according to the trial plan. participants in pps group who seriously violate trial protocol or take a forbidden drug or do not obey a schedule can be excluded. ittm analysis set and the ppsm set will be applied to sensitivity analysis to check for overall robustness of the primary analyses. safety set (ss) the safety set group will include participants who took the iguratimod and underwent safety assessment at least once. per protocol set will pick up from the full analysis set for analysis. statistical analysis of the efficacy of the study will be performed using statistical data sets that meet the protocol. we will calculate the average, standard deviation, minimum value and maximum value for continuous data, and we will calculate the frequency and percentage for categorical data. for the purpose of group comparisons, we will compare the demographic data for the two groups by using the χ 2 test, fisher's exact test, t test, non-parametric statistical method, or cmh analysis. efficacy analysis will be performed to compare the efficacy of each group. continuous variables will be compared by using t test and covariance analysis model considering central effect (two sides: processing factor and baseline factor). data of steroid reduction will be compared using survival analysis, and kaplan-meier curve method will be used for survival analysis. descriptive statistics (presented in table form) will be used for safety and tolerability data. if necessary, fisher's exact test will be conducted for the percentage of adverse events between the two groups. laboratory test results will be used to describe conditions when abnormal results happen and the connection with the trial agents. a two-sided test with a 0.05 significance level will be applied, p values ≤ 0.05 will be considered statistically significant. all statistical analyses will be performed with sas software, version 9.13 (sas institute, cary, nc, usa). the treatment arms will be masked to both outcome assessors and data analysts and maintained according to standard practices. subjects will suffer from side effects of drugs and treatment failure during the trial. adverse events reported in clinical trials of iguratimod for rheumatoid arthritis patients are as follows: the most common adverse effect is impaired liver function. common adverse effects include leukopenia, stomach discomfort, nausea, bloating, stomachache, anorexia, rash, nausea, abdominal distension, thrombocytopenia, sour regurgitation, abdominal pain, blurred vision, skin itching, duodenitis, gastritis, fecal occult blood, hair loss, insomnia, abnormal electrocardiogram, menstrual disorders, and anemia. rare adverse effects include diarrhea, dyspepsia, belch, gastric ulcer, reflux esophagitis, duodenal ulcer, gastric antrum bleeding, vomiting, fever, cough, dry mouth, oral ulcers, facial edema, skin edema, fatigue, chest tightness, chest pain, positive urine protein, elevated total bilirubin, flu-like symptoms, upper respiratory tract infection, and acne-like gastritis. most of the adverse effects mentioned above will alleviate or disappear spontaneously after withdrawal. it is possible that new adverse events occur during the trial. the research team will assess all participants at the end of 24 weeks. participants will withdraw from the trial and switch to another therapy if they fail to achieve remission. the research team will advise early termination of the trial in the event of safety concerns or lack of any treatment effect. all aes will be recorded at trial visits for the 52 weeks of by the member of the research team and filled in in the case report form. all adverse events must be judged for their character, severity, and potential relationship to the study treatment. all aes will be judged by a medically qualified member of the research team or the sponsor will be followed until resolution or the event is considered stable, clinically insignificant, or asymptomatic. all related aes that result in a participant's withdrawal from the study or are present at the end of the study should be followed up until a satisfactory resolution occurs. the correlation between adverse events and study treatment is divided into five levels: definitely related, probably related, possibly related, possibly unrelated, and definitely unrelated. the adverse reactions are divided into 3 levels: classification i: mild (+), participants can continue trial agents without special treatment. classification ii: moderate (++), participants will continue agents after the break or other treatments. classification iii: severe (+++), participants need to suspend medications. the principles for adverse events are as follows: 1. abnormalities in blood cell counts or liver function tests hepatoprotective drugs and whitening drugs can be added as appropriate for the above conditions. the research team will adjust the medication regimen according to the relevance of the adverse event and the trial agents. participants will be prescribed trial agents with the original dose. the treatment can be repeated 3 times, and agents will not be added to the original dosage for the fourth time. 2. participants will withdraw from the trial when they are allergic to agents and treated according to the clinician's experience 3. other adverse events: participants will be diagnosed and treated following related routine the present study is being conducted in accordance with the declaration of helsinki, quality management standard of drug clinical trials (gcp), and relevant clinical study research regulations in china. the protocol was approved by the ethics committee of the renji hospital, shanghai, china. systemic lupus erythematosus (sle) is an autoimmune disease of unknown cause involving various tissues and organs damaged, and lupus nephritis (ln) is one of the most severe organ manifestations of sle. most patients diagnosed with sle will develop ln within 5 years [25] [26] [27] . some patients will progress to end-stage renal disease, ln remains a major cause of morbidity and death [27] [28] [29] . the main goal of treatment is to palliate symptoms, defer progression, reduce disease progression-related complications and mortality rate, and also preserve fertility for female patients. glucocorticoids combined with either cyclophosphamide or mmf currently is used as first-line agents. on top of this, there are some other medications recommended such as calcineurin inhibitors, leflunomide, and other biological agents [30] . however, some patients still suffer from relapse or ineffective treatment and medication intolerance is also frequent. therefore, the demand for new drugs continues. igu is a novel small-molecule immunomodulatory agent. igu can effectively inhibit expression of various inflammatory factors, inhibit b cells from producing immunoglobulins and autoantibodies, downregulate t cellmediated cellular immunity, accelerate bone formation, and exert some activity against anti-pulmonary fibrosis. igu is widely used in the treatment of ra; both monotherapy and combination therapy suggest good efficacy and safety. in addition, the clinical studies suggest that igu has a good effect on other rheumatic diseases, such as sjögren's syndrome, ankylosing spondylitis, and igg4related diseases [31] . our study was designed as a head-to-head comparison between iguratimod with a classic ln therapy, cyc ensued with aza. in the first 24 weeks of the trial, igu is compared with cyclophosphamide as an induction therapy, and in the second 24 weeks, igu is compared with azathioprine as a maintenance therapy. in the previous observatory ln study, over 90% refractory ln patients responded to iguratimod monotherapy without any steroids increasing in the first 24 weeks of treatment [18] . given to this outstanding effectiveness of igu, the result of this study is expectable and promising. the availability and use of an unproven, but relatively safe, drug for people with ln and the reluctance of potential subjects are the major obstacles. also, it might be a hindrance when we persuade patients to underwent kidney biopsy. a limitation of this study is that the longtime effect of the agent or therapy was unable to confirm due to the limited study duration. however, participants who achieve renal remission at the end of 52-week visit will undergo extended follow-up visits (not reflected in the data). the igelu trial is designed to demonstrate whether iguratimod can induce and maintain remission thereby slowing the progression of disease in subjects who have lupus nephritis. data from this study will provide an evidence on whether or not iguratimod should be recommended to all lupus nephritis patients, and those patients will be able to make an informed choice about the risks and benefits of this medication. the current igelu study protocol version is 2.1 dated 7 july 2017. enrollment was initiated in august 2017. the outbreak of covid-19 forced us to change the protocol and delayed the progress of our trial. igutimod is used as an induction therapy for lupus nephritis for the first time, which decreases enthusiasm on the patient side, and also caused difficulties in recruiting. therefore, enrollment completion is anticipated for november 2022. and the estimated study completion date is november 2023. gender differences in the pathogenesis and outcome of lupus and of lupus nephritis pediatric lupus-are there differences in presentation, genetics, response to therapy, and damage accrual compared with adult lupus? update on lupus nephritis 2019 update of the eular recommendations for the management of systemic lupus erythematosus risk of end-stage renal disease in patients with lupus nephritis, 1971-2015: a systematic review and bayesian meta-analysis long-term outcomes in systemic lupus erythematosus: trends over time and major contributors american college of rheumatology guidelines for screening, treatment, and management of lupus nephritis histopathologic and clinical outcome of rituximab treatment in patients with cyclophosphamide-resistant proliferative lupus nephritis use of rituximab in the treatment of refractory systemic lupus erythematosus: singapore experience long-term efficacy and safety of rituximab in refractory and relapsing systemic lupus erythematosus op0277 aurora phase 3 study demonstrates voclosporin statistical superiority over standard of care in lupus nephritis (ln) op0164 bliss-ln: a randomised, double-blind, placebocontrolled phase 3 trial of intravenous belimumab in patients with active lupus nephritis t-614): a novel disease modifying anti-rheumatic drug efficacy and safety of iguratimod compared with placebo and salazosulfapyridine in active rheumatoid arthritis: a controlled, multicenter, double-blind, parallel-group study safety and efficacy of t-614 in the treatment of patients with active rheumatoid arthritis: a double blind, randomized, placebo-controlled and multicenter trial multicenter, randomized, double-blind, controlled trial of treatment of active rheumatoid arthritis with t-614 compared with methotrexate prevention of immune nephritis by the small molecular weight immunomodulator iguratimod in mrl/lpr mice iguratimod as an alternative induction therapy for refractory lupus nephritis: a preliminary investigational study inhibitory effects of an anti-rheumatic agent t-614 on immunoglobulin production by cultured b cells and rheumatoid synovial tissues engrafted into scid mice t-614, a novel immunomodulator, attenuates joint inflammation and articular damage in collagen-induced arthritis a novel disease-modifying antirheumatic drug, iguratimod, ameliorates murine arthritis by blocking il-17 signaling, distinct from methotrexate and leflunomide iguratimod represses b cell terminal differentiation linked with the inhibition of pkc/egr1 axis systemic lupus erythematosus disease activity index the development and initial validation of the systemic lupus international collaborating clinics/american college of rheumatology damage index for systemic lupus erythematosus epidemiology and management of refractory lupus nephritis morbidity and mortality in systemic lupus erythematosus during a 10-year period: a comparison of early and late manifestations in a cohort of 1,000 patients. medicine (baltimore) assessment of a lupus nephritis cohort over a 30-year period mortality in systemic lupus erythematosus molecular mechanisms and clinical application of iguratimod: a review publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the online version contains supplementary material available at https://doi. org/10.1186/s13063-021-05475-3. authors' contributions cdb, qry, and fd participated in study conception, design, and supervision. md, xwd, jhx, jpt, nsw grj, and zjl were involved in literature search, study design, data collection, statistical analysis, interpretation, and writing and revision of the manuscript. xw, qx, xfh, xyz, and yz participated in data collection. qry, py, and df were involved in the data collection and supervision of the study. qry and ynk were involved in data interpretation, writing, and revision. all authors revised the manuscript and approved the final report. this is an investigator-initiated trial (iit). this work is supported by shanghai shenkang promoting project (16cr1027b), national natural science foundation of china (81601401 and 81373207), and shanghai municipal commission of health and family planning (20204y0088). the funding body has no active had no active role in the design of the study, collection, analysis, and interpretation of data, and in writing the manuscript. the clinical study report of this trial would be published with the research paper as an appendix. the raw data would be accessible for all the investigators. ethics approval and consent to participate this study and all relevant data have been approved by the medical ethics committee of renji hospital shanghai jiaotong university (number 2016[128 k]). the study progress is quickly reviewed by the committee yearly. the investigators will inform the ethics committee of any major changes to the protocol or consent materials. any amendments would be approved by the committee. by far, there are no major changes to the protocol. we ensure that this study is conducted in accordance with the principles of the declaration of helsinki and the principles of good clinical practice. central ethics approval has been confirmed from the medical ethics committee of renji hospital and we will not begin recruiting at other centers in the trial until local ethics approval has been obtained. this trial was registered at the china clinical trial registry website under the identification number clinicaltrials.gov, nct 02936375 on october 26, 2016. signed informed consent forms will be obtained from all qualified participants before enrollment. not applicable. the authors declare that they have no competing interests. key: cord-0034237-zyiqigck authors: diulio, maggie title: veterinary immunology and serology: clinical laboratory diagnostics date: 1999-01-01 journal: lab med doi: 10.1093/labmed/30.1.36 sha: c7f6319a2a9e7bfa8d90568d6fd1e85131c0c9f2 doc_id: 34237 cord_uid: zyiqigck methods used in clinical laboratory diagnosis in the veterinary laboratory closely parallel the common techniques used in the human laboratory. immunology procedures include immunohematology, autoimmune testing, and assays for detection of immune deficiencies and infectious diseases. veterinary immunohematology procedures deal with immune-mediated hemolysis, as well as blood typing, cross matching, and transfusion. diseases of the immune system in animals include rheumatoid arthritis, systemic lupus erythematosus (sle), and immunodeficiency disorders. the number of infectious diseases that can be diagnosed in a veterinary laboratory is almost limitless, but perhaps two of the most prevalent and significant are heartworm disease and feline infectious peritonitis (fip). immunology and serology in a veterinary diagnostic laboratory are much the same as in a human diagnostic laboratory. standard immunology methods such as particle agglutination, indirect fluorescent antibody (ifa) and direct fluorescent antibody (dfa) tests, enzyme-linked immunosorbent assay (elisa), radioimmunoassay, enzyme immunoassay, immunohistochemistry, and chemiluminescence are used in contemporary veterinary antinuclear antibody lupus erythematosus cell preparation immunohematology blood typing, crossmatching coombs' test diagnostics. polymerase chain reaction (pcr) is used for definitive identification of many infectious organisms. veterinary assays range from immunology and immunohematology (table 1) to infectious serology (table 2) to endocrinology and oncology. the focus of this article is on diagnostic procedures in veterinary immunology, serology, and infectious diseases. the number of species that might be included in veterinary diagnostics is almost limitless, but for the sake of brevity only the most common (canine, feline, and equine) are included. although crossmatching of donor and recipient blood is common practice before transfusion in domestic animals, blood typing usually is performed only in dogs (fig 1) , in large part because of the lack of naturally occurring antibodies in nonhuman species. at least 11 blood group systems are found in dogs; only one, the dog erythrocyte antigen (dea)-l system, is polymorphic. only dea-7 has a significant frequency of naturally occurring antibody in nonsensitized animals. efforts are made during blood typing to assure that donor dogs do not have dea-1 or dea-7. dea-1 is immunogenic and frequently can sensitize a recipient lacking that antigen, and dea-7 is associated with naturally occurring anti-dea-7 in approximately 50% of animals negative for that antigen. the rbc antigen system in cats is simple, with only a and b antigens defined. it appears these antigens have reciprocal antibodies; that is, cats with type a blood have anti-b antibodies, and those with type b blood have anti-a antibodies. clinically, this requires crossmatching before transfusion to prevent these naturally occurring antibodies. the rbc antigen system in horses is more complex, demonstrating at least seven systems, with 1 to 11 antigens in each. however, none of these systems seems to support naturally occurring antibodies. only crossmatching is performed before transfusion, and the complex antigen systems are used primarily as a genetic marker system for pedigree substantiation. hemolytic disease of the newborn (hdn) is virtually nonexistent in utero in domestic animals, but can occur as the result of antibodies passed postpartum after the newborn animal has nursed the immunoglobulin-rich colostrum. that natural antibodies occur so infrequently indicates maternal immunization by previous transfusion, traumatic parturition, or rbc-contaminated vaccine. postpartum hdn and other immune-mediated hemolytic anemias in domestic animals require that veterinary diagnostic laboratories offer coombs' testing for a variety of domestic animals. the test requires antiserum raised against the species-specific immunoglobulin, which is commonly multivalent against igg, igm, and c3. a wide variety of autoimmune disorders occur in domestic animals, including skin disease, blood, joint, and systemic organ diseases. the common veterinary laboratory diagnostic tools for autoimmune disease are virtually identical to those used in human diagnostics, but modified for the specific species. rheumatoid arthritis is relatively common in small canine breeds. the commonly used assay detects antibody (rheumatoid factor) that reacts with the animal's own igg. the rheumatoid factor is most commonly igg, but may be iga or igm. the assay uses a particle-agglutination method, which utilizes either sheep rbcs or latex particles coated with canine igg. the particles are reacted with canine serum. agglutination indicates the presence of antibody against canine igg (rheumatoid factor). the ana assay uses an ifa technique to detect anas in animals with systemic lupus erythematosus (sle). this disease has been diagnosed in canines since 1964, and has now been described in virtually all breeds, with a disproportionate prevalence in poodles and german shepherds. feline sle has also been documented. in the common ana assay, substrate cells (usually human laryngeal tumor cells [hep-2 cells]) are affixed to a microscopic slide and flooded with the serum from the affected animal. if ana is present, it will affix to a variety of structures in the nucleus of the substrate cells. after rinsing, an antiglobulin conjugated with fluorescein is reacted with the substrate cells. the presence and pattern of ana attachment can then be viewed under a fluorescent microscope. the le cell preparation is another tool used to diagnose sle. as in humans, this test in veterinary diagnostics has poor sensitivity and specificity, but can be performed inexpensively and easily and does not require species-specific reagents. the method requires that some of the cells within a blood or body fluid specimen be traumatized to expose nuclear material. if ana is present, it will attach to the exposed nucleus, causing swelling and loss of chromatin structure. this sensitized nuclear material is then phagocytized by the intact macrophages or neutrophils in the specimen, creating the diagnostic le cells. dfa techniques can be used to diagnose a variety of autoimmune diseases in animals. affected tissue is harvested at biopsy. the tissue is sectioned and mounted on slides, and flooded with species-specific fluorescein-conjugated anti-immunoglobulin. tissues with autoimmune antibody attachment can then be viewed under the fluorescent microscope. ifa techniques can be used to demonstrate the presence of autoimmune antibody in animal plasma. the ana test is a variant of this technique. purchased or prepared substrate cells (commonly skin, renal, or hepatic cells) from a specific affected tissue are flooded with plasma from the affected animal. after washing, the cells are flooded with species-specific fluorescein-conjugated anti-immunoglobulin. the presence of autoantibody directed against specific tissue components, such as cutaneous basement membrane or renal glomerular basement membrane, can be viewed under the fluorescent microscope. direct and indirect coombs' testing is used to assist with the diagnosis of immune-mediated hemolytic anemia in animals. the application of this test in veterinary diagnostics requires the use of species-specific antiglobulin. failure of passive transfer of maternal antibody in neonates is the most common immune deficiency of the animal kingdom. the effects of this condition depend on the relative contribution of placental transfer of maternal antibody to the neonate vs colostral transfer. canine and feline placenta types allow limited transplacental immunoglobulin transfer. equine and bovine placenta types do not allow immunoglobulin transfer from mother to neonate, and patent neonatal immunity depends totally on successful postpartum suckling in these animals. intestinal absorption of colostral immunoglobulin is most efficient in the first 6 hours postpartum, and is virtually nonexistent after 24 hours. lack of colostrum appears to have little effect in puppies, and perhaps slight effect in kittens. its greatest effect is in cattle and horses, and it is extremely difficult to rear these young animals successfully without colostrum. a veterinary laboratory can diagnose failure of passive transfer with a variety of quantitative or semiquantitative immunoglobulin assays. time is critical. although the animal should be at least 18 hours old, to allow maximum transfer of colostral immunoglobulin, deficient animals must be treated within 72 hours. semiquantitative stat or point-of-care methods to measure neonatal immunoglobulin levels such as particle agglutination and precipitation in inorganic salt solution are usually adequate, and are preferred. defects in almost every aspect of the immune system have been described in animals, including severe combined immunodeficiency disease, cellular dysfunctions, complement dysfunctions, cliidiak-higashi syndrome, pelger-huet nuclear anomaly, hypogammaglobulinemia, defective barrier systems, and infection-induced immune system suppression (fig 2) . the immunology laboratory can assist with the diagnosis of immune deficiencies in the humoral system with quantitative and qualitative immunoglobulin assays. immunoglobulin quantitation is usually accomplished with radial immunodiffusion, rocket immunoelectrophoresis, elisa, or radioimmunoassay. the primary challenge with these techniques is development of species-specific reagent systems. each infectious disease presents its own set of diagnostic challenges for the veterinary laboratory. two, however, are particularly worthy of further discussion because of their prevalence, diagnostic significance, and developing technology. heartworm disease is caused by infection with dirofilaria itnmitis, which is endemic in all parts of the united states and is particularly common in atlantic and gulf coast saltwater marshes, where mosquitoes are prevalent. the life cycle of this parasite begins with an infected mosquito that passes larvae to a healthy mammal during a bite. the larvae migrate to muscle or fat, and develop into young adult worms within 90 days. the adult migrates to the right ventricle and pulmonary arteries, where it matures within 4 months. in a patent infection, females become gravid by 5 months, and shed microfilaria 6 to 7 months after the initial infection. canines are the definitive host for d immitis, but felines and other small mammals can become infected. noncanine hosts generally mount a vigorous immune response to infection, and the development of a patent infection with adult worms is uncommon and often self-limiting. in the past, detection of heartworm infection depended on detection of either the microfilaria by direct examination of stained blood smears (knott test , fig 3) or the shedding antigen from adult worms in the serum of infected animals (elisa method). the specificity of the heartworm antigen method is good, and a positive result is an excellent predictor of a patent infection with productive female worms. although the test is marketed for use in canines, clinical experience has demonstrated the antigen test to be useful in felines and other small mammals. however, several factors create a problem with the sensitivity of this assay. the heartworm antigen method can only detect antigen shed from adult female worms. therefore this method will produce a negative result if the infection is early in development or includes only male worms. also, if the worm burden is low, the antigen level may not rise above the lower sensitivity limit of this elisa method. these limiting conditions are commonly present early in canine infection and in infections in noncanine species. heartworm antibody testing (ifa method) also is available. the antibody assay has a much higher sensitivity than the antigen assay, demonstrating somatic antibodies (against larval antigens) within 1 to 2 weeks of exposure and cuticular antibody (against adult and filarial antigens) within 2 to 3 months. the assay commonly yields.positive results during the prepatent period, in infection with male-only worms, and in infections with low worm burden. the antibody assay is often the assay of choice in noncanine species, because worm burdens are often low or unisex. however, this assay also yields positive results after any exposure to heartworm, even if the exposure fails to develop into an infection. after an exposure or infection has cleared, the antibody assay generally remains positive for 9 to 12 months. this test is also limited by the availability of species-specific fluorescein-conjugated antiglobulin. fip is caused by infection with a feline coronavirus that causes thoracic and abdominal effusions, characteristic multifocal fibrinonecrotic plaques, and wasting and death. the virus is passed by the fecal-oral route, and will spread rapidly through a breeding colony or domestic household. like the common rhinovirus in humans, many coronaviruses can infect felines, but only a few cause clinical fip. the laboratory's ability to assist with the diagnosis of fip was severely limited by the nonspecific nature of the immunofluorescent antibody assay used to detect anti-coronavirus antibody. the assay demonstrates a positive titer in a cat that is or was infected with any coronavirus. the majority of adult cats demonstrate some level of anti-coronavirus antibody. the clinician is faced with determining what level is significant, whether the titer is rising or falling, and whether the clinical symptoms are consistent with fip or with some other benign coronavirus infection. pcr is of help in making this difficult diagnosis. pcr techniques focus on detection of a specific region on the virus genome that is present only in the coronavirus subset known to cause fip. this specific region is known as the 7b gene, and pcr techniques are capable of determining whether this gene is present in the infecting coronavirus. however, while it is true that all flp-causing coronaviruses demonstrate the 7b region, not all 7b-positive coronaviruses cause fip. the key to fip infection seems to be the ability of the 7b region to produce and secrete a specific protein, the 7b-region specific protein. if the coronavirus is secreting this specific protein, it is capable of causing fip. a new elisa method has recently been developed that can detect the presence of this 7b-region specific protein. the method has been described as the feline infectious peritonitis-specific elisa (fipse), and appears to have good predictive value in the diagnosis of the disease. 1 this test is currently available in a limited market while clinical correlation studies are carried out. immunology and serology procedures in a veterinary diagnostic laboratory closely parallel those in human laboratories. however, because of the number of nonhuman animal species and the number of congenital and acquired diseases found in each species, the number of diagnostic laboratory procedures that can be performed in a veterinary laboratory is almost infinite. i have summarized some of the most common immunology procedures performed in a veterinary laboratory in the most common companion animal species.® new diagnostic serology for fip halliwell rew, gorman nt. veterinary clinical immunology small animal clinical diagnosis by laboratory methods key: cord-0006691-f7y2o5tf authors: calder, philip c. title: n−3 polyunsaturated fatty acids and inflammation: from molecular biology to the clinic date: 2003 journal: lipids doi: 10.1007/s11745-003-1068-y sha: 06edaa3506b7c70f4380ae3e727c668aae1d6f6c doc_id: 6691 cord_uid: f7y2o5tf the immune system is involved in host defense against infectious agents, tumor cells, and environmental insults. inflammation is an important component of the early immunologic response. inappropriate or dysfunctional immune responses underlie acute and chronic inflammatory diseases. the n−6 pufa arachidonic acid (aa) is the precursor of prostaglandins, leukotrienes, and related compounds that have important roles in inflammation and in the regulation of immunity. feeding fish oil results in partial replacement of aa in cell membranes by epa. this leads to decreased production of aa-derived mediators, through several mechanisms, including decreased availability of aa, competition for cyclooxygenase (cox) and lipoxygenase (lox) enzymes, and decreased expression of cox-2 and 5-lox. this alone is a potentially beneficial anti-inflammatory effect of n−3 fa. however, n−3 fa have a number of other effects that might occur down-stream of altered eicosanoid production or might be independent of this effect. for example, dietary fish oil results in suppressed production of proinflammatory cytokines and can modulate adhesion molecule expression. these effects occur at the level of altered gene expression. fish oil feeding has been shown to ameliorate the symptoms of some animal models of autoimmune disease and to protect against the effects of endotoxin. clinical studies have reported that oral fish oil supplementation has beneficial effects in rheumatoid arthritis and among some asthmatics, supporting the idea that the n−3 fa in fish oil are anti-inflammatory. there are indications that the inclusion of fish oil in enteral and parenteral formulae is beneficial to patients. inflammation is the body's immediate response to infection or injury. its role is to begin the immunological process of elimination of invading pathogens and toxins and to repair damaged tissue. these responses must be ordered and controlled. the movement of cells into the inflammatory/infected site is induced by the upregulation of adhesion molecules such as intercellular adhesion molecule-1 (icam-1), vascular cell adhesion molecule-1 (vcam-1), and e-selection on the surface of endothelial cells, allowing leukocyte binding and subsequent diapedesis. the activity of leukocytes is induced by certain triggers. one important exogenous trigger is bacterial endotoxin (also known as lipopolysaccharide or lps), a component of the cell wall of gram-negative bacteria. lps can directly activate monocyte/macrophages, inducing them to form cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)-1, il-6 and il-8; eicosanoids, such as prostaglandin (pg)e 2 ; nitric oxide; matrix metalloproteinases (mmp); and other mediators. lps also induces adhesion molecule expression on the surface of endothelial cells and leukocytes. the cytokines produced by monocyte/macrophages also serve to regulate the whole-body response to infection and injury (see fig. 3 of ref. 1). thus, inflammation and the inflammatory response are part of the normal, innate immune response; inflammatory mediators also provide a link between the innate and acquired immune responses (see fig. 3 of ref. 1). however, when inflammation occurs in an uncontrolled manner, disease ensues. high levels of tnf-α, il-1β, and il-6 are particularly destructive. chronic overproduction of tnf-α and il-1 may cause muscle wasting and loss of bone mass. tnf-α, il-1, and il-6 are implicated in causing some of the pathologic responses that occur in endotoxic shock, in adult respiratory distress syndrome, and in chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. the fa composition of inflammatory cells. the link between fa and inflammation relates to the composition of inflammatory cell membrane phospholipids. this is because the fa composition of membrane phospholipids can influence various membrane activities, which can, in turn, influence cellular responses (fig. 1) . for example, the physical nature of the membrane, often referred to as fluidity, is regulated in part by the fa composition of its constituent phospholipids (2) . membrane fluidity affects the activity of membrane-bound proteins including receptors, transporters, and enzymes (3); thus, it can alter the responsiveness of inflammatory cells to a stimulus (for a review, see ref. 4) . intracellular signals, such as dag, inositol phosphates, and ceramide, are produced from membrane phospholipids in response to a suitable cell stimulus, and there is evidence that the ability of the phospholipase enzymes to generate these signaling molecules can be altered by the fa composition of the substrate phospholipids (for references, see ref. 5) . another group of mediators, the eicosanoids, are generated from fa liberated from membrane phospholipids; the ability to produce these mediators is therefore strongly influenced by the fa composition of membrane phospholipids (i.e., substrate availability) (see below). thus, the fa composition of inflammatory cells is important in terms of regulating the functional responses of those cells. inflammatory cells typically contain a high proportion of the n-6 pufa arachidonic acid (aa; 20:4n-6) and low proportions of n-3 pufa, especially epa (20:5n-3). the exact proportion of aa in human inflammatory cells varies according to cell type and the lipid fraction examined (see, for example, refs. 6,7). increased consumption of fish oil, which is rich in long-chain n-3 pufa such as epa and dha (22:6n-3), results in increased proportions of those fa in inflammatory cell phospholipids, partly at the expense of aa (see, for example, refs. 6,7). aa as an eicosanoid precursor. the principal functional role for aa is as a substrate for synthesis of the eicosanoid family of bioactive mediators [e.g., pg, thromboxanes (tx), leukotrienes (lt), or hydroxyeicosatetraenoic acids (hete); see fig. 4 of ref . 1] . aa in cell membranes can be mobilized by various phospholipase enzymes, especially phospholipase a 2 , and the free aa can subsequently act as a substrate for the enzymes that synthesize eicosanoids (fig. 2) . metabolism of aa by cyclooxygenase enzymes (cox) gives rise to the 2-series pg and tx (fig. 2 ). there are two isoforms of cox: cox-1 is a constitutive enzyme and cox-2 is induced in inflammatory cells as a result of stimulation and is responsible for the markedly elevated production of pg that occurs upon cellular activation. pg are formed in a cell-specific manner. for example, upon activation monocytes and macrophages produce large amounts of pge 2 and pgf 2α , neutrophils produce moderate amounts of pge 2 , and mast cells produce pgd 2 . metabolism of aa by the 5-lipoxygenase (5-lox) pathway gives rise to hydroxy and hydroperoxy derivatives [5-hete and 5-hydroperoxyeicosatetraenoic acid (5-hpete), respectively], and the 4-series lt, lta 4 , b 4 , c 4 , d 4 , and e 4 (fig. 2) . 5-lox is found in mast cells, monocytes, macrophages, and granulocytes. aa-derived eicosanoids and inflammation. eicosanoids are involved in modulating the intensity and duration of inflammatory responses (for reviews, see refs. [8] [9] [10] . for example, pge 2 has a number of proinflammatory effects including inducing fever, increasing vascular permeability and vasodilation, and enhancing pain and edema caused by other agents such as bradykinin and histamine. pge 2 also promotes ige production by b lymphocytes; ige is a mediator of allergic inflammation. pge 2 suppresses production of tnf-α, il-1, and il-6, which, in these respects, is an anti-inflammatory action. ltb 4 increases vascular permeability, is a vasoconstrictor, enhances local blood flow, is a potent chemotactic agent for leukocytes, induces release of lysosomal enzymes, enhances the generation of reactive oxygen species, and enhances production of tnf-α, il-1, and il-6. thus, ltb 4 is proinflammatory in nature. in inflammatory conditions, increased rates of production of aa-derived eicosanoids occur, and elevated levels of these eicosanoids are observed in blood and tissues from patients with trauma, burns, and a variety of inflammatory disorders (8, 10) . interestingly, recent studies have shown that pge 2 inhibits 5-lox, thereby preventing the generation of the inflammatory 4-series lt (11) . recent studies have also reported novel anti-inflammatory effects of certain lipoxins generated from aa by 15-lox (12, 13) . pge 2 was found to induce generation of one of these, lipoxin a 4 (11) , indicating that pge 2 is involved in mediating the resolution of inflammation through effects on the generation of other eicosanoids. n-3 pufa and eicosanoid production. because significantly increased consumption of long-chain n-3 pufa results in a decrease in the amount of aa in the membranes of inflammatory cells, there will be less substrate available for synthesis of eicosanoids from aa. however, it is now apparent that the ability of long-chain n-3 pufa to influence pro-duction of eicosanoids extends beyond simply decreasing substrate availability. for example, epa competitively inhibits the oxygenation of aa by cox (14) . recent cell culture studies have demonstrated that n-3 pufa suppress cytokine-induction of cox-2 and 5-lox gene expression (15, 16) . owing to these various actions, fish oil feeding results in a decreased capacity of inflammatory cells to synthesize cox-and 5-lox-derived eicosanoids from aa (see, for example, refs. 7, [17] [18] [19] [20] . the reduction in the generation of aa-derived mediators that accompanies fish oil consumption has led to the idea that fish oil is anti-inflammatory. in addition to inhibiting the metabolism of aa, epa is able to act as a substrate for both cox and 5-lox (fig. 2) , giving rise to derivatives that differ in structure from those produced from aa (i.e., 3-series pg and tx, and 5-series lt). thus, the epa-induced suppression in the production of aa-derived eicosanoids may be accompanied by an elevation in the production of epa-derived eicosanoids. this is most evident for the 5-lox products of epa metabolism (7, 20) . the eicosanoids produced from epa are considered to be less biologically potent than the analogs synthesized from aa, although the full range of biological activities of these compounds has not been investigated. additionally, n-3 pufa have been shown to give rise to novel anti-inflammatory eicosanoids generated via cox-2 (21). epa and dha can inhibit the production of il-1β and tnf-α by cultured monocytes (see ref. 22) , and the production of il-6 and il-8 by cultured venous endothelial cells (23, 24) . more recent studies have extended such findings to include suppression of tissue factor production by n-3 pufa (25). consistent with cell culture studies involving n-3 pufa, fish oil feeding decreased ex vivo production of tnf-α, il-1β, and il-6 by rodent macrophages (26) (27) (28) and decreased circulating tnf-α, il-1β, and il-6 concentrations in mice injected with lps (29) . supplementation of the diet of healthy human volunteers with fish oil providing more than 2.4 g/d epa plus dha decreased production of tnf-α, il-1, and il-6 by mononuclear cells (17, 19, 30, 31) . these inhibitory effects of n-3 pufa on inflammatory cytokine production are not those that would be predicted on the basis of antagonism of pge 2 production, suggesting some other mechanism of action. this might be via effects on production of eicosanoids other than pge 2 or via eicosanoid-independent effects. culture of murine macrophages with n-3 pufa decreased their ability to bind to various surfaces (32) ; how this effect occurred was not investigated. later studies showed that culture of human venous endothelial cells with epa or dha decreased cytokine-or lps-induced surface expression of eselectin, icam-1, and vcam-1 (23, 33) , and diminished the adhesion of ligand-bearing monocytes (33, 34) . in another cell culture study, epa decreased surface expression of icam-1 on monocytes stimulated with interferon (ifn)-γ (35) . dietary fish oil decreased expression of icam-1 on the surface of murine macrophages (36) . supplementing the diet of healthy humans with fish oil providing ~1.5 g/d epa plus dha resulted in a lower level of expression of icam-1 on the surface of blood monocytes stimulated ex vivo with ifn-γ (37). more recently, dietary fish was found to decrease circulating levels of soluble vcam-1 in elderly subjects (38) , but it is not clear whether this represents decreased surface expression of vcam-1. if so, then this effect might be indicative of decreased endothelial inflammation in vivo. de caterina et al. (23) demonstrated that the downregulation of vcam-1 expression on the surface of endothelial cells caused by dha was exerted at the level of vcam-1 gene expression, and that this effect was independent of any effects on eicosanoid production and on antioxidant status. this was among the first demonstrations of an effect of n-3 pufa on the expression of inflammatory genes. more recently, it was shown that culturing bovine chondrocytes with α-linolenic acid, epa, or dha dramatically decreased cytokine-mediated induction of expression of the cox-2 (but not cox-1), tnf-α, il-1α, and aggrecanase-1 and -2 genes (15) . recently, this study was extended by a study using cultured explants of human osteoarthritic cartilage (16) . including αlinolenic acid, epa, or dha in the culture medium markedly decreased the cytokine-induced upregulation of expression of the cox-2, il-1α, il-1β, tnf-α, 5-lox, 5-lox activating protein (flap), mmp-3, mmp-13, and aggrecanase-1 genes in these cells. the n-3 pufa did not affect expression of the cox-1, 12-lox, or 15-lox genes, which were not induced by cytokines (16) . also, there was little effect of n-3 pufa on the expression of genes for the tissue inhibitor of metalloproteinase (timp)-1, -2, or -3, which again were not cytokine inducible (16) . these studies indicate a marked capacity of n-3 pufa to suppress the expression of inflammatory genes, with little effect on the expression of housekeeping (e.g., cox-1) or anti-inflammatory (timp) genes. they also indicate that an important, hitherto unrecognized contributor to the reduction in the generation of aa-derived eicosanoids after fish oil feeding may be decreased expression of the enzymes and proteins responsible (e.g., cox-2, 5-lox, flap). these observations provide exciting new understanding of the anti-inflammatory effects of n-3 pufa. a limited number of feeding studies have demonstrated an effect of dietary fish oil on inflammatory gene expression. dietary fish oil completely abolished mrna for tnf-α, il-1β, and il-6 in the kidneys of autoimmune disease-prone mice (39) . feeding mice a fish oil-rich diet significantly decreased the level of il-1β mrna in lps-stimulated spleen lymphocytes (40) . this study further demonstrated that the lower il-1β mrna level was not due to accelerated mrna degradation but to impaired mrna synthesis (40) . fish oil lowered lps-stimulated tnf-α mrna levels in murine peritoneal macrophages (27) . icam-1 mrna levels were lower in peritoneal macrophages from mice fed fish oil (36) . thus, significant evidence is emerging of an effect of dietary fish oil on inflammatory gene expression. because eicosanoids derived from aa regulate inflammatory gene expression, the effects of n-3 pufa might come about through antagonism of the effects of aa-derived mediators. however, at least some of these effects have been demonstrated to occur in an eicosanoid-independent manner (see, for example ref. 23) . recent studies indicate that n-3 pufa exert some effects through direct actions on the intracellular signaling pathways that lead to activation of one or more transcription factors. nuclear factor κb (nfκb) is a transcription factor involved in the induction of numerous inflammatory genes including cox-2, icam-1, vcam-1, e-selectin, tnf-α, il-1β, il-6, nitric oxide synthase, acute phase proteins, and mmp in response to inflammatory stimuli (41-43) (fig. 3) . nfκb exists as an inactive heterotrimer in the cytosol of resting inflammatory cells; one of the subunits is called the inhibitory subunit of nfκb (iκb). upon stimulation, a signaling cascade activates a protein complex known as iκb kinase (iκk). activated iκk phosphorylates iκb, causing its dissociation from the rest of the inactive nfκb trimer (44, 45) . the phosphorylated iκb is degraded. the remaining nfκb heterodimer is rapidly translocated to the nucleus where it binds to response elements in target genes, thus regulating their transcription. recent studies suggest that one aspect of the anti-inflammatory action of fish oil is decreased activation of nfκb. for example, dietary fish oil resulted in a lower level of nfκb in the nucleus (i.e., activated nfκb) of lps-stimulated murine spleen lymphocytes compared with feeding corn oil (46) . how n-3 pufa decreases the activation of nfκb is not clear. however, incubating human monocytes with epa decreased lps-induced activation of nfκb, and this was associated with decreased phosphorylation of iκb (47) . this suggests an effect of n-3 pufa on the signaling process leading to activation of iκk. incubation of a pancreatic cell line with tnf-α markedly upregulated degradation of iκb, and this could be totally abolished by prior incubation of the cells with epa, but not with aa (48) . this effect could be due to inhibition of phosphorylation of iκb, thereby preventing it from being targeted for degradation, or to inhibition of the degradation process itself. a second group of transcription factors currently eliciting much interest because of their potential role in inflammation is the peroxisome proliferator-activated receptors (ppar). although pparα and -γ play important roles in liver and adipose tissue, respectively (49) , they are also found in inflammatory cells (50, 51) . ppar act through dimerization with the retinoid-x-receptor and subsequent regulation of gene expression. ppar can bind, and appear to be regulated by pufa and eicosanoids (52, 53) . mice deficient in pparα have a prolonged response to inflammatory stimuli (53), leading to the suggestion that pparα activation might be "anti-inflammatory." activators of both pparα and -γ have been shown to inhibit the induction of a range of inflammatory genes including tnf-α, il-1β, il-6, il-8, cox-2, vcam-1, nitric oxide synthase, mmp, and acute phase proteins (51, (54) (55) (56) (57) (58) (59) (60) . two mechanisms for the anti-inflammatory actions of ppar have been proposed (for reviews, see refs. 61,62). the first is that ppar stimulate the breakdown of inflammatory eicosanoids through induction of peroxisomal βoxidation. the second is that ppar might interfere with or antagonize the activation of other transcription factors, including nfκb. expression of pparα and -γ in liver and adipose tissue, respectively, is increased by feeding mice fish oil (63) . the effect of fish oil on ppar expression in inflammatory cells has not been reported. however, it is possible that n-3 pufa might act by increasing the level of these anti-inflammatory transcription factors in such cells. a number of other transcription factors are activated by inflammatory signals and play a role in the expression of inflammatory genes (for a review, see ref. 64 ). possibly n-3 pufa might affect the activation of these factors, but this has not been studied in detail. however, effects of n-3 pufa on signaling processes that lead to activation of various transcription factors have been reported. for example, incubation of murine macrophages with epa was found to decrease lpsinduced phosphorylation and activation of mitogen-activated protein kinase (65) . chronic inflammatory diseases. chronic inflammatory diseases are characterized by a dysregulated t-cell response that drives an ongoing immune response to normally benign, often host, antigens. there is a genetic predisposition to such diseases. the response has a strong inflammatory component, and inflammatory mediators are responsible for damage to host tissues and for the metabolic changes seen at the wholebody level. rheumatoid arthritis is an example of such a disease. it is characterized by a dysregulated t helper 1-type response that promotes the production of inflammatory cytokines, such as tnf-α, il-1β, il-6, and il-8 (66, 67) . high levels of tnf-α, il-1β, il-6, il-8, and granulocyte/macrophage-colony stimulating factor (gm-csf) are present in synovial biopsies from patients with rheumatoid arthritis (68) . furthermore, cultured synovial cells produce tnf-α, il-1β, il-6, il-8, and gm-csf without any additional stimulus (68), suggesting chronic stimulation. cox-2 expression is increased in the synovium of rheumatoid arthritis patients, and in the joint tissues in rat models of arthritis (69) . pge 2 , ltb 4 , 5-hete, and platelet activating factor are found in the synovial fluid of patients with active rheumatoid arthritis (70) . the efficacy of nonsteroidal anti-inflammatory drugs in rheumatoid arthritis indicates the importance of proinflammatory cox pathway products in the pathophysiology of the disease. increased expression of e-selection, vcam-1, and icam-1 is found in patients with arthritis, and blocking icam-1 or vcam-1 with antibodies reduces leukocyte infiltration into the synovium and synovial inflammation in animal models of the disease (for references, see ref. 71 ). the effects of fish oil on inflammatory eicosanoid and cytokine production and on adhesion molecule expression suggest that it might have a role in prevention and therapy of rheumatoid arthritis (and other chronic inflammatory diseases). certainly, dietary fish oil has been shown to have beneficial effects in animal models of arthritis. for example, dietary fish oil delayed the onset and reduced the incidence and severity of type ii collagen-induced arthritis in mice (72) . it was recently reported that both epa and dha suppress streptococcal cell wallinduced arthritis in rats, but that epa was more effective (73) . numerous (at least 14) randomized, placebo-controlled, double-blind studies of fish oil in rheumatoid arthritis have been reported. these trials were reviewed in some detail elsewhere (74, 75) and are summarized briefly here. the trials used between 1 and 7.1 g/d epa plus dha (average dose was 3.3 g/d) with a duration of 12-52 wk. various improvements in clinical outcomes were reported. these included reduced duration of morning stiffness, reduced number of tender or swollen joints, reduced joint pain, reduced time to fatigue, increased grip strength, and decreased use of nonsteroidal anti-inflammatory drugs. in an editorial commentary discussing the use of fish oil in rheumatoid arthritis, it was concluded that "the findings of benefit from fish oil in rheumatoid arthritis are robust," "that dietary fish oil supplements in rheumatoid arthritis have treatment efficacy," and that "dietary fish oil supplements should now be regarded as part of the standard therapy for rheumatoid arthritis" (76) . the efficacy of dietary fish oil has been examined in other chronic inflammatory diseases including ulcerative colitis, crohn's disease, lupus, and multiple sclerosis (reviewed in ref. 22) . although there are examples of trials reporting clinical improvement in each of these conditions, in general, these studies show little benefit (22) . this may be because the dose of n-3 pufa used was too low, the duration of the studies was too short, the studies were insufficiently powered to detect a significant effect, or a number of other reasons. atopic disease. there is currently considerable interest in the relative effects of n-3 and n-6 pufa in asthma (and other atopic diseases). the discussions center on the roles of various eicosanoids produced from aa in mediating allergic inflammation and in programming t lymphocytes to a phenotype that predisposes to such inflammation. aa-derived eicosanoids such as pgd 2 , ltc 4 , d 4 , and e 4 are produced by the cells that mediate pulmonary inflammation in asthma (mast cells) and are believed to be the major mediators of asthmatic bronchoconstriction. thus, provision of n-3 pufa to asthmatics might be beneficial because of the resulting decrease in production of 4-series lt (7, 20) and other aaderived mediators. however, the situation is complicated by the fact that different eicosanoids have different effects, some antagonizing others. for example, the observations that pge 2 inhibits 5-lox (11) and promotes the generation of lipoxins that act as inflammation "stop signals" (11) (12) (13) indicate that pge 2 could, in fact, be protective in active asthma. thus, interventions that aim to suppress pge 2 production could be counterproductive, at least in some asthmatics. nevertheless, numerous trials of fish oil in asthma and related atopic diseases have been performed (for reviews, see refs. 77, 78) . most of these studies reveal limited clinical effect, despite significant biochemical changes (e.g., reduced 4-series lt production), although some have shown some significant clinical improvements at least in some patient groups (78) . however, a study performed by broughton et al. (79) suggests that fish oil should be used cautiously in asthmatics. these researchers found that although n-3 pufa ingestion resulted in markedly improved lung function in >40% of adult asthmatic subjects, some patients did not respond favorably to the high n-3 pufa intake (79) . this study suggests that there are patients who respond positively to fish oil intervention and patients who are nonresponders. another area of current interest relates to the putative predisposing role of pge 2 toward atopic disease, particularly in childhood. there has been a rapid increase in the prevalence of childhood atopic disease in developed countries over the last 30 years (80, 81) , and this coincides with the period of increase in the intake of n-6 pufa. because pge 2 regulates t-lymphocyte differentiation, promoting the development of the t helper 2-type phenotype that underlies sensitization to environmental allergens (82) , it is suggested that the pattern of change in dietary fa intake over the last 40 yr is responsible for the increase in childhood atopic disease (83) (84) (85) . certainly there are biochemical measurements suggesting an inverse relationship between n-3 fa status and atopic disease. the proportions of epa, docosapentaenoic acid, and dha were higher in umbilical cord serum phospholipids from nonallergic compared with allergic mothers (86) . higher n-3 pufa in breast milk were associated with a decreased likelihood of atopy in the infants (87) . the proportions of dha and of total n-3 pufa were higher in the serum phospholipids from 12-to 15-yr-old nonatopic controls than in those from children with asthma and/or atopic dermatitis (88) . there is also epidemiologic evidence to support a protective role of long-chain n-3 pufa in atopic disease. data from the first and second national health and nutrition surveys in the united states found that dietary fish intake was positively associated with lung function (89) and protected against wheezing (90), respectively. australian schoolchildren who included oily fish in their diet had a much lower likelihood of having asthma than children who did not consume oily fish (91) . schoolchildren who went on to develop atopy had previously consumed more margarine and less butter (and thus more n-6 pufa) than those who did not develop atopy (92) . despite a biologically plausible mechanism and the supportive biochemical and epidemiologic data, the key to demonstrating a protective effect of increased long-chain n-3 pufa consumption toward atopic disease must come from well-designed, placebocontrolled intervention studies. it is now recognized that sensitization to allergens occurs early in life (93) ; thus, the characteristics of the maternal diet may be very important in determining predisposition to atopy, and studies addressing this question should be performed in pregnant women. several such studies are currently under way, and their findings are eagerly anticipated. systemic inflammatory response to surgery and injury. it is now recognized that a hyperinflammatory response, characterized by overproduction of tnf-α, il-1β, il-6, and il-8, is important in the progression of very ill patients toward sepsis, i.e., markedly elevated circulating concentrations of these mediators are seen in sepsis (see, for example, refs. 94,95); vervloet et al. (96) stated that "these mediators are largely, if not completely, responsible for the clinical signs and symptoms of the septic response to a bacterial infection." enhanced production of aaderived eicosanoids such as pge 2 is also associated with such a pathophysiology (97, 98) . the inflammatory effects of infection can be mimicked by administration of lps, which causes an elevation of circulating concentrations of inflammatory cytokines and an upregulation of adhesion molecule expression (for references, see ref. 43) . laboratory animals can be protected against bacterial-and lps-induced shock by neutralizing these cytokines or by blocking or gene deletion of vcam-1 or icam-1 (for references, see ref. 43 ). the ability of n-3 pufa to decrease production of inflammatory cytokines and eicosanoids and to decrease adhesion molecule expression suggests that fish oil might be a useful agent to aid the control of endotoxemia and the so-called systemic inflammatory response syndrome (sirs). fish oil feeding or infusions enhanced the survival of guinea pigs after lps challenge and decreased the accompanying metabolic perturbations in guinea pigs and rats (for references, see ref. 43) . mice fed fish oil and then injected with lps had lower plasma tnf-α, il-1β, and il-6 concentrations than mice fed safflower oil (29) , whereas fish oil-containing parenteral nutrition decreased serum tnf-α, il-6, and il-8 concentrations in burned rats (99, 100) . postsurgery patients administered parenteral fish oil after major abdominal surgery had lower serum concentrations of tnf-α and il-6 than those given a standard lipid mix (101) . in another study in postsurgical patients, parenteral fish oil decreased tnf-α production by lps-stimulated whole blood and decreased serum il-6 concentration compared with the control group that was given standard parenteral nutrition (102) . postoperative stay in the intensive care unit and in hospital tended to be shorter in the fish oil group (102) . these studies indicate the potential for significant modification of the inflammatory changes induced by major surgery by infusion of n-3 pufa in the form of fish oil (101, 102) . however, larger studies are required to evaluate the effects on complication rates, hospital stay, and mortality. numerous clinical trials (at least 20) have been performed in intensive care or surgical patients using enteral formulae containing n-3 pufa. the majority of these trials used formulae that also contained other nutrients proposed to be beneficial such as arginine and nucleotides. many of these trials report beneficial outcomes, including decreased numbers of infections and infectious or wound complications, decreased severity of infection, decreased need for mechanical ventilation, decreased progression to sirs, and decreased length of intensive care unit and/or total hospital stay (for a meta-analysis, see ref. 103) . a number of these studies also measured circulating inflammatory cytokine levels or ex vivo cytokine production. these studies reported lower plasma concentrations of inflammatory cytokines (especially il-6) in patients given n-3 pufa-containing enteral formula pre-or postsurgery than in those administered standard enteral nutrition (104) (105) (106) (107) . although this is in agreement with the effects of n-3 pufa reported in other settings, and could be used as evidence of their efficacy in the trauma and postsurgery settings, the complex nature of the formulae prevents such a clear interpretation. the effects could be due to any one of the specified nutrients (i.e., arginine, nucleotides, n-3 pufa) or to the combination of these nutrients. a trial performed in patients with moderate and severe acute respiratory distress syndrome used an enteral formula claimed to differ only in lipid source from the control (32% canola oil + 25% medium-chain tag + 20% borage oil + 20% fish oil + 3% soy lecithin vs. 97% corn oil + 3% soy lecithin) (108) . however, in addition to the difference in fa composition between the formulae, the n-3 pufa-rich formula contained more vitamin c and e than the control and contained β-carotene, taurine, and carnitine, whereas the control did not. the study included a number of patients with surgical trauma, sepsis, and pneumonia; all patients had respiratory failure and about one third had failure of at least one other organ system. patients were given ~7 g epa, 3 g dha, 6 g γ-linolenic acid, 1.1 g vitamin c, 400 iu vitamin e, and 6.6 mg β-carotene daily for up to 7 d. by day 4, the numbers of leukocytes and neutrophils in the alveolar fluid had significantly declined in the treatment group and were lower than in the control group. furthermore, arterial oxygenation and gas exchange were improved in the treatment group. patients in the treatment group had decreased requirement for supplemental oxygen, reduced time on ventilation support, and shorter length of intensive care unit stay. total length of hospital stay also tended to be shorter. fewer patients in the treatment group developed new organ failure. mortality was 19% in the control group and 12% in the treatment group, but this was not a significant difference. nevertheless, this study suggests the efficacy of n-3 pufa (in combination with γ-linolenic acid, medium-chain tag, antioxidant vitamins, taurine, and carnitine) in this group of patients. inflammation is a component of a range of acute and chronic human diseases, and is characterized by the production of inflammatory cytokines, aa-derived eicosanoids, other inflammatory mediators (e.g., platelet-activating factor), and adhesion molecules. the n-3 pufa decrease the production of inflammatory mediators and the expression of adhesion molecules. they act both directly (e.g., by replacing aa as an eicosanoid substrate and inhibiting aa metabolism) and indirectly (e.g., by altering the expression of inflammatory genes through effects on transcription factor activation). thus, n-3 pufa are potentially potent anti-inflammatory agents. as such, they may be of therapeutic use in a variety of acute and chronic inflammatory settings. evidence of their clinical efficacy is stronger in some settings (e.g., in rheumatoid arthritis) than others (e.g., in asthma, in trauma patients). polyunsaturated fatty acids, inflammation and immunity the modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function dietary fatty acids and membrane function dietary lipids and the inflammatory response modulation of immune function by dietary fatty acids the effects of short-and long-term supplementation with fish oil on the incorporation of n-3 polyunsaturated fatty acids into cells of the immune system in healthy volunteers dietary ω-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils leukotrienes and other products of the 5-lipoxygenase pathn-3 fa and inflammation way: biochemistry and relation to pathobiology in human diseases mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes dietary polyunsaturated fatty acids and eicosanoids: potential effects on the modulation of inflammatory and immune cells: an overview lipid mediator class switching during acute inflammation: signals in resolution endogenous anti-inflammatory mediators from arachidonate in human neutrophils lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis eicosapentaenoic acid inhibits prostaglandin d 2 generation by inhibiting cyclo-oxygenase-2 in cultured human mast cells n-3 fatty acids specifically modulate catabolic factors involved in articular cartilage degradation pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids the effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells immunologic effects of national cholesterol education panel step-2 diets with and without fish the effect on human tumor necrosis factor α and interleukin 1β production of diets enriched in n-3 fatty acids from vegetable oil or fish oil effects of dietary enrichment with eicosapentaenoic acid and docosahexaenoic acid on in vitro neutrophil and monocyte leukotriene generation and neutrophil function anti-inflammatory lipid signals generated from dietary n-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for nsaid and n-3 pufa therapeutic actions n-3 polyunsaturated fatty acids and cytokine production in health and disease the omega-3 fatty acid docosahexaenoate reduces cytokine-induced expression of proatherogenic and proinflammatory proteins in human endothelial cells docosahexaenoic and eicosapentaenoic acids inhibit in vitro human endothelial cell production of interleukin-6 blockade by polyunsaturated n-3 fatty acids of endotoxin-induced monocytic tissue factor activation is mediated by the depressed receptor expression in thp-1 cells fatty acid uptake and kupffer cell function: fish oil alters eicosanoid and monokine production to endotoxin stimulation dietary n-3 polyunsaturated fatty acids prevent the development of atherosclerotic lesions in mice: modulation of macrophage secretory activities effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women cytokine secretion and eicosanoid production in the peripheral blood mononuclear cells of ms patients undergoing dietary supplementation with n-3 polyunsaturated fatty acids uptake of saturated and unsaturated fatty acids into macrophage lipids and their effect upon macrophage adhesion and phagocytosis dietary fish oil added to a hyperlipidemic diet for swine results in reduction in the excessive number of monocytes attached to the arterial endothelium control of endothelial leukocyte adhesion molecules by fatty acids n-3) polyunsaturated fatty acids modulate the expression of functionally associated molecules on human monocytes in vitro dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages fish oil supplementation inhibits the expression of major histocompatibility complex class ii molecules and adhesion molecules on human monocytes influence of age and dietary fish oil on plasma soluble adhesion molecule concentrations decreased proinflammatory cytokines and increased antioxidant enzyme gene expression by ω-3 lipids in murine lupus nephritis dietary marine lipids suppress continuous expression of interleukin-1β gene expression nuclear factor-κb: a pivotal role in systemic inflammatory response syndrome and new target for therapy new insights into the role of nuclear factor-κb, a ubiquitous transcription factor in the initiation of diseases dietary modification of inflammation with lipids phosphorylation meets ubiquination: the control of nf-κb activity the iκb kinase (iκk) and nf-κb: key elements of proinflammatory signalling fish oil suppressed cytokines and nuclear factor κb induced by murine aids virus infection eicosapentaenoic acid decreases lipopolysaccharide-stimulated tumor necrosis factor-α expression by inhibiting nuclear factor κb activation the anti-catabolic effects of n-3 fatty acids the peroxisome proliferator activated receptors (ppars) and their effects on lipid metabolism and adipocyte differentiation activation of peroxisome-activated receptors α and γ induces apoptosis of human monocyte-derived macrophages the peroxisome proliferator-activated receptorγ is a negative regulator of macrophage activation a prostaglandin j 2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation the pparγ-leukotriene b 4 pathway to inflammation control ppar-γ agonists inhibit production of monocyte inflammatory cytokines peroxisome proliferator-activated receptor α activation modulates cellular redox status, represses nuclear factor κb signalling, and reduces inflammatory cytokine production in aging peroxisome proliferator-activated receptor activators target human endothelial cells to inhibit leukocyte-endothelial cell interaction, arterioscler pparα activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells peroxisome proliferator-receptor activators inhibit lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal rat cardiac myocytes inhibition of the transcription factors ap-1 and nf-κb in cd4 t cells by peroxisome proliferator-activated receptor γ ligands ppar α and gr differentially down-regulate the expression of nuclear factor-κb-responsive genes in vascular endothelial cells peroxisome proliferator-activated receptors (ppars): nuclear receptors at the crossroads between lipid metabolism and inflammation peroxisome proliferator-activated receptors in inflammation control the effect of dietary fatty acids on the expression of genes involved in lipid handling receptor-mediated signaling pathways: potential targets of modulation by dietary fatty acids fish oil modulates macrophage p44/42 mitogen-activated protein kinase activity induced by lipopolysaccharide targeting of cells involved in the pathogenesis of rheumatoid arthritis the role of cytokines in the pathogenesis of rheumatoid arthritis role of cytokines in rheumatoid arthritis in vivo cyclooxygenase expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and streptococcal cell wall arthritis eicosanoids in rheumatoid arthritis adhesion molecules in health and disease dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility in mice the eicosapentaenoic to docosahexaenoic acid ratio of diets affects the pathogenesis of arthritis in lew/ssn rats n-3 fatty acids and rheumatoid arthritis, in food and nutritional supplements in health and disease n-3 fa and inflammation polyunsaturated fatty acids and rheumatoid arthritis fish oil and rheumatoid arthritis: anti-inflammatory and collateral health benefits omega-3 fatty-acids in respiratory diseases-a review fatty acids and atopic disease fatty acid ingestion are related to 5-series leukotriene production has the prevalence of asthma increased in children? evidence from the national survey of health and growth trends in prevalence of atopic diseases and allergic sensitization in children in eastern germany the role of lymphocytes in allergic disease increased consumption of polyunsaturated oils may be a cause of increased prevalence of childhood asthma dietary fat and asthma: is there a connection? dietary fatty acids and allergy phospholipid fatty acids in cord blood: family history and development of allergy atopic sensitization during the first year of life in relation to long-chain polyunsaturated fatty acid levels in human milk polyunsaturated fatty acids in school children in relation to allergy and serum ige levels the relationship of dietary fish intake to level of pulmonary function in the first national health and nutrition survey dietary factors and their relation to respiratory symptoms: the second national health and nutrition survey consumption of oily fish and childhood asthma risk diet, serum fatty acids, and atopic diseases in childhood fetal peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic stimuli during gestation circulating interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever predictive value of nuclear factor κb activity and plasma cytokine levels in patients with sepsis derangements of coagulation and fibrinolysis in critically ill patients with sepsis and septic shock the role of prostaglandin e 2 in immune suppression following injury ibuprofen restores cellular immunity and decreases susceptibility to sepsis following hemorrhage n-3 versus n-6 polyunsaturated fatty acids in critical illness effects of intravenous omega-3 and omega-6 fat emulsion on cytokine production and delayed type hypersensitivity in burned rats receiving total parenteral nutrition influence of a total parenteral nutrition enriched with ω-3 fatty acids on leukotriene synthesis of peripheral leukocytes and systemic cytokine levels in patients with major surgery immunomodulation by perioperative administration of n-3 fatty acids should immunonutrition become routine in critically ill patients? a systematic review of the evidence immune and nutritional effects of early enteral nutrition after major abdominal operations perioperative immunonutrition in patients undergoing cancer surgery a prospective, randomized clinical trial on perioperative feeding with an arginine-, omega-3 fatty acid-, and rna-enriched enteral diet: effect on host response and nutritional status effect of preoperative oral immune-enhancing nutritional supplement on patients at risk of infection after cardiac surgery: a randomised placebo-controlled trial and the enteral nutrition in ards study group (1999) effect of enteral feeding with eicosapentaenoic acid, γ-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome key: cord-0021984-j1rnm9eo authors: hedar, ahmed m.; stradner, martin h.; roessler, andreas; goswami, nandu title: autoimmune rheumatic diseases and vascular function: the concept of autoimmune atherosclerosis date: 2021-09-27 journal: j clin med doi: 10.3390/jcm10194427 sha: b54740f5dc200baf224baa0bff4152d45d1d4751 doc_id: 21984 cord_uid: j1rnm9eo autoimmune rheumatic diseases (airds) with unknown etiology are increasing in incidence and prevalence. up to 5% of the population is affected. airds include rheumatoid arthritis, system lupus erythematosus, systemic sclerosis, and sjögren’s syndrome. in patients with autoimmune diseases, the immune system attacks structures of its own body, leading to widespread tissue and organ damage, which, in turn, is associated with increased morbidity and mortality. one third of the mortality associated with autoimmune diseases is due to cardiovascular diseases. atherosclerosis is considered the main underlying cause of cardiovascular diseases. currently, because of finding macrophages and lymphocytes at the atheroma, atherosclerosis is considered a chronic immune-inflammatory disease. in active inflammation, the liberation of inflammatory mediators such as tumor necrotic factor alpha (tnfa), interleukine-6 (il-6), il-1 and other factors like t and b cells, play a major role in the atheroma formation. in addition, antioxidized, low-density lipoprotein (ldl) antibodies, antinuclear antibodies (ana), and rheumatoid factor (rf) are higher in the atherosclerotic patients. traditional risk factors like gender, age, hypercholesterolemia, smoking, diabetes mellitus, and hypertension, however, do not alone explain the risk of atherosclerosis present in autoimmune diseases. this review examines the role of chronic inflammation in the etiology—and progression—of atherosclerosis in autoimmune rheumatic diseases. in addition, discussed here in detail are the possible effects of autoimmune rheumatic diseases that can affect vascular function. we present here the current findings from studies that assessed vascular function changes using state-of-the-art techniques and innovative endothelial function biomarkers. until now, no evidence about the true mechanism of the atherosclerosis process in autoimmune rheumatic diseases (airds) is known [1] . current research findings are often contradictory. whether traditional risk factor like age, gender, smoking, or hypertension contribute solely to the atherosclerosis, or whether it is a consequence of a nontraditional states like chronic inflammation or changes in cytokines, antibodies that accompany atherosclerosis, or a combination of both aspects is not yet clear and is debated [1] . moreover, it is currently not known which factors play a major role in process of development of premature atherosclerosis in autoimmune rheumatic disease patients. atherosclerosis can be classified into primary simple atherosclerosis, which occurs with age, and secondary autoimmune atherosclerosis, which was also coined as accelerated atherosclerosis [2] . atherosclerosis is the main cause of cardiovascular diseases in autoimmune rheumatic diseases. this review examines the role of chronic inflammation in the etiology-and progression-of atherosclerosis in autoimmune rheumatic diseases. in addition, discussed here in detail are the possible effects autoimmune rheumatic diseases can have on vascular function, as well as how their effects on vascular function could potentially be assessed using state-of-the-art techniques and innovative biomarkers. the current literature on the subject of "autoimmune disease and vascular function" was systematically reviewed. both primary and secondary sources of literature on the topic were examined. pubmed and web of science were used as search engines to access the relevant literature. the initial search criteria included the following keywords: autoimmune atherosclerosis, connective tissue autoimmune diseases and atherosclerosis, premature atherosclerosis in autoimmune diseases, vascular dysfunction, vascular dysfunction in autoimmune diseases, rheumatoid arthritis and cardiovascular diseases, rheumatoid arthritis and atherosclerosis, systemic lupus erythematosus and cardiovascular diseases, lupus and atherosclerosis and premature atherosclerosis, sjögren's syndrome and cardiovascular diseases, sjögren's syndrome and atherosclerosis, mortality and morbidity in autoimmune diseases, mechanism of atherosclerosis in autoimmune diseases, measurement of endothelial function, endothelial dysfunction, vasculitis, autoimmune vasculitis, atherosclerosis as immune diseases, new theory of atherosclerosis, blood marker of endothelial dysfunction, oxidized ldl anti oxidized ldl antibodies, amyloidosis in autoimmune diseases, traditional risk factor of atherosclerosis, nontraditional risk factor in atherosclerosis, cryoglobulinemia and cryoglobulinemic vasculitis, cryoglobulin and autoimmune diseases. the search resulted in 16,000 articles from pubmed and web of science. after exclusion of animal studies and duplicate studies and studies in languages other than english language, and after restricting the search to only the last 5 years (2016-2020), 500 relevant papers were identified. next, articles that did not fulfil our research concern were removed. this resulted in 180 papers. finally, the reference lists of the selected papers were examined for any relevant papers ( figure 1 ). figure 2 below provides an overview of the traditional and nontraditional factors that play important roles in the development of atherosclerosis and/or hypertension in auto-immune rheumatic diseases. these are discussed in detail below. cardiovascular diseases are considered as one of the most common causes of morbidity and mortality in autoimmune rheumatic diseases [19] . recent studies, however, show contradictory results, and there is currently a debate about the role of traditional and nontraditional risks in the development of atherosclerosis processes, which, in turn, are believed to play important role in the development of cardiovascular diseases in autoimmune rheumatic diseases [12, 20] . traditional risk factors include age, gender, smoking, obesity, dyslipidemia, hypertension, diabetes, and a sedentary lifestyle [12, 21] . interestingly, there is some controversy about body weight in relation to disease activity, especially in rheumatoid arthritis. some studies named it "inflammatory cachexia" [22] , in active inflammation, the liberation of inflammatory mediators, such as tnfa, il6, il1, and others that affect the body metabolism, leads to a state of hypercatabolism. this hypercatabolism is in addition to the anorexia associated with acute inflammation in these patients, thus leading to decreases in body weight (mostly in muscle mass) rather than obesity [23, 24] . however, most studies show an increased prevalence of obesity in ra patients [25] [26] [27] . other studies reported that even though obesity is a contributing risk factor in development of atherosclerosis process, obesity has an otherwise good prognostic and protective effect in cardiovascular mortality in comparison to that of low body weight in rheumatoid patients. for instance, a bmi more than 30 was reported to have a better prognostic outcome as compared to that of a bmi of less than 20 [28] . similarly, "lipid paradox" was also reported in patients with autoimmune rheumatic diseases. "lipid paradox" is a term applied for lipid profile in airds, and perhaps even in all inflammatory conditions, where, during the active inflammatory state, there is a decrease in all lipid profiles: total cholesterol, ldl, and hdl cholesterol (that is, there is a state of hypercatabolism). however, once the active inflammatory subsides (spontaneously or with drugs), there is a state of normal or increased serum cholesterol levels [29] . smoking is a well-known independent risk factor for atherosclerosis [30] . in addition, most studies show that it increases the risk of cardiovascular diseases in ra patients [31] , especially if smoking is accompanied by the presence of rheumatoid factor (rf) and/or anticitrullinated peptide antibody (acpa) [32] . hyperhomocysteinemia, known as a traditional risk factor in cardiovascular diseases [33] , is also present in airds. it is considered one of the independent risk factors in the severity of the autoimmune diseases [34] . although there is debate about obesity and cachexia in airds, all studies suggest that metabolic syndrome is associated with disease severity and increased atherosclerosis risk in autoimmune diseases [29, 35] . hyperuricemia is considered a risk factor in cardiovascular diseases, and also in airds [36] . finally, insulin resistance was also suggested to play a role in autoimmune rheumatic diseases [29] . nontraditional risk factors for atherosclerosis refer to those risk factors that are not related to the usual framingham cardiovascular risk factors, such as age, gender, etc., and are specific to certain diseases. these classically include cells, cytokines, chemokines, antibodies, and drugs (see figure 2 ). endothelial cells have a major role in the state of endothelial dysfunction. during inflammation, there is a release of many cytokines, which influence the action of nitric oxide and lead to vasoconstriction, as well as increase the risk of thrombosis and atheroma formation [37] . also, the cells of the immune system especially t lymphocytes and b lymphocytes are implicated in this process. for instance, t cells are involved in the processes of atherosclerosis, and are a major contributor to airds [38] . t helper cells, whether t helper 1 [39] or t helper 17 (th17) cells, have a stimulatory effect on atherosclerosis formation [14] . on the other hand, regulatory t cells (treg) have a protective function on the atherosclerosis process [40] . unsurprisingly, the ratio of helper th17 cells to regulator t reg cells is important in atherosclerosis formation and in controlling the disease during therapeutical interventions (e.g., with medications) [39] . with regards to immune mediators, tnfa is one of the most important mediators in autoimmune atherosclerosis and autoimmune diseases. tnfa is secreted from macrophages, endothelial cells, and t lymphocytes. as tnfa initiates or potentiates acute inflammatory states, anti-tnfa could play a major role in the control of the inflammatory conditions and disease control [41] . il6 is also considered an important cytokine in inflammatory conditions. il6 is found in ra patients with premature atherosclerosis [42] . furthermore, il1 was reported to have a role in the development of atherosclerosis in airds [43] . antibodies are basic contributors in the pathogenesis of autoimmune diseases. many antibodies were reported to be implicated in the development of autoimmune rheumatic diseases and autoimmune atherosclerosis [44] . rheumatoid factor (rf) is an antibody associated with rheumatoid arthritis, and its levels correlate with arthritis activity and prognosis; its presence is also associated with increase in cardiovascular risk [45, 46] . also, anticitrullinated protein antibody (acpa) is associated with aggressive rheumatoid diseases; their presence is associated with worsening of the prognosis in ra and greater cardiovascular manifestations [47] . antinuclear antibodies (ana) are secreted in many airds, and their levels are also related to diseases activity and formation of atherosclerotic plaques [48] . anti-dna antibody, which is specific to systemic lupus patients, also relates to disease activity [49] . anti-ldl antibodies are antibodies formed against oxidized ldl, and its presence in the blood of the patients indicates an increased incidence of vascular dysfunction and cardiovascular deconditioning [50] . however, other studies indicate that antioxidized ldl confers protection against cardiovascular risk [51, 52] . interestingly, studies on the effect of anti-ldl antibodies showed that igm is protective against atherosclerosis, while igg has harmful effects [50] . the presence of antiphospholipid antibodies (e.g., in antiphospholipid antibody syndrome or its presence as secondary antiphospholipid in other connective tissue diseases) indicates a major risk for development of vascular thrombosis and cardiovascular diseases [53] . cryoglobulin, immunoglobulins that precipitate in vitro at lower temperatures, was shown to be associated with many autoimmune diseases, especially sjögren's syndrome, systemic lupus, and hepatitis c viral infection [54] . its presence in the blood does not indicate the presence of disease, but it can cause many vascular symptoms due to its precipitation and obstruction of blood vessels of multiple organs ranging from critical vascular ischemia to autoimmune vasculitis [55] , which aggravate endothelial dysfunction [56] . although anti-ro anti-la antibodies are associated with congenital heart block in neonatal lupus disease [57] , the presence of anti-ro antibodies in adults may be associated with complete heart block in primary sjögren's syndrome patients [58, 59] . normal cardiac output heart failure is the type of heart failure that is seen in rheumatoid arthritis patients [60] . previously, researchers attributed heart failure in ra to ischemia of coronary arteries, but subsequently, heart failure in these patients was seen with preserved systolic function, which was not explained by coronary ischemia [60, 61] . others reported that heart failure may arise due to deposition of protein fibril between cardiac muscle; this protein material deposit in the heart occurs due to a chronic inflammatory state [62] . this refers to the possibility of amyloid deposits in cardiac muscle (secondary amyloidosis, aa). amyloidosis is classified as primary amyloidosis (al), which is caused by hematological diseases such as multiple myeloma and secondary amyloidosis, which is a result of chronic inflammation a state and deposition of serum amyloid a in a chronic inflammatory situation like immune diseases or chronic infection [63] . primary amyloidosis affects mainly the heart but secondary amyloidosis, although it affects mainly the kidney leading to proteinuria and nephrotic syndrome, can affect any organ in the body including the heart [64, 65] . as secondary amyloidosis results from a chronic inflammatory state, and most airds persist for more than 10 years, we speculate that cardiac amyloidosis could have led to heart failure in airds. this could lead to increased mortality in these patients. the prevalence of hypertension in autoimmune rheumatic diseases is high. indeed, there are several studies that showed that hypertension is very common in rheumatoid arthritis patients [66] . there is a wide range of prevalence of hypertension in these studies [67] [68] [69] [70] [71] : with some studies reporting hypertension in rheumatoid patients between 3-70% [72] . however, other studies reported no differences in blood pressure between patients and controls [73] [74] [75] [76] [77] [78] . therefore, there is still a debate about the percentage of hypertension in autoimmune diseases [3, 70, 71] . however, some study-related hypertension in rheumatoid arthritis was seen when age and disease duration of the patients are considered [79] . as hypertension and autoimmune rheumatic diseases can be influenced by genetic and environmental factors, it is often difficult to identify which of these started first. for instance, hypertension leads to mechanical injury of the endothelium, which then starts the sequence of endothelial dysfunction, loss of endothelial vasodilation substances, arterial stiffness, and atherosclerosis, which is most probably helped by the state of chronic inflammation in autoimmune patients. on the other hand, in chronic autoimmune rheumatic diseases, endothelial injury arises due to chronic inflammation, antigen-antibody reaction, complement fixation, and/or disturbance in the normal equilibrium of cytokines and chemical mediators, which can lead to vasoconstriction (especially in renal vasculature), and increase vascular resistance and subsequent hypertension [80] . roman and colleagues illustrated that there is no difference regarding hypertension prevalence between the control group and rheumatoid group [77] . in general, hypertension is the single most important cardiovascular risk factor that can accelerate the development of premature atherosclerosis, and consequently, cardiovascular diseases [21] . overall, age and hypertension are the main factors responsible for arterial stiffness; both are considered as the most important factors responsible for atherosclerosis and development of subsequent cardiovascular diseases [80] . the combination of autoimmune rheumatic diseases and hypertension is more dangerous than autoimmune rheumatic diseases alone [81] . for example, the treatment of autoimmune diseases with drugs, especially corticosteroids, could potentially influence hypertension control in these patients [81] . all studies from systemic lupus erythematosus patients show that the prevalence of hypertension is very high in sle, especially when the kidneys are also affected. renal diseases (both glomerulonephritis or tubular diseases) lead to elevation of blood pressure via salt-water retention and/or ischemia to the glomerulus and consequent activation of the renin-angiotensin system [82] [83] [84] [85] [86] . hypertension is now considered an immune disease, as data from experimental animals show that the deletion of b cells by anti-cd 20 influences blood pressure control [87, 88] . also, the change in the ratio of helper t cells 1 and 17 and t regulator cells modulates the control of blood pressure [89, 90] . the problem of hypertension in autoimmune diseases is that the true mechanism of hypertension development is not known; no guidelines for hypertension control in autoimmune diseases are available, thus making the control of hypertension in autoimmune diseases very difficult [81] . furthermore, the effect of corticosteroid therapy on hypertension is still unclear as it produces salt and water retention, thus leading to increases in blood pressure on the one hand, but as its anti-inflammatory activity decreases, cytokine production consequently decreases blood pressure on the other hand [72, 91] . most of the antihypertensive medications show little effects on control of the blood pressure in autoimmune rheumatic diseases. however, angiotensin converting enzyme inhibitors (aceis) and selective angiotensin receptor blockers (arbs) are effective in blood pressure regulation in these patients as they have both immunomodulatory and antihypertensive effects [81, 92] . cardiovascular signs in rheumatoid arthritis-due to the accompanying accelerated atherosclerosis-present earlier in the course of the disease. these signs may sometimes appear before even a diagnosis is established [93] . also, the number of rheumatoid arthritis patients with cardiovascular manifestations can double within one-year of follow-up [94] . common classical ("traditional") risk factors related to cardiovascular disease in ra include male sex, age, hypertension, hypercholesterolemia, smoking, and obesity. however, the increased incidence of vascular dysfunction, premature atherosclerosis, and cardiovascular diseases in rheumatoid arthritis is not fully explained by these traditional factors [95] . overall, up to 70% of cases of ra can be attributed to classical risk factors, but the remaining 30% of ra patients have no known relevant risk factors associated with development of cardiovascular diseases [96, 97] . it is possible that a combination of nonclassical risk factors and classical risk factors predispose ra patients towards development of cardiovascular diseases. or perhaps there remains another risk factor that is not yet identified [98] . ischemic heart diseases-arising due to atherosclerosis and narrowing of coronary vesselsare considered common presentations, as well as leading cause of death, in rheumatoid arthritis patients. the risk of myocardial infarction in rheumatoid patients is similar to that in diabetic patients [99] . humphreys and colleagues (2014) suggested that, despite current advances in diagnosis and management of rheumatoid arthritis, including biological therapy, the mortality rate did not fall within the last 20 years [100] . on the other hand, myasoedova and colleagues (2017) reported that in the last 20 years the mortality of rheumatoid arthritis decreased when compared to that of the normal population. they suggested that this decrease in mortality possibly arose due to the effects of modulatory drugs (e.g., traditional immunomodulatory or biological therapy) [101] . the average life expectancy of patients with rheumatoid arthritis can decrease by up to 10 years when compared to that of the normal population [102] . in fact, cardiovascular disease is considered a major cause of death in rheumatoid arthritis patients worldwide [103] : one-third or more of mortality in ra is due to cardiovascular disease. there is also a 3-fold mortality rate in ra patients as compared to that of the normal population [104] . as compared to that of the normal population, there is a doubling of sudden cardiac death in rheumatoid arthritis [48] . senescent cd4 + cd28 − t helper cells play a role in the pathogenesis of atherosclerosis in rheumatoid patients. the patients who increased amount of cd4 + cd28 − t helper cells have higher probability of developing increased intima-media thickness [44] . also, the level of rf and acpp are correlated with cardiovascular risk [45, 46] . all studies in which the effect of rheumatoid arthritis on flow-mediated dilatation was investigated show impairments in brachial artery reactive hyperemia [105, 106] . in addition, increases in arterial stiffness and carotid intima-media thickness were also seen thus suggesting premature atherosclerosis [107] [108] [109] [110] [111] in these patients. many of the biological drugs used in ra antagonize the effect of inflammatory cytokines like tnf alpha or il-6. these antagonists of the inflammatory cascade improve flow-mediated dilation, and subsequently, decrease atherosclerosis and cardiovascular events [111] . there seems to be no correlation between macrovascular and microvascular endothelial function in rheumatoid arthritis (that is, they appear to be independent of each other) [112] . in sle, there is a loss of immune tolerance and spontaneous activation of the immune system with the production of the autoantibody against nuclear proteins, termed antinuclear antibodies (ana). deposition of antigen-antibody immune complexes in blood vessels leads to organ injury [113] . sle affects women predominantly, with a proportion of female-to-male 12:1 during the childbearing period (this ratio decreases as females age). the disease can start at any age of life, but early diagnosis is associated with poor prognosis. in organs such as the heart, brain, and kidney, failure is associated with high mortality. vasculitis, a common presentation in lupus patients, arises when direct antibodies against endothelial cells as well antigen-antibodies complex become deposited in blood vessels [114, 115] ; it could also arise due to vasculitis in situ. vasculitis aggravates the state of endothelial dysfunction with the rapid development of premature atherosclerosis [12, 44] . besides the common traditional risk factors associated with atherosclerosis, other associated factors related to lupus disease itself appear to be more important towards the aggravation of atherosclerosis [20, 116] . it is related to chronic inflammation and immune complex deposition as well as the disease or therapy related complications. zhu and his colleagues found that the serum level of il-10 in sle patients with atherosclerosis was less than in that of the sle patients without atherosclerosis. also, they found elevated levels of serum il-6 and il-17 in the atherosclerotic group. in addition, they reported that the amount of regulatory t cells (treg) in atherosclerotic sle was lower than those found in sle patients without atherosclerosis. on the other hand, the number of t helper 17 cells (th17) in atherosclerotic sle group was higher as compared to that of patients without atherosclerosis [40] . drugs used in the treatment of systemic lupus erythematosus and other autoimmune diseases such as corticosteroids in high doses could lead to hypertension and diabetes mellitus [117] . on the other hand, corticosteroids in low doses (acting as antiinflammatory agents) can prevent atherosclerosis [118] . other immunosuppressive drugs such as hydroxyl-chloroquine, which may be protective on vessels as potent immunomodulatory agents, and drugs used like nonsteroidal anti-inflammatory drugs, especially anti cox-2 used as analgesics, could have side effects on cardiac muscle functions. coronary arterial disease is a major cardiovascular risk in sle [119] . women aged 35-44 years are more likely to have a high risk of developing myocardial infarction and acute coronary syndrome, with an increase in mortality, as compared to that of women of a similar age [11, 20] . in general, the risk of developing myocardial infarction in sle is doubled as compared to the normal population [119] . interestingly, in women aged 40-49 having sle, the risk of myocardial infarction increases by 8-times during a 7-year follow-up [115, 120] . females with sle below age 45 years have an increased incidence of cardiovascular disease as compared to that of same-age women. these diseases include hypertension, especially with involvement of the kidneys [121, 122] , pulmonary hypertension, myocarditis, and stroke [123] . cardiovascular disease risk in lupus patients is two-fold as compared to that of the normal population [124] . classically, there are two peaks in sle mortality, with the first peak appearing in the first 3 years of the disease diagnosis. this peak is due to activity of the disease, the complication of immunosuppressant drugs decreasing immunity, increased infections, and kidney involvement. the second peak of mortality ends with death, occurring after a long period lasting from 4-20 years following diagnosis and treatment, resulting mainly from cardiovascular diseases [20, 116] . aviña-zubieta and his colleagues (2017) reported that the mortality from cardiovascular disease and myocardial infarction in the first year of diagnosis is possible [120] . fmd is impaired in lupus erythematosus patients; interestingly, the impairment is mostly found in older age patients with hypertension, diabetes, and renal impairments [125, 126] . arterial stiffness is increased in lupus patients with more relation to age, hypertension, and glucocorticoid use [127] [128] [129] . carotid plaque is 21% in patients with lupus under 35 years, and the percentage increased and reached 100% for women over age 65 years [20, 130] , which may be related to age or disease activity or both. pss is an autoimmune rheumatic disease that affects mostly women above 40 years. it is characterized by lymphocytic infiltration, chronic inflammation, and destruction of the salivary and lacrimal glands, leading to xerostomia and xerophthalmia, respectively [131] . pss can also affect other extra glandular organs, such as blood vessels, thyroid, joint, kidney, and lung [132] . although studies related to the investigation of cardiovascular risk with pss are limited, pss is associated with some degree of vascular dysfunction, rigidness of blood vessels, and arterial stiffness that can lead to subclinical atherosclerosis. however, these cardiovascular effects of pss cannot be explained by traditional risk factors alone [133] . demirci et al., (2016) suggest that the increase in vascular stiffness in pss may not be related to the disease per se but rather to the usage of corticosteroids, accompanying hypertension, and/or abnormal lipid profile, which may accompany the disease [134] . chronic inflammation, cytokine production, and antibodies present in pss patients could all contribute towards vascular dysfunction [135] . antioxidized ldl antibodies, which are atheroprotective in nature, are low in pss. this finding in pss patients is in contrast to what is seen in sle patients, indicating that the antibodies have a role in atherosclerosis formation [51] . for several years, lymphoma was considered the leading cause of mortality and morbidity in pss patients, but currently, cardiovascular diseases are identified as the major causes for mortality in pss patients [136] . overall, pss appears to be associated with some degree of vascular dysfunction, rigidness of blood vessels, and arterial stiffness that can lead to subclinical atherosclerosis [133, 134] . thus, it is expected that drugs that interfere with inflammatory processes can reduce inflammation and subsequently reduce disease activity and the development of atherosclerosis [111] . table 1 below provides an outline of different studies that examined the effects of autoimmune rheumatic diseases on vascular function and assessed the risk of accompanying atherosclerosis and the development of cardiovascular diseases. arterial stiffness in pss patients may be due to the associated hypertension, steroidal usage, and hyperlipidemia in these patients, and may not be due to the disease itself dysfunction of the endothelium initiates vascular injury and commences the process of atherosclerosis. the following are the physiological effects of nitric oxide (no): it keeps the tone of vessels, ensures laminar blood flow, prevents leucocyte migration and adhesion, plays important role in the maintenance of homeostasis, and prevents platelet aggregation, and, consequently, prevents atherosclerosis [137] [138] [139] . asymmetric dimethylarginine (adma), an analogue of l-arginine, is a naturally occurring product of metabolism found in human circulation. adma is known to antagonize the action of nitric oxide on blood vessels via inhibition of the enzyme nitric oxide synthase. hence, adma contributes towards dysfunction of endothelium, and consecutively, vessel diseases [140, 141] . current literature indicates that endothelial dysfunction is an early sign of atherosclerosis and all cardiovascular events and future diseases [142, 143] . defects in nitric oxide synthesis, release or its function are often associated with the commencement of endothelial dysfunction and atherosclerosis. nitric oxide is synthesized in the endothelial cell layer by enzyme nitric oxide synthase (nos) from amino acid arginine. adma is a competitive inhibitor of nos that leads to a decrease in nitric oxide formation [144, 145] . atherosclerosis was previously considered a degenerative disease and was always associated with the aging process. however, new research has shown that atherosclerosis is not a disease of only older persons and is also not an inevitable disease. there are immune and inflammatory factors associated with its pathogenesis and lipoprotein metabolism, which play an important role in atheroma formation through activation of the immune system [16, 146] and participation of oxidized ldl and antioxidized ldl [147] [148] [149] [150] [151] . the innate immune mechanisms were shared in the initiation of vascular dysfunction, hypertrophy of smooth muscle cells layer, atheroma formation, and arterial narrowing through the production of cytokines and inflammatory mediators [152] . also, the adaptive immune system through b and t cell lymphocytes [16, 152] , whether t helper (t4) or t cytotoxic (t8) [153] , are found in plaque section with antibodies [154] . atherosclerosis consists of fatty degeneration, which is responsible for arthrosis with plaque formation, and vessel stiffening, which refers to sclerosis. plaque is formed of the precipitation of lipoprotein, macrophage (foam cells), antibodies, and cytokines within the vessel wall [155] . all these factors are responsible for the gradual thickening of the intima and media with stiffness of the vessel wall, resulting in rigidity and narrower vessels with turbulence blood flow, rupture of the plaque, and cardiovascular complications. figure 3 below provides an overview of the possible underlying mechanisms of immune-atherosclerosis. the measurements of endothelial dysfunction (vascular function) are important, as they are considered early predictors of cardiovascular risk, future morbidity, and mortality [143] . mortality can be increased due to cardiovascular diseases like myocardial infarction and cerebrovascular stroke [143] . assessment of vascular function can be done by showing the arteries themselves directly, which means we measure its direct function or can be indirect with measuring blood marker, which denotes their function. both atherosis and sclerosis processes can be measured with noninvasive ultrasound devices. in atherosclerosis in the arteries, for example, carotid artery can be measured with ultrasound, and plaque and thickness of intima-media assessed [156] . arterial plaque and intima-media reflect the anatomical structure, while stiffness reflects the function of the arteries. all these are subclinical detectors for cardiovascular disease and a good way to predict future cardiovascular events [157, 158] . there are several techniques that can be used to assess vascular function and endothelial health [159] . figure 4 below provides an overview of these selected but important measurements of vascular function assessments, including some biomarkers, that are widely used in the literature. some of these assessment techniques of vascular function are detailed below: endothelial (dys) function can be assessed using flow-mediated dilatation. fmd involves ultrasonic assessment of the percentage increase in brachial artery diameter from baseline conditions to maximum vessel diameter during hyperemia induced by inflation and deflation of a sphygmomanometer cuff to supra systolic levels for 5 min [160] [161] [162] . the principle of this technique is that when there is a cutting off blood supply to the tissues, there is a collection of metabolites (no, for example) that lead to compensatory vasodilation after the return of circulation. in normal healthy endothelium, occlusion of the artery liberates these metabolites, which, in turn, leads to dilation of the vessel. in unhealthy persons, who often have low levels of no, no increases in vascular diameter are seen when the arterial occlusion is released. the gold standard of noninvasive endothelial function testing is fmd of the brachial artery [163] [164] [165] . as the fmd measurements are operator-dependent, strict physiological and methodological should be used [166] [167] [168] [169] . vascular stiffness of big blood vessels measured using pulse-wave velocity which detects the rate of arterial pulse waves move along the vessel wall [170] . the vicorder instrument (smt medical gmbh & co. kg, würzburg, germany) used to investigate changes in arterial stiffness during rest. pwv needs two-point to measure the speed of wave transmission between them through the arterial system, and the measure between carotid and femoral is the best. for the recording, the subject needs to lie down in the bed. one cuff has to be fixed around the neck so that the probe lies above the carotid. the second cuff should be fixed as high as possible around the right thigh for femoral artery detection [171] . the measure of intima-media thickness is an easy way to find endothelial dysfunction [172] . as in normal endothelium, there is no plaque, and the average thickness on intima-media is 0.9 mm [164] , but when the endothelium does not function well and becomes unhealthy, there is an increase in its thickness, and atheromatous plaque forms [173] . carotid or femoral artery can easily accept big arteries for detecting either plaque or thickness of intima-media by ultrasound device. the call-out box 1 below summarizes the current literature related to assessment of fmd, pulse wave velocity, and carotid intima media thickness: box 1. summary of the findings related to assessment of vascular function and atherosclerosis in different autoimmune rheumatic diseases. rheumatoid arthritis: all studies regarding the effect of rheumatoid arthritis on flow-mediated dilatation showing impairment in its reactive hyperemia [105, 106] and increase in arterial stiffness and carotid intima media thickness showing the evidence of premature atherosclerosis [107] [108] [109] [110] [111] . there is no correlation between macrovascular and microvascular endothelial function in rheumatoid arthritis (independent of each other) [112] . • systemic lupus erythematosus: fmd is impaired in lupus erythematosus patients; interestingly, the impairment is mostly found in older age patients with hypertension, diabetes, and renal impairments [125, 126] . arterial stiffness is increased in lupus patients with more relation to age, hypertension, and glucocorticoid use [127] [128] [129] . carotid plaque is 21% in patients with lupus under 35 years, and the percentage increased and reached 100% for women over age 65 years [20, 130] , which may be related to age or disease activity or both. • primary sjögren's syndrome (pss): pss appears to be associated with some degree of vascular dysfunction, rigidness of blood vessels, and arterial stiffness, which can lead to subclinical atherosclerosis [133, 134] . another noninvasive technique involving retinal microvasculature assessment can also be used to assess vascular function. retinal microvasculature showed that venules are not only passive vessels, but they represent the state of dynamic components responsive to changes in the microcirculation [174] . other recent studies reported that dilated retinal venules are denoting inflammation, endothelial dysfunction, and cerebral hypoxia. on the other side, constricted retinal arterioles correspond to endothelial dysfunction and elevated blood pressure [175] . it was reported that aging affects the microvasculature of the retina [176] . digital retinal camera scans can be proposed for noninvasive retinal vessels' assessment. the portable or table mounted retinal cameras are designed for a comfortable, painless, and quick examination of the retina. retinal images are stored on a laptop and data analysis is performed. this analysis is done using a commercially available retinal imaging and analysis software. during analysis of the retinal microvascular, the largest six arterioles and venules appearing through a zone between half and one-disc diameter from the optic disc margin are measured [177] . the measurements are described as central arteriolar equivalent (crae), which denotes the diameter of retinal arteries and central retinal venular equivalent (crve), which denotes diameter of retinal veins. the ratio between the crae and crve is also measured. this test assesses endothelial function. it depends on peripheral arterial tone (pat) signal, which is measured from the fingertip by measuring arterial pulsatile volume changes [178] . it is a reliable, objective, and not dependent on human factors (e.g., skill of the ultrasonographer). furthermore, the derivation of the reactive hyperemia index (rhi) is automated. there are several blood markers that can detect the function of the endothelium directly or indirectly (figure 4 ). oxidized ldl can be measured in the blood. similarly, antibodies to oxidized ldl, which may be responsible for the protection or harming endothelium during the process of atherosclerosis (wither igm or igg), can be measured. therefore, antibodies to oxidized ldl were introduced as new biomarker for assessment of endothelial health [148] [149] [150] [151] . interestingly, high-density lipoprotein (hdl), which is anti-atherosclerosis and a scavenger for ldl in the blood, when ldl becomes oxidized, hdl lost its function and converted to proinflammatory hdl (pihdl) [179] . also, adma, which is considered the most important indicator for endothelial dysfunction, can be used as a biomarker for endothelial health assessment. cryoglobulin is a marker for evaluation of blood vessel vasculitis, and it is a good indicator, especially in sjögren's syndrome, to detect vasculitis and malignant lymphoma transformation risk [180] . similarly, antiphospholipid antibodies are very important markers of vascular function, especially in patients with antiphospholipid syndrome [181] . additionally, pentraxin-3 is an inflammatory marker that increases in cardiovascular diseases and airds [182] . finally, endocan is another mediator of endothelial dysfunction and is related to the development/monitoring of atherosclerosis and cardiovascular diseases [183, 184] . mortality can increase in autoimmune rheumatic diseases due to cardiovascular diseases, especially myocardial infarction and cerebrovascular stroke. therefore, there is a need to diagnose early on the state of accelerated atherosclerosis in autoimmune diseases. this review provides an overview of selected autoimmune rheumatic diseases and discusses the roles of traditional and nontraditional factors in the development of cardiovascular diseases in these patients. comprehensive studies including state of the art vascular function measurements in different population groups and stages of the diseases are required in the future. atherosclerosis in patients with autoimmune disorders atherosclerosis as autoimmune disease epidemiology of cvd in rheumatic disease, with a focus on ra and sle autoimmune diseases and their manifestations on oral cavity: diagnosis and clinical management autoimmune diseases: the failure of self-tolerance how does age at onset influence the outcome of autoimmune diseases? autoimmune dis lung infections in systemic rheumatic disease: focus on opportunistic infections cardiovascular disorders and rheumatic disease. rev. española de cardiol serious infection risk in rheumatoid arthritis compared with non-inflammatory rheumatic and musculoskeletal diseases: a us national cohort study agespecific incidence rates of myocardial infarction and angina in women with systemic lupus erythematosus: comparison with the framingham study rojas-villarraga, a. cardiovascular involvement in autoimmune diseases heart involvement in inflammatory rheumatic diseases: a systematic literature review autoimmune atherosclerosis in 3d: how it develops, how to diagnose and what to do the role of cytokines in the development of atherosclerosis atherosclerosis-an inflammatory disease mechanisms of immune complex-mediated neutrophil recruitment and tissue injury environmental factors in autoimmune diseases and their role in multiple sclerosis cardiovascular disease in systemic lupus erythematosus: a comprehensive update risk factors for coronary artery disease: historical perspectives rheumatoid cachexia revisited: a metabolic co-morbidity in rheumatoid arthritis mediators and patterns of muscle loss in chronic systemic inflammation inflammation induced loss of skeletal muscle body mass index and clinical response to infliximab in rheumatoid arthritis obesity is independently associated with impaired quality of life in patients with rheumatoid arthritis high prevalence of obesity in rheumatoid arthritis patients: association with disease activity, hypertension, dyslipidemia and diabetes, a multi-center study a high body mass index has a protective effect on the amount of joint destruction in small joints in early rheumatoid arthritis rheumatoid arthritis and metabolic syndrome nicotine dependence as an independent risk factor for atherosclerosis in the national lung screening trial new insights to the mechanisms underlying atherosclerosis in rheumatoid arthritis association between antibodies to carbamylated proteins and subclinical atherosclerosis in rheumatoid arthritis patients hyperhomocysteinemia as a risk factor and potential nutraceutical target for certain pathologies metabolic syndrome and systemic lupus erythematosus: the connection a marker of increased cardiovascular risk in rheumatic patients: analysis of the act-cvd cohort atherosclerosis in rheumatoid arthritis: is it all about inflammation? mechanisms of disease: atherosclerosis in autoimmune diseases of mice and men: how animal models advance our understanding of t-cell function in ra th17/treg imbalance induced by increased incidence of atherosclerosis in patients with systemic lupus erythematosus (sle) drugs for autoimmune inflammatory diseases: from small molecule compounds to anti-tnf endothelial dysfunction and atherosclerosis in rheumatoid arthritis: a multiparametric analysis using imaging techniques and laboratory markers of inflammation and autoimmunity is atherosclerosis an autoimmune disease atherosclerosis and autoimmunity: a growing relationship the clinical value of autoantibodies in rheumatoid arthritis anti-citrullinated protein antibody specificities, rheumatoid factor isotypes, and incident cardiovascular events in patients with rheumatoid arthritis association of rheumatoid factors with subclinical and clinical atherosclerosis in african american women: the multiethnic study of atherosclerosis autoimmunity and atherosclerosis: the presence of antinuclear antibodies is associated with decreased carotid elasticity in young women. the cardiovascular risk in young finns study autoantibodies associated with connective tissue diseases: what meaning for clinicians? antibodies against β2-glycoprotein i complexed with an oxidised lipoprotein relate to intima thickening of carotid arteries in primary antiphospholipid syndrome autoantibodies to ox-ldl in sjögren's syndrome: are they atheroprotective? oxidized low-density lipoprotein and β2-glycoprotein i in patients with systemic lupus erythematosus and increased carotid intima-media thickness: implications in autoimmune-mediated atherosclerosis diagnosing antiphospholipid syndrome: 'extra-criteria' manifestations and technical advances cryoglobulins: identification, classification, and novel biomarkers of mysterious proteins vasculitic syndromes in hepatitis c virus: a review diffuse endothelial dysfunction is common to anca associated systemic vasculitis and polyarteritis nodosa anti-ssa/ro and anti-ssb/la autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of tnf-α by macrophages anti-ro antibodies and complete heart block in adults with sjögren's syndrome complete atrioventricular block in adult sjögren's syndrome with anti-ro autoantibody increased prevalence of diastolic heart failure in patients with rheumatoid arthritis correlates with active disease, but not with treatment type association between rheumatoid arthritis and risk of ischemic and nonischemic heart failure five-year changes in cardiac structure and function in patients with rheumatoid arthritis compared with the general population the systemic amyloidoses. new engl secondary amyloidosis as a life-ending event in multifocal motor neuropathy sarcoid, amyloid, and acute myocardial failure characteristics of rheumatoid arthritis and its association with major comorbid conditions: cross-sectional study of 502 649 uk biobank participants prevalence of the metabolic syndrome is increased in rheumatoid arthritis and is associated with coronary atherosclerosis early atherosclerosis in rheumatoid arthritis: effects of smoking on thickness of the carotid artery intima media cardiovascular disease and risk factors in patients with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis prevalence and associations of hypertension and its control in patients with rheumatoid arthritis atherosclerotic cardiovascular disease in patients with chronic inflammatory joint disorders hypertension in rheumatoid arthritis metabolic syndrome is common among middle-to-older aged mediterranean patients with rheumatoid arthritis and correlates with disease activity: a retrospective, cross-sectional, controlled, study cardiovascular risk in rheumatoid arthritis versus osteoarthritis: acute phase response related decreased insulin sensitivity and high-density lipoprotein cholesterol as well as clustering of metabolic syndrome features in rheumatoid arthritis preclinical carotid atherosclerosis in patients with rheumatoid arthritis insulin resistance is an independent risk factor for atherosclerosis in rheumatoid arthritis arterial stiffness in chronic inflammatory diseases cardiovascular risk factors in women with and without rheumatoid arthritis inflammation and hypertension in rheumatoid arthritis arterial stiffness and hypertension-which comes first? maedica autoimmune disease-associated hypertension persistent hypertension in lupus nephritis and the associated risk factors risk factors for coronary heart disease in women with systemic lupus erythematosus: the toronto risk factor study prevalence of and factors associated with hypertension in young and old women with systemic lupus erythematosus predictors of cardiovascular events in patients with systemic lupus erythematosus (sle): a systematic review and meta-analysis cumulated organ damage is associated with arterial stiffness in women with systemic lupus erythematosus irrespective of renal function the immune system in hypertension immune mechanisms of hypertension dysregulation of t cell subsets in the pathogenesis of hypertension role of the immune system in hypertension beneficial cardiovascular effects of low-dose glucocorticoid therapy in inflammatory rheumatic diseases mechanisms of hypertension in autoimmune rheumatic diseases how early in the course of rheumatoid arthritis does the excess cardiovascular risk appear? impact of risk factors associated with cardiovascular outcomes in patients with rheumatoid arthritis cardiovascular disease in rheumatoid arthritis: a systematic literature review in latin america nontraditional risk factors and biomarkers for cardiovascular disease: mechanistic, research, and clinical considerations for youth the association between microvascular and macrovascular endothelial function in patients with rheumatoid arthritis: a cross-sectional study prediction of cardiovascular events in rheumatoid arthritis using risk age calculations: evaluation of concordance across risk age models increased cardiovascular events and subclinical atherosclerosis in rheumatoid arthritis patients: 1 year prospective single centre study mortality trends in patients with early rheumatoid arthritis over 20 years: results from the norfolk arthritis register decreased cardiovascular mortality in patients with incident rheumatoid arthritis (ra) in recent years: dawn of a new era in cardiovascular disease in ra? mortality in rheumatoid arthritis: relationship to single and composite measures of disease activity cardiac involvement in systemic rheumatic diseases: an update increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: a population-based cohort study assessment of arterial stiffness variables in patients with rheumatoid arthritis: a mediation analysis ab0296 arterial stiffness in rheumatoid arthritis patients: do disease activity and duration of illness matter? clinical significance of aortic stiffness, carotid inti-ma-media thickness and serum osteoprotegerin level in rheumatoid arthritis patients impaired brachial artery flow-mediated dilation and increased carotid intima-media thickness in rheumatoid arthritis patients endothelial function in rheumatoid arthritis endothelial injury in rheumatoid arthritis: a crosstalk between dimethylarginines and systemic inflammation infliximab improves vascular stiffness in patients with rheumatoid arthritis impact of race and ethnicity in the course and outcome of systemic lupus erythematosus an update on antibodies to necleosome components as biomarkers of sistemic lupus erythematosus and of lupus flares immune complex-induced, nitric oxide-mediated vascular endothelial cell death by phagocytes is prevented with decoy fcγreceptors vascular stiffness in women with systemic lupus erythematosus the bimodal mortality pattern of systemic lupus erythematosus the anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights glucocorticoids are active players and therapeutic targets in atherosclerotic cardiovascular disease cardiovascular disease in systemic lupus erythematosus: the role of traditional and lupus related risk factors risk of myocardial infarction and stroke in newly diagnosed systemic lupus erythematosus: a general population-based study high-frequency ultrasound of multiple arterial areas reveals increased intima media thickness, vessel wall appearance, and atherosclerotic plaques in systemic lupus erythematosus premature morbidity from cardiovascular and cerebro-vascular diseases in women with systemic lupus erythematosus the epidemiology of atherosclerotic cardiovascular disease among patients with sle: a systematic review cardiovascular disease in systemic lupus erythematosus: recent data on epidemiology, risk factors and prevention reduced flow-mediated vasodilation as a marker for cardiovascular complications in lupus patients endothelial dysfunction in systemic lupus erythematosus-a case-control study and an updated meta-analysis and meta-regression increased arterial stiffness in systemic lupus erythematosus (sle) patients at low risk for cardiovascular disease: a cross-sectional controlled study accelerated age-related arterial stiffness in systemic lupus erythematosus patients endothelial dysfunction and arterial stiffness in patients with systemic lupus erythematosus: a systematic review and meta-analysis progression of carotid intima-media thickness and plaque in women with systemic lupus erythematosus dysfunction of lacrimal and salivary glands in sjögren's syndrome: nonimmunologic injury in preinflammatory phase and mouse model articular manifestations in primary sjogren's syndrome: clinical significance and prognosis of 188 patients association between primary sjogren's syndrome, arterial stiffness, and subclinical atherosclerosis: a systematic review and meta-analysis is there an increased arterial stiffness in patients with primary sjögren's syndrome? primary sjogren's syndrome: new clinical and therapeutic concepts predictable biomarkers of developing lymphoma in patients with sjögren syndrome: a nationwide population-based cohort study fundamentals of endothelial function for the clinical cardiologist the relationship between shear stress and flow-mediated dilatation: implications for the assessment of endothelial function linking endothelial dysfunction with endothelial cell activation asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the "l-arginine paradox" and acts as a novel cardiovascular risk factor review on uremic toxins: classification, concentration, and interindividual variability endothelial dysfunction as an early sign of atherosclerosis relationship between endothelial function, an-tiretroviral treatment and cardiovascular risk factors in hiv patients of african descent in south africa: a cross-sectional study covid-19 and its effects on endothelium in hiv-positive patients in sub-saharan africa: cardiometabolic risk, thrombosis and vascular function (endocovid study) accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure vascular macrophages in atherosclerosis the dynamics of oxidized ldl during atherogenesis autoantibodies to oxidized low-density lipoprotein in patients with aortic regurgitation: association with aortic diameter size are anti-oxidized low-density lipoprotein antibodies pathogenic or protective? circulation anti-oxidized ldl antibodies and coronary artery disease: a systematic review anti-oxidized low-density lipoprotein antibodies in chronic heart failure inflammation, atherosclerosis, and coronary artery disease immunohistochemical and ultrastructural demon-stration of the lymphocytemacrophage interaction in human aortic intima association of coronary plaque rupture and atherosclerotic in-flammation prevalence of foam cells and helper-t cells in atherosclerotic plaques of korean patients with carotid atheroma use of sonography to evaluate carotid atherosclerosis in the elderly. the cardiovascular health study cardiometabolic risk factors and early indicators of vascular dysfunction: a cross-sectional cohort study in south african adolescents cardiovascular risk factors and their relationship with vascular dysfunction in south african children of african ancestry effects of an innovative head-up tilt protocol on blood pressure and arterial stiffness changes physical therapy affects endothelial function in lymphedema patients aids: emerging threat to cardiovascular health in sub-saharan africa endothelial function in patients with chronic obstructive pulmonary disease: a systematic review of studies using flow mediated dilatation vascular health assessment with flow-mediated dilatation and retinal image analysis: a pilot study in an adult population from cape town microvascular reactivity in rehabilitating car-diac patients based on measurements of retinal blood vessel diameters vascular endothelial dysfunction in the wake of hiv and art cardiovascular risk and endothelial function in people living with hiv/aids: design of the multi-site, longitudinal endoafrica study in the western cape province of south africa assessment of flow-mediated vasodilatation (fmd) of the brachial artery: effects of technical aspects of the fmd measurement on the fmd response non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis assessment of arterial distensibility by automatic pulse wave velocity measurement measurement of pulse wave velocity in children and young adults: a comparative study using three different devices endothelial dysfunction, intima-media thickness and coronary reserve in relation to risk factors and framingham score in patients without clinical atherosclerosis measurement of intima media thickness of carotid artery by b-mode ultrasound in healthy people of india and bangladesh, and relation of age and sex with carotid artery intima media thickness: an observational study strengthening tight junctions of retinal microvascular endothelial cells by pericytes under normoxia and hypoxia involving angiopoietin-1 signal way determinants of retinal microvascular architecture in normal subjects retinal vessel diameters and their associations with age and blood pressure the metabolic syndrome and retinal microvascular signs in a japanese population: the funagata study effect of orthostasis on endothelial function: a gender comparative study anti-inflammatory hdl becomes pro-inflammatory during the acute phase response. loss of protective effect of hdl against ldl oxidation in aortic wall cell cocultures cryoglobulinaemic vasculitis at diagnosis predicts mortality in primary sjögren syndrome: analysis of 515 patients a importância de reconhecer a síndrome antifosfolípide na medicina vascular pentraxin 3 in cardiovascular disease endocan: a novel circulating proteoglycan endocan: a novel inflammatory indicator in cardiovascular disease? acknowledgments: bianca brix from physiology division for her essential help in preparing this review. no conflict of interest. key: cord-0006470-yqh4exz0 authors: aruoma, okezie i. title: free radicals, oxidative stress, and antioxidants in human health and disease date: 1998 journal: j am oil chem soc doi: 10.1007/s11746-998-0032-9 sha: 321bea20628962f3ad1c15fbff33e00aad46c4eb doc_id: 6470 cord_uid: yqh4exz0 free radicals and other reactive oxygen species (ros) are constantly formed in the human body. free-radical mechanisms have been implicated in the pathology of several human diseases, including cancer, atherosclerosis, malaria, and rheumatoid arthritis and neurodegenerative diseases. for example, the superoxide radical (o(2)(·−)) and hydrogen peroxide (h(2)o(2)) are known to be generated in the brain and nervous system in vivo, and several areas of the human brain are rich in iron, which appears to be easily mobilizable in a form that can stimulate free-radical reactions. antioxidant defenses to remove o(2)(·−) and h(2)o(2) exist. superoxide dismutases (sod) remove o(2)(·−) by greatly accelerating its conversion to h(2)o(2). catalases in peroxisomes convert h(2)o(2) into water and o(2) and help to dispose of h(2)o(2) generated by the action of the oxidase enzymes that are located in these organelles. other important h(2)o(2)-removing enzymes in human cells are the glutathione peroxidases. when produced in excess, ros can cause tissue injury. however, tissue injury can itself cause ros generation (e.g., by causing activation of phagocytes or releasing transition metal ions from damaged cells), which may (or may not, depending on the situation) contribute to a worsening of the injury. assessment of oxidative damage to biomolecules by means of emerging technologies based on products of oxidative damage to dna (e.g., 8-hydroxydeoxyguanosine), lipids (e.g., isoprostanes), and proteins (altered amino acids) would not only advance our understanding of the underlying mechanisms but also facilitate supplementation and intervention studies designed and conducted to test antioxidant efficacy in human health and disease. humans and other aerobes are able to tolerate oxygen (o 2 ) because, at the same time that organisms were evolving electron transport chains and other enzyme systems to use it, antioxidant defenses to protect against the toxic effects of o 2 were evolving in parallel. the predecessors of the anaerobic bacteria that exist today followed the "blind" evolutionary path of restricting themselves to environments that o 2 did not penetrate. hence, the evolution of multicellular aerobes and of antioxidant defense mechanisms is intimately related. interest in free radicals began with the work of moses gomberg (1) , who in 1900 demonstrated the existence of the triphenylmethyl radical (ph 3 c·). a free radical is any chemical species (capable of independent existence) that possesses one or more unpaired electrons, an unpaired electron being one that is alone in an orbital. radicals are generally less stable than nonradicals, although their reactivities vary. the simplest free radical is an atom of the element hydrogen. the hydrogen atom contains one proton and a single unpaired electron, which qualifies it as a free radical-often denoted by the insertion of the radical dot (·) to indicate that one or more unpaired electrons is present. once radicals form, they can react either with another radical or with another molecule by various interactions. the rate and selectivity of these types of reactions depend on high concentrations of the radicals, on delocalization of the single electron of the radical (thus increasing its life time), and on the absence of weak bonds in any other molecules present with which the radical could interact (see table 1 ). most biological molecules are nonradicals that contain only paired electrons. extensive discussions on free-radical chemistry may be found elsewhere (2) (3) (4) (5) (6) (7) . gerschman et al. (8) proposed that "oxygen poisoning and radiation injury have at least one common basis of action, possibly the formation of oxidizing free radicals." this pioneering idea soon began to capture the imaginations of scientists. in the early 1960s, superoxide was found to be associated with a number of enzymes, including xanthine oxidase. however, it was believed that this free radical was "bound" to the enzyme. in 1968, it was discovered that superoxide was secreted into solution (9) (10) (11) , allowing superoxide to mediate cellular toxicity. the role of free-radical reactions in human disease, biology, toxicology, and the deterioration of food has become an area of intense interest. in the main, the free-radical reaction of lipid peroxidation is an important issue in the food industry where manufacturers minimize oxidation in lipid-containing foods by use of antioxidants during the manufacturing process; foods are produced that maintain their nutritional quality over a defined shelf life. by contrast, biomedical sci-entists and clinicians are interested in antioxidants because they protect the body against damage by reactive oxygen species (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) . the rest of this article will focus on biomedical aspects of free radicals. free radicals of importance in living organisms include hydroxyl (oh·), superoxide (o 2 · − ) nitric oxide (no·), and peroxyl (ro 2 · ). peroxynitrite (onoo − ), hypochlorous acid (hocl), hydrogen peroxide (h 2 o 2 ), singlet oxygen 1 ∆g (often written as 1 o 2 ), and ozone (o 3 ) are not free radicals but can easily lead to free-radical reactions in living organisms. the term "reactive oxygen species" (ros) is often used to include not only the radicals oh·, ro 2 ·, no· and o 2 · − but also the nonradicals hocl, 1 extensive reviews on the role of free radicals in biology and medicine abound (22) (23) (24) (25) (26) (27) (28) (29) (30) . scheme 1 summarizes the mechanisms of cellular damage in oxidative stress. "oxidative stress" is the term that refers to the imbalance between reactions result from the instability of the first formed radical. the radicals may completely decompose or rearrange before reaction with other molecules or radicals present. reaction in which the radical decomposes to give a stable molecule and a new radical. rearrangement: 1. breaking of an adjacent c-c bond in a cyclic system with concomitant formation of a new bond, usually carbonyl and a new isomeric radical. 2. migration of an atom, via intramolecular abstraction by the radical center, thus creating a new, isomeric radical. addition to unsaturated systems: 1. addition of a radical to an olefinic double bond to give a new, saturated radical. typical reaction is the radical-induced polymerization of olefins. 2. addition of a radical to an aromatic double bond. this intermediate step is widespread in free-radical chemistry, e.g., in the radical substitution of aromatic compounds (homolytic aromatic substitution). the net overall reaction is displacement of an aromatic substituent by a radical: ar-x + y· → ar-y + x· abstraction or displacement: s h 2 reactions b : 1. biomolecular reaction involving homolytic attack of a radical on a molecule. the radical attacks a univalent atom, usually a terminal halogen or hydrogen in an abstraction reaction to give rise to a new radical, e.g., ph· + cbrcl 3 → ·ccl 3 + phbr 2. homolytic substitution at multivalent atoms also occurs, but both do not normally occur at saturated carbon centers. radicals readily undergo 1-electron oxidations with oxidizing reagents of suitable redox potential to give positive ions. example is the meerwein reaction, which involves the oxidation of cinnamyl-derived radicals by cupric ions: phc · hchrco 2 et + cu 2+ → phc + hchrco 2 et + cu + dimerization or radical coupling: localized radicals (methyl, phenyl radicals) react readily with little chance of dimerization. only delocalized radicals have a high probability of dimerization in solution. thus, when r' = r", the reaction is dimerization; and when r' ≠ r", the reaction is radical coupling or combination. involves collision of the radicals, resulting in the abstraction of an atom, usually hydrogen, by one radical from the other. this leads to the formation of two stable molecules, with the atom abstracted being β to the radical center: c e.g., the disproportionation of two phenylethyl radicals to give styrene and ethylbenzene. a the reader is referred to references 2-7 for an extensive overview of the reaction sequences highlighted in this table and an examination of the complex chemistry of organic radicals. b s h 2 stands for substitution homolytic biomolecular. c the disproportionation reaction derives its driving force from the formation of two new strong bonds and from the fact that the β-ch bonds in radicals are usually weak. generation of ros and the activity of the antioxidant defenses. severe oxidative stress can cause cell damage and death. it has been implicated in numerous human diseases ( table 2 ). in mammalian cells, oxidative stress appears to lead to increases in the levels of free ca 2+ and iron within cells (31, 32) . indeed, orrenius et al. (31) have suggested that excessive rises in intracellular free ca 2+ may cause dna fragmentation by activating endonucleases. the importance of oxidative stress injury depends on the molecular target, the severity of the stress, and the mechanism by which the oxidative stress is imposed, i.e., drug-induced, fenton chemistry, trauma, enzyme activation [e.g., no· synthase (nos) activity], etc. brief comments on some of the ros are worthwhile. ozone. this pale blue gas, which is not produced in vivo, serves as an important protective shield against solar radiation in the atmosphere. close to the earth's surface, o 3 is an unwanted oxidant and is often regarded as the most toxic air pollutant (33) . o 3 can form in laboratory equipment that has high-energy ultraviolet (uv)-producing lamps and in urban air as a result of photochemical reactions and pollution. the tissue most susceptible to damage upon exposure to o 3 is the lung. the biological effect of o 3 is often attributed to its ability to cause oxidation or peroxidation of biomolecules either directly or via free-radical mechanisms (34) (35) (36) . nitric oxide (no). no· is a free radical. it is widely thought that the endothelium-derived relaxing factor (edrf) produced by vascular endothelium, which is an important mediator of vascular responses induced by several pharmacological agents (including bradykinin and acetylcholine), is identical to no· (37, 39) . vascular endothelial cells that line the blood vessels also seem to produce small amounts of o 2 · − , some of which could react with no to produce nonradical products. thus, variation in the production of no· and o 2 · − by endothelium might provide one mechanism for the regulation of vascular tone and hence blood pressure. a role for no· has also been demonstrated in such human diseases as malaria, where no· appears to be partly involved in resistance to malaria infection, in cardiovascular disease, acute inflammation, cancer, neurodegenerative diseases, and diabetes. no· exerts potent actions in the regulation of cell function and tissue viability-this includes the recognized ability to mediate signal transduction via stimulation of guanylate cyclase-mediated cgmp synthesis (reviewed in refs. 37-43). sessa et al. (44) advocated that in dogs, "chronic exercise, presumably by increasing endothelial shear stress, increases edrf/no· production and endothelial cell no· synthase gene expression, and may contribute to the beneficial effects of exercise on the cardiovascular effort." the reaction between no · and o 2 · − leads to dna oxidative damage owing to the formation of onoo − , which may have oh·like potential, leading to the formation of nitroguanine and other loose products (45) (46) (47) (48) (49) (50) (51) . onoo − has the propensity to damage supercoiled dna in plasmid pbr322 (52) . a suggested toxicity of no· involves its reaction with o 2 · − to give onoo − , a reaction that occurs with a rate constant of 6.7 × 10 9 m −1 s −1 (53) . onoo − has been suggested to mediate nodependent toxicity. in addition to dna base nitration mentioned above, onoo − potentiates the activation of guanylate cyclase; has bactericidal activity and trypanocidal activity, converting low-density lipoprotein (ldl) to a form that may be recognized by the macrophage scavenger receptor; induces peroxidation of lipids; oxidizes methionine and sh residues in proteins; depletes antioxidants (e.g., ascorbate, glutathione), nitrates tyrosine residues; and inactivates α 1 -antiproteinase (α 1 ap) (the major inhibitor of serine proteases in human body fluids). indeed, nitration of tyrosine residues appears to be a "marker" of onoo − -dependent damage in vivo (42, (54) (55) (56) (57) (58) (59) (60) (61) (62) (63) . skin injury due to solar radiation, porphyria, contact dermatitis, and photosensitizers may also involve free-radical mechanisms. tiated by any species that has sufficient reactivity to abstract a hydrogen atom from a polyunsaturated fatty acid (pufa) side chain (such as those of arachidonic acid and linolenic acid, for example) in membrane lipids. arachidonic acid is a precursor of prostaglandins and leukotrienes. it contains a number of methylene-interrupted double bonds, which are particularly prone to hydrogen atom abstraction (64) . although much has been written about the mechanism and biological significance of lipid peroxidation, there seems to be no unanimity in the methods for its measurement. delineation of lipid peroxidation as a major pathway in most degenerative diseases ( table 2 ) would depend on adequate standardization and control of measurement conditions. the link between dna damage, faulty repair of dna, proto-oncogene activation, and the ability of some of the end-products of lipid peroxidation to act as promoters of carcinogenesis is widely discussed (65, 66) . end-products of lipid peroxidation could have profound effects on vascular function because of their ability to mimic or antagonize the actions of some of the stereospecific products formed by cyclooxygenase and lipoxygenase enzymes. for example, the f 2 -isoprostanes are generated by the peroxidation of arachidonic acid via the generation of ro 2 · isomers, which undergo endocyclization to prostaglandin-like compounds. their formation in vivo appears to be enhanced under conditions of oxidative stress, such as smoking, exposure to xenobiotics, and pathological conditions associated with inflammation (67, 68) . the mechanism of ldl oxidation possesses the general characteristics of lipid peroxidation reactions and free-radical reactions (69) . hypochlorous acid. hocl is produced by the neutrophilderived enzyme myeloperoxidase at sites of inflammation and when activated neutrophils infiltrate reoxygenated tissue (28) . the enzyme oxidizes chloride ions, cl − , in the presence of h 2 o 2 . a total of 5 × 10 6 activated human neutrophils in 1 ml produces 88 µm hocl in 2 h at 22°c (70) . hocl is not a free radical, but it is a potent chlorinating and oxidizing agent. it has recently been suggested that the formation of cholesterol chlorohydrins could disrupt cell membranes and lead to cell lysis and death (71) . on the basis of this observation, the cholesterol chlorohydrins have been suggested to be potential biomarkers for oxidative damage associated with neutrophil/monocyte activation (71) . hocl can attack many other biological molecules. for example, the proteolytic inhibitor alpha-1-antiproteinase (α 1 ap) is the major inhibitor in human plasma of proteolytic enzymes, such as elastase. α 1 ap accounts for about 90% of the elastase-inhibitory capacity of the human serum (72) . thus, its inactivation by hocl might greatly potentiate tissue damage because elastase is also released from activated neutrophils. hocl attacks primary amines and sulfhydryl groups in proteins and chlorinates purine bases in dna (73, 74) . kozumbo et al. (75) have demonstrated that physiological levels of hocl can react with substituted aryl amine (e.g., aniline, 1-naphthylamine, and 1-naphthol) to form long-lived products that bind dna and are genotoxic to human cells. in the context of food handling, hocl is a major active constituent of chlorine-based bleaches that are often used to disinfect equipment with which foods will come into contact. perhaps most significant is the observation that phenolic compounds can react effectively with hocl, hence protecting susceptible targets against oxidation (76, 77) . this may have physiological significance, given the interest in the use of dietary natural antioxidants to manipulate disease states (78) . superoxide radicals (o 2 · − ). o 2 · − is an oxygen-centered radical with selective reactivity. this species is produced by a number of enzyme systems, by autooxidation reactions, and by nonenzymatic electron transfers that univalently reduce molecular oxygen. in aqueous solution, o 2 · − can oxidize ascorbic acid. it can also reduce certain iron complexes such as cytochrome c and ferric-ethylenediaminetetraacetic acid (fe 3+ -edta hydroxyl radicals. oh· is a highly reactive oxygen-centered radical with an estimated half-life in cells of only 10 −9 s. most studies on oh·, prior to the discovery of sod, were done by radiation chemists. one feature of oh· is that it begets another radical, i.e., when it reacts with a molecule, the result is the formation of another radical species. the resulting species usually has lower reactivity than oh·. oh· attacks all proteins, dna, pufa in membranes, and almost any biological molecule it touches. in the case of oh· generation by fenton-type chemistry, the extent of oh· formation is largely determined by the availability and location of the metal ion catalyst. in an in vitro study, in which the technique of gas chromatography-mass spectrometry with selected ion monitoring (gc/ms/sim) was used to compare the role of copper and iron ions in promoting damage to dna by h 2 o 2 , added copper ions (80) were significantly more reactive in causing dna damage in the presence of h 2 o 2 than were equimolar iron ions. the availability of "free" iron and copper in vivo is controlled. iron ions are absorbed from the gut and transported to iron-requiring cells by the protein transferrin. iron specifically bound to transferrin does not participate in freeradical reactions. excess iron is stored as ferritin and hemosiderin in an attempt to keep the iron pool as small as possible. most or all of the plasma copper in humans is attached to the protein ceruloplasmin, which will not stimulate free-radical reactions under normal conditions (81, 82) . hydroxyl radical generation can take place when the hemostasis is altered. for example, copper and iron ions released into perfusates can cause ischemia/reperfusion injury (83) . ramos et al. (84) , using the electron spin resonance (esr) spin-trapping system, detected oh· as the α-hydroxyethyl spin-trapped adduct of 4-pyridyl 1-oxide n-tert-butylnitrone, formed from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. in systems that lacked myeloperoxidase, it is necessary to add iron to detect the oh· adduct. thus, human neutrophils and monocytes can generate oh· through a myeloperoxidase-dependent mechanism (85), which could have microbicidal implications. tissue injury can itself cause ros generation (e.g., by causing activation of phagocytes or releasing transition metal ions from damaged cells), which may (or may not, depending on the situation) contribute to a worsening of the injury. for example, the sequelae of traumatic brain injury and stroke may involve a postinjury stimulation of iron ion-dependent free-radical reactions. parkinson's disease is caused by death of cells in the substantia nigra. lysis of dead cells could cause iron ion release. thus, patients with parkinson's disease may be under oxidative stress, and free-radical reactions probably contribute to the degeneration of the substantia nigra (86) . some generation of ros in vivo is unavoidable and may be deliberate; no· is a typical example. the most important route for the formation of no· is the oxidation of arginine by nos of which there are two types: the constitutive calcium/calmodulin-dependent forms (cnos), which produce low levels of no·, and the inducible forms (inos). the inos, which have tightly bound calmodulin, are permanently active and are capable of generating high levels of no· (37, 38, 42, 43, 87) . the phagocytes (neutrophils, monocytes, macrophages, eosinophils) that defend against foreign organ-isms are also involved in this good and bad scenario. they generate o 2 · − , h 2 o 2 , and (in the case of neutrophils) hocl as one of their mechanisms for killing foreign organisms (26, 27) . this essential defense mechanism can go awry: several diseases (such as rheumatoid arthritis and inflammatory bowel disease) are accompanied by excessive phagocyte activation and resulting tissue damage, to which ros contribute. human defenses against ros-induced damage include the enzymes catalase and glutathione peroxidase (both of which remove h 2 o 2 , as well as sod, which catalyzes the dismutation of o 2 · − to form h 2 o 2 and o 2 · − ). glutathione peroxidase is generally thought to be more important than catalase as a h 2 o 2 -removing system in humans. catalase is located in peroxisomes, whereas glutathione peroxidase is localized in the mitochondria and cytosol, a similar distribution to that of sod. interestingly, in mammalian (including human) tissues, sod is mainly an intracellular enzyme. only small amounts are present in extracellular fluids, such as plasma, cerebrospinal fluid or synovial fluid. in addition, some low-molecular-mass substances, such as uric acid, ascorbate (vitamin c), glutathione, tocopherol (vitamin e), ubiquinol, ergothioneine, hypotaurine, and lipoic acid, may act as antioxidants in the human body. much has been written about the antioxidant status of the biological system. references 17, 19, 78, and 88-98 are illustrative. the rates of free-radical generation probably increase in most diseases, although we still do not know how important they are in promoting tissue injury, mainly because of the limitations of the assays used to measure them. the development and validation of assays, indicated in table 3 for humans, should allow us to rapidly evaluate the role of free radicals in disease pathology and establish a logical basis for the therapeutic use of antioxidants. numerous antioxidant supplementation studies (17, 18, (99) (100) (101) (102) (103) (104) (105) (106) (107) (108) (109) (110) (111) (112) (113) (114) (115) (116) (117) for the primary prevention of chronic diseases have been undertaken. the principal endpoint has always been "incidence" of the respective disease, i.e., incidence of cancer or cardiovascular disease. in a departure from this, duthie et al. (20) investigated the extent of oxidative dna damage in scottish men aged 50-59 years. their result suggests that long-term antioxidant supplementation can decrease both endogenous and exogenous oxidative dna damage in lymphocytes. i suggest that the application and validation of the biochemical markers in table 3 should be built into future supplementation studies to evaluate the pharmacology of antioxidants (drug-or plant-derived antioxidants). it is necessary to balance this with the choice of population, formulation and dose of antioxidants being used, the expected outcome variables, and the pathologic end points. the remainder of this article will focus on the technology (emerging and established) for assessing the products of oxidative damage to dna, proteins, and lipids in context human diseases. measurement of oxidative dna damage. oxidative damage to dna seems to occur continuously in vivo, in that low levels (presumably a "steady-state" balance between dna damage and repair) have been detected in dna isolated from human cells and tissues. the pattern of damage to the purine and pyrimidine bases bears the chemical fingerprint of oh· attack, suggesting that oh· formation occurs within the nucleus in vivo (117-123) (scheme 2, dna base products). however, this raises the question of how oh· could be produced in the nucleus. if oh· is attacking dna, it must be produced close to the dna because oh· is so reactive that it cannot diffuse from its site of formation. background radiation may be one source, but radiation-generated oh· is formed over the whole cell and only a small fraction hits dna (119) . other potential sources of oh· or oh·-like species include the decomposition of onoo − , the reaction of o 2 · − with hocl, the attack of hocl on dna bases, and generation of chlorinated products. the greatest interest has been in reactions of transition-metal ions with h 2 o 2 as a source of oh· (80, (120) (121) (122) . the key question is whether "catalytic" transition-metal ions (e.g., iron and copper ions) are really in close proximity to dna in vivo. although iron and copper seem to be present in the nucleus (123, 124) , it remains to be established why and how they reach the dna because these ions are normally carefully sequestered by the human body. however, oxidative stress and cell death can liberate metal ions from their normal sequestration sites and might then bind to dna, a powerful metal ion chelator. extensive reviews on the chemistry and measurement of oxidative dna damage may be found in references 120 and 125. base products may be measured essentially as described in there are two types of measurement of oxidative dna damage. steady-state damage can be measured when dna is isolated from human cells and tissues and analyzed for base damage products: it presumably reflects the balance between damage and dna repair. hence, a rise in steady-state oxidative dna damage could be due to increased damage and/or decreased repair. the measurement of baseline levels of oxidatively modified dna bases, although important, does not necessarily provide information as to whether this damage is in active genes or quiescent dna. it is important also to have an index of total dna damage in the human body, i.e., the "input" side of the steady-state equation. the most common approach has been to assess the "output" side, i.e., trying to estimate the rate of repair of oxidized dna. several dna base damage products are excreted in human urine, including the nucleoside 8-hydroxy-deoxyguanosine (8-ohdg), 8-hydroxy-adenine, and 7-methyl-8-hydroxyguanine (131) (132) (133) , but the one most exploited is 8-ohdg, usually measured by a method involving high-performance liquid chromatography (hplc) with electrochemical detection. gc/ms has also been used to measure 8-ohdg in urine, and the limit of detection was 1.8 pmol, corresponding to a level of 8-ohdg in urine of 35 nm (134) . the level of 8-ohdg in urine is presumably unaffected by the diet because nucleosides are not absorbed from the gut. the question of whether any 8-ohdg is metabolized to other products in humans has not been rigorously addressed. it is also possible that some or all of the 8-ohdg excreted in urine may arise not from dna, but from deoxygtp (dgtp) in the dna precursor pool of nucleotides. an enzyme has been described that hydrolyzes dgtp-containing oxidized guanine to prevent its incorporation into dna (135, 136) . the development of alternative urinary markers of total-body oxidative damage, to address these uncertainties and to validate measurements of guanine damage products in dna by hplc and gc/ms is the focus of current investigations (reviewed in ref. 137) . lipid peroxidation is important in vivo. it contributes to the development of cardiovascular diseases, such as preeclampsia and atherosclerosis, and the end-products of this process [particularly cytotoxic aldehydes, such as malondialdehyde (mda) and 4-hydroxynonenal (hne)] can cause damage to proteins and to dna. peroxidation causes impairment of biological membrane functioning, e.g., decreases fluidity, inactivates membrane-bound enzymes and receptors, and may change nonspecific calcium ion permeability (31, 32) . the more unsaturated a fatty acid side-chain, the greater its propensity to undergo lipid peroxidation. but do pufa really peroxidize faster than saturated fatty acids in vivo? this question is particularly important in relation to proposals that increases in dietary polyunsaturate/saturate ratios are beneficial to health (reviews in 64, 66, 137) . the ability to measure levels of isoprostanes and hydroxyeicosatetraenoic acids (hete) represents an important development in attempts to measure clinically relevant oxidative lipid damage. the hydroperoxides hete and isoprostanes are biologically active. hete are chemotactic for neutrophils and facilitate calcium uptake and protein kinase c mobilization (138, 139) . the 12-hete are involved in adrenocorticotropin and parathyroid secretion, modulation of mitogenic processes, and lymphocyte function (140) (141) (142) , while the 15-hete may inhibit neutrophil migration across cytokine-activated endothelium (143) . hete can be measured by gc-ms, operated in the negative ion chemical ionization mode (144) (145) (146) . isoprostanes (scheme 3) are a series of prostaglandin-like compounds that are formed during peroxidation of arachidonic acid, although probably similar products are formed from other pufa. because they are structurally similar to prostaglandin f 2α , the compounds are collectively referred to as f 2 -isoprostanes. these compounds can be measured in plasma (35 ± 6 pg/ml) and urine (1600 ± 600 pg/mg creatinine) of healthy volunteers, indicative of ongoing lipid peroxidation even in healthy human subjects. most of the plasma isoprostanes is esterified to phospholipids, but some are "free." one of the isoprostanes, 8-iso pgf 2α , is a powerful renal vasoconstrictor and decreases kidney blood flow and glomerular filtration rate by almost half at low nanomolar concentrations. it has been suggested that elevated circulating concentrations of f 2 -isoprostanes may contribute to the pathology of hepatorenal syndrome, an almost uniformly fatal disorder characterized by the development of kidney failure in patients with severe liver disease. urinary excretion of isoprostanes is elevated in patients with scleroderma and in smokers (extensive reviews may be found in refs. 67, 68, 144, 147, 148) . isoprostanes and their metabolites can be measured in human urine, and this may prove to be a valuable assay of whole-body lipid peroxidation if a confounding effect of diet can be ruled out. the mechanism of formation of the isoprostanes is shown in scheme 3, including the pathways (without the sterochemical orientation for clarity) that lead to the formation of four regioisomers (i-iv) of isoprostanes. the endoperoxides can undergo reduction to produce f 2 -isoprostanes or rearrangements to form the e 2 -isoprostanes, d 2 -isoprostanes, and/or isothromboxanes (68) . oxidative damage to proteins in vivo may affect the function of receptors, enzymes, transport proteins, etc. and perhaps generate new antigens that provoke immune responses. products of oxidative protein damage can contribute to secondary damage to other biomolecules, e.g., inactivation of dna repair enzymes and loss of fidelity of dna polymerases in replicating dna (149) (150) (151) . the chemical reactions that result from attack of ros upon proteins are complex (152) . free-radical attack upon proteins generates radicals from amino acid residues, and electrons can be transferred between different amino acids (scheme 4, products of oxidative protein damage) (137, 149) . the levels of any one (or, preferably, of more than one) of these products in proteins could in principle be used to assess the balance between oxidative protein damage and the repair of (or, more likely, hydrolytic removal of) damaged proteins. the only product exploited to date has been hydroxylated phenylalanine. for example, levels of ortho-tyrosine and dityrosine in human lens proteins have been reported in relation to age (153) . rns in scheme 4 refers to reactive nitrogen species, such as no and peroxynitrite. the carbonyl assay (154), a general assay of oxidative protein damage, to assess steady-state protein damage in human tissues and body fluids is based on the fact that several ros can attack histidine, arginine, lysine, and proline residues in proteins to produce carbonyl functions that can be measured after reactions with 2,4-dinitrophenylhydrazine (dnph) (149, (155) (156) (157) (158) . immunochemical methods involving anti-dnph antibodies also have been described (159, 160) . nevertheless, the reader is referred to the seminal discussions of the mechanisms and the molecular biology of oxidative damage to proteins (in refs. [161] [162] [163] . ros are formed in the human body. when endogenous antioxidant defenses are inadequate for the purpose of scavenging the ros completely, ongoing oxidative damage to dna, lipids, proteins and other molecules can be demonstrated. although ros have been implicated in pathology of several human diseases, it is important to appreciate that ros may not be the primary cause of these diseases. although diet-derived and drug-derived antioxidants may be particularly important in protecting against a number of human diseases (see table 2 ), the physiological relevance of these effects is uncertain. the effects of dietary antioxidants and/or antioxidant supplements in vivo are best studied using a combination of validated methods (see table 3 ) for measuring oxidative damage and where a particular disease is being investigated, the respective disease end point (164) . this should now become part of the strategy for future antioxidant supplementation and intervention studies of any kind. i thank nestec sa switzerland, the uk ministry of agriculture, fisheries and food, and the world cancer research fund for research support. an incidence of trivalent carbon trimethylphenyl some organic reactions involving the occurrence of free radicals in solution principles of free radical chemistry investigations of the radical ho 2 in solution the chemistry of free radical polymerization a chemical interpretation of the mechanism of oxidation by dehydrogenase enzymes oxygen poisoning and x-irradiation: a mechanism in common an enzymatic function for erythrocuprein (hemocuprein) free radicals and foods paradoxical behaviour of antioxidants in food and biological systems lipid oxidation oil-soluble antioxidants in foods the use of antioxidants in food antioxidant nutrients and disease prevention: an overview fruit, vegetables and cancer prevention: a review of the epidemiological evidence characterization of drugs as antioxidant prophylactics antioxidant supplementation decreases oxidative dna damage in human lymphocytes plant-derived anticancer agents free radical biology: xenobiotics, cancer, and aging free radicals in medicine ii free radicals in biology and medicine free radicals in tropical diseases oxidants from phagocytes: agents of defense and destruction oxygen metabolism and the toxic properties of phagocytes tissue destruction by neutrophils, new engl an approach to free radicals in medicine and biology for a collection of review articles, see oxygen radicals and lung injury role of ca 2+ in toxic cell killing oxidative stress and calcium homeostasis ozone toxicology biochemical basis of ozone toxicity mechanism of radical formation from reactions of ozone with target molecules in the lung singlet oxygen production from the reactions of ozone with biological molecules vascular endothelium cell synthesize nitric oxide from l-arginine endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide endothelium-derived relaxing factor reduces platelet adhesion to bovine endothelium cells the biology of nitrogen oxide in the airways nitric oxide: mediator, murderer and medicine nitric oxide regulation of tissue free radical injury the biological chemistry of nitric oxide chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression nitric oxide induces oxidative damage in addition to deamination in macrophage dna peroxynitrite mediated oxidation of purine bases of nucleosides and isolated dna reactions of peroxynitrite with 2′-deoxyguanosine,7,8-dihydro-8-oxo-2′-deoxyguanosine, and calf-thymus dna deamination of single-stranded dna cytosine residues in aerobic nitric oxide solution at micromolar total no exposures an adduct between peroxynitrite and 2′-deoxyguanosine: 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-deoxyguanosine formation of 8-nitroguanine in dna treated with peroxynitrite in vitro and its rapid removal from dna by depurination base modification and strand breakage in isolated calf thymus dna and in dna from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholinosydnonimine peroxynitrite causes dna nicks in plasmid pbr322 the reaction of no with superoxide interactions of peroxynitrite with human plasma and its constituents: oxidative damage and antioxidant depletion leishmanicidal activity of peroxynitrite peroxynitrite-dependent chemuliminescence of amino acids, proteins and intact cells peroxynitrite oxidation of sulfhydryls. the cytotoxic potential of superoxide and nitric oxide peroxynitrite stimulates vascular smooth muscle cell cyclic gmp synthesis role of apolipoprotein-derived radical and α-tocopheroxyl radical in peroxidase-dependent oxidation of low density lipoprotein the chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide peroxynitrite modification of low-density lipoprotein leads to recognition by the macrophage scavenger receptor the role of lipid peroxidation and antioxidants on oxidative modification of ldl pro-oxidant states and tumor activation lipid peroxidation and cancer a series of prostaglandin f 2 like compounds are produced in vivo by humans by a non-cyclooxygenase, free radical catalyzed mechanism the isoprostanes: current knowledge and directions for future research the chemistry of oxidation of lipoproteins, in oxidative stress, lipoproteins and cardiovascular dysfunction generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants chlorination of cholesterol in cell membranes by hypochlorous acid human plasma proteinase inhibitors the reaction of nucleotides with aqueous hypochlorous acid the nature of the reactions between chlorine and purine and pyrimidine bases: products and kinetics breakage and binding of dna by reaction products of hypochlorous acid with aniline, 1-naphthylamine or 1-naphthol antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid evaluation of the antioxidant actions of gallic acid and its derivatives nutrition and health aspects of free radicals and antioxidants superoxide dismutases. an adaptation to the paramagnetic gas copper-ion dependent damage to the bases in dna in the presence of hydrogen peroxide superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron: are lactoferrin and transferrin promoters of hydroxyl radical generation role of free radicals and catalytic metal ions in human disease: an overview copper and iron are mobilized following myocardial ischemia: possible productive criteria for tissue injury spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes effect of antiinflammatory drugs on myeloperoxidase-dependent hydroxy radical generation by human neutrophils neurodegeneration and neuroprotection in parkinson's disease the l-arginine-nitric oxide pathway, new engl oxidants, antioxidants, and the degenerative disease of aging antioxidant therapy in congestive heart failure: is there any advantage? natural antioxidants in human health and disease human disease, free radicals and the oxidant/antioxidant balance antioxidant therapy in haematological disorders antioxidants: health implications lipid-soluble antioxidants: biochemistry and clinical applications antioxidants in nutrition, health and disease natural antioxidants and food quality in atherosclerosis and cancer prevention antioxidants in human health and disease antioxidants in disease mechanisms and therapy vitamin e consumption and the associated risk of coronary disease in women antioxidant vitamin intake and coronary mortality in a longitudinal population study dietary antioxidant flavonoids and risk of coronary heart disease: the zutphen elderly study long term adequacy of all major antioxidants, presumably in synergy with other vegetable-derived nutrients may help to prevent early stages of cardiovascular disease and cancer respectively consumption of antioxidant vitamins and incidence of stroke in women vitamin supplement use and reduced risk of oral and pharyngeal cancer nutrient intake and cataract extraction in women: a prospective study poor plasma status of carotene and vitamin c is associated with higher mortality from ischemic heart disease and stroke: basel prospective study effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein nutrition intervention trials in linxian, china, supplementation with specific vitamin/mineral combinations, cancer incidence and disease specific mortality in the general population are antioxidants or supplements protective for age related macular degeneration a clinical trial of antioxidant vitamins to prevent colorectal adenoma the effect of vitamin e and beta carotene on the incidence of lung cancer and other cancers in male smokers (the alpha-tocopherol lack of effect of long-term supplementation with β-carotene on the incidence of malignant neoplasma and cardiovascular disease protective effect of fruits and vegetables on development of stroke in men randomised controlled trial of vitamin e in patients with coronary diseases: cambridge heart antioxidant study (chaos) ten year retrospective on the antioxidant hypothesis of atherosclerosis: threshold plasma levels of antioxidant micronutrients related to minimum cardiovascular risk effect of a combination of β-carotene and vitamin a on lung cancer and cardiovascular disease oxidative dna damage in human respiratory tract epithelial cells. time course in relation to dna strand breakage repair of products of oxidative dna base damage in human cells dna base damage in chromatin of γ-irradiated cultured human cells reactions of oxyl radicals with dna evidence for involvement of multiple iron species in dna single-strand scission by h 2 o 2 in hl-60 the role of oxidative processes in metal carcinogenesis structure of binary complexes of mono and polynucleotides with metal ions of the first transition group partitioning of zinc and copper within subnuclear nucleoprotein particles iron ion dependent modification of bases in dna by the superoxide radical generating system hypoxanthine/xanthine oxidase chemical determination of free radical induced damage to dna halliwell, intense oxidative dna damage promoted by l-dopa and its metabolites. implications for neurodegenerative disease direct enzymic detection of endogenous oxidative base damage in human lymphocyte dna a novel hplc procedure for the analysis of 8-oxoguanine in dna urinary 8-hydroxy 2′deoxyguanosine as a biological marker of in vivo oxidative dna damage jeding, 8-hydroxydeoxyguanosine as a urinary marker of oxidative dna damage analysis of methylated and oxidized purines in urine by capillary gas chromatography-mass spectrometry method for the analysis of oxidized nucleosides by gas chromatography/mass spectrometry cloning and expression of cdna for a human enzyme that hydrolyzes 8-oxo-dgtp, a mutagenic substrate for dna synthesis hydrolytic elimination of a mutagenic nucleotide, 8-oxodgtp, by human 18-kilodalton protein; sanitization of nucleotide pool free radicals and antioxidants: the need for in vivo markers of oxidative stress stimulation of human eosinophil and neutrophil polymorphonuclear leukocyte chemotaxis and random migration by 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid 5-hydroxyeicosatetraenoate promotes ca 2+ and protein kinase mobilisation in neutrophils the role of lipoxygenase metabolite of arachidonic acid in the regulation of adrenocorticotropin secretion by perfused rat anterior pituitary cells prigent, esterification of 12(s)-hydroxy-5,8,10,14-eicosatetraenoic acid into the phospholipids of human peripheral blood mononuclear cells: inhibition of the proliferative response effects of lipoxygenase products of arachidonate metabolism on parathyroid hormone secretion 15-hydroxyeicosatetraenoic acid inhibits neutrophil migration across cytokine-activated endothelium analysis of f 2 -isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure quantitation of hydroperoxy-eicosatetraenoic acids and hydroxy-eicosatetraenoic acids as indicators of lipid peroxidation using gas chromatography-mass spectrometry formation of novel non-cyclooxygenase derived prostanoids (f 2 -isoprostanes) in carbontetrachloride hepatotoxicity: an animal model of lipid peroxidation measurement of urinary 8-epi-prostaglandin f 2α , a novel index of lipid peroxidation in vivo, by immunoaffinity extraction/gas chromatography-mass spectrometry free radical-induced generation of isoprostanes in vivo. evidence for the formation of d-ring and e-ring isoprostanes oxidative stress, nutrition and health. experimental stratgegies for optimization of nutritional antioxidant intake in humans biochemical mechanisms accounting for the toxic action of oxygen on living organisms. the key role of superoxide dismutase enzymes that repair oxidative damage to dna free radical damage to proteins: the influence of the relative localization of radical generation, antioxidants and target proteins oxidized amino acids in lens proteins with age. measurement of o-tyrosine and dityrosine in the aging human lens oxidative damage to proteins: spectrophotometric method for carbonyl assay conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions protein oxidation and aging. difficulties in measuring reactive protein carbonyls in tissues using 2,4-dinitrophenylhydrazine oxidative damage and motor neurone disease. difficulties in the measurement of protein carbonyls in human brain tissue carbonyl assays for determination of oxidatively modified proteins immunochemical detection of oxidized proteins age-related changes in oxidized proteins oxidative damage to amino acids, peptides and proteins by radiation biochemistry and pathology of radical mediated protein oxidation molecular aspects of free radical damage to proteins extracts as antioxidant prophylactic agents key: cord-0037759-eiyqwgu2 authors: bhavsar, ishita; miller, craig s.; al-sabbagh, mohanad title: macrophage inflammatory protein-1 alpha (mip-1 alpha)/ccl3: as a biomarker date: 2015-06-01 journal: general methods in biomarker research and their applications doi: 10.1007/978-94-007-7696-8_27 sha: 1bd27e8bda56233613dcdb18364da1b7b41a8fab doc_id: 37759 cord_uid: eiyqwgu2 macrophage inflammatory protein-1 alpha (mip-1α/ccl3) is a chemotactic chemokine secreted by macrophages. it performs various biological functions, such as recruiting inflammatory cells, wound healing, inhibition of stem cells, and maintaining effector immune response. it activates bone resorption cells and directly induces bone destruction. cells that secrete mip-1α/ccl3 are increased at sites of inflammation and bone resorption. mip-1α/ccl3 plays an important role in the pathogenesis of various inflammatory diseases and conditions that exhibit bone resorption, such as periodontitis, multiple myeloma, sjögren syndrome, and rheumatoid arthritis. biological fluids from patients with these diseases exhibit elevated levels of mip-1α/ccl3. this finding indicates that mip-1[formula: see text] /ccl3 protein may have diagnostic potential for the detection of several inflammatory diseases and conditions. this chapter discusses the biological functions of mip-1α/ccl3; describes several diseases associated with mip-1α/ccl3, particularly periodontitis; and delineates the potential application of mip-1α/ccl3 as a biomarker. macrophage inflammatory protein-1 alpha (mip-1α/ccl3) is a chemotactic chemokine secreted by macrophages. it performs various biological functions, such as recruiting inflammatory cells, wound healing, inhibition of stem cells, and maintaining effector immune response. it activates bone resorption cells and directly induces bone destruction. cells that secrete mip-1α/ccl3 are increased at sites of inflammation and bone resorption. mip-1α/ccl3 plays an important role in the pathogenesis of various inflammatory diseases and conditions that exhibit bone resorption, such as periodontitis, multiple myeloma, sjögren syndrome, and rheumatoid arthritis. biological fluids from patients with these diseases exhibit elevated levels of mip-1α/ccl3. this finding indicates that mip-1α/ccl3 protein may have diagnostic potential for the detection of several inflammatory diseases and conditions. this chapter discusses the biological functions of mip-1α/ccl3; describes several diseases associated with mip-1α/ccl3, particularly periodontitis; and delineates the potential application of mip-1α/ccl3 as a biomarker. key facts of mip-1a/ccl3 • mip-1α/ccl3 is a type of chemotactic cytokine. • mip-1α/ccl3 belongs to the cc subfamily of chemokines. • mip-1α/ccl3 was first discovered by stephen d. wolpe in 1988. • mip-1α/ccl3 is a multifunction peptide that is secreted by a variety of hematopoietic and non-hematopoietic cells upon stimulation. • mip-1α/ccl3-expressing cells are usually found in areas of inflammation and bone resorption. • mip-1α/ccl3 recruits macrophages, lymphocytes, and eosinophils via the ccr1 or ccr5 receptor. • mip-1α/ccl3 preferentially attracts activated cd8 + t cells. • mip-1α/ccl3 exerts strong hiv-suppressive activity. • mip-1α/ccl3 plays an important role in the bone remodeling process by inducing osteoclastogenesis. • mip-1α/ccl3 is associated with periodontitis and multiple myeloma because of mip-1α/ccl3's biological functions of recruiting inflammatory cells and osteoclastogenesis activity. • increased concentrations of mip-1α/ccl3 can be detected in biological fluids of persons suffering from inflammatory and osteoclastogenic diseases. chemokine chemokines are type of cytokines that play an important role in recruiting various cells. the mediators responsible for recruitment, activation, and migration of immune and inflammatory cells to the site of inflammation have been studied for years. many of these mediators are cytokines and chemokines. cytokines belong to a group of signaling proteins that are secreted by various cells after cellular activation (foster 2001) . growth factors, inflammatory cytokines, and chemokines are types of cytokines. chemokines play an important role in recruiting inflammatory and immune cells to the site of infection or inflammation. they are also known as chemoattractant cytokines (foster 2001) . the typical structure of a chemokine consists of a configuration of four cysteine residues. depending on the presence or absence of amino acid between the first two cysteine residues, chemokines are classified into two main subfamilies, cxc (alpha) and cc (beta), with the x denoting one amino acid that separates the two terminal cysteines (fig. 1) . the configuration of chemokines in the cc subfamily consists of two adjacent cysteines (fernandez and lolis 2002) . chemokines are associated with various biological functions such as homeostasis, leukocyte recruitment, wound healing, tumor metastasis, angiogenesis, and activation of the innate and adaptive immune responses (murphy et al. 2000; rossi and zlotnik 2000; fernandez and lolis 2002) . the biological functions of chemokines occur after activating specific g protein-coupled protein receptors found on the surface of a variety of cells including monocytes/macrophages, neutrophils, basophils, eosinophils, dendritic cells, lymphocytes, and platelets (menten et al. 2002b) . upon activation, chemokines induce migration and accumulation of inflammatory cells at the source of chemokine production. it is interesting to note that chemokines can bind to more than one chemokine receptor (fig. 2) . for example, chemokine macrophage inflammatory protein-1 alpha (mip-1α/ccl3), a chemoattractant cytokine associated with the recruitment of leukocytes in response to tissue injury or inflammation, can bind to chemokine receptors ccr1 and ccr5 (maurer and von stebut 2004) . owing to these dual receptors, multiple cells can be recruited by this protein. this chapter focuses on the chemokine mip-1α/ccl3, its biological functions, its association with several inflammatory diseases, and its potential use as a biomarker for the diagnostic assessment of various diseases, particularly periodontal disease. macrophage inflammatory protein (mip)-1α/ccl3 is an inflammatory chemokine produced by cells during infection or inflammation. it belongs to the cc chemokine family, which displays potent chemotactic properties. the mip-1 protein was first in cc chemokine, the first and second conservation cysteine residues are adjacent to each other. in cxc chemokine, the first two cysteine residues are separated by an amino acid identified by stephen d. wolpe in 1988. his research group reported the appearance of a new heparin-binding protein from the murine macrophage cell line in response to endotoxin stimulation. this protein was called macrophage inflammatory protein (mip) because of its biological function of inducing an inflammatory response characterized by neutrophil infiltration ). the initial report of the separation of mip-1 on sds-page gel was described by sherry et al. (1988) . their findings suggested that the mip-1 protein was composed of two peptides. partial sequencing revealed two proteins: mip-1α and mip-1β. although these proteins exhibited slight variation in the sequence of amino acid residue at their nh 2 terminals, they share approximately 56.7 % sequence homology ). the genomic nucleotide sequence of murine mip1 α is highly homologous to human counterparts ld78alpha/gos19-1/scya3 (widmer et al. 1991 ). many researchers have independently isolated human and murine mip-1α and mip-1β in their laboratories. for this reason, mip-1α has more than one name in published reports. however, with the introduction of the new nomenclature, human mip-1α is currently called chemokine (c-c motif) ligand 3 (ccl3), and mip-1β is called chemokine (c-c motif) ligand 4 (ccl4) (zlotnik and yoshie 2000) . mip-1α/ccl3 is synthetized as a precursor protein with 92 amino acids. the premature protein splits at several sites, resulting in the formation of biologically active mature protein. chemokines exhibit a high affinity toward proteoglycans. an increased tendency of mip-1α/ccl3 to bind with heparin (a common proteoglycan) has been demonstrated experimentally by wolpe et al. (1988) , and this binding can enhance their activity (ali et al. 2000) . most mature hematopoietic cells can induce the synthesis of mip-1α/ccl3 and mip-1β/ccl4. monocytes, t lymphocytes, b lymphocytes, neutrophils, dendritic cells, and natural killer cells are known to secrete mip-1α/ccl3 (fig. 3 ) fig. 2 a chemokine can bind to more than one chemokine receptor. (a) schematic representation of mip-1α/ccl3 (chemokine) approaching toward the cell expressing the chemokine receptors ccr1, ccr2, and ccr5. (b) mip-1α/ccl3 binds to both ccr1 and ccr5 (chemokine receptors) (menten et al. 2002a, b) . under normal conditions, synthesis of mip-1α/ccl3 occurs at very low levels. however, upon stimulation of receptive cells with endotoxins such as lipopolysaccharide (lps) (suzuki et al. 2000) , virus proteins (melchjorsen et al. 2003) , or pro-inflammatory cytokines (il-1β) (standiford et al. 1993a) , cellular signaling events are activated, and this activation induces increased production of mip-1α/ccl3. mip-1α levels are regulated by certain transcriptional factors. mip-1 alpha nuclear protein (mnp) is one such transcriptional factor that induces the transcription of the mip-1α/ccl3 gene (ritter et al. 1995) . on the other hand, prostaglandin e 2 (pge2) has been shown to inhibit the expression of mip-1α/ccl3 from cells (dendritic cells) previously exposed to lps (jing et al. 2003) . non-hematopoietic cells such as osteoblasts (kukita et al. 1997 ) and epithelial cells (ryu et al. 2007) can secrete mip-1α/ccl3 upon stimulation. mip-1α/ccl3 is found to be one of the chemokines copiously expressed in inflamed gingival tissue and by oral epithelial keratinocytes (gemmell et al. 2001; kabashima et al. 2002; ryu et al. 2007 ). the biological function of mip-1α/ccl3 are broadly illustrataed in fig. 4 and discussed below. the inflammatory process consists of sequential cellular events that are associated with the expression of several mediators (cytokines and chemokines, including mip-1 α /ccl3) that induce the recruitment of leukocytes to the site of the interaction between activated endothelial cells and leukocytes migrating in the circulatory system into the site of inflammation depends on cytokine production and endothelial adhesion molecules (luscinskas et al. 1991) . unrestricted production of cytokines and chemokines can lead to tissue destruction (fig. 5 , step 3). activated adhesion molecules help to retain leukocytes and transmigrate them into the inflamed tissues after injury (fig. 5, step 4) . it has been reported that intracellular adhesion molecules (icam-1) play an important role in the interaction between monocytes and endothelial cells to produce mip-1α/ccl3 (lukacs et al. 1994 ). in vitro studies have shown a strong inflammatory reaction characterized by neutrophil accumulation after subcutaneous injection of mip-1α/ccl3 into mice footpads ). thus, mip-1α/ccl3 is suggested to play an important role in the inflammatory process by promoting the recruitment of leukocytes (pmns, macrophages, and lymphocytes) to the site of inflammation. mip-1α/ccl3 is important for inducing the inflammatory response against viruses. for example, upon exposure of lung tissue to respiratory syncytial virus (rsv), a strong inflammation response is induced by mip-1α/ccl3. mip-1α/ccl3 induces recruitment and migration of eosinophils and basophils. eosinophil degranulation results in severe tissue damage and is suggested to contribute to the pathological process triggered by viral infections (garofalo et al. 1992) . mip-1α/ccl3 is the most abundantly produced chemokine in mice infected with rsv, and respiratory epithelial cells and adjacent endothelial cells have been shown to be the major source of mip-1α/ccl3. furthermore, transgenic mice lacking the mip-1α/ccl3 gene showed a marked decreased inflammatory response when infected with rsv (haeberle et al. 2001 ). an increased level of mip-1α/ccl3 has been detected in bronchoalveolar lavage fluid (balf) from patients with different lung diseases such as allergen-mediated asthma (holgate et al. 1997) , acute respiratory distress syndrome (ards) (goodman et al. 1996) , and pulmonary fibrosis (ziegenhagen et al. 1998 ). genetically altered mip-1α/ccl3 null mice infected with coxsackie virus fail to develop myocarditis, and when inoculated with influenza virus, the degree of pulmonary inflammation was reduced (cook 1996) . these findings suggest that mip-1 α /ccl3 plays a significant role in promoting inflammation in response to different viral infections. the transmission and progression of human immunodeficiency virus (hiv) infection is greatly controlled by chemokines and chemokine receptors. while cd4 + t lymphocytes are the primary target cells, chemokine receptors ccr5 and cxcr4 act as primary coreceptors for entry of the virus (alfano and poli 2005) . because mip-1α/ccl3 binds to the chemokine receptor ccr5, it can play an important role in the etiopathogenesis of hiv infection. some cd4 + t cells secrete hiv-suppressive factors; hence, they are not preferentially infected with hiv (abdelwahab et al. 2003) . apart from t lymphocytes, macrophages are wellknown target cells that are infected with hiv. macrophages infected with hiv exhibit increased levels of mip-1α/ccl3 expression, and this expression correlates with viral replication (canque et al. 1996) . various chemokines such as regulated on activation normal t cell expressed and secreted (rantes/ccl5), mip-1α/ ccl3, and mip-1β/ccl4 demonstrate hiv-suppressive activity (cocchi et al. 1995) . furthermore, experimental studies have shown antibodies against rantes/ccl5, mip-1α/ccl3, and mip-1β/ccl4 can entirely block hiv-suppressive activity (cocchi et al. 1995) . these findings suggest that chemokines have a potential role in the prevention and treatment of hiv infection. at the site of injury, macrophages perform multifunctional roles to promote wound healing. they secrete various growth factors that encourage the proliferation of cells such as fibroblasts, endothelial cells, and keratinocytes. they also produce proteins that promote extracellular matrix remodeling (collagenase) and proteases. macrophages can produce several enzymes that degrade infectious agents and induce phagocytosis (fig. 5, step 6 ). delayed wound healing and decreased phagocytic activities are found when the numbers of macrophages and monocytes are reduced (leibovich and ross 1975) . mediators such as mip-1α/ccl3 may help promote wound healing activity by recruiting macrophages to the site of tissue injury. an in vitro study by dipietro et al. reported an increase in mip-1α/ccl3 mrna expression and protein levels after excisional wound injury. furthermore, they also reported that neutralization of mip-1α/ccl3 by anti-mip-1a antiserum decreases collagen synthesis activity, angiogenic activity, and the number of infiltrating macrophages (dipietro et al. 1998 ). these findings reflect the important role of mip-1α/ ccl3 in mediating macrophage migration into wounds to mediate tissue repair. mip-1α/ccl3 is implicated in the inhibition of hematopoietic stem cell proliferation. this function was not revealed until a molecule derived from macrophage (j774.2 cell culture) designated as a stem cell inhibitor (sci) was detected by graham et al. it was shown that sci inhibits the proliferation of both primitive cells (cfu-a assay) and hematopoietic stem cells (cfu-s assay). further studies showed that sci is identical to mip-1α/ccl3, as demonstrated by its amino acid sequence (graham et al. 1990 ). furthermore, mip-1α/ccl3 may play a pivotal role in protecting the hematopoietic stem cell from negative effects triggered by cytotoxic drugs during cancer treatment. mip-1α/ccl3 promotes the orientation and migration of osteoclasts (fuller et al. 1995) . it stimulates the process of osteoclastogenesis by directly activating osteoclast cells (kukita et al. 1992; watanabe et al. 2004) . increased mip-1α/ccl3 mrna expression has been detected in human bone marrow myelocytes and human bone tissues (kukita et al. 1997) . moreover, ccr1, ccr3, and cx3cr1 are some of the chemokine receptors expressed by osteoclast cells (lean et al. 2002) . it is suggested that mip-1α/ccl3 plays a key role in the bone remodeling process because it binds with the ccr1 receptor, which is predominantly expressed by osteoclasts. multiple myeloma (mm) is characterized by the accumulation of malignant plasma cells in the bone marrow and is associated with increased osteoclast activity. consistent with the role of mip-1α/ccl3, the expression of mip-1α/ccl3 mrna is higher in bone marrow samples from patients with mm than healthy control subjects (choi et al. 2000) , and levels of mip-1α/ccl3 from isolated bone marrow plasma of patients with active mm are increased compared with bone marrow plasma from patients with stable disease (choi et al. 2000) . mip-1α/ccl3 binds to ccr1 or ccr5 to induce osteoclast formation, and neutralizing antibodies of ccr1 and ccr5 inhibit the osteoclast-induced bone resorption activity of mip-1α/ccl3 (oba et al. 2005) . a strong positive correlation exists between bone resorption markers such as urine deoxypyridinoline (udpd), serum bone-specific alkaline phosphatase (s-balp), and serum mip-1 levels (mip-1α/ccl3 and mip-1β/ ccl4) in patients with multiple lytic bone lesions (hashimoto et al. 2004) . also, serum mip-1α/ccl3 levels correlate with the severity and prognosis of mm (terpos et al. 2003 (terpos et al. , 2005 . therefore, it can be concluded that bone destruction in mm is a process highly associated with mip-1α/ccl3. on the other hand, il-1β, a potent osteoclast-activating factor, has been implicated in the etiopathogenesis of mm. however, mm patients have shown either low to no detectable levels of il-1 β protein (choi et al. 2000) . thus, it is suggested that mip-1α/ccl3 is more potent than il-1β in inducing bone resorption in mm patients. since the concentrations of the mediators causing bone destruction are elevated, evaluating their levels can serve as a diagnostic tool for the detection of disease activity. mip-1α/ccl3 has been recently implicated in the regulation of tumor metastasis. oral squamous cell carcinoma (oscc) patients with metastatic lymph nodes have increased mrna expression of mip-1α/ccl3 in comparison with same patients with non-metastatic lymph nodes, thereby suggesting a possible role of mip-1α/ ccl3 in tumor migration (silva et al. 2007 ). mip-1α/ccl3 is also produced by stimulated leukocytic cell lines or lymphoblasts from pediatric patients with acute lymphoblastic leukemia or acute myeloid leukemia (struyf et al. 2003) . in a study involving a microchemotaxis chamber, mcf-7 breast cancer cell line responded to mip-1α/ccl3 and mip-1β/ccl4 suggesting their potential role in the migration of breast cancer cells (youngs et al. 1997) . wu et al. reported that ccl3-ccr5 axis may play an important role in enhancing various tumor cell functions including tumor metastasis (wu et al. 2008) . they found increased expression of the ccr5 receptor and not the ccr1 receptor in mice injected with renal cell carcinoma, a cancer known to metastasize to the bone. their reports suggest that ccl3-ccr5 axis contributes to neovascularization by enhancing mmp-9 expression which can help degrade extracellular matrix. furthermore, the ccl3-ccr5 axis may help in tumor progression by increasing hgf-expressing fibroblasts (angiogenic factor). the concept of tumor migration is based on the ability of chemokines to attract tumor cells to secondary sites. thus, the quantity of chemokines produced by lymphoma or leukemia cells and the types of chemokines present at distant sites of metastasis may determine the migration of tumor cells (menten et al. 2002a ). mip-1α/ccl3 acts as a potent chemoattractant for t lymphocytes and therefore plays an important role in recruiting t cells during the cell-mediated immune response (taub et al. 1993; cook et al. 1999 ). the two main subsets of t lymphocytes are cd4 (helper t cells) and cd8 (cytotoxic t cells). these cells are known to produce generous amount of cytokines and are broadly categorized as th-1-type cytokines and th-2-type cytokines. th-1 cytokines are potent pro-inflammatory mediators and are responsible for maintaining autoimmune responses. however, a large quantity of these pro-inflammatory cytokines can induce unobstructed tissue damage. thus, there is a need to counterbalance the mediators that promote unrestricted tissue destruction. th-2-type cytokines induce an anti-inflammatory response, thereby counteracting the functions of th-1 cytokines. in general, mip-1α/ccl3 and mip-1β/ccl4 play a key role in determining the responses of lymphocytes. for example, mip-1β/ccl4 selectively attracts activated cd4 + t cells (helper), and mip-1α/ccl3 preferentially attracts activated cd8 + t cells (cytotoxic) (taub et al. 1993 ). mip-1α/ccl3 is associated with the activation and migration of leukocytes, and it can induce osteoclastogenesis by activating osteoclasts. as a result, mip-1α/ccl3 has been implicated in the pathogenesis of various inflammatory diseases and conditions such as multiple myeloma (mm), aggressive periodontitis, sarcoidosis, papillon-lefèvre syndrome, rheumatoid arthritis (ra), sjögren syndrome (ss), and cardiovascular diseases. the periodontium includes the gingiva and the attachment apparatus surrounding each erupted tooth. the gingiva is comprised of keratinized oral squamous epithelium and the underlying connective tissue, whereas the attachment apparatus consists of the periodontal ligament (connective tissue), cementum, and the supporting alveolar bone. their function is to attach the tooth to the alveolar bone. periodontitis is a chronic disease that results from the interaction between oral bacteria and the host inflammatory response. it is reported to affect nearly 47 % of adults in the united states (eke et al. 2012) . poor oral hygiene methods contribute to the accumulation of bacterial plaque formation at the interface between the tooth and the gingiva, which when chronically present causes gingivitis, a reversible inflammation of the gum tissue (i.e., gingiva). if bacterial plaque is removed daily through effective oral hygiene measures, health is quickly restored. periodontitis is a more serious condition than gingivitis and is characterized by the destruction of the connective tissue attachment and bone surrounding teeth. unlike gingivitis, periodontitis requires more time and gram-negative bacteria in subgingival sites for its development. the presence of periodontal pathogens is sufficient to cause but will not predictably cause disease in all infected patients. differential susceptibility to the bacterial by-products elicits host responses that are contributory (seymour 1991) . although tooth-associated bacterial biofilm is the precipitating cause of periodontitis, many risk factors predispose a person to these diseases. these factors include genetic influences (il-1β polymorphism), aging, diabetes, stress, and smoking. periodontal pathogens have a unique advantage in that the dentogingival interface is an ideal substrate upon which a biofilm can be formed. the tooth penetrates the integument and is the only interface in the body where a calcified structure is in contact with the external environment and is subjected to colonization by bacteria. this tooth-associated biofilm confers a degree of protection and allows some degree of metabolic cooperation between the members of the community. microbes are held in contiguity with the delicate epithelium that lines the gingival sulcus (a potential space between the tooth and gum). if allowed to remain in situ, the increasing anaerobic and gram-negative flora will elicit an inflammatory host response. influx of polymorphonuclear (pmn) cells and macrophages takes place after activation of the immune reaction (fig. 6) . the exposure of the bacterial lipopolysaccharide (lps) induces the initiation of various inflammatory cells to stimulate the expression of inflammatory cytokines such as interleukin-1 (il-1), interleukin-6 (il-6), and tumor necrosis factor alpha (tnf-α) in the gingival connective tissue. chemokines are upregulated during the course of periodontal disease as a result of the interaction between the bacterial proteins and cellular surface receptors. experiments have shown that mip-1α/ccl3 expression is induced by polymorphonuclear (pmn) cells and gingival epithelial cells and not by gingival fibroblasts and human mg63 osteosarcoma cells (osteoblastic cells) when subjected to bacterial lipopolysaccharide (lps) and interleukin (il-1β) (ryu et al. 2007) . epithelial keratinocytes in the oral cavity can produce various chemokines such as mcp-1/ccl2, rantes/ccl5, mip-1α/ccl3, and interferon-γ-inducible protein-10 (ip-10/cxcl10) (gemmell et al. 2001 ). an immunohistochemistry study showed that the number of cells secreting mip-1α/ccl3 and chemokine receptor ccr5 and cxcr3 is higher in inflamed gingival tissue than in normal healthy gingival tissue (kabashima et al. 2002) . examination of gingival tissue biopsy samples from patients with various degrees of inflammation fig. 6 role of mip-1α/ccl3 in periodontal disease. schematic representation of tooth surface and accumulation of dental plaque. advanced periodontal lesions are associated with an acute inflammation reaction characterized by pmn, macrophages, and plasma cells. increased production of cytokines and chemokines takes place in response to bacterial lps. mip-1α/ccl3 binds to the cells presenting its receptors and induces recruitment of macrophages to induce inflammatory and immune response in the periodontal connective tissue. mip-1α/ccl3 also activates osteoclasts and promotes bone destruction. the numbers of b lymphocytes and th1 cells are increased with the progression of the advanced periodontal lesion (gingivitis and periodontitis) showed that cells from the periodontitis samples contained more mip-1α/ccl3 than mcp-1/ccl2. moreover, mip-1 α /ccl3 exhibited higher levels with increase in inflammation (gemmell et al. 2001) . mip-1α/ccl3 plays a significant role in recruiting inflammatory cells that are associated with later stages of inflammation. a correlation has been reported between mip-1α/ccl3-positive cells and an increased proportion of cd8 + t cells (cytotoxic cells) and b cells in association with increased inflammation (gemmell et al. 2001) . correspondingly, microchemotaxis experiments have shown that mip-1α/ccl3 has an increased tendency to attract cytotoxic t cells and b cells (schall et al. 1993) . mip-1α/ccl3 functions by recruiting inflammatory cells to the inflamed tissue, upregulating the expression of mip-1 α /ccl3 by gingival epithelial cells to promote an acute inflammation response, and recruiting b lymphocytes in the later stages of periodontal disease. on the other hand, relative amounts of rantes/ccl5 and ip-10/cxcl10 (specific for activated t cell) from the gingival tissues of periodontitis and gingivitis samples remained stable or reduced suggesting lack of recruitments of t-cell subsets by these chemokines. these findings indicate that mip-1 α /ccl3 plays a crucial role in recruiting leukocytes at early and later stages of periodontitis (gemmell et al. 2001) . previous reports have suggested that inflammatory mediators can temporarily alter the gene expression of a subset of cells. interestingly, fibroblasts from gingival tissue express higher levels of chemokine (mip-1α/ccl3) and cytokine (il-6) than periodontal ligament (pdl) fibroblasts when they are subjected to lps stimulation (morandini et al. 2010) . thus, the differential response between gingival and pdl fibroblasts appears due to different surface receptors that bind to chemokines. aggressive periodontitis is a specific type of periodontitis that is characterized by a genetic component and rapid attachment loss and severe bone destruction. it predominantly affects children and usually involves molars and incisors. patients often exhibit levels of bacterial plaque that are inconsistent with the amount of periodontal tissue destruction. an increased incidence of aggregatibacter actinomycetemcomitans (aa), a gram-negative bacteria, is a predominant feature of this disease. the local host inflammatory response is characterized by intense infiltration of pmns, macrophages, and lymphocytes, predominantly plasma cells (about 70 %) (liljenberg and lindhe 1980) . activated cells produce unrestricted expression of cytokines and chemokines that can further stimulate cells to produce prostaglandin e2 and tissue-degrading enzymes (masada et al. 1990; offenbacher et al. 1993; okada and murakami 1998) . mip-1α/ccl3 has been suggested to play an important role in aggressive periodontitis by inducing the activation of osteoclasts and mediating th-1 cytokines that promote tissue destruction (fig. 6) . mip-1α/ccl3 can also induce osteoclast formation and stimulate osteoclastogenesis (kukita et al. 1992; kukita et al. 1997) . mip-1α/ccl3 elicits bone resorption primarily through its receptor ccr1, which is predominantly expressed by osteoclast cells. as a result, increased level of mip-1α/ccl3 is one of the diagnostic features of this disease. fine et al. performed a longitudinal study to evaluate salivary levels of mip-1α/ccl3 from periodontally healthy aa + and aa à subjects. three subject groups were studied: seven subjects who developed localized aggressive periodontitis (lagp) with clinical signs of bone loss, seven healthy subjects with no bone loss (aa + ), and seven healthy subjects with no bone loss (aa à ). fine et al. found significantly higher levels (about 50 fold) of mip-1α/ccl3 from subjects in the lagp aa + group than the healthy control groups. furthermore, levels of mip-1α/ccl3 were found to be higher 6-9 months before the detection of bone loss. although the number of study subjects was small, this study demonstrates the potential role of mip-1α/ccl3 in promoting macrophage recruitment and osteoclast activation. the fact that mip-1α/ccl3 levels were higher before the detection of bone loss suggests that this protein may play a role as a biomarker for detecting patients at risk of bone loss during the course of periodontitis. mip-1α/ccl3 rna and its receptor ccr5 have also been detected at higher levels in gingival biopsy samples from patients with aggressive periodontitis than in samples from patients with chronic periodontitis (garlet et al. 2003) . consistent with these results was the finding that the chemokine receptor ccr5 is preferentially expressed on th1 cells rather than on th2 cells (loetscher et al. 1998 ). thus, the expression of mip-1α/ccl3 and its receptor ccr5 in aggressive periodontitis suggests an inclination toward the production of the th1 cell response. one study showed that a higher level of ifn-γ (th-1-type cytokine) is found in aggressive periodontitis, a finding suggesting the possibility of differential patterns of cytokine expression in periodontal tissue (garlet et al. 2003) . thus, depending on the recruitment of lymphocyte subsets, differential cytokine expression is expected in areas of aggressive periodontitis. visual evaluation, periodontal pocket depth, clinical attachment level, plaque index, bleeding upon probing, and demonstration of alveolar bone loss by radiographic images are some of the traditional parameters for diagnosing periodontal disease. however, these conventional diagnostic measures fail to recognize highly susceptible subjects who are at risk of future tissue breakdown. also, a substantial amount of damage must take place before these diagnostic measures can detect disease activity. hence, scientists are attempting to develop new diagnostics for detecting ongoing periodontal disease at an early stage. new diagnostics should be able to provide information about the extent of current disease activity as well as determine which patients are highly susceptible for future disease progression. recently, the use of serum, saliva, and gingival crevicular fluid (gcf) in clinical research has provided insight into the pathogenesis of periodontitis and potential biomarkers that may have diagnostic utility. currently, oral fluid biomarkers such as enzymes (collagenase), host cells (pmn), bacteria (aa, porphyromonas gingivalis), bacterial products (lps), and immunoglobulin (iga) show potential for detecting periodontal disease (khashu et al. 2012 ). periodontitis and mip-1a/ccl3: findings from oral fluids saliva from patients with periodontal disease contains higher concentrations of mip-1α/ccl3 than healthy controls. similar to the findings of fine et al. (2009) , levels of mip-1α/ccl3 were 18-fold higher in the saliva of 40 subjects with periodontitis than in 40 healthy control subjects (al-sabbagh et al. 2012) . salivary concentrations of mip-1α/ccl3 also demonstrate a strong positive correlation with clinical parameters of periodontal disease. in addition to correlating with disease severity, salivary levels of mip-1α/ccl3 have been shown to reflect response to therapy (sexton et al. 2011) . tymkiv et al. reported that the level of mip-1α/ccl3 is lower in subjects with periodontitis who smoke than in similar subjects who do not smoke (tymkiw et al. 2011) . this finding could be because smoking impairs the immune response (palmer et al. 2005) . although mip-1α/ccl3 appears to be a good salivary biomarker of periodontitis, one study reports that concentrations of mip-1α/ccl3 in gingival crevicular fluid were not different in subjects presenting with increasing severities of periodontal diseases (i.e., gingivitis or chronic and aggressive periodontitis) (emingil et al. 2005) . thus, this finding indicates more research is needed and could reflect the episodic nature of periodontal destruction activity (socransky et al. 1984 ; table 1 ). in dentistry, the concept of osseointegration is revolutionizing the success of dental implants as replacements for missing teeth. the demand for dental implants has grown considerably. however, implants can fail because of peri-implant disease. peri-implant mucositis is the inflammation of the soft tissue surrounding the implant, whereas peri-implantitis is a irreversible inflammation surrounding an osseointegrated implant that leads to severe bone loss caused either by bacteria or by technical (mechanical trauma) complications (lindhe and meyle 2008) (albrektsson and isidor 1994) . several studies have shown that the progression of peri-implantitis results in a markedly higher rate of implant failure (sakka et al. 2012) . in as much as peri-implantitis is a major cause of implant failure, various salivary biomarkers have been investigated for the detection of periimplantitis (arakawa et al. 2012; fonseca et al. 2012) . reports of cytokine and chemokine level in peri-implant crevicular fluid (picf) provide valuable information about the health status of dental implants. to date, at least one study has shown that levels of picf mip-1α/ccl3 and il-1β are significantly higher in patients with peri-implantitis than in patients with healthy implants (petkovic et al. 2010) . a significant reduction in mip-1α/ccl3 levels was found in patients who responded to periodontal therapy (srp + ohi) compared to those who did not sexton et al. 2011 than the healthy control group, but not statistically higher levels (de queiroz et al. 2008) . in contrast to serum studies, freshly isolated bone marrow plasma from patients with active mm exhibited higher concentrations of mip-1α/ccl3 than plasma from patients with stable disease (choi et al. 2000) . likewise, mm patients with more than three lytic lesions exhibit higher serum mip-1α/ccl3 concentrations than mm patients with fewer (less than 3) lytic lesions (terpos et al. 2005) suggesting that serum mip-1α/ccl3 levels and markers of bone resorption correlate with disease severity of mm (terpos et al. 2003; hashimoto et al. 2004 ), but less so with periodontal disease. sarcoidosis is a chronic granulomatous disease characterized by the accumulation of t lymphocytes and macrophages; this accumulation produces inflammatory reactions in various organs. because mip-1α/ccl3 is a potent macrophage chemotaxin, its role in the pathogenesis of sarcoidosis is significant. patients with sarcoidosis exhibit higher concentrations of mip-1α/ccl3 from bronchoalveolar lavage fluid (balf) and a 2.5-fold increase in chemotactic activity when compared to control subjects. also, reductions in chemotaxis of approximately 22 % were observed in the presence of anti-mip-1α antibodies (standiford et al. 1993b ). these findings suggest that the plasma concentration of mip-1α/ccl3 is related to the disease activity of sarcoidosis. patients with active sarcoidosis, including both systemic and pulmonary involvement, showed a marked increase in plasma levels of mip-1α/ccl3 (hashimoto et al. 1998 ). however, patients with predominantly pulmonary involvement without any systemic manifestation also showed elevated concentrations of mip-1α/ccl3. thus, the plasma concentration of mip-1α/ccl3 can provide valuable information about potential extrathoracic disease activity in patients with sarcoidosis and can correlate with disease activity. papillon-lefèvre syndrome (pls) is a rare autosomal recessive disorder initially reported by papillon and lefèrve in 1924. it is characterized by early loss of both deciduous and permanent dentition and by palmar-plantar keratosis. reports have shown that a genetic mutation of the chromosome (11q14-21) that results in complete loss of cathepsin c activity is responsible for the development of pls (hart et al. 1999 ). these patients exhibit severe periodontitis, which involves uncontrolled destruction of connective tissue and alveolar bone resorption. this occurs because of the downregulation of cathepsin c that impedes the activation of neutrophil serine proteinases such as cathepsin g, proteinase 3, and elastase (de haar et al. 2004) . neutrophil serine proteinases are important for maintaining host immune functions. patients with pls demonstrate impaired proteolysis of mip-1α/ccl3 because of the inactivity of neutrophil serine proteinases resulting in severe periodontal destruction attributed to excessive buildup of mip-1α/ccl3 (ryu et al. 2005) . rheumatoid arthritis (ra) is a chronic, inflammatory autoimmune disease that predominantly affects synovial joints. it is genetically associated with hla antigen dr4 (gran et al. 1983) . during the progression of this disease, inflamed synovial joints are infiltrated by various inflammatory cells, such as macrophages, lymphocytes, and plasma cells. the activated cells produce pro-inflammatory cytokines such as tnf-α, which further mediates inflammatory synovitis and induces matrix metalloproteinase to promote collagen fiber destruction. macrophages also play an important role in secreting chemokines involved in the pathogenesis of rheumatic arthritis. mip-1α/ccl3 expression is higher in synovial fluid and serum from ra patients than in these fluids from patients with osteoarthritis (koch et al. 1994) . increased levels of mip-1α/ccl3 mrna and protein also have been found in macrophages from the synovial tissue from ra patients. the higher expression can be attributed to an increase in the recruitment of macrophages and t cells into the areas of inflamed synovium by mip-1α/ccl3. likewise, increased expression of the chemokine receptor for mip-1α/ccl3, ccr5, occurs in ra patients (patel et al. 2001 ). the pathogenesis of sjögren syndrome (ss) is unclear; it is characterized by a chronic inflammation of the salivary glands, which is driven by interferon-related dysregulation resulting in damage to the glands and impaired excretion of saliva (lessard et al. 2013) . clinically, patients exhibit dry eyes and a dry mouth; however, other organs can be involved. biopsy samples from salivary glands of patients with ss express higher concentrations of both mip-1α/ccl3 and mip-1β/ ccl4 than that of rantes/ccl5 and il-8. in these studies, the ductal epithelial cells express higher concentrations of mip-1β/ccl4 than mip-1α/ccl3 (cuello et al. 1998 ). it has been suggested that the main causative factor in the development of ss is either the recruitment of leukocytes into the ductal epithelium by chemokines or the secretion of various pro-inflammatory cytokines by these cells. recent studies indicate the pathogenic role of c-c chemokines in the development of several cardiovascular diseases such as atherosclerosis (braunersreuther et al. 2007; de jager et al. 2013) , myocardial ischemia (de jager et al. 2008) , and congestive heart failure (aukrust et al. 1998 ). this is due to their biological function of activation and migration of leukocytes into areas of inflammation. clinical studies have recently identified elevated concentrations of circulating chemotactic chemokines, thereby suggesting their potential as diagnostic markers of cardiovascular diseases. for example, serum concentrations of mcp-1/ccl2, mip-1α/ccl3,, and rantes/ccl5 are elevated in patients with congestive heart failure (aukrust et al. 1998) . patients with acute myocardial infarction associated with heart failure complication and severe left ventricular dysfunction also show a higher concentration of circulating mip-1 α /ccl3 in comparison with healthy control patients (parissis et al. 2002) . furthermore, a strong correlation between myocardial infarction and mip-1α/ccl3 has been reported suggesting its strong prognostic potential to identify high-risk patients (de jager et al. 2008 (de jager et al. , 2012 . increasing evidence suggest that c-c chemokines (mip-1 α /ccl3) and their receptors play a significant role in the etiopathogenesis of the cardiovascular disease. therefore, inhibition of these chemokines and their receptors could provide novel therapeutic strategies. in conclusion, mip-1α/ccl3 plays a key role in the activation and regulation of inflammatory and host defense responses. mip-1α/ccl3 is crucial for the recruitment of macrophages and t lymphocytes from the circulation to sites of infection or injury; thus, it orchestrates acute and chronic inflammatory host responses. it also acts as a potent osteoclast activator and induces bone resorption. hence, mip-1α/ ccl3 is a key protein that plays an important pathological role in the development of several inflammatory and autoimmune diseases such as multiple myeloma, rheumatoid arthritis, sjögren syndrome, sarcoidosis, respiratory disease, cardiovascular disease, and periodontitis. evaluating the levels of mip-1α/ccl3 in biological fluids provides a great opportunity for diagnosing various inflammatory diseases and conditions at their early stages. moreover, inhibitors of mip-1α/ ccl3 and its receptors could be potential therapeutic targets for the treatment of these diseases. • mip-1α/ccl3 is a key chemokine that plays an important role in recruiting inflammatory cells (macrophages, lymphocytes, eosinophil, and basophils) to the site of injury or inflammation. • mip-1α/ccl3 is expressed by several cells upon stimulation of endotoxins such as lps, viral proteins, and pro-inflammatory cytokines. • mip-1α/ccl3 performs important biological functions, such as the initiation of the inflammatory response by the recruitment of macrophages, wound healing, tumor metastasis, promoting osteoclast activation, hiv-suppressive activity, and inhibition of hematopoietic stem cells, principally by binding to their selective chemokine receptors ccr1 and ccr5. • mip-1α/ccl3 induces osteoclastogenesis by directly activating osteoclasts; therefore, it is suggested to be a main etiological factor in diseases such as multiple myeloma and periodontitis that are characterized by severe tissue destruction and bone destruction. • mip-1α/ccl3 is associated with several inflammatory diseases including papillon-lefèvre syndrome, peri-implantitis, sarcoidosis, sjögren syndrome, and rheumatoid arthritis. • detection of increased concentrations of mip-1α/ccl3 in bodily fluids (serum, saliva, gcr, bronchial lavage) is a promising approach for detecting disease risk, disease activity, and response to therapy regarding several inflammatory and osseous destructive diseases. hiv-1-suppressive factors are secreted by cd4 + t cells during primary immune responses consensus report of session iv role of cytokines and chemokines in the regulation of innate immunity and hiv infection examination of the function of rantes, mip-1alpha, and mip-1beta following interaction with heparin-like glycosaminoglycans bone remodeling-associated salivary biomarker mip-1alpha distinguishes periodontal disease from health matrix metalloproteinase-8 is the major potential collagenase in active peri-implantitis elevated circulating levels of c-c chemokines in patients with congestive heart failure the specific role of chemokines in atherosclerosis macrophage inflammatory protein-1alpha is induced by human immunodeficiency virus infection of monocytederived macrophages macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma identification of rantes, mip-1 alpha, and mip-1 beta as the major hiv-suppressive factors produced by cd8 + t cells the role of mip-1 alpha in inflammation and hematopoiesis cd8 + t cells are a biologically relevant source of macrophage inflammatory protein-1 alpha in vivo chemokine expression and leucocyte infiltration in sjogren's syndrome loss-of-function mutations in cathepsin c in two families with papillon-lefevre syndrome are associated with deficiency of serine proteinases in pmns ccl3 (mip-1 alpha) levels are elevated during acute coronary syndromes and show strong prognostic power for future ischemic events chemokines ccl3/mip1alpha, ccl5/rantes and ccl18/parc are independent risk predictors of short-term mortality in patients with acute coronary syndromes leukocyte-specific ccl3 deficiency inhibits atherosclerotic lesion development by affecting neutrophil accumulation inflammation markers in healthy and periodontitis patients: a preliminary data screening mip-1alpha as a critical macrophage chemoattractant in murine wound repair prevalence of periodontitis in adults in the united states: 2009 and 2010 gingival crevicular fluid emap-ii, mip-1alpha and mip-1beta levels of patients with periodontal disease structure, function, and inhibition of chemokines macrophage inflammatory protein-1alpha: a salivary biomarker of bone loss in a longitudinal cohort study of children at risk for aggressive periodontal disease? cytokines expression in saliva and peri-implant crevicular fluid of patients with peri-implant disease the functions of cytokines and their uses in toxicology macrophage inflammatory protein-1 alpha and il-8 stimulate the motility but suppress the resorption of isolated rat osteoclasts patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection chemokines in human periodontal disease tissues inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome identification and characterization of an inhibitor of haemopoietic stem cell proliferation the association between rheumatoid arthritis and the hla antigen dr4 inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of mip-1alpha in lung pathology mutations of the cathepsin c gene are responsible for papillon-lefevre syndrome correlation of plasma monocyte chemoattractant protein-1 (mcp-1) and monocyte inflammatory protein-1alpha (mip-1alpha) levels with disease activity and clinical course of sarcoidosis ability of myeloma cells to secrete macrophage inflammatory protein (mip)-1alpha and mip-1beta correlates with lytic bone lesions in patients with multiple myeloma release of rantes, mip-1 alpha, and mcp-1 into asthmatic airways following endobronchial allergen challenge prostaglandin e2 inhibits production of the inflammatory chemokines ccl3 and ccl4 in dendritic cells the presence of chemokine (mcp-1, mip-1alpha, mip-1beta, ip-10, rantes)-positive cells and chemokine receptor (ccr5, cxcr3)-positive cells in inflamed human gingival tissues salivary biomarkers: a periodontal overview macrophage inflammatory protein-1 alpha. a novel chemotactic cytokine for macrophages in rheumatoid arthritis recombinant ld78 protein, a member of the small cytokine family, enhances osteoclast differentiation in rat bone marrow culture system macrophage inflammatory protein-1 alpha (ld78) expressed in human bone marrow: its role in regulation of hematopoiesis and osteoclast recruitment ccl9/mip-1gamma and its receptor ccr1 are the major chemokine ligand/receptor species expressed by osteoclasts the role of the macrophage in wound repair. a study with hydrocortisone and antimacrophage serum variants at multiple loci implicated in both innate and adaptive immune responses are associated with sjogren's syndrome juvenile periodontitis. some microbiological, histopathological and clinical characteristics peri-implant diseases: consensus report of the sixth european workshop on periodontology ccr5 is characteristic of th1 lymphocytes intercellular adhesion molecule-1 mediates the expression of monocyte-derived mip-1 alpha during monocyteendothelial cell interactions cytokineactivated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 measurement of interleukin-1 alpha and -1 beta in gingival crevicular fluid: implications for the pathogenesis of periodontal disease macrophage inflammatory protein-1 expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function role of the autocrine chemokines mip-1alpha and mip-1beta in the metastatic behavior of murine t cell lymphoma macrophage inflammatory protein-1 differential production of macrophage inflammatory protein-1alpha, stromal-derived factor-1, and il-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide from p. gingivalis international union of pharmacology. xxii. nomenclature for chemokine receptors mip-1alpha utilizes both ccr1 and ccr5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells modulation of host pge2 secretion as a determinant of periodontal disease expression cytokine expression in periodontal health and disease mechanisms of action of environmental factors-tobacco smoking serum profiles of c-c chemokines in acute myocardial infarction: possible implication in postinfarction left ventricular remodeling cxcr3 and ccr5 ligands in rheumatoid arthritis synovium proinflammatory cytokines (il-1beta and tnf-alpha) and chemokines (il-8 and mip-1alpha) as markers of peri-implant tissue condition mip1 alpha nuclear protein (mnp), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human mip-1 alpha gene the biology of chemokines and their receptors proteolysis of macrophage inflammatory protein-1alpha isoforms ld78beta and ld78alpha by neutrophilderived serine proteases gingival epithelial cell expression of macrophage inflammatory protein-1alpha induced by interleukin-1beta and lipopolysaccharide factors associated with early and late failure of dental implants human macrophage inflammatory protein alpha (mip-1 alpha) and mip-1 beta chemokines attract distinct populations of lymphocytes salivary biomarkers of periodontal disease in response to treatment importance of the host response in the periodontium resolution of the two components of macrophage inflammatory protein 1, and cloning and characterization of one of those components, macrophage inflammatory protein 1 beta dual role of ccl3/ccr1 in oral squamous cell carcinoma: implications in tumor metastasis and local host defense new concepts of destructive periodontal disease gene expression of macrophage inflammatory protein-1 alpha from human blood monocytes and alveolar macrophages is inhibited by interleukin-4 macrophage inflammatory protein-1 alpha expression in interstitial lung disease parc/ccl18 is a plasma cc chemokine with increased levels in childhood acute lymphoblastic leukemia comprehensive gene expression profile of lps-stimulated human monocytes by sage preferential migration of activated cd4 + and cd8 + t cells in response to mip-1 alpha and mip-1 beta serum levels of macrophage inflammatory protein-1 alpha (mip-1alpha) correlate with the extent of bone disease and survival in patients with multiple myeloma significance of macrophage inflammatory protein-1 alpha (mip-1alpha) in multiple myeloma influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis direct stimulation of osteoclastogenesis by mip-1alpha: evidence obtained from studies using raw264 cell clone highly responsive to rankl genomic structure of murine macrophage inflammatory protein-1 alpha and conservation of potential regulatory sequences with a human homolog, ld78 macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties ccl3-ccr5 axis regulates intratumoral accumulation of leukocytes and fibroblasts and promotes angiogenesis in murine lung metastasis process chemokines induce migrational responses in human breast carcinoma cell lines increased expression of proinflammatory chemokines in bronchoalveolar lavage cells of patients with progressing idiopathic pulmonary fibrosis and sarcoidosis chemokines: a new classification system and their role in immunity key: cord-0066404-19n62hi1 authors: lambrecht, antonia; vuillerme, nicolas; raab, christina; simon, david; messner, eva-maria; hagen, melanie; bayat, sara; kleyer, arnd; aubourg, timothée; schett, georg; hueber, axel; knitza, johannes title: quality of a supporting mobile app for rheumatic patients: patient-based assessment using the user version of the mobile application scale (umars) date: 2021-07-22 journal: front med (lausanne) doi: 10.3389/fmed.2021.715345 sha: 56f62b27c4ea48c3fea4440f5ca5511f55f699c5 doc_id: 66404 cord_uid: 19n62hi1 introduction: mobile applications promise to improve current health care. however, current mobile app quality ratings are mostly physician-based. the aim of this study was (1) to assess the quality of the self-management app rheuma auszeit using the validated umars (user version of the mobile app rating scale) app quality assessment tool and (2) to evaluate the association between umars scores and patients' characteristics. materials and methods: consecutive patients with rheumatoid arthritis, psoriatic arthritis and spondyloarthritis were seen at the rheumatology clinic at university hospital erlangen, germany. they were asked to test rheuma auszeit, evaluate its quality using umars and complete a paper-based survey evaluating the individual preferences, attitudes and ehealth literacy. the association between umars scores and patients' characteristics was further explored. results: between december 2018 and january 2019, a total of 126 patients evaluated rheuma auszeit using umars and filled out the paper-based survey. the median umars score was 3.9, iqr 0.7. functionality was the domain with the highest rating (median 4.8, iqr 0.8), followed by aesthetics (median 4.0, iqr 0.7), information (median 3.5, iqr 0.8), and engagement (median 3.2, iqr 1.0). subjective quality was average (median 3.0, iqr 1.0). the lowest scoring individual item was customization with a median of 2.5/5. lower functionality scores were reported among older female rheumatic patients (p < 0.004). older male rheumatic patients reported a higher subjective quality score (p < 0.024). perceived disease activity and disease duration did not significantly correlate with any umars subdomain scores. ehealth literacy significantly correlated with functionality umars subdomain ratings (rho = 0.18; p < 0.042). preferred time of app usage significantly correlated with engagement (rho = 0.20; p < 0.024), functionality (rho = 0.19; p < 0.029), total umars score (rho = 0.21; p < 0.017) and subjective quality score (rho = 0.21; p < 0.017). the vast majority of rheumatic patients would consider recommending rheuma auszeit to other patients (117/126; 92.9%). conclusion: rheuma auszeit was well-accepted by german patients suffering from rheumatoid arthritis, psoriatic arthritis and ankylosing spondyloarthritis. lacking customization could lead to low app compliance and should be improved. lower functionality scores among older female rheumatic patients highlight the need for patient education. the study underlines the potential and feasibility of therapeutic complementary digital solutions in rheumatology. introduction: mobile applications promise to improve current health care. however, current mobile app quality ratings are mostly physician-based. the aim of this study was (1) to assess the quality of the self-management app rheuma auszeit using the validated umars (user version of the mobile app rating scale) app quality assessment tool and (2) to evaluate the association between umars scores and patients' characteristics. consecutive patients with rheumatoid arthritis, psoriatic arthritis and spondyloarthritis were seen at the rheumatology clinic at university hospital erlangen, germany. they were asked to test rheuma auszeit, evaluate its quality using umars and complete a paper-based survey evaluating the individual preferences, attitudes and ehealth literacy. the association between umars scores and patients' characteristics was further explored. results: between december 2018 and january 2019, a total of 126 patients evaluated rheuma auszeit using umars and filled out the paper-based survey. the median umars score was 3.9, iqr 0.7. functionality was the domain with the highest rating (median 4.8, iqr 0.8), followed by aesthetics (median 4.0, iqr 0.7), information (median 3.5, iqr 0.8), and engagement (median 3.2, iqr 1.0). subjective quality was average (median 3.0, iqr 1.0). the lowest scoring individual item was customization with a median of 2.5/5. lower functionality scores were reported among older female rheumatic patients (p < 0.004). older male rheumatic patients reported a higher subjective quality score (p < 0.024). perceived disease activity and disease duration did not significantly correlate with any umars subdomain scores. ehealth literacy significantly correlated with functionality umars subdomain ratings (rho = 0.18; p < 0.042). preferred time of app usage significantly correlated with engagement (rho = 0.20; p < 0.024), functionality (rho rheumatology encompasses a variety of chronic inflammatory diseases that require immunosuppressive treatment. despite more effective medical treatment becoming available to patients, many patients are unable to reach clinical remission. an integrative, holistic treatment approach might improve patients' quality of life. due to the wide adoption of smartphones (1), medical apps could become an effective tool (2) (3) (4) (5) to promote predictive, preventive, personalized and participatory ("p4") medicine (6) . the european alliance of associations for rheumatology (eular) anticipated the considerable potential of mobile apps to aid rheumatic patient disease self-management and recently published guidelines (3) to support the implementation into clinical care. medical apps are already being used in clinical routine by an increasing number of rheumatologists (7) . however, little knowledge of the quality of existing apps (8) and evidence (9) supporting their effectiveness and acceptance currently prevents widely adoption (10) . in a recent physicianbased appstore analysis (4), rheuma auszeit received the highest rating among sixteen identified apps meeting inclusion criteria. notably, rheuma auszeit was the only app, mainly developed by rheumatic patients (german league against rheumatism). most of the apps provided symptom tracking, whereas rheuma auszeit delivers video and audio instructions for mental and physical exercises. mobile apps created for patient use are currently primarily assessed exclusively by physicians (4, 5) , yet it has been repeatedly stressed to include end-user (patient) evaluations (1, 4, (11) (12) (13) . the perceived app quality is subjective and could significantly differ between patients and physicians, similar to perceived disease activity (14) . to enable a user-centric app evaluation, the user version of the mobile app rating scale (umars) has been created (15) . it assesses the dimensions of engagement, functionality, aesthetics, information, and subjective quality on 5-point scales and has been used to evaluate the usability of dengue fever apps (16) , mental health apps (17) and wearable sensor-based biofeedback systems (18) . user-centered app evaluations are crucial to improve app design, effectiveness and importantly app adherence (4) . in order to maximize app effectiveness and long-term usage, continuous improvement and personalization are necessary (1, 4, 9, 12, 13) . the aim of this study was to assess the quality of rheuma auszeit by rheumatic patients using umars and to explore associations between umars scores and patient characteristics. consecutive patients with rheumatoid arthritis (ra), psoriatic arthritis (psa) and spondyloarthritis (spa) were recruited between december 2018 and january 2019 at the rheumatology clinic at university hospital erlangen, germany. the study was approved by the local ethics committee (no. 418-18b) and conducted referring to good clinical practice. all patients provided informed consent and were then asked to (1) evaluate the app rheuma auszeit using umars and to (2) complete a paper-based survey evaluating the individual preferences, attitudes and ehealth literacy. the association between umars scores and patient characteristics were further explored. rheuma auszeit (figure 1 ) has been developed by the german rmd patient organization rheuma-liga and is available to download for free in the german app store and google play store. it offers rheumatic patients, irrespective of the exact rheumatic disease, practical self-management recommendations that can immediately be applied to support patients coping with rheumatic pain. the app consists of audio, images and videos that should guide patients. exercises manuals include muscle relaxation, self-massage, mind journeys, movement training and cold/hot treatments. there is no predetermined order of exercises and patients are free to select their preferred exercises. importantly, symptom tracking/monitoring offered by various other apps (4) is not offered by this app. all material can be downloaded to support offline usage. local study personnel explained the goal and functions of the app and gave a short demonstration. as recommended by the umars developers (15) , patients tested rheuma auszeit for at least 10 min using tablets (ipads). patients were then asked to evaluate the app quality employing the paper version of the german version of the umars (15) , which is a simplified user version of the mars (19, 20) including 20 items instead of the original 23. mars has been used to evaluate the quality of a multitude of apps and its construct validity, reliability and objectivity has been recently validated in a multicenter study (19) . both rating scales assess the five dimensions: engagement, functionality, aesthetics, information, and subjective quality on 5-point scales. instead of the original 7 information quality mars items, umars consists of four information quality items: quality of information, quantity of information, visual information, credibility of source. the subjective quality dimension is based on four questions also using 5-point scales: willingness to recommend the app, anticipated app usage frequency, willingness to pay for the app, and overall rating. besides the app evaluation, patients were asked to complete a paper-based survey regarding individual sociodemographic details, mhealth preferences, attitudes and ehealth literacy. patients' ehealth literacy was measured using the validated german version (21) of the ehealth literacy scale (eheals) (22) . it is based on a 5-point likert scale and includes eight statements concerning self-perceived ehealth literacy. we previously published exact survey details and results from a larger patient collective (1) . in this study survey data was only used from patients that evaluated rheuma auszeit. the statistical analysis was split into three distinct steps: (i) patient characteristics were summarized using medians, interquartile ranges, means, standard deviations, counts, and percentages as appropriate. descriptive statistics were computed for describing distributions of variables related to umars score and subjective quality among patients. in details, boxplots and distribution histograms were used to summarize scores distributions from the whole population regarding engagement, functionality, aesthetics, information, total score and subjective quality variables. among these variables, (1) engagement, (2) functionality, (3) aesthetics and (4) information variables resulted from a combination of several other variables averaged together, respectively (1) entertainment, interest, customization, interactivity, target group, (2) performance, ease of use, navigation, gestural design (3) layout, graphics, visual appeal, (4) quality of information, quantity of information, visual information and credibility of source. thus, supplementary boxplots were computed for summarizing the distribution of each of these combination variables. finally, boxplots computed from the whole population for describing the main variables, i.e., engagement, functionality, aesthetics, information, total score and subjective quality, were visually compared with subgroups of the patient populations related to gender and disease. as such, violin plots were used to compare the whole patient population with (1) axial spondyloarthrisis, (2) patient demographic information is displayed in and only a small minority was aware of useful rheumatology apps/websites (10/126; 7.9%) or previously participated in an online health program (2/126; 1.6%). usability and data security were rated as more essential app features (10) than design (5) and interactivity (5) by patients. were significantly lower for spa patients (p < 0.047) and psa patients (p < 0.016) compared to ra. subjective quality was average (median 3.0, iqr 1.0) and was significantly lower in spa compared to psa patients (p < 0.013). figure 3 shows no significant gender-based differences regarding umars subdomain ratings, total score and subjective quality. figure 4 presents the single umars items of the four subdomains, umars total score and subjective quality. the highest individual item scores were the functionality subdomain items (performance, ease of use, navigation and gestural design), each with a median of 5/5. the lowest scoring item was customization with a median of 2.5/5. table 3 lists the subjective app quality umars item ratings. only a minority of patients would not at all recommend rheuma auszeit (9/126; 7.1%) and most patients (61/126; 48.4%) would maybe recommend it. similarly, only 14/126 (11.1%) patients think that they would never use rheuma auszeit. 10/126 (7.9%) patients would definitely pay for this app. the majority of patients assigned an average rating (68/126; 54.0%). supplementary table 1 displays the correlations between umars ratings, users' socio-demographics, health characteristics and ehealth literacy. we observed a significant correlation with increasing age and lower functionality umars subdomain ratings for female patients (rho = −0.33; p < 0.004). increasing age correlated positively with subjective quality (rho = 0.32; p < 0.024) and total umars score (rho = 0.25; p < 0.083) for male patients. perceived disease activity and disease duration did not significantly correlate with any umars items. ehealth literacy significantly correlated with functionality umars subdomain ratings (rho = 0.18; p < 0.042). preferred time of app usage significantly correlated with engagement (rho = 0.20; p < 0.024), functionality (rho = 0.19; p < 0.029), total umars score (rho = 0.21; p < 0.017) and subjective quality score (rho = 0.21; p < 0.017). importance of usability correlated with functionality scores (rho = 0.18; p < 0.048) and total scores (rho = 0.19; p < 0.035). in a previous german app store analysis (4), rheuma auszeit received the highest physician-based mars rating (4.2/5). it was however unclear if rheumatic patients share this positive perception. this study now complements the previous evaluation by presenting a detailed patient perspective, as demanded in the published eular recommendations (3). to our knowledge, rheuma auszeit is the first german rheumatology app that has been tested by both, physicians and patients. our study provides valuable patient insights regarding the app's usability in terms of its functions, engagement, design, information, and subjective qualities. this study shows that rheuma auszeit is well-accepted among rheumatic patients. in particular, the app received high functionality and aesthetics ratings. most patients stated that they would use the app 3-50 times (91; 72.2%) during the next 12 months and only a minority of patients (14/126; 11.1%) would not use the app at all. we did not observe significant correlations between disease activity, disease duration and total umars score. the overall umars score was 3.9/5 compared to the physician-based mars score (4.2/5) (4). similar to the previous physician-based mars analysis (4), subjective quality was significantly (p < 0.0001) lower compared to the overall umars score. the lower subjective ratings could be due to a general german conservativeness. importantly our patient results are in line with physician evaluations rating the engagement subdomain as the worst umars subdomain (4) and showing no significant gender rating differences. we believe that different disease specific app expectations could have caused the differences regarding the umars engagement subsection. for example spa patients are likely to be more engaged if spine associated features are addressed in the app. patients were reluctant to pay for the app and actively recommend it to other patients. most patients would welcome such app recommendations from the national society of rheumatology. the german digital healthcare act (digital versorgung gesetz-dvg) allows to partially overcome these implementation barriers, as physicians can now prescribe designated medical apps to patients, which are then reimbursed by insurance companies. rheuma auszeit (translated: rheuma timeout) is unique, as it was the only app in a recent review mainly developed by rheumatic patients offering coping techniques (4). the top two app features rheumatic patients named in a previous survey were information (medication and disease) and exercises to reduce stress and pain (1) . rheuma auszeit addresses the latter. most other previously identified german rheumatic apps focus on symptom tracking (4), however offer no techniques to reduce stress and pain. lalloo et al. showed that symptom tracking alone can lead to a clinically meaningful reduction in pain intensity in a population with juvenile idiopathic arthritis (23) . on the other hand, seppen et al. showed that pain positively mediated app usage in ra patients but some patients identified symptom tracking (reminding patients of their disease) as a barrier for app usage (24) . this suggests that symptom tracking frequency should be discussed with patients via shared-decisionmaking (sdm). shaw et al. recently showed that patient-reported outcome results should be discussed during face-to-face time to enhance the patient-provider interaction (25) . we believe that rheumatic patients need to receive proper instructions on how to use medical apps similar to other medical products. this personalized training will likely improve app adherence and effectiveness, however will most likely not be carried out by the treating physician due to the pressing time constraints. the lack of information on available tools and supporting evidence are main barriers that delay wide adoption. our study showed lower functionality scores among older female patients (p < 0.004) and higher subjective quality scores among older male patients (p < 0.024). this suggests that not all rheumatic patients might benefit equally from mhealth solutions and instructions need to be personalized as well. the high overall umars score and number of rheumatic patients regularly using smartphones imply that a longitudinal clinical study is feasible. the low customization score suggests however that such a trial will likely result in low app adherence, especially since app retention in general is an imminent mhealth barrier (26) also present in rheumatology (24, 27) . in our opinion, customizability should be improved by gathering additional qualitative feedback from rheumatic patients before initiating a longitudinal clinical study evaluating clinical benefits of the app. this study has some limitations. the analysis was solely based on quantitative feedback from a single evaluation tool based on short app usage. no sample size calculation was carried out. qualitative feedback, similar to herbuela et al. (16) , would have added valuable depth and details to our analysis and are planned to be carried out. furthermore, the disease selection limits the generalizability to other rheumatic diseases and only patientreported items were collected in the study. we received a lot of positive feedback especially from older patients that would not have come in contact with rheuma auszeit otherwise. this feedback was however not properly measured. furthermore, rheuma auszeit is currently only available in german, limiting the potential international impact. the german umars version which we used has not been published yet, however the items are identical to the published german mars translation (28) . the system usability scale (sus) (24, 29) and net promoter score (nps) (24) are increasingly being used in app evaluation studies and could have enhanced our work. rheuma auszeit is well-accepted among patients suffering from different rheumatic diseases, different ages and digital competencies. physicians and other health care professional should be encouraged to integrate mhealth into their clinical routine. lacking customizability of rheuma auszeit diminished the total umars score and will likely limit long-term app usage by patients. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by ethics committee of the friedrich-alexander university erlangen-nürnberg (fau); (no. 418-18b). the patients/participants provided their written informed consent to participate in this study. al and jk wrote the draft manuscript. al, jk, ta, and nv performed the statistical analysis. all authors reviewed the draft and provided comments for changes, approved the final manuscript. mobile health usage, preferences, barriers, and ehealth literacy in rheumatology: patient survey study effectiveness of mobile apps to promote health and manage disease: systematic review and meta-analysis of randomized controlled trials eular points to consider for the development, evaluation and implementation of mobile health applications aiding self-management in people living with rheumatic and musculoskeletal diseases german mobile apps in rheumatology: review and analysis using the mobile application rating scale (mars) mobile health technologies for the management of rheumatic diseases: a systematic review of online stores in brazil p4 medicine: how systems medicine will transform the healthcare sector and society nutzung von medizin-apps und online-plattformen unter deutschen rheumatologen acceptance, usage, and barriers of electronic patient-reported outcomes among german rheumatologists: survey study digital rheumatology in the era of covid-19: results of a national patient and physician survey digital health transition in rheumatology: a qualitative study response to "development and validation of the user version of the mobile application rating scale (umars) apps for people with rheumatoid arthritis to monitor their disease activity: a review of apps for best practice and quality association between user engagement of a mobile health app for gout and improvements in self-care behaviors: randomized controlled trial discrepancies between patients and physicians in their perceptions of rheumatoid arthritis disease activity development and validation of the user version of the mobile application rating scale (umars) early detection of dengue fever outbreaks using a surveillance app (mozzify): cross-sectional mixed methods usability study clinicians' perceptions of ptsd coach australia wearable sensor-based exercise biofeedback for orthopaedic rehabilitation: a mixed methods user evaluation of a prototype system validation of the mobile application rating scale (mars) mobile app rating scale: a new tool for assessing the quality of health mobile apps the concept of ehealth literacy and its measurement eheals: the ehealth literacy scale the icancope pain self-management application for adolescents with juvenile idiopathic arthritis: a pilot randomized controlled trial feasibility of self-monitoring rheumatoid arthritis with a smartphone app: results of two mixed-methods pilot studies impact of assessing patient-reported outcomes with mobile apps on patient-provider interaction incorporating a static versus supportive mobile phone app into a partial meal replacement program with face-to-face support: randomized controlled trial patient adherence with a smartphone app for patient-reported outcomes in rheumatoid arthritis the german version of the mobile app rating scale (mars-g): development and validation study patient and clinician views on an app for rheumatoid arthritis disease monitoring: function, implementation and implications the authors thank all participating patients and the whole team of medizinische klinik 3 for their support of this project. the present work was performed in fulfillment of the requirements for obtaining the degree dr. med for the first author, al, and is part of the phd thesis of the last author, jk (ageis, université grenoble alpes, grenoble, france). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2021.715345/full#supplementary-material key: cord-0009982-or5nzvw3 authors: davies, janet m title: molecular mimicry: can epitope mimicry induce autoimmune disease? date: 1997-04-01 journal: immunol cell biol doi: 10.1038/icb.1997.16 sha: 0ef8953414af2b480b5dec741a4a913ed1380321 doc_id: 9982 cord_uid: or5nzvw3 mimicry of host antigens by infectious agents may induce cross‐reactive autoimmune responses to epitopes within host proteins which, in susceptible individuals, may tip the balance of immunological response versus tolerance toward response and subsequently lead to autoimmune disease. epitope mimicry may indeed be involved in the pathogenesis of several diseases such as post‐viral myocarditis or chagas disease, but for many other diseases in which it has been implicated, such as insulin‐dependent diabetes mellitis or rheumatoid arthritis, convincing evidence is still lacking. even if an epitope mimic can support a cross‐reactive t or b cell response in vitro, its ability to induce an autoimmune disease in vivo will depend upon the appropriate presentation of the mimicked host antigen in the target tissue and, in the case of t cell mimics, the ability of the mimicking epitope to induce a proliferative rather than anergizing response upon engagement of the mhc‐peptide complex with the t cell receptor. b cell presentation of mimicking foreign antigen to t cells is a possible mechanism for instigating an autoimmune response to self antigens that in turn can lead to autoimmune disease under particular conditions of antigen presentation, secondary signalling and effector cell repertoire. in this review evidence in support of epitope mimicry is examined in the light of the necessary immunological considerations of the theory. epitope mimicry is widely thought to be the mechanism for the induction of autoimmune disease. the thcor\' is that an infectious agent (parasite, bacteria, yeast or virus) displays epitopes immunologicaily resembling host determinants and due to the minor antigenie diticrences between the two. the pathogen's epitope is able to induce an immune response that breaks tolerance to the host epitope. the cross-reactive t or b cell is then able to induce a pathogenic autoimmune response that leads to disease.' bacterial urinary tract infections have been suggested to induce cross-reactive immune responses to antigens in the liver epithelium that contribute to the development of primary biliary cirrhosis (pbc).-mimicr\ ofa self antigen, the zp3 zona pellucida antigen, by another unrelated self molecule, the nicotinic acetylcholine receptor delta chain, has even been proposed as an instigator of autoimmune reactivity to ovary cells.-m olecular mimicry is a widely adopted term but it is more correct when considering the induction of autoimmune disease by mimicking pathogens to use the term epitope mimicry. this distinguishes it from other types of correspondence: dr janet m davics, department of biochemistry and molecular biology. monash university. clayton vic. 3168. australia. received l9june 1996; accepted 1 october 1996. molecular mimicp.' such as ligand-reccptor messenger rna mimicr>'' and mimicr\ of cytokines and chemokines." all types of mimicr\ may be strategies used by pathogens to gain entry to target cells, to manipulate inter-and intra-cellular processes or to avoid immune responses that limit their life cycle in the host. autoimmunity resulting from epitope mimicrv may be an unfortunate by-product of the anti-pathogen immune response that ean lead to disease in prc-disposed individuals. the scope of this review does not permit a comprehensive account of all studies on epitope mimicr>' that have emerged recently. other reviews on mimicr>^ and its role in arthritis,'' aids" and diabetes'" arc available. in this paper, the evidence needed to support the case for epitope mimicrs' in the induction of autoimmune disease will be addressed. the type of data which is presented to support the case for epitope mimicry and the necessary immunological considerations will be examined. there are three points to be made to establish the phenomenon of antibody cross-reactivity: (i) some antibodies can react with self; (ii) some auto-antibodies also react with a non-self antigen; and (iii) some antibodies to foreign antigens can cross-react to self antigens. it has been observed in a panel of 600 anti-viral mab that as many as 3.5% cross-react with mammalian proteins," in experiments using mice constitutively expressing the hcl-2 gene that inhibits apoptosis and hence, clonal deletion, antibodies to dsdna were generated after immunization with the bacterial hapten pneumococcal cell wall.'-many experimental or clinical examples of auto-antibodies could have been cited here to support the existence of autoantibodies in the peripheral b cell repertoire but this paper'-is particularly pertinent to the topic of mimicry since the elicitor of the anti-dsdna antibodies was a bacterial hapten. among the normal human population there is a prevalence of natural auto-antibodies to intracellular molecules such as dna, nuclear and cytoskeletal proteins which for the major part arc not pathogenic but may contribute to the normal regulation of the immune system.'-"* the presence of auto-reactive t cells in the periphery is exemplified by the ability to expand t cells of multiple sclerosis (ms) patients to the auto-antigens of myelin, for example myelin basic protein (mbp).'"^ a new study of graves" disease has revealed y5 t cells reactive to self epitopes expressed by thyroid cells.'^ demonstration of cross-reactivity of t cells is relatively new but several papers provide evidence for its occurrence. plasticity in the recognition of mbp-specific t cell clones for various analogues of the immunodominant mbp epitope in ms has been shown by bhardwaj e( al.^'' wucherpfennig and strominger'^ tested the ability of 128 viral and bacterial peptides identified by data base searching as having sequence similarity to a motif of the immunodominant mbp epitope in ms for reactivity with t cell clones restricted by the ms-assoeiated hla dr2 presenting celts and found eight of these peptides induced cross-reactive proliferative responses.'^ these studies show that a degree of difference between peptides can be accommodated by the trimolecular tcr-mhc-peptide interaction. the ability to find cross-reactive t cells or antibodies which can recognize self epitopes does not necessarily mean an autoimmune disease will develop. immunological tolerance to self molecules is a balance that is normally maintained by mechanisms such as thymic and peripheral cional deletion via apoptosis. direct t cell suppression, or antigen specific anergy.'^"-" the capability of crossreactive immune responses to break tolerance and induce autoimmune disease depends on numerous intrinsic and extrinsic factors such as the ability of host mhc molecules to bind and present particular antigens, the likelihood of a peptide being processed for binding to the mhc, the existence in the periphery of b cells with antibody or t cells with receptors specific for self epitopes and the level of cytokines induced after an immune recognition event. epitope mimicry may be one means of breaking immunological non-responsiveness to an endogenous tissue an-tigen. the route by which a foreign antigen is presented may be quite different to that of an endogenous molecule which is likely to be presented on the cell surface in the groove of mhc class i. an antigen of an infectious agent is likely to be presented in the groove of mhc class ii on the surface ofa professional apc. such as a dendritic cell. or an activated b cell that could provide the appropriate secondary signalling, such as b7. for stimulating a proliferative response. this difference needs to be considered as does the effect of interaction ofa peptide mimic with the tcr. the significance of t cell stimulation with epitope mimics or altered peptide ligands that fail to induce proliferation will be discussed later. both cross-reactive antibody and t cell responses may contribute to the pathogenesis of an autoimmune disease. t cell pathogenesis is exemplified by several experimental models of autoimmune disease which can be transferred to naive laboratorv animals using t cells primed in vimno auto-antigens such as mbp for experimental allergic encephalitis-' and heat shock protein (hsp) for adjuvant arthritis (aa).--b cell-mediated autoimmune pathogenesis and indeed epitope mimicr\ is demonstrated in a murine model of myocarditis. antibodies generated after cmv infection were affinity purified against cardiac myosin. these antibodies cross-reacted with murine cmv polypeptides and could passively transfer myocarditis to naive mice.^-b esides their contribution to the pathogenesis of an autoimmune disease another link between b cell and t ceil activity is in antigen presentation. mamula el a!.-'* showed that murine b cells primed in vivo to the human small nuclear ribonucleoprotein (snrnp) antigen can be transferred to naive mice whose t cells subsequently proliferated in response to both the human and the murine snrnp antigen. presumably, the b cells reactive with the foreign snrnp antigen acted as apc. processing and presenting self snrnp to t cells in the recipients previously unable to proliferate to self snrnp and enabling their t cells to respond to self snrnp protein. an early experimental study of epitope mimicry by fujinami and oldstone-'* elucidates a number of important features of the theon of autoimmune disease induction by epitope mimicry. rabbits were injected with a hepatitis b virus polymerase peptide (igcygslpqe) that mimicked an epitope of mbp (tthvgslpqk) and which was able to induce antibodies to mbp in five out of seven rabbits. central nervous system lesions resembling some aspects ofthe demyelinating disease experimental allergic encephalomyelitis (eae) were observed in some rabbits. since then several lines of evidence support the theory of epitope mimicry. the first is exemplified by a paper describing primary sequence homologies between various viruses and the peptides ofthe auto-antigen la (ss-b) that were most frequently recognized by antibodies in sera of sjogren's syndrome (ss) and sle patients.-*^ in this paper and many others, only primary sequence similarity is listed as a suggestion of epitope mimicry. data on immune recognition ofthe potential mimic is lacking. the second line of evidence is the demonstration of cross-reactivity of disease relevant mab or atfinity purified antibodies to a different antigen of unrelated origin. this is exemplified by another murine model of myocarditis. a mab to streptococcus pyogenes cross-reacts with high molecular weight cardiac and skeletal myosin and this reaction can be absorbed by s. pyogenes. further to this a mab to ventricular myosin was found to crossreact with s pyogenes antigen.-t he third type of data presented for epitope mimicry is tcell cross-reaction. forexampic, the ability of the arthritogenic anti-hsp t cell clone of aa to cross-react with host proteoglycan.-^ another is a recent paper presenting data on t cell lines from 14 ms patients which were raised in vitro against mbp. some of which cross-react with a coronavirus epitope.-'^ further examples of data in support of the theory of epitope mimicry are listed in table 1 . if epitope mimicry by an infectious agent is to trigger an autoimmune response that leads to an autoimmune disease then several facts need to be established: (i) the presence ofthe pathogen needs to associate with the onset of symptoms or disease in a convincing proportion of cases: {ii) a clinically detectable immune response to the pathogen should be demonstrable at least at the onset of the disease and this response should be shown to cross-react with host antigens of the atfected tissue; (iii) also, the pathology of the disease ought to be consistent with the proposed immune response. these criteria are often hard to fulfil since the pathology inducing autoimmune disease may have commenced long before the onset of clinical symptoms which hampers research into the triggers of autoimmunity. a disease in which there is convmcing evidence for epitope mimicry is chagas disease induced by the parasite trypanosoma cruzi. first there is a real association of the parasite with the disease. during infection of the host, various antibody idiotypes arc elicited by a parasite, some of which directly cross-react with host proteins and may contribute to the pathogenicity ofthe infection.^^ autoantibodies and t cell responses arc directed agamst both cardiac and nervous tissue antigens.-^^-^'^ the prevalence of antibody cross-reactivity is high; 30 out of 30 chronic and 11 out of 13 acute chagas patients have antibodies to a 1 ?0 kda parasitic antigen which lyscs the blood stage of the parasite and cross-reacts with human and murinc striated and smooth muscle.""' more than one epitope ofa 160 kda parasite antigen induces cross-reactive antibodies to ditfercnt epitopes ofa 48 kda antigen of myentric and sciatic ner� cells."" since an autoimmune inflamma-tor\ pathology is evident in chagas patients, the pathology occurs in the absence of parasites in the local region and since there are several parasitic antigens which appear to induce antibodies to nerve and muscle antigens^^"" it is reasonable to suggest that molecular mimicry may be an initiating mechanism. there is a similarly strong case for mimicry of the 's" active site pcptidc oi aeet\l eholine receptor (achr) has sequence similarity 30 with the hsv gpd and antibodies to the gpd peptide x-r with achr and inhibit binding of ligand to achr. mab to tmev vp-1 protein x-r with oligodendrocytcs and augments 31 demyelination. mah to coxsackic b4 vp-i protein x-r with mouse a-cardiac myosin. 32 neutralizing antibodies to conformational epitopes ofthe coxsackie b3 capsid 33 protein induce myocarditis in naive mice; these neutralizing antibodies x-r with the surface of cultured cardiac fibroblasts and induce complement mediated lysis in vitro. mab to hsv 1 ribonucleotide reduetase x-r with p^ myelin protein. 34 patient derived igm mab to myelin associated glycoprotcin x-r with 35 90-100 kda protein of cttrohaitcr jtvcr^u.s and prcteus nuiri^uni. 23 of 63 patients" antibody reacts with ribonucleoprotein smd peptide 95-119 36 with sequence similarity to ebv nuclear antigen i (ebnal) polypeptide. three patient sera atfinity purified against the smd peptide x-r with ihe ebnal polypeptide and ebv transfected cells. ebv epstein barr virus. hsv herpes simplex virus. tmev theilcrs murine encephalitis vims. x-r cross-reacts. systemic lupus erythematosus antigen ofthe optic nerve and the retina being the cause of the eye pathology of river blindness in onchocerca volvulus patients. antibodies to the ov39 antigen ofthe parastte, which is recognized by o. volvulus patient sera, erossreact with a 44 kda antigen isolated from a human cdna librar\' that localizes to neural tissue in the retina. the two mimicking antigens are also able to stimulate crossreactive t cell responses.•*-the case for the epitope mimi-c17 leading to the pathogenesis ofthe eye disease is supported by the work with experimental uveitis in which a similar disease can be induced in lewis rats by immunization with the s antigen epitope"^^ and t cells specific for the epitope can adoptively transfer the disease.''•' the disease guillain-barre syndrome (gbs) deserves attention here since: (i) it fulfils the first criterion for epitope mimicr>-association with the pathogen; (ii) it is not frequently considered despite the observation that the postinfectious syndrome is clearly associated with antibodies to peripheral nerves; and (iii) it exemplifies the fact that epitope mimicr\' need not be confined to peptide epitopes but may include molecules such as glycolipids. there is a good association with intestinal infection with campylobacicr jcjtmi and the onset of gbs. a disease ofthe peripheral nervous system involving t cell and antibody reactivity to the myelin antigens p,,. p, and gangliosides gm,. gd,3 and gd^,, gt and/or gq.^ as well as increased expression of mhc class ii molecules on schwann cells. ifny. icam-1. and increased macrophage infiltration."^m imicry of the myelin gangliosides by the lps of the intestinal bacteria c. jejuni which can be isolated from patients with gbs. has been postulated to cause the antimyelin autoimmunity."*^ yuki et al.'^'' described the association of prior infection with c. jejuni serotype 19 and the development of gbs and noted that the carbohydrate of its lps is structurally identical to part of the gm, ganglioside. they also showed that c. jejuni can be isolated from patients with fisher syndrome (fs), the acute onset form of gbs which is characterized by antibodies to the gqih ganglioside. and that mab to the gq^, ganglioside and auto-antibodies to gqn, from fs cross-react to c jejuni lps. antibodies from c jejuni infected enteritis patients without neurological symptoms did not react with gangliosides. in a high percentage of gbs patients (9 out of 11) with previous c jejuni infections antibodies to gm i can be competitively inhibited by two strains of c. jejuni.'^'^ this data provides good circumstantial support for the hypothesis of mimicry inducing the neurological symptoms that develop after intestinal infection with c jejuni. an observation that some people who had clinical signs of enteritis but no neurological symptoms were infected with the same strain as those who develop fs"*"* implies that induction of neurological pathology is restricted by host factors, such as hla type, which would be in keeping with an autoimmunological mechanism of disease. the ability to show that antibodies to the glycolipids can cause the pathology of gbs would be necessary to support the case for epitope mimicry. a case for epitope mimicry in insulin-dependent diabetes mellitis (iddm) is emerging in the literature but reasonable association of the purported agent. coxsackie b virus and iddm has been difficult to establish. the 65 kda isoform of glutamic acid decarboxylase (gad) is believed to be the primary target antigen in iddm and auto-antibodies to gad are well documented in iddm^^ and sequence homology to the p2-c replication protein of coxsackie b enteroviruses has been noted.^' the coxsackie viruses have been associated with iddm by the ability to demonstrate t cell proliferation to three coxsackie strains in about 50% of 22 newly diagnosed iddm patients^-and increased prevalence of anti-coxsackie antibodies in newly diagnosed children with iddm.^"* t cell proliferation and antibodies to both the gad 65 in the region of amino acids 247-279 which encodes the sequence similar to the coxsackie p2-c antigen and the p2-c mimic have been demonstrated in four iddm patients.^"* antibodies to both the gad 65 and similar p2-c peptides have been found in both coxsackie b4 infected diabetic mice and iddm patients and response to each antigen can be competitively inhibited by the other.^^ other studies found that the t cell epitope of gad 65 lies in the region of amino acids 473-555, which is inconsistent with the hypothesis of mimicry of gad amino acids 250-273-^'' and mab to gad derived from one patient with iddm were not cross-reactive with the proposed mimicking sequences.^r abbit antisera against the coxsackie virus b4 p2-c peptide can cross-react and precipitate gad 65. however, these authors also showed that only 18% of iddm chidren and 10% of normal subjects have antibodies to this peptide.""' "^ none the less. coxsackie b viruses can potentiate diabetes in laboratory animals. anti-p2-c peptide antibodies react with gad 65 and prior infection with coxsackie b viruses, and in particular strain b4. is found in a number of iddm patients.^"'^'' it has been proposed that expression of p2-c during coxsaekie virus infection primes t cells to both the exogenous and endogenous gad antigen on p-cclls of the pancreas. whether gad is presented on the surface of p-cells in mhc molecules such that t cells could access and bind them has yet to be shown. microbial infections have long been blamed for the development of rheumatic fever via the induction of crossreactive antibodies to heart antigens."'^^"''-a popular theory which has subsequently been partially substantiated-'^ ''^ that molecular mimicry by so called prominent group a streptococcal antigens, now known to be epitopes in the m surface protein were inducing cross-reactions to antigens in human tissue.^' this is further supported by a report describing structural similarity between the streptococci sialyl lewis oligosaccharide and host cardiac selectin which could facilitate binding of bacteria to the heart endocardium^"* and possibly be a target for autoimmunity. expansion of the epidemiological evidence for the involvement of bacteria in rheumatic fever has been reviewed in smiley and in the 1980s extensive investigation was undertaken to show that mimicry by antigens of a number of enteric bacteria such as klebsiella, shigella and streptococcus caused the pathogenesis of reactive arthritis or ankylosing spondylitis. this area has been thoroughly reviewed elsewhere (for example smith et al}) and is not discussed here. however, it is noteworthy that the suspected target of mimicry was the hla-b27 molecule and this is the first implication that the target of mimicry was a molecule directly involved in antigen presentation. it is fair to state that a firm link between arthritic diseases and enteric bacteria remains controversial. a subsequent study has revealed two further candidates for mimicry by k. pneumoniae for the pathogenesis of ankylosing spondylitis (as). fielder el al.*^'^ propose that the observed sequence similarity between the puld gene product and b27. and the pula gene product and type 1 and iv collagen are involved via epitope mimicrv. in the induction of as. a significant igg and iga response to synthetic peptides corresponding to these regions of similarity was demonstrated by elisa with as patient sera but it can be noted that the antibody response to collagen was low. many have noted that the target of mimicry may be the hla molecules themselves. rheumatoid factor igm has been shown to recognize peptides of hla class i a2 a2 peptides and 53% of 30 rf positive ra patients reacted with these peptides.''^ t cells specific for m. leprae hsp 3-13 epitope are also activated by the v3 peptide ofthe hla class ii dr2 chain."ô f the tens of thousands of peptides associated with mhc class ii molecules on the surface of b cells, peptides derived from mhc domains are dominant.""^" it is plausible that mhc peptides would make good targets of autoimmune reactions because of this relatively high level of expression on the cell surface. an organism wishing to evade an immune response may seek to mimic epitopes which should be tolerized by the host (i.e. its own tissue type antigens). the fact that an attempted mimicry is incomplete and only similarity results, the epitope created by a mimic may be sufficient to overcome the tolerance mechanisms and prime the immune response against self the puzzle for this theory being the scenario for the induction of organ specific or non-systemic disease such as rheumatoid arthritis (ra) and iddm is that the target hla antigen would be expressed on cells systemically. it has been suggested that a viral antigen or bacterial hsp which mimics host hsp could be the initiating ra by molecular mimicry."" initially the proposed mimic was mycobacterial hsp 65 and the target was the human homologue hsp 65. now there is another level of complexity to the mimicry since not only is the host hsp 65 a target but there is also proposed mimicry by bacterial hsp and other antigens of infectious agents and the hla dr4 p-chain allele associated with ra susceptibility in the region where this allele differs from other non-ra associated alleles (eqrraa).^-one mimic postulated to be involved in the development of ra is the epitope of the e. coli hsp jnaj (qkraa) which has been used to induce rabbit antibodies that recognize the hla protein.^' during microbial infection, the immunological responses against the infectious agent generate expression of hsp of host origin and hsp of the infectious agent may also be expressed and responded to by auto-reactive t cells.^"' this process could initiate an autoimmune response against host synovium and thus commence the pathogenesis of ra. in the experimental model aa. responses to an epitope of mycobacterial hsp 65 by a specific t cell clone induces arthritis in lewis rats presumably by mimicr\' ofthe host hsp 65.-^-' numerous studies have shown that patients with ra have both humorat'' and cell-mediated immune responses to hsp 65 from both niycobactcriiil and human origin.^'•"^'^ in support ofa pathogenic role for immune responses to hsp in arthritis. born et al.^"* show th;it the recognition of hsp 65 is b\ yd t cells and antibodies to hsp have been observed to react with synovial tissues of arthritic rats and rheumatoid and ostcoarthritis in humans.^"* a case against the role of cither microbial hsp mimicr\' of hsp and/or of hla epitopes is made in an extensive study using elisa with hsp antigens of various sources. jarjour el a/.*" found increased reactivity to human hsp60 only in 20% of patients with mixed connective tissue disease not as. reiter's syndrome (rs) or ra patients. similarly. karopoulos et al.^^ found no difference in the serum antibody response to hsp 65 between ra. sle. mycobactcrially infected and multisystemic autoimmune disease patients and normal control subjects. in contrast. albani ct al.^-report that t cells from 18 out of 21 ra patients proliferated in response to the jnajpi peptide that contains the region of similarity to the hla molecule and that ra patient antibody but not control subject antibody responses to dnaj were inhibited by the c///(/ypl peptide. however, it should be noted that in their study anti-hsp antibodies were found in both ra and normal sera. it is possible that diberent epitopes are recognized by ra than other sera but in the study of karopoulos (va/.^' no difference between the two sera sets was found in the recognition of truncated forms of the hsp gene expressed in e coli and therefore this seems an unlikely explanation. the second sequence similarity with the ra-associaled dr4 haplotype (fqkraa) is with the 110 kda glycoprotein of ebv (balf-4). affinity purified antibodies from ebv-infected mononucleosis patients will cross-react with the hla dr4 antigen.^-'' ebv is a virus often implicated in the pathogenesis of ra and other rheumatic autoimmune diseases like slh due to serological reactivity to ebv antigens in some patients.**"^ also epitope mim-icry between ebna-1 and type i collagen has also been suggested as being implicated in the pathogenesis of ra.**ŵ ilson cl al.^^' report two further examples of sequence similarity and antibody reactivity to peptides mimicking hla dr4*0401 and not dr4*0402. ra patient sera and not as or normal sera reacted with peptides of: (i) p. mtrahtlis haemolysin (esrral) and the ra susceptibility sequence of dr4*0401 (eorraa);and(ii)/'. mirabili's urease (lrrel) and collagen xi (lrret). the implications of these studies are that hla association with ra maybe more than simply the effect of hla diff"erences on antigen presentation*^^ and linkage to other unknown co-segregating genes. however the case for epitope mimicry in the pathogenesis of arthritis is hindered by the fact that ihe proposed pathogens are fairly common and it is difficult to establish an exclusive association of specific infections with the disease. there is increasing evidence that mimicry of mhc molecules by the hiv contributes to the depletion of t cells in aids. many examples of mimicry between the hiv cnv proteins and hla molecules have been found and some of these are described in table 2 . it has also been shown that monoclonal anti-idiotypes against the gpl20 of hiv were able to compete with the peptide antigen defining the epitope and inhibit binding of the peptide with its antibody.'*-it is feasible that gpl20 itself could induce idiotypes that bind to the host cell determinants like the mhc and initiate an immune response to the cells bearing those molecules. if mimicry of mhc class ii is occurring in aids, as patients develop antibodies to the hiv cm\ t cells could be destroyed by autoimmune reactions. evidence for this aspect of pathogenesis is supported by the observation that the frequency of antibodies to (32 microglobulin free hla increase with higher disease severity scores."^ similarly, silvestris el al.'* report that antibodies to the hla molecule correlate with lymphocytopenia. evidence to support an alternative theory of direct viral effects is that plasma virons incorporate mhc molecules from their host cell into their envelope. the virus panicle may then be seen as self and avoid immune response, or act to prime the immune system against the aberrantly expressed mhc.^"* mimicry ofthe mhc class ii molecule at the cd4 binding site by gp 120 may result in partial stimulation of t cells binding through cd4 to the gpl20 without co-stimulation of t cell cd3 or cd28 by other normal apc molecules thus resulting in anergy or apoptosis of the t cell.''-'' the results discussed so far has arisen from either: (i) primar>' sequence similarities between autoimmune epitopes or key proteins; or (ii) discover\-of cross-reactive antibody or t cell reactions involved in the autoimmune disease. there are uncertainties in interpreting these results and other sequence similarities listed in the literature as evidence for epitope mimicry. the degree of similarity in mimicking peptides necessary for interaction of epitopes with antibody or tcr and mhc molecule, and the degree of divergence that will allow the mimic to escape the normal down-regulating mechanisms are the unknown entities. though the effects of amino acid substitutions in linear epitopes can to an extent be predicted by use of matrices such as that prethe letters in bold type represent identical amino acids present in both sequences. take into account the position of anchor residues for mhc binding or tcr binding sites which are more constrained than other amino acids in the epitope. the method of discovering mimics of an epitope defined by a primary sequence involves database searching for similar sequences then consideration as to the likely significance ofthe similar sequences. database search programs such as fastp''"^ were originally devised to find evolutionaryrelated protein families, hence matches with low level similarity over longer stretches rank higher than high levels of homology over short sequences. a factor to consider is the bias of the databases for human. e. coli and viral genomes. hence the sequences in the data bases, though they are constantly expanding in number, are limited to targets of interest to researchers. an essential consideration to be made when database searching for epitope similarity is that ofthe significance ofthe similarity. the probability of chance similarity in primar> sequences decreases in proportion with the length over which the similarity extends. the probability of incorporating by chance the same six amino acids in a row in two unrelated proteins is 1 in 20^ if each amino acid is considered equally likely to be associated next to any other amino acid. however, amino acids tend to pair together and not associate by random assortment."^ ""' many short oligopeptide motifs ranging in length from di-to octapeptides have been abundantly found in protein similarity searches of totally unrelated sequences."" '"-the frequency of homologous pentamers found in databases was determined to be 7.9 x 10"^ "" and of all possible tetrapeptides. 20% did not occur at all and 10% only occurred once in databases. weise and carnegie chose the value of greater than 60% similar over 6-15 amino acids for consideration as a peptide with potential physiological mimicry. not all linear sequence similarities give rise to antibody or t cell reactivity to the similar or mimicked sequence despite the ability to generate cross-reactive antibodies in experimental animals. for example, similar peptides with conservative amino acid substitutions of mbp and human t cell leukaemia virus-i (htlv-1) which induces j demyelinating encephalitis named htlv-i associated myelopathy (ham) were able to induce cross-reactive antibodies in rabbits yet neither peptide was recognized by ham patient sera or t cells.'""* a similar study investigating the role of mbp sequence similarity in the pathogenesis ofthe encephalitic condition of \ isna in macdi visna virus (mvv) infected sheep demonstrated crossreactivity of antibodies raised against the mvv peptide to the similar mbp peptide and sheep mbp but only the mbp peptide was recognized by the mvv-infected sheep antibodies and no t cell response could be detected to either peptide.'"mn the study of dyrberg et al. serological eross-reactivity was generated between some but not all hsv gpd peptides that shared sequence similarity with the aeetylcholine receptor a-in the previously mentioned study of wucherpfennig and strominger only 8 out of 128 peptides that had primary' sequence similarity to the mbp epitope motif were able to stimulate the t cell line, despite peptide selection based on structurally conserative amino acid substitutions.'^ peptides with 9 out of 12 identical amino acids of the grave's disease antigen thyroid peroxidase (tpo) did not support cross-reactivity of t cell clones specific for the tpo antigen because anchor residues binding to the mhc (dob1*0602/dqai*0102) groove resulted in a different conformation presented to the t cell clone.'"t hese data demonstrate that factors other than primary sequence similarity are needed for the generation of crossreactive b and t cell responses. the suspected mimics need to elicit t or b cells specific for their potential epitope. epitope prediction strategics have been designed for b cell epitopes""* and t cell epitopes'"" based on mhc binding motifs, amphipathy of alpha helices, surface probability, tlexibility and hydropathy which may provide an indication as to the epitopes within a protein sequence. epitope prediction programs are not universally applicable or reliable.'"t he need for epitope presenlai ion and antigen accessibility for t cell epitopes the similar sequences need to be presented in the appropriate mhc molecule associated with disease. hla binding studies ha� now enabled the prediction of peptides that will bind particular mhc motifs.''' the hla associations for particular autoimmune diseases are well characterized now and part of this association can be attributed to the ability of particular mhc haplotype to bind key epitopes through specificanchor residues."-'" the ra-associated and nonassociated dr types differ at four sites in the p-chain and binding specificity to peptides also differ.'^' the non-r.a drb 1*0402 mhc allcle which is associated with the autoimmune skin disease pemphigus vulgaris. differs in the p4 pocket ofthe mhc from the ra-associated drbi allele and this results in its ability to present the skin disease antigen dcsmoglein 3.""' the binding of hla a 0201 and b27 to ebv ebna-i protein peptides was found to be limited not because there are no mhc" binding motifs present in ebna-i but for other reasons not accounted for by the ability of the mhc to bind the peptides.' '^ after examination ofthe data in the paper by stuber el t//."^ it was evident that prediction of peptides which might bind the particular mhc does not always correspond to the experimental result of binding studies. more than mhc binding is necessary for ehective presentation of antigen to t cells. as well as an epitope being able to bind an mhc molecule, factors such as the proteolytic processing ofthe antigen in the specific cell and the resulting conformation ofthe mhc and bound peptide are important. in line with this. englehard and colleagues observed that the frequency of peptides eluted from mhc molecules did not correlate with the atfinity of those peptides for the mhc." ^ some amino acid changes in epitopes have been shown to actually enhance the reactivity of t cells."^"^ alternatively. t cell recognition of an epitope bound in mhc need not result in a proliferative immune response that is auto-degenerative. the effect of amino acid substitutions in sequence similarities is dependent upon numerous factors which need to be determined empirically for each sequence similarity identified or epitope being assessed for mimicry. for example, an epitope of mbp which induces eae in mice was substituted with alanine in up to five places and the corresponding peptide was still able to induce disease'' "^ which is convincing evidence in support of a degree of ffexibility in the interaction of tcr for mhc class il-epitope complexes. amino acid substitutions may alternatively result in analogues ofthe epitope which are presented by the mhc molecule, bind the tcr but deliver only partial secondary signals resulting in altered cytokine production and inhibition of proliferation.'-'^'-'. analogues ofthe influenza haemagglutinin 301-319 epitope bound the mhc class ii complex but t cell response was specifically and non-competitively inhibited due to lack of induction of intracellular inositol phosphate production, calcium accumulation and il-2 production.'--substitution of an anchor residue in the influenza matrix protein epitope resulted in the analogue's ability to bind the hla-b35 mhc complex but it was not able to induce t cell proliferation and also antagonized the proliferation of the t cells presented with the original epitope.'"^ therefore epitope mimics, which are effectively analogues of self epitopes. may likewise inhibit a reaction or fail to induce proliferation of t cells. analogues may act antagonistically and much lower concentrations ofthe analogue may inhibit the response normally resulting from the original epitope. fpitope analogues of the hsp 65 iso-iss'--* for aa and a combination of antagonist peptides ofthe encephalitic proteolipid protein epitope 139-151'^^ or mbp epitope 97-99' ^^ for eae have been used in therapy for prevention of the autoimmune disease at concentrations up to 10-fold lower than the original epitope concentration required to induce disease. mimicking peptide epitopes from pathogens may therefore act to anergize or down-regulate a response to self epitope. there are natural examples of viruses which contain and express altered epitopes that abrogate the immune response to its natural or dominant antigen version. natural antagonistic epitope analogues have been found in isolates of hiv'^^'^^ and hbv.'-t he ability to induce a cross-reactive t cell response to an hla allele by an ebv peptide'^^ was found to result in absence ofa particular t cell response to that epitope in individuals with that particular hla allele.'" therefore rather than a proliferative response being induced by the presence ofa similar epitope in the virus, the response had been abrogated and the people were tolerant to that epitope. page et ai.^^~ showed that though an altered peptide ligand was unable to stimulate t cell proliferation it was functional in the ability to induce negative selection of t cells in the thymus. in the light of the research presented in this paragraph it is evident that primary sequence similarity and even t cell recognition of similar peptides should be considered with guarded optimism when searching for potential instigators of autoimmunity through cross-reactive immune responses. a mimic may be as likely to tolerize as to induce a proliferative response depending on other factors such as the genetic haplotype and presence of co-stimulatory signals. a further consideration for the theor\' of mimicry is that the mimicking antigen need not be similar at the primary' amino acid sequence level. cross-reactivity of t cells between peptides from unrelated proteins with only marginal sequence similarity has been shown.'^''-'•^^ antibody cross-reactivity is often demonstrated without primary sequence similarity. a similar antigenie surface can be made up of associations of amino acids from distal regions of the same or different peptide chains ofa complex protein antigen or totally dissimilar molecules such as carbohydrate or nucieotide structures, or linear stretches of amino acids which display the same conformation to the antibody or tcr. for example, synthetic peptide libraries have been used to define the polyspecificity of mab to an oncogene peptide and it was found that certain peptides were mimotopes but had no amino acid homology to the original epitope and some peptides even had higher affinity binding to the mab than the original antigen.'^'^ in mice, the in vno expression ofthe polyoma virus t antigen from a plasmid carrying the t antigen gene was sufficient to generate antibodies reactive to double strand dna and histones.'"*^^ the mechanism by which expression ofa viral dna binding protein induced antibodies to either dna and dna binding proteins was not elucidated but it was stated by the authors that anti-dna antibodies in some sle patients are expressed simultaneously with polyoma virus production. another study showed that the anti-dna antibodies in sle may be directed against protein antigens or at least be crossreactive with a protein, hp8. selected from a cdna library by antibodies to dna.' ^^ hence there is molecular mimicry between the shape ofthe dna and this protein to which these antibodies also bind. it may be possible that the polyoma t antigen and other viral proteins act as mimics of host dna and are a priming target for the development of auto-antibodies in diseases like sle and ss. much ofthe data on mimicry is in reference to antibody cross-reactivity and one criticism ofthe theory of mimicry is that t cells mediate many ofthe autoimmune diseases. an hypothesis could be that b cells are intrinsically involved in autoimmune pathogenesis mediated by mimicry due to their role as apc and thus cross-reactive b cell responses may be crucial to the pathogenesis of autoimmune disease because they present mimicking epitopes to t cells. mamula and colleagues described two experi-ments in which either human snrnp or cytochrome c was used to prime an immune response in mice against the non-self protein and as a result, an immune response to the self protein was elicited. immunization with the murine protein could not induce a t or b cell response to the murine protein but immunization with the human homologue caused antibody production to the murine protein. transfer of self reactive b cells to naive mice facilitated a t cell response to the murine protein in recipients. ^"^ ' ^'' the importance of this research is that an autoimmune response was induced by a foreign molecule with only slight antigenie differences from the self molecule. similarly, mimicking epitopes of pathogens could in theory induce a b cell response to the mimic that can present antigen to t cells and break the mechanisms that are normally maintaining the self tolerance. james el al,^^^ injected a single peptide of auto-antigen rnp sm b/b' into numerous rabbits and antibodies to a range of epitopes from that molecule and other rnp were induced over time. it has been noted that six regions ofthe 60 kda ro antigen in sle share sequence similarity with the nucleocapsid (n) protein ofthe vesicular stomatitis virus (vsv) purported to be implicated in sle. immunization of rabbits with the vsv n protein induced antibodies reactive with the n protein and the similar ro antigen. repeated immunization with the n protein induced antibodies to numerous ro peptides including ones that did not share sequence similarity.'^"^ it was inferred that the n protein activated cross-reactive b cells that recognized the ro antigen and then acted as antigen presenting cells priming t cells to other ro peptides which in turn provided help to other b cells thus resulting in epitope spreading. it would follow then that a mimicking antigen, similar in only one epitope, may initiate a primary crossreactive response to that epitope that subsequently results in recognition of numerous epitopes in the mimicked host protein. if this were to be the case, one would expect that autoimmune diseases would be characterized by responses to many epitopes and the initial target would be difficult to determine. recognition of multiple antigens and epitopes is evident in iddm. sle, ra, pbc and probably most autoimmune diseases. clinical studies arc usually conducted in the phase of pathogenesis when multiple epitopes and antigens are recognized by polyclonal t and b cell responses. it is conceivably difficult to investigate the hypothesis of mimicry at this stage since the initial event leading to autoimmunity may have long since passed. a useful model for the induction of autoimmune disease based on the observations presented in this review is shown in figure 1 . a mimicking antigen sufficiently different from self proteins as to avoid tolerance, yet similar enough to self epitopes as to cross-react, primes a b cell that may produce antibodies that cross-react with a self protein. in addition or alternatively, the primed b cell presents the mimicking epitope bound in mhc class 11 to figure 1 mechanisms of autoimmune disease mduced by epitope mimicr\, b cells present mimicking epitopes of infcclious agents to t cells via a mhc class ii pathwa\. in addition, they produce antibodies with specificity for the instigating epitope that cross-react with host proteins combining the mimicked epitope. t cells interact with host tissue expressing the mimicked self epitope in the context of mhc class i and. depending on the appropriate secondar\ signalling, a pathogenic autoimmune response develops. as the immune response persists new epitopes arc processed from the target tissue and epitope spreading occurs. t cells in a manner that results in stimulation of the t cells that then interact with the antigen bound by mhc class 1 m situ. in this way a disease process could be initiated. as the disease progresses, new epitopes from the original and related molecules may emerge and mask the response to the instigating epitope. this model would account for the oligo or polyclonality seen in the t cell responses of autoimmune disease patients, for example ms or ra patients.'"*" '•^' this model does not explain why disease develops in a particular tissue if the target auto-antigen is common to all cells such as the mitochondrial pyruvate dehydrogenase complex which is the pri-mar>' auto-antigcn in pbc.'"*-this model depends on a self epitope being expressed in the context of mhc class i on the cell surface with the necessary secondary signallmg for t cell reactivity or in the instance of antibody crossreactivity the accessibility ofa b cell epitope. it is known that stimulation of inactive t cells with peptide epitopes complexed with soluble mhc class ii and no secondary signals undergo apoptosis.'"^^ the 'out clause' in this model, then, is that t cells inappropriately stimulated with antigen mimics would be likely to apoptose. or anergize, and thus an autoitnmune response would not ensue. a combination of factors such as local increased expression of cytokines, vascular adhesion molecules, hsp and mhc induced by infection may create an environment in which mimicry of host epitopes could initiate an ongoing autoimmune response in patients with the permissive genetic background. fortunately, the requirements for the induction of an autoimmune disease are multiple and the probability of an individual having the combination of all the environmental and genetic factors that lead to an autoimmune disease is relatively low. the existence of natural auto-antibodies and t cells that will cross-react with self molecules supports the notion that the immune system is able to cope with peripheral presence of auto-reactive b and t cells and that tolerance to self antigens is normally maintained in such an environment. mimicry may be one mechanism to break the balance of tolerance and trigger the autoimmune response but the mere presence of self determinants in a virus or bacteria need not result in pathogenesis. molecular mimicn.' and autoimmune disease detection of m2 antibodies in patients with recurrent urinary tract infection using an elisa and punfied pbc specific antigens. evidence for a molecular mimicrs-mechanism in the pathogenesis of primary biliary cirrhosis? antigen mim-icr>' in autoimmune disease sharingof amino acid residues critical for pathogenic t cell activation antigenie mimicry of an immunoglobulin a epitope by a hepatitis b virus cell attachment site viral mimicry and disease. today's human cytomegalovirus open reading frame i's2^ encodes a functional p chemokine receptor the autoimmune diseases ii walls 1. relationship between foodbomc bacterial pathogens and the reactive arthritides autoreactivity in hiv-1 infection: role of molecular mimicry coxsackieviruses and diabetes prabhakar bs et al molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues pathogenic autoantibodies are routinely generated during the response to foreign antigen; a paradigm for autoimmune disease natural autoamibodies t cells responsive to myelin basic protein in patients with multiple sclerosis self-reactive cytotoxic y5 t lymphocytes in graves' disease specifically recognize thyroid epithelial cells degenerate recognition ofa dissimilar antigenie peptide by myelm basic protein-reactive t cells molecular mimicry in t cell-mediated autoimmunity: viral petides activate human t cell clones specific for myelin basic protein tolerance, danger and the extended family clonal deletion versus clona! anergy; the role of the thymus in inducing self tolerance negative selection of lymphocytes a myelin basic protein specific t lymphocyte line which mediates experimental autoimmune encephalomyelitis a cartilage-mimicking t-cell epitope on a 65 k mycobacterial heat-shock protein: adjuvant arthritis as a model for human rheumatoid arthritis mouse cytomegalovirus infection induces antibodies which cross-react with virus and cardiac myosin: a model for the study of molecular mimicry in the pathogcnsis of viral myocarditis b cells process and present lupus autoantigcns that initiate autoimmune t cell responses. 7 amino acid homology between the encephalitogcnic site of myelin basic protein and virus: mechanism for autoimmunity serum antibodies from patients with primary sjogren's syndrome and systemic lupus erythematosus reeognise multiple epitopes on the la(ss-b) autoantigen resembling viral protein sequences myosin; a link between streptococci and heart myelin basic protein and human eoronavirus 229e crossreactive t cells in multiple sclerosis molecular mimicry and lyme borreliosis molecular mimiery and myasthenia gravis: an autoantigenic site ofthe acetylcholine receptor a-subunit that has biological activity and reacts immunologicaily with herpes simplex virus monoclonal antibody to theiler's murine encephalomyelitis virus defines a determinant on myelin and oligodendrocytes. and augments demyelination in experimental allergie encephalomyelitis identification of a putative shared epitope between coxsackie virus b4 and alpha cardiac myosin heavy chain molecular mimicry, anti-coxsackievirus b3 neutrahzing monoclonal antibodies, and myocarditis olsson t. a monoclonal antibody against hsv type 1 ribonucleotide reduetase cross-reacts with the p^, protein of peripheral nerve myelin monoclonal igm from patients with peripheral demychnating neuropathies cross-react with bacterial polypeptides auloantibodies from patients with systemic lupus erythematosus bind a shared sequence of smd and epstein-barr virus-encoded nuclear antigen ebna-i wallukat g ct al molecular mimicry between the immunodominant ribosomal protein po of trypanosinna cruzi and a functional epitope on the human p,-adrenergic receptor molecular mimicry by trypanosoma cruzt\ the fl-i 60 epitope that mimics mammalian nerve can be mapped to a i2-amino acid peptide trypanosoma cruzi infection enhances polyreactive antibody response in an aeute case of human chagas" disease a lytic monoclonal antibody to trypanosoma cruzt bloodstream trypomastigotes which recognizes an epitope expressed in tissue affected in chagas" disease fl-160 proteins of trypanosoma cruzi are expressed from a multigene family and contain two distinct epitopes that mimic nervous tissues molecular and immunological characterization of hr44. a human oeular component immunologicaily cross-react ice with antigen ov39 of onchocerca volvulus the use of synthetic peptides in the study of experimental autoimmune uveitis molecular mimicry between uveitopathogenic site of retinal s-antigen and escherichia coli protein: induction of experimental autoimmune uveitis and lymphocyte cross-reaction immunopathogenesis and treatment of the guillain-barre syndrome-part i immunopathogenesis and treatment of the guillain-barre sytidrome-part il a bacterium lipopolysaccharide that elicits guillain-barre syndrome has a gml gangliosidc-like structure igg antibodies and campylobacter bacteria in guillain-barre syndrome: evidence of molecular mimicry takahashi m el al molecular mimicry between gqi^, ganglioside and lipopolysaccharides of campylohacter jetunt isolated from patients with fisher's syndrome autoantibodies to glutamate decarboxylase in insulin dependent diabetes mellitub autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes melhtus proliferative lymphocyte responses to viruses containing sequence similarity to glutamie aeid decarboxylase in insulin-dependent diabetes a prospective study of the role of coxsackic b and other cntero\ irus infections in the pathogenesis of iddm cellular immunit\ to a determinant common to glutamate dccarboxylase and coxsackie virus in insulin-dependent diabetes antibodies to glutamic acid decarboxylase and p2-c peptides in sera from coxsackie virus b4 infected mice and iddm patients immunodominant epitopes of glutamic acid decarboxylase 65 and 67 m insulin-dependent diabetes mellitus sehoel b et al sequence homology ofthe diabetes-associated autoantigen glutamate decarbox-\lase with coxsackie b4-2c protein and heat shock protein 60 mediates no molecular mimicry-of autoantibodies antibody crossreactivity induced by the homologous regions in glutamic aeid decarboxylase (gad^s) and 2c protein ot coxsackievirus b4 coxsackie b virus infection and onset of childhood diabetes a theory of the pathogenesis of rheumatic fever, glomerulo-nephritis and other autoimmune disease triggered by infection rheumatic fever and the strepioi^occus. another look at molecular mimicry sequence of myosin-crossreactive epitopes of streptococcal m protein cytotoxic and viral neutralizing antibodies crossreact with streptococcal m protein, cntcrovlruses, and human cardlae myosin crossreactivity between human sialyl lewis oligosaccharide and common causative oral bacteria of infective endocarditis southwestern internal medicine conference: the role of infections in the rheumatic diseases: molecular mimicry between bacterial and human stress proteins^^l/?? molecular mimicry and ankylosing spondylitis: possible role ofa novel sequence in pullulanase of klebsiella pneumoniae igm rheumatoid factors react with human class i hla molecules mycobacterium /c7jrat'-speeific t cells from a tubereuloid leprosy patient suppress hla-dr3-restricted t cell responses to an immunodominant epitope in 65-kda hsp of mycobacteria structure of peptides associated with class 1 and class ii mhc molecules self-peptides bound to the type i diabetes associated class ii mhc moleeules hla-dql and hla-uq)^ from the shared epitope hypothesis to a peptidie model of rheumatoid arthritis hla-d region antigens in patients with rheumatoid arthritis the susceptibility sequence to rheumatoid arthritis is a eross-reactive b cell epitope shared by the escherichia coli heat shock protein dnaj and the histoeompatibility leukocyte antigen drb10401 molecule recognition ofa peptide antigen by heat-shock-reactive ys t lymphocytes antibody levels to mycobacteria in relation to hla type: evidenee for non-hla-linked high levels of antibody to the 65 kd heat shock protein of m. bovis in rheumatoid arthritis proliferative response to synovia! fluid and peripheral blood mononuclear cells to arthritogenic and non-arthritogenic microbial antigens and to the 65-kda myeobacterial heat-shoek protein stimulatory response towards the 65 kda heat shock protein and other mycobaeterial antigens in patients with rheumatoid arthritis reeognition of human 60 kd heat shock protein by mononuelear cells from patients with juvenile chronie arthritis antibodies to the mycobacterial 65-kd heat-shock protein are reactive with synovial tissue of adjuvant arthritic rats and patients with rheumatoid arthritis and osteoarthritis autoantibodies to human stress proteins: a survey of various rheumatic and other infiammatory diseases antibody reaetivity to myeobaeterial 65 kda heat shock protein: relevance to autoimmunity positive selection in autoimmunity: abnormal immune responses to a bacterial dnaj antigenie determinant in patients with early rheumatoid arthritis susceptibility to rheumatoid arthritis maps to a t-cell epitope shared by the hla-dw4 dr p-i chain and the epstein-barr virus glycoprotein gpl 10 potential role of epstein-barr virus in sjogren's syndrome and rheumatoid arthritis sulitzeanu d. crossreactivity between the ebna-1 pi07 peptide. collagen, and keratin: implications for the pathogenesis of rheumatoid arthritis shared amino acid sequences between major histoeompatibility complex class ii glycoproteins. type xi collagen and proteus mifabilis in rheumatoid arthritis peptide binding speciticity of hla-dr4 molecules: correlation with rheumatoid arthritis association identitication of homologous regions in human immunodeficiency virus i gp41 and human mhc class ii p 1 domain autoimmune response in aids kozhich at ct al. antibodies to mhc class ii peptides are present in hi v-1 -positive sera human immunodeficiency virus type i gpl20 c5 region mimics the hla class i al peptide-binding domain induction of antibodies to the envelope protein ofthe human immunodeficiency virus by immunization with monoclonal antiidiotypes autoantibodies against p2-microglobulin-free hla antigens in hla class 11 on hiv particles is functional in 113 superantigen presentation to human t eells: implications for hiv pathogenesis molecular mimicry in the pathogenesis of aids the hiv/mhc/mycoplasma triangle cognitive features of eontinuous antigenie determinants./. mol rccoi^ predicting isomorphie residue 1 15 replacements for protein design searehing for molecular mimicry in lentiviral diseases of sheep and goats rapid and sensitive protein similarity searehes repeating sequenees and gene duplication 11 7 in proteins oligopeptide biases in protein sequenees and their use in predicting protein coding regions in nucieotide sequences to be or not to be a responder in t-cell responses: ubiquitous oligopeptides in all proteins an approaeh to searching protein 119 sequences for superfamily relationships or chance similarities relevant to the molecular mimicry hypothesis: application to the basie protein of myelin. / neurochem mimicry between htlv-i and myelin basic protein: no response in htlv-i-assoeiated myelopathy patients mim-121 iery of a 21.5 kda myelin basic protein peplide by a maedi visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep. \'el peptides as probes to study molecular mimicr\ and virus-induced auloimmunity similar antigenie surfaces, rather than sequence homology. dictate i 24 t-cell epitope molecular mimiery the antigenie index: a novel algorithm for predicting antigenie deteminants t-cell antigenie sites tend to be amphipathicstructures./'rf)t predicting antigenie sites on proteins new methods to predict mhc-bindmg .sequences within protein antigens endogenous peptides bound to hla-a3 possess a specific combination of anchor residues that permit identification of potential antigenie peptides high-affinity binding of short peptides to major histoeompatibility complex class ii moleeules by anchor combinations structural basis for major histoeompatibility complex (mhc)-linked susceptibility to autoimmunity: charged residues ofa single mhc binding pocket confer selective presentation of self-peptides in pemphigus \ulgaris hla-a020i and hla-b7 binding peptides in the ebv-encoded ebna-1 ebna-2 and bzlf-i proteins delecled in the mhc class i alleles in ebna-l southwood s ct al natural!) processed peptides longer than nine amino acid residues bind to the class i mhc molecule hla-a2.1 with high affinity and in differeni conformations a single amino acid change in a myelin basic protein peptide confers the capacity to prevcnl rather than induce experimental autoimmune encephalomyelitis the importanee of dominant negative effects of amino acid side chain substitution in pcptidc-mhc molecule interactions and t cell recognition peptidemajor histoeompatibility complex class ii complexes with mi�d agonist/antagonist properties provide evidence for ligand-related differences in t cell receptor-dependent intracellular signaling significance of t-ccll stimulation b>' altered peptide ligands in t cell biology antigen analog-major histocompatibilitv complexes act as antagonists ofthe t cell receptor b35-restrictcd epitope modified at an anchor residue results in an antagonist peptide disease inhibition by major histoeompatibility complex binding peptide analogues of diseaseassociated epitopes: more than blocking alone arrhenius t et al 1 cell receptor antagonist peptides are highly effcetive inhibitors of experimental allergic encephalomyelitis allegretta m et at. treatment of experimental encephalomyelitis with a peptide analgoue of myelin basic protein genetic variability in hiv-1 gpl20 affects intcraetions with hla moleeules and teell reeeptor t-cell activity antagonized by naturally occurring hiv-1 gag variants natural variants of cytotoxic epitopes are t-cell receptor antagonists lor antiviral cytotoxic t cells an alloresponsc in humans is dominated by cytotoxic t lymphocytes (ctl) cross-reactive with a single epstein-barr virus ctl epitope: implications for gratvverus-host disease argaet vp. t cell receptor repertoire for a viral epitopc in humans is diversified by tolerance to background major histoeompatibility complex antigen snoke k i7 f^(/. negative selection of cd4 * cds * thymocytes by t-eell receptor peptide antagonists. prcc nail.-had sci cross-recognition by t cells of an epitope shared by two unrelated mycobaeterial antigens elucidation of monoclonal antibody polyspecificity using a synthetic combinatorial librara seternes 0-m. hey aw et al in vivo expression ofa single viral dna-binding protein generates systemic lupus erythematosus-related autoimmunity to doublestranded dna and histoncs dna mimies a self-protem that may be a target for some anti-dna antibodies in synthetic lupus erythematosus breaking t cell tolerance with foreign and self co-immunogens: a study of autimmune b and t cell epitopes of c>1oehrome c immunoglobulin epitope spreading and autoimmune disease after peptide immunization: sm b/b'-derived pppgmrpp and ppp-girgp induce splieeosome autoimmunity immunisation with vesicular stomatitis virus nucleoeapsid protein induces autoantibodies to the 60 kd ro ribonucleoprotein particle heterogeneity ofthe t-cell receptor p gene rearrangements generated in myelin basic protein-speeific t-eell clones isolated from a patient with multiple selcrosis characterization of t-eell receptor ap repertoire in synovial tissue from different temporal phases of rheumatoid arthritis is primar�iliar\'cirrhosis an autoimmune disease antigen-speeific apoptosis in immortalized t cells by soluble mhc class ll-peptide complexes key: cord-0029472-ttytoz3v authors: torequl islam, muhammad; quispe, cristina; herrera-bravo, jesús; rahaman, md. mizanur; hossain, rajib; sarkar, chandan; raihan, md abdur; chowdhury, md. mashrur; uddin, shaikh jamal; shilpi, jamil a.; marcelo de castro e sousa, joão; melo-cavalcante, ana amélia de carvalho; mubarak, mohammad s.; sharifi-rad, javad; calina, daniela title: activities and molecular mechanisms of diterpenes, diterpenoids, and their derivatives in rheumatoid arthritis date: 2022-03-25 journal: evid based complement alternat med doi: 10.1155/2022/4787643 sha: 94bf9fbd6a6dd5c5924e49861f488ff7e9d0b9e7 doc_id: 29472 cord_uid: ttytoz3v diterpenes and their derivatives have many biological activities, including anti-inflammatory and immunomodulatory effects. to date, several diterpenes, diterpenoids, and their laboratory-derived products have been demonstrated for antiarthritic activities. this study summarizes the literature about diterpenes and their derivatives acting against rheumatoid arthritis (ra) depending on the database reports until 31 august 2021. for this, we have conducted an extensive search in databases such as pubmed, science direct, google scholar, and clinicaltrials.gov using specific relevant keywords. the search yielded 2708 published records, among which 48 have been included in this study. the findings offer several potential diterpenes and their derivatives as anti-ra in various test models. among the diterpenes and their derivatives, andrographolide, triptolide, and tanshinone iia have been found to exhibit anti-ra activity through diverse pathways. in addition, some important derivatives of triptolide and tanshinone iia have also been shown to have anti-ra effects. overall, findings suggest that these substances could reduce arthritis score, downregulate oxidative, proinflammatory, and inflammatory biomarkers, modulate various arthritis pathways, and improve joint destruction and clinical arthritic conditions, signs, symptoms, and physical functions in humans and numerous experimental animals, mainly through cytokine and chemokine as well as several physiological protein interaction pathways. taken all together, diterpenes, diterpenoids, and their derivatives may be promising tools for ra management. arthritis is a long-term musculoskeletal illness marked by inflammation of the joints. rheumatoid arthritis (ra) is one of the most common kinds of arthritis [1] . it is a long-term condition marked by inflammatory synovitis. joint asymmetry and invasive inflammation are common symptoms of ra, which can lead to joint deformity, dysfunction, and even loss of function. adults in rich countries have a prevalence of 0.5-1.0%, with 5-50 new cases per 100,000 persons each year. women and the elderly, on the other hand, are the ones who suffer the most [2] . although the exact origin of ra is unknown, medicinal therapy is a common and effective treatment option for ra patients. treatments for ra include nonsteroidal anti-inflammatory medications (nsaids), corticosteroids, diseasemodifying antirheumatic medicines (dmards), and biological response modifiers [3] . all of these anti-ra drugs, unfortunately, have numerous negative effects. nsaids may endanger patients' lives by increasing the risk of upper gastrointestinal (gi) haemorrhage, liver, and kidney damage [4, 5] . furthermore, headaches, cognitive impairments, and allergic reactions are common reasons for patients to stop taking nsaids, limiting their usage. infection, hypersplenism, hypertension, osteoporosis, and fractures are all possible side effects of long-term corticosteroid usage [6, 7] . vomiting, diarrhea, rashes, low white blood cell (wbc) counts, and impaired liver and renal function are also side effects of dmards [8, 9] . biological therapies with high pharmacological selectivity and fewer side effects provide novel ra treatment alternatives [10] . regrettably, these are pricey. as a result, many patients may be unable to afford these drugs [11] . as a result, it is critical to seek out treatments that have a positive therapeutic benefit, few side effects, and are affordable. many ailments are treated according to conventional medical principles. many major studies on therapeutic items with natural origins have been conducted by modern scientists. plants or their derivatives, marine items, and so forth are examples. ese natural items have been discovered as a promising treatment option for ra [12] . aside from that, several conventional pharmaceutical formulae for ra care have been difficult. two significant features of this method are the use of nutraceuticals and polyherbal approaches [13, 14] . however, when these preparations are used in combination with other drugs, they may cause health problems. as a consequence, researchers devised a novel strategy for extracting active chemicals from some of these things. terpenes, flavonoids, catechins, quinones, alkaloids, anthocyanins, and anthoxanthins are just a few of the plant-derived phytochemicals that can alter t cell development, inflammatory signaling pathways, and synoviocyte death. as a result, they can be utilized to treat rheumatoid arthritis [15] . diterpenes are a diverse group of structurally diverse natural chemicals abundant in nature [16, 17] . ese are c20 compounds containing four isoprene (c 5 h 8 ) units that may be found in both terrestrial and marine settings in plants, fungi, bacteria, and animals [18, 19] . several diterpenes are potential pharmaceutical candidates due to their exceptional pharmacological effect [20] [21] [22] [23] . some diterpenes are considered to be the defining traits of a genus, making them taxonomically significant [24] . natural diterpenoid compounds come in a wide range of chemical forms and contain many medicinal and economically relevant molecules. all diterpenoids are made from the same substrate, (e, e, e)-geranylgeranyl diphosphate, which is then cyclized into one of the multiple scaffolds by a diterpene synthase [25] . secondary metabolites with 20 carbon atoms result from the condensation of four isoprene units. diterpenoids are divided into approximately 45 distinct categories, and they are also present in marine animals, where their skeletons are fascinating [26] . based on their skeletal nucleus, diterpenes are classified as linear, bicyclic, tricyclic, tetracyclic, pentacyclic, or macrocyclic. ey are usually found in nature polyoxygenated, with keto and hydroxyl groups that are commonly esterified by tiny aliphatic or aromatic acids [27] . diterpenoids have a variety of biological functions, including antioxidant [23, 28] , anti-inflammatory [29, 30] , and immune-modulatory action [31] . given the significance, the goal of this study is to outline the effects of diterpenes, diterpenoids, and their derivatives on ra based on current understanding. a search with the keywords "diterpene and rheumatoid arthritis," "diterpenoid and rheumatoid arthritis," "diterpene and arthritis," "diterpenoid and arthritis," "diterpene derivative and rheumatoid arthritis," and "diterpene derivative and arthritis" was conducted in the pubmed, science direct, google scholar, and clinicaltrials. gov databases. a total of 2708 records were found. after screening, among them, this study used 48 published records that are related to its aim. is study includes only the records of having antiarthritic or anti-ra along with anti-inflammatory activities of the diterpenes, diterpenoids, and their derivatives obtained from various sources (e.g., medicinal plants and marine origins) on various test systems (e.g., humans), laboratory animals (e.g., mice, rats, and rabbits), and their derivatives (e.g., cells, tissues or organs). most of the diterpenes, diterpenoids, and their derivatives have antioxidant and anti-inflammatory properties, and for this reason, this study does not include them. reports on crude extracts or fractions without chemical characteristics having antiarthritic or anti-ra effects were also excluded in this study. is study mainly focuses on the anti-ra activities of diterpenes, diterpenoids, and their derivatives. however, it will also focus on the antiarthritic effects of these substances based on updated database records (till 31 august 2021). inflammatory cytokines according to their antigen response activities. tnf-α, interleukins (ils) (e.g., il-1β, -6, -7, -15, -17, -18, and -23), interferon-gamma (ifn-c), and granulocyte-macrophage colony-stimulating factor (gm-csf) have all been found to limit inflammation in the progression of ra. in the synovium, synovial fluid, serum, and peripheral blood of ra patients, these cytokines were detected in high amounts [32] [33] [34] [35] [36] [37] [38] . t cell trafficking and proinflammatory cytokines such as tnf-α, il-1β, il-6, and mmps are reduced when il-7 is blocked, which lowers joint inflammation [39] . e major cause of il-23-induced synovial inflammation (rors) is the activation of janus kinase (jak)/signal transducer and activator of transcription (stat), tyrosine kinase 2, nf-κb, and retinoic acid receptor-related orphan receptors [40] . macrophages can produce a variety of cytokines such as tnf-α, il-1β, -6, -7, -15, -18, and -23. in this regard, tnf-α may stimulate fibroblast-like synoviocytes (fls) and synovial cell proliferation through nuclear factor kappa-b (nf-κb) and extracellular regulated protein kinases (erk)-1/2-e26 transformation-specific (ets)-1 regulatory pathways [37] . consequently, several inflammatory mediators such as il-6 and matrix metalloproteinases (mmp), mmp-1 and mmp-3, are secreted and increase inflammation [41] . small molecular metabolites such as pgs, lipoxins (lxs), platelet-activating factor (paf) and leukotrienes (lts), nitric oxide (no), and ros play important roles in the physiopathology of ra [42] . pg expressions such as pgd2, pge2, pgf2a, pgi2, pgj2, and txa2 are aberrant in ra [42] . lxs derived from arachidonic acids, such as lxa4 and lxb4, possess anti-inflammatory properties. lxa4 can reduce memory b cell response in ra patients' synovial tissues by engaging the lipoxin a4 receptor (alx)/formyl peptide receptor-2 (fpr-2) and, therefore, reduce inflammation [43, 44] . circulating platelet activation affects leukocyte activity and contributes to inflammation development in ra patients [45] . tnf-α-regulated pathways are known to control paf, and tnf-α antagonists decrease platelet activation in active ra [46] . chemokines have a role in the underlying pathophysiology of ra by attracting leukocytes and influencing angiogenesis. published research indicated that xc chemokines and their receptors (such as xcl1 and xcr1) and cx3c chemokines and their receptors (e.g., cx3cl1 and cx3cr1) are upregulated in ra patients' mononuclear cells (mncs) and fls, respectively [47, 48] . numerous inflammatory chemokines are mostly generated in the joints of ra patients by synovial macrophages and fls, while cx3cl1 is produced by synovial endothelial cells. e chemokines xc and cx3c are linked to the recruitment of t lymphocytes and synovial fibroblasts. furthermore, cx3cl1 and xcl1 stimulate the migration of monocytes and subchondral mesenchymal progenitor cells into the ra synovium, respectively [49] . cc chemokines including ccl2-5, ccl7, ccl13, ccl14, ccl16, ccl18-21, and ccl-25 are differentially expressed in ra plasma and synovium [50] . an upregulated cc chemokine ccl5 is significantly correlated with swollen joints, erythrocyte sedimentation rate (esr), and c-reactive protein (crp) in ra patients [51] . on the other hand, cxc chemokines, such as cxcl1, cxcl2, cxcl5, cxcl8, cxcr1, and cxcr2, are generally involved in neutrophil chemotaxis [52] . e chemokine cxcl10 promotes effector t cells into the joint [53] . e expression of peroxisome proliferator-activated receptor-gamma (pparc or pparg) in human monocytes/ mdms may be an indication of disease activity and treatment effectiveness in ra. several studies have shown that key cell types in the joints [54, 55] express pparc at both the mrna and protein levels. long noncoding rnas (lncrnas) are more than 200 nucleotides in length and are extensively expressed in many organs of the human body. several researchers have shown that lncrna could be used to diagnose ra [56, 57] . in ra patients with active synovitis, osteoprotegerin (opg) expression on macrophage type synovial lining cells and also endothelial cells is low. as a result, addressing opg expression in ra patients' inflamed joints may be an essential approach for the treatment of ra in humans [58] . e rankl/opg pathway is the connecting factor between bone production and bone resorption in the complicated system of bone remodeling. rankl promotes the activation and differentiation of preosteoclasts and mature osteoclasts by binding to their receptors (rank). certain hormones, growth factors, and cytokines affect the synthesis of rankl and opg by osteoblasts in various ways. us, the level of proliferation and activity of osteoclasts are determined by the balance of rankl and opg. bone erosions in ra are caused by osteoclastic bone resorption in synovitis sites, in which rankl expression is also observed [59] . currently, available anti-ra agents focus on targeting cytokines, chemokines, and various physiological proteins in humans. adalimumab is an anti-ra medication that prevents tnf and its receptors from binding, thus lowering cytokines (e.g., mmp-1 and mmp-3)-mediated inflammatory mechanisms and cartilage and bone degradation [41] . on the other hand, (5r)-5-hydroxytriptolide can systemically affect the fls and, in particular, in the process of immune-related processes at 100 nm concentration through a genome-wide microarray assay in ra patients [60] . il-1 activates the extracellular signal-regulated kinase (erk), c-jun n-terminal kinase (jnk), apetala (ap)-1, and nf-b activating pathways, which stimulate mmp production and leukocyte adhesion to ra fls [61] , whereas oridonin (2-10 m for 24-72 h) suppress ra fls proliferation in ra [62] . in ra fls, (5r)-5hydroxytriptolide (50 and 100 nm) reduced proliferation and invasion, as well as cytokine production (mmp-3, il-1, and -6) [63] . by stimulating the synthesis of mmps and nf-κb ligand (rankl) receptors, il-6 promotes bone resorption and cartilage degradation [64, 65] . nsaids work by reducing the enzymatic activity of the cyclooxygenase (cox) enzymes, which are involved in the production of prostaglandins (pgs). nsaids inhibit cox-2, which limits pg synthesis at sites of inflammation; however, inhibiting cox-1 in other tissues (e.g., platelets and mucosa) results in classic nsaid side effects such as bleeding and gi ulcers [66] . summarized scheme of the physiopathology of ra is shown in figure 1 . diterpenoids are the most prominent source of anti-ra agents with potential pharmacological effects. a recent study has been claimed that diterpenes isolated from caesalpinia minax (hance) substantially reduced the change in paw swelling perimeter, arthritic score, and increased bodyweight loss in vivo study [67] . furthermore, the primary components of the extract were 14 cassane derivatives, such as caesalpins a-h, caesalminaxin a-l, and others, which exhibit a promising effect on the expression of mrna of the cytokines il-1β and il-6 and tnf-α generated by macrophage cells. moreover, some other diterpenoids (rhodojaponin iii, rhodojaponin vi, 2-o-methylrhodojaponin, and 5′-β-d-glucopyranosyloxyjasmonic acid) in rhododendron molle fruits at 0.6 mg/kg dose dramatically reduced ra symptoms in cia rats [68] by strongly preventing aberrant t and b lymphocyte proliferation and substantially decreased levels of the proinflammatory cytokines il-1β and il-6, as well as tnf-α. it has been seen that (5r)-5-hydroxytriptolide can inhibit il-1β, il-6, and il-21 secretion and elevated il-10 secretion in peripheral blood and synovial fluid of ra patients [69] . andrographolide in bone marrow macrophages cells and mice inhibited rankl-stimulated osteoclastogenesis via downregulating nf-κb and erk/mapk expression, thereby averting bone loss [70] . cryptotanshinone at 6 and 18 mg/kg (p.o., for 16 days) in type ii collagen-induced arthritis in female wistar rats, and 5 and 20 μm concentration inhibits the degradation of nf-κb (iκb)-α blocker [71] . research findings indicated that triptolide (1-4 nm/l) inhibited rankl-induced nf-κb activation and rankl and tumor cell-induced osteoclastogenesis [72] . additionally, a derivative of triptolide (5r)-5-hydroxytriptolide downregulated the expression of (p)-iκb, a major regulator of the rankl-signaling pathway in ra patients' peripheral blood and synovial fluid [69] . another study suggested that triptolide (2.5-40 nm) enhanced the inhibitory effects of tregs on osteoclast differentiation and bone resorption through an increase in the secretion of il-10 and transforming growth factor-beta 1 (tgf-β1) in mice bone marrow macrophages [73] . table 1 provides the list of various diterpenes, diterpenoids, and their derivatives, which act in various ra models. triptolide in collagen-induced arthritis rats significantly inhibited triggering receptors expressed on myeloid cells (trem)-1 mrna and dap12 mrna expression and activation of jak2 and stat3 in the ankles of test animals and in lps-stimulated u937 cells [82] . in mh7a cells, retinoic acid-platinum (ii) complex (0.25-12 μm) downregulated the activation of the mek/nf-κb pathway [90] , whereas sclareol exhibited anti-ra potential in collagen-induced arthritis in vivo and in vitro models [74] . furthermore, through inhibiting nf-b translocation and mapk pathway activation, sclareol inhibited the il-1β-induced production of tnf-α, mmp-1, and il-6. furthermore, sclareol at doses of 5 and 10 mg/kg (i.p.) reduced the number of 17 cells in mice and improved edema and bone erosion. in a cytokine-stimulated expression of the major cartilage damaging proteases, mmp-3, mmp-13, and adamts[80] . it also prevented mmp-13 production by il-1 in human and bovine cartilage explants and il-1, il-17, and tnf-α induced expression of adamts-4 in bovine chondrocytes. triptolide inhibited the il-1-induced phosphorylation of erk, p38, and jnk at protein levels in bovine type ii collagen-induced arthritis da rats treated with 11-45 g/kg/day (i.g.) for 28 days and significantly decreased the expression of angiogenic activators such as tnf-α, il-17, vascular endothelial growth factor (vegf), vegf receptor (vegfr), and ang-1 [81] . triptolide also lowered the production of tnf-α, il-1β, and il-6 in blood and joints of collagen-induced arthritic rats [82] . similarly, kirenol, isolated from herba siegesbeckiae, at 100-200 μg/ml inhibited the migration, invasion, and proinflammatory il-6 secretion in ra-associated synovial fibroblasts [87] . moreover, it inhibited the production of proinflammatory cytokines (e.g., il-6) and synovium hyperplasia and cartilage erosion in a dose-dependent manner in collagen-induced arthritis male dba/1 mice, whereas tanshinone iia suppressed il-6 and tnf-α expression and release in neutrophils and promoted neutrophil apoptosis in adjuvant-induced arthritis in female c57bl/6 mice [94] . phlomisoside f (5, 10, and 20 mg/kg, p.o., for 28 days) inhibited the expression of tnf-α, il-1β, il-6, cox-2, and 5-lipoxygenase (5-lox), and increased the expression of il-10 in complete freund's adjuvant-induced arthritis male wistar rats [94] . 11-epi-sinulariolide acetate (9 mg/kg, s.c., once every 2 days from day 7 to day 28 postimmunization) reduced the expression of cathepsin k, mmp-9, trap, and tnf-α in ankle tissues in adjuvant-induced ra in female lewis rats [89] . research findings indicated that retinoic acid-pt (ii) complex (2 and 5 mg/kg, i.g.) drastically decreased il-1β, il-6, il-8, mmp-1, and mmp-13 levels in synovial fluid dosedependently in sprague-dawley rats [90] . it also significantly inhibited the expression of inos and cox-2 mrna proteins in ra rats. furthermore, retinoic acid-platinum (ii) complex (0.25-12 μm) reduced tnf-α-induced proliferation in a concentration-dependent manner in mh7a cells. in collagen ii-induced arthritis male dba/1j mice, ginkgolide b (10, 20, and 40 μm, i.p., for 43 days) decreased the serum levels of il-1β, il-6, tnf-α, mmp-3, and mmp-13 and increased the anti-inflammatory cytokine il-10 [99] . e synovial production of monocyte chemoattractant protein-1 (mcp-1) may be crucial in the recruitment of mononuclear phagocytes during ra inflammation [100] . xie et al. [99] demonstrated that ginkgolide b significantly decreases the serum levels of chemokine mcp-1 in arthritis animals. in animal studies, wb2086, a human paf receptor antagonist, reduces paf-induced platelet aggregation [101] . findings showed that sclareol exhibits significant anti-inflammatory effects in experimental animals; it inhibits no production and upregulates inducible nitric oxide synthase (inos) and cox-2 expression in lipopolysaccharide (lps)stimulated macrophages [102] . it also decreased paw edema and neutrophil infiltration in the λ carrageenan-induced paw edema animal model. on the other hand, aphamines a-c isolated from aphanamixis polystachya exhibited inhibitory effects on no production (ic 50 : 6.71-15.36 μmol/l) and reduced the expression of inos in lps-induced raw 264.7 macrophages [103] . serralabdanes a-e isolated from the whole plant of chloranthus serratus also showed inhibitory effects on lpsinduced no production in raw264.7 cells [104] . other compounds such as tripterycoside a-c, 11-o-β-d-glucopyranosyl-neotritophenolide, and wilfordoside a at 10 μm exerted substantial inhibition of il-1β secretion in lpsinduced rat primary synovial fibroblasts [105] . similarly, researchers found that secoferruginol isolated from the heartwood of cryptomeria japonica modulates human dc function in a fashion that favors 2 cell polarization [106] , whereas songorine, a c 20 diterpenoid alkaloid and 12-keto analogue of napelline, isolated from aconitum soongaricum, exhibited anti-inflammatory and antiarthritis activities [107] . some of the important proteins involved in ra include jak, p38 mitogen-activated protein kinase (mapk), extracellular receptor kinase (erk), jnk, il-1 receptor-associated kinase (irak)-4, mmps, toll-like receptor 4 (tlr-4), g protein-coupled receptor kinase (grk)-2, bruton's tyrosine kinase (btk), cd3, cd11a, cd19, cd20, and cd80. jak is a component of the jak/stat signaling [108] . published research showed that excavatolide b (2.5 and 5 mg/kg, s.c.) in adjuvant (aia) and type ii collagen-induced arthritis in rats attenuate the protein expression of cd11b and nuclear factor of activated t cells 1 (nfatc1) in ankle tissues [75] . giannelli et al. [109] reported that evaluation of synovial fluid concentrations of timps (e.g., timp-1 and timp-2) is more reliable than that determined in serum when remodeling cartilage ecm proteins, besides mmps evaluated. ese researchers suggested that both timps and mmp inhibitors might be a potential target for novel ra treatments administered directly into the joint area. in this respect, triptolide (10, 30, and 50 nm) in ra fls from 7 ra patients reduced the tnfα-induced expression of phosphorylated jnk [83] . additionally, it has been demonstrated that a jnk-specific inhibitor reduces the migration and invasion of ra fls. research by zhong and colleagues showed that sclareol exhibits antiosteoarthritic properties in il-1β-induced rabbit chondrocytes and a rabbit model of osteoarthritis induced by aclt [110] . sclareol also inhibited mmp, inos, and cox-2 expression and increased timp-1 expression and ameliorated cartilage degradation in the test systems. similarly, 11-epi-sinulariolide acetate significantly inhibited the expression of the proinflammatory proteins inos and cox-2 in lps-stimulated murine macrophages [89] . moreover, oridonin (2-10 μg/ml for 24-72 h) increased apoptosis, protein levels of bax, and cleaved caspase-3 in ra fls. however, it significantly decreased il-1β levels in the test system [52] . meanwhile, excavatolide b at 10 μm inhibited multinucleated cell and actin ring formation and also tartrate-resistant acid phosphatase (trap or trapase), mmp-9, and cathepsin k expression in lps-stimulated raw 264.7 cells [75] . researchers also demonstrated that the soft coral-derived diterpene at 2.5 and 5 mg/kg (s.c.) significantly attenuated the characteristics of ra, improved histopathological features, decreased the number of trap-positive multinucleated cells, and attenuated the protein expression of cathepsin k, mmp-2, and mmp-9 in ankle tissues as well as the level of il-17a and macrophage colony-stimulating factor in adjuvant (aia) and type ii collagen-induced arthritis in rats. figure 2 shows the possible mechanisms of diterpenes and their derivatives targeting cytokines. a clinical trial of e6011 (an anti-cx3cl1 mab) is currently underway, and it has been shown to have a potential function in active ra patients [111] . in lps-induced fls, ginkgolide b (5-80 μm) remarkably inhibited ra fls viability in a concentration-dependent fashion. it also reduced the apoptotic ratio and enhanced the expression of cleaved caspase-3 and bax. furthermore, it reduced bcl-2 expression in ra fls, decreased the development of inflammation by regulating inflammatory cytokine secretion and mmp gene expression, and reduced expression levels of wnt5a, (p)-jnk, and p-p65 in synovial tissues and ra fls [88] . other diterpenes, diterpenoids, or their derivatives that inhibit ra fls include triptolide [79, 83] , tanshinone iia [93] , (5r)-5-hydroxytriptolide [60] , kirenol [87] , oridonin [52] , and triptolide (tp) loaded with mir-30-5p inhibitor [84] . findings showed that carnosol, carnosic acid, carnosic acid-12-methyl ether, 20-deoxocarnosol, and abieta-8,11,13triene-11,12,20-triol significantly blunt gene expression levels of inos, cytokines/interleukins (il-1α, il-6), and chemokines including ccl5/rantes, cxcl10/ip-10 in murine macrophages (raw264.7 cells), and human chondrocytes [112] . figure 3 shows the possible mechanisms of diterpenes and their derivatives targeting chemokines. triptolide (0.01-10 μm) downregulated ppar-c activation and induced dna fragmentation in rsf in rheumatoid synovial fibroblasts from ra patients [79] . it also decreased arthritis scores and significantly reduced capillaries, small, medium, and large vessel density in the synovial membrane tissues of inflamed joints in bovine type ii collagen-induced arthritis da rats [81] . moreover, triptolide inhibited matrigel-induced cell adhesion of hfls-ra, and huvec as well as disrupted tube formation of huvec on matrigel, and suppressed the vegf-induced chemotactic migration of hfls-ra and huvec, respectively, in arthritis rats. (5r)-5-hydroxytriptolide increased the rate of osteoprotegerin (opg) expression in cd3 + t leukomonocytes in peripheral blood and the ratio of opg/rankl in both peripheral blood and synovial fluid in peripheral blood and synovial fluid of ra patients [69] . it also inhibited il-23 secretion in the supernatants of pbmcs and sfmcs in peripheral blood and synovial fluid of ra patients [69] . it additionally prevented collagen-induced arthritis via inhibiting opg/rank/rankl signaling in osteoclastogenesis and ifn-c signaling in t cells [113, 114] . recently, zhou et al. [63] demonstrated that it exerts an anti-ra effect through the wakmar2/mir-4478/e2f1/p53 dependent pathway in ra fls. meg3 lncrna overexpression reduces inflammation by affecting the akt/mtor signaling pathway [115] . tanshinone iia (1-80 μm) exerted cytotoxically apoptosis effects through upregulating lncrna gas5, possibly with an increase in cleaved caspase-3/9 expression and inhibiting the pi3k/akt signaling pathway in fls from ra patients [93] . in addition, numerous studies indicated that pgs play an important role in physiological immune responses and in pathological diseases related to inflammation and tissue damage. in murine macrophages (raw264.7 cells) and human chondrocytes, carnosol, carnosic acid, carnosic acid-12methyl ether, 20-deoxocarnosol, and abieta-8,11,13-triene-11,12,20-triol reduced no and pge 2 production in a concentration-dependent manner. ey also significantly reduced inos and cytokine (il-1α and il-6) gene expression levels in the test systems [112] . additionally, these substances altered the expression of catabolic and anabolic evidence-based complementary and alternative medicine genes in the chondrosarcoma cell line sw1353 and primary human chondrocytes, stimulated by il-1β, where catabolic genes such as mmp-13 and adam metallopeptidase with thrombospondin type 1 motif 4 (adamts-4) that contribute to cartilage erosion were downregulated, whereas anabolic gene expression, particularly col2a1 and aggrecan, was moved towards prepathophysiological equilibrium. furthermore, carnosol exhibited the greatest overall impact on inflammatory mediators as well as macrophage and chondrocyte gene expression. it significantly inhibited il-1β-induced nuclear translocation of nf-κb-p65, suggesting that it is primarily regulated through the nf-κb signaling pathway. lobolide, a cembrane diterpene, also acts through the nf-κb signaling pathway [116] . moreover, andrographolide attenuated mouse cortical chemokine levels from the cc and cxc subfamilies in lps-induced chemokine upregulation in a mouse model [117] . table 2 provides the list of diterpenes, diterpenoids, and their derivatives that interact with various proteins involved in inflammatory and ra processes. for 28 days) significantly delayed the onset of arthritis. in addition, the arthritis incidence, clinical arthritis severity score, histopathological arthritis severity score, and in vivo cell-mediated immunity to collagen were all reduced [78] . in bovine collagen type ii and complete freund's adjuvant-induced arthritis in dba/1 mice, cryptotanshinone (20 and 60 mg/kg, p.o., for 6 weeks) ameliorated the inflammation and joint destruction [98] . it also suppressed p300-mediated stat3 acetylation in test animals. similarly, carnosic acid at 30 and 60 mg/kg (i.p., 4 weeks) in collagen-induced arthritis in male c57bl/ksj-db/db mice and at 10 or 20 μm in mouse bone marrow cells reduced osteoclast formation and bone loss through suppression of inflammation by regulating the rosdependent p38 pathway [99] . on the other hand, xylopic acid nanoformulation showed anti-inflammatory and anti-ra effects in raw 264.7 cells and complete freund's adjuvantinduced arthritis in male sprague-dawley rats [97] . research findings showed that 7β-hydroxycalcaratarin a, a labdane-type diterpenoid derived from hedychium coronarium, inhibits superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-lphenylalanine/cytochalasin b (fmlp/cb). it also inhibited fmlp/cb-induced elastase release [118] . chemotherapy with docetaxel (60 mg/m 2 ) and carboplatin dosed every 3 weeks for 4 cycles to an ovarian carcinoma patient (66-yearold woman) was found to mask ra [119] . andrographolide (25 μm, for 16 h) in lps-stimulated neutrophils accelerated apoptosis and inhibited autophagydependent extracellular traps formation [77] . it also reduced neutrophil infiltration and netosis in the ankle joints and relieved the systematic inflammation in adjuvant-induced arthritis c57bl/6 mice. tanshinone iia inhibited the net formation of neutrophils in adjuvant-induced arthritis in female c57bl/6 mice [92] . on the other hand, triptolide (0.01-10 μm) reduced viability and proliferation and induced apoptosis of rsf in a concentration-dependent manner in fls from ra patients [79] . it also upregulated caspase-3 activity in the test system, whereas retinoic acidplatinum (ii) complex (0.25-12 μm) in mh7a cells induced apoptosis and caused the arrest of the cell cycle [90] . andrographolide (50 mg/kg/day) combined with methotrexate (2 mg/kg/week) for 35 days (injection) in complete freund's adjuvant-induced arthritis in wistar rats improved the serum marker. is may be attributed to the antioxidant activity of this compound, as evidenced by histological alterations in the liver [76] . andrographolide when combined with methotrexate in complete freund's adjuvant-induced arthritis in wistar rats strengthened the antiarthritic capacity of methotrexate, reduced the inflammatory symptoms evidence-based complementary and alternative medicine in animals, showed hepatoprotective activity, and significantly reduced serum tnf-α, il-6, and il-1β levels [76] . triptolide loaded by a poly-c-glutamic acid-grafted l-phenylalanine ethyl ester copolymer at 6.25-200 nm reduced the damaging effects on the liver, kidney, and spleen of mice [85] . in addition, triptolide (100 μg/kg, i.p., 21 days) improved clinical arthritic conditions and joint destruction in collagen-induced arthritis in male dba/1 mice [83] , whereas triptolide-loaded poly(d,l-lactic acid) nanoparticles (0.05-0.2 mg/kg, p.o., for 14 days) in complete freund's adjuvant-induced arthritis in male wistar rats significantly inhibited arthritis and exerted a preferable antiinflammatory effect with long-time administration [86] . in another study, triptolide loaded with mir-30-5p inhibitor significantly inhibited ra synovial fibroblast proliferation and increased apoptosis in collagen-induced arthritis female sprague-dawley rats [84] . is nanopreparation also downregulated immune system activation in rats. phytol (acyclic diterpene alcohol derived from chlorophyll) at 200 μl (injection in the tail, for 10 days) was found to restore the oxidative-burst effect and induce a strikingly similar ifn-β-dependent pathway in da rats [95] . researchers suggested that it may be effective against naturally occurring genetic polymorphisms in the ncf-1 gene that modulated the activity of the nadph oxidase complex, which is potentially regulated in the severity of arthritis, whereas 11-epi-sinulariolide acetate significantly inhibited ra characteristics in adjuvant-induced arthritis in female lewis rats [89] . resiniferatoxin (10 μl of 0.001 or 0.003%, injection) significantly improved arthritis with monoarticular inflammatory arthritis in evoked pain scores in arthritic male c57bl6 mice [96] . similarly, tanshinone iia (30 mg/kg, i.p., for 30 days) alleviated cartilage erosion and neutrophil infiltration in the ankle joints and reduced proinflammatory cytokine expression levels in sera in adjuvant-induced arthritis in female c57bl/6 mice [92] . in the complete freund's adjuvant-induced arthritis rat model, phlomisoside f (5, 10, and 20 mg/kg, p.o., for 28 days) markedly offset the body weight loss, inhibited the paw edema, and reduced the arthritis scores and indices of the thymus and spleen [92] . leflunomide (20 mg once daily) in combination with methotrexate improved signs, symptoms, and physical function in ra patients [91] , while ginkgolide b (10, 20, and 40 μm, i.p., for 43 days) in collagen ii-induced arthritis male 14 evidence-based complementary and alternative medicine dba/1j mice significantly decreased arthritis scores, synovial hyperplasia, and cartilage and bone destruction [88] . e chemical structures of some anti-ra diterpenes and their derivatives are shown in figure 4 . diterpenes and their derivatives are gaining popularity due to their intriguing biological and pharmacological properties. us far, hundreds of natural diterpene compounds from terrestrial and marine species have been described. many of these compounds have become clinically effective. plants are an important source of diterpenes. diterpenes can be linear, bicyclic, tricyclic, tetracyclic, pentacyclic, or macrocyclic. ey are typically found in nature in a polyoxygenated form with keto and hydroxyl groups, which are frequently esterified by small-sized aliphatic or aromatic acids. for example, the anticancer drug taxol is used as a promising anticancer agent for ovarian, breast, and lung cancers. in addition, many of its synthetic derivatives are also examples of medicinal agents in the management of various diseases in humans. docetaxel, sold under the brand name taxotere ® , is a taxoid antineoplastic drug used to treat a variety of malignancies, including locally advanced or metastatic breast cancer, metastatic prostate cancer, gastric adenocarcinoma, and head and neck cancer. moreover, carboplatin, when combined with this drug, was found to mask ra in an ovarian carcinoma patient [119] . similarly, ginkgolides are other promising diterpenes that have strong and specific antagonistic action against platelet-activating factors rising in shock, burns, ulceration, and inflammatory skin disorders [120] . additionally, ginkgolide b exhibits multiedge-like anti-ra effects in in vitro and in vivo test models [88] . meanwhile, the anti-ra diterpene resiniferatoxin (an ultrapotent vanilloid derived from the latex of euphorbia resinifera) is promising for bladder hyperreflexia and diabetic neuropathy [120] . in short, diterpenes, diterpenoids, and their derivatives might be promising tools to manage ra and its consequences. according to current knowledge [50] , the most promising therapeutic targets in ra include the following: it appears that diterpenes and their derivatives have multiedge-like actions on different ra models. ese compounds exerted anti-ra effects through the cytokine, chemokine, inflammatory/noninflammatory proteins, and small molecular metabolites pathways. among the diterpenes, triptolide and its derivative (5r)-5-hydroxytriptolide have been found to display promising anti-ra activity in various test systems. other hopeful anti-ra diterpenes and diterpenoids found in this updated review include carnosol and carnosic acid and their derivatives, excavatolide b, kirenol, evidence-based complementary and alternative medicine ginkgolide b, 11-epi-sinulariolide acetate, oridonin, phlomisoside f, phytol, retinoic acid, resiniferatoxin, sclareol, and xylopic acid among others. a novel triptolide derivative (also known as lldt-8), which exhibited anti-ra therapeutic properties, is currently in phase ii clinical studies in china [63] . diterpenes and their derivatives act through multidimensional pathways in different ra animal models. moreover, triptolide-loaded nanocomplexes also improved anti-ra potential in experimental modalities. besides these compounds/formulations, andrographolide, tanshinone iia, and its derived compound cryptotanshinone also displayed promising anti-ra effects in test systems. to date, many natural products that have the anti-ra capacity, including those obtained from medicinal plants and marine organisms, have been identified. e sources of diterpenes and diterpenoids are widely distributed. natural products, including medicinal plant-derived chemicals, are a prominent source of semisynthetic and synthetic derivatives. hence, nature and modern medicinal science are capable of providing new and more effective diterpene derivatives. diterpenes and their derivatives have been shown to possess promising immunomodulatory properties in various experimental models; therefore, these natural bioactive compounds are a promising adjuvant pharmacotherapy in ra. e data used to support the findings of this study are available from the corresponding author upon request and are cited within the article as references. e global prevalence of rheumatoid arthritis: a meta-analysis based on a systematic review rheumatoid arthritis: still a chronic disease novel therapeutic targets in rheumatoid arthritis adverse reactions to aspirin and nonsteroidal antiinflammatory drugs (nsaids) upper gastrointestinal bleeding associated with the use of nsaids: newer versus older agents infections induced by low-dose corticosteroids in rheumatoid arthritis: a systematic literature review multiple adverse effects of systemic corticosteroids: a case report adverse effects of low dose methotrexate in rheumatoid arthritis patients overview of safety of non-biologic and biologic dmards use of leflunomide plus tnf-α inhibitors in rheumatoid arthritis e cost-effectiveness of tnf-inhibitors for the treatment of rheumatoid arthritis in swedish clinical practice chinese herbal medicines for rheumatoid arthritis: text-mining the classical literature for potentially effective natural products dietary habits and nutrition in rheumatoid arthritis: can diet influence disease development and clinical manifestations scientific validation of anti-arthritic effect of kashayams-a polyherbal formulation in collagen induced arthritic rats e newly discovered natural compounds against rheumatoid arthritis-an overview extraction, purification, and nmr analysis of terpenes from brown algae diterpenes/ diterpenoids and their derivatives as potential bioactive leads against dengue virus: a computational and network pharmacology study naturally occurring diterpenoid dimers: source, biosynthesis, chemistry and bioactivities diterpenoids of terrestrial origin diterpenes and their derivatives as potential anticancer agents effect of diterpenes on hepatic system immunomodulatory effects of diterpenes and their derivatives through nlrp3 inflammasome pathway: a review amentoflavone, new hope against sars-cov-2: an outlook through its scientific records and an in silico study diterpenes from the brown alga dictyota crenulata platensimycin and platencin biosynthesis in streptomyces platensis, showcasing discovery and characterization of novel bacterial diterpene synthases diterpenoids from the medicinal plants of africa terpenoids interactions between carotenoids from marine bacteria and other micronutrients: impact on stability and antioxidant activity effects of radix curcumae-derived diterpenoid c on helicobacter pylori-induced inflammation and nuclear factor kappa b signal pathways diterpenoid lactones with anti-inflammatory effects from the aerial parts of andrographis paniculata natural diterpenoid oridonin ameliorates experimental autoimmune neuritis by promoting anti-inflammatory macrophages through blocking notch pathway historical insights into cytokines progression to rheumatoid arthritis in early inflammatory arthritis is associated with low il-7 serum levels increased interleukin-23 is associated with increased disease activity in patients with rheumatoid arthritis enhanced and persistent levels of interleukin (il)-17 + cd4 + t cells and serum il-17 in patients with early inflammatory arthritis anti-tnf in rheumatoid arthritis: an overview granulocyte macrophage colony-stimulating factor receptor α expression and its targeting in antigen-induced arthritis and inflammation cytokines (il-15, il-21, and ifn-c) in rheumatoid arthritis: association with positivity to autoantibodies (rf, anti-ccp, anti-mcv, and anti-padi4) and clinical activity blockade of the interleukin-7 receptor inhibits collagen-induced arthritis and is associated with reduction of t cell activity and proinflammatory mediators rankl is necessary and sufficient to initiate development of antigensampling m cells in the intestinal epithelium adalimumab in the treatment of arthritis e roles of small-molecule inflammatory mediators in rheumatoid arthritis antiinflammatory mediator lipoxin a4 and its receptor in synovitis of patients with rheumatoid arthritis lipoxin a 4 modulates adaptive immunity by decreasing memory b-cell responses via an alx/fpr2-dependent mechanism platelet-neutrophil-interactions: linking hemostasis and inflammation anti-tnfα agents curb platelet activation in patients with rheumatoid arthritis upregulation of circulating inflammatory biomarkers under the influence of periodontal disease in rheumatoid arthritis patients blockade of xcl1/ lymphotactin ameliorates severity of periprosthetic osteolysis triggered by polyethylene-particles chemokine profile of synovial fluid from normal, osteoarthritis and rheumatoid arthritis patients: ccl25, cxcl10 and xcl1 recruit human subchondral mesenchymal progenitor cells promising therapeutic targets for treatment of rheumatoid arthritis expression of cc chemokine ligand 5 in patients with rheumatoid arthritis and its correlation with disease activity and medication complement c5a receptor is the key initiator of neutrophil adhesion igniting immune complex-induced arthritis pathogenic roles of cxcl10 signaling through cxcr3 and tlr4 in macrophages and t cells: relevance for arthritis e peroxisome proliferator-activated receptor-c is a negative regulator of macrophage activation peroxisome proliferator-activated receptor-c (ppar-c) ligands as potential therapeutic agents to treat arthritis long noncoding rnas expression profile and functional networks in rheumatoid arthritis analysis of lncrna expression profiles by sequencing reveals that lnc-al928768.3 and lnc-ac091493.1 are novel biomarkers for disease risk and activity of rheumatoid arthritis osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls e role of rank ligand/osteoprotegerin in rheumatoid arthritis (5r)-5-hydroxytriptolide (lldt-8) induces substantial epigenetic mediated immune response network changes in fibroblast-like synoviocytes from rheumatoid arthritis patients interleukin-1β induces icam-1 expression enhancing leukocyte adhesion in human rheumatoid arthritis synovial fibroblasts: involvement of erk, jnk, ap-1, and nf-κb oridonin suppresses autophagy and survival in rheumatoid arthritis fibroblastlike synoviocytes erapeutic effects of (5r)-5-hydroxytriptolide on fibroblast-like synoviocytes in rheumatoid arthritis via lncrna wakmar2 e2f1/p53 axis e balance between soluble receptors regulating il-6 trans-signaling is predictive for the rankl/osteoprotegerin ratio in postmenopausal women with rheumatoid arthritis matrix metalloproteinase 13-deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development use of nsaids in treating patients with arthritis erapeutic effects of caesalpinia minax hance on complete freund's adjuvant (cfa)-induced arthritis and the anti-inflammatory activity of cassane diterpenes as main active components anti-rheumatoid arthritis potential of diterpenoid fraction derived from rhododendron molle fruits (5r)-5-hydroxytriptolide (lldt-8) inhibits osteoclastogenesis via rankl/rank/ opg signaling pathway andrographolide suppresses rankl-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo erapeutic effect of cryptotanshinone on collagen-induced arthritis in rats via inhibiting nuclear factor kappa b signaling pathway triptolide, a diterpene, inhibits osteoclastogenesis, induced by rankl signaling and human cancer cells triptolide inhibits osteoclast differentiation and bone resorption in vitro via enhancing the production of il-10 and tgf-β1 by regulatory t cells erapeutic potential of sclareol in experimental models of rheumatoid arthritis excavatolide b attenuates rheumatoid arthritis through the inhibition of osteoclastogenesis tanshinone iia promotes the apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis by up-regulating lncrna gas5 andrographolide ameliorates rheumatoid arthritis by regulating the apoptosis-netosis balance of neutrophils inhibition of type ii collagen-induced arthritis in rats by triptolide triptolide, an active compound identified in a traditional chinese herb, induces apoptosis of rheumatoid synovial fibroblasts triptolide suppresses proinflammatory cytokine-induced matrix metalloproteinase and aggrecanase-1 gene expression in chondrocytes anti-angiogenic effect of triptolide in rheumatoid arthritis by targeting angiogenic cascade triptolide modulates trem-1 signal pathway to inhibit the inflammatory response in rheumatoid arthritis triptolide inhibits the migration and invasion of rheumatoid fibroblast-like synoviocytes by blocking the activation of the jnk mapk pathway efficient delivery of triptolide plus a mir-30-5p inhibitor through the use of near infrared laser responsive or cady modified msns for efficacy in rheumatoid arthritis therapeutics fabrication of novel vesicles of triptolide for antirheumatoid activity with reduced toxicity in vitro and in vivo anti-inflammatory effects of triptolide loaded poly (d,l-lactic acid) nanoparticles on adjuvant-induced arthritis in rats kirenol inhibits the function and inflammation of fibroblast-like synoviocytes in rheumatoid arthritis in vitro and in vivo ginkgolide b attenuates collagen-induced rheumatoid arthritis and regulates fibroblast-like synoviocytes-mediated apoptosis and inflammation a soft coral-derived compound, 11-epi-sinulariolide acetate suppresses inflammatory response and bone destruction in adjuvant-induced arthritis retinoic acidplatinum (ii) complex [rt-pt (ii)] protects against rheumatoid arthritis in mice via mek/nuclear factor kappa b (nf-κb) pathway downregulation chinese herbal formula huayu-qiangshen-tongbi decoction compared with leflunomide in combination with methotrexate in patients with active rheumatoid arthritis: an open-label, randomized, controlled, pilot study investigation of the effect of phlomisoside f on complete freund's adjuvant-induced arthritis evaluation of the effect of andrographolide and methotrexate combined therapy in complete freundʼs adjuvant induced arthritis with reduced hepatotoxicity tanshinone iia ameliorates chronic arthritis in mice by modulating neutrophil activities arthritis suppression by nadph activation operates through an interferon-β pathway e effect of treatment with resiniferatoxin and capsaicin on dynamic weight bearing measures and evoked pain responses in a chronic inflammatory arthritis murine model metabolomic and transcriptomic analyses of the antirheumatoid arthritis potential of xylopic acid in a bioinspired lipoprotein nanoformulation erapeutic effect of cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-stat3 acetylation carnosic acid (ca) attenuates collagen-induced arthritis in db/db mice via inflammation suppression by regulating ros-dependent p38 pathway enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis e specific binding of the platelet-activating factor (paf) receptor antagonist web 2086 and the benzodiazepine flunitrazepam to rat hepatocytes sclareol exhibits anti-inflammatory activity in both lipopolysaccharidestimulated macrophages and the λ-carrageenan-induced paw edema model aphamines a-c, dimeric acyclic diterpene enantiomers from aphanamixis polystachya labdane diterpenes from chloranthus serratus ree new abietane-type diterpene glycosides from the roots of tripterygium wilfordii diterpenes inhibit il-12 production by dc and enhance 2 cells polarization erapeutic potential of songorine, a diterpenoid alkaloid of the genus aconitum e role of the jak/stat signal pathway in rheumatoid arthritis mmp-2, mmp-9, timp-1 and timp-2 levels in patients with rheumatoid arthritis and psoriatic arthritis sclareol exerts anti-osteoarthritic activities in interleukin-1β-induced rabbit chondrocytes and a rabbit osteoarthritis model a phase 2 study of e6011, an anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis inadequately responding to biological disease-modifying antirheumatic drugs carnosol and related substances modulate chemokine and cytokine production in macrophages and chondrocytes (5r)-5-hydroxytriptolide attenuated collagen-induced arthritis in dba/1 mice via suppressing interferon-c production and its related signaling (5r)-5-hydroxytriptolide (lldt-8) prevents collagen-induced arthritis through opg/rank/rankl signaling in a rat model of rheumatoid arthritis lncrna meg3 inhibits rheumatoid arthritis through mir-141 and inactivation of akt/mtor signalling pathway lobolide, a diterpene, blockades the nf-κb pathway and p38 and erk mapk activity in macrophages in vitro andrographolide attenuates lps-stimulated up-regulation of cc and cxc motif chemokines in rodent cortex and primary astrocytes new labdane-type diterpenoids and anti-inflammatory constituents from hedychium coronarium rheumatoid arthritis masked by docetaxel chemotherapy in a patient with ovarian carcinoma diterpenes for therapeutic use e authors declare that they have no conflicts of interest. all authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas, that is, revising or critically reviewing the article, giving final approval of the version to be published, agreeing on the journal to which the article has been submitted, and confirming to be accountable for all aspects of the work. key: cord-0029814-v6t9trz7 authors: watanabe, ryu; hashimoto, motomu title: perspectives of jak inhibitors for large vessel vasculitis date: 2022-03-30 journal: front immunol doi: 10.3389/fimmu.2022.881705 sha: b48443f2309fadd1f851d95bdf251bad6dc57ab8 doc_id: 29814 cord_uid: v6t9trz7 vasculitis is an inflammation of the blood vessels caused by autoimmunity and/or autoinflammation, and recent advances in research have led to a better understanding of its pathogenesis. glucocorticoids and cyclophosphamide have long been the standard of care. however, b-cell depletion therapy with rituximab has become available for treating antineutrophil cytoplasmic antibody-associated vasculitis (aav). more recently, avacopan, an inhibitor of the complement 5a receptor, was shown to have high efficacy in remission induction against aav. thus, treatment options for aav have been expanded. in contrast, in large vessel vasculitis (lvv), including giant cell arteritis and takayasu arteritis, tocilizumab, an il-6 receptor antagonist, was shown to be effective in suppressing relapse and has steroid-sparing effects. however, the relapse rate remains high, and other therapeutic options have long been awaited. in the last decade, janus kinase (jak) inhibitors have emerged as therapeutic options for rheumatoid arthritis (ra). their efficacy has been proven in multiple studies; thus, jak inhibitors are expected to be promising agents for treating other rheumatic diseases, including lvv. this mini-review briefly introduces the mechanism of action of jak inhibitors and their efficacy in patients with ra. then, the pathophysiology of lvv is updated, and a rationale for treating lvv with jak inhibitors is provided with a brief introduction of our preliminary results using a mouse model. finally, we discuss the newly raised safety concerns regarding jak inhibitors and future perspectives for treating lvv. vasculitis syndrome is an autoimmune and/or autoinflammatory condition that causes inflammation of the blood vessels, and the resultant tissue ischemia causes damage to various organs. it is classified as large-, medium-, and small-vessel vasculitis according to the size of the affected blood vessels. the mainstay of treatment for vasculitis has long been glucocorticoids (gcs) and immunosuppressive agents such as cyclophosphamide, azathioprine, and methotrexate (mtx). however, new treatment options have long been awaited because of the significant burden of side effects of the treatment. treatment options for antineutrophil cytoplasmic antibody (anca)-associated vasculitis, particularly in microscopic polyangiitis and granulomatosis with polyangiitis, have expanded considerably in recent years. for example, b-cell depletion therapy using rituximab is as effective and safe as cyclophosphamide (1, 2) . more recently, the efficacy and safety of avacopan, a complement 5a (c5a) receptor inhibitor that blocks neutrophil chemoattraction and activation, has been examined in ancarelated vasculitis (3) . this study showed that the c5a receptor blockade was superior to standard steroid therapy in remission induction at week 52, suggesting that avacopan may have the potential to replace standard steroid therapy (4) . in contrast, in large vessel vasculitis (lvv), including giant cell arteritis (gca) and takayasu arteritis (tak), tocilizumab (tcz), an il-6 receptor antibody, is effective in preventing recurrence and reducing the dose of gcs (5, 6) . however, the primary endpoint was not met in tak (5) , and many patients experienced relapse after discontinuation of tcz (7), necessitating treatment that fundamentally improves vascular inflammation. moreover, blockade of t cell costimulation signals using abatacept is effective and safe in gca (8) , but failed to show its efficacy in tak (9) . tak often affects young women, and the side effects of accumulated steroids owing to multiple relapses are immense (10) . many of the drugs used in real-world clinical practice for tak lack sufficient evidence in randomized controlled trials (11) . thus, unmet clinical needs remain for lvv, particularly in tak. in the last 10 years, janus kinase (jak) inhibitors have emerged as promising agents for rheumatology (12) . jak inhibitors are low-molecular-weight compounds that can be orally administered to patients with rheumatoid arthritis (ra), unlike biological disease-modifying antirheumatic drugs (bdmards) (13) . their efficacy and safety have been compared with those of bdmards and have been proven in multiple studies in patients with ra. this mini-review briefly explains the mechanism of action of jak inhibitors and their efficacy in patients with ra. then, we update the pathophysiology and provide a rationale for treating lvv with jak inhibitors. finally, we discuss the safety and future perspectives of jak inhibitors for lvv treatment. cytokine receptors are grouped into several superfamilies based on their shared structural elements of the receptors (14) . type i and type ii cytokines utilize the jak-signal transducer and activation of transcription (stat) pathway ( figure 1 ). when type i and ii cytokines bind to their receptors on the cell surface, jaks bound to the intracellular domains are phosphorylated by adenosine triphosphate binding, which in turn phosphorylates the receptor end. the transcription factor stat binds to the receptor end, and phosphorylated stats form a dimer, which is then transferred to the nucleus to regulate gene expression (15) . there are four isoforms of jaks (jak1, jak2, jak3, and tyk2). type i and type ii cytokines include the common g chain family (il-2, 4, 7, 9, 13, and 15), gp130 cytokines (il-6, oncostatin m), granulocyte colony-stimulating factor (g-csf), granulocyte macrophage colony-stimulating factor (gm-csf), interferon (ifn)-a, b, g, il-12, and others, but not tumor necrosis factor a (tnf-a), il-1, il-17, and tgf-b (12) . the efficacy of jak inhibitors has been tested in head-to-head comparisons with adalimumab, a representative tnf-a inhibitor, in multiple trials involving patients with ra. the results demonstrated that jak inhibitors are non-inferior or even superior to adalimumab in controlling disease activity (16) (17) (18) . based on these results, jak inhibitors have been placed equal to bdmards in the most updated ra treatment recommendations (19) . in other words, when methotrexate fails to induce remission, ra patients can choose either bdmards or jak inhibitors. thus, jak inhibitors are an essential therapeutic option for the treatment of ra. both gca and tak affect the aorta and its major branches and are characterized by granulomatous vascular inflammation (20) . ifn-g and il-17 derived from th1 and th17 cells are the dominant cytokines (21) (22) (23) , and neoangiogenesis or new formation of vasa vasorum in the adventitia and lumen occlusion due to intimal hyperplasia can be observed in both diseases (24) . although many disease mechanisms are shared, several differences exist. for example, granulomatous lesions mainly contain cd4 + t cells and macrophages in gca, whereas cd8 + t and nk cells are also involved in tak (25) . in the peripheral blood, the follicular helper t cell-b cell signature, which promotes immunoglobulin production, is highly enriched in tak, but not in gca (26) . adventitial fibrosis is more prominent in tak than in gca (25) . thus, from a pathomechanistic point of view, tak is more complex than gca and a single therapeutic target alone may not be sufficient to achieve remission. in this context, jak inhibitors are expected to be effective because of the simultaneous blockade of multiple cytokines, especially in tak. our previous work showed enhanced activity of the jak-stat pathway in the vascular lesions of patients with gca (27) . compared with biopsy-negative temporal arteries, biopsypositive temporal arteries showed elevated transcripts of stat1, stat2, and stat4, as well as target genes corresponding to each stat. moreover, cytokine production in cd4 + t cells from patients with gca was dependent on the jak-stat pathway, as demonstrated by an experiment showing that tofacitinib, an inhibitor of jak1 and jak3, inhibited ifn-g production in a dose-dependent manner (27) . in line with this report, a recent study from a french group demonstrated that stat1 and stat2 transcripts were highly upregulated in aortic lesions of gca by using microarray analysis (28) . in addition, both cd4 + and cd8 + t cells in the peripheral blood of patients with gca showed increased activity of the jak-stat pathway. the same group also identified upregulated jak-stat signals in both cd4 + and cd8 + t cells in the peripheral blood of patients with tak (29) . what is the mechanism underlying the enhanced activity of the jak-stat pathway in lvv (figure 2) ? this question is equivalent to asking which type i and ii cytokines are implicated in gca and tak. undoubtedly, il-6 plays a key role in both diseases, as suggested by the clinical effects of tcz (5, 6) . il-6 is mainly derived from cd68 + tissue macrophages in both gca and tak (30, 31) , and il-6 primarily utilizes stat3 as a downstream transcription factor (32). since the abovementioned studies have demonstrated that stat3 is highly activated in cd4 + and cd8 + t cells in both diseases (28, 29) , this il-6-stat3 axis substantially contributes to the pathogenesis of both diseases. however, this axis alone does not explain the increased activity of stat1 and stat2 signals in the vascular lesions of the gca (27, 28) . in recent years, type i ifns have attracted attention in the pathophysiology of lvv. upregulation of type i ifns, particularly ifn-a, has been reported in the serum, temporal arteries, and aortic lesions of gca (27, 28) . a highly enriched type i ifn signature has been observed in both cd4 + and cd8 + t cells from tak patients (29) . the binding of type i ifns to their receptors activates jak1 and tyk2, which is followed by the phosphorylation of stat1 and stat2 (33) , which fits perfectly into the context of what has been reported so far. although plasmacytoid dendritic cells are the main source of type i ifns in systemic lupus erythematosus, those in gca and tak remain unknown (34) . in addition to type i ifns, the role of gm-csf in the promotion of vascular inflammation in gca has been reported (35) . thus, il-6, type i ifns, and gm-csf are involved in the pathogenesis, all of which utilize the jak-stat pathway, making it highly likely that jak inhibitors are effective against gca. furthermore, a genome-wide association study identified il-12b as a susceptibility gene for tak (36, 37) . serum il-12 levels are elevated in tak patients (38) , and the risk allele of il-12b is closely associated with vascular damage in tak (39) . il-12 uses the jak-stat pathway, and jak2 and tyk2 are located in the downstream signaling pathway (40) . thus, jak inhibitors are expected to be effective against tak (41) . based on these findings, we examined the effects of tofacitinib, which blocks jak1 and jak3, on lvv in a mouse model (27) . in this mouse model, human medium-sized arteries were engrafted into immunodeficient mice, and vascular inflammation was induced by injecting lipopolysaccharide and peripheral blood mononuclear cells from patients with gca. in this model, tofacitinib not only inhibited t-cell activation and cytokine production but also inhibited macrophage activation, resulting in the efficient suppression of vascular inflammation. analysis of t cells in vasculitic lesions identified a highly proliferative population, called "tissue-resident memory t (trm) cells". trm cells express cd69 and cd103 and show a rapid response to antigens once encountered. these cells may have the potential to induce a relapse of vascular inflammation in gca (42) . our results demonstrate that these cells can be targeted by tofacitinib as well (27) . in line with these data from basic research, several case reports describing the efficacy of jak inhibitors on lvv have been published (29, (43) (44) (45) (46) (47) (48) . very recently, baricitinib, an inhibitor of jak1 and jak2, was reported to be effective against relapsing gca in a prospective open-label study (49) . although the number of enrolled patients was small, the high remission induction and steroid withdrawal rates suggest that this treatment is promising for gca. in addition, the efficacy and safety of tofacitinib and mtx were prospectively evaluated in active takayasu arteritis (50) . compared to mtx-treated group, complete remission and steroid reduction rates were higher in the tofacitinib-treated group, but relapse and imaging improvement rates did not reach the statistical significance. other clinical trials of jak inhibitors for gca (nct03725202, upadacitinib) and tak (nct04161898, upadacitinib) are ongoing. tak may be less likely to produce good results than gca because of the complexity of the disease mechanism; however, we are awaiting promising results. once the efficacy of jak inhibitors has been experimentally demonstrated in lvv, they are expected to be effective in other forms of vasculitis. some pilot studies and case reports demonstrated the efficacy of jak inhibitors for ancaassociated vasculitis (51) , polyarteritis nodosa (52), cutaneous leukocytoclastic vasculitis (53) , and vascular behcet's disease (54) ; however, data on other forms of vasculitis are very limited (55) , and we cannot get any conclusion from such limited data. so far, we have focused on the efficacy of jak inhibitors for rheumatic diseases. as for safety, data are accumulating on the treatment of ra. the use of jak inhibitors is associated with a higher risk of developing shingles, reactivation of varicella-zoster virus (vzv), than bdmards (56) . an increased risk of serious infections compared to bdmards has also been reported in some trials (57) . in addition, new safety concerns emerged after the results of an oral surveillance trial were published (58) . in this trial, patients with active ra who were at risk for cardiovascular events, such as smoking, were assigned to one of three treatment groups: tnf inhibitors, or 5 mg of tofacitinib twice daily, or 10 mg twice daily, and observed for 5 years. the results showed an increased risk of death, malignancy, major adverse cardiac events (mace), and venous thromboembolism (vte) in tofacitinib-treated patients (both 5mg and 10 mg arms) compared to those treated with tnf inhibitors (58) . in september 2021, the food and drug administration issued a warning regarding the use of jak inhibitors. subsequently, the use of jak inhibitors for patients with ra is, in principle, limited to patients who are refractory to at least one tnf inhibitor. although selection bias, which only recruited patients at risk of cardiovascular events, cannot be ruled out in the study, and realword data from a large cohort do not support the increased risk of such serious adverse events (59), we agree that screening before administration and regular monitoring during administration are essential for the treatment with jak inhibitors. gca patients are often older than ra patients and are at higher risk of serious infection, mace, and vte (60) (61) (62) . therefore, it is recommended that jak inhibitors be administered only after adequate risk management and cardiovascular prevention. with regard to shingles, it has been reported that vzv is a contributing factor in the development of gca (63, 64) and is considered extremely high-risk in elderly patients with gca. in the study of baricitinib for gca described above, it was reported that the live-attenuated zoster vaccine did not prevent the onset of shingles (49) . it has been reported that recombinant adjuvanted zoster vaccine can suppress the onset of herpes zoster at a high rate in ra patients (65) . therefore, administration of this recombinant vaccine prior to the use of jak inhibitors is desirable in patients with gca. in conclusion, the efficacy of jak inhibitors in treating rheumatic diseases is promising. given their pathophysiology, jak inhibitors should have high efficacy for gca and tak. therefore, the results of these clinical trials are awaited. however, new safety concerns have emerged that may be limited to treatment-resistant cases. there is an urgent need to establish the long-term safety of jak inhibitors. rw drafted the manuscript. mh revised and finalized the manuscript. all authors contributed to the article and approved the submitted version. rituximab versus cyclophosphamide in anca-associated renal vasculitis rituximab versus cyclophosphamide for anca-associated vasculitis avacopan for the treatment of anca-associated vasculitis avacopan -time to replace glucocorticoids? efficacy and safety of tocilizumab in patients with refractory takayasu arteritis: results from a randomised, double-blind, placebo-controlled, phase 3 trial in japan (the takt study) trial of tocilizumab in giant-cell arteritis use of 18f-fluorodeoxyglucose positron emission tomography to monitor tocilizumab effect on vascular inflammation in giant cell arteritis double-blind trial of abatacept (ctla-4ig) for the treatment of giant cell arteritis double-blind trial of abatacept (ctla-4ig) for the treatment of takayasu arteritis reducing the toxicity of long-term glucocorticoid treatment in large vessel vasculitis disease-modifying anti-rheumatic drugs for the management of takayasu arteritis-a systematic review and meta-analysis janus kinase-targeting therapies in rheumatology: a mechanisms-based approach small molecules to the rescue: inhibition of cytokine signaling in immune-mediated diseases type i/ii cytokines, jaks, and new strategies for treating autoimmune diseases impact on human disease and therapeutic intervention safety and effectiveness of upadacitinib or adalimumab plus methotrexate in patients with rheumatoid arthritis over 48 weeks with switch to alternate therapy in patients with insufficient response baricitinib versus placebo or adalimumab in rheumatoid arthritis tofacitinib or adalimumab versus placebo in rheumatoid arthritis eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update large-vessel vasculitis th17 and th1 t-cell responses in giant cell arteritis th1 and th17 cytokines drive inflammation in takayasu arteritis pro-inflammatory and anti-inflammatory t cells in giant cell arteritis immune mechanisms in medium and large-vessel vasculitis pathogenesis of giant cell arteritis and takayasu arteritis-similarities and differences specific follicular helper t cell signature in takayasu arteritis inhibition of jak-stat signaling suppresses pathogenic immune responses in medium and large vessel vasculitis interferon signature in giant cell arteritis aortitis targeting jak/stat pathway in takayasu's arteritis the critical role of il-6 in the pathogenesis of takayasu arteritis functional profile of tissue-infiltrating and circulating cd68+ cells in giant cell arteritis. evidence for two components of the disease il-6 revisited: from rheumatoid arthritis to car t cell therapy and covid-19 interferon target-gene expression and epigenomic signatures in health and disease cellular signaling pathways in medium and large vessel vasculitis blocking gm-csf receptor alpha with mavrilimumab reduces infiltrating cells, pro-inflammatory markers and neoangiogenesis in ex vivo cultured arteries from patients with giant cell arteritis two susceptibility loci to takayasu arteritis reveal a synergistic role of the il12b and hla-b regions in a japanese population genetic determinants and an epistasis of lilra3 and hla-b*52 in takayasu arteritis a novel susceptibility locus in the il12b region is associated with the pathophysiology of takayasu arteritis through il-12p40 and il-12p70 a susceptibility locus in the il12b but not lilra3 region is associated with vascular damage in takayasu arteritis signaling by il-12 and il-23 and the immunoregulatory roles of stat4 inhibitors as promising agents for refractory takayasu arteritis growth factors and proteases in medium and large vessel vasculitis successful remission with tofacitinib in a patient with refractory takayasu arteritis complicated by ulcerative colitis tofacitinib in patients with refractory takayasu's arteritis giant-cell arteritis with large-vessel involvement assessed by 18f-fdg pet-ct a systematic review of clinical and preclinical evidences for janus kinase inhibitors in large vessel vasculitis efficacy of baricitinib for refractory large-vessel vasculitis a case of takayasu arteritis complicated by refractory ulcerative colitis successfully treated with tofacitinib baricitinib for relapsing giant cell arteritis: a prospective open-label 52-week pilot study treatment efficacy and safety of tofacitinib versus methotrexate in takayasu arteritis: a prospective observational study tofacitinib for the treatment of antineutrophil cytoplasm antibody-associated vasculitis: a pilot study tofacitinib for polyarteritis nodosa: a tailored therapy tofacitinib treatment of refractory cutaneous leukocytoclastic vasculitis: a case report a pilot study of tofacitinib for refractory behcè©t's syndrome contribution of janus-kinase/signal transduction activator of transcription pathway in the pathogenesis of vasculitis: a possible treatment target in the upcoming future risk of herpes zoster (shingles) in patients with rheumatoid arthritis under biologic, targeted synthetic and conventional synthetic dmard treatment: data from the german rabbit register trial of upadacitinib or abatacept in rheumatoid arthritis cardiovascular and cancer risk with tofacitinib in rheumatoid arthritis postapproval comparative safety study of tofacitinib and biological disease-modifying antirheumatic drugs: 5-year results from a united states-based rheumatoid arthritis registry an updated review of cardiovascular events in giant cell arteritis giant cell arteritis treatment patterns and rates of serious infections temporal trends of venous thromboembolism risk before and after diagnosis of giant cell arteritis prevalence and distribution of vzv in temporal arteries of patients with giant cell arteritis decreased immunity to varicella zoster virus in giant cell arteritis efficacy and serious adverse events profile of the adjuvanted recombinant zoster vaccine in adults with pre-existing potential immune-mediated diseases: a pooled post hoc analysis on two parallel randomized trials conflict of interest: rw receives speaker's fee from eli lilly we would like to thank editage for the language review (https:// www.editage.jp/). key: cord-0031594-udo2obt1 authors: athanassiou, lambros; mavragani, clio p.; koutsilieris, michael title: the immunomodulatory properties of vitamin d date: 2022-03-31 journal: mediterr j rheumatol doi: 10.31138/mjr.33.1.7 sha: 9dedc9982808dbe3008b1db88ad60eed067ca0b6 doc_id: 31594 cord_uid: udo2obt1 since its discovery, vitamin d was shown to have both immunostimulatory and immunomodulatory effects on the immune system. a growing body of evidence so far linked vitamin d deficiency with the development and severity of several systemic and organ specific autoimmune/inflammatory diseases, such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. in the present report, the multiple and diverse effects of vitamin d on the immune system are reviewed. while vitamin d is well-known for its actions on bone and mineral metabolism, 1,2 extraskeletal effects are increasingly recognized 3, 4 ; its influences on the immune system have been the focus of intense research. [5] [6] [7] in earlier years, immunostimulatory effects were recognised, 8 followed by subsequent observations revealing the relationship of vitamin d deficiency 9,10 with the development of autoimmune diseases, 5, 10 given the ability of vitamin d to induce immune tolerance. 11, 12 in rheumatoid arthritis, vitamin d deficiency has been found to be prevalent in patients with rheumatoid arthritis [13] [14] [15] [16] and inflammatory bowel disease 17 in association with increased disease activity. 14, 16 similar observations were made in patients with systemic lupus erythematosus 18-20 and systemic sclerosis, 21 with the reported associations with disease activity being rather conflicting. 18,22-24 vitamin d deficiency has been also observed in patients with multiple sclerosis (ms), 25-28 and vitamin d administration may be a complementary agent in ms treatment. 26 vitamin d deficiency has also been reported in patients with diabetes mellitus type 1 29-32 and has been implicated in the development of the disease, 30,33 potentially through modulating inflammatory pathways. 34 vitamin d receptors have been found in many cells of the immune system, [35] [36] [37] [38] such as t lymphocytes 36, 39, 40 and macrophages, 41 among others. moreover, 1a-hydroxylase, the enzyme responsible for the formation of the active compound of the vitamin d system, namely 1,25(oh)2d3, has been found to be expressed in cells of the immune system, 42 amongst them extremely important are its effects on the immune system (figure 1) . cells of the immune system harbour the vitamin d activating enzyme 1-α-hydroxylase and express the vitamin d receptor (vdr). 43, 44 the extra-renal 1-α-hydroxylase is not regulated by pth and thus production of 1,25(oh)2d3 is dependent on concentrations of the substrate 25(oh)d3 and it may be regulated by inflammatory signals, such as lipopolysaccharide and cytokines. 42, 49 cells of the immune system which express the vdr and harbour 1-α-hydroxylase are macrophages, t cells, dendritic cells, monocytes, and b cells 36,50 (figure 2 ). vitamin d is involved both in the regulation of the innate immunity as it enhances the body defence system against microbes and other pathogenic organisms, as well as in the modulation of the adaptive immune system through direct effects on t cell activation and on the phenotype and function of antigen-presenting cells; in particular, dendritic cells. vitamin d regulates the innate immune system. 2,5,51 the innate immune system -an older evolutionary defence strategy-is a first line of defence against infection, 52, 53 and one of the two main immunity arms in vertebrates, including humans. 53 its major functions include recruitment of immune cells, activation of the complement cascade, identification and removal of foreign substances, activation of the adaptive immune response, and the utilization of physical and chemical barriers against infectious agents. 53 the vitamin d receptor (vdr) is expressed both in the keratinocytes 54, 55 and cells of the innate immune system such as macrophages and monocytes, 56-59 thus ensuring its action on two lines of body defence. the beneficial effects of vitamin d on the innate immune system were appreciated early on, as it was implemented as a treatment of infections for a period longer than 150 years, including mycobacterial diseases, such as tuberculosis and leprosy. [60] [61] [62] [63] thus, in 1849, williams reported favourable results after the administration of cod liver oil, an excellent source of vitamin d, in the treatment of patients with tuberculosis. 64 half a century later, niels finsen successfully used uv light, an effective method to increase vitamin d levels, for the treatment of lupus vulgaris, a form of skin sarcoidosis-receiving the third nobel prize in medicine. 6, 65 moreover, alfred windaus, contributed to the discovery of the chemical structure of vitamin d2 and vitamin d3 found in codliver-oil, also receiving the nobel prize. 7, 8, 66 thereafter, several groups used vitamin d2 and d3 as a treatment for tuberculosis. 7, 67 rook et al. 68 demonstrated in the 1980s that 1,25(oh)2d3 inhibited the proliferation of m. tuberculosis in cell cultures. vitamin d enhances the production of defensin β2 and cathelicidin in response to infection by macrophages, monocytes, and keratinocytes. 49 humans have only one cathelicidin, 69 which is produced by cells of the immune system, including neutrophils, macrophages, and cells lining epithelial surfaces that are constantly exposed to potential pathogens such as the skin, the respiratory, and the gastrointestinal tract. [70] [71] [72] cathelicidin has broad antimicrobial activity against gram-positive and gram-negative bacteria, an effect mediated through cell lysis via cell membrane destabilization, 73 as well as activity against certain viruses and fungi. 74 treatment with 1,25(oh)2d3 upregulates cathelicidin mrna in several cell lines, ensuring antimicrobial peptide production on a variety of different cells. 75 25(oh)d3 is the major circulating form of vitamin d used to determine vitamin d status and is important for local production of 1,25(oh)2d3, which upregulates cathelicidin production in both skin and macrophages. exposing human monocytes to pathogens, increases the expression of both 1,25(oh)2d3 and vdr, thus increasing both the local production of 1,25(oh)2d3 and the ability of the cell to respond to it. 49 as keratinocytes possess 25-α-hydroxylase, uv light may directly stimulate cathelicidin production by providing the substrate 25(oh)d3 directly from vitamin d3 produced within the skin. 76, 77 macrophages are phagocytic antigen-presenting cells, which are involved in the first line of defence against pathogens. 1,25(oh)2d3 has various roles in macrophage differentiation and activation. macrophage exposure to 1,25(oh)2d3 can enhance the differentiation of macrophages from monocytes and upon exposure to inflammatory immune signals the expression of 1a-hydroxylase is enhanced, thus allowing the macrophage to locally produce the bioactive metabolite of vitamin d, namely 1,25(oh) 2d3, 42,78 which is necessary for immune modulation. macrophages respond to vitamin d increasing their antimicrobial activity in an heterogeneous manner; thus, those activated after an interleukin-15 stimulus respond adequately, in contrast, interleukin-10 stimulus leads to weak responses. 79, 80 taken together, the ability of the immune cells to hydroxylate 25(oh)d3 locally, suggests that in patients with infections it may be better to administer 25(oh)d3 rather than hydroxylated metabolites to allow for local production and the feedback system to function. neutrophils are the most abundant white blood cell population in the human, and they contribute to a line of defence against microbial pathogens. neutrophils can clear microbes through many mechanisms including phagocytosis and generation of reactive oxygen species and express a functional vitamin d receptor. 38 in accordance, 1,25(oh)2d3 administration has been shown to reduce the production of inflammatory cytokines and reactive oxygen species 81 and to downregulate neutrophil function and activity. monocytes and in particular dendritic cells represent antigen presenting cells, which are important in the initiation of the adaptive immune response. both cell types can be either immunogenic or tolerogenic and thereby modulate t cell responses. 82, 83 tolerogenic antigen presenting cells are characterised by a reduced expression of co-stimulatory molecules and a cytokine production favouring regulatory t cell (treg) induction. 84 dendritic cells are antigen presenting cells, which survey the microenvironment and are specialised in antigen uptake and processing. dendritic cells are crucial regulators of the delicate balance between immunogenicity and immune tolerance. 85 in dendritic cells 1,25(oh)2d3 can interfere with the differentiation and maturation process, thus resulting in an altered morphology, phenotype and function leading to a semimature or tolerogenic phenotype. 86, 87 vitamin d has been shown to manipulate monocytes and dendritic cells at different levels enabling them to exert tolerogenic activities, which could be exploited to better control autoimmune diseases. 86 although primarily an activator of the innate immune system to enhance immediate response to infection, vitamin d also acts to regulate the adaptive immune system. the adaptive immune system includes both humoral immunity components and cell mediated immunity components, both directed against invading pathogens. adaptive immunity leads to immunological memory after an initial response to a specific pathogen, resulting in an enhanced response to future encounters with that pathogen 88 through faster and enhanced production of neutralising antibodies. 89 treg cells (tregs) are an immunosuppressive subpopulation of t cells, which modulate the immune system, maintaining self-tolerance, and preventing autoimmunity. 90 vitamin d can promote development and function of tregs in vitro. 91 effector t cells are directly and indirectly affected leading to a shift in the th1/th2 balance toward th2 and a reduction of the th17 response. 91 once t cells are activated, 1,25(oh)2d3 inhibits il-2 production. 92 t cells harbour the vitamin d receptor. 36 the behaviour of t cells is modulated by vitamin d indirectly via its effects on dendritic cells. the vitamin d receptor is expressed at low levels in freshly isolated cd8+ and cd4+ t cells. 36, 40, 93, 94 following activation and addition of 1,25(oh)2d3 the expression of the vitamin d receptor is induced. in addition, activated cd8+ cells can produce 1a-hydroxylase, which can convert 25(oh)d3 to the active 1,25(oh)2d3. 95 thus, the regulation of t cells responsiveness to vitamin d is a late event. 96 vitamin d and 1,25(oh)2d3 inhibit t cell proliferation and cytokine production, an event occurring after activation. 36,93 it has been hypothesised that following an infection, t cells are induced which are important for clearing the pathogen. the effect of vitamin d does not occur until after the t cell response to the infectious organism has begun. in the infection models, t cells eliminate the pathogen, and the antigen is removed from the system, whereas in an immune mediated disease the antigen persists and t cells are chronically activated, producing inflammatory cytokines. 97 it has been proposed that vitamin d deficiency results in a reduced capacity to turn off t cells following activation. 96 in a previous study, peripheral blood mononuclear cells which were stimulated with t-cell specific mitogens in the presence of 1,25(oh) 2d3 proliferated less and produced less inflammatory cytokines, including interferon-γ. 98 b cells express immunoglobulin receptors in their plasma membrane, recognising antigenic epitopes. they pro-1 2022 10 mediterranean journal of rheumatology 33 1 2022 duce autoantibodies and form b cell follicles with germinal centre activity. once activated, b cells can upregulate the expression of vitamin d receptor and 1a-hydroxylase. 99 1,25(oh)2d3 in b cells can induce apoptosis, inhibiting memory b cell formation and preventing differentiation of b cells to immunoglobulin-producing plasma cells. 100 vitamin d has immunomodulatory properties, 50, 101, 102 and early on after its discovery, it was shown to have immunostimulatory effects as well. 7 in the course of the years, and as the autoimmune diseases were found to increase in prevalence, 103 a worldwide prevalence of vitamin d deficiency was observed, 1,104 implying a significant role of vitamin d in inducing immune tolerance, 11, 12, 86 (figure 1 ) and a potential role of vitamin d deficiency in the development of autoimmune diseases. 10, 105, 106 extensive research provided evidence that vitamin d deficiency may induce the development of rheumatoid arthritis [13] [14] [15] [16] [107] [108] [109] and that it is related to its activity and severity 14, 16 (table 1) . a cross-talk between oestrogen and vitamin d has been postulated, suggesting a sex-specific effect of vitamin d in autoimmunity. 110 research also provided evidence that vitamin d deficiency may be related to systemic lupus erythematosus 18-20,22,23 and multiple sclerosis. 25, 27, [111] [112] [113] vitamin d deficiency appears to be also highly prevalent in patients with inflammatory bowel disease 17 (crohn's disease and ulcerative colitis) in relation to disease activity. 114 vitamin d supports the integrity of the intestinal barrier and is related to microbiota homeostasis in this cohort of patients 115, 116 and may contribute to the prevention of inflammatory bowel disease by supporting the integrity of the intestinal barrier, ensuring bacterial homeostasis and ameliorating disease progression via anti-inflammatory action. 117 vitamin d deficiency in inflammatory bowel disease is aggravated by decreased absorption of the vitamin via the gastrointestinal tract. 116 additionally, vitamin d seemed to induce remission in a cohort of patients with crohn's disease. 118 it has been postulated that vitamin d resistance may be observed in some patients necessitating an individualised approach in the treatment of vitamin d deficiency. 119 in conclusion, vitamin d is a likely immunomodulatory agent. it has immune stimulating properties, as it enhances the function of the innate immune system, and it may induce immune tolerance. vitamin d deficiency may be related to the development of autoimmune diseases. there is no conflict of interest. vitamin d deficiency new developments in our understanding of vitamin metabolism, action and treatment vitamin d and health: beyond bone vitamin d: metabolism, molecular mechanism of action, and pleiotropic effects mechanisms underlying the regulation of innate and adaptive immunity by vitamin d how finsen's light cured lupus vulgaris adolf windaus--nobel prize for research on sterols evolving role of vitamin d in immune-mediated disease and its implications in autoimmune hepatitis vitamin d and autoimmune diseases vitamin d, immune tolerance, and prevention of type 1 diabetes immunomodulatory effects of vitamin d in pregnancy and beyond vitamin d and immunomodulation in early rheumatoid arthritis: a randomized double-blind placebo-controlled study vitamin d and rheumatoid arthritis vitamin d deficiency and rheumatoid arthritis vitamin d level in rheumatoid arthritis and its correlation with the disease activity: a meta-analysis vitamin d deficiency is associated with increased disease activity in patients with inflammatory bowel disease human t lymphocytes are direct targets of 1,25-dihydroxyvitamin d3 in the immune system the targets of vitamin d depend on the differentiation and activation status of cd4 positive t cells 1,25-dihydroxyvitamin d modulates antibacterial and inflammatory response in human cigarette smoke-exposed macrophages immune regulation of 25-hydroxyvitamin-d3-1al-pha-hydroxylase in human monocytes vitamin d status and gene transcription in immune cells the active metabolite of vitamin d3 as a potential immunomodulator activation of the type i interferon pathway in primary sjogren's syndrome type i interferons in autoimmune disease brief report: vitamin d deficiency is associated with endothelial dysfunction and increases type i interferon gene expression in a murine model of systemic lupus erythematosus vitamin d -effects on skeletal and extraskeletal health and the need for supplementation toll-like receptor triggering of a vitamin d-mediated human antimicrobial response vitamin d: nutrient, hormone, and immunomodulator differential immunomodulatory effect of vitamin d (1,25 (oh)(2) d(3)) on the innate immune response in different types of cells infected in vitro with infectious bursal disease virus how the innate immune system senses trouble and causes trouble introduction to the immune system vitamin d/vdr signaling inhibits lps-induced ifnγ and il-1β in oral epithelia by regulating hypoxia-inducible factor-1α signaling pathway the vitamin d receptor as tumor suppressor in skin toll-like receptor triggering of a vitamin d-mediated human antimicrobial response vitamin d's effect on immune function vitamin d: modulator of the immune system effects of vitamin d on macrophages and myeloid-derived suppressor cells (mdscs) hyperinflammatory response in the lungs of covid-19 patients vitamin d receptor expression and hepcidin levels in the protection or severity of leprosy: a systematic review vitamin d modulates human macrophage response to mycobacterium tuberculosis dna vdr polymorphism, gene expression and vitamin d levels in leprosy patients from north indian population vitamin d as an adjunctive treatment to standard drugs in pulmonary tuberculosis patients: an evidence-based case report on the use and administration of cod-liver oil in pulmonary consumption nobel prize presentation speech by professor the count k.a.h. morner, rector of the royal caroline institute on the discovery of vitamin d: the contribution of adolf windaus vitamin d and tuberculosis: where next? vitamin d3, gamma interferon, and control of proliferation of mycobacterium tuberculosis by human monocytes the human cathelicidin ll-37--a pore-forming antibacterial peptide and host-cell modulator vitamin d induces the antimicrobial protein hcap18 in human skin the peptide antibiotic ll-37/hcap-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface identification of cramp, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse the human antimicrobial and chemotactic peptides ll-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity cutting edge: 1,25-dihydroxyvitamin d3 is a direct inducer of antimicrobial peptide gene expression conversion of vitamin d3 to 1alpha,25-dihydroxyvitamin d3 in human skin equivalents expression of vitamin d3 25-hydroxylase (cyp27) mrna after induction by vitamin d3 or uvb radiation in keratinocytes of human skin equivalents--a preliminary study 1,25-dihydroxyvitamin d3 curtails the inflammatory and t cell stimulatory capacity of macrophages through an il-10-dependent mechanism il-15 links tlr2/1-induced macrophage differentiation to the vitamin d-dependent antimicrobial pathway vitamin d status contributes to the antimicrobial activity of macrophages against mycobacterium leprae decreased anti-inflammatory responses to vitamin d in neonatal neutrophils decreased accessory cell function and costimulatory activity by 1,25-dihydroxyvitamin d3-treated monocytes taking dendritic cells into medicine tolerogenic dendritic cells some interfaces of dendritic cell biology induction of tolerogenic dendritic cells by vitamin d receptor agonists dendritic cells as key targets for immunomodulation by vitamin d receptor ligands adaptive immunity evolving adaptive immunity taking regulatory t cells into medicine effects of vitamin d on the peripheral adaptive immune system: a review vitamin d receptor expression controls proliferation of naïve cd8+ t cells and development of cd8 mediated gastrointestinal inflammation 1,25-dihydroxyvitamin d3 receptors in human leukocytes interactions of 1,25-dihydroxyvitamin d3 and the immune system murine cd8+ t cells but not macrophages express the vitamin d 1α-hydroxylase vitamin d regulation of immune function in the gut: why do t cells have vitamin d receptors? the vitamin d receptor turns off chronically activated t cells regulation of lymphokine production and human t lymphocyte activation by 1,25-dihydroxyvitamin d3. specific inhibition at the level of messenger rna modulatory effects of 1,25-dihydroxyvitamin d3 on human b cell differentiation 1 alpha,25-dihydroxyvitamin d3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells skeletal and extraskeletal actions of vitamin d: current evidence and outstanding questions extraskeletal actions of vitamin d the world incidence and prevalence of autoimmune diseases is increasing the vitamin d deficiency pandemic: approaches for diagnosis, treatment and prevention cistromic and genetic evidence that the vitamin d receptor mediates susceptibility to latitude-dependent autoimmune diseases emerging role of vitamin d in autoimmune diseases: an update on evidence and therapeutic implications role of vitamin d in rheumatoid arthritis vitamin d and rheumatoid arthritis: an ongoing mystery association between vitamin d intake and the risk of rheumatoid arthritis: a meta-analysis the role of vitamin d in autoimmune diseases: could sex make the difference? vitamin d and its role in immunology: multiple sclerosis, and inflammatory bowel disease vitamin d, multiple sclerosis and inflammatory bowel disease evidence for a causal relationship between low vitamin d, high bmi, and pediatric-onset ms an update on inflammatory bowel disease immunopathogenesis of ibd: current state of the art the role of vitamin d in inflammatory bowel disease: mechanism to management clinical evaluation of vitamin d status and its relationship with disease activity and changes of intestinal immune function in patients with crohn's disease in the chinese population therapeutic effect of vitamin d supplementation in a pilot study of crohn's patients vitamin d resistance as a possible cause of autoimmune diseases: a hypothesis confirmed by a therapeutic high-dose vitamin d protocol key: cord-0028192-a3ahbdp1 authors: park, dong won; kim, yun jin; sung, yoon-kyoung; chung, sung jun; yeo, yoomi; park, tai sun; lee, hyun; moon, ji-yong; kim, sang-heon; kim, tae-hyung; yoon, ho joo; sohn, jang won title: tnf inhibitors increase the risk of nontuberculous mycobacteria in patients with seropositive rheumatoid arthritis in a mycobacterium tuberculosis endemic area date: 2022-03-07 journal: sci rep doi: 10.1038/s41598-022-07968-w sha: 3fca320b80763deeb6f9adb52c1b3a7a17dd9875 doc_id: 28192 cord_uid: a3ahbdp1 the aim of this study is to examine the impact of tumor necrosis factor inhibitors (tnfi) on nontuberculous mycobacterium (ntm) infection in rheumatoid arthritis (ra) patients in a mycobacterium tuberculosis (mtb) endemic area. we selected 1089 tnfi-treated ra patients and 4356 untreated ra patients using propensity-matching analysis according to age, gender, and charlson comorbidity index using the korean national health insurance service database from july 2009 to december 2010. both groups were followed-up until the end of 2016 to measure the incidence of mycobacterial diseases. the incidence rate of ntm in tnfi-treated ra group was similar to those of mtb (328.1 and 340.9 per 100,000 person-years, respectively). the adjusted hazard ratio (ahr) of ntm for tnfi-treated ra compared to untreated ra was 1.751(95% ci 1.105–2.774). the risk of tnfi-associated ntm in ra was 2.108-fold higher among women than men. the age-stratified effects of tnfi on ntm development were significantly high in ra patients aged 50–65 years (ahr 2.018). ra patients without comorbidities had a higher incidence of ntm following tnfi treatment (ahr 1.742). this real-world, observational study highlights the need to increase awareness of ntm in tnfi-treated ra patients in an mtb endemic area. the risk of developing ntm in ra with and without tnfi treatment. among 5445 ra patients, 86 (1.58%) and 107 (1.97%) were found to be have ntm and mtb during the 86.0 months (median) follow-up, respectively. a total of 26 ntm and 27 mtb cases occurred after starting tnfi treatment in the 1089 tnfitreated ra patients. the median duration from initiation of tnfi therapy to development of ntm and mtb was 29.4 and 16.4 months, respectively. the incidence rate of ntm disease in tnfi-treated ra group was similar to those of mtb disease (328.1 and 340.9 per 100,000 person-years, py, respectively). the incidence of ntm disease was 1.750 times greater in tnfi-treated ra than untreated ra (328.1 vs. 187.4 per 100,000 py, respectively), with an adjusted hr (ahr) of 1.751 (95% ci 1.105-2.774). the risk of developing ntm after tnfi treatment was significantly greater for female ra patients than male patients (ahr 2.108, 95% ci 1.287-3.453). the risk of ntm after tnfi treatment was also significantly increased in ra patients in the age ranges of 50-65 years (ahr 2.018, 95% ci 1.062-3.833). patients without comorbidity had a higher ahr of 1.742 (95% ci 1.019-2.978) for ntm disease in tnfi-treated ra than in untreated ra ( table 2 ). the incidence of mtb was 1.358 times greater in tnfi-treated ra than untreated ra (340.9 vs. 251.1 per 100,000 py, ahr 1.352, 95% ci 0.874-2.091), but this difference was not significant ( table 2 ). as shown in fig. 2 , kaplan-meier analysis 17 . in western countries, the incidence rate of mtb is relatively low and the burden of ntm far outstrips that of mtb 18 . this trend seems to hold true in the impact of tnfi on development of mycobacterial infection in ra patients. previous studies in the us reported that ntm cases are nearly twice as frequent as mtb cases in patients receiving tnfi 14, 19 . however, we showed ntm www.nature.com/scientificreports/ incidence (328.1 per 100,000 py) was similar to those of mtb (340.9 per 100,000 py), which is not surprising considered that our study was conducted in mtb endemic area. moreover, another us study showed that the incidence of ntm was significantly higher in tnfi-treated ra than untreated ra 14 . a case-control study also reported that senior canadians with ntm and mtb were more likely to be under current tnfi (adjusted ors 5.04, and 2.19, respectively) compared with non-tnfi users 13 . on the other hand, a case-control study in an mtb endemic area by liao et al. found that although tnfi-treated ra patients had an increased risk of ntm infections compared to untreated ra patients, there was no statistical significance after adjusting for variables 15 . this decreased statistical power might be related to small case numbers. extending those findings with a large population-based longitudinal ra cohort study, we showed that ntm is significantly more common in tnfiexposed ra patients than unexposed patients. these findings suggest that tnfi poses a risk to the occurrence of ntm regardless of the prevalence of mtb. the risk of ntm increases with age, both worldwide 20 , and in south korea 21 , as failure to control infection is more likely in elderly patients with comorbidities. previous studies indicated that advanced age (≥ 65 years) was a significant risk factor for the development of ntm diseases in the ra cohort 22 , even in patients receiving tnfi 14 . however, this study showed that the risk of ntm disease significantly increased in ra patients aged 50-65 years. interestingly, the impact of tnfi on ntm development was found to be significantly high in middle-aged ra patients as well as in elderly ra patients. patient-specific risk factors for developing ntm may vary with geographical area and disease characteristics. ra may be associated with increased risk of all forms of ntm, including pulmonary and extrapulmonary forms 12 . by reviewing the medwatch database for ntm cases in the us, winthrop et al. reported that 44% of extrapulmonary ntm occurred in patients receiving tnfi 23 . in western countries, disseminated ntm diseases seem to occur at a considerable rate in ra patients with tnfi 12, 14 . however, in south korea, pulmonary ntm diseases seem to predominate over extrapulmonary cases in ra patients. lim et al. evaluated ntm prevalence in south korean ra patients and reported no extrapulmonary involvement in any of the cases 24 . another south korean study on the clinical characteristics of ntm disease during tnfi therapy reported only six ntm patients with pulmonary complications 8 . considering these results, our study seems to reveal a relatively large proportion of pulmonary ntm diseases, although, due to study methodologies, it was not possible to determine the involvement of ntm. ntm lung disease almost www.nature.com/scientificreports/ exclusively occurs in individuals over 45 years old with underlying lung disease, such as bronchiectasis and interstitial lung disease 25 . recently, bronchiectasis has been observed in up to 30% of ra patients 26 , and such modifications of the pulmonary structure may provide a favorable environment for infection and colonization with ntm organisms 27 . a previous population-based study showed that younger bronchiectasis patients had a higher risk of ntm growth 28 . this may have affected the risk of tnfi-induced ntm in our ra cohort. further research is required to explore the association of ntm with young and middle aged tnfi-exposed ra patients. we found that the ra cohort without cci displayed a 1.742-fold higher risk of developing ntm disease than the comparison cohort, supporting the theory that tnfi could play a critical role for development of ntm disease 11 . our study also showed that the tnfi-associated risk of ntm disease in ra was significantly increased in females. reports of gender differences in the incidence of ntm vary in the literature. previous reports have shown that women predominated the ntm cases in ra patients receiving tnfi 8, 23 . however, other studies have shown no difference in the development of tnfi-associated ntm between men and women 14 . one study in taiwan reported that the risk of ntm disease was even higher among male ra patients 22 . more studies are therefore needed to address the impact of gender on ntm development in tnfi-treated ra patients. the tnfi treatment could increase the risk of progression of both latent and newly acquired mtb infection by restricting macrophage function to control the growth of intracellular mtb 29 . however, regarding ntm, for which latent infection is not recognized, harmful effects of tnfi are likely limited to promoting the acquisition of new ntm infection or the progression of an existing infection 30 . the difference in the tnfi related risks between mtb and ntm provide clinical implications that strategies to prevent mtb and ntm should be different. screening and treating ltbi prior to use of tnfi have clear evidence to reduce the risk of mtb infection 7 . current evidence might not yet be sufficient to recommend screening and prevention for ntm infection prior to starting tnfi in ra patients. we demonstrated tnfi treatment in ra patients could be a risk factor for ntm disease, which may promote the progression of preexisting unrecognized ntm infection. ntm screening including chest imaging and systemic symptom review might result in early detection of ntm disease that require antimicrobial therapy in tnfi-treated ra patients. this study was intended to focus on the impact of tnfi on the burden of incident ntm infection in ra patients. regarding study design, however, several issues should be considered when interpreting our results. although we implemented a relatively long washout period of > 2 years to exclude cases previously treated tnfi, enrolled subjects could have taken tnfi before the washout period or in clinical trials. we could not exclude patients who had received diagnoses of ntm and mtb before washout period (2007-2009) because we only have data for 2007-2016. our study applied the different definition of index date between tnfi-treated ra group and untreated ra group. tnfi treated ra group had a slightly longer follow-up period compared to untreated ra group (89.7 ± 7.6 and 84.4 ± 7.9 months, respectively), which may affect the incidence of ntm infection. our study had several limitations. first, the nhis database does not provide detailed individual patient information, such as data on diagnosis year of ra or rheumatic disease activity, which has been associated with anti-rheumatic medication and opportunistic infection 31 . the american college of rheumatology guidelines request that clinicians consider various factors, including comorbid conditions, disease activity, and previous use of dmards and other immunosuppressive agents, before prescribing tnfi to ra patients 32 . in clinical practice, tnfis have restricted applications in patients with comorbid conditions such as heart failure, malignancy, or infection, including mycobacterial infections, which may affect the decision to prescribe tnfi for ra. additionally, patients with more severe ra may be more likely to use biologics. tiippana-kinnunen et al. observed that comorbidities increased during the 15 years of ra and there was significant relationship between comorbidities and disease activity in both early ra disease and at end-point 33 . the cci score might be related to the risk of mycobacterial infection in the ra cohort 11 .various comorbidities in ra patients may also influence the discontinuation of tnfi 34 . high baseline cci in ra patients also correlated with higher disease activity both in early ra disease and at end-point 33 . considering these findings, we controlled for cci, as well as age and gender, using propensity score matching. second, since drug data such as data regarding treatment with non-tnf biologics, dmards, and corticosteroids were not collected in our study, they were not considered in determining the outcomes in this study, which could be another limitation. as potent immunosuppressive drugs, corticosteroids therapy may increase the risk of ntm disease in ra patients in a dose-dependent manner 15 . exposure to leflunomide and other anti-rheumatic drugs with high immunosuppressive potential also was associated with both tb and ntm disease in older ra patients 13 . further investigation exploring the impact of tnfi on the incidence of ntm disease considering the administered drugs other than tnfi, which could shed more light on our speculation, will be needed. third, we identified ra cases using ra diagnosis code from the icd10 and the rare intractable disease (rid) database. this study may therefore include more severe ra patients restricted to those who visited secondary or tertiary medical institutions and required treatment in the rid database. moreover, our study focuses only on seropositive ra patients identified using the rid database, which includes seropositive, but not seronegative ra. about 85% of ra patients in korea are expected to have rheumatoid factor and anti-ccp antibody 35 . thus, our data may not be generalizable to seronegative ra. fourth, we defined ntm infection using only icd-10 codes assigned by health care providers. ntm infection was mainly confirmed by ntm isolation from a respiratory source. this coding-based analysis may not include laboratory results, radiological findings, or bacteriologic results, and instead relies on physician judgment for diagnosis. recent ntm epidemiological studies in south korea have demonstrated the reliability of using this method to define ntm disease 21, 36 . despite, we could not confirm whether the patients completely met the diagnostic criteria of ntm infection and underestimate the actual incidence of ntm disease. thus, our data should be interpreted with caution. fifth, this study did not compare the individual risk for ntm or mtb diseases between various types of tnfi. differences in risk could be caused either by different types of tnfi, differences in patient characteristics, or use of other immunosuppressive agents. the nhis database is limited because physician case reports www.nature.com/scientificreports/ are voluntary and no data on exposure to other drugs are collected. for these reasons, we did not attempt to calculate or compare rates of ntm disease among different tnfi. nonetheless, our study deserves to be highlighted as the first population-based, longitudinal cohort study in an mtb endemic area using propensity score matching to assess the association of ntm disease with tnfi usage in ra patients. with more than 6 years of follow-up, our analysis showed that the cumulative incidence of ntm was higher in tnfi-treated ra than in untreated ra. we have demonstrated that tnfi treatment is associated with the risk of developing ntm in ra patients in the mtb endemic area. unlike mtb infection, there are no formal recommendations for screening and prevention for ntm infection prior to starting tnfi in ra patients. thus, this study can serve to increase awareness of the necessity for early detection of ntm disease in tnfi-treated ra even in younger patients and those without high cci scores. data source and study population. this study is a retrospective population-based cohort study which retrieved data from the nhis, provided by the korean government. the nhis, as an obligatory health insurance system in south korea, provides mandatory healthcare for the vast majority of the korean population and collects healthcare database information on patient demographics, inpatient and outpatient usage, prescription records, and deaths 37, 38 . for certain rare and chronic diseases, such as ra and mtb, the nhis operates the rid registration program and offers financial support (up to 90% copayment) to patients who met the diagnostic criteria after the rid program registration. to qualify for enrollment in the rid program, patients had to meet the diagnostic criteria provided by the nhis for each rid, and assessments had to be approved by specialized physicians. the diagnostic codes were defined according to the international classification of diseases, 10th revision (icd-10), and a special code (v code) was assigned in the rid database 39 . since the nhis could refuse to pay hospital costs if diagnosis did not meet specific criteria, cases are reviewed by medical institutions prior to submission to the nhis and a reliable diagnosis can be presumed. we identified ra patients using special code (v code) in the rid database, as this method has been shown to have higher accuracy than using only the icd code 40, 41 . there have also been several prior studies on the incidence and prevalence of other rare incurable disease using the same rid registration database [42] [43] [44] . registration of ra in the rid database requires both a positive rheumatoid factor test and an official doctor's report documenting that the patient fulfills the classification criteria of ra. study design and definitions. from january 1, 2007 to december 31, 2016 we retrieved data from the nhis database. we selected seropositive ra patients > 18 years of age fulfilling both the ra diagnosis code from the icd10 (m05) and the v223 code in the rid database between july 1, 2009 and december 31, 2010 for inclusion in this study. the tnfi-treated ra group contained ra patients who had received at least one treatment with tnfis, including infliximab, etanercept, or adalimumab and the untreated ra group contained patients not treated with any tnfis during the study period. we decided to establish a washout period to excluded cases previously treated with tnfis or diagnosed with ntm and mtb. previous studies reported that the risk of infection requiring hospitalization associated with tnfi was not statistically significant after two years of tnfi administration 45 , and that most cases of ntm and mtb occurred within three years of tnfi treatment 8, 46, 47 , and those with other immunocompromised condition including malignancy (c00-c97), hiv (b20-b24) during the washout period. we also excluded a further 1946 patients who were treated with tnfi during the follow-up period, giving a total of 27,844 enrolled ra patients (fig. 1) . we followed any newly diagnosed mycobacterial infection (including ntm and mtb) until december 31, 2016 (supplementary figure s1 ). mtb infection was detected based on its icd-10 codes (a15-a19) and the mtb rid registration code, while ntm infection was defined as at least one principal diagnosis of ntm (icd-10 code: a31). the index date was defined as the date of the first prescription of tnfi in the tnfi-treated ra group and the first detection date of ra diagnosis in the untreated ra group during period of inclusion between july 1, 2009 and december 31, 2010. there were no cases previously treated with tnfi or diagnosed with ntm and mtb between july 1, 2009, and the index date in either the tnfi-treated ra group or untreated ra group. the follow-up period was from the index date to the date of detection of new mycobacterial infection or december 31, 2016, whichever was sooner. baseline patient demographic data obtained from the nhis claims database were: age at index date, gender, and diagnosis of comorbidities using icd-10 codes during the washout period. we calculated the charlson comorbidity index (cci), which encompasses 22 comorbid conditions, including cardiovascular disease, malignancy 48 . the scores for connective tissue disease were omitted from the cci score in this study. the study protocol was approved by the institutional review board (irb) of hanyang university hospital, seoul, south korea (irb no. 2018-12-015) according to the ethical guidelines of the declaration of helsinki. the requirement for informed consent from the participants was waived because the nhis database was constructed after anonymization. all data used in this study were approved and provided in a blinded form by the nhis (nhis-2019-1-201). www.nature.com/scientificreports/ statistical analysis. data analysis compared distributions of demographic variables and the cci score between the tnfi-treated and untreated ra groups. descriptive statistics included means ± standard deviations for continuous variables and frequencies and percentages for categorical variables. results were compared using t-test or mann-whitney u-test and the χ 2 test or fisher's exact test, as appropriate. incidence rates of ntm and mtb were calculated for both groups (per 100,000 py), and incidence rates of ntm were stratified by demographic variables and cci scores. controls for each tnfi-treated ra patient (1:4 pairs) were identified through propensity score matching based on gender, age, and cci scores. propensity score matching was performed by greedy matching with using a caliper of 0.2 standard deviations of the logit of the propensity score. to assess the effect of tnfi on incidence of mycobacterial infection, the follow-up py was calculated for each patient. we used the poisson regression model to test the different incidence density of outcome (ntm or mtb) between tnfi-treated and untreated ra groups and presented the result as the incidence rate ratio (irr) and 95% confidence interval (95% ci) between groups. we also used multivariable cox proportional hazards regression analysis to measure the adjusted hazard ratio (ahr) of ntm and mtb with 95% ci for tnfi-treated ra compared to untreated ra, while controlling for age, gender, and cci score. the kaplan-meier method was used to plot the cumulative proportions of ntm cases during the follow-up period, and the log-rank test was used to assess the differences between both groups. all analyses were conducted using sas version 9.4 (sas institute, cary, nc, usa). all tests were two-sided and p-values < 0.05 were considered statistically significant. frequency of infection in patients with rheumatoid arthritis compared with controls: a population-based study infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase iii trial. att rac t study group efficacy and safety of infliximab in patients with ankylosing spondylitis: results of a randomized, placebocontrolled trial (assert) tumour necrosis factor antagonists: structure, function, and tuberculosis risks tuberculosis and targeted synthetic or biologic dmards, beyond tumor necrosis factor inhibitors risk of tuberculosis in patients with immune-mediated diseases on biological therapies: a population-based study in a tuberculosis endemic region effectiveness of recommendations to prevent reactivation of latent tuberculosis infection in patients treated with tumor necrosis factor antagonists mycobacterial diseases developed during anti-tumour necrosis factor-alpha therapy an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases abnormal nasal nitric oxide production, ciliary beat frequency, and toll-like receptor response in pulmonary nontuberculous mycobacterial disease epithelium rheumatoid arthritis increases the risk of nontuberculosis mycobacterial disease and active pulmonary tuberculosis risk of mycobacterial infections associated with rheumatoid arthritis in ontario increased risk of mycobacterial infections associated with anti-rheumatic medications mycobacterial diseases and antitumour necrosis factor therapy in usa risk factors and outcomes of nontuberculous mycobacterial disease among rheumatoid arthritis patients: a case-control study in a non-tuberculous mycobacteria in tb-endemic countries: are we neglecting the danger the epidemiologic relationship between tuberculosis and non-tuberculous mycobacterial disease: a systematic review the growing challenge of nontuberculous mycobacteria infectious diseases society of america emerging infections, n. mycobacterial and other serious infections in patients receiving anti-tumor necrosis factor and other newly approved biologic therapies: case finding through the emerging infections network nontuberculous mycobacterial lung disease prevalence at four integrated health care delivery systems incidence, comorbidities, and treatment patterns of nontuberculous mycobacterial infection in south korea risk for mycobacterial disease among patients with rheumatoid arthritis nontuberculous mycobacteria infections and anti-tumor necrosis factor-alpha therapy nontuberculous mycobacterial infection in rheumatoid arthritis patients: a single-center experience in south korea pulmonary nontuberculous mycobacterial disease prevalence and clinical features: an emerging public health disease comparison of pulmonary abnormalities on high-resolution computed tomography in patients with early versus longstanding rheumatoid arthritis nontuberculous mycobacterial lung infections in ontario prevalence, risk factors and prognosis of nontuberculous mycobacterial infection among people with bronchiectasis: a population survey tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death bedfellows: mycobacteria and rheumatoid arthritis in the era of biologic therapy high disease activity is associated with an increased risk of infection in patients with rheumatoid arthritis american college of rheumatology guideline for the treatment of rheumatoid arthritis co-morbidities in finnish patients with rheumatoid arthritis: 15-year follow-up impact of comorbidities on tnf inhibitor persistence in rheumatoid arthritis patients: an analysis of korean national health insurance claims data korean observational study network for arthritis (korona): establishment of a prospective multicenter cohort for rheumatoid arthritis in south korea prevalence, incidence, and mortality of nontuberculous mycobacterial infection in korea: a nationwide populationbased study data resource profile: the national health information database of the national health insurance service in south korea thirty years of national health insurance in south korea: lessons for achieving universal health care coverage risk of incident mycobacterium tuberculosis infection in patients with inflammatory bowel disease: a nationwide population-based study in south korea incidence and natural course of inflammatory bowel disease in korea temporal trends of the prevalence and incidence of atrial fibrillation and stroke among asian patients with hypertrophic cardiomyopathy: a nationwide population-based study effect of hydroxychloroquine pre-exposure on infection with sars-cov-2 in rheumatic disease patients: a population-based cohort study epidemiology of antiphospholipid syndrome in korea: a nationwide population-based study prevalence and incidence of sarcoidosis in korea: a nationwide population-based study time-dependent increase in risk of hospitalisation with infection among swedish ra patients treated with tnf antagonists incidence of tuberculosis in patients receiving anti-tnf therapy for rheumatic diseases: a systematic review mycobacterial infections in patients treated with tumor necrosis factor antagonists in south korea updating and validating the charlson comorbidity index and score for risk adjustment in hospital discharge abstracts using data from 6 countries this work was supported by the research fund of hanyang university (hy-201700000000454). the authors declare no competing interests. the online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-022-07968-w.correspondence and requests for materials should be addressed to d.w.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-0034758-vqeeaagp authors: hoang, mai p.; park, joonsoo title: vasculitis date: 2020-02-29 journal: hospital-based dermatopathology doi: 10.1007/978-3-030-35820-4_7 sha: ecbfa4c9776e81f65cc68960bbdf3e895035cdb2 doc_id: 34758 cord_uid: vqeeaagp vasculitis, characterized by inflammation and necrosis, manifests a wide spectrum of presentation by involving a vasculature of various sizes and locations. a definitive diagnosis of vasculitis invariably requires histologic confirmation since there are no diagnostic clinical, imaging, or laboratory findings. the most widely adopted vasculitis classification is the chapel hill consensus conference (chcc) nomenclature of systemic vasculitis which integrated clinical symptoms, histopathologic features, and laboratory findings. this classification accounts for the size of the involved vessels. this chapter outlines the clinical and histologic features of the small-vessel vasculitis including the immune complex vasculitis and antineutrophil cytoplasmic antibody-associated vasculitis; medium-vessel vasculitis such as polyarteritis nodosa and kawasaki disease; large-vessel vasculitis, namely, giant cell arteritis and takayasu arteritis; variable-vessel vasculitis such as behcet disease and cogan syndrome; and vasculitis associated with systemic diseases including rheumatoid arthritis, lupus vasculitis, and sarcoid vasculitis. vasculitis can also be secondary to drugs, infection, underlying systemic disease, or trauma. therefore, a diagnosis of vasculitis cannot be based on histologic ground alone. clinical pathologic correlation is necessary. vasculitis, characterized by inflammation and necrosis, manifests a wide spectrum of presentation by involving a vasculature of various sizes and locations. it can present clinically as urticaria, purpura, papules, nodules, erythema, ulcer, infarct, or livedo reticularis. a definitive diagnosis of vasculitis invariably requires histologic confirmation since there are no diagnostic clinical, imaging, or laboratory findings. a skin biopsy would provide information regarding the size of the involved vessels and the nature of the inflammatory infiltrate (neutrophils, lymphocytes, or histiocytes). the optimal time frame would be 24-48 hours after lesion onset. vasculitis can be primary or secondary to drugs, infection, underlying systemic disease, or trauma. therefore, a diagnosis of vasculitis cannot be based on histologic ground alone. it requires clinical pathologic correlation. several classifications have been proposed to differentiate one type of vasculitis from other vasculitides. the initial classification scheme was proposed by the american college of rheumatology (acr) in 1990 based mainly on clinical symptoms [1] . the subcommittee on classification of vasculitides analyzed 1000 consecutive patients with definitive vasculitis and proposed classification criteria for hypersensitivity vasculitis, henoch-schonlein purpura, churg-strauss syndrome, polyarteritis nodosa, wegener granulomatosis, takayasu arteritis, and giant cell arteritis (table 7 .1) [1] [2] [3] . the criteria present in one type of vasculitis but absent or infrequent in the other types were excluded; thus, sensitivity and specificity were low for hypersensitivity vasculitis. in a recent series of 1095 patients with primary vasculitis and 415 with comparable diseases, using acr criteria the sensitivity of each type of vasculitis has decreased although specificity remains high [4] . in 2010 the european league against rheumatism and pediatric rheumatology european society have outlined consensus criteria for the classification of childhood vasculitis [5] . the most widely adopted vasculitis classification is the chapel hill consensus conference (chcc) nomenclature of systemic vasculitis which integrated clinical symptoms, histopathologic features, and laboratory findings [2] . this classification allows differentiation of immune complex-mediated vasculitis such as henoch-schonlein purpura and essential cryoglobuline-mic vasculitis from nonimmune complex ones. it also classifies vasculitides based on the size of the involved vessels: small vessel (<50 um), medium vessel (50-100 um) , and large vessel (>150 um) ( fig. 7.1 ). however, classification by vessel size is imperfect due to overlapping vessel sizes. there is an overlap with arterial involvement since all three major categories of vasculitis can affect any artery size. while microscopic polyangiitis affects mainly the small vessels, polyarteritis nodosa involves medium-sized arteries. the criteria have been modified by the chcc in 2012 (table 7. 2) [1] [2] [3] . these criteria are for classification and they are not for diagnostic purposes. recently several entities have been proposed to be included under the category of cutaneous single-organ vasculitis. these include igm/igg immune complex vasculitis, nodular vasculitis (erythema induratum of bazin), erythema elevatum diutinum, recurrent macular arteritis in hypergammaglobulinemia (hypergammaglobulinemic purpura of waldenstrom), and normocomplementemic urticarial vasculitis [6] . the frequency of various types of vasculitis is summarized in table 7 .3 [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . while wegener granulomatosis is more common in the northern hemisphere, polyarteritis nodosa and microscopic polyangiitis are more common in southern europe and especially in arab countries. hlasouthern norway 1999-2012 [23] drb1 alleles have been shown to be associated with disease susceptibility in giant cell arteritis and henoch-schonlein purpura. cutaneous small-vessel vasculitis or leukocytoclastic vasculitis (lcv) affects postcapillary venules and is limited to the skin. it can be idiopathic or associated with an underlying disease such as connective tissue diseases, rheumatoid arthritis, infections, medications, or malignancies. approximately 10-24% of lcv cases are caused by drug hypersensitivity [24] . it affects both children and adults, with a predilection for women. the most common cutaneous presentation is palpable purpura on sites of dependency, but also livedo reticularis, urticarial lesions, and ulcers [24] . extravasation of erythrocytes from dermal blood vessel results in purpura which does not blanch under pressure ( fig. 7. 2). resolution of purpura progresses to postinflammatory hyperpigmentation. patients with lcv confined to the skin have better prognosis than those with systemic vasculitis. most episodes of cutaneous lcv are self-limited and resolve over 3-4 weeks with residual hyperpigmentation. features characteristics of lcv including fibrinoid necrosis of the vessel walls and perivascular infiltrate of neutrophils and erythrocytes are seen ( fig. 7.3 ). on direct immunofluorescence studies, perivascular c3 and igm can be seen. it is important to exclude associated systemic diseases and identifiable offending agent. the main differential diagnosis for a primary small-vessel vasculitis would be the immune complex smallvessel vasculitis and anca-associated vasculitis. systemic disease (lupus erythematosus) and infection-associated (hepatitis virus) vasculitis can present as lcv. localized forms of chronic cutaneous small-vessel vasculitis include granuloma faciale and erythema elevatum diutinum (see case study 1). the small-vessel vasculitis can also be a process secondary to infections, insect bites, and ulceration; therefore, clinicopathologic correlation is important. 99 .2%) and specificity (87% and 86%) for children and adults, respectively [5] . purpura or petechiae with lower limb predominance is a mandatory criterion plus at least one of the following: abdominal pain, leukocytoclastic vasculitis or proliferative glomerulonephritis on histopathology with iga deposits, arthritis or arthralgia, and proteinuria or hematuria [5] . the consensus to replace "henoch-schonlein purpura" with iga vasculitis is based on evidence indicating that abnormal vascular iga deposits are the defining pathophysiologic feature. genetic predisposition may play a role with a reported strong association with hla-drb1 * 01 phenotype [25] . the risk of hsp development and snp7 subunit polymorphisms of the c1galt1 gene as well as mcp1/ccl1-2518 t polymorphisms has been proposed. in addition, an association between disease severity and nephritis with rantes/ccl5 and rantes/ • palpable purpura on dependent areas, urticarial lesions, and ulcers. • extracutaneous manifestations are uncommon. • leukocytoclastic vasculitis. • direct immunofluorescence studies would be negative. • immune complex small-vessel vasculitis • anca-associated vasculitis • secondary vasculitis due to infections, insect bites, and ulceration clinical relevant pearls • cutaneous small-vessel vasculitis or leukocytoclastic vasculitis is a diagnosis of exclusion. it is important to exclude associated systemic diseases and identifiable offending agent. • clinicopathologic pathologic correlation is essential to exclude the secondary small-vessel vasculitis. ccl5-403t polymorphisms has been reported, respectively [25] . it is the most common form of vasculitis in children presenting between the ages of 2 and 10 years (median of 4 years). the patients typically present in autumn or winter with a tetrad of symptoms: cutaneous palpable purpura, abdominal pain, joint pain, and renal involvement. the cutaneous lesions present in all cases often start as petechiae and palpable purpura on the lower extremities and buttocks, likely attributed to gravity-dependent areas ( fig. 7.4) . about one-third of the patients have trunk and upper extremity involvement. glomerulonephritis indistinguishable from iga nephropathy may occur. a majority of cases have preceding upper respiratory tract infections. the hemorrhagic skin lesions will resolve into skin discoloration between several weeks to a few months. iga vasculitis is generally selflimited in the pediatric population; however, 20-80% of adult patients have renal involvement, and end-stage renal failure can develop in 1% of cases. the risk of chronic renal failure is related to the development of nephrotic syndrome. therefore, although iga vasculitis is more common in children, it has a more severe course in adults. vasculitis affects small vessels (predominantly capillaries, venules, or arterioles). skin biopsy would typically demonstrate fibrinoid necrosis of the vessel walls and perivascular infiltrate of neutrophils, leukocytoclasis, and extravasated erythrocytes in 92% of cases ( fig. 7 .5) [26] . on direct immunofluorescence (dif) studies, vascular iga and c3 would be identified in 81% of cases ( fig. 7.6 ) [26] . although there is a strong correlation between iga deposition on dif and iga vasculitis, there is a subset of patients in which there is absent cutaneous vascular iga deposition [27] . acute hemorrhagic edema, hypersensitivity vasculitis, wegener disease, and microscopic polyangiitis can have similar clinical presentation. in addition, features of leukocytoclastic vasculitis can be seen on histologic sections of skin biopsies. vascular iga deposition can be seen in dermatitis herpetiformis, iga nephropathy, and chronic alcohol intake [28] . cryoglobulinemic vasculitis (cv) is a vasculitis with cryoglobulin immune deposits within small vessels (capillaries, venules, or arterioles) and associated serum cryoglobulins [3] . skin, peripheral nerves, joints, and kidneys are often involved. cryoglobulins are circulating antibodies that precipitate below core body temperature. based on the composition of the cryoprecipitate, cryoglobulinemia is divided into three types: monoclonal igm or igg (rarely iga) in type i, a mixture of polyclonal igg and monoclonal igm with rheumatoid factor activity in type ii, and polyclonal igg and polyclonal igm with rheumatoid activity in type iii. type ii and iii cryoglobulins are mixed cryoglobulinemia that frequently lead to systemic vasculitis due to immune complex deposition. type i is invariably associated with b-cell lymphoproliferative disorders such as waldenstrom's macroglobulinemia and multiple myeloma. the main etiology of mixed cryoglobulinemia is hepatitis c virus (hcv) infection accounting for more than 90% of all cases of cv. although circulating cryoglobulins can be detected in 25-30% of chronic hcv-positive patients, cv would develop in only 10-15% of these patients. there is no correlation between hcv genotype and the development of cv. others include connective tissue diseases (sjogren's syndrome, systemic lupus erythematosus, rheumatoid arthritis) or human immunodeficiency virus (hiv) infection. cv is classified as essential or idiopathic when there is no apparent underlying disease process [29] . • purpura or petechiae with lower limb predominance is a mandatory criterion plus at least one of the following: abdominal pain, leukocytoclastic vasculitis or proliferative glomerulonephritis on histopathology with iga deposits, arthritis or arthralgia, and proteinuria or hematuria. • leukocytoclastic vasculitis. • vascular iga and c3 deposition on direct immunofluorescence studies would be identified in 81% of cases. • hypersensitivity vasculitis • granulomatosis with polyangiitis (wegener) and microscopic polyangiitis clinical relevant pearls • a tetrad of symptoms: cutaneous palpable purpura, abdominal pain, joint pain, and renal involvement. • although iga vasculitis is more common in children, it has a more severe course in adults. • 20-80% of adult patients have renal involvement, and end-stage renal failure can develop in 1% of cases. • there is a subset of patients in which there is absent cutaneous vascular iga deposition. the disease severity varies ranging from petechial eruptions to life-threatening presentation. fatigue, the main symptom, is noted in 80-90% of patients. the characteristic cutaneous presentation is palpable purpura, but also ecchymoses, erythematous papules, and nodules on the lower extremities and less often the trunk and upper extremities. the cutaneous purpura is sporadic and intermittent, lasts for 3-10 days, and resolves with post-inflammatory hyperpigmentation. raynaud's phenomenon, acral cyanosis, and livedo reticularis are common cutaneous lesions seen in type i. arthralgia is noted in 40-60% of patients and usually as intermittent mono-or oligo-arthralgia affecting large joints. glomerulonephritis and symmetric peripheral neuropathy can be seen in type ii and iii cryoglobulinemias, respectively [29] . neurologic manifestations can be seen in 60-70% of patients and affect sensory nerves more frequently than motor nerves. renal manifestations are seen in 20-30% and associated with a poor prognosis. significant morbidity and mortality is seen with cv. the 10-year survivals for type i, hcvnegative mixed cv, and hcv-positive mixed cv are 87%, 65%, and 63%, respectively [29] . the presence of liver fibrosis, central nervous system (cns) involvement, kidney involvement, and heart involvement are associated with poor prognosis. in patients with mixed cryoglobulinemia due to hcv, viral eradication was associated with clinical improvement. hcv-positive patients with mixed cryoglobulinemia had a poorer course than non-hcv type ii and iii cryoglobulinemia due to infections and end-stage liver disease. therefore, the main treatment for hcv-positive cv is antiviral therapy [30] . the use of immunosuppressant is associated with poor outcome [29] . both pure and mixed cv have an increased risk of developing b-cell non-hodgkin lymphoma [31] . thrombotic occlusion of vascular lumens is seen in type i (see chap. 8), whereas a necrotizing vas-culitis is seen in type ii and type iii cryoglobulinemias ( fig. 7.7 ). vascular igm, igg, and c3 deposition would be seen on dif studies. immunoexpression of hcv-related proteins within vascular walls supports the etiologic role of hcv. the cryoglobulin level is considered significant when greater than 0.05 g/l at two separate testings performed with at least a 12-week interval. immunoblotting is a sensitive and specific method which detects cryoglobulin in 98% of cases. ex vivo cryoprecipitation can result in artifacts; therefore, serum should be transported and tested at 37 °c. in the second phase of testing, serum should be incubated at 4 °c for 3 to 7 days. in the typing of cryoglobulin phase, immunofixation or immunoelectrophoresis allows classification as types i-iii. other surrogate laboratory indicators include decreased complement c4 levels and the presence of an immunoglobulin with rheumatoid activity [29] . a monoclonal b-cell lymphocytosis can be seen. hcv-related polyarteritis nodosa affects mainly the medium-sized vessels; therefore, lifethreatening vasculitis, severe multifocal sensorimotor mononeuropathies, malignant hypertension, cerebral angiitis, and kidney and liver microaneurysms would be present more frequently in comparison to cv. hypocomplementemic urticarial vasculitis (huv) is an uncommon systemic and relapsing immune complex-mediated vasculitis of unknown etiology. huv is characterized by urticaria, hypocomplementemia, and vasculitis affecting small vessels (capillaries, venules, or arterioles) and anti-c1q antibodies [3] . schwartz et al. [32] have proposed two major criteria (chronic urticaria and low complement levels) together with at least two minor criteria (leukocytoclastic vasculitis, arthralgias or arthritis, uveitis or episcleritis or conjunctivitis, glomerulonephritis, abdominal pain, and/or positive anti-c1q antibody). these symptoms should be present for at least a 6-month duration. urticarial vasculitis can be either normocomplementemic (nuv) or hypocomplementemic (huv). although most huv cases are idiopathic, approximately 25% of cases may be associated with systemic diseases such as systemic lupus erythematosus (sle), primary sjogren's syndrome, serum sickness reaction, monoclonal gammopathy, hematologic disorders, and drug hypersensitivity [33] . in a study of a family with three affected children with huv, ozcakar et al. [34] reported mutations in dnase1l3, encoding an endonuclease that has previously been associated with sle. therefore, it is not surprising that some authors have proposed that huv syndrome is a subset of sle. the pathophysiology of urticarial vasculitis has been thought to be a type iii immune complex-mediated hypersensitivity reaction. c1q is the subunit of the c1 complex of the complement activation cascade. the c1q antibodies and associated immune complexes activate the complement pathway resulting in mast cell degranulation, subsequent increased vascular permeability, and urticaria and/or angioedema. • a small-vessel vasculitis with cryoglobulin immune deposits within small vessels and associated serum cryoglobulins. • it affects skin, peripheral nerves, joints, and kidneys. • a necrotizing vasculitis is seen in type ii and type iii cryoglobulinemias. • thrombotic vasculopathy is seen in type i. clinical relevant pearls • in hcv-negative patients with cryoglobulinemic vasculitis, pulmonary, gastrointestinal, and renal involvement and age >65 years are associated with death. • in hcv-positive patients with cryoglobulinemic vasculitis, antivirals and immunosuppressant are associated with good and poor outcome, respectively. • there is an increased risk of b-cell non-hodgkin lymphoma. • surrogate laboratory indicators include decreased complement c4 levels and the presence of an immunoglobulin with rheumatoid activity. there is a female predominance and affects patients in the fourth to fifth decade of life. glomerulonephritis, arthritis, obstructive pulmonary disease, and ocular inflammation are common clinical manifestations. urticarial lesions in this setting are often painful, associate with angioedema, persist more than 24 hours, and resolve with hyperpigmentation ( fig. 7.8 ). in addition to urticaria, annular lesions, targetoid lesions, palpable purpura, livedo reticularis, and bullae can be other cutaneous manifestations [33] . pruritic urticarial lesions can be seen in 51% of patients, purpura in 35%, and livedo reticularis in 14% [33] . extracutaneous manifestations include constitutional symptoms (fever, fatigue, malaise) (56%), musculoskeletal (82%), ocular (56%), pulmonary (19%), gastrointestinal (18%), and kidney (14%) involvement [33] . arthralgias and arthritis frequently affect the joints of the hands, wrists, elbows, knees, and ankles. often mild in adults, renal involvement manifested as proteinuria and microscopic hematuria can be severe in children. pulmonary manifestations include dyspnea, coughing, hemoptysis, pleural effusion, and chronic obstructive pulmonary disease which is the most frequent cause of death. gastrointestinal symptoms include nausea, vom-iting, diarrhea, and ascites. uveitis, episcleritis, are conjunctivitis are common ocular involvements. laboratory studies reveal low complement levels (c1q, c2, c3, and c4), low c1q levels, normal c1 inhibitor levels, and anti-c1q antibodies in 55% [33] . the detection of anti-c1q antibodies is not a major criterion since it can be negative [33] . high morbidity and mortality are observed due to chronic obstructive pulmonary disease. patients with hypocomplementemic urticarial vasculitis have a greater risk than those with normocomplementemic urticarial vasculitis for multiorgan involvement. histologic features of leukocytoclastic vasculitis such as fibrinoid necrosis of the vessel walls, leukocytoclasis, and perivascular erythrocytes are typically seen. dermal infiltrate of eosinophils and edema can be seen. direct immunofluorescence studies show granular igg, less commonly igm and c3 at the basement membrane zone and c3 in walls of blood vessels. the differential diagnosis of urticarial vasculitis includes common urticaria, serum sickness, systemic lupus erythematosus (sle); neoplasia; mixed cryoglobulinemia; cogan syndrome; muckle-wells syndrome; arthritis, hives, and angioedema (aha) syndrome; and schnitzler syndrome. although the presence of anti-c1q antibody has often been used to distinguish huv from connective tissue disease, the antibody can be seen in both primary and secondary vasculitis [35] . inflammatory ocular disease, a prominent feature of huv, would be unusual in sle. cogan syndrome, a disease of young adults, presents with interstitial keratitis and meniere's-like episodes [36] . cogan syndrome can be accompanied by a systemic vasculitis resembling either takayasu arteritis or polyarteritis nodosa, depending on the size of the involved vasculature. muckle-wells syndrome is a dominantly inherited autoinflammatory disease characterized by urticarial rashes, fever, arthralgia, progressive sensorineural deafness, and frequent association with systemic aa amyloidosis [37] . in aha syndrome, urticarial vasculitis is accompanied by angioedema [38] . monoclonal igm gammopathy, intermittent fever, and hyperostoses would be noted in schnitzler syndrome. anti-glomerular basement membrane (anti-gbm) disease, previously known as goodpasture's disease, is a rare yet often lifethreatening small-vessel vasculitis caused by in situ immune complex deposition. anti-gbm affects glomerular capillaries, pulmonary capillaries, or both, with gbm deposition of anti-gbm autoantibodies [3] . lung involvement causes pulmonary hemorrhage, and renal involvement causes glomerulonephritis with necrosis and crescents. anti-gbm is a misnomer since anti-gbm antibodies react not only with gbm but also with pulmonary alveolar capillary membranes. "goodpasture's syndrome" has been used in the past for combined pulmonary and renal expression of anti-gbm disease. type ii antigen-antibody reaction plays the role in this disease. the autoantibodies bind to epitopes in the basement membrane, activate the complement cascade, and cause subsequent tissue damage. this interaction between antigen and antibody can be visualized as linear igg deposition along glomerular basement membrane and on occasion the alveolar basement membrane on direct immunofluorescence studies. a 28-kd monomeric subunit of the non-collagenous-1 (nc1) domain of the α3 chain of type iv collagen, α345nc1, has been identified as the target • key diagnostic features are urticarial vasculitis and hypocomplementemia. • to render a diagnosis, one would need two major criteria (chronic urticaria and low complement levels) together with at least two minor criteria (leukocytoclastic vasculitis, arthralgias or arthritis, uveitis or episcleritis or conjunctivitis, glomerulonephritis, abdominal pain, and/or positive anti-c1q antibody). • in contrast to urticaria, urticarial vasculitis lasts more than 24 hours. • the systemic vasculitis of this syndrome involves skin, eyes, joints, kidneys, and gastrointestinal tract. • pulmonary complication is the most life-threatening complication. • the detection of anti-c1q antibodies is not a major criterion since it can be negative. antigen of anti-gbm disease [39] . conformational alteration of the quaternary structure of α345nc1 at residues 17-31 and 127-141 of α3(iv)nc1 is likely the responsible trigger of immune response [39] . the α3(iv) chain is expressed in few specialized basement membranes including the glomerular, alveolar, testicular, inner ear, and eye [40] . only the glomerular and alveolar basement membranes are preferentially affected, likely attributed to the greater accessibility of epitopes to the circulating antibodies [39] . the principal component of the glomerular filtration barrier is type iv collagen. in the absence of any of the type iv collagen chains, progressive renal failure develops. anti-gbm disease has a strong association with human leukocyte antigen (hla)-drb1 * 1501 and a lesser extent with hla-drb1 * 1502, suggesting that additional factors such as genetic or environmental are necessary for disease progression [41] . the disease affects 0.5-1 per million persons per year [41] . the disease has a bimodal presentation, 20-30 years and 60-70 years, and affects preferentially men in the younger group and equally men and women over 60 years of age. it is characterized by both pulmonary hemorrhage and renal failure, ranging from mild to lethal outcome, in 60-80% of the patients. solely renal symptoms are seen in 20-40%, whereas less than 10% have only pulmonary symptoms. the patients invariably have hematuria in addition to constitutional symptoms such as fever, weight loss, and arthralgias. lung involvement is manifested clinically as exertional dyspnea and hemoptysis. anemia secondary to iron deficiency can develop. the titer of the circulating autoantibodies correlates with disease severity [41] . anca positivity is present in patients with extrarenal and extrapulmonary manifestations and recurrent renal or pulmonary disease. in a series of 221 chinese patients with anti-gbm disease in 1998-2008, the authors reported milder renal damage and less frequent pulmonary involvement in patients older than 65 years of age [42] . a combination treatment composed of plasmapheresis, corticosteroids, and immunosuppressive drugs has helped to improve the 1-year survival to 70-90% [41] . the 5-year survival is more than 80%, and less than 30% of the patients would require longterm dialysis. percutaneous renal biopsy provides much higher diagnostic yield than transbronchial or open lung biopsy. renal biopsy typically demonstrates a necrotizing crescentic glomerulonephritis ( fig. 7.9 ). histologic hallmark is linear igg deposition along the glomerular basement membrane seen on direct immunofluorescence studies ( fig. 7 .10) [41] . serologic testings such as radioimmunoassays or enzyme-linked immunosorbent assays (elisa) for anti-gbm antibodies are highly sensitive and specific. elisa invariably shows the presence of anti-gbm antibodies in serum of these patients. one-third of the patients can have circulating anca, mainly mpo-anca, in addition to anti-gbm antibody [43] . anca can precede the development of anti-gbm antibody by months or years. the patients can be doubly positive for anti-gbm and c-anca or p-anca [43] . double-positive patients have characteristics similar to those of anca-associated vasculitis including older age and longer symptom duration before diagnosis and features of anti-gbm disease such as kidney and lung involvement at presentation [43] . the clinical course however is similar to those with anti-gbm only. conditions that cause pulmonary-renal syndromes include anca-associated vasculitides (mpa, gpa, and epga), iga vasculitis, sle, undifferentiated connective tissue disease, and rarely rapidly progressive glomerulonephritis. anti-gbm antibodies can be detected in gpa and other anca-associated vasculitides and inflammatory conditions characterized by renal and pulmonary involvement. since mpo-anca can be detected in patients with anti-gbm disease, distinction from gpa would be important. careful review of the clinical history, physical examination, and laboratory studies would be needed to correctly render the diagnosis. antineutrophil cytoplasmic antibody (anca)associated vasculitides are rare systemic diseases which comprise of microscopic polyangiitis • a rare autoimmune disease characterized by renal involvement and sometimes with lung involvement. • the non-collagenous domain-1 of the α3 chain of type iv collagen is the autoantigen. clinical relevant pearls • without prompt diagnosis and treatment, the patient can develop alveolar hemorrhage, kidney failure, and subsequently death. • the key to better prognosis is timely diagnosis. • since 20-35% have both anti-gbm and mpo-anca simultaneously, anti-gbm and anca should be tested in parallel in patients with renal disease. • kidney biopsy would provide a definitive diagnosis. • serologic studies for anti-gbm antibodies to confirm the diagnosis and to monitor therapeutic response. • crescentic glomerulonephritis on kidney biopsy • a linear igg deposition along glomerular basement membrane on direct immunofluorescence • presence of circulating anti-gbm antibodies, specifically the anti-α3(iv) nc1 antibodies on solid-phase immunoassay • other conditions that cause pulmonaryrenal syndromes such as systemic lupus erythematosus and anca-associated vasculitides • granulomatosis with polyangiitis in those with anti-gbm and mpo-anca (mpa), eosinophilic granulomatosis with polyangiitis (egpa, churg-strauss syndrome), and granulomatosis with polyangiitis (gpa, wegener's). they have few or no immune deposits and predominantly affect small vessels (such as capillaries, venules, arterioles, and small arteries). anca-associated vasculitides are systemic diseases, but limited forms confined to single organs may occur. renal biopsy is still the best for rendering diagnosis, and it is an important predictor of renal outcome. the anca antibodies can be specific for either myeloperoxidase (mpo-anca) or proteinase 3 (pr3-anca). by using indirect immunofluorescence on ethanol-fixed neutrophils, c-anca is characterized by diffuse cytoplasmic granular fluorescence, and the antigen is generally pr3. p-anca is characterized by perinuclear neutrophil staining pattern, and the major target antigen is myeloperoxidase. a-anca or atypical anca exhibits both cytoplasmic and perinuclear/nuclear staining. pr3-ancas account for majority of anca with cytoplasmic immunofluorescence (canca) and are associated with gpa (wegener's) in 60-80% of the cases. mpo-ancas account for perinuclear immunofluorescence pattern (panca) and are associated with mpa (80-90%) and egpa (churg-strauss) (35-40%) [3] . currently enzyme-linked immunosorbent assay (elisa) is commonly used to detect these antibodies. anca status is important since patients with c-anca and p-anca have different organ manifestations, likelihood of therapy response, and risk of relapse ( fig. 7.3) . anca specificity predicts differences in long-term prognosis. patients with pr3-ancas are at a higher risk of relapse than patients with mpo-ancas [44] . anca specificity has been suggested to be better than clinical diagnosis for defining homogeneous groups of patients, since pr3-anca and mpo-anca are associated with different genetic backgrounds and epidemiology [44] . patients with pr3-aav and mpo-aav do not share the same genetic background and have only some pathophysiologic mechanisms in common. anca specificity predicts response to induction therapies; rituximab is more effective than cyclophosphamide in patients with pr3-aav. on the contrary, both treatments are similarly effective in patients with mpo-aav. the pathogenesis of anca-associated vasculitis remains unclear, and it is likely due to a variety of factors such as genetic susceptibility, environmental agents, and innate and adaptive immune responses. in a genome-wide association study (gwas) of 2687 northern european caucasian patients, gpa was reported to be associated with genetic variants within hla-dp, serpina1 (encoding alpha-1-antitrypsin), and prtn3 (encoding pr3), while mpa is associated with hla-dq [45] . of interest, these genetic backgrounds were more closely associated with mpo-or pr3-anca specificity than with the clinical syndrome. proteomic analyses have identified timp1 as a marker of anca-associated vasculitis activity and tkt and cd93 as markers of renal involvement and outcome in anca-associated vasculitis [46] . a recent meta-analysis identified 33 genetic variants, supporting a role for alpha-1-antitrypsin, the major histocompatibility complex system, and inflammatory processes in the pathogenesis of anca-associated vasculitis [47] . initially reported as microscopic polyarteritis, microscopic polyangiitis (mpa) affects mainly small vessels (capillaries, venules, and arterioles), but can involve the medium arteries. it is with few or no immune deposits and lack of granulomatous inflammation. necrotizing glomerulonephritis and pulmonary capillaritis are common symptoms. it is associated with p-anca due to antibodies against mpo in 50-75% of cases [48] . although environmental factors such as silica exposure have been implicated, the etiology of mpa remains unknown. mpa is a systemic vasculitis that can affect multiple organs; however, it can be restricted to only the kidneys. renal involvement often as rapidly progressive glomerulonephritis is invariably seen in all patients [48] . renal symptoms seen in 80-100% of patients can range from an asymptomatic urinary sediment to end-stage renal disease necessitating dialysis. glomerulonephritis is the only symptoms in some cases. pulmonary involvement can be seen in 25-55% with diffuse alveolar hemorrhage resulting in hemoptysis, dyspnea, cough, and pleuritic chest pain as the classic presentation [48] . cutaneous involvement as palpable purpura, livedo reticularis, nodules, urticarial lesions, and skin ulcers on bilateral extremities can be seen in 30-60% of patients. skin lesions can be the initial presenting sign in 15-30% of the patients [49] . abdominal pain, the most common gastrointestinal symptom, can be seen in 30-58% of patients. neurologic involvement can be seen in 37-72% of the patients and commonly comprises peripheral neuropathy including mononeuritis multiplex and distal symmetrical polyneuropathy [49] . if untreated the prognosis is very poor due to pulmonary hemorrhage and rapidly progressive glomerulonephritis with a 10% 1-year survival. with aggressive immunosuppressive treatment, the 1-year and 5-year survival rates are 82% and 76%, respectively [50] . serum creatinine level, african american ethnic background, and arterial sclerosis on kidney biopsy are predictors for end-stage renal failure [50] . response to induction therapy in japanese patients with mpa can be predicted by monitoring the altered gene expression of 16 candidates in the peripheral blood [51] . skin biopsies often show only leukocytoclastic vasculitis (figs. 7.11 and 7.12). histologic confirmation of necrotizing vasculitis of small vessels including arterioles, capillaries, and venules, usually with either kidney or lung biopsy, is still the gold standard. since p-anca due to antibodies against myeloperoxidase is seen in 50-75% of cases, a negative anca test does not exclude the diagnosis of mpa. the presence or absence of small-vessel involvement rather than the presence of medium-sized arteries is the distinguishing feature between polyarteritis nodosa and mpa. in a comparison study in children, pulmonary manifestations were less frequent and less severe in patients with mpa versus those with gpa [52] . however, renal involvement with greater severity (nephroticrange proteinuria, dialysis, and end-stage renal disease) was noted in patients with mpa versus gpa. genome-wide association analyses of mpa and gpa cases have demonstrated correlation between the patients' genotypes and anca specificity [45] . • a systemic vasculitis that affects small vessels of multiple organs, mainly the lungs and kidneys causing diffuse alveolar hemorrhage and rapidly progressive glomerulonephritis, respectively eosinophilic granulomatosis with polyangiitis (epga) (formerly churg-strauss syndrome) is a rare necrotizing vasculitis affecting small to medium vessels with eosinophil-rich and necro-tizing granulomatous inflammation involving the respiratory tract, associated with asthma and peripheral blood eosinophilia [3] . the acr criteria are most commonly used for diagnosis which can be based on clinical findings with or without histologic confirmation. the diagnosis can be made with a sensitivity of 85% and specificity of 99.7% [53] when four of the following six criteria are present: asthma, eosinophilia greater than 10%, neuropathy, pulmonary infiltrates, paranasal sinus abnormality, and extravascular eosinophils on biopsy [1] . egpa is considered a systemic disease; however, limited expressions of egpa confined to the upper or lower respiratory tract may occur. anca with a perinuclear immunofluorescence staining pattern is positive in only about 40-60% of patients [48] . those with anca were more likely to have definitive features or surrogates of small-vessel vasculitis, purpura, peripheral neuropathy, myalgia, arthralgia, and glomerulonephritis [54] , whereas those without anca developed myocarditis, lung infiltrates, and gastrointestinal symptoms frequently. in addition, a recent study reported that not all egpa patients had definite vasculitis features [54] . hypereosinophilic asthma with (any) systemic (nonvasculitic) manifestations (hasm) has been proposed for patients with asthmas, blood hypereosinophilia, and systemic symptoms [54] . tissue eosinophil proliferation and activation result in granule cytotoxic protein release contributing to tissue damage including eosinophilic pneumonitis and myocarditis. production of il-25 maintains the cycles of th2-mediated disease. the clinical presentations appear to segregate into two subsets: vasculitic and eosinophilic manifestations. asthma with or without allergic rhinitis is seen in 96-100% of patients in the initial prodromal phase. other symptoms can be present in this phase including arthralgias, myalgias, malaise, fever, and weight loss which can last from months to years. the eosinophilic phase is characterized by peripheral eosinophilia and organ (lung, cardiac, and gastrointestinal) • small-vessel vasculitis • p-anca due to antibodies against myeloperoxidase seen in 50-75% of cases • polyarteritis nodosa • granulomatosis with polyangiitis (gpa) • renal disease is the most frequent clinical presentation, followed by systemic features, musculoskeletal, cutaneous, lower respiratory tract, and gastrointestinal involvement. • diagnosis relies on clinical findings, anca antibody, kidney, and lung biopsies. • in the setting of pulmonary-renal syndrome antineutrophil cytoplasmic antibodies (anca), testing is useful for the diagnosis of anca-associated vasculitis. • ancas are predominantly directed against myeloperoxidase (mpo-anca), but against proteinase 3 (pr3-anca) in 20-30% of mpa cases. • anca can be negative in a small subset of mpa patients. • anca can be falsely positive in connective tissue diseases, infection, and malignancies. involvement. constitutional symptoms (fever, weight loss, fatigue) and skin lesions are common in the vasculitic phase. cutaneous involvement, seen in 40-50% of cases, can be papular and nodular, purpuric, erythematous, or vesiculopapular arising on a background of purpura on extremities, trunk, neck, and face. purpura and petechiae on the lower extremities are the most common (fig. 7.13 ). other skin manifestations including urticarial, erythematous papule, cutaneous or subcutaneous nodules, livedo reticularis, digital gangrene, and bullous lesion can be seen [55] . both vessel inflammation and eosinophilic proliferation are thought to contribute to organ damage, but the clinical presentations are heterogeneous. neurologic symptoms can be present including peripheral neuropathy (polyneuropathy or multiple mononeuropathy) in 55% of cases, cns such as ischemic lesions and intracerebral hemorrhages, cranial nerve palsies, and loss of visual acuity in 8% of cases [56] . cns involvement was noted at diagnosis in 86%, before the diagnosis in 2%, and during follow-up in 12% of cases [57] . cerebral infarction and subarachnoid hemorrhage, the main cns manifestations, are thought to be induced by vasculitis and/or eosinophil-mediated injury [57] . in general egpa is considered a milder form of systemic vasculitis with lower mortality compared to other types of vasculitis. long-term out-comes are generally good, and relapse was noted in 26-28% of patients in remission [58] . the overall 5-year and 10-year survival rates were 88.9% and 78.6%, respectively [48] . ancapositive patients more frequently had a "vasculitic" phenotype, ent involvement, peripheral neuropathy, and/or renal involvement, whereas the anca-negative patients would have an eosinophilic "tissue" phenotype -more frequently cardiomyopathy [56] . the 5-year relapsefree survival was 58% and 68% for anca-positive and anca-negative patients, respectively [56] . cardiomyopathy and older age at diagnosis were independent risk factors for death and lower eosinophil count at diagnosis as predictive of relapse in multivariate analyses [56] . myocardial and gastrointestinal tract involvement are indicators of frequent relapses in a series of 121 japanese patients with egpa [59] . the french vasculitis study group has proposed the five-factor score (ffs). these include (1) elevated serum creatine levels (>1.58 mg/dl), (2) proteinuria (>1 g per day), (3) gastrointestinal tract involvement, (4) cardiomyopathy, and (5) central nervous system involvement [60] . those with ffs of 0 have better survival than those with ffs greater than 1. extravascular tissue eosinophils are seen in any organ during the early phase. features of vasculitis (fibrinoid necrosis, neutrophils, and eosinophils infiltration of vessel walls) are seen in small-to medium-sized vessel walls in the vasculitic phase. the presence of numerous eosinophils is often a diagnostic clue (fig. 7.14) . due to the widespread of glucocorticoid therapy and available small biopsy specimens, granulomatous inflammation is rarely observed histopathologically. since the skin is most accessible, biopsy of a cutaneous lesion might result in early diagnosis of epga. the presence of asthma and eosinophilia distinguishes egpa from mpa and gpa. although egpa belongs to the anca-associated vasculitis group, ancas are detected in only 40% of the a vesiculopapular eruption on a background of purpura is seen on the lower extremities cases. in addition, its clinical presentation and pathophysiology are different from those of mpa and gpa. eosinophils in egpa are the main responsible inflammatory cells in contrast to neutrophils in mpa and gpa. although idiopathic hypereosinophilic syndrome (hes) is characterized by tissue infiltration by eosinophils, there is absence of asthmas, vasculitis on biopsy, and serum anca. venous thrombosis (75%) occurs more frequently than arterial thrombosis (39%) in egpa, whereas arterial thrombosis (72%) is more frequent than venous thrombosis (28%) in hypereosinophilic syndrome [61] . the american college of rheumatology, the american society of nephrology, and the european league against rheumatism have recommended to replace "wegener granulomatosis" • egpa is a necrotizing vasculitis affecting small to medium vessels and associated with asthma and eosinophilia. • the features of the clinical phases of egpa include asthma in prodromic phase, peripheral eosinophilia and organ involvement in eosinophilic phase, and symptoms due to small-vessel vasculitis in vasculitic phase. • vasculitis can be diagnosed as egpa when four of the following six criteria are met: asthma, eosinophilia greater than 10%, neuropathy, migratory pulmonary infiltrates, paranasal sinus abnormality, and biopsy-proven extravascular eosinophils. • skin involvement can be seen in 40-50% of cases. • anca-positive patients more frequently had peripheral neuropathy or renal involvement, but less frequently had cardiomyopathy. • cardiomyopathy and older age at diagnosis were independent risk factors for death and lower eosinophil count at diagnosis as predictive of relapse in multivariate analyses. • pediatric cases more frequently have cardiomyopathy which can account for the higher mortality rates. • anca can be falsely negative in patients with egpa. with "granulomatosis with polyangiitis." granulomatosis with polyangiitis (gpa) is necrotizing vasculitis affecting small to medium vessels (capillaries, venules, arterioles, arteries, and veins), necrotizing granulomatous inflammation involving the upper and lower respiratory tract [3] . necrotizing glomerulonephritis is common. there are two phenotypes of gpa, the limited or localized form and the systemic form. the localized gpa is characterized by eye and ear, nose, and throat (ent) involvement, small percentage of anca positivity, severe local damages, and frequent relapses. the age of the patients at presentation is often between 50 and 75 years. nasal sinus involvement is reported in 70-100% of cases at the time of diagnosis and might be the only symptom in the localized form of the disease. saddle nose deformity is due to the destruction of the nasal cartilage ( fig. 7.15 ). the lungs can be affected in 50-90% of patients resulting in alveolar hemorrhage and/or parenchymal nodules [48] . the kidney is often affected in 40-100%. skin involvement is seen in 50% of patients and presents as polymorphic papules, nodules, vesicles, and blisters on a background of livedo reticularis. they most commonly occur on the lower extremities but also the face and scalp. cytoplasmic-pattern antineutrophil cytoplasmic autoantibodies (c-anca) with antigen specificity for proteinase 3 (pr3-anca) are sensitive serologic marker for gpa. ets1 (ets protooncogene 1) polymorphism was suggested to be associated with gpa and c-anca in japanese population [62] . however, mpo-anca can be seen in 10-20% of patients in europe and greater than 50% in asia. patients with mpo-ancapositive gpa are predominantly females. they had limited disease, high incidence of subglottic stenosis, less need for immunosuppressive therapy, and lower relapse rates in comparison to patients with pr3-anca [63] . infections and renal failure are the main causes of mortality [48] . the prognosis of untreated patients is very poor with only 10% 1-year survival [48] . with the introduction of glucocorticoids and cyclophosphamide, the clinical course has become a chronic relapsing and remitting disease with remission noted in 90% of the patients. increased risk for relapse is associated with pr3-anca positivity and lung or ent involvement [63] . the anca titer often rises in active disease. leukocytoclastic vasculitis is seen up to 50% of skin biopsies. granulomatous inflammation around the vessels or palisading necrotizing granulomatous can be seen in biopsies of internal organ infiltrates, but rarely noted in the skin (fig. 7.16) . neutrophilic dermatoses and pyoderma gangrenosum or sweet's syndrome can be seen in association of anca-associated vasculitis, most commonly with gpa [64] . gpa can be distinguished from mpa by the involvement of the respiratory tract. however, it is difficult to distinguish gpa from microscopic polyangiitis (microscopic polyarteritis) with respiratory tract involvement. drug exposure must be excluded in patients with localized gpa to rule out levamisole-induced/cocaineassociated vasculitis and midline destructive lesion due to cocaine use. in the setting of a solitary nodule, opportunistic infection such as blastomycosis, aspergillosis, and mycobacterial infection rather than localized gpa is the likely etiology. there can be overlap between gpa and igg4-related disease. chronic periaortitis, tubulointerstitial nephritis, and prevertebral fibrosis are characteristics of igg4-related disease rather than of anca-associated vasculitis [65] . rarely extranodal natural killer/t-cell lymphoma can present with midline destructive lesion. the types of vasculitis that affect the medium vessels include polyarteritis nodosa and kawasaki disease. • typically characterized by ear-nosethroat involvement followed by systemic symptoms, renal, lower respiratory tract, musculoskeletal, and cutaneous involvement • associated with proteinase 3 anca (c-anca) • leukocytoclastic vasculitis is seen up to 50% of skin biopsies. • vascular granulomatous inflammation or palisading necrotizing granulomatous can be seen in biopsies of internal organ infiltrates. clinical relevant pearls • gpa can be distinguished from mpa by the involvement of respiratory tract. • c-anca directed against proteinase 3 is a sensitive serologic marker for gpa. • patients with mpo-anca-positive gpa exhibit a different clinical course than those with pr3-anca-positive gpa. • increased risk for relapse is associated with pr3-anca positivity and lung or ent involvement. • granulomatous inflammation is rarely noted in the skin. polyarteritis nodosa (pan) has been divided into two major subtypes, the systemic pan and cutaneous pan [66] . a recent study proposed that cutaneous pan and macular lymphocytic arteritis are related and not distinct entities [67] . based on the 1990 acr classification criteria, systemic pan is diagnosed if at least three of the following ten criteria are met: significant weight loss, livedo reticularis, testicular pain, myalgia or muscle weakness, neuropathy, elevated diastolic blood pressure, increased blood urea nitrogen or creatinine, hepatitis b infection, unexplained abnormal angiogram, and biopsy-proven neutrophilic vasculitis [1] . based on the eular classification criteria, childhood systemic pan is diagnosed by the presence of either a biopsy-proven medium-sized artery necrotizing vasculitis or angiographic abnormalities together with at least two of the following seven criteria: skin involvement, myalgia or muscle weakness, systemic hypertension, neuropathy, abnormal urine analysis and/or impaired renal function, testicular pain, and signs or symptoms suggesting vasculitis of other major organ systems [68] . after systemic manifestations have been excluded, both clinical and histologic criteria must be present to diagnose cutaneous pan. cutaneous pan can occur at any age, ranging from childhood to the elderly, with a mean age of 40 years at presentation [69] . cutaneous lesions such as livedo reticularis, painful subcutaneous nodules (fig. 7.17) , and large "punched-out" ulcers are present often over the lower extremities (knee, anterior lower leg, malleoli, and dorsal foot) in 20-50% of cases. although cutaneous pan is uncommon in the pediatric population, group a beta-hemolytic streptococcus infections are the most frequent infections associated with cutaneous pan in this population. in adults, hepatitis b and c, hiv, parvovirus b19, and mycobacterium tuberculosis infections have been implicated. association with systemic diseases such as inflammatory bowel diseases and rheumatoid arthritis has been reported [69] . the following factors were significantly associated with poorer 5-year survival in systemic pan: age greater than 65 years, cardiac manifestations, gastrointestinal involvement, and renal insufficiency [70] . the french vasculitis study group has proposed the five-factor score (ffs). those with ffs of 0 have better survival than those with ffs greater than 1 [guillevin 2011 ] [70] . while systemic pan is a potentially lifethreatening disorder, cutaneous pan is a chronic benign disease with frequent relapses [71] . cutaneous pan rarely progresses to the systemic form. comorbidities may be present in 60% of cases of cutaneous pan [72] . both localized and systemic pan are characterized by necrotizing vasculitis of arterioles or medium-sized arteries (figs. 7.18 and 7.19 ). fibrinoid necrosis of the muscular wall accompanied by an infiltrate rich in neutrophils is seen in the initial phase. later on fibrous endarteritis can result in vascular occlusion. macular lymphocytic arteritis (mla) is characterized clinically by erythematous or pigmented reticulated patch on the lower extremities and histopathologically by prominent lymphocytic infiltrate. it has been proposed that mla represents the early stage of cutaneous pan [67] . the absence of anca is a distinguishing clinical feature from microscopic polyangiitis. in addition, pan does not involve the lungs, while microscopic polyangiitis can cause pulmonary hemorrhage. kawasaki disease is associated with mucocutaneous lymph node syndrome in addition to arteritis. kawasaki disease (kd) is an arteritis associated with the mucocutaneous lymph node syndrome and affecting predominantly medium and small arteries. kd is the most common medium-vessel vasculitis and second most common pediatric vasculitis after hsp. it affects mainly the pediatric population in children below the age of 5 and with predilection for the coronary artery. it is the leading cause of pediatric acquired heart disease in asian countries such as japan, korea, china, and taiwan but also north america and europe [17] . the kawasaki disease research committee guidelines (japanese ministry of health guidelines) were introduced in 2002 [73] . five of the following six criteria must be met for diagnosis: fever persisting greater than 5 days, bilateral conjunctival congestion, changes of lips and oral cavity, polymorphous exanthema, changes of peripheral extremities, and acute non-purulent cervical lymphadenopathy [73] . the 2004 american heart association (aha) guidelines are the widely used ones and have been revised in 2017 [74] . fever is essential for the diagnosis per aha criteria but not per the japanese criteria. the diagnosis is a clinical one and relies on the presence of fever greater than 5 days and four or more of the following features: bilateral bulbar conjunctival injection, mucosal changes involving the lips and oral cavity, unilateral cervical lymphadenopathy, polymorphous exanthema, and extremity changes [74] . as there is seasonal variation, viral infections have been suspected as etiologic agents. various organisms (streptococcus, staphylococcus, epstein-barr virus, coronavirus, and parvovirus), bacterial super antigens (staphylococcal and streptococcal), and genetic factors have been proposed as possible etiology; however, the cause remains unknown [gupta 2016 ] [75] . potential susceptibility genes include single nucleotide polymorphism in itpkc (1,4,5-inositol trisphosphate 3-kinase c), casp3, fcgr2a (fc gamma receptor iia), and kcnn2 genes; b lymphoid tyrosine kinase; and the transforming growth factor-beta signaling pathway. the disease typically affects infants and young children less than 5 years of age. coronary arteries are often involved. aorta and large arteries may be involved. the disease appears to have three phases. an acute febrile phase lasts for 10-14 days and is characterized by fever, polymorphous rash (fig. 7.20) , mucosal changes, extremity changes, and cervical lymphadenopathy. the eruption can be macular to maculopapular or morbilliform. there can be vertically cracked lips, "strawberry" tongue, and exanthema of oral and pharyngeal mucosa. the subacute phase from week 2 to 4 is characterized by periungual desquamation and at the tips of fingers and toes. coronary artery abnormalities are most commonly detected by echocardiography during the subacute phase. the patients become asymptomatic during convalescent phase. when there is fever yet with less than four principal clinical features, the patient would be diagnosed to have "incomplete kd." risk of coronary abnormalities is increased in this group due to delayed diagnosis. when a child exhibits seizures, stroke, pneumonia, myositis, nephritis, and acute hepatitis in addition to features of kd, he/she would be diagnosed with "atypical kd." [75] although rare, adult-onset kd has a high incidence of cardiovascular complication. although it is a self-limiting disease in the majority of cases, coronary dilatations and aneurysms can be developed in a quarter of untreated patients. timely diagnosis and implementation of intravenous immunoglobulin are important to decrease the risk of coronary artery complications. even those with treatment coronary artery abnormalities can develop down the road; therefore, long-term follow-up is important [75] . the diagnosis remains a clinical one, and there is no laboratory test to confirm the clinical diagnosis. the absence of involvement of vessels smaller than arteries distinguishes kawasaki disease from microscopic polyangiitis. twodimensional transthoracic echocardiography has long been the traditional diagnostic tool. dualsource computed tomography coronary angiography is increasingly employed to assess the coronary arteries [76] . since the disease occurs mainly in children under 5 years of age and fever more than 5 days is a required diagnostic criteria, viral infection is a main clinical differential diagnosis. the various infections can include adenovirus, measles, parvovirus, human herpes viruses, rocky mountain spotted fever, and leptospira, streptococci, and staphylococci. although scarlet fever is in the differential diagnosis, the lip involvement and conjunctival injection are features seen only in kd. the differential diagnosis also includes immune system reactions (toxic shock syndrome, serum sickness) and rheumatic diseases (systemic juvenile idiopathic arthritis, polyarteritis nodosa). • an arteritis associated with the mucocutaneous lymph node syndrome and affecting predominantly medium and small arteries. • kawasaki disease is the most common vasculitic disorder in children and affects patients less than 5 years of age. • the diagnosis is clinical and includes the presence of fever greater than 5 days and four or more of the following features: -bilateral bulbar conjunctival injection -mucosal changes involving the lips and oral cavity -unilateral cervical lymphadenopathy -polymorphous exanthema -extremity changes giant cell arteritis (gca) is a systemic, often granulomatous vasculitis affecting large vessels branching from the aorta, with a predilection for branches of the external carotid and vertebral arteries. giant cell arteritis is the most common vasculitis in the elderly in western countries, while it is uncommon in asia, africa, and south america. association with hla-drb1 * 04 alleles has been implicated in disease susceptibility and risk of visual complications [77] . gca involves large-and medium-sized vessels with predilection for branches of the external carotid and vertebral arteries [69] . it frequently involves the temporal artery of patients older than 50 years of age, women more than men, and has associated polymyalgia rheumatica. symptoms include headaches in the temporal and occipital areas, temporal artery tenderness, jaw claudication, malaise, and fever [69] . red and tender nodules may be palpable over the temporal arteries with diminished pulse (fig. 7.21) . a minority of patients experienced partial or complete loss of vision. cutaneous manifestations of giant cell arteritis are rare as the result of arterial occlusions. they include scalp induration, erythema, or necrosis; tongue necrosis; purpura; periorbital ecchymosis; edema of the face and neck; and nodules of the head or limbs [69] . • currently the diagnosis remains a clinical one and there is no laboratory test to confirm the clinical diagnosis. • children with incomplete and atypical kawasaki disease may have higher rate of coronary complications since the diagnosis often is delayed. • timely diagnosis and appropriate treatment will decrease the risk of coronary artery complications. a risk of partial or complete loss of vision can be seen in 13-19% of patients. visual complications were seen in 35-60% of patients prior to the introduction of corticosteroids [gonzalez-gay 2000] [77] ; however, their frequency has significantly diminished with this therapy. it is characterized by granulomatous inflammation of the internal elastic lamina of the aorta and major branches best diagnosed on a temporal artery biopsy (figs. 7.22 and 7.23) . the histologic features of giant cell arteritis can be identical to those of takayasu's disease. the serum igg4 level is elevated in patients with polymyalgia rheumatica in comparison to patients with giant cell arteritis [78] . although it has been suggested that both diseases represent ends of the same disease spectrum [79] , giant cell arteritis and takayasu disease are classified as two diseases based on differences in genetics, age, ethnicity, signs, symptoms, and vascular distribution. giant cell arteritis is associated with hla-dr4, while takayasu arteritis is associated with hla-bw52 [80] . while takayasu arteritis affects patients younger than 50 years, giant cell arteritis affects patients older than 50 years. takayasu arteritis is more common in asian populations; on the contrary, giant cell arteritis would be more common in caucasians. although both have contiguous aortic involvement and symmetric in paired branch vessels, left carotid and mesenteric arteries have more significant disease in takayasu arteritis and more left and right axillary artery disease in gca [79] . the shape of the stenotic lesions in the subclavian and carotid arteries can be a useful discriminator [79] . • a vasculitis of large-and medium-sized vessels with predilection for branches of the external carotid and vertebral arteries. • frequently involves the temporal artery of patients older than 50 years of age. • symptoms include headaches in the temporal and occipital areas and partial or complete loss of vision. takayasu arteritis is a rare chronic large-vessel vasculitis affecting predominantly the aorta, its major branches, and the pulmonary arteries. large arteries including the ascending or descending aorta and subclavian and carotid arteries are involved in 60-90% of cases [81] . this disease is common in southeast asia, india, mexico, or africa and rare in european countries. this could be due to underlying genetic differences. patients with hla-b52 have susceptibility to takayasu arteritis, and those with leukocyte antigen bw52 have a higher rate of complications than those without [80] . doppler ultrasound (us), magnetic resonance angiograph (mra), computed tomography angiograph (cta), and 18 f-fluorodeoxyglucose positron emission tomography ( 18 f-fdg-pet) have the potential to replace conventional x-ray angiography as noninvasive diagnostic method [barra 2018 ] [82] . 18 f-fdg-pet is helpful in the setting of absent vascular symptoms, fever of unknown origin, or unexplained acute-phase response. there is a predilection for women during the second and third decades of life. onset is typically in patients younger than 50 years of age. nonspecific symptoms such as fever, malaise, and weight loss are noted in the initial inflammatory stage. it is followed by an occlusive stage characterized by inflammation of the medial and adventitial layers of large-vessel walls resulting in stenosis and/or aneurysm formation (figs. 7.24 and 7.25). symptoms related to ischemia of involved vessels are extremity pain, claudication, absent or diminish pulse, and/or asymmetric blood pressure. while most japanese patients present with pulselessness, indian patients would be hypertensive [83] . a subclassification was created based on angiographic findings: type i, branches of the aortic arch; type iia, ascending aorta, aortic arch, and its branches; type iib, ascending aorta, aortic arch its branches, and thoracic descending aorta; type iii, descending thoracic aorta, abdominal aorta, and/or renal arteries; type iv, abdominal aorta and/or renal arteries; and type v, combined features of types 2b and 4 [83] . cutaneous manifestations vary according to geographical locations. erythema nodosum-like and acute inflammatory lesions are most commonly seen in europe and north america, whereas pyoderma gangrenosum is frequently observed in japan. livedo reticularis, papular or papulonecrotic lesions, and superficial phlebitis may also be observed [69] . it follows an indolent course. acute visual loss and stroke are rare events in takayasu arteritis [81] . the hla-b52 allele has been implicated in disease severity of takayasu arteritis [84] . the presence of aortic regurgitation, renal arterial stenosis, aortic coarctation and aneurysms are significant prognostic factors. it is often a granulomatous arteritis. rendering the diagnosis is a clinical challenge since there is a lack of a tissue biopsy or a "gold standard." diagnostic criteria were outlined by the american college of rheumatology in 1990 [1] . since the histopathologic features of takayasu arteritis and giant cell arteritis are similar and aortic involvement can be seen in giant cell arteritis, it has been suggested that both diseases represent ends of the same disease spectrum [79] . behcet disease and cogan syndrome can affect either small vessel or medium vessel; thus, the category of variable-vessel vasculitis has been proposed. described in 1937 by hulusi behcet as a clinical triad of oral ulcers, genital ulcers, and uveitis, behcet disease is now recognized to be a systemic condition having clinical features of • a chronic large-vessel vasculitis affecting predominantly the aorta, its major branches, and the pulmonary arteries. • the disease is common in southeast asia, india, mexico, or africa and rare in european countries. • it has a predilection for women during the second and third decades of life. • an initial inflammatory stage is followed by an occlusive stage. clinical relevant pearls • while takayasu arteritis affects patients younger than 50 years in asian populations, giant cell arteritis affects patients older than 50 years in caucasians. both autoinflammatory disease and vasculitis [85] . there is an increased incidence in individuals from japan and the middle east with the highest prevalence in turkey [85] ; therefore, behcet disease is known as silk road disease. in 1990, the international study group for behcet disease evaluated 914 patients from 7 different countries and proposed the following diagnostic criteria: presence of oral ulceration that recur at least 3 times in a 12-month period plus 2 of the following which are recurrent genital ulcers, uveitis, cutaneous lesions, and positive pathergy test. a minimum of three out of six outlined categories was defined as having pediatric behcet disease [86] . although its pathogenesis remains currently unclear, a combination of hormonal factors, genetic background (predisposing genes), and environmental factors (especially infection) plays a role. infectious factors can trigger the disease in genetically predisposed patients. genetic studies have identified hla-b * 51 to be the important risk factor. genome-wide association studies (gwas) have cited an association between behcet disease and the following loci: il23r-il12rb2, il10, stat4, ccr1-ccr3, klrc4, erap1, tnfaip3, and fut2. rare nonsynonymous variants of il23r, tlr4, nod2, and mefv have been implicated in pathogenesis by targeted next-generation sequencing [87] . the mean age of onset was 27 years with the most common clinical presentations being oral aphthous lesions (fig. 7.26 ), genital ulcers ( fig. 7.27) , and skin lesions. in a recent prospective cohort of 230 pediatric patients from 42 centers from 12 countries, the male-to-female ratio was 1:1. although men and women are equally affected worldwide, women are more frequently affected in europe and the usa. papulopustular lesions, pathergy positivity, and vascular, eye, and renal involvement were commonly seen in men, whereas genital ulcers, arthritis, and arthralgia were more commonly seen in women [88] . oral aphthosis invariably presents in most patients, up to 98%, and can precede other manifestations in an average of 7-8 years. genital ulcers (57-93%) commonly affect the scrotum in males and vulva, vagina, and cervix in females [89] . ocular disease (30-70%) is the main cause of blindness in a quarter of the patients. it is characterized by anterior and posterior uveitis, iridocyclitis, keratitis, episcleritis, vitritis, retinal vasculitis, and optic neuritis [89] . cutaneous the prognosis of severe forms of the disease has significantly improved in recent years due to the use of aggressive immunosuppressant therapy and new treatment modalities. however, when ocular, cardiovascular, neurological, and gastrointestinal systems are involved, the prognosis remains unfavorable. vasculitis in behcet disease can affect arteries or veins. small-vessel vasculitis, thromboangiitis, thrombosis, arteritis, and arterial aneurysms may occur. the cutaneous histologic features of behcet disease are often nonspecific. approximately 50% of biopsies show vasculitis, either lymphocytic or leukocytoclastic (fig. 7.29) . the following histopathologic features can be seen: leukocytoclastic vasculitis, lymphocytic vasculitis, superficial and/or deep perivascular inflammation, and folliculitis and/or perifolliculitis. although papulopustular lesions are common cutaneous lesions in behcet disease, they can also be seen in acne vulgaris. in a comparative study by kalkan et al., [90] papulopustular lesions from 42 patients with behcet disease were compared to those of 21 patients with acne vulgaris; vasculitis was seen significantly more frequent in the behcet disease patients. in another study by ilknur et al., [91] the histologic features of papulopustular lesions of 18 behcet disease patients were compared to those of 16 control lesions (11 bacterial folliculitis and 5 acne vulgaris), and lymphocytic vasculitis was more frequently noted in behcet disease group (p = 0.046) and folliculitis or perifolliculitis more frequently in the control group (p = 0.038). in a series of 26 cases of erythema nodosum-like lesions in behcet disease, misago et al. [92] reported the presence of vasculitis in 73% of cases. thus, although the histology of behcet disease is nonspecific, the finding of vasculitis appears to be very helpful to render the diagnosis. cogan syndrome is a rare autoimmune systemic disease which is characterized by a triad of inflammatory eye disease, vestibuloauditory dysfunction, and vasculitis. it was first described by morgan and baumgartner as a non-syphilitic interstitial keratitis associated with vestibuloauditory dysfunction in 1934. an ophthalmologist, david cogan, was the one who defined the entity in his 1945 report of five cases [93] . haynes et al. in 1980 proposed to classify the syndrome as "typical" and "atypical" which is characterized by grasland and colleagues in 2004 [94] . although the pathogenesis of cogan syndrome is currently unknown, there is mounting evidence toward autoimmunity. in 27% of cases, there is preceding upper respiratory tract infection [94] . the disease affects individuals in early adulthood; however, it can occur in children and older patients, characterized by ocular inflammatory lesions, including interstitial keratitis, uveitis, and episcleritis, and inner ear disease, including sensorineural hearing loss and vestibular dysfunction (tinnitus and vertigo). vasculitic manifestations may include arteritis (affecting small, medium, or larger arteries), aortitis, aortic aneurysms, and aortic and mitral valvulitis. approximately 70% of the patients have systemic disease likely attributed to vasculitis. either the eye (41%) or the ear (43%) alone is the first affected site. the most common eye disease is interstitial keratitis which can result in photophobia, pain, redness, tearing, and blurring of vision. the autoimmune inner ear diseases can result in deafness in 30-50% of patients. the symptoms of typical cogan syndrome include (1) ocular involvement (interstitial keratitis, iritis, conjunctivitis, subconjunctival hemorrhage), (2) audiovestibular involvement similar to meniere's disease, and (3) less than a 2-year interval between the onset of ocular and audiovestibular manifestations [95] . those of atypical cogan syndrome include (1) chronic and recurrent conjunctivitis, scleritis, uveitis, optic disk edema, and retinal vasculitis and audiovestibular symptoms not resembling meniere's disease [95] . delay in diagnosis can result in debilitating outcome. • vascular manifestations can include venous claudication, bronchial arterial collaterals, and "silent" budd-chiari syndrome. • eye disease or vascular involvement might be more specific than other organ manifestations. • there are no unique histologic or laboratory criteria; therefore, the diagnosis relies mainly on clinical features. • although the histology of behcet disease is nonspecific, the finding of vasculitis can be helpful to render the diagnosis. • diagnostic criteria include the presence of oral ulceration that recurs at least three times in a 12-month period plus two of the following: recurrent genital ulcers, uveitis, cutaneous lesions, and positive pathergy test. • approximately 50% of biopsies show vasculitis, either lymphocytic or leukocytoclastic. histologic sections of corneal tissue show nonspecific lymphocytic and plasma cell infiltrate, suggesting a cell-mediated reaction. vasculitis involving the dura, brain, gastrointestinal system, kidneys, spleen, aorta, and the coronary arteries was observed at autopsies [96] . the systemic features can resemble pan. rheumatoid arthritis (ra) is a chronic, inflammatory, and debilitating disease that can present with articular as well as extra-articular symp-toms. the disease preferentially affects females prior to 50 years of age. the cutaneous manifestations of ra include neutrophilic dermatosis, palisading granuloma, and vascular lesions. rheumatoid vasculitis, a rare yet serious complication, can affect 2-5% of patients with at least 10 years of severe disease [97, 98] . male gender, multiple rheumatoid nodules, joint erosions, higher rheumatoid factor titer, and treatment with biological agents are risk factors of developing rheumatoid vasculitis [97, 98] . rheumatoid vasculitis likely represents a type iii hypersensitivity reaction due to immune complex deposition [98] . rheumatoid vasculitis can affect small, medium, and large vessels of any organ. a peri-and/or epineural arteritis causing mononeuritis multiplex or sensory symmetric neuropathy can be seen in half of the cases [99] . in less than 1% of the patients, systemic vasculitis involving the heart, lungs, central nervous system, eyes, kidneys, and gastrointestinal tracts can be observed [99] . clinical presentation of a small-vessel involvement includes petechiae, palpable purpura, and hemorrhagic vesicles. bywaters' lesions are small, 0.5-1 mm, painless, purpuric papules on the pulp or nail fold of the distal finger due to small-vessel vasculitis. urticarial vasculitis can be seen which is characterized by wheals lasting greater than 24 hours. subcutaneous nodules, livedo reticularis, atrophie blanche, and ulceration would be the clinical presentation of an involvement of medium-sized vessels. other vasculopathic lesions observed in ra include pauci-inflammatory vascular thrombosis, reactive angioendotheliomatosis with glomeruloid neovascularization, perifollicular vasculitis, and granulomatous vasculitis [100] . vasculitis is associated with an increase in morbidity and mortality. prognosis is determined by the size of involved blood vessels and severity of systemic diseases [98] . isolated cutaneous rheumatoid vasculitis has a better clinical course, whereas a poorer prognosis would be observed when cutaneous lesions are followed by peripheral clinical relevant pearls • the possibility of large-vessel involvement must be considered in all patients. nerve (mononeuritis multiplex), digital gangrene, bowel, or cardiac involvement [101] . involvement of the small vessel would show a leukocytoclastic vasculitis on skin biopsy. direct immunofluorescence studies would show igm as well as c3 deposition within vascular walls of small-and medium-sized vessels [100] . involvement of medium-sized vessels by rheumatoid vasculitis resembles polyarteritis nodosa histopathologically ( fig. 7.30 ). systemic lupus erythematosus (sle) is a systemic autoimmune disease that can affect any organ (see chap. 11). affecting any vessel sizes in 11-36% of sle patients, vasculitis has been considered one of the leading causes of death in sle patients due to mesenteric vasculitis, pulmonary hemorrhage, or mononeuritis multiplex [102] . deposition of immune complexes within the vascular wall has thought to trigger the vascular inflammatory process. vasculitis was documented in 11% of sle patients [102] . 89% of the patients presented with cutaneous vasculitis, while 11% presented with visceral vasculitis. in another cohort of 540 sle patients with 10 years of follow-up, 82% had cutaneous involvement, 12% with visceral involvement, and 5% with both [103] . the patients with vasculitis experienced longer disease duration, presented at younger age, and were frequently males [103] . vasculitis would occur in association with fever, fatigue, weight loss, anemia, elevated erythrocyte sedimentation rate, and autoantibodies [104] . the clinical presentation would depend on the size of the involved vessels and affected organs. small-vessel vasculitis (86%) is more frequently seen in cutaneous lesions, whereas medium-and large-vessel vasculitis (14%) would affect internal organs [102] . cutaneous vasculitic . 7.31 ). some presented with non-blanching erythematous or violaceous punctate lesions on the fingertips and/or palms ( fig. 7.32) . urticarial vasculitis is a nonspecific finding. hypocomplementemia can be seen in one-fifth of patients. patients with medium-sized arterial involvement have a greater risk for thrombotic events and higher mortality than those without. vasculitis was found to be associated with antiphospholipid syndrome, myocarditis, raynaud's phenomenon, serositis, pleuritis, lymphopenia, and leukopenia [103] . small-vessel vasculitis characterized by neutrophilic infiltrate, fibrinoid necrosis of vessel wall, and leukocytoclasis is seen affecting the entire dermis with associated epidermal necrosis ( fig. 7.33 ). lymphocytic vasculitis can be seen. the differential diagnosis includes other forms of small-vessel vasculitides. sarcoidosis is a granulomatous disease of unknown etiology that can affect multiple organs including the lymph nodes, lungs, skin, and eyes. sarcoid vasculitis was defined in the 2012 chapel hill consensus conference (chcc) as vasculitis that is associated or secondary to sarcoidosis [3] . as with sarcoidosis, the associated vasculitis can be a single-organ or systemic process. the presence of vasculitis in patients with sarcoidosis is exceedingly rare with few cases with takayasu-like large-vessel vasculitis, and cases of sarcoidosis associated with giant cell arteritis or granulomatosis with polyangiitis have been reported in the literature [105] . the onset of cutaneous vasculitis was simultaneous with sarcoidosis in majority of the patients, preceded (12.5%), or subsequent to (25%) sarcoidosis [106] . lower extremities were the most commonly involved site. the clinical presentation would reflect the size of affected vessels. african american and asian patients are more common than caucasians to have large-vessel involvement. in a french series of seven cases of both sarcoidosis and takayasu vasculitis, these patients represented 0.7% of the sarcoidosis cases and 4% of the takayasu cohort [105] . less than 15 cases of concurrent sarcoidosis and takayasu have been reported, and majority of the patients were females with the mean age at diagnosis of 36 and 37 years for sarcoidosis and takayasu vasculitis, respectively. the diagnosis of sarcoidosis often precedes the diagnosis of takayasu vasculitis; however, takayasu vasculitis is diagnosed before sarcoidosis supportive of their association [105, 107] . there is often a significant time lapse between the two diagnoses. the patients with leukocytoclastic vasculitis would more likely have a complete resolution in comparison to those with granulomatous vasculitis. the vasculitis can be either leukocytoclastic vasculitis or granulomatous vasculitis in the setting of acute or chronic sarcoidosis, respectively. sarcoidal granulomas were observed at postmortem in cases of sarcoidosis affecting the aorta and its branches. although granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis are in the clinical differential diagnosis, neutrophils and eosinophils would be seen, respectively, in contrast to granulomas in sarcoid vasculitis. • visceral vasculitis is associated with increased mortality. • there appears to be a correlation between the presence of vasculitis and lupus activity. • the clinical presentation would depend on the size of the involved vessels and affected organs. • small-vessel vasculitis is more frequently seen in cutaneous lesions, whereas medium-and large-vessel vasculitis would affect internal organs. clinical relevant pearls • as with sarcoidosis, the associated vasculitis can be a single-organ or systemic process. a 60-year-old woman presented with recurrent erythematous plaques on her face especially the cheek (fig. 7.34 ). a skin biopsy of the cheek lesion was performed. a dense dermal infiltrate with overlying grenz zone is noted (fig. 7.35) . the dermal infiltrate is comprised of neutrophils, lymphocytes, and eosinophils. plump endothelial cells, fibrinoid necrosis of small-vessel walls, and leukocytoclasis are characteristic features of a small-vessel vasculitis (fig. 7.36 ). granuloma faciale is most often seen in middle-aged men [108] . the lesions present clinically as recurrent erythematous plaques on the forehead, ears, nose, and cheeks. the majority, up to 92%, of the patients has only facial lesions. however, the presence of extrafacial lesions do not exclude the possibility of granuloma faciale. although the etiology remains unknown, ultraviolet light exposure, radiation, trauma, and allergy have been implicated as predisposing factors. it is currently thought by many to be a chronic form of cutaneous vasculitis. the documentation of perivascular igg, iga, igm, and c3 deposition on direct immunofluorescence studies is supportive of the speculation that classical pathway activation of complement is causative of vasculitis in granuloma faciale [109] . the clinical differential diagnosis of granuloma faciale includes sarcoidosis, lupus erythematosus, and cutaneous lymphoma. the reddish-brown color of the clinical lesion and the presence of dilated follicular ostia are helpful features to distinguish granuloma faciale from sarcoidosis and lymphoma [108] . the histologic differential diagnosis includes erythema elevatum diutinum (eed), angiolymphoid hyperplasia with eosinophilia, and kimura's disease. granuloma faciale has been suggested to represent a localized form of eed to the face. eed, another distinctive form of chronic recurrent form of cutaneous leukocytoclastic vasculitis, often presents as symmetrical, firm, tender, reddish-brown papules and nodules on the extensor aspects of the extremities of middle-aged and older adults. there is no gender predilection. they are located on the fingers, hands, elbows, ankles, and knees. eed often follows a chronic clinical course. although the etiology is unknown, the thought is that it is likely secondary to vascular immune complex deposition due to its association with rheumatoid arthritis, ulcerative colitis, crohn's disease, and diabetes mellitus. associated infections include tuberculosis, syphilis, hepatitis b virus, and human immunodeficiency virus [110] . early histopathologic changes of eed include leukocytoclastic vasculitis and a mixed dermal infiltrate containing neutrophils, histiocytes, lymphocytes, and eosinophils. as the lesion ages, histiocytes become more prominent. these early histologic changes can mimic granuloma faciale and sweet's syndrome. on the other hand, angiocentric fibrosis ranging from perivascular to storiform would be seen in older lesions [111] . the changes of the late-stage lesion can resemble a fibrous neoplasm such as sclerotic fibroma or inflammatory pseudotumor [ 111] . a 9-year-old girl presented to the emergency room with left hip pain and a skin rash. she had a history of sore throat, fever, and lethargy several days ago with multiple subcutaneous, erythematous, and indurated nodules. fibrinoid necrosis and neutrophilic infiltrate affect a medium vessel at the deep dermis and subcutaneous tissue junction (figs. 7.37 and 7.38). although pan is a disease of adults, this form of medium-sized vasculitis can be seen in children after an episode of streptococcal infection, implicated by positive throat swab or a significant titer of either antistreptolysin o or antihyaluronidase. the relationship between streptococcal infection and a medium-sized vasculitis was initially proposed by fink in a series of seven children with pan [112] . poststreptococcal pan can be limited to the skin or exhibits widespread systemic involvement. the clinical findings can comprise fever, arthralgias, myalgias, arthritis, cutaneous eruption, abdominal pain, hematuria, hypertension, and peripheral neuropathy. the cutaneous presentation includes tender erythematous subcutaneous nodules on the lower extremities [112] . the palms and soles can be involved. although mild systemic symptoms were present in the majority of patients, the clinical course was typically benign. poststreptococcal pan follows a relapsing and remitting clinical course [113] . recognition of this form of pan is important for prompt antibiotic therapy and prophylaxis. the main differential diagnosis would be pan associated with hepatitis b and a systemic form of pan in childhood [114] . laboratory testings to detect streptococcal infection should be performed routinely in children presenting with fever, painful subcutaneous nodules, and arthralgias. a 42-year-old woman presented with multiple pin-sized purpura on both of her lower extremities (fig. 7.39 ). a skin biopsy demonstrated a perivascular infiltrate of lymphocytes and erythrocytes in the superficial dermis (fig. 7.40 ). an iron stain highlighted the prominent hemosiderin deposition in the dermis (fig. 7.41 ). pigmented purpuric dermatoses (ppd) comprise a spectrum of chronic and relapsing purpuric rash. there are six groups including schamberg's disease, purpura annularis telangiectodes, lichen aureus, lichenoid purpura of gougerot and blum, itching purpura, and eczematoid purpura of doucas and kapetanakis [115] . the clinical presentation varies with the different types of ppd. schamberg's disease would present with asymptomatic punctate purpura on lower extremities, whereas bluish-red annular macules would be seen in purpura annularis telangiectodes. a golden-brown eruption is typically seen in lichen aureus. the eruption is intensely pruritic and affects more sites in eczematoid purpura of doucas and kapetanakis [115] . although the etiology remains unknown, medications, chronic venous hypertension, thyroid dysfunction, and diabetes mellitus have been implicated [115] . in a recent retrospective review of 107 asian cases, five histopathologic patterns were observed: lichenoid (42%), perivascular (37%), interface (10%), spongiotic (7%), and granulomatous (4%). of interest, lymphocytic vasculitis was noted in 16% of cases [116] . schamberg, eczematoid, and lichen aureus clinical variants correlate with perivascular, spongiotic, and lichenoid histopathologic patterns [116] . a 35-year-old woman presented with a 3-year history of painful nodules on her lower extremity (fig. 7.42 ). a skin biopsy showed a necrotizing and granulomatous vasculitis of medium-sized vessels (figs. 7.43 and 7.44). nodular vasculitis is most commonly associated with a lobular panniculitis with medium-vessel vasculitis. it is known as erythema induratum of bazin in the presence of tuberculosis [117] . tuberculosis is the most frequently identified causative agent, and mycobacterium tuberculosis dna was detected by polymerase chain reaction in 77% of 74 skin biopsies from 65 patients [118] . in countries with low prevalence of tuberculosis, nodular vasculitis could represent a reactive process to cold, chronic venous insufficiency, or thrombophlebitis. in a retrospective series of 101 skin biopsies from 86 patients with the diagnosis of nodular vasculitis, vasculitis affecting the small vessels as well as the medium vessels was evident in 90% of the cases [117] . a lobular granulomatous panniculitis with associated vasculitis was seen in 12% and mainly lobular panniculitis without associated vasculitis in 10%. the vasculitis can be neutrophilic, granulomatous, or lymphocytic depending on the stage of the the american college of rheumatology 1990 criteria for the classification of vasculitis nomenclature of systemic vasculitides. proposal of an international consensus conference 2012 revised international chapel hill consensus conference nomenclature of vasculitides are the 1990 american college of rheumatology vasculitis classification criteria still valid? rhematology eular/ printo/pres criteria for henoch-schonlein purpura, childhood polyarteritis nodosa, childhood wegener granulomatosis and childhood takayasu arteritis: ankara 2008. part ii: final classification criteria nomenclature of cutaneous vasculitis: dermatologic addendum to the 2012 revised international chapel hill consensus conference nomenclature of vasculitides cutaneous vasculitis in a defined population: clinical and epidemiological associations henoch-schonlein purpura in children from northwestern spain: a 20-year epidemiologic and clinical study epidemiology of systemic vasculitis: a tenyear study in the united kingdom epidemiology of vasculitis in europe the epidemiology of antineutrophil cytoplasmic autoantibody-associated vasculitis in olmsted county, minnesota: a twenty-year us population-based study prevalence of wegener's granulomatosis, microscopic polyangiitis, polyarteritis nodosa and churg-strauss syndrome within a defined population in southern sweden epidemiology of wegener's granulomatosis in northern norway giant cell arteritis is more prevalent in urban than in rural populations: results of an epidemiological study of primary systemic vasculitides in germany prevalences of polyarteritis nodosa, microscopic polyangiitis, wegener's granulomatosis, and churg-strauss syndrome in a french urban multiethnic population in 2000: a capture-recapture estimate descriptive epidemiology of kawasaki disease in japan epidemiological observations of kawasaki disease in japan epidemiology of kawasaki disease in canada 2004 to 2014: comparison of surveillance using administrative data vs periodic medical record review the epidemiology of kawasaki disease: a global update the incidence of polymyalgia rheumatic and temporal arteritis in the county of aust adger, south norway: a prospective study 1987-1994 epidemiology of biopsy proven giant cell arteritis in northwestern spain: trend over an 18 year period incidence of giant cell arteritis in western norway 1972-2012: a retrospective disease. neutrophils are prominent in the early stage, whereas histiocytes and multinucleated giant cells impart a granulomatous pattern in the established lesion. large zones of caseous necrosis would be seen when there is much vascular damage. the accompanied lobular panniculitis distinguishes nodular vasculitis from cutaneous pan. the primary location of the vasculitis in the panniculus separates nodular vasculitis from gpa and egpa. cohort study prevalence, incidence, and disease characteristics of takayasu arteritis by ethnic background: data from a large, population-based cohort resident in southern norway skin involvement in cutaneous and systemic vasculitis genetics of immunoglobulin-a vasculitis (henoch-schonlein purpura): an updated review henoch-schonlein purpura: a literature review immunoglobulin-a-associated small-vessel vasculitis: a 10-year experience at the massachusetts general hospital the clinical and histological spectrum of 37 cases of immunoglobulin a-associated vasculitis virologic, clinical, and immune response outcomes of patients with hepatitis c virus-associated cryoglobulinemia treated with direct-acting antivirals hepatitis c virus and b-cell non-hodgkin lymphomas: an italian multicenter case-control study hypocomplementemic urticarial vasculitis: association with chronic obstructive pulmonary disease the clinical spectrum and therapeutic management of hypocomplementemic urticarial vasculitis: data from a french nationwide study of fifty-seven patients dnase1l3 mutations in hypocomplementemic urticarial vasculitis syndrome clinical utility of anti-c1q antibody in primary and secondary vasculitic conditions cogan's syndrome spectrum of clinical features in muckle-wells syndrome and response to anakinra the aha syndrome: arthritis, hives and angioedema proteolysis breaks tolerance toward intact α345(iv) collagen, eliciting novel anti-glomerular basement membrane autoantibodies specific for α345nc1 hexamers human tissue distribution of novel basement membrane collagen diagnosis and classification of goodpasture's disease (anti-gbm) clinical features and outcomes of anti-glomerular basement membrane disease in older patients patients double-seropositive for anca and anti-gbm antibodies have varied renal survival, frequency of relapse, and outcomes compared to single-seropositive patients anca-associated vasculitis -clinical utility of using anca specificity to classify patients genetically distinct subsets within anca-associated vasculitis targeted proteomics reveals promising biomarkers of disease activity and organ involvement in antineutrophil cytoplasmic antibody-associated vasculitis genetic variants in anca-associated vasculitis: a meta-analysis cutaneous manifestations of ancaassociated small vessels vasculitis microscopic polyangiitis: advances in diagnostic and therapeutic approaches the diagnosis and classification of microscopic polyangiitis prediction of response to remission induction therapy by gene expression profiling of peripheral blood comparing presenting clinical features in 48 children with microscopic polyangiitis to 183 children who have granulomatosis with polyangiitis (wegener's): and archive cohort study the american college of rheumatology 1990 criteria for the classification of churg-strauss syndrome (allergic granulomatosis and angiitis) revisiting the systemic vasculitis in eosinophilic granulomatosis with polyangiitis cutaneous manifestation of churg-strauss syndrome: a clinicopathologic correlation eosinophilic granulomatosis with polyangiitis (churg-strauss): clinical characteristics and long term followup of the 383 patients enrolled in the french vasculitis study group cohort central nervous system involvement in eosinophilic granulomatosis with polyangiitis (churg-strauss): report of 26 patients and review of the literature churg-strauss syndrome long-term prognosis of 121 patients with eosinophilic granulomatosis with polyangiitis in japan prognositc factors in polyarteritis nodosa and churg-strauss syndrome. a prospective study in 342 patients hypereosinophilic syndrome, churg-strauss syndrome and parasitic disease: possible links between eosinophilia and thrombosis association of ets1 polymorphism with granulomatosis with polyangiitis and proteinase 3-anti-neutrophil cytoplasmic antibody positive vasculitis in a japanese population myeloperoxidase-antineutrophil cytoplasmic antibody (anca)-positive granulomatosis with polyangiitis (wegener's) is a clinically distinct subset of anca-associated vasculitis: a retrospective analysis of 315 patients from a german vasculitis referral center neutrophilic dermatoses in antineutrophil cytoplasmic antibody-associated vasculitis: a french multicenter study of 17 cases and literature review antineutrophil cytoplasmic antibody-associated vasculitides and igg4-related disease: a new overlap syndrome early-onset stroke and vasculopathy associated with mutations in ada2 relationship between cutaneous polyarteritis nodosa (cpan) and macular lymphocytic arteritis (mla): blinded histologic assessment of 35 cpan cases the changing face of polyarteritis nodosa and necrotizing vasculitis cutaneous manifestations of medium-and large-vessel vasculitis the five-factor score revisited: assessment of prognoses of systemic necrotizing vasculitides based on the french vasculitis study group (fvsg) cohort polyarteritis nodosa: a contemporary overview cutaneous periarteritis nodosa: a clinicopathological study of 79 cases kawasaki disease research committee. revision of diagnostic guidelines for kawasaki disease (the 5th revised edition) american heart association rheumatic fever, endocarditis, and kawasaki disease committee of the council on cardiovascular disease in the young; counsil on cardiovascular and stroke nursing; council on cardiovascular surgery and anesthesia; and council of epidemiology and prevention. diagnosis, treatment, and long-term management of kawasaki disease: a scientific statement for health professionals from the american heart association kawasaki disease for dermatologists visual manifestations of giant cell arteritis. trends and clinical spectrum in 161 patients serum immunoglobulin g4 in giant cell arteritis and polymyalgia rheumatica distribution of arterial lesions in takayasu's arteritis and giant cell arteritis revisted hla and non-hla genetics of takayasu arteritis -where are we? clinical assessment in takayasu's arteritis: major challenges and controversies imaging modalities for the diagnosis and disease activity assessment of takayasu's arteritis: a systematic review and meta-analysis clinical manifestations of takayasu arteritis in india and japan -new classification of angiographic findings the severity of takayasu arteritis is associated with the hla-b52 allele in japanese patients clinical immunology review series: an approach to the patient with recurrent orogenital ulceration, including behcet's syndrome consensus classification criteria for paediatric behcet's disease from a prospective observational cohort: pedbd the immunogenetics of behcet's disease: a comprehensive review patient characteristics in behcet disease: a retrospective analysis of 213 turkish patients during 2001-4 behcet's disease physiopathology: a contemporary review a histopathological approach: when papulopustular lesions should be in the diagnostic criteria of behcet's disease? histopathologic and direct immunofluorescence findings of the papulopustular lesions in behcet's disease erythema nodosum-like lesions in behcet's disease: a clinicopathological study of 26 cases syndrome of non-syphilitic interstitial keratitis and vestibule-auditory symptoms typical and atypical cogan's syndrome: 32 cases and review of the literature cogan's syndrome: an oculo-audiovestibular disease vasculitis and cogan's syndrome. rheumatic dis clin north am skin manifestations of rheumatoid arthritis, juvenile idiopathic arthritis, and spondyloarthritides new insight into the rheumatoid vasculitis systemic rheumatoid arthritis: a review the spectrum of cutaneous lesions in rheumatoid arthritis: a clinical and pathological study of 43 patients clinical and histopathological spectrum of cutaneous vasculitis in rheumatoid arthritis vascular disease in systemic lupus erythematosus vasculitis in systemic lupus erythematosus vasculitis in systemic lupus erythematosus: prevalence and clinical characteristics in 670 patients sarcoidosis with takayasu arteritis: a model of overlapping granulomatosis. a report of seven cases and literature review circumscribed cicatricial alopecia due to localized sarcoidal granulomas and single-organ granulomatous arteritis: a case report and systematic review of sarcoidal vasculitis sarcoidosis with pulmonary hypertension exacerbated by takayasu-like large vessel vasculitis granuloma faciale: a clinicopathologic study of 66 patients direct immunofluorescence in granuloma faciale: a case report and review of the literature erythema elevatum diutinum latestage nodular erythema elevatum diutinum the role of streptococcus in poststreptococcal reactive arthritis and childhood polyarteritis nodosa long-term follow-up of juvenileonset cutaneous polyarteritis nodosa associated with streptococcal infection systemic polyarteritis nodosa in the young: a single centre experience over 32 years characteristics and clinical manifestations of pigmented purpuric dermatosis the pathological spectrum and clinical correlation of pigmented purpuric dermatosis -a retrospective review of 107 cases vasculitis in erythema induratum of bazin: a histopathologic study of 101 biopsy specimens from 86 patients detection of mycobacterium tuberculosis dna in lobular granulomatous panniculitis (erythema induratum-nodular vasculitis) key: cord-0037678-qo30oajs authors: lee, eun young; lee, yun jong title: glucocorticoids (as an etiologic factor) date: 2014-04-09 journal: osteonecrosis doi: 10.1007/978-3-642-35767-1_11 sha: 69baeaddd806c35139a562d624853b136bbd0369 doc_id: 37678 cord_uid: qo30oajs adrenocortical hormones were first prepared from the adrenal gland as a new compound by kendall in 1935 [1]. thirteen years later, hench et al. [2] observed a miraculous effect of cortisone in a patient with severe rheumatoid arthritis, which opened new doors to innovative treatments for a variety of inflammatory diseases. against this background, kendall, hench, and reichstein were awarded the 1950 nobel prize for physiology or medicine. since then, glucocorticoids have been used as a first-line therapy for immune-mediated conditions or as an adjunctive therapy in many inflammatory, infectious, or malignant diseases. adrenocortical hormones were fi rst prepared from the adrenal gland as a new compound by kendall in 1935 [ 1 ] . thirteen years later, hench et al. [ 2 ] observed a miraculous effect of cortisone in a patient with severe rheumatoid arthritis, which opened new doors to innovative treatments for a variety of infl ammatory diseases. against this background, kendall, hench, and reichstein were awarded the 1950 nobel prize for physiology or medicine. since then, glucocorticoids have been used as a fi rst-line therapy for immunemediated conditions or as an adjunctive therapy in many infl ammatory, infectious, or malignant diseases. however, the janus-faced effects of glucocorticoids became apparent a few years after their introduction into clinical practice. today, it is well known that glucocorticoids can have harmful effects on many tissues and can produce numerous adverse effects throughout the body, mainly depending on the dose, the administration route, and the duration of treatment (table 11 .1 ). since 1957 when the fi rst case of glucocorticoid-induced femoral head osteonecrosis (gi-fhon) was reported [ 3 ] , many human and animal studies have reported an association between glucocorticoids and fhon. to date, glucocorticoids have been considered the most common cause of nontraumatic fhon [ 4 ] . nevertheless, pathophysiological mechanisms and effective preventive or therapeutic strategies against gifhon have not yet been completely established. gi-fhon may develop in patients receiving glucocorticoids in short-term high doses or in long-term doses and even after intra-articular injection or application of topical preparations. since glucocorticoids have been widely used as therapeutic drugs to treat numerous diseases in various regimens, the reported prevalences of gi-fhon differ among investigators. most reports have documented the prevalences in patients with systemic lupus erythematosus (sle), renal transplantation, or leukemia. in prospective studies where 50 or more patients were included and an mri was used for diagnosis [ 5 -23 ] , 14.5-34.6 % of sle patients were diagnosed as having gi-fhon (table 11 .2 ). of patients undergoing kidney transplantation, 6.0-24.7 % were reported to suffer from gi-fhon. on the other hand, gi-fhon occurred in 4.3-11.3 % of patients with hematologic diseases, including acute lymphoblastic leukemia. because gi-fhon develops in patients with different underlying conditions, risk factors are not homogeneous in all disease states. the dose of the glucocorticoids has been the primary focus of research on risk factors for gi-fhon. many human studies have described associations between glucocorticoid doses and the prevalences of gi-fhon, while others have not. for example, a study analyzing 24 cohorts receiving glucocorticoids reported a 4.6 % increase in gi-fhon development as the doses of oral prednisone increase by 10 mg/day [ 24 ] . animal models have shown that higher doses are more likely to develop gi-fhon [ 25 ] . it has been reported that rabbits are more susceptible to the development of gi-fhon when treated with methylprednisolone than to prednisolone or triamcinolone in [ 26 ] , although there is no similar evidence in humans. among rabbits receiving methylprednisolone, those with increased low-density lipoprotein (ldl) cholesterol to high-density lipoprotein (hdl) cholesterol, increased free fatty acids, or decreased hepatic cytochrome p4503a activity are at high risk of developing gi-fhon [ 27 , 28 ] . in sle patients, a daily prednisone dose of >30-40 mg has been reported to be associated with gi-fhon [ 6 , 29 ] . however, cumulative or the highest dose has been heterogeneously reported as a risk factor by different investigators. additionally, methylprednisolone pulse therapy and cytotoxic agents have also been found to be related to gi-fhon [ 29 -32 ] . generally, sle patients with more active diseases are more likely to receive glucocorticoids in higher doses of methylprednisolone pulse therapy or cytotoxic agents. however, there are discrepancies regarding results on sle disease activity between patients with and without the development of gi-fhon [ 33 , 34 ] . the majority of gi-fhon cases develop within 3-12 months of initiation of glucocorticoid treatment [ 8 , 31 ] . increases in glucocorticoid doses secondary to a relapse of sle may be associated with gi-fhon [ 34 ] . in addition to the use of glucocorticoids, several features of sle have been identifi ed as risk factors: thrombophlebitis, vasculitis, raynaud's phenomenon, renal dysfunction, arthritis, smoking, preeclampsia, and so on [ 6 , 30 ] . in contrast, nested case-control studies have revealed that the use of glucocorticoids is the only risk factor for gi-fhon [ 32 , 33 ] . studies on the association between antiphospholipid antibodies and gi-fhon have reported different results [ 35 -37 ] . therefore, a specifi c manifestation may not be a risk factor for gi-fhon in sle patients. because not all sle patients do develop gi-fhon when receiving high-dose glucocorticoids, the presence of additional regional risk factors or individual variations of glucocorticoid sensitivity can be involved in the occurrence of gi-fhon. for example, age at the time of initial glucocorticoid administration can affect the development of gi-fhon. a prospective study has revealed that gi-fhon did not develop in sle patients at the age of <14 years, suggesting that children may tolerate ischemia due to their abundant vascularity with growth plates and red marrow [ 9 ] . in addition, posttreatment presentation of cushingoid body phenotype, which is a characteristic feature of glucocorticoid excess and does not develop in patients with glucocorticoid resistance, is reported to be a risk factor [ 29 , 30 , 35 ]. kidney transplantation is the treatment of choice for patients with end-stage renal diseases. just after transplantation, immunosuppressive therapy, including glucocorticoids, is needed to prevent and treat acute graft rejection and to avoid chronic graft damage. it has been shown that the risk of gi-fhon is associated with glucocorticoid doses [ 14 , 38 , 39 ] . in particular, the total dose of glucocorticoids during the fi rst 7 months can have a signifi cant risk [ 14 , 38 ] . introduction of immunosuppressants, such as cyclosporine or tacrolimus, can decrease the incidence of gi-fhon in kidney transplant patients [ 38 ] because these immunosuppressants reduce glucocorticoids doses. an association of the incidence of gi-fhon with a history of acute rejection is not consistently reported [ 14 , 39 ] . several pretransplantation conditions can impact on the development of gi-fhon: iron overload, hypophosphatemia secondary to hyperparathyroidism, or poor renal function [ 39 -41 ] . additionally, apoprotein(a) molecular weight phenotype was reported to be an independent risk factor [ 42 ] . glucocorticoids successfully treat acute lymphoblastic leukemia (all), lymphoma (nhl), and multiple myeloma (mm) because they can kill hematologic malignant cells. thus, many treatment regimens include glucocorticoids, such as dexamethasone. since the prevalence of all is higher in children than in adults, most gi-fhon studies in all patients have been conducted on pediatric patients. all patients at the age of >10 years increase the risk of gi-fhon [ 16 , 20 -22 , 43 , 44 ] , and old age is also an independent risk factor in adult patients with mm [ 18 ] . additionally, female patients have a higher risk of gi-fhon than males [ 20 , 21 , 43 , 44 ] . intensive therapy with dexamethasone confers a high incidence of gi-fhon [ 20 , 22 ] , and alternate-week scheduling of dexamethasone can reduce the development of gi-fhon [ 43 , 44 ] . in some studies, patients receiving dexamethasone more frequently developed fhon than those receiving prednisone [ 43 ] . an association between fhon and poor dexamethasone clearance has been reported [ 22 ] . in patients undergoing stem cell transplantation, both an episode of graft-versus-host disease (gvhd) and the use of glucocorticoid are risk factors [ 17 ] . the incidence of gi-fhon has been reported to be higher in patients receiving unrelated donor transplants than in those receiving allogenic matched related donor or autologous transplants [ 17 , 45 ] . the pathophysiology of gi-fhon is multifactorial, complex, and poorly understood. although host factors and underlying diseases have been shown to play a signifi cant role in the development of gi-fhon, investigators have failed to explain why only a fraction of patients are at greater risk than others. additionally, the multisystemic effects of glucocorticoids and their interactions make the pathological mechanisms more complicated. in this context, the multi-hit theory proposed by several investigators is a plausible explanation for the development of gi-fhon [ 46 , 56 ] . in susceptible patients who have a genetic predisposition or an underlying disease that threatens bone and vascular tissues, the accumulative glucocorticoid effects may result in the occurrence of gi-fhon ( fig. 11.1 ) . genetic factors will be discussed in another chapter. animal and human studies have shown that glucocorticoids can induce apoptosis of osteoblasts and suppress the production of osteoblasts in the bone marrow [ 47 ] . osteoblast-/osteocyte-specifi c 11β-hydroxysteroid dehydrogenase type 2 (hsd2, the enzyme metabolizes glucocorticoids into inactive metabolites) transgenic mice protect from prednisolone-induced decrease in osteoblast survival, osteoblast number, and bone formation [ 48 ] . on the other hand, the survival of osteoclasts can be prolonged by glucocorticoids [ 49 ] . the interaction of receptor activator of nf-κb ligand (rankl) and osteoprotegerin (opg) is a major determinant of osteoclastogenesis. rankl and macrophage colony-stimulating factor (m-csf) are essential for osteoclastogenesis, while opg prevents rankl from binding to rank, resulting in inhibition of osteoclastogenesis. glucocorticoids increase rankl and m-csf expression and decrease opg expression by human osteoblastic and stromal cells in culture [ 50 , 51 ] . from the results of studies using mice with conditional osteoclast-specifi c deletion of glucocorticoid receptors [ 52 ] , glucocorticoids inhibit proliferation of osteoclast precursors and mature osteoclast-mediated bone resorption. such glucocorticoid-induced suppression of osteoclasts induces blunting of osteoblast function in the context of osteoclast-osteoblast interactions throughout bone remodeling [ 53 ] . also, glucocorticoids suppress the secretion of sex hormones and the expression of bone morphogenetic protein-2, insulin growth factor 1, and osteocalcin [ 54 , 55 ] . furthermore, calcium absorption from the kidney and intestine is disturbed by glucocorticoids [ 54 ] . osteocytes are the most abundant cells (>95 %) in the bone and embedded in mineralized bone matrices. they are thought to play a pivotal role as mechanosensors and initiators of the bone remodeling process [ 56 ] . living osteocytes directly stimulate osteoblastogenesis and inhibit osteoclastic resorption through opg expression. the femurs from patients with gi-fhon show many apoptotic osteocytes, anatomically juxtaposed to the osteonecrotic fractures [ 57 ] . glucocorticoid-induced osteocyte apoptosis results in the mechanosensory dysfunction of the osteocyte network and consequently leads to impairment of bone repair processes. additionally, hypoxia-inducible factor (hif)-1α, a critical regulator of cellular response in hypoxic condition, can alter the mechanosensitivity of osteocytes and suppress load-induced bone formation [ 58 ] . glucocorticoids promote differentiation of preadipocytes to mature adipocytes via upregulation of the peroxisome proliferator-activated receptor (ppar)-γ and downregulation of runt-related transcription factor 2 (runx2). in addition, glucocorticoids can cause adipocyte hypertrophy through increased synthesis and storage of lipids [ 59 ] . it has been shown that the number and area of fat cells increase in the bone marrow after exposure to glucocorticoids [ 60 , 61 ] . these changes can generate compression of venous sinusoids and congestion in the bone marrow. as a result, adequate arterial blood fl ow may not be achieved due to increases in intraosseous pressures, eventually leading to osteonecrosis [ 62 ] . patients with cushing syndrome have been reported to have increased cardiovascular morbidity and mortality. chronic administration of glucocorticoids inhibits the synthesis of vasorelaxants, such as prostaglandin e1, prostacyclin, and endothelial nitric oxide (no), via a suppression phospholipase a2 and endothelial nitric oxide synthase (enos) [ 63 ] . in addition, glucocorticoids indirectly increase vascular tone through the upregulation of angiotensin ii type i receptor and α-1 adrenergic receptors in vascular smooth muscle cells [ 64 ] . furthermore, glucocorticoids can stimulate the synthesis of vasoconstrictor endothelin-1 and potentiate its vasoconstricting effect in vascular smooth muscle cells [ 65 ] . there is some evidence that glucocorticoids increase reactive oxidative stress species (ros) which decrease no availability [ 66 ] . thus, glucocorticoids can cause dysregulation of endothelium-dependent and endothelium-independent vasodilatation. furthermore, higher doses of dexamethasone were reported to result in microvascular endothelial cell apoptosis and could lead to capillary rarefaction in a glucocorticoid-induced hypertension model [ 67 ] . this may be involved in glucocorticoid-induced hypertension and hypercoagulability. the effect of glucocorticoids on the coagulation system is discussed below. new blood vessel formation is essential for the repair of ischemia-damaged tissues when fhon develops. however, glucocorticoids have been shown to inhibit angiogenesis. in a dexamethasone-induced fhon rabbit model, vascular endothelial growth factor (vegf) gene therapy improved the repair process of osteonecrosis [ 68 ] . glucocorticoids suppress expression of vegf and increase expression of antiangiogenic thrombospondin-1 [ 69 , 70 ] . glucocorticoidinduced suppression of matrix metalloproteinases and plasminogen activators may affect the proangiogenic process through impairment of basement membrane turnover [ 71 ] . it has also been shown that gcs can inhibit capillary growth by reducing collagen synthesis by myofi broblasts [ 72 ] . because cartilage components released from subchondral fracture sites have been considered antiangiogenic, the negative effects of glucocorticoids on angiogenesis may be potentiated in the subchondral bone tissue in fhon cases [ 73 ] . fibrinolytic activity is balanced by tissue plasminogen activator (t-pa) and plasma plasminogen activator inhibitor-1 (pai-1). the accumulated evidence of in vitro studies as well as studies of patients with cushing syndrome suggests that glucocorticoids increase the pai-1 activity, resulting in a relatively hypercoagulable state [ 74 ] . the plasma level of the von willebrand factor (vwf) is a marker of endothelial cell damage because the vwf is synthesized and stored in the endothelial cells. plasma vwf levels are reported to increase in subjects with exogenous or endogenous excess glucocorticoids, and dexamethasone induces the expression of vwf, cell adhesion molecules, and tissue factor in vascular endothelial cells [ 75 ] . since vwf is involved in platelet aggregation and adhesion, glucocorticoid-induced endothelial damage may contribute to thrombi formation. the clinical features of gi-fhon are not different from those in other fhon. patients with gi-fhon usually experience pain primarily in the groin but occasionally the buttocks. the pain onset often can be described as acute. the pain is usually deep and throbbing and becomes worse with ambulation. patients frequently complain of a catching or popping sensation with motion. in patients with a history of glucocorticoid treatment, especially in higher doses, persistent hip pain with joint movement, tenderness, or reduced range of motions indicates the need for a prompt workup for gi-fhon. on physical examination, limitation of internal rotation in both fl exion and extension is observed, with passive internal rotation in extension being particularly painful. a trendelenburg gait is often present. additionally, some characteristic features of chronic glucocorticoid users can be seen: moon face, central obesity, and buffalo hump. anteroposterior radiographs and frog lateral radiographs of both hips are the primary diagnostic modalities, while plain radiographic fi ndings are frequently normal. magnetic resonance imaging (mri) is ideal if x-ray fi ndings are normal and clinical suspicion is high [ 9 , 37 ] . the sensitivity and specifi city of mri are both greater than 98 % for the diagnosis of osteonecrosis, which is higher than those of other diagnostic modalities. therefore, mri should be performed in all patients with osteonecrosis to assess the extent of the disease. three-dimensional mri with image registration can be used to assess changes in lesion size [ 76 , 88 ] . bone scan may be helpful when x-ray fi ndings are normal and when mri cannot be performed. it may be low-cost alternative when the suspicion index is low [ 77 , 89 ] . most studies on the natural history of fhon have been conducted on patients with heterogeneous subtypes of nontraumatic fhon. however, the natural history of nontraumatic fhon cannot represent that of gi-fhon because subtypes of nontraumatic fhon may have different pathogenic mechanisms and underlying conditions necessitating glucocorticoids can affect the course of fhon. the results of studies using mri to diagnose asymptomatic gi-fhon are summarized in table 11 .3 [ 7 , 11 , 78 -83 ] . about one-third of patients with asymptomatic early gi-fhon have symptomatic or radiological progression over a period of several years. thus, clinical progression or subchondral collapse rate seems to be lower than the other subtypes of nontraumatic fhon, especially fhon associated with sickle cell anemia. sometimes small lesions of gi-fhon are reported to be spontaneously resolved [ 81 -83 ] . however, as with other subtypes, femoral head collapse develops most often in gi-fhon patients with larger osteonecrosis areas (>15-30 %), lesions occupying the weight-bearing surface, higher radiographic stages, or hip pain [ 84 ] . there is no established risk stratifi cation system to exactly estimate outcomes in gi-fhon patients. because proper therapeutic decisions depend on the natural course of fhon, more extended studies are needed to develop an improved risk classifi cation system. until now, no specifi c prevention strategy has been developed. because the risk of gi-fhon is usually associated with the dose and duration of glucocorticoid treatment, the effort to use the minimal effective dose and to appropriately taper glucocorticoid doses is needed. because introduction of immunosuppressants may decrease the incidence of gi-fhon in renal transplant patients [ 38 ] , 28 ) should be made since the choice of treatment depends on the staging. to reduce weight bearing using canes, crutches, or a walker for about 6 weeks is the fi rst acceptable treatment. simple analgesics or nonsteroidal antiinfl ammatory drugs can be prescribed for symptomatic treatments. however, no conservative or medical therapy has been proven to benefi t patients with gi-fhon, and surgical treatment is inevitable in most symptomatic cases [ 84 ] . based on hypothetical mechanisms, various therapeutic agents have been attempted in gi-fhon animal models. among them, the preventive effect of statin and warfarin was evaluated in patients receiving glucocorticoids; the results concerning statin effects were controversial [ 85 , 86 ] , and warfarin treatment only tended to decrease symptomatic gi-fhon cases in sle patients [ 87 ] . additionally, enoxaparin, a low molecular weight heparin, is not helpful in gi-fhon patients [ 88 ] . a single-arm study reported that intravenous infusion of prostaglandin i 2 for 5 days decreased pain and radiographic outcomes in patients with bone marrow edema or early nontraumatic fhon [ 89 ] . however, some evidence suggests that alendronate is a candidate drug for the treatment of early gi-fhon. although 7 (18 %) of the 40 study subjects had gi-fhon, a randomized controlled study demonstrated that alendronate (70 mg/week for 25 weeks) could signifi cantly prevent the collapse of the femoral head in fhon patients with steinberg stage iic or iiic [ 90 ] . a prospective open-label study, where 28 (47 %) of the 60 patients had gi-fhon, showed symptomatic improvement and minimal or no radiographic progression after 1 year of follow-up [ 91 ] . in another prospective study that included 26 (79 %) of the 33 patients with gi-fhon, alendronate (5 mg/day for 1 year) decreased the frequency of the femoral head collapse [ 92 ] . when the add-on effect of alendronate was studied for 4 years, combination therapy of multiple drilling and alendronate showed a signifi cant benefi t in gi-fhon patients with ficat stages ii and iii [ 93 ] . however, a 2-year randomized double-blind study where 12 (23 %) of the 52 patients developed gi-fhon reported no signifi cant effects in the 52 patients at stage iic or iiic [ 94 ] . thus, further studies are warranted to confi rm that alendronate is a potential option to postpone the need for hip surgery in patients with gi-fhon. recently, stem cell-based therapy has attracted attention. because gi-fhon is believed to have limited reparative capacity partially secondary to decreased proliferation of mesenchymal stem cells, the approach could be acceptable [ 95 ] . in rabbit gi-fhon models, cell-based therapies using autologous bone marrow or mesenchymal stem cells, peripheral blood-derived mononuclear cells, or endogenital progenitor cells showed benefi cial effects on progression prevention, vascularization, and bone regeneration of the femoral head [ 96 -99 ] . a pilot controlled study was performed using autologous bone marrow mononuclear cells in 18 patients with early stage (i and ii) fhon. direct implantation of bone marrow-derived mononuclear cells into the femoral head resulted in a signifi cant decrease in symptoms and collapse rate at 2 years [ 100 ] . in an observational study of sle patients with gi-fhon, concentrated autologous bone marrow aspirate transplantation improved joint pain and resulted in a 40 % radiographic progression rate over a period of 41 months in patients with type c2 lesions [ 101 ] . although several preclinical results showed promising benefi ts, cellbased therapies have not been widely used to treat gi-fhon, and there are no long-term and large-scale studies. further clinical studies are needed to evaluate the availability and safety of the stem cell-based therapies in human patients. various joint-preserving treatments have been proposed because of the failure of nonoperative treatments and the limited durability of prosthetics. core decompression, vascularized fi bular grafts, bone grafting, bone marrow grafting, and osteotomy have been applied to delay total hip replacement with variable outcomes (for detailed information, refer to chap. 28 ). in advanced or symptomatic gi-fhon, total hip replacement is the treatment of choice. glucocorticoids are considered the most common cause of nontraumatic fhon and gi-fhon, which can develop in less than 30 % of patients receiving higher doses of glucocorticoids. although the pathophysiological mechanisms have not yet been fully deduced, accumulation of glucocorticoid effects can lead to the development of gi-fhon in subjects with a genetic predisposition or an underlying disease that threatens bone and vascular tissue. the clinical features of gi-fhon are not different from those of other fhon. clinical progression or subchondral collapse rates may be similar to or lower in asymptomatic gi-fhon than in other subtypes of nontraumatic fhon. total hip replacement is eventually needed in many symptomatic cases. efforts to use the minimal effective dose and to appropriately taper doses of glucocorticoids are essential to preventing gi-fhon. more studies are necessary to establish the effects of pharmacologic and non-pharmacologic joint-preserving therapies in patients with early stage gi-fhon. the chemistry of crystalline substances isolated from the suprarenal gland the effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone: compound e) and of pituitary adrenocorticotropic hormone on rheumatoid arthritis osteopatia da prolongato trattamento cortisono nontraumatic avascular necrosis of the femoral head. past, present, and future corticosteroid therapy associated with ischemic necrosis of bone in systemic lupus erythematosus risk factors of avascular necrosis of the femoral head in patients with systemic lupus erythematosus under high-dose corticosteroid therapy prognostication of osteonecrosis of the femoral head in patients with systemic lupus erythematosus by magnetic resonance imaging osteonecrosis in patients with systemic lupus erythematosus develops very early after starting high dose corticosteroid treatment age at time of corticosteroid administration is a risk factor for osteonecrosis in pediatric patients with systemic lupus erythematosus: a prospective magnetic resonance imaging study incidence of osteonecrosis associated with corticosteroid therapy among different underlying diseases: prospective mri study apparent avascular necrosis of the hip: appearance and spontaneous resolution of mr fi ndings in renal allograft recipients clinically occult avascular necrosis of the hip: prevalence in an asymptomatic population at risk osteonecrosis of the femoral head after solid organ transplantation: a prospective study degree of corticosteroid treatment within the fi rst 2 months of renal transplantation has a strong infl uence on the incidence of osteonecrosis of the femoral head fractures and avascular necrosis before and after orthotopic liver transplantation: long-term follow-up and predictive factors magnetic resonance imaging detection of avascular necrosis of the bone in children receiving intensive prednisone therapy for acute lymphoblastic leukemia or non-hodgkin lymphoma avascular necrosis in long-term survivors after allogeneic or autologous stem cell transplantation: a single center experience and a review avascular necrosis of femoral and/ or humeral heads in multiple myeloma: results of a prospective study of patients treated with dexamethasone-based regimens and highdose chemotherapy low pretransplant bone-mineral density and rapid bone loss do not increase risk for avascular osteonecrosis after allogeneic hematopoietic stem cell transplantation high body mass index increases the risk for osteonecrosis in children with acute lymphoblastic leukemia veerman aj, van den heuvel-eibrink mm. prospective study on incidence, risk factors, and long-term outcome of osteonecrosis in pediatric acute lymphoblastic leukemia pharmacokinetic, pharmacodynamic, and pharmacogenetic determinants of osteonecrosis in children with acute lymphoblastic leukemia risk of avascular necrosis following short term megadose methylprednisolone treatment across-study evaluation of association between steroid dose and bolus steroids and avascular necrosis of bone dose effects of corticosteroids on the development of osteonecrosis in rabbits effects of different corticosteroids on the development of osteonecrosis in rabbits onset of steroid-induced osteonecrosis in rabbits and its relationship to hyperlipaemia and increased free fatty acids increased hepatic cytochrome p4503a activity decreases the risk of developing steroid-induced osteonecrosis in a rabbit model high-dose intravenous methylprednisolone therapy associated with osteonecrosis in patients with systemic lupus erythematosus predictive factors for symptomatic osteonecrosis in patients with systemic lupus erythematosus very early development of steroid-associated osteonecrosis of femoral head in systemic lupus erythematosus: prospective study by mri systemic lupus erythematosus in a multiethnic us cohort (lumina): xxiv. cytotoxic treatment is an additional risk factor for the development of symptomatic osteonecrosis in lupus patients: results of a nested matched case-control study the role of noncorticosteroid related factors in osteonecrosis (on) in systemic lupus erythematosus: a nested case-control study of inception patients investigation of occurrence of osteonecrosis of the femoral head after increasing corticosteroids in patients with recurring systemic lupus erythematosus risk factors for osteonecrosis in systemic lupus erythematosus association of osteonecrosis in systemic lupus erythematosus with abnormalities of fi brinolysis magnetic resonance imaging-detected avascular osteonecrosis in systemic lupus erythematosus: lack of correlation with antiphospholipid antibodies avascular osteonecrosis after renal transplantation aseptic bone necrosis after renal transplantation avascular necrosis following bone marrow transplantation: a casecontrol study risk factors for nontraumatic osteonecrosis of the femoral head after renal transplantation low molecular weight phenotype of apo(a) is a risk factor of corticosteroid-induced osteonecrosis of the femoral head after renal transplant management of osteonecrosis in children and young adults with acute lymphoblastic leukaemia effect of alternate-week versus continuous dexamethasone scheduling on the risk of osteonecrosis in paediatric patients with acute lymphoblastic leukaemia: results from the ccg-1961 randomised cohort trial predictors of avascular necrosis of bone in long-term survivors of hematopoietic cell transplantation accumulative cell stress: the multifactorial etiology of idiopathic osteonecrosis glucocorticoid-induced osteoporosis and osteonecrosis glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoidinduced osteoporosis dexamethasone promotes expression of membrane-bound macrophage colony-stimulating factor in murine osteoblast-like cells glucocorticoids suppress bone formation via the osteoclast new understanding of glucocorticoid action in bone cells mechanisms of glucocorticoid action in bone glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells osteocyte: the unrecognized side of bone tissue apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip hypoxia-inducible factor-1α protein negatively regulates load-induced bone formation the effects of glucocorticoids on adipose tissue lipid metabolism bone marrow fat-cell enlargement in early steroidinduced osteonecrosis -a histomorphometric study of autopsy cases effects of glucocorticoid on adipocyte size in human bone marrow bone marrow fat cell enlargement and a rise in intraosseous pressure in steroid-treated rabbits with osteonecrosis does altered glucocorticoid homeostasis increase cardiovascular risk? the role of corticosteroids in the regulation of vascular tone glucocorticoids induce endothelin release from vascular smooth muscle cells but not endothelial cells glucocorticoid excess induces superoxide production in vascular endothelial cells and elicits vascular endothelial dysfunction microvascular endothelial cell death and rarefaction in the glucocorticoid-induced hypertensive rat vascular endothelial growth factor gene transfection to enhance the repair of avascular necrosis of the femoral head of rabbit endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged mice glucocorticoid-mediated inhibition of angiogenic changes in human endothelial cells is not caused by reductions in cell proliferation or migration inhibition of proliferation, migration and proteolysis contribute to corticosterone-mediated inhibition of angiogenesis the effects of glucocorticoids on angiogenesis in vitro glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action systematic review on the effect of glucocorticoid use on procoagulant, anti-coagulant and fi brinolytic factors effect of high-dose dexamethasone on endothelial haemostatic gene expression and neutrophil adhesion longitudinal quantitative evaluation of lesion size change in femoral head osteonecrosis using three-dimensional magnetic resonance imaging and image registration bone scanning of limited value for diagnosis of symptomatic oligofocal and multifocal osteonecrosis initial mri fi ndings of non-traumatic osteonecrosis of the femoral head in renal allograft recipients osteonecrosis of the femoral head: a prospective study with mri long-term observation of avascular necrosis of the femoral head in systemic lupus erythematosus: an mri study spontaneous resolution of osteonecrosis of the femoral head spontaneous repair of asymptomatic osteonecrosis associated with corticosteroid therapy in systemic lupus erythematosus: 10-year minimum follow-up with mri osteonecrosis of the femoral head in sars patients: seven years later the natural history of untreated asymptomatic osteonecrosis of the femoral head: a systematic literature review does statin usage reduce the risk of corticosteroid-related osteonecrosis in renal transplant population? statin therapy decreases the risk of osteonecrosis in patients receiving steroids prevention of steroid-induced osteonecrosis of femoral head in systemic lupus erythematosus by anti-coagulant enoxaparin prevents progression of stages i and ii osteonecrosis of the hip rationale for prostaglandin i2 in bone marrow oedema: from theory to application the use of alendronate to prevent early collapse of the femoral head in patients with nontraumatic osteonecrosis. a randomized clinical study effi cacy of alendronate, a bisphosphonate, in the treatment of avn of the hip. a prospective open-label study does alendronate prevent collapse in osteonecrosis of the femoral head? are the results of multiple drilling and alendronate for osteonecrosis of the femoral head better than those of multiple drilling? a pilot study alendronate in the prevention of collapse of the femoral head in nontraumatic osteonecrosis: a two-year multicenter, prospective, randomized, double-blind, placebo-controlled study decreased proliferation of mesenchymal stem cells in corticosteroid-induced osteonecrosis of femoral head prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells regenerative effects of transplanting autologous mesenchymal stem cells on corticosteroid-induced osteonecrosis in rabbits prevention of osteonecrosis by intravenous administration of human peripheral blood-derived cd34-positive cells in a rat osteonecrosis model benefi cial effect of autologous transplantation of endothelial progenitor cells on steroid-induced femoral head osteonecrosis in rabbits treatment of osteonecrosis of the femoral head with implantation of autologous bone marrow cells. a pilot study concentrated autologous bone marrow aspirate transplantation treatment for corticosteroid-induced osteonecrosis of the femoral head in systemic lupus erythematosus key: cord-0002114-scbku3ce authors: cheng, brian chi yan; yu, hua; guo, hui; su, tao; fu, xiu-qiong; li, ting; cao, hui-hui; tse, anfernee kai-wing; wu, zheng-zhi; kwan, hiu-yee; yu, zhi-ling title: a herbal formula comprising rosae multiflorae fructus and lonicerae japonicae flos, attenuates collagen-induced arthritis and inhibits tlr4 signalling in rats date: 2016-02-10 journal: sci rep doi: 10.1038/srep20042 sha: 8aca747989e024abacecae2fcc95186972c3f5df doc_id: 2114 cord_uid: scbku3ce rl, a traditional remedy for rheumatoid arthritis (ra), comprises two edible herbs, rosae multiflorae fructus and lonicerae japonicae flos. we have reported that rl could inhibit the production of inflammatory mediators in immune cells. here we investigated the effects and the mechanism of action of rl in collagen-induced arthritis (cia) rats. rl significantly increased food intake and weight gain of cia rats without any observable adverse effect; ameliorated joint erythema and swelling; inhibited immune cell infiltration, bone erosion and osteophyte formation in joints; reduced joint protein expression levels of tlr4, phospho-tak1, phospho-nf-κb p65, phospho-c-jun and phospho-irf3; lowered levels of inflammatory factors (tnf-α, il-6, il-1β, il-17a and mcp-1 in sera and tnf-α, il-6, il-1β and il-17a in joints); elevated serum il-10 level; reinvigorated activities of antioxidant sod, cat and gsh-px in the liver and serum; reduced th17 cell proportions in splenocytes; inhibited splenocyte proliferation and activation; and lowered serum igg level. in conclusion, rl at nontoxic doses inhibited tlr4 signaling and potently improved clinical conditions of cia rats. these findings provide further pharmacological justifications for the traditional use of rl in ra management. prescribed by chinese medicine practitioners for treating various inflammatory disorders including ra [14] [15] [16] . extracts/constituents of these two herbs attenuate animal arthritis and inhibit tlr4 signalling [17] [18] [19] [20] [21] [22] . we have previously reported that rl, a herbal formula composed of these two herbs, could inhibit various components of the tlr4 signalling pathways 23, 24 . therefore, here we examined if rl could improve clinical conditions in collagen-induced arthritis (cia) rats, a model that is well established for studying human ra, and investigated the involvement of tlr4 signalling in the effects of rl. in the present study, we investigated the in vivo efficacy of rl in cia rats. rats immunized with bovine type ii collagen began to develop arthritis in the first week. the initial manifestation of arthritis was erythema and swelling of ankle joints, followed by the inflammation of the metatarsal and inter-phalangeal joints. disease progression can be evaluated by measuring paw swelling volume, which is an indicator of the degree of inflammation. in order to evaluate the anti-arthritic efficacy of rl, the paw volume changes were quantified using plethysmometry. at all three doses, rl significantly ameliorated paw swelling. at the end of the experiment, more significant reductions of paw volume were observed in groups treated with rl (660 mg/kg) and indomethacin (2.5 mg/kg), a nonsteroidal anti-inflammatory drug (nsaid) used as the positive control (fig. 1a) . overall arthritis severity in cia rats is commonly assessed by macroscopic clinical scoring. beginning from day 7, the arthritis score of cia rats increased progressively and reached about 6.5 in the last week. administration of rl significantly attenuated all clinical signs of arthritis indicated by the significant reduction in the mean arthritis score (fig. 1b) . representative photographs showing morphological changes of hind paws of rats from different groups on day 57 are shown in fig. 1c . beginning from day 21, cia rats gained significantly less weight than the normal rats. the average food intake of cia rats was significantly reduced. rl effectively improved the food intake and ameliorated weight loss caused by cia. moreover, no observable adverse reaction was found in rl-treated groups during the entire experimental period. (fig. 1d ,e). rl attenuated the radiographic damage of cia rats. radiographic examination of the talocrural joints revealed tissue swelling, bone matrix resorption, osteophyte formation and bone erosion at the joint margins in cia rats. no pathogenic change was observed in normal rats. rl markedly ameliorated soft tissue swelling and the degree of joint damage ( fig. 2a ). significant reduction in radiographic score was observed in rl-treated groups (fig. 2b ). rl improved the histological parameters of cia rats. in order to determine whether rl prevented articular destruction, talocrural joints were analysed histologically. histological examinations showed massive infiltration of inflammatory cells, pannus invasion, cartilage damage, and subchondral bone erosion in cia rats (fig. 3a) . histological scores of individual groups were shown in (fig. 3b ). rl inhibited tlr4 signalling in cia rats. in a previous study, we have demonstrated that rl could inhibit the tak1/nf-κ b, tak1/ap-1, tbk1/irf3 pathways in lps-stimulated murine raw 264.7 and human thp-1 cells. increased expression/activities of these tlr4 signalling components (tak1, nf-κ b, ap-1 and irf3) in cia rats demonstrated their involvement in the pathology of cia. rl reduced the upregulated protein levels of tlr4, phospho-tak1, phospho-nf-κ b p65, phospho-c-jun and phospho-irf3 in cia joint tissues (fig. 4a,b) . upon ligand binding, tlr4 recruits several adaptors which lead to activation of tak1. transcription of nf-κ b, ap-1 and irf3 initiates the production of pro-inflammatory cytokines and effector cytokines such as tnf-α , il-1β and il-6, which contribute to the pathogenesis of cia. rl mitigated the upregulated levels of pro-inflammatory tnf-α , il-1β and il-6, while elevated the levels of anti-inflammatory il-10 in both sera and joint tissue lysates (figs 4c and 5a-d). rl also lowered the level of mcp-1 in sera (fig. 5e ). activated tlr4 signalling induces oxidative stress, resulting in imbalance of antioxidant system 25, 26 . excessive production of pro-inflammatory mediators exaggerates the oxidative stress in ra, resulting in immune cells activation 27 . since the two herbs in rl contain various natural antioxidants 21, 28, 29 , we determined the effects of rl on the activities of the endogenous antioxidant enzymes in cia rats. rl treatment at high dose significantly rejuvenated the declined activities of the antioxidant enzymes (sod, cat and gsh-px) in sera (fig. 6a ) and liver tissues of cia rats (fig. 6b ). and adaptive immune responses. inhibition on tlr4 signalling has been shown to lower the synovial expression of il-17 and serum concentrations of il-17 in cia models 6, 30 . in order to characterize the effect of rl on tlr4-signalling-related immune responses, levels of il-17 in joint tissues and sera were determined. rl significantly mitigated the levels of il-17 in both sera and joint tissue lysates of cia rats (fig. 7a,b) . studies showed that the splenic proportion of pathogenic th17 cells, which play an important role in the pathogenesis of ra 31 , correlated directly with the levels of serum il-17 in arthritic mice 32 . in line with decreasing serum il-17, rl also reduced th17 cell proportion in splenocytes (fig. 7c ,d) and inhibited il-17a production in pma-and-ionomycin-activated splenocytes isolated from cia rats (fig. 7e ). the humoral response against autoantigen is essential for the development of erosive arthritis 33 . rl inhibited cii-stimulated proliferation of splenocytes isolated from cia rats (fig. 8a ) and significantly reduced the igg antibody level in sera (fig. 8b ). discussion current drug treatment options against ra like nsaid and disease-modifying anti-rheumatic drugs are not satisfactory because of their low efficacy, adverse effects and toxicity. new therapeutic agents with low toxicity and high efficacy should be explored. researchers are seeking alternative, complementary therapeutic agents from multi-target chinese medicines. rl, a traditional remedy for inflammatory disorders including ra, is composed of yingshi and jinyinhua, which are of low toxicity. yingshi can be used to make various food products 28, 34, 35 , and jinyinhua is treated as food in asian countries 36 . it has been shown that oral administration of an extract of yingshi attenuated the severity of cia rats without showing any observable adverse effects 21 . jinyinhua together with other herbs have been demonstrated to improve conditions in cia mice 37 . in the present study, we demonstrated that the herbal formula rl significantly improved the clinical conditions throughout the course of disease in cia rats without observable adverse effects. these evidences support rl to be a potential safe and effective anti-ra agent. several studies have proposed novel strategies for the treatment of ra and cia by regulating the activities of immune cells as well as the levels of certain cytokines (e.g. tnf-α , gmcsf, il-17) 38, 39 . the tlr4 signalling pathway, which regulates a wide range of proinflammatory cytokines and the activities of various immune cells, is believed to be one of the novel therapeutic targets in ra management 11, 12 . we previously reported that rl could inhibit various tlr4 signalling components 23 . therefore, we investigated rl's effects on tlr4 signalling-related events in cia rats, in which tlr4 can be activated by ligands from damaged synovial tissues 6 . upon ligand binding, tlr4 signalling activates transcription factors nf-κ b (p50/p65 heterodimer), ap-1 (c-fos/c-jun heterodimer) and irf3 40, 41 . activation of these transcription factors results in the production of inflammatory mediators e.g. il-6, mcp-1, tnf-α and il-1β , providing a positive feedback to the tlr4 signalling pathways, which results in persistent inflammation and favours the induction of autoimmunity 42 . in this study, we found that rl could inhibit tlr4 signalling. this was demonstrated by the suppressed protein expressions of tlr4, phospho-tak1, phospho-nf-κ b p65, phospho-c-jun, and phospho-irf3 in joint tissues. phosphorylation of the key transcription factors of tlr4 signalling is essential to the production of pro-inflammatory cytokines. rl's inhibition on tlr4 signalling was also evidenced by the reduced production of pro-inflammatory cytokines regulated by the transcription factors nf-κ b, ap-1 and irf3 in sera and joint tissues of cia rats. these pro-inflammatory cytokines could induce oxidative stress, leading to declined activities of endogenous antioxidants. rl rejuvenated the declined activities of the antioxidant enzymes (sod, cat and gsh-px) in sera and liver tissues of cia rats. tlr4 signalling also takes part in immune responses 43 . production of autoimmune antibodies and il-17 upon collagen immunization was found to be less in tlr4-deficient mice than in wild type mice 6 . in the present study, rl was found to be able to lower the levels of il-17 in sera and joint tissues of cia rats. this observation was supported by the reduction of pathogenic th17 cells and il-17 production in spleen cells. in addition, rl could reduce the igg antibody production and inhibit antigen-stimulated proliferation of splenocytes. these suggest that attenuation of tlr4 signalling is possibly one of the molecular mechanisms for the anti-arthritic effects of rl in cia rats (fig. 9) . reduction in the proportions of th17 cells may also be responsible for the effects of rl. several studies have demonstrated that besides the th17 cells, modulating the activities of other immune cells (e.g. macrophages, monocytes, b cells, dendritic cells, treg cells) may also be a potential therapeutic approach in ra management [44] [45] [46] [47] . because of the multi-component and multi-target natures of chinese medicines, we believe that this herbal formula may also affect other immune cells. further studies are needed to investigate the effects of this formula on other signalling pathways as well as other components of the immune system. in conclusion, rl improved the clinical conditions of cia rats without overt adverse effects, which was associated with the inhibition of tlr4 signalling. these findings provide further pharmacological basis for the traditional use of this formula in controlling ra. further studies should be performed to develop rl into a safe, effective and modern anti-ra agent. preparation of rl. the herbal material was prepared as described previously 23 . briefly, 500 g yingshi and 300 g jinyinhua were mixed, minced, macerated with 12 l absolute ethanol for 24 h, and refluxed for 2 h twice. supernatants were filtered, concentrated, freeze-dried and produced rl (yield: 15.2%). to control its quality, we established an hplc chromatogram for rl and quantified the contents of gallic acid and chlorogenic acid (supplementary figure 1) , which is the same as the supplementary material of a previous report 23 . animal treatments. male wistar rats (140 ± 15 g) were supplied by the chinese university of hong kong and housed under standard conditions (25 ± 2 °c, humidity: 60 ± 10%, 12 h-light:12 h-dark) with free access to water and chow. forty-eight rats were randomly divided into six groups of eight. five groups were immunized intradermally, at the base of the tail and the back, with 200 μ g bovine type ii collagen (chondrex, redmond, wa, usa) in 0.05 m acetic acid emulsified with equal volume incomplete freund's adjuvant (ifa) (chondrex, redmond, wa, usa) on day 0. a boost injection of 100 μ g collagen-ifa suspension was given in the same manner on day 7. from day 14 to 56, immunized groups were intragastrically (i.g.) administered with 165, 330, 660 mg rl/kg/day, 2.5 mg indomethacin/kg/day and saline, respectively; while normal controls were i.g. administered with saline. retro-orbital blood samplings under anaesthesia were performed at various time points. sera were collected by centrifugation (1500 g, 20 min). at day 57, the rats were fasted overnight and killed by anaesthetic overdose. after taking radiographs of hind limbs, left paw tissues (whole joints including synovium, adjacent tissues and bones) were pulverized using a mortar and pestle filled with liquid nitrogen, and homogenized in 50 mm tris-hcl, 1% np-40, 0.35% sodium deoxycholate, 150 mm nacl, 1 mm edta of ph 7.4, 1 mm phenylmethylsulfonyl fluoride, 1 mm naf, 1 mm na 3 vo 4 , 10 μ g/ml aprotinin, 10 μ g/ml leupetin and 10 μ g/ml pepstatin a. right hind limbs were collected for histological examinations. single cell suspensions of splenocytes were prepared from spleens as described 6 . red blood cells in the suspensions were removed by treatment with 0.16 m tris-nh 4 cl solution. liver tissues were dissected and homogenized in ice-cold saline using an ultra-turrax t-25 homogenizer. the experimental protocol was approved by the ethics committee of hong kong baptist university. all experimental procedures were conducted according to the principles expressed in the declaration of helsinki and the guide for the care and use of laboratory animals published by the us national institutes of health. every effort was made to reduce the number of animals used and minimize their pain and distress. macroscopic scoring of cia. each hind limb was scored on a 0 ~ 4 scale: 0 = normal; 1 = mild, but definite ankle erythema and swelling, or limited to individual digits; 2 = moderate ankle erythema and swelling; 3 = entire limb erythema and swelling including digits; 4 = maximally inflamed limb with multiple joints involvement. the arthritis score was the sum of the scores of the two hind limbs (maximum: 8). to quantify oedema, hind paw volumes were measured using a plethysmometer. hind limbs radiographs taken on day 57 were evaluated using a 0 ~ 3 scale (maximum: 6): 0 = normal; 1 = soft tissue swelling only; 2 = soft tissue swelling and early joint erosions; 3 = severe bone erosion or significant osteophyte formation. histological analysis. right hind limbs were fixed in 4% neutral formalin for one week and then decalcified in 0.5 m edta and 0.5% paraformaldehyde for 20 days. decalcified samples were dehydrated with alcohol, embedded in paraffin and sectioned (10 μ thick). talocrural joint sections were stained with hematoxylin and eosin and mounted on glass slides for histological analysis by light microscopy. histopathological changes of the joint were evaluated based on histologic parameters (inflammation, synovial hyperplasia, pannus formation, and erosion of cartilage/bone) using a 0 ~ 4-point scale: 0 = normal; 1 = synovium hypertrophy with cell infiltrates; 2 = pannus and cartilage erosions; 3 = major cartilage and subchondral bone erosions; 4 = loss of joint integrity and ankylosis. immunoblotting. the quantified joint tissue lysates were assessed by western blot analysis as described previously 23 . briefly, protein lysates were subjected to 10% sds-page and then electro-transferred onto nitrocellulose membrane. after blocking with 5% non-fat milk in tbst, the membrane was incubated with designated primary antibodies overnight. the membrane was then incubated with anti-mouse or anti-rabbit secondary antibodies 48, 49 . the specific immunoreactive bands were detected using enhanced chemiluminescence ecl detection kit (invitrogen, carlsbad, ca, usa) following the manufacturer's instruction. splenocytes culture. splenocytes isolated from animals were maintained in rpmi 1640 medium containing 10% heat-inactivated foetal bovine serum (fbs) and 1% antibiotics of penicillin/ streptomycin at 37 °c under 5% co 2 . splenocytes proliferation assay. splenocytes suspensions (5 × 10 6 cells/well in 96 well plate) were cultured in rpmi 1640 medium containing 50 μ g/ml heat-denatured bovine cii (pre-treated at 80 °c, 10 min) for 72 h. mtt (0.5 mg/ml) was then added for 1 h. supernatants were removed after centrifugation (500g, 10 min), and 100 μ l of dmso were added. absorbance at 570 nm, measured using microplate spectrophotometer, was expressed as percentage of the normal control. splenocytes activation assay. splenocytes (2 × 10 6 cells/ml) were stimulated with pma (0.05 μ g/ml) and ionomycin (1 μ g/ml) at 37 °c for 48 h. il-17a concentrations in the supernatants were determined by elisa (ebioscience, inc., ca, usa) following manufacturer's instructions. flow cytometric analysis. splenocytes (2 × 10 6 cells/ml) were stimulated with pma (0.05 μ g/ml) and ionomycin (1 μ g/ml) for 4 h and then brefeldin a (bfa, 5 μ g/ml) for 2 h. cells were extracellularly stained with fitc-conjugated anti-cd4, fixed, permeabilized, then labelled intracellularly with pe-conjugated anti-il-17a 50, 51 . percentages of stained cells were measured using facs instruments. cytokine, chemokine, antioxidant and anti-collagen antibody assays. cytokine and chemokine levels were quantified by milliplex map rat cytokine/chemokine panel (merck millipore), while antioxidants and anti-mouse cii igg levels were determined by elisa kits (ebioscience, inc., ca, usa) following manufacturer's instructions 52 . the results are presented as the means ± sem. data were analyzed by one-way anova. comparisons between two groups were performed using the dunnett's multiple comparisons test or post-hoc analysis. statistical analyses were carried out using graphpad prism version 5.0 (graphpad software, san diego, ca, usa). p < 0.05 was considered statistically significant. life expectancy, standardized mortality ratios, and causes of death in six rheumatic diseases in hong kong, china safety and efficacy of on-demand versus continuous use of nonsteroidal antiinflammatory drugs in patients with inflammatory arthritis: a systematic literature review intrinsic danger: activation of toll-like receptors in rheumatoid arthritis overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: toll-like receptor expression in early and longstanding arthritis presence of bacterial dna and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides toll-like receptor 4 is involved in inflammatory and joint destructive pathways in collagen-induced arthritis in dba1j mice understanding how lipopolysaccharide impacts cd4 t-cell immunity toll-like receptor 4 signaling in t cells promotes autoimmune inflammation toll-like receptors control activation of adaptive immune responses essential roles of toll-like receptor-4 signaling in arthritis induced by type ii collagen antibody and lps negative regulatory approaches to the attenuation of toll-like receptor signaling the role of toll-like receptors in rheumatoid arthritis scientific basis of botanical medicine as alternative remedies for rheumatoid arthritis earliest report of flos lonicerae japonicae and its medical organ chinese materia medica: combinations and applications wound repair and anti-inflammatory potential of lonicera japonica in excision wound-induced rats anti-inflammatory activities and mechanisms of action of the petroleum ether fraction of rosa multiflora thunb. hips lonicera japonica thunb. extract inhibits lipopolysaccharide-stimulated inflammatory responses by suppressing nf-kappab signaling in bv-2 microglial cells anti-inflammatory effect of lonicera japonica in proteinase-activated receptor 2-mediated paw edema petroleum ether extractive of the hips of rosa multiflora ameliorates collagen-induced arthritis in rats anti-inflammatory and analgesic effects of the ethanol extract of rosa multiflora thunb. hips a herbal formula consisting of rosae multiflorae fructus and lonicerae japonicae flos inhibits inflammatory mediators in lps-stimulated raw 264.7 macrophages a herbal formula comprising rosae multiflorae fructus and lonicerae japonicae flos inhibits the production of inflammatory mediators and the irak-1/tak1 and tbk1/irf3 pathways in raw 264.7 and thp-1 cells tlr costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production toll-like receptor 4 senses oxidative stress mediated by the oxidation of phospholipids in extracellular vesicles rheumatoid arthritis: the role of reactive oxygen species in disease development and therapeutic strategies detection of vitamin c in fruits of rosa multiflora with hplc evaluation of antioxidant activities of extracts from 19 chinese edible flowers shift from toll-like receptor 2 (tlr-2) toward tlr-4 dependency in the erosive stage of chronic streptococcal cell wall arthritis coincident with tlr-4-mediated interleukin-17 production the role of the th17 cytokines il-17 and il-22 in rheumatoid arthritis pathogenesis and developments in cytokine immunotherapy toll-like receptor 2 signaling in cd4 + t lymphocytes promotes t helper 17 responses and regulates the pathogenesis of autoimmune disease chronic development of collagen-induced arthritis is associated with arthritogenic antibodies against specific epitopes on type ii collagen antioxidant and antimicrobial activities of native rosa sp. from british columbia, canada research development and application of edible herbal resources in xinjiang acute and subacute toxicity study of the ethanol extract from lonicera japonica thunb anti-arthritic activity of fu-fang-lu-jiao-shuang on collagen-induced arthritis in balb/c mice and its underlying mechanisms eye-mediated immune tolerance to type ii collagen in arthritis-prone strains of mice type ii collagen induces peripheral tolerance in balb/c mice via the generation of cd8 + t regulatory cells fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis cytokines in the pathogenesis of rheumatoid arthritis toll-like receptors: linking innate and adaptive immunity dual role of gm-csf as a pro-inflammatory and a regulatory cytokine: implications for immune therapy excretory-secretory products secreted by paragonimus westermani delay the spontaneous cell death of human eosinophils through autocrine production of gm-csf gm-csf: an immune modulatory cytokine that can suppress autoimmunity immunity to onchocerciasis: cells from putatively immune individuals produce enhanced levels of interleukin-5, gamma interferon, and granulocyte-macrophage colony-stimulating factor in response to onchocerca volvulus larval and male worm antigens human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay a novel pancreatic beta-cell targeting bispecific-antibody (bsab) can prevent the development of type 1 diabetes in nod mice the novel role of il-7 ligation to il-7 receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis inhibition of inflammatory response by artepillin c in activated evid based complement alternat med this work is partially supported by grants jcyj20120829154222473 and jcyj20140807091945050 from the science, technology and innovation commission of shenzhen, hkbu 262512 from the research grants council of hong kong, hmrf11122521 from food and health bureau of hong kong, and frg2/14-15/056, frg2/15-16/020 from the hong kong baptist university. key: cord-0032691-sgucvmzt authors: dijkshoorn, bas; raadsen, reinder; nurmohamed, michael t. title: cardiovascular disease risk in rheumatoid arthritis anno 2022 date: 2022-05-11 journal: j clin med doi: 10.3390/jcm11102704 sha: ffaab45363496c7d953a217ed3661e7cbf23106b doc_id: 32691 cord_uid: sgucvmzt the risk for developing cardiovascular diseases (cvd) in rheumatoid arthritis (ra) patients is 1.5 times higher compared to the general population. this risk is partly due to the contribution of systemic inflammation in increased atherogenesis, while an increased prevalence of “traditional” cardiovascular risk factors, such as hypertension and dyslipidemia, is also attributed to nearly 50% of the total cvd risk. most anti-rheumatic medication partly reduces this cvd risk, primarily by reducing inflammation. the increased risk is recognized by most guidelines, which advise consequent screening and multiplying calculated risk scores by 1.5. however, screening in daily clinical practice is poorly done, and ra patients often have undiagnosed and untreated risk factors. in conclusion, even nowadays, ra patients still have an increased risk of developing cvd. advances in anti-inflammatory treatment partly mitigate this risk, but ra patients need mandatory screening for cv risk factors to turn their cvd risk towards that of the general population. rheumatoid arthritis (ra) is the most prevalent chronic inflammatory joint disease, with a prevalence of 460 per 100,000 people [1] . ra is characterized by chronic and destructive synovitis, leading to irreversible joint damage. however, the systemic inflammation characteristic of the disease also causes problems beyond the joints, such as interstitial lung disease, vasculitis and cardiovascular disease (cvd). in this review, we focus on cvd, discussing the cvd risk, the impact of anti-rheumatic treatment on cvd and the current guidelines regarding cardiovascular risk management in patients with ra. the etiology of ra is still not completely understood; however, both genetic and environmental factors contribute to the development of the disease. several genetic and epigenetic components have been linked to ra, along with several environmental factors such as cigarette smoke, dust exposure and our microbiome. other environmental factors, such as hormones, might explain the higher risk in women. progression of the disease often starts years before the onset of symptoms with the development of certain autoantibodies, such as rheumatoid factor (rf) and anti-citrullinated protein antibodies (acpa). acpapositive ra patients have more severe disease activity and increased cv mortality, and the presence of these antibodies might also contribute to the atherosclerotic process. acpa were also found in non-ra patients with cvd and were again associated with worse cvd outcomes [2] . after this initial, abnormal immune response has been established, which may take several years, large quantities of rf and acpa are produced by plasma cells. these auto-antibodies subsequently activate macrophages through complement and fc receptor binding, primarily in the joints. the local inflammation, enhanced by activated t cells, causes damage to the synovium, inducing pain, swelling and stiffness of the joints. however, the inflammatory reaction is not limited to the synovium but is systemic, occurring throughout the bloodstream. several pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-alpha (tnf-α) and interleukins 1 and 6 (il-1/il-6), are produced, which attract activated b and t cells, monocytes and macrophages. recently, the jak/stat signaling pathway, which regulates cytokine signaling, has been found to play a role in the pathogenesis of ra [3] . this pathway is activated by il-6, among other cytokines, and increases the production of pro-inflammatory cytokines [4] . this all leads to more systemic inflammation, causing extra-articular comorbidities, such as atherosclerosis [5] . the formation of atherosclerotic plaques is the main mechanism in the development of cvd in ra patients. the pathogenesis is multifactorial and has vascular, metabolic and inflammatory components [6] . dyslipidemia, hypertension, smoking, and inflammation, among other risk factors, contribute to atherogenesis by causing damage to the endothelium of the arteries. dysfunctional endothelial cells have increased permeability, enabling ldl to become trapped in the intima of the arterial walls, where it becomes oxidized by phospholipases and free radicals [7] . increases in adhesion molecules on the endothelium cause monocytes to migrate to the intima, which subsequently differentiate into macrophages. macrophages then ingest the accumulated lipids, turning into "foam cells", and can microscopically be seen as a "fatty streak" in the arteries. the foam cells, along with damaged endothelial cells, produce several cytokines, such as tnf-α and il-6, which attract and activate more leukocytes, endothelial and smooth muscle cells [8] . some foam cells eventually die from apoptosis, creating a necrotic nucleus in the atherosclerotic plaque. smooth muscle cells, macrophages and t-lymphocytes form a fibrous cap on the endothelial side of the plaque [9] . atherosclerotic plaques can either gradually grow and cause chronic ischemia or become unstable and cause acute cardiovascular events [10] . it is widely accepted that chronic inflammation, such as in ra, can amplify the process of atherosclerosis [11] . the systemic inflammation causes endothelial dysfunction, leading to the earlier and faster progression of atherosclerotic plaques. several of the underlying mechanisms are shared by ra and atherosclerosis [8] . tnf-α, one of the most important pro-inflammatory cytokines in ra, is also produced by foam cells and contributes to endothelial dysfunction. it also upregulates the expression of adhesion molecules, resulting in the accumulation of more immune cells in the plaques. il-6 promotes fatty streak development, and il-1 plays a role in the regulation of macrophages and t-helper 17 cells. in addition to directly influencing the development of atherosclerotic plaques, systemic inflammation also enhances the cv risk factors. by contributing to endothelial dysfunction and increased oxidative stress, inflammation leads to increased systemic vascular resistance and, thus, to hypertension [12] . inflammation in ra also leads to the increased catabolism of lipoproteins, often resulting in a decrease in the hdl and ldl levels while still associated with the future cvd risk in what is called the "lipid paradox". furthermore, inflammation causes the hdl levels to become pro-atherogenic by reducing its cholesterol efflux capacity, the ability to accept cholesterol from macrophages, which is an independent risk factor for cvd [13, 14] . higher levels of lipoprotein(a), a pro-inflammatory and pro-atherogenic modified ldl particle, are also associated with ra and are an independent risk factor for cvd [15] . treatment with anti-inflammatory medication largely reverses these effects, with a subsequent cvd risk reduction in ra patients. it has become increasingly known that cvd is a common comorbidity in ra patients, resulting in a more severe disease burden. the risk of developing cvd in ra patients is 1.5 times higher compared to the general population [16, 17] . a very recent cohort study demonstrated that, even nowadays, this risk still approaches that of diabetes patients [18] . several meta-analyses have indicated an increased chance of cv death by 50-60% [19, 20] . this risk is partly due to the contribution of systemic inflammation to increased atherogenesis, while the "traditional" cv risk factors are also attributed to nearly 50% of the total cvd risk [21] . several of these risk factors have increased the prevalence in patients with ra. smokers are more often observed in ra patients, since smoking is associated with a higher incidence of ra [21] [22] [23] . crowson et al., found that smoking, along with hypertension, have the highest population attributable risk (par), with non-smokers having a 23% lower risk compared to smokers. ra patients with the active disease have significantly lower highand low-density lipoprotein cholesterol (hdl-c/ldl-c) levels compared to the healthy population [24] . smoking was also significantly associated with a higher disease activity score, leading to worse clinical outcomes [25] . hypertension and diabetes are also more prevalent in ra patients compared to healthy controls [24] . treating patients with anti-rheumatics who also suffered from hypertension with medication resulted in a greatly reduced cardiovascular risk [21] . while hypertension is established as risk factor for cardiovascular disease, there is increasing evidence that systemic inflammation, as seen in ra patients, plays a central role in maintaining hypertension [26] . in addition to the traditional risk factors, patients with ra are more likely to have metabolic syndrome [27] . metabolic syndrome is highly correlated with traditional risk factors such as hyperlipidemia, hypertension and diabetes mellitus and results in a relative risk for cardiovascular disease of 1.93 [28] . higher disease activity or duration are also important, independent predictors of metabolic syndrome in ra patients [29] . better weight control through dieting could improve this burden and improve some of the associated risks. many observational trials have shown that higher disease activity leads to increased cardiovascular risk, e.g., a dutch study, where the researchers found that an increased das-28 score of 1 unit resulted in a 28% increased cv risk, irrespective of the disease duration [30] . vice versa, one can expect a reduced cvd risk with lower disease activity, and in a large-scale trial done in 2015, solomon et al., showed that a 10-point reduction in the cdai (clinical disease activity index) resulted in a 26% reduction in cv events [31] . methotrexate (mtx) is the anchor conventional synthetic disease-modifying antirheumatic drug (csdmard) in the treatment of ra and reduced cardiovascular events by 20% in a recently updated meta-analysis that included nearly 200,000 ra patients [32] . the main mode of action of mtx in ra is the inhibition of the enzyme dihydrofolate reductase, which produces a key ingredient needed for dna and rna synthesis. this, in turn, causes a reduced amount of proliferated lymphocytes [33] . these proliferated lymphocytes excrete several key cytokines that are essential in maintaining the pro-inflammatory environment. by reducing these lymphocytes, the inflammation subsides and the patient experiences less pain and swelling. however, the mechanisms that lead to its cardioprotective effects have not yet been entirely elucidated. mtx seems to reduce tnf-α, il-6 and c-reactive protein while also downregulating foam cell production [34, 35] . the reduction of these pro-inflammatory molecules most likely signals the reduced systemic inflammation caused by mtx, which, in turn, leads to a reduced cv risk. the significant reduction in the cvd risk in patients with ra led to a randomized double-blinded placebo-controlled trial with low-dose mtx on general population patients that previously had a myocardial infarction or multi-vessel disease combined with either diabetes or metabolic syndrome [36] . patients in the mtx arm received 15 mg once per week at the start that was increased to 20 mg once per week after 4 months. in contrast to the expectations, the investigators found no difference between the mtx and placebo arms with respect to the cvd endpoints. possible explanations could be the relatively low dose of mtx and/or the low systemic inflammatory load in comparison to ra. there have been relatively few studies on the cv risk of csdmards compared to mtx. a study done by our group showed a reduced risk for ra patients that used either methotrexate, sulfasalazine, or methotrexate, sulfasalazine and hydroxychloroquine [37] . given the case-control design of the study, it was not optimally designed to give an effect estimation. antimalarial drugs such as hydroxychloroquine and chloroquine might have a positive effect on the reduction of cv events. in a retrospective study done by sharma et al., a 72% decrease of cv events was seen [38] . although, it must be noted that these results have not been replicated by other studies. a meta-analysis looking at hydroxychloroquine and chloroquine use in all rheumatic diseases found a significantly reduced cv risk of 28%. possible causes of this reduced risk are reduced systemic inflammation and a reduction of the traditional risk factors such as hyperglycemia and hypercholesterolemia [39] . while there is not enough data to make a definitive conclusion, the first signs are positive. glucocorticoids such as prednisolone might increase the cardiovascular risk in ra patients in a dose-and duration-dependent manner. in a very large retrospective registry study, ocon et al. showed that ra patients that used <5 mg daily or had a cumulative dose of <750 mg total had no increased cv risk [40] . patients with higher daily doses, higher cumulative doses or longer treatment durations did show an increased cv risk. during the recent gloria trial, patients were given low-dose glucocorticoids (5 mg prednisolone) for 24 months. this resulted in an increased incidence of cv events of approximately 35% (2.4 cases per 100 patient years in the prednisolone group vs. 1.7 in the placebo group) [41] . selective cox-2 inhibitors are known to increase the cv risk in the general population by approximately 35-40% compared to the placebo [42, 43] . this increase is comparable to that of traditional nsaids such as ibuprofen or diclofenac [44] . there have not been any prospective studies investigating the cv risk of celecoxib or other selective cox-2 inhibitors specifically in ra patients. one could speculate that, on the one hand, this could be similar to the general population, while, on the other hand, in ra, nsaids might somewhat suppress inflammation and improve mobility, thus having favorable effects on the cv risk. studies investigating the effect of leflunomide on cv risk are rare. two retrospective database cohort studies from the same research group showed a decreased relative risk for acute myocardial infarction and congestive heart failure in ra patients using leflunomide [45, 46] . no prospective studies have been performed. during the last 20 years, numerous biological dmards (bdmards) have become available for ra treatment. most of these bdmards specifically target proteins associated with inflammation, such as tumor necrosis factor (tnf) and interleukin 6 (il-6), while others target the development of t and b cells (abatacept and rituximab, respectively). newer targeted synthetic dmards, such as the janus kinase (jak) inhibitors, affect the cytokine pathways of the immune system more specifically. as the fundamental research in the pathophysiology of atherosclerosis shows, the immune system has an important role in the development of atherosclerotic plaques [47] . tnf-α especially, but also il-6, seem to contribute to endothelial dysfunction. thus, it is hypothesized that treatment with medication inhibiting these proteins might reduce the risk of cvd in ra patients. tnf inhibitors specifically target tnf-α, a pro-inflammatory cytokine associated with the systemic inflammation in ra. tnf-a is produced by macrophages and foam cells during atherosclerotic plaque formation to maintain and progress plaque development [48, 49] . treatment with tnf inhibitors thus seems to impact both systemic inflammation and atherosclerotic plaque formation. several studies have shown that the incidence of cvd in ra patients treated with these medications is reduced compared to patients without anti-inflammatory treatment or with csdmards [50] [51] [52] [53] [54] . a longer treatment duration with tnf inhibitors also seems to further reduce the cvd risk. additionally, tnf inhibitors seem to increase hdl cholesterol in patients, which might contribute to its cardio-protective effects [55, 56] . the decrease in systemic inflammation caused by tnf inhibitors also improves the hdl efflux capacity and reduces the risk of cvd independent of the hdl levels [14, 57] . a proof-of-concept study also found a potential blood pressure-lowering effect [58] . however, these effects still need to be confirmed in prospective-controlled studies. interleukin inhibitors used in ra treatment primarily focus on il-6, another proinflammatory cytokine involved in ra pathology. like tnf-a, its production is upregulated during the early stages of atherosclerotic plaque formation [48] . il-6 inhibition results in a decrease in inflammation in most ra patients, but an increase in ldl cholesterol is also often measured. while this seems paradoxical, it still results in a decreased cvd risk comparable with tnf inhibition, partly because of an improvement in the hdl efflux capacity [59, 60] . an il-6 blockade might also reduce the lipoprotein(a) levels [61] . the decrease in inflammation thus outweighs the increase in ldl cholesterol. il-6 inhibition also has a direct effect on the endothelium, improving endothelium function in high-risk ra patients [62] . this effect was also replicated in non-ra patients with atherosclerosis, in whom treatment with an il-6 inhibitor resulted in reduced cv events and mortality [63] . abatacept reduces the inflammation in ra patients by inhibiting the activation of t cells, which restricts its ability to produce an immune response. several studies indicated that abatacept might be more effective in decreasing cardiovascular risk in ra patients than csdmards and tnf inhibitors [64, 65] . abatacept also seems to have favorable effects on the insulin sensitivity, especially in diabetes patients [66] . hypertension has also been described as a possible side effect of 1-10% [67]. however, the cvd reduction due to the anti-inflammatory effect of abatacept seems to not be affected [68, 69] . rituximab is a cd-20 monoclonal antibody that reduces the b-cell count in patients. it increases the hdl cholesterol and improves endothelial function and arterial stiffness [70, 71] . while its overall effect on the incidence of cardiovascular diseases is still unclear, myocardial ischemia and infarction have both been described as possible rare adverse events in rituximab users during infusion, although only in a few case reports [72] . it has yet to be elucidated whether its positive effect on the risk factors outweighs the possible cv side effects. jak inhibitors are new targeted synthetic dmards used in treatment for ra that reduce inflammation by inhibiting the jak-stat pathway to decrease the production of several pro-inflammatory cytokines. the jak-stat pathway has a function in atherosclerotic cvd; however, more long-term studies are required to understand jak inhibitor effects on the cvd risk [73, 74] . the reported side effects of jak inhibitors include hypercholesterolemia and thromboembolic events, such as deep vein thrombosis (dvt) and pulmonary embolism (pe) [75] . caution is advised in patients with preexisting risk factors for thromboembolic events, such as higher age, obesity, smoking, history of dvt/pe or long periods of immobilization [76] . in several trials, the jak inhibitors did not show an increased cvd risk compared to the placebo, cs-/bdmards or other jak inhibitors [77, 78] . however, more recently, the fda issued a safety warning concerning the results of the "oral surveillance" safety trial [79] . in this trial, patients were randomized to either tofacitinib 5 mg or 10 mg twice daily or a tnf inhibitor. other inclusion criteria were ages over 50 and one or more cardiovascular risk factors [80] . during the trial, an increased risk of pulmonary embolism was found in the 10 mg group, and therefore, the dose was reduced to 5 mg twice daily during the remainder of the trial [81] . the incidence of mace in the tofacitinib 5 mg group was 0.91 per 100 patient years vs. 0.73 per 100 patient years in the tnf inhibitor group, resulting in a hazard ratio of 1.33 (ci 95% 0.91-1.94). non-inferiority was not proven, as the upper boundary of the confidence interval of the hazard ratio was higher than 1.8, indicating a higher cvd risk with the uses of tofacitinib in comparison to the tnf inhibitor. another recent study suggested no significant differences in mace and thrombosis incidence after 60 days of treatment between tofacitinib and etanercept. however, the number of studied patients, length of follow-up and number of arterial and venous thrombotic events were too low to reach valid conclusions [82] . the european medicines agency has also recommended that patients over 65 years of age, patients who are current or past smokers, patients with other cv risk factors and patients with other malignancy risk factors should only use tofacitinib if no other treatment is available [83]. the increased cardiovascular risk in ra patients has been acknowledged by the current guidelines and has been implemented in several risk calculators [84] . the european league against rheumatism (eular) in their most recent guideline recommends screening every patient with ra at least every five years and more often if an increased risk was previously found [85] . screening should be performed according to the national guidelines. for a 10-year cvd risk prediction, the use of a score model is recommended. calculated risk scores should be multiplied by 1.5 if the algorithm does not already account for ra as an independent risk factor. the eular recommends using the same treatment targets for the lipid levels and blood pressure as for the general population, with an additional emphasis on lifestyle interventions, and advises caution with prolonged nsaids and glucocorticoids usage. the european society of cardiology (esc) also acknowledges the increased risk in ra but is less firm in its recommendations and suggests a low threshold for the assessment of the total cvd risk in adult patients and multiplying the calculated risk based on the disease activity by 1.5 [86] . the esc also advises treating the cvd risk with similar interventions as for the general high-risk population. while some well-known cardiovascular risk calculators, such as framingham, have yet to include ra as a risk factor, several others have heeded these recommendations. the score calculator includes a 1.5 multiplication factor for ra patients, and the most recent qrisk calculator multiplies the risk in ra patients by around 1.2. however, these calculators still seem to underestimate the risk in many ra patients, especially those categorized as low and intermediate risk. only a small proportion of patients gets relocated to a more appropriate risk category by utilizing these multiplication factors. to correct this underestimation, an ra-specific calculator for cv risk screening was in development by the atacc-ra group; however, the model could not be validated [87] . despite such explicit advice and the availability of tools incorporating the increased cardiovascular risk of ra patients, screening in daily clinical practice is generally poorly done. ra patients often have undiagnosed or untreated risk factors, such as hypertension or hypercholesterolemia [88] . a study surveying general practitioners (gps) about the cardiovascular risk of ra patients found that the majority did not recognize ra as an independent risk factor for cardiovascular diseases and did not perform the recommended screening assessments on ra patients in their practice [89] . they also found that only a small minority calculated cardiovascular risk scores using the 1.5 multiplication factor. even after screening, when general practitioners and internists received notice of risk stratification, a more recent study found that only one-third of patients with an indication of treatment received this [90] . even nowadays, ra patients still have an increased risk of developing cvd, due to both an increased prevalence of "traditional" cv risk factors, as well as systemic inflammation. advances in anti-inflammatory treatment partly mitigate this risk by reducing the inflammation. currently, screening (and treatment, if indicated) for cv risk factors is still insufficient, resulting in undiagnosed and untreated risk factors. ra patients need optimal control of their systemic inflammation and mandatory regular screening for cv risk factors to turn their cvd risk towards that of the general population. the global prevalence of rheumatoid arthritis: a meta-analysis based on a systematic review long-term mortality in patients with st-segment elevation myocardial infarction is associated with anti-citrullinated protein antibodies stat pathway in pathology of rheumatoid arthritis the role of the jak/stat signal pathway in rheumatoid arthritis extra-articular manifestations in rheumatoid arthritis immune and inflammatory mechanisms of atherosclerosis pathophysiology and treatment of atherosclerosis: current view and future perspective on lipoprotein modification treatment the role of cytokines in the development of atherosclerosis atherosclerosis: process, indicators, risk factors and new hopes inflammation and plaque vulnerability cellular-molecular mechanisms of atherosclerosis development (scientific legacy of academician rams v. a. nagornev) roles of inflammation, oxidative stress, and vascular dysfunction in hypertension inflammation, remodeling and other factors affecting hdl cholesterol efflux ferraz-amaro, i. hdl cholesterol efflux capacity in rheumatoid arthritis patients: contributing factors and relationship with subclinical atherosclerosis lipoprotein (a) and its role in inflammation, atherosclerosis and malignancies patients with rheumatic diseases adhere to covid-19 isolation measures more strictly than the general population cardiovascular risk and mortality in rheumatoid arthritis compared with diabetes mellitus and the general population cardiovascular disease risk in inflammatory arthritis patients still substantially elevated in 2020 risk of cardiovascular mortality in patients with rheumatoid arthritis: a meta-analysis of observational studies trends in cardiovascular mortality in patients with rheumatoid arthritis over 50 years: a systematic review and meta-analysis of cohort studies impact of risk factors associated with cardiovascular outcomes in patients with rheumatoid arthritis cigarette smoking and risk of rheumatoid arthritis: a dose-response meta-analysis impact of smoking as a risk factor for developing rheumatoid arthritis: a meta-analysis of observational studies traditional cardiovascular risk factors in rheumatoid arthritis: a meta-analysis smoking is associated with higher disease activity in rheumatoid arthritis: a longitudinal study controlling for time-varying covariates the role of inflammation in hypertension: novel concepts metabolic syndrome and its components among rheumatoid arthritis patients: a comprehensive updated systematic review and meta-analysis risks for all-cause mortality, cardiovascular disease, and diabetes associated with the metabolic syndrome: a summary of the evidence metabolic syndrome and atherogenic indices in rheumatoid arthritis and their relationship with disease activity: a hospital-based study from northeast india the effect of disease duration and disease activity on the risk of cardiovascular disease in rheumatoid arthritis patients disease activity in rheumatoid arthritis and the risk of cardiovascular events methotrexate can prevent cardiovascular events in patients with rheumatoid arthritis: an updated meta-analysis the mechanism of action of methotrexate signalling pathways of the tnf superfamily: a double-edged sword atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human thp-1 monocyte/macrophages low-dose methotrexate for the prevention of atherosclerotic events disease-modifying antirheumatic drugs are associated with a reduced risk for cardiovascular disease in patients with rheumatoid arthritis: a case control study hydroxychloroquine use is associated with decreased incident cardiovascular events in rheumatoid arthritis patients hydroxychloroquine and risk of diabetes in patients with rheumatoid arthritis short-term dose and duration-dependent glucocorticoid risk for cardiovascular events in glucocorticoid-naive patients with rheumatoid arthritis favorable balance of benefit and harm of long-term, low dose prednisolone added to standard treatment in rheumatoid arthritis patients aged 65+: the pragmatic, multicenter, placebo-controlled gloria trial vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials do selective cyclo-oxygenase-2 inhibitors and traditional non-steroidal anti-inflammatory drugs increase the risk of atherothrombosis? meta-analysis of randomised trials cardiovascular safety of celecoxib, naproxen, or ibuprofen for arthritis antirheumatic drug use and the risk of acute myocardial infarction anti-rheumatic drug use and risk of hospitalization for congestive heart failure in rheumatoid arthritis how the immune system shapes atherosclerosis: roles of innate and adaptive immunity cardiovascular effects of approved drugs for rheumatoid arthritis macrophages in atherosclerosis: a dynamic balance the effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis the effects of tumor necrosis factor inhibitors on cardiovascular risk in rheumatoid arthritis longer durations of antitumour necrosis factor treatment are associated with reduced risk of cardiovascular events in patients with rheumatoid arthritis tumour necrosis factor antagonists and the risk of cardiovascular disease in patients with rheumatoid arthritis: a systematic literature review cardiovascular risk in rheumatoid arthritis: comparing tnf-α blockade with nonbiologic dmards effect of tnf inhibitors on lipid profile in rheumatoid arthritis: a systematic review with meta-analysis influence of anti-tumour necrosis factor therapy on cardiovascular risk factors in patients with active rheumatoid arthritis the association between reduction in inflammation and changes in lipoprotein levels and hdl cholesterol efflux capacity in rheumatoid arthritis effects of anti-tnf alpha therapy on blood pressure in resistant hypertensive subjects: a randomized, double-blind, placebo-controlled pilot study cardiovascular safety of tocilizumab versus tumor necrosis factor inhibitors in patients with rheumatoid arthritis: a multi-database cohort study. arthritis rheumatol tocilizumab and the risk of cardiovascular disease: direct comparison among biologic disease-modifying antirheumatic drugs for rheumatoid arthritis patients il-6 blockade by monoclonal antibodies inhibits apolipoprotein (a) expression and lipoprotein (a) synthesis in humans interleukin 6 inhibition and coronary artery disease in a high-risk population: a prospective community-based clinical study canakinumab for residual inflammatory risk the risk of cardiovascular events associated with disease-modifying antirheumatic drugs in rheumatoid arthritis comparative cardiovascular risk of abatacept and tumor necrosis factor inhibitors in patients with rheumatoid arthritis with and without diabetes mellitus: a multidatabase cohort study the effect of non-tnf-targeted biologics and small molecules on insulin resistance in inflammatory arthritis factors associated with risk of major adverse cardiovascular events in patients with rheumatoid arthritis: a nationwide, population-based, case-control study biologic agents reduce cardiovascular events in rheumatoid arthritis not responsive to tumour necrosis factor inhibitors: a national cohort study depletion of b-cells with rituximab improves endothelial function and reduces inflammation among individuals with rheumatoid arthritis the effects of rituximab on lipids, arterial stiffness and carotid intima-media thickness in rheumatoid arthritis anterior st-elevation myocardial infarction induced by rituximab infusion: a case report and review of the literature an emerging target for cardiovascular disease in rheumatoid arthritis and myeloproliferative neoplasms role of janus kinase inhibitors in rheumatoid arthritis treatment jak inhibitors in rheumatoid arthritis: an evidence-based review on the emerging clinical data risk of venous thromboembolism associated with janus kinase inhibitors for rheumatoid arthritis: case presentation and literature review efficacy and safety of tofacitinib versus baricitinib in patients with rheumatoid arthritis in real clinical practice: analyses with propensity score-based inverse probability of treatment weighting safety profile of upadacitinib in rheumatoid arthritis: integrated analysis from the select phase iii clinical programme cardiovascular and cancer risk with tofacitinib in rheumatoid arthritis oral surveillance and jak inhibitor safety: the theory of relativity comparison of cardiovascular, cancer and herpes zoster risk of tofacitinib versus etanercept: single centre observational study best practices for cardiovascular disease prevention in rheumatoid arthritis: a systematic review of guideline recommendations and quality indicators eular recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders esc guidelines on cardiovascular disease prevention in clinical practice: developed by the task force for cardiovascular disease prevention in clinical practice with representatives of the european society of cardiology and 12 medical societies with the special contribution of the european association of preventive cardiology (eapc) rheumatoid arthritis-specific cardiovascular risk scores are not superior to general risk scores: a validation analysis of patients from seven countries castro cabezas, m. marked underdiagnosis and undertreatment of hypertension and hypercholesterolaemia in rheumatoid arthritis the recognition and assessment of cardiovascular risk in people with rheumatoid arthritis in primary care: a questionnaire-based study of general practitioners suboptimal cardiovascular risk management in rheumatoid arthritis patients despite an explicit cardiovascular risk screening programme key: cord-0037486-qs7ztlor authors: matta, bassem n.; nayfe, rabih; taher, ali t.; uthman, imad title: b-cell targeted therapies in autoimmune cytopenias and thrombosis date: 2013-10-29 journal: drugs targeting b-cells in autoimmune diseases doi: 10.1007/978-3-0348-0706-7_11 sha: b3d177bd58d442986c25eebc466b7db43032c9ba doc_id: 37486 cord_uid: qs7ztlor ever since the advent of rituximab and subsequently the emergence of other compounds targeting b cells, a cornucopia of medical applications have been found for this family of compounds. after their establishment as standard of care in many conditions such as rituximab in lymphoma and rheumatoid arthritis, they have been progressively found to aid in the treatment of many other conditions. this area constituted a fertile area of research in the past 12 years. physicians have investigated the b-cell depleting agents use in cases of autoimmune hematologic cytopenias such as immune thrombocytopenia, evans syndrome, cold and warm autoimmune hemolytic anemia, and other thrombophilic disorders such as the antiphospholipid syndrome and thrombocytopenic purpura. this chapter presents a historical perspective reviewing the various studies that have been published in this field. in addition, it offers a current assessment of the evidence regarding the use of b-cell depleting agents in the aforementioned conditions. ever since the advent of rituximab and subsequently the emergence of other compounds targeting b cells, a cornucopia of medical applications have been found for this family of compounds. after their establishment as standard of care in many conditions such as rituximab in lymphoma, they have been progressively found to aid in the treatment of many other conditions. this area constituted a fertile area of research in the past 12 years (zimmer et al. 2004) . physicians have investigated the b-cell depleting agents use in cases of autoimmune hematologic cytopenias such as itp (immune thrombocytopenia), evans syndrome, cold and warm autoimmune hemolytic anemia, and other thrombophilic disorders such as the antiphospholipid syndrome and ttp (thrombocytopenic purpura). immune thrombocytopenia is an autoimmune disease of platelet destruction and subsequent thrombocytopenia. the disease has been witnessed to many changes all the way from understanding its pathophysiology to the evolution in its management options. initially called idiopathic thrombocytopenic purpura and thus the acronym itp, it went on to be named immune thrombocytopenic purpura, and more recently immune thrombocytopenia (stasi et al. 1995) . changes in nomenclature emerged due to advances in understanding the disease. it was primarily thought of as a disease of peripheral platelet destruction of unknown origin. itp's definition progressively evolved to include peripheral platelet destruction due to autoantibodies in addition to an inadequate production by megakaryocytes through disturbed level of erythropoietin (neunert et al. 2011; nugent et al. 2009 ). itp is classified into primary and secondary. no test has been yet developed that can accurately pinpoint the diagnosis. the primary form is usually diagnosed by exclusion of a secondary form which is usually part of another disease process such as: antiphospholipid syndrome, systemic lupus erythematosus, a hematologic or non-hematologic malignancy, common variable immune deficiency or might be due to hepatitis c or helicobacter pylori, cytomegalovirus, varicella zoster or human immunodeficiency virus infection or to a vaccination side effect (cines et al. 2009 ). secondary itp accounts for around 20 % of the total number of patients diagnosed with immune thrombocytopenia (cines et al. 2009 ). of relevance to this chapter is the primary form since this is where b-cell depleting agents come into play. the management of itp varies between children and adults. this stems from the fact that children have a more acute disease with a higher tendency for spontaneous remission as opposed to adults (neunert et al. 2011; stasi et al. 1995) . initial therapy in an emergency setting for symptomatic patients mainly consists of glucocorticoids, ivig, or alternatively anti-d in select cases (non-splenectomized, rh-positive patients). high dose dexamethasone can also be considered in patients who do not respond to the above-mentioned treatments or as an alternative to splenectomy in chronic itp patients. splenectomy is exclusively a second-line therapy however, should be delayed until 12 months after the diagnosis since patients might spontaneously remit within this period (sailer et al. 2006 ). treatment is usually administered in this population when patients are bleeding or are at increased risk of bleeding, such as premenopausal women or when they have coexistent risk factors, lifestyle, preference and pros versus cons of treatment (stasi et al. 1995; cohen et al. 2000; daou et al. 2008) . it has been proposed that a platelet count of 30 â 10 9 l à1 can be adopted as a threshold for treatment as it was shown to improve mortality in newly diagnosed itp patients (li et al. 2005; neylon et al. 2003; neunert et al. 2011) . glucocorticoids, ivig, and anti-d have all been proposed as treatment options for itp and are given alone or in combination based on the patients' tolerability of every treatment or the need for a quicker response in some cases (nugent et al. 2009; thota et al. 2012; godeau et al. 1999; newman et al. 2001; zimmer et al. 2004) . high dose dexamethasone has also been reported to be highly effective however, due to paucity of head to head studies with other treatments, it is not yet considered a standard of care (cheng et al. 2003) . splenectomy, rituximab, and tpo agonists are all considered adequate second-line therapies (provan et al. 2010; arnold 2013; neunert et al. 2011) . splenectomy is the option physicians have the most experience with not to mention the fact that it has the most studies to support it since it was the first and only second-line treatment option for a long time (ghanima et al. 2012 ). the first study exploring b depleting agents in itp was reported in 2000. it was a retrospective one that included patients who had already been treated with steroids. it showed a 30 % response rate to rituximab when given at 375 mg/m 2 weekly for 4 weeks (as per the lymphoma protocol) (saleh et al. 2000) . the first prospective trial using rituximab in itp was reported in 2001 (stasi et al. 2001 ). this study included 25 patients who had chronic itp and had failed prior treatments. all of the patients received the 375 mg/m 2 dose. the results from that trial showed an encouraging 52 % response rates in otherwise treatment refractory patients. with the minimal adverse event profile of this drug, it was found to be a safe alternative to more traditional treatments. a large systematic review exploring the efficacy of rituximab as a second-line treatment in patients older than 15 years of age with roughly half of them having undergone splenectomy found that treatment with rituximab gave a complete response rate (cr) of 46.3 % (95 % ci: 29.5-57.7) and a partial response rate (pr) of 24.0 % (95 % ci: 15.2-32.7). in that study pr and cr were defined as exceeding 50 â 10 9 cells/l and 150 â 10 9 cells/l, respectively (arnold et al. 2007) . this is different than the most recent ash guidelines that define a cr as one that leads to a platelet count !100 â 10 9 cells/l without bleeding and a response as one that leads platelet count !30 â 10 9 cells/l accompanied with a greater than twofold increase of platelet count from baseline with absence of bleeding (neunert et al. 2011 ). the median time to response and response duration in the above-mentioned review were 5.5 weeks and 10.5 months, respectively. a recent meta-analysis confirmed rituximab's efficacy in itp revealing an overall response and complete response of 59.7 and 45.7 %, respectively (barcellini and zanella 2011) . previous studies have shown that the response rate does not vary whether rituximab is used before or after splenectomy or used after a previous trial of rituximab (auger et al. 2012 ). it has also been shown that the 100 mg/m 2 dose can be as effective as the much higher ones used in the lymphoma protocol giving an overall response rate of 71 % (stasi 2010) . a recent systematic review recently published that included 18 observational studies with a total number 323 children with itp concluded that cr for these patients was 39 % (ci: 30-49 %). the response rate was, however, 68 % (ci: 58-77 %). this is very similar to the response rates observed in adults; however, the definitions of cr (!100 â 10 9 cells/l) and response (!30 â 10 9 cells/l) differed from their adult counterparts (liang et al. 2012) . a multicenter prospective trial of chronic itp patients, who are candidates for splenectomy and were treated with rituximab, has shown promising results. at 1 year, 40 % of patients had responded (95 % ci: 28-52 %). this group, however, decreased over time with only 6.7 % of the patients experiencing sustained response after a single course of rituximab using the lymphoma protocol. this proved that rituximab could be used to delay or prevent splenectomy (godeau et al. 2008 ) a recent large meta-analysis exploring rituximab as an option prior to splenectomy demonstrated a 57 % overall response rate after rituximab treatment with younger patients responding the most (auger et al. 2012 ). another study using rituximab earlier in the course of treatment of patients with immune thrombocytopenia evaluated 103 patients that were treatment naïve. it consisted of randomizing patients to either dexamethasone alone or to dexamethasone with concomitant rituximab (lymphoma protocol). results showed a significantly better sustained response by 27 % in patients receiving the steroid and rituximab regiment; 63 % versus 36 % in the steroid only arm. the occurrence of side effects nonetheless (grade 3 and 4) was much more pronounced however in the combined treatment group . a newer trial demonstrated that there was no significant difference in the composite outcome of reaching a platelet count less than 50 â 10 9 cells/l, significant bleeding or need for rescue treatment, between a group of untreated itp patients receiving their standard treatment and another receiving additional adjuvant rituximab (arnold et al. 2012) . one prohibitive factor in the use of rituximab has been the elevated cost. on average, a splenectomy would cost $20,000 whereas thrombopoietin and rituximab would each cost $2,500-$4,500 per month, and $10,000-$50,000, respectively (ghanima et al. 2012 ). three mechanisms of action of rituximab have been proposed. the first involves a decrease in macrophage phagocytosis and peripheral destruction of platelet coated with autoantibodies, when the latter are bound by rituximab. this would explain the early response (after 4 weeks) which occurs in the majority of cases after rituximab treatment. it is thought to be mediated by an inhibition of the fc receptor portion of macrophages. the second mechanism involves b-cell depletion and accounts for the late response by decreasing the number of autoantibody producing b cells. the third mechanism is probably through t cell modulation effect of the drug since in some cases no correlation is observed between the disease severity and the antibody level (stasi 2010). it seems that rituximab dominates the biologics field in itp. a recent noteworthy trial has combined low rituximab with alemtuzumab yielded an impressive 100 % overall response rate and a complete response rate of 56 % (gomez-almaguer et al. 2010). thrombotic cytopenic purpura is a rare hematological disease that has an estimated annual incidence of 11.3 cases/1,000,000 people. it has been classically defined as patients presenting with the pentad of thrombocytopenia, uremia, microangiopathic hemolytic anemia, fever, and neurologic symptoms. this continues to be the classic teaching even though these symptoms are present all together in a minority of patients: 5 % of 64 patients from the oklahoma registry of ttp patients (george 2010) . although ttp shares the same histological appearance with the hemolytic uremic syndrome, it is important to differentiate it from that latter since the management differs radically (claus et al. 2010) . it is considered to be an ancient disease that was first described as early as 1923 (moschcowitz 2003) . it has been proposed that it is due to aggregation of single von willebrand factors units into a large multimer due to the absence of a metalloproteinase that usually breaks it down in order to keep the system in check (galbusera et al. 1999) . later on, this metalloproteinase was discovered to be adamts 13 (zheng et al. 2001) . the direct result of this abnormally large multimer is platelet aggregation, injury to rbcs, thrombosis, and subsequent end organ damage. adamts 13's deficiency was considered the culprit of the disease up until recently when some ttp patients were discovered not to have a deficiency in this metalloproteinase (kremer hovinga et al. 2010; vesely et al. 2003) . ttp has been classified as either congenital or acquired. patients having the congenital form of the disease (upshaw-schulman syndrome) constitute 5 % of people with ttp whereas the people with the acquired type make up the remaining 95 %. the acquired type is further subdivided into an autoimmune idiopathic subtype that makes up 70-80 % of those patients versus a secondary subtype that constitutes the rest (rizzo et al. 2012) . some of the secondary causes that have been described are infections such as hiv, tumors, autoimmune diseases, pregnancy, and stem cell transplant recipients (kremer hovinga et al. 2010) . the diagnosis of the disease continues to be mainly a clinical one due to the absence of a specific lab test that could pinpoint the diagnosis. adamts 13 as mentioned earlier maybe negative in a subset of patients due mainly to the variability in the testing methods however, when present, it offers supporting evidence of diagnosis (shah and sarode 2013; george 2010). plasma exchange is the sinequanone of treatment in ttp since untreated, this disease is usually fatal (ghanima et al. 2012) . it has been shown to be superior to plasma infusion and can reduce mortality to 20 % or less (rock et al. 1991) . nonetheless, plasma exchange is not without risk. with a death rate that can reach 3 % not to mention fatal arrests, hypotension, catheter-related complications, and venous thrombosis, plasmapheresis should be used with caution (cohen et al. 2000; mcminn et al. 2003) . in plasma exchange non-responders glucocorticoids and other immunosuppressive agents such as vincristine, cyclosporine, cyclophosphamide, and even splenectomy have been tried with varying degrees of success. in addition, a large numbers of initial responders (>30 %) relapsed at a later time (george 2000 (george , 2010 sadler et al. 2004 ). the investigation of rituximab as a potential treatment for ttp started in 2002 after two women with refractory ttp were reported to respond after addition of rituximab to their plasma exchange therapy (chemnitz et al. 2002) . many studies later on observed the same effect of rituximab in treating refractory ttp patients in conjunction with plasmapheresis. a recent review reviewing six studies each of which included five or more patients reported clinical remission in 97 % from a total of 67 patients treated with rituximab given as per lymphoma protocol (stasi 2010) . this result should be interpreted with caution, since due to the rarity of the disease, many of the studies included represented small case series with no adequate controls (sallah et al. 2004; reddy et al. 2005; heidel et al. 2007; ling et al. 2009 ). another review that included 118 patients with either refractory of relapsing ttp treated with rituximab came to a conclusion that 85 % of patients achieved remission and considered rituximab to be a safe and efficient treatment option in this subset of patients (caramazza et al. 2010 ). rituximab has also been shown to be a good first-line treatment in conjunction with plasma exchange. it has been shown to decrease hospital stay by a mean of 7 days in non-icu admitted patients. moreover, it markedly decreased relapse to 10 % versus 56 % in controls (scully et al. 2011 ). rituximab has successfully been used as a preemptive maintenance therapy in patients with recurrent disease and has also been shown to be equally effective in patients with long-standing and recently diagnosed ttp (herbei and venugopal 2006; stasi 2010) . it has been postulated that rituximab acts not only by depleting antibodies against adamts 113 but also by decreasing cytokine production. this stems from the fact that patients with normal adamts 13 levels still respond to the biologic therapy (kameda et al. 2007; reddy et al. 2005) . the regular lymphoma dose of rituximab has been used in most studies however, some have used lower or more numerous dosing regiments with success (newman et al. 2001; kivity and agmon-levin 2011) . furthermore, some authors advise for performing plasma exchange 24 h after rituximab infusion whereas others recommend doing it after 72 h (boctor and smith 2006) . no other b-cell depleting agent has been investigated in ttp, but given the success of rituximab, we should be expecting increasing interest in this field in the near future. evan's syndrome is defined as autoimmune hemolytic anemia coexisting with itp. patients usually suffer from intermittent exacerbations and remissions in their lifetime. its diagnosis is usually confirmed by the direct antiglobulin test which is usually positive (norton and roberts 2006) . corticosteroids were historically found to be the cornerstone of treatment. this poses a challenge to the treating physician especially when considering corticosteroids side effects on the long term owing to the chronicity of the disease. research tackling second-line treatments has been scarce in this field and relied mostly on immunosuppressant such as danazol, mycophenolate mofetil, cyclosporine, or splenectomy (norton and roberts 2006) . we have recently witnessed an emergence of studies employing rituximab as a second-line or even first-line treatment (barcellini and zanella 2011). early reports have shown encouraging results. a response rate of 83 % was first reported which subsequently increased to 94 % in new reports (norton and roberts 2006; barcellini and zanella 2011) . the problem with those numbers however is that they are based on case reports and case series with no adequate controls not to mention the eventual publication bias that predominates such studies. most of the studies deal with relapsed patients with evan's syndrome but dosing has been highly variable between different patients. a recent retrospective study looked at the charts of 11 patients having received rituximab, seven for refractory itp, three for relapsing hemolytic anemia, and one for refractory itp and hemolytic anemia. an encouraging 82 % response rate was achieved with a 64 % long-term response rate after a 1 year mean follow-up ). the antiphospholipid syndrome (aps) is an acquired autoimmune disease characterized by a hypercoagulable state that leads to arterial and/or venous thrombosis, recurrent pregnancy loss, and persistently positive antiphospholipid (apl) antibodies, namely anticardiolipin (acl), lupus anticoagulant (la), and anti-β 2 -glycoprotein i antibodies (anti-β2gpi). the latest classification criteria for diagnosing aps are the 2006 updated sapporo criteria that require the presence of at least one clinical manifestation and one positive laboratory criteria (table 1) (miyakis et al. 2006) . in a small subset of aps patients, the disease can have an accelerated progression resulting in multiorgan failure, called "catastrophic" aps (caps). caps is characterized by multiple organ involvement with histopathologic evidence of small-vessel thrombosis developing over a very short period of time, in the presence of laboratory confirmation of apl antibodies (asherson et al. 2003) . in 2003, hughes and khamashta described another group of patients who present with clinical manifestations highly suggestive of aps but with persistently negative la, acl, and anti-β2gpi antibodies. this group was collectively referred to as seronegative aps (snaps) (hughes and khamashta 2003; nayfe et al. 2013) . taking into consideration the diverse clinical manifestations of aps, it is suggested that more than one pathological process may be involved. despite this fact, the current therapeutic approaches are mostly restricted to anticoagulation therapy, which does not happen to benefit all patients (pierangeli et al. 1995 . moreover, recurrent thrombotic events can occur in up to 30 % of aps patients (gharavi et al. 1999) , and 2-3 % might experience bleeding complications (pierangeli et al. 1996) . the best treatment for these aps patients who are !1 fetal death (at or beyond the 10th week of gestation) !1 premature birth before the 34th week of gestation because of eclampsia, severe preeclampsia or placental insufficiency !3 consecutive (pre) embryonic losses (before the 10th week of gestation) laboratory criteria lupus anticoagulant positivity on !2 occasions at least 12 weeks apart anticardiolipin antibody (igg and/or igm) in medium or high titer (i.e., >40, or above the 99th percentile), on two or more occasions at least 12 weeks apart anti-β 2 -glycoprotein-1-antibody (igg and/or igm) in medium or high titer (i.e., above the 99th percentile) on two or more occasions at least 12 weeks apart definite aps is present if at least one of the clinical criteria and one of the laboratory criteria are met intolerant or resistant to long-term anticoagulation remains unclear. new pathogenic mechanisms in aps are under investigation by ongoing research, including apl-induced activation of platelets, endothelial cells, monocytes, complement and coagulation cascade, leading to the discovery of potential targets and therapies for aps (comarmond and cacoub 2012) . new data indicates a link between high titers of apl antibodies and elevated circulating cd5+ b cells, suggesting that aps may be responsive to b cell targeted therapies (youinou and renaudineau 2004) . the pathogenic mechanisms behind the clinical symptoms of aps are not fully understood, and many factors contribute in the apl-induced manifestations of the disease (comarmond and cacoub 2012). apl antibodies promote thrombus formation in both the venous and arterial circulation, and their thrombogenic properties have been shown in several in vitro and in vivo animal studies to be responsible for the pathogenesis of aps (domenico sebastiani et al. 2003; dagenais et al. 1992; zhou et al. 2011) . nonetheless, how these antibodies are produced and the exact mechanism by which they mediate thrombosis is not fully elucidated. apls comprise a heterogeneous family of autoantibodies; yet, similar hla class alleles are identified to be consistent with aps patients, namely hla-dr4, -dr7, and -drw53 (doring et al. 2010) . apl antibodies bind to their target cells (i.e., monocytes, platelets, endothelial cells, and trophoblasts) through a mediator plasma apolipoprotein called β2gpi, the main autoantigen for apl antibodies. consequently, up-regulation of tissue factor expression on monocytes and endothelial cells takes place leading to thrombosis and fetal loss through a series of signal transduction events (romay-penabad et al. 2007 ). since apls play a central role in the pathogenesis of aps, special interest has been put on b cells, as they are the source of these pathogenic autoantibodies. over the past 10 years, the role of autoreactive b cells in aps and the breakdown of b-cell tolerance have been extensively studied (rand et al. 2008; edwards et al. 1997 ). in addition to antibody production, b cells have other pathogenic mechanisms in aps, such as modifying their b-cell receptor specificity and acting as antigen presenting cells for self-antigens; besides differentiating into b effector cells (be-1 and be-2) which regulate helper t cells and their functions (wallace 1994; khattri et al. 2012 ). because accumulated data support the pathogenic role of b cells in the development and progression of aps, b-cell targeting therapies have been investigated in both human and murine aps. to date, literature review reveals only a limited number of case reports and series published regarding the use of rituximab in the treatment of aps. no randomized clinical trials were retrieved. rituximab is a chimeric (murine/human) monoclonal antibody that targets cd20 on peripheral b cells, depleting them from the circulation and consequently decreasing disease activity (willems et al. 2006; youinou et al. 2009 ). rituximab is fda approved for the treatment of rheumatoid arthritis, b-cell non-hodgkin's lymphoma (higashida et al. 2005) and recently for antineutrophilic cytoplasmic antibody (anca)-associated vasculitis (cohen tervaert 2011) . in addition to these indications, rituximab is being used as off-label treatment in a number of inflammatory and systemic autoimmune diseases (butterly et al. 2010) . no reports on other b-cell directed therapies in aps patients were found. rituximab was used in the treatment of 27 reported cases of aps (primary, secondary, and aps with concomitant malignancies), including 18 females and 9 male patients, whose age ranged from 3 months to 69 years (khattri et al. 2012 ). of the 27 aps patients, four suffered from concomitant lymphomas; two had non-hodgkin's lymphoma for which rituximab was administered as part of the r-chop chemotherapy regimen (veneri et al. 2005; erre et al. 2008) , and two received rituximab for marginal zone lymphoma (manner et al. 2008; harner et al. 2004 ), one of which had sjogren's syndrome also (harner et al. 2004) . five patients had sle with secondary aps, where rituximab was used after failure of treatment with anticoagulation and/or immunosuppression for lupus (cianciulli et al. 2008; weide et al. 2003; tomietto et al. 2004; ahn et al. 2005; anandacoomarasamy et al. 2006) . one patient had evans syndrome (ruckert et al. 2008 ). the rest of the patients had primary aps (iglesias-jimenez et al. 2010; nageswara rao et al. 2009; tsagalis et al. 2010; adamson et al. 2008; van wissen et al. 2008; chalam et al. 2007; ames et al. 2007; rubenstein et al. 2006; trappe et al. 2006; binstadt et al. 2003; erdozain et al. 2004; asherson et al. 2008; sciascia et al. 2011) . table 2 summarizes the clinical and serological manifestations of the reported aps patients along with their outcomes after treatment with rituximab (khattri et al. 2012) . the rituximab dosing regimen used in the majority of the cases was 375 mg/m 2 body surface area, given weekly for 4 weeks. four patients received rituximab 1,000 mg given 15 days apart. the reported patients were not treatment naïve prior to rituximab administration; most received anticoagulation unless otherwise contraindicated, eight patients were treated with cyclophosphamide earlier (ahn et al. 2005; anandacoomarasamy et al. 2006; iglesias-jimenez et al. 2010; ames et al. 2007; rubenstein et al. 2006; binstadt et al. 2003; asherson et al. 2008) , and all but one case (tsagalis et al. 2010 ) used corticosteroids. the treatment regimen contained other immunosuppressants too, including azathiprine, mycophenolate mofetil, dapsone, and cyclosporine (khattri et al. 2012) . improvement in the serological markers of aps was noticed in the majority of the cases, where decrease or normalization of la, acl, and anti-β2gpi antibodies was seen. furthermore, multiple systemic clinical manifestations associated with aps improved after starting rituximab regardless whether the patient suffered from primary or secondary aps. there was a uniformly good response to rituximab in primary aps and in aps associated with sle (weide et al. 2003; cianciulli et al. 2008; tomietto et al. 2004; ahn et al. 2005; anandacoomarasamy et al. 2006; ruckert et al. 2008; tsagalis et al. 2010; adamson et al. 2008; chalam et al. 2007; ames et al. 2007; rubenstein et al. 2006; trappe et al. 2006; binstadt et al. 2003; erdozain et al. 2004; sciascia et al. 2011; vianna et al. 1994; danowski et al. 2009 ) or lymphoma (erre et al. 2008; veneri et al. 2005; manner et al. 2008; harner et al. 2004) in the case reports reviewed. moreover, six out of seven caps patients benefited from rituximab treatment, knowing the severity and frequent fatality faced in this entity in spite of standard treatment with anticoagulants and immunosuppressant agents (manner et al. 2008; iglesias-jimenez et al. 2010; nageswara rao et al. 2009; van wissen et al. 2008; rubenstein et al. 2006; asherson et al. 2008) . in the biogeas registry, a multicenter, national registry in spain, rituximab was shown to have beneficial therapeutic effects in aps, with 92 % response rate in 12 aps patients (ramos-casals et al. 2008) . despite the promising data from case reports and the biogeas registry on the beneficial effect of rituximab in aps patients, the literature is still limited on this topic as there are no clinical trial data available yet. moreover, the abovementioned published case reports have several limitations including their small number, besides the fact that in all the cases, other immunosuppressants were used including steroids and cyclophosphamide, which creates confusion on whether it was rituximab-induced b-cell depletion by itself or the combination of immunosuppressants used that caused improvement in aps patients. also, it is worth mentioning that the treated population was diverse including patients with primary or secondary aps or malignancy (khattri et al. 2012) . finally, in a pilot open-label phase ii trial aimed primarily to evaluate the safety of rituximab in apl-positive patients with non-criteria manifestations of aps, and secondarily to evaluate the effect on the apl profile and efficacy of treatment, it was suggested that rituximab may be effective in controlling some but not all non-criteria manifestations of aps with a safety profile in apl-positive patients consistent with that of rituximab (erkan et al. 2013) . (manner et al. 2008; iglesias-jimenez et al. 2010; van wissen et al. 2008; rubenstein et al. 2006; asherson et al. 2008; nageswara rao et al. 2009) acl, la, anti-β2gpi acl, la, anti-β2gpi normalized all patients improved except for one death due to complications (sepsis, subdural hematoma) (asherson et al. 2008) and one had minor selflimiting episodes associated with thrombocytopenia (asherson et al. 2008 ) primary aps (ruckert et al. 2008; tsagalis et al. 2010; chalam et al. 2007; ames et al. 2007; rubenstein et al. 2006; trappe et al. 2006; erdozain et al. 2004; sciascia et al. 2011; binstadt et al. 2003; adamson et al. 2008) acl, la, anti-β2gpi acl, la, anti-β2gpi normalized improvement in thrombocytopenia, no further thrombotic events (continued) rituximab, a chimeric anti-cd20 monoclonal antibody, is fda approved for the treatment of rheumatoid arthritis, b-cell non-hodgkin's lymphoma and ancaassociated vasculitis. the off-label use of b-cell depleting agents in several systemic autoimmune diseases has been studied. data on the use of rituximab in the treatment of aps is limited to case reports, the biogeas registry and a pilot openlabel phase ii trial, and suggests a beneficial role in the therapeutic approach of aps. however, well-designed randomized clinical trials are needed to evaluate the use of rituximab, alone or in combination with other immunosuppressants, in improving the clinical and serological manifestations of the disease. autoimmune hemolytic anemia (aiha) is an uncommon disorder characterized by autoantibodies directed against self red blood cells (rbcs) (gehrs and friedberg 2002) . consequently, the normal 100-120 days lifetime of the rbcs is reduced to just a few days in serious cases (sawitsky and ozaeta 1970) . aiha can be idiopathic or secondary to infections, other autoimmune conditions or lymphoproliferative disorders, and depending on the thermal range of the autoantibodies involved, the disease can be classified into warm, cold (which includes cold agglutinin disease and paroxysmal nocturnal hemoglobinuria) or mixed (gehrs and friedberg 2002). whether warm-or cold-type secondary aiha, each can result from its own more common secondary causes. for instance, secondary warmtype aiha mostly results from lymphoproliferative disorders (e.g., chronic lymphocytic leukemia, lymphoma) and other autoimmune disorders, including sle, ra, scleroderma, and ulcerative colitis. less commonly, it can be caused by neoplasms other than lymphoid and infection. similarly, secondary cold-type aiha is primarily caused by lymphoproliferative disorders, but also occurs secondary to infection, especially by mycoplasma, viral pneumonia, infectious mononucleosis, and other respiratory infections, and infrequently due to concomitant autoimmune disorders (sokol et al. 1981 ). 6.2 treatment of aiha in warm aiha, the first-line therapy has been the administration of corticosteroids, where the response rate reaches 70-85 %, of which only one third remain in longterm remission after drug discontinuation, 50 % necessitate maintenance doses, and around 20-30 % require second-line therapies including immunosuppressants and splenectomy (wahl et al. 2008) . splenectomy is probably the most effective second-line treatment with a response rate of 50 %, especially in relapsing patients on corticosteroids or those requiring the equivalent of 10-15 mg prednisone per day to maintain adequate hemoglobin levels. we have to bear in mind the surgical and infective complications, particularly gram-negative sepsis in patients above 65 (gehrs and friedberg 2002; valent and lechner 2008; newland et al. 2005) . moreover, patients who are unresponsive to or do not fit for splenectomy, have limited options including cytotoxic or immunosuppressive medications such as azathioprine, cyclophosphamide, or cyclosporine, with a response rate of 40-60 % and associated side effects (52). on the other hand, cold-type aiha has failed to demonstrate a convincing response to standard therapy, particularly cold hemaglutinin disease (barcellini and zanella 2011). in a recent review carried by garvey et al. collecting data from studies using rituximab in the treatment of aiha, rituximab (375 mg/m 2 given weekly for 4 weeks) was found to be effective in treating both warm aiha and cold hemaglutinin disease, with a median response rate of 60 % and lasting responses for more than 3 years (garvey 2008) . furthermore, in three more recent studies (dierickx et al. 2009; bussone et al. 2009; penalver et al. 2010) , the response rate was higher and ranged from 77 to 93 % with a disease-free survival at 1 and 2 years in 72 and 56 % of cases, respectively (dierickx et al. 2009 ). in several case series, rituximab has been shown to be effective in both patient groups with idiopathic and secondary aiha, even in those associated with autoimmune and lymphoproliferative disorders, and bone marrow transplant (shanafelt et al. 2003; penalver et al. 2010; quartier et al. 2001; zecca et al. 2003; narat et al. 2005; berentsen et al. 2006; schollkopf et al. 2006; d'arena et al. 2007; gupta et al. 2002; trape et al. 2003; d'arena et al. 2006) . moreover, rituximab proved to be effective whether used as a monotherapy or in combination with corticosteroids, immunosuppressants and interferon (zecca et al. 2003; narat et al. 2005; berentsen et al. 2006; d'arena et al. 2007; gupta et al. 2002) , and irrespective or prior therapy (penalver et al. 2010; shanafelt et al. 2003; quartier et al. 2001; zecca et al. 2003; narat et al. 2005; berentsen et al. 2006; schollkopf et al. 2006; gupta et al. 2002; trape et al. 2003; d'arena et al. 2006 ). time to maximum response varied between the studies from quick response to weeks or even months, where in two recent studies, the median time to response was 3 and 6 weeks, respectively (bussone et al. 2009; penalver et al. 2010) . it is noteworthy that re-treatment with rituximab is effective with both warm aiha and cold hemaglutinin disease (rao et al. 2008; zecca et al. 2003; berentsen et al. 2006; gupta et al. 2002) , with re-treatment benefiting some patients more than once (zecca et al. 2003; berentsen et al. 2006; penalver et al. 2010) . when comparing rituximab to the next best therapeutic regimen which includes alkylating agents with or without corticosteroids, rituximab was the only treatment able to induce a complete response in cold hemaglutinin disease with a response of 60 % (10 % complete response and 50 % partial response), compared with 16 % (all partial responses) (berentsen et al. 2006; berentsen et al. 2004; barcellini and zanella 2011) . regarding rituximab safety when given to patients with aiha, the drug was well tolerated and no adverse events were reported in most cases, besides mild to moderate infusion-related side effects (e.g., fever, chills, hypotension, and upper airway edema) (rao et al. 2008; zecca et al. 2003; schollkopf et al. 2006; gupta et al. 2002; trape et al. 2003) . some patients (around 7 %) experienced possible rituximab-related infections (quartier et al. 2001; zecca et al. 2003; narat et al. 2005; trape et al. 2003; schollkopf et al. 2006) , and few (roughly 2 %) had grade 4 neutropenia (berentsen et al. 2006; gupta et al. 2002) . moreover, low dose rituximab (100 mg â 4 weeks) was tried in patients unresponsive to standard therapy in an attempt to reduce side effects and costs, and it was found effective as a monotherapy (provan et al. 2007) and in combination with alemtuzumab (gomez-almaguer et al. 2010) . in a study conducted by barcellini et al, low dose rituximab along with standard oral prednisone was able to induce an overall response rate of 86 % (complete 67 % and partial 19 %) in 21 patients with warm and cold aiha; response was sustained (hb >10 g/ dl) in 13/14 patients at 6 months and in 11/11 evaluable patients at 1 year (barcellini et al. 2010). rituximab proved to be an effective therapeutic alternative to standard therapy of splenectomy and/or chemotherapy in patients with primary or secondary warm aiha with a significant response rate and sustained remissions. furthermore, rituximab induced durable responses in patients with cold hemaglutinin disease, once a disease with very limited therapeutic options (barcellini and zanella 2011) . clinical improvement in antiphospholipid syndrome after rituximab therapy long-term remission from life-threatening hypercoagulable state associated with lupus anticoagulant (la) following rituximab therapy limited effect of rituximab on thrombocytopaenia and anticardiolipin antibodies in a patient with primary antiphospholipid syndrome cure' of life-threatening antiphospholipid syndrome with rituximab positioning new treatments in the management of immune thrombocytopenia systematic review: efficacy and safety of rituximab for adults with idiopathic thrombocytopenic purpura a pilot randomized trial of adjuvant rituximab or placebo for nonsplenectomized patients with immune thrombocytopenia catastrophic antiphospholipid syndrome: international consensus statement on classification criteria and treatment guidelines relapsing catastrophic antiphospholipid syndrome: report of three cases rituximab before splenectomy in adults with primary idiopathic thrombocytopenic purpura: a meta-analysis rituximab therapy for autoimmune haematological diseases low-dose rituximab in idiopathic autoimmune haemolytic anaemia rituximab for primary chronic cold agglutinin disease: a prospective study of 37 courses of therapy in 27 patients primary chronic cold agglutinin disease: a population based clinical study of 86 patients rituximab therapy for multisystem autoimmune diseases in pediatric patients timing of plasma exchange and rituximab for the treatment of thrombotic thrombocytopenic purpura efficacy and safety of rituximab in adults' warm antibody autoimmune haemolytic anemia: retrospective analysis of 27 cases off-label use of rituximab in a tertiary queensland hospital relapsing or refractory idiopathic thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: the role of rituximab rituximab effectively reverses papilledema associated with cerebral venous sinus thrombosis in antiphospholipid antibody syndrome successful treatment of severe thrombotic thrombocytopenic purpura with the monoclonal antibody rituximab initial treatment of immune thrombocytopenic purpura with high-dose dexamethasone right atrial thrombus mimicking myxoma with pulmonary embolism in a patient with systemic lupus erythematosus and secondary antiphospholipid syndrome the itp syndrome: pathogenic and clinical diversity the balance between von-willebrand factor and its cleaving protease adamts13: biomarker in systemic inflammation and development of organ failure? rituximab in anca-associated vasculitis: a revolution? the bleeding risk and natural history of idiopathic thrombocytopenic purpura in patients with persistent low platelet counts antiphospholipid syndrome: from pathogenesis to novel immunomodulatory therapies rituximab for warm-type idiopathic autoimmune hemolytic anemia: a retrospective study of 11 adult patients rituximab therapy for chronic lymphocytic leukemia-associated autoimmune hemolytic anemia a family study of the antiphospholipid syndrome associated with other autoimmune diseases determinants of risk for venous and arterial thrombosis in primary antiphospholipid syndrome and in antiphospholipid syndrome with systemic lupus erythematosus idiopathic thrombocytopenic purpura in elderly patients: a study of 47 cases from a single reference center rituximab in auto-immune haemolytic anaemia and immune thrombocytopenic purpura: a belgian retrospective multicentric study hla class ii alleles and genetic predisposition to the antiphospholipid syndrome human antiphospholipid antibodies induce tnfalpha in monocytes via toll-like receptor 8 hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice sustained response to rituximab of autoimmune hemolytic anemia associated with antiphospholipid syndrome a pilot open-label phase ii trial of rituximab for non-criteria manifestations of antiphospholipid syndrome effect of rituximab on clinical and laboratory features of antiphospholipid syndrome: a case report and a review of literature unrecognized pattern of von willebrand factor abnormalities in hemolytic uremic syndrome and thrombotic thrombocytopenic purpura rituximab in the treatment of autoimmune haematological disorders autoimmune hemolytic anemia how i treat patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome how i treat patients with thrombotic thrombocytopenic purpura bussel jb (2012) how i treat immune thrombocytopenia: the choice between splenectomy or a medical therapy as a second-line treatment gdkvinduced antiphospholipid antibodies enhance thrombosis and activate endothelial cells in vivo and in vitro intravenous immunoglobulin for adults with autoimmune thrombocytopenic purpura: results of a randomized trial comparing 0.5 and 1 g/kg b.w rituximab efficacy and safety in adult splenectomy candidates with chronic immune thrombocytopenic purpura: results of a prospective multicenter phase 2 study low-dose rituximab and alemtuzumab combination therapy for patients with steroid-refractory autoimmune cytopenias rituximab-based chemotherapy for steroid-refractory autoimmune hemolytic anemia of chronic lymphocytic leukemia normalization of anticardiolipin antibodies following rituximab therapy for marginal zone lymphoma in a patient with sjogren's syndrome addition of rituximab to standard therapy improves response rate and progression-free survival in relapsed or refractory thrombotic thrombocytopenic purpura and autoimmune haemolytic anaemia recurrent thrombotic thrombocytopenic purpura treated repeatedly and successfully with the monoclonal antibody rituximab safety and efficacy of rituximab in patients with rheumatoid arthritis refractory to disease modifying antirheumatic drugs and anti-tumor necrosis factor-alpha treatment seronegative antiphospholipid syndrome infant with probable catastrophic antiphospholipid syndrome successfully managed with rituximab two cases of refractory thrombotic thrombocytopenic purpura associated with collagen vascular disease were significantly improved by rituximab treatment b-cell directed therapies in antiphospholipid antibody syndrome-new directions based on murine and human data rituximab for thrombotic thrombocytopenic purpura survival and relapse in patients with thrombotic thrombocytopenic purpura chronic idiopathic thrombocytopenic purpura in adult chinese patients: a retrospective single-centered analysis of 1791 cases rituximab for children with immune thrombocytopenia: a systematic review sustained response with rituximab in patients with thrombotic thrombocytopenic purpura: a report of 13 cases and review of the literature successful treatment of catastrophic antiphospholipid antibody syndrome (caps) associated with splenic marginal-zone lymphoma with low-molecular weight heparin, rituximab and bendamustine complications of plasma exchange in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: a study of 78 additional patients the spectrum of evans syndrome in adults: new insight into the disease based on the analysis of 68 cases international consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (aps) an acute febrile pleiochromic anemia with hyaline thrombosis of the terminal arterioles and capillaries: an undescribed disease rituximab for successful management of probable pediatric catastrophic antiphospholipid syndrome rituximab in the treatment of refractory autoimmune cytopenias in adults seronegative antiphospholipid syndrome the american society of hematology 2011 evidence-based practice guideline for immune thrombocytopenia preventing severe infection after splenectomy a dose of 75 microg/kg/d of i.v. anti-d increases the platelet count more rapidly and for a longer period of time than 50 microg/kg/d in adults with immune thrombocytopenic purpura clinically significant newly presenting autoimmune thrombocytopenic purpura in adults: a prospective study of a population-based cohort of 245 patients management of evans syndrome pathogenesis of chronic immune thrombocytopenia: increased platelet destruction and/or decreased platelet production rituximab is an effective and safe therapeutic alternative in adults with refractory and severe autoimmune hemolytic anemia antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo thrombogenic properties of murine anti-cardiolipin antibodies induced by beta 2 glycoprotein 1 and human immunoglobulin g antiphospholipid antibodies induction of thrombosis in a mouse model by igg, igm and iga immunoglobulins from patients with the antiphospholipid syndrome activity and safety profile of low-dose rituximab for the treatment of autoimmune cytopenias in adults international consensus report on the investigation and management of primary immune thrombocytopenia treatment of childhood autoimmune haemolytic anaemia with rituximab a systematic review of the off-label use of biological therapies in systemic autoimmune diseases hydroxychloroquine directly reduces the binding of antiphospholipid antibody-beta2-glycoprotein i complexes to phospholipid bilayers safety, efficacy, and immune reconstitution after rituximab therapy in pediatric patients with chronic or refractory hematologic autoimmune cytopenias rituximab in the treatment of relapsed thrombotic thrombocytopenic purpura thrombotic thrombocytopenic purpura: a review of the literature in the light of our experience with plasma exchange comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenic purpura. canadian apheresis study group c5a receptor-deficient mice are protected from thrombophilia and endothelial cell activation induced by some antiphospholipid antibodies rituximab treatment for resistant antiphospholipid syndrome successful treatment of life-threatening evans syndrome due to antiphospholipid antibody syndrome by rituximab-based regimen: a case with long-term follow-up recent advances in thrombotic thrombocytopenic purpura the course of severe autoimmune thrombocytopenia in patients not undergoing splenectomy a pilot study of the anti-cd20 monoclonal antibody rituximab in patients with refractory immune thrombocytopenia rituximab in patients with refractory thrombotic thrombocytopenic purpura disease-associated autoimmune hemolytic anemia rituximab in chronic cold agglutinin disease: a prospective study of 20 patients treatment-induced downregulation of antiphospholipid antibodies: effect of rituximab alone on clinical and laboratory features of antiphospholipid syndrome a phase 2 study of the safety and efficacy of rituximab with plasma exchange in acute acquired thrombotic thrombocytopenic purpura thrombotic thrombocytopenic purpura-what is new? rituximab for immune cytopenia in adults: idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, and evans syndrome autoimmune haemolysis: an 18-year study of 865 cases referred to a regional transfusion centre rituximab in autoimmune hematologic diseases: not just a matter of b cells rituximab chimeric anti-cd20 monoclonal antibody treatment for adults with chronic idiopathic thrombocytopenic purpura long-term observation of 208 adults with chronic idiopathic thrombocytopenic purpura immune thrombocytopenia in adults: an update b cell depletion may lead to normalization of anti-platelet, anti-erythrocyte and antiphospholipid antibodies in systemic lupus erythematosus rituximab chimeric anti-cd20 monoclonal antibody treatment for refractory hemolytic anemia in patients with lymphoproliferative disorders successful treatment of thrombocytopenia in primary antiphospholipid antibody syndrome with the anti-cd20 antibody rituximab-monitoring of antiphospholipid and anti-gp antibodies: a case report effective treatment of antiphospholipid syndrome with plasmapheresis and rituximab diagnosis and treatment of autoimmune haemolytic anaemias in adults: a clinical review catastrophic antiphospholipid syndrome mimicking a malignant pancreatic tumour-a case report remission of severe antiphospholipid syndrome associated with non-hodgkin's b-cell lymphoma after combined treatment with rituximab and chemotherapy adamts13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients comparison of the primary and secondary antiphospholipid syndrome: a european multicenter study of 114 patients need for additional trials of primary prophylaxis in patients with high-risk antiphospholipid antibody profiles: comment on the article by erkan et al antimalarial agents and lupus successful long-term treatment of systemic lupus erythematosus with rituximab maintenance therapy rituximab therapy for childhood-onset systemic lupus erythematosus the antiphospholipid syndrome as a model for b cell-induced autoimmune diseases b lymphocyte cytokines and rheumatic autoimmune disease dexamethasone plus rituximab yields higher sustained response rates than dexamethasone monotherapy in adults with primary immune thrombocytopenia rituximab for the treatment of refractory autoimmune hemolytic anemia in children structure of von willebrand factor-cleaving protease (adamts13), a metalloprotease involved in thrombotic thrombocytopenic purpura toll-like receptor (tlr)-4 mediates anti-beta2gpi/beta2gpi-induced tissue factor expression in thp-1 cells current management of adult idiopathic thrombocytopenic purpura in practice: a cohort study of 201 patients from a single center key: cord-0030225-wddgamb0 authors: han, maozhen; zhang, na; mao, yujie; huang, bingbing; ren, mengfei; peng, zhangjie; bai, zipeng; chen, long; liu, yan; wang, shanshan; huang, shenghai; cheng, zhixiang title: the potential of gut microbiota metabolic capability to detect drug response in rheumatoid arthritis patients date: 2022-04-08 journal: front microbiol doi: 10.3389/fmicb.2022.839015 sha: ffa9603d37aa6b4943dc804cf7956e82fdfbb786 doc_id: 30225 cord_uid: wddgamb0 gut microbiota plays an essential role in the development of rheumatoid arthritis (ra) and affects drug responses. however, the underlying mechanism remains elusive and urgent to elucidate to explore the pathology and clinical treatment of ra. therefore, we selected methotrexate (mtx) as an example of ra drugs to explore the interactions between the gut microbiota and drug responses and obtain an in-depth understanding of their correlation from the perspective of the metabolic capability of gut microbiota on drug metabolism. we identified 2,654 proteins and the corresponding genes involved in mtx metabolism and then profiled their abundances in the gut microbiome datasets of four cohorts. we found that the gut microbiota harbored various genes involved in mtx metabolism in healthy individuals and ra patients. interestingly, the number of genes involved in mtx metabolism was not significantly different between response (r) and non-response (nr) groups to mtx, but the gene composition in the microbial communities significantly differed between these two groups. particularly, several models were built based on clinical information, as well as data on the gene, taxonomical, and functional biomarkers by using the random forest algorithm and then validated. our findings provide bases for clinical management not only of ra but also other gut microbiome–related diseases. first, it suggests that the potential metabolic capability of gut microbiota on drug metabolism is important because they affect drug efficiency; as such, clinical treatment strategies should incorporate the gene compositions of gut microbial communities, in particular genes involved in drug metabolism. second, a suitable model can be developed to determine hosts’ responses to drugs before clinical treatment. gut microbiota plays an essential role in the development of rheumatoid arthritis (ra) and affects drug responses. however, the underlying mechanism remains elusive and urgent to elucidate to explore the pathology and clinical treatment of ra. therefore, we selected methotrexate (mtx) as an example of ra drugs to explore the interactions between the gut microbiota and drug responses and obtain an in-depth understanding of their correlation from the perspective of the metabolic capability of gut microbiota on drug metabolism. we identified 2,654 proteins and the corresponding genes involved in mtx metabolism and then profiled their abundances in the gut microbiome datasets of four cohorts. we found that the gut microbiota harbored various genes involved in mtx metabolism in healthy individuals and ra patients. interestingly, the number of genes involved in mtx metabolism was not significantly different between response (r) and non-response (nr) groups to mtx, but the gene composition in the microbial communities significantly differed between these two groups. particularly, several models were built based on clinical information, as well as data on the gene, taxonomical, and functional biomarkers by using the random forest algorithm and then validated. our findings provide bases for clinical management not only of ra but also other gut microbiome-related diseases. first, it suggests that the potential metabolic capability of gut microbiota on drug metabolism is important because they affect drug efficiency; as such, clinical treatment strategies should incorporate the gene compositions of gut microbial communities, in particular genes involved in drug metabolism. second, a suitable model can be developed to determine hosts' responses to drugs before clinical treatment. the human microbial community consists of more than 100 trillion microbes, most of which are unculturable and mainly colonize the intestinal tract (manzo and bhatt, 2015) . these microbes evolved with the hosts and become an inalienable part of the hosts (glasner, 2017) . increasing lines of evidence have demonstrated that the human gut microbiota is associated with human health and plays an essential role in maintaining the homeostasis and health of hosts. for instance, numerous studies have proven that human gut microbiota is involved in the metabolism and immune system development of hosts; it also contributes to host physiology and is associated with the development of inflammatory diseases, such as rheumatoid arthritis (ra) (manasson et al., 2020) , juvenile idiopathic arthritis (tejesvi et al., 2016) , systemic lupus erythematosus (chen et al., 2021) , crohn disease, ulcerative colitis, and irritable bowel syndrome (wu and wu, 2012; carding et al., 2015) , and other diseases, such as hypertension (li et al., 2017) , cholestatic liver disease (isaacs-ten et al., 2020) , and cancers (amy et al., 2020; slowicka et al., 2020) . however, although many studies investigated the pathology of diseases and established the correlation between the dynamic changes of human gut microbiota and diseases (bäckhed et al., 2015; ghaisas et al., 2016; gaulke and sharpton, 2018) , the correlation analysis was conducted only in a certain cohort and the causality of the disease remains elusive and urgent to elucidate using a large of metagenome datasets. besides, databases of reference genes and proteins that are associated with a certain disease, particularly those involved in drug metabolism, are needed for rapid analysis of metagenome datasets. rheumatoid arthritis is an autoimmune disease that has chronic inflammation symptoms and disorders. in general, ra can cause joint pain, swelling, and stiffness, as well as damages throughout the body, not only on the skin, eyes, lungs, and heart (majithia and geraci, 2007; bullock et al., 2018) . to date, although the pathology of ra remains not fully understood, many studies have reported that the occurrence and development of ra are associated with genetic and environmental factors (majithia and geraci, 2007; aletaha and smolen, 2018) , including oral and gut microbiota (maeda and takeda, 2017) , smoking, age, body mass index, and so on (rydell et al., 2018) . the experiment results in germ-free mice revealed that the gut microbiota can shape the intestinal immune system and has a strong positive association with the diseases of the immune system (round and mazmanian, 2009; torres et al., 2020) , including ra. the dysbiosis of human gut microbial communities can cause disorders of the immune system in ra patients; numerous studies have also reported the dynamic changes of oral and gut microbiome between ra patients and healthy controls (zhang et al., 2015; horta-baas et al., 2017; brown et al., 2020) . among gut microorganisms, prevotella copri was significantly dominant in ra patients and strongly correlated with the absence of human leukocyte antigen-drb1 (scher et al., 2013) . besides, the hidden links in the gut-joint axis for ra have been demonstrated, and the ascorbate degradation of gut microbes contributes to the development of ra has been proposed . this finding provides new avenues for the prevention and treatment of ra (cassotta et al., 2021) . together, these results suggest that the gut microbiota plays an important role in the occurrence and development of ra; hence, the clinical treatment of ra should consider gut microbiome approaches. at present, although the pathology of ra remains unknown, many drugs have been developed and applied to its clinical treatment; these drugs include methotrexate (mtx), ibuprofen, naproxen, prednisolone, sulfasalazine, leflunomide, and other disease-modifying antirheumatic drugs (dmards) (aletaha and smolen, 2018) . mtx, as one of the dmards, has been recommended as the first-line drug for the clinical treatment of ra among several treatment guidelines (bello et al., 2017) . however, mtx is insufficient to control the symptoms of patients regardless of the mode of delivery, and up to 50% of patients do not obtain a clinically effective outcome (bello et al., 2017; yamakawa et al., 2021) . the phenomenon of different responses for the same drug in ra patients could be due to variations in their gut microbiome (zhang et al., 2015; kishikawa et al., 2020; artacho et al., 2021) . however, although several studies focused on responses to drugs, and the dynamic changes of the gut microbiome have been conducted, the underlying mechanism remains elusive, and the potential metabolic capability of the gut microbiota on drug metabolism is unclear. hence, together with several recent studies (zhang et al., 2015; kishikawa et al., 2020; artacho et al., 2021) , there is no doubt that the gut microbiota plays an essential role in the occurrence and development of ra, and the drug responses in ra patients depend on the gut microbiome of the host. however, the detailed mechanism, particularly the potential metabolic capability of the gut microbiome, remains elusive and urgent to elucidate to explore ra pathology and develop a microbiome-based approach for detecting hosts' responses to drugs before clinical treatment of ra. in this present work, we collected the gut metagenome datasets related to ra and the corresponding clinical information of the host. only the bioproject prjna682730 has the information on drug response. considering the clinical application of mtx, we selected it as an example of ra drugs to explore the relationship between gut microbiota and drug response. specifically, we collected the gut metagenome datasets of healthy individuals and ra patients from four cohorts. we crossassembled the gut metagenome datasets of the training dataset of prjna682730, identified the proteins and genes involved in mtx metabolism, calculated their abundance and presence in the gut microbiome datasets of the four cohorts, constructed several machine models with the random forest algorithm, and verified the models in the testing data of prjna682730. our findings suggest that the potential metabolic capability of gut microbiota evaluated by the composition of the genes involved in drug metabolism affected the efficiency of drugs and can be applied to detect the drug response of ra patients, which elicits effects on clinical management of gut microbiomerelated diseases. several clinical drugs, including mtx, leflunomide, sulfasalazine, ibuprofen, naproxen, and other dmards (aletaha and smolen, 2018) , have been widely applied to the clinical treatment of ra. although mtx, a famous clinical dmard, has been recommended as the first-line drug for the clinical treatment of ra among several treatment guidelines, more than 50% of ra patients who received it do not achieve a clinically effective outcome. the reason and detailed mechanism remain unclear but may be dependent on the gut microbiome. hence, we selected mtx, which is a typical and preferred drug in the clinical treatment of ra, to obtain an in-depth understanding of the interactions between the gut microbiota and drug response and reveal the potential metabolic capability of gut microbiota on drug metabolism in ra treatment. specifically, we collected proteins that used mtx as a ligand and were involved in its metabolism from the pdb database (length > 50 amino acid residues). and then we extracted the protein sequences from the nr databases and evaluated the protein database by using diamond (similarity > 90% and query coverage > 90%; figure 1a ). finally, we collected 10,756 protein sequences and used them to construct the database for downstream analysis of mtx ( figure 1a ). protein databases for other drugs were also built (figure 1) . these databases can be obtained and downloaded from github. 1 in this present study, we collected the gut metagenome datasets from four human cohorts to explore the interactions between the gut microbiota and drug response of the host from the perspective of the potential metabolic capability of gut microbiota on drug metabolism. (artacho et al., 2021) ; prjeb6997 (healthy chinese and chinese ra patients who underwent pretreatment and posttreatment, 232 fecal samples, the group information is unclear) (zhang et al., 2015) ; prjna356102 (ra patients and healthy individuals, 168 fecal samples); and the healthy individuals of prjeb13870 (41 fecal samples, these samples were used as control; figure 1b ) (li et al., 2017) . thus, a total of 533 human gut metagenome datasets were collected to profile taxonomical and functional compositions, and the abundances of potential genes involved in mtx metabolism of gut microbial communities. particularly, the gut metagenome datasets of the training and testing data of the bioproject prjnz682730 were used to investigate differences between the r and nr groups, construct, and verify the models, respectively. quality control was first conducted on the human gut metagenome datasets collected from the four cohorts against the human genome (version hg38) by using bowtie2 to remove the contaminations of humans. then, the quality of the metagenome datasets was controlled with trimmomatic (version 0.32) (bolger et al., 2014) with the following parameters: truseq3-pe.fa: 2:30:10, leading: 3, trailing: 3, sliding window: 5:20, and min length: 25, to remove adaptors and filter the low-quality sequences. the high-quality sequences were used for downstream analysis. to evaluate the potential metabolic capability of gut microbiota on mtx metabolism across the four cohorts, we selected the metagenome datasets of the training data in the bioproject prjnz682730 and cross-assembled the datasets of 49 samples ( figure 1b and supplementary table 1 ). de novo metagenome assembly was performed with megahit (version 1.2.9) (li et al., 2016) , with option -meta-large and with a k-mer list of 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127 . contigs larger than 1,000 bp were kept for further analysis. prodigal (version 2.6.2) in the "meta" model was applied to detect the complete open reading frames and proteins in the assembled contigs (hyatt et al., 2010) . diamond was applied to identify the protein involved in mtx metabolism with the following parameters: similarity >30% based on the threshold of homology modeling and query coverage >70% (kurella and gali, 2014) . thus, we identified 2,654 proteins that potentially involved in mtx metabolism. subsequently, we obtained the corresponding genes and the assembled contigs carrying these genes. the abundance of each gene was calculated according to the equation of reads per kilobase per million mapped reads and then transformed to relative abundance. the compositions of genes involved in mtx metabolism were calculated for all samples. the presence of the 2,654 genes was investigated in the gut metagenome datasets of the four cohorts based on the abundance of genes. in addition, the taxonomy of the assembled contigs carrying genes involved in mtx metabolism was annotated with contig annotation tool (cat) 2 (von meijenfeldt et al., 2019) . besides, the functions of genes attached to the assembled contigs were obtained by annotating the corresponding proteins against the nr database with diamond (e value < 1e-5), figure 1 | construction of protein databases for clinical drugs in rheumatoid arthritis (ra) treatment and the human gut metagenome datasets used in the present study. (a) construction of databases for detection of proteins and the corresponding genes involved in drug metabolism in the gut microbial communities of ra patients. (b) gut metagenome datasets used in this study were collected from four cohorts, namely, bioproject prjna682730, prjeb6997, prjna356102, and prjeb13870 (only the dataset of healthy individuals was used). and the top annotation was selected as the best functional hit for each gene. to profile the taxonomical and functional compositions of gut microbial communities in the training and testing data of bioproject prjnz682730 (supplementary table 1) , metaphlan3 and humann3 were applied to obtain the taxonomical composition at phylum, class, order, family, genus, and species levels, and functional composition with kegg level 2 of gut microbial communities with default settings (beghini et al., 2021) , respectively. high-quality paired-end reads were combined into a single document and used as an input for humann3. linear discriminant analysis (lda) effect size (lefse, version 1.0) (segata et al., 2011) was applied to identify taxonomical and functional biomarkers and biomarkers of genes involved in mtx metabolism between the r and nr groups based on their composition in the training data of prjnz682730. the p-value for the kruskal-wallis test among groups was set at 0.05. the thresholds for the logarithmic lda score for different gene, taxonomical, and functional features were set at 3.0, 2.5, and 3.0, respectively. statistical analysis was conducted on the r platform. based on the bray-curtis distance matrix, the difference in the profiles of genes involved in mtx metabolism between the r and nr groups was estimated. lda, a supervised learning approach, was applied to maximize the separation of r and nr groups. particularly, random forest models using 10-fold cross-valuation were built with the "caret" package in r based on the training data and verified on the testing data of prjnz682730. the accuracy and area under the curve (auc) values of the models built using different strategies were calculated and compared (han et al., 2020a) . after the cross-assembly of the 49 metagenome datasets collected from the training data of the bioproject prjnz682730, 552,518 contigs were obtained, and 1,642,925 complete genes/proteins were predicted. based on the constructed protein database involved in mtx metabolism, we identified 2,654 proteins and the corresponding genes from the gut microbial communities of r and nr samples. we found that the gut microbiota harbored various genes involved in mtx metabolism in healthy individuals and ra patients (figures 2a,b) . interestingly, we found that although the number of genes involved in mtx metabolism was not significantly different between the r and nr groups (p > 0.05, wilcoxon test; figure 2b ), the compositions of the genes in the microbial communities of the r and nr groups were significantly different (figures 2c,d; p < 0.05, bray-curtis dissimilarity). particularly, the result of lda showed the distribution of the samples had a distinct separation between the r and nr groups ( figure 2e) . this finding indicates that the compositions of genes involved in mtx metabolism of the gut microbial community are differed and suggests that the potential metabolic capability of gut microbiota on mtx metabolism is associated with the host's response to mtx. to distinguish the samples between the r and nr groups, lefse was applied to identify the gene biomarkers involved in mtx metabolism, taxonomical biomarkers, and functional biomarkers between the r and nr groups. specifically, a total of 25 genes (figure 3a) , including the dihydrofolate reductase gene and methylenetetrahydrofolate dehydrogenase gene, were identified as gene biomarkers. we found that more gene biomarkers were identified in the r group (15 genes) than those in the nr group (10 genes), especially the genes k127_4726130_1 and k127_2379523_1 exhibiting an obvious difference between the two groups ( figure 3a) . moreover, 15 taxonomical species, including p. copri, parabacteroides distasonis, dorea longicatena, bacteroides intestinalis, and so on, were identified as taxonomical biomarkers ( figure 3b) . in contrast to the distribution of the number of gene biomarkers between the r and nr groups, more taxonomical biomarkers were identified in the nr group ( figure 3b) . besides, 25 functional traits, including the biosynthesis of several kinds of amino acids and the pathway of pyruvate fermentation to acetate and lactate, were identified as functional biomarkers ( figure 3c) . moreover, to highlight the potential applications of gene biomarkers involved in mtx metabolism, as well as taxonomical and functional biomarkers in clinical treatment, we built several models based on clinical information and gene, taxonomical, and functional biomarkers by using the random forest algorithm and verified them in the testing dataset of prjna682730 ( figure 3d) . we found that the accuracy of the model built based on gene biomarkers (61.90%, auc = 0.611) was higher than that based on clinical information (57.14%, auc = 0.5714). particularly, the highest accuracy was found in the model built based on clinical information (rf titer), gene biomarkers (k127_4431000_2, k127_5955546_27, k127_932549_1), taxonomical (p. figure 3d ; 80.95%, auc = 0.8333); the auc value is roughly equal to that reported in a previous study (auc = 0.84, models built with different features) (artacho et al., 2021) . the results suggest that the potential metabolic capability of gut microbiota on mtx metabolism should be considered to determine the drug response of ra patients before the clinical treatment of ra. furthermore, to explore the sources of genes involved in mtx metabolism and their functions, host-tracking and functional annotations were conducted. the host-tracking results revealed that the genes were mainly affiliated with firmicutes (1,524 [57.42%]) and bacteroidetes (525 [19.78%]; figure 4a and supplementary table 2 ). the functional traits of these genes were mainly annotated as methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (19.56%) and dihydrofolate reductase (14.13%; figure 4b ) that can metabolize mtx (rajagopalan et al., 2002) . besides, the distribution of these genes in different contigs was diverse, even for the same genes that have the same function and highly similar sequences. for example, the occurrence of dihydrofolate reductase gene is strong with thymidylate synthase gene, and its distribution in different microbiota (assembled contigs) was diverse and complex ( figure 4c and supplementary table 3) , which reflected that the potential metabolic capability of different gut microorganisms on mtx metabolism is diverse. in the past decades, many studies have proven that the gut microbiota plays an essential role in maintaining human health and participates in the occurrence and development of diseases, including inflammatory diseases [e.g., ra manasson et al. (2020) ] and other diseases (wu and wu, 2012; carding et al., 2015) . researchers have proven that the gut microbiome is in a state of homeostasis under normal circumstances, and the alterations of the human gut microbiome can be caused by several factors, such as environmental factors, host genetics, diet, and drugs. in particular, the theory of "affinal drug and diet" of traditional chinese medicine and the declaration of hippocrates ("let medicine be the food and let food be the medicine") have highlighted the importance of diet and drugs in the composition of the human gut microbiota. in recent years, researchers have focused on the interactions between diseases and gut microbiota to develop effective treatment methods to treat the diseases related to the gut microbiota, such as fecal microbiota transplantation and probiotics (han et al., 2020b) . several studies have proven that modulating the homeostasis of the gut microbiota can treat joint diseases (nayak, 2021) and ra (pan et al., 2019; fan et al., 2021) . in particular, the relationship between drugs and gut microbiota has become another important research hotspot. for example, by integrating the multi-omics datasets of 2,173 european residents from the metacardis cohort, a previous study reported the explanatory power of drugs for the variability in host and the gut microbiome that contributed to the disease and provided a new hypothesis for drug-hostmicrobiome interactions in cardiometabolic diseases (forslund et al., 2021) . obtaining an in-depth understanding of the relationship between drugs and the gut microbiome, in particular, the mechanism of drug metabolism by gut microorganisms and the alterations in the gut microbiome affected by drugs, would provide a new view on the drug-host-microbiome axis and has potential implications for treatment of diseases (enright et al., 2016; walsh et al., 2018; weersma et al., 2020) . the occurrence and development of ra, as a widely recognized autoimmune disease, are associated with alterations in the gut microbiota (zhang et al., 2015; horta-baas et al., 2017; brown et al., 2020) . however, the pathology of ra remains elusive, and the effect of clinical treatment is still unsatisfactory. for example, up to 50% of patients treated with mtx could not obtain a clinically effective outcome; the possible reason might be associated with the gut microbiota (artacho et al., 2021) , which suggests that the metabolism of drugs is associated with the gut microbiota. to date, despite the development of several effective therapies, the field of rheumatology lacks tools to help clinicians decide on which drugs are most likely to benefit the patients. identifying such a tool is urgent and essential for the clinical treatment of ra. hence, to address this issue, we searched and downloaded the gut metagenome datasets related to ra patients in a public database. three available datasets were obtained (zhang et al., 2015; artacho et al., 2021) , and only bioproject prjnz682730 recorded the response to mtx, so mtx was selected as the model drug. in addition, to compare the taxonomical and functional compositions of gut microbiota and the potential metabolic capability of gut microbiota in healthy individuals and ra patients, the gut metagenome datasets were also collected from a healthy chinese cohort (li et al., 2017) . as a result, a total of 533 human gut metagenome datasets were obtained to investigate the relationship among ra, drug response, and gut microbiota and to reveal the underlying mechanism of different effects of the same drug. to obtain additional potential genes/proteins involved in mtx metabolism, we used those proteins obtained from the pdb database as seed sequences and searched in nr database, which significantly increased the number of potential proteins. these proteins were used to construct a database to profile the compositions of the proteins involved in mtx metabolism and the corresponding genes in healthy individuals and ra patients. we found that the genes involved in mtx metabolism were detected in healthy individuals and ra patients (figures 2a,b) , indicating that the universality of genes related to mtx metabolism in the human gut microbial community and gut microbiota harbors the potential metabolic capability on mtx. however, the number of genes involved in mtx metabolism differed among the cohorts, which suggests that the construction of a comprehensive nonredundant protein set should be established in future study. furthermore, we also calculated the relative abundance of the 2,654 genes in the training and testing data of the bioproject prjnz682730. we found that the compositions of these genes in the gut microbial communities significantly differed between the r and nr groups ( figure 2c) . this finding suggests that the response of mtx in ra patients could be associated with the gut microbiota, particularly the compositions of genes involved in mtx metabolism. hence, these results revealed that gut microbiota possibly participates in the metabolism of drugs in the clinical treatment of ra patients. whether the structure of gut microbial communities, including taxonomical, functional, and gene compositions, can serve as a classifier to predict the effectiveness of a clinical medication should be verified. subsequently, we compared the composition of the genes involved in mtx metabolism between the r and nr groups, and our results revealed that the potential metabolic capability of gut microbiota on mtx metabolism has a significant difference between the gut microbial communities of the r and nr groups. in addition, it should be noted that the potential metabolic capability of gut microbiota on mtx metabolism was evaluated based on the abundance of the genes; as such, the results might be not consistent with the real abundance of proteins involved in mtx metabolism. in this regard, transcriptome and protein sequencing are optional in future studies. moreover, taxonomical, functional, and gene biomarkers were identified with lefse (figures 3a,b) . interestingly, p. copri, belonging to the family prevotellaceae, has been proven to be significantly altered in individuals with preclinical ra stages compared with the healthy controls (drago, 2019) , which was identified as a fecal biomarker for ra diagnosis (alpizar-rodriguez et al., 2019) and a taxonomical biomarker for the r and nr groups. furthermore, to better serve the clinical medication, we established models based on clinical information, as well as taxonomical, functional, and gene biomarkers by using the random forest algorithm and then verified them in the testing data of bioproject figure 3d) . our results showed that the accuracy of the models reached 80.95%, with the auc value of 0.8333, which is approximately equal to the value reported earlier (auc = 0.84, models built with different features) (artacho et al., 2021) , suggesting that the proposed model can guide clinical application. in addition, to better understand the sources of genes involved in the mtx metabolism, we conducted the hosttracking analysis and explored the distribution of the host taxonomy of these genes. our results showed that the taxonomies of these genes are mainly classified into firmicutes and bacteroidetes ( figure 4a ). previous studies have demonstrated that the ratio of firmicutes to bacteroidetes is a biomarker that represents the dysbiosis of the gut microbiota (grigor'eva, 2020), and firmicutes and bacteroidetes play an important role in the metabolism of substrates. for example, foods rich in fibers are mainly degraded by firmicutes and bacteroidetes into short-chain fatty acid such as butyrate, which could have a positive effect on gut dysbiosis by reducing intestinal permeability, promoting bacterial translocation, and limiting inflammation (de santis et al., 2015; mitsou et al., 2017) . in particular, in terms of the pathogenic factors of ra, smoking affects the relative abundance of firmicutes and bacteroidetes (savin et al., 2018) , which suggests that the taxonomical members of these two phyla play a key role in the development and clinical treatment of ra. these results remind us that the gut microbiota plays an essential role in the metabolism of substrates, including drugs. nevertheless, it should be noted that the taxonomical classification of each gene cannot be identified at the species level, which is a challenge to gain insight into which bacteria harbor the metabolic ability of mtx. overall, our results demonstrate that the response mechanism to mtx in ra patients is highly probably related to the catabolic ability of the drug in the gut microbiota and suggest that the profile of potential metabolic capability is an important feature for determining whether the host responds to mtx. our findings provide a solid foundation for addressing individual differences in drug responses in ra or even other diseases and could elicit effects on clinical management not only for ra but also other gut microbiome-related diseases in the following contexts: first, it suggests that the profile of the potential genes involved in drug metabolism is an important feature for affecting the efficiency of drugs, and clinical treatment strategy should incorporate the compositions of these genes of the gut microbial communities. second, a suitable model could be constructed by combining gene profiles and other valuable information in the gut microbiome to determine whether the host responds to drugs before clinical treatment (onuora, 2021) . finally, this work provides an idea that other diseases related to gut microbiota can be studied by similar methods to determine the clinical role of various drugs. the gut microbiota plays an essential role in the occurrence and development of diseases, such as inflammatory disease, including ra. the interactions between the gut microbiota and clinical treatment have become a research hotspot. however, the mechanism underlying the effect of microbiota on drug efficiency remains unclear, and a decision model should be established to guide the selection of drugs during the clinical treatment of diseases. in this study, we collected the gut metagenome datasets related to ra patients treated with mtx from a public database, as well as the metagenome datasets of healthy individuals and other ra patients. a total of 533 human gut metagenome datasets were collected from four cohorts. we profiled the taxonomical and functional compositions and identified the genes involved in mtx metabolism between the r and nr groups. our results showed that the composition of genes involved in mtx metabolism significantly differed between the r and nr groups. these genes were mainly affiliated with firmicutes and bacteroidetes. based on the taxonomical, functional, and gene biomarkers, a random forest model with high accuracy was constructed to provide a decision model for the clinical application of drugs. in addition, the constructed databases can be available at the website: https://github.com/ labhanmz/drug_response_in_ra. overall, our study highlights that the potential metabolic capability of gut microbiota on drug metabolism is associated with the drug response and provides a solid foundation for exploring the interactions among drug metabolism, drug response, and gut microbiota and deepens the understanding of the mechanism of drug metabolism mediated by gut microbiota. our work provides a basis for the clinical management of diseases related to gut microbiota. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors. mh, zc, and sh designed the study. mh, nz, and ym downloaded the datasets and conducted the data analysis. mh, zc, nz, ym, bh, mr, and sh wrote the initial draft of the manuscript. all authors read, modified, and approved the final manuscript. diagnosis and management of rheumatoid arthritis: a review prevotella copri in individuals at risk for rheumatoid arthritis gut microbiota modulate cd8 t cell responses to influence colitis-associated tumorigenesis the pretreatment gut microbiome is associated with lack of response to methotrexate in new-onset rheumatoid arthritis dynamics and stabilization of the human gut microbiome during the first year of life integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with biobakery recommendations for optimizing methotrexate treatment for patients with rheumatoid arthritis trimmomatic: a flexible trimmer for illumina sequence data intestinal dysbiosis and tryptophan metabolism in rheumatoid arthritis: a brief overview of the treatment dysbiosis of the gut microbiota in disease nutrition and rheumatoid arthritis in the the gut microbiota of non-treated patients with sle defines an autoimmunogenic and proinflammatory profile nutritional keys for intestinal barrier modulation prevotella copri and microbiota in rheumatoid arthritis: fully convincing evidence? the impact of the gut microbiota on drug metabolism and clinical outcome lactobacillus casei ccfm1074 alleviates collagen-induced arthritis in rats via balancing treg/th17 and modulating the metabolites and gut microbiota combinatorial, additive and dosedependent drug-microbiome associations the influence of ethnicity and geography on human gut microbiome composition gut microbiome in health and disease: linking the microbiome-gut-brain axis and environmental factors in the pathogenesis of systemic and neurodegenerative diseases finding enzymes in the gut metagenome gallstone disease, obesity and the firmicutes/bacteroidetes ratio as a possible biomarker of gut dysbiosis stratification of athletes' gut microbiota: the multifaceted hubs associated with dietary factors, physical characteristics and performance utilizing microbiome approaches to assist source tracking, treatment and prevention of covid-19: review and assessment intestinal dysbiosis and rheumatoid arthritis: a link between gut microbiota and the pathogenesis of rheumatoid arthritis prodigal: prokaryotic gene recognition and translation initiation site identification intestinal microbiome-macrophage crosstalk contributes to cholestatic liver disease by promoting intestinal permeability in mice metagenome-wide association study of gut microbiome revealed novel aetiology of rheumatoid arthritis in the japanese population structure guided homology model based design and engineering of mouse antibodies for humanization megahit v1. 0: a fast and scalable metagenome assembler driven by advanced methodologies and community practices gut microbiota dysbiosis contributes to the development of hypertension role of gut microbiota in rheumatoid arthritis rheumatoid arthritis: diagnosis and management the microbiome in rheumatology: where are we and where should we go? the human microbiome in hematopoiesis and hematologic disorders adherence to the mediterranean diet is associated with the gut microbiota pattern and gastrointestinal characteristics in an adult population western diet and psoriatic-like skin and joint diseases: a potential role for the gut microbiota gut microbiome could predict drug response in ra a single bacterium restores the microbiome dysbiosis to protect bones from destruction in a rat model of rheumatoid arthritis interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics the gut microbiota shapes intestinal immune responses during health and disease smoking, body mass index, disease activity, and the risk of rapid radiographic progression in patients with early rheumatoid arthritis smoking and the intestinal microbiome expansion of intestinal prevotella copri correlates with enhanced susceptibility to arthritis metagenomic biomarker discovery and explanation zeb2 drives invasive and microbiota-dependent colon carcinoma faecal microbiome in new-onset juvenile idiopathic arthritis infants born to mothers with ibd present with altered gut microbiome that transfers abnormalities of the adaptive immune system to germ-free mice robust taxonomic classification of uncharted microbial sequences and bins with cat and bat drug-gut microbiota interactions: implications for neuropharmacology interaction between drugs and the gut microbiome the role of gut microbiota in immune homeostasis and autoimmunity decision-making strategy for the treatment of rheumatoid arthritis-associated interstitial lung disease (ra-ild) the oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment hidden link in gut-joint axis: gut microbes promote rheumatoid arthritis at early stage by enhancing ascorbate degradation the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2022.839015/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.copyright © 2022 han, zhang, mao, huang, ren, peng, bai, chen, liu, wang, huang and cheng. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0045298-fawll2v7 authors: fletcher, ashley; lassere, marissa; march, lyn; hill, catherine; carroll, graeme; barrett, claire; buchbinder, rachelle title: oral complementary medicine use among people with inflammatory arthritis: an australian rheumatology association database analysis date: 2020-06-05 journal: int j rheumatol doi: 10.1155/2020/6542965 sha: 2bb24cbb0f48921d6ecd351e4357a3c56aada8b2 doc_id: 45298 cord_uid: fawll2v7 objectives: to describe oral complementary medicine (cm) use in people with inflammatory arthritis, associations with use, and changes in use over time. methods: demographic, clinical, and patient-reported outcome data from 5,630 participants with rheumatoid arthritis (ra), ankylosing spondylitis (as), psoriatic arthritis (psa), and juvenile idiopathic arthritis (jia) were extracted from the australian rheumatology association database (arad), a national observational database. cm use at entry into arad was ascertained for participants recruited between 2002 and 2018. cm was categorised according to the nih/cochrane schema (fatty acids, herbs, or supplements). logistic regression was used to assess associations between demographic characteristics and cm use. change in cm use between 2006 and 2016 was investigated using a nonparametric test for trend of rate by year. results: 2,156 (38.3%) arad participants were taking cm at enrolment (ra: 1,502/3,960 (37.9%), as: 281/736 (38.2%), psa: 334/749 (44.6%), and jia: 39/185 (21.1%)). cm use was more prevalent in women (or 1.3; 95% ci: 1.13-1.50), those with tertiary education (or 1.32; 95% ci: 1.13-1.55), private health insurance (or 1.26; (95% ci: 1.10-1.44), drinking alcohol sometimes (or 1.22; 95% ci: 1.05-1.43), poorer function (haq) (or 1.13; 95% ci: 1.02-1.24), use of nsaid (or 1.32; 95% ci 1.17-1.50), weak (or 1.21; 95% ci 1.05-1.41) but not strong opioids, and less prevalent in current smokers (or 0.76; 95%: ci 0.63-0.91). cm use was not associated with pain, disease activity, or quality of life. the most common cms were fish oils (n = 1,489 users) followed by glucosamine (n = 605). both declined in use over time between 2006 and 2016 (27.5% to 21.4%, trend p = 0.85 and 15.5% to 6.4%, trend p < 0.01), respectively. conclusion: oral cm use is common among australians with inflammatory arthritis. its use is greater among women and those with tertiary education. fish oil and glucosamine, the most common cms, both declined in use over time. inflammatory arthritis comprises a range of conditions that affect joints and other tissues. these conditions include rheu-matoid arthritis (ra), psoriatic arthritis (psa), ankylosing spondylitis (as), and juvenile idiopathic arthritis (jia). in australia, the prevalence of ra ranges between 0.46% from a systematic review that included doctor-diagnosed ra only [1] to 1.9% according to the 2017-18 national health survey which relies solely upon self-report [2] . up to 0.4% of school children are reported to have jia [3] , and as and psa have a reported prevalence of 0.25% and 0.19%, respectively [4] . treatment for inflammatory arthritis depends on the type of disease, its symptoms, and severity. when indicated, conventional disease-modifying antirheumatic drugs (csdmards) and biologic/targeted synthetic dmards (b/tsdmards) are prescribed to slow or stop the progression of the disease and limit or prevent joint damage [5] . nonsteroidal anti-inflammatory drugs (nsaids) and glucocorticoids are also sometimes indicated to manage pain and inflammation and may also have disease-modifying effects for some types of inflammatory arthritis [6, 7] . complementary medicine (cm) is a broad term that encompasses a range of therapies, usually natural products, and also mind and body practices. natural products include fatty acids (e.g., fish oil, new zealand (nz) green-lipped mussels, and evening primrose oil), herbs (e.g., ginger, celery, and chinese herbs), vitamins (e.g., vitamin c) and multivitamins, minerals (e.g., magnesium and zinc), complex compounds (e.g., glucosamine and chondroitin), and probiotics, all commonly available as dietary supplements [8] . cm may or may not be recommended by health professionals and may be used alone or in combination with conventional medicine. different forms of cm are frequently used by patients with chronic diseases including inflammatory arthritis [9] . two separate population-based studies performed in australia reported that over a third of those with arthritis use oral cm [9, 10] . a national health survey reported that 36.1% of 25,906 adults with self-reported arthritis use oral cm [10] , while 38.4% of 3,161 participants in the north west adelaide health study with self-reported doctor-diagnosed arthritis reported cm use [9] . clinic-based estimates vary widely depending upon the setting. for example, 23% were found to be using oral cm in a hospital-based early inflammatory arthritis clinic in singapore [11] , while the rate has been estimated to vary from 35% to 63% in ra clinics in japan [12] and australia [13] , respectively. in an american general practice study 26.6% of patients with ra were taking supplements, 20.8% were taking vitamins and minerals, and 10.1% were taking herbs [14] . studies have also shown that oral cm use is common in as. in a cross-sectional study of 75 attendees of an australian as clinic, 72.1% were taking a dietary cm at the time of the study [15] . the prevalence of use is reportedly lower in jia; 17% of 235 attendees of a canadian arthritis clinic were reported to be using dietary changes or supplements [16] , while 29% of 134 patients in us paediatric rheumatology clinics report current herbal medicine use [17] . no studies have looked at the prevalence of dietary cm use in people with psa. the aim of this study was to describe the use of oral cm among people with ra, psa, as, and jia contributing to the australian rheumatology association database (arad), a large national prospective observational registry. secondary aims were to determine any associations with oral cm use and the demographic and clinical characteristics of arad participants and investigate trends in types of oral cm used over time. arad is a voluntary national registry that collects longitudinal health outcome data from people with inflammatory arthritis. it includes participants with ra, psa, as, and jia [18] . most participants enroll when they commence a b/tsdmard. enrolment is also encouraged for those not starting b/tsdmards but active targeting of this group has only occurred on an ad hoc basis. based on residential postcode, demographic and clinical characteristics participants appear to be nationally representative, with rheumatologists from all states and territories having contributed patients [19] . arad has received ethical approval from committees and organisations across australia. all participants provide written permission to be contacted by arad investigators and informed consent to participate in the registry as well as have their data linked with other national databases including state and territory cancer registries, death registry, and the pharmaceutical and medical benefits schemes. as described previously [18] [19] [20] , at enrolment, details of diagnosis, disease status, and b/tsdmard prescribed (if applicable) are obtained from the treating rheumatologist. all arad participants complete detailed entry and 6-to 12-monthly follow-up questionnaires (paper-based or online). data collected from the participants include demographic and socioeconomic details, disease duration and severity, self-reported past, and current medical history including malignancies and other chronic conditions, use of antirheumatic drugs with the date commenced and ceased, other medications including cm, and smoking and alcohol history. details concerning the level of pain experienced (0 to 100 scale where 0 is no pain and 100 is as bad as it could be) and overall arthritis activity (0 to 100 scale where 0 is none and 100 is extreme) in the preceding week are also ascertained. arthritis-specific disability is assessed by the health assessment questionnaire (haq) (0 to 3 where higher scores indicate greater disability) [21] , while quality of life is measured with the assessment of quality of life (aqol) (0 to 1 where higher score indicates better quality of life) [22] , european quality of life (euroqol or eq-5d (uk)) (0 to 1 where 0 is worst imaginable health state and 1 is the best imaginable health state) [23] , and the medical outcomes survey short form (sf-36) (0 to 100 where a higher score indicates better health) [24] . in the event of missing or ambiguous data, arad staff contact the participant, the rheumatologist, and/or other treating doctor to verify the data. for the purpose of this study, arad participants were eligible if they enrolled between 1 september 2001 and 31 may 2018. medication. self-reported use of oral cm therapy for arthritis treatment is collected at each questionnaire. current use of ginger, st john's wort, celery seed, 2 international journal of rheumatology pennywort, chinese herbs, glucosamine, fish oil, homeopathic remedies, nz green-lipped mussels, kelp, and shark fin are asked about specifically. the use of other cms is obtained from two free text fields. for the purpose of this study, we grouped cms, as defined by nih [25] and cochrane [8] , into three categories: fatty acids, herbal medicine, and supplements. fatty acids are animal and vegetable oils that contain omega-3 (e.g., fish oil and evening primrose oil); herbal medicines are plant preparations (e.g., ginger and chinese herbs); and supplements are vitamins, minerals (e.g., magnesium and zinc), and complex compounds such as amino sugars (e.g., glucosamine and chondroitin). we excluded calcium, iron, and vitamin d supplements as they are not specific for inflammatory arthritis and/or could have been prescribed for other reasons, while folic acid use was excluded as this is prescribed with methotrexate. selected baseline characteristics of the arad cohort were assessed by disease type using descriptive statistics, frequency, and chi-squared for categorical variables, anova, or kruskal-wallis tests for continuous variables. analyses were performed with and without jia to assess if the jia population influenced the results. baseline was defined as the entry date into arad (first questionnaire completed). baseline responses were compared for cm users and those not taking any cms. logistic regression models were used to assess associations between baseline demographic characteristics and cm use. multivariable modelling was performed to ascertain independent predictors of cm use. employment was coded as working, not working, and permanently unable to work due to illness. not working includes home duties, student, retired, and others from a free text field that includes casual work, volunteer work, and carers. the physical component summary (pcs) and mental component summary (mcs) scores of the sf-36 were calculated using standard algorithms based on population norms for australia. for socioeconomic status (ses), we assigned an australian bureau of statistics (abs) socio-economic indexes for areas (seifa) score. we coded ses according to the index of relative socio-economic advantage and disadvantage (irsad). the location was based on the participant's baseline address and classified at statistical areas level 1 (sa1). seifa 1 is the lowest quintile with relatively greater disadvantage and a lack of advantage in general; seifa 5 is the highest quintile with a relative lack of disadvantage and greater advantage in general. potential explanatory variables included in the model were age, sex, disease duration, haq, aqol, eq-5d (uk), pain level in the last week, overall arthritis activity in the last week, education (tertiary/secondary/did not completed secondary), employment (working, not working/home duties/student/retired/other, or permanently unable/ill), ses, current private health insurance, current smoking status, alcohol status (never, sometimes, or every day), current b/tsdmard/prednisolone/methotrexate status, opioid, and nsaid use. opioids were classified as high (morphine, oxycodone, hydromorphone, methadone, buprenorphine, fenta-nyl, sufentanil, tapentadol, and tramadol), and low (aspirin/codeine, paracetamol/codeine, dextropropoxyphene) potency. variables were retained in the multivariable model if they remained significant at p = 0:05. results are expressed as odds ratios (ors) and 95% confidence interval (ci). cm use between 2006 and 2016, the period with high numbers at baseline, is represented graphically and statistically using a nonparametric test for trend of rate by year. all analyses were performed using stata 14. table 1 ). overall participants were predominantly caucasian (94.1%). there were significant differences between the disease populations. compared with ra participants, other disease groups were significantly younger (p < 0:01 for each comparison); a higher proportion of females had ra, jia, and psa, but a lower proportion had as; and disease duration was shorter on average for jia than other diagnoses. in addition, a lower proportion of ra participants had completed tertiary education and they were less likely to be in current employment and have private health insurance in comparison to psa and as participants, while almost three-quarters of jia participants were still students. ra participants also had higher pain levels and greater disability and poorer health-related quality of life compared with the other disease groups, as indicated by higher mean haq, lower mean sf-36 (pcs and mcs scores), and aqol scores. the majority of participants across all diagnosis categories were taking b/tsdmards at baseline (55.6% overall), while current methotrexate and prednisolone use was higher among participants with ra. overall, 2156 (38.3%) of the cohort were taking at least one cm at baseline ( table 2) . homeopathic remedies (n = 47) were included in the overall analysis, but not grouped by category because they had many ingredients across categories. participants with psa had the greatest use (44.6%), followed by as (38.2%), ra (37.9%), and jia (21.1%). among those with cm use, 57.4% were taking one, 27.7% were taking two, and 14.9% were taking three or more cms. table 2 shows the type of cm in use across all arad participants at baseline. the most common category of cm was fatty acids (n = 1,564), most commonly fish oil (n = 1,489, 95.2% of fatty acids). there were 808 participants using supplements, most commonly glucosamine (n = 605, 74.9% of supplements), and 296 participants using herbal medicines, most commonly ginger (n = 148, 50.0% of herbs). table 3 presents the results of the univariate and multivariable analysis investigating differences between cm users and nonusers. in the univariate analysis, cm use was associated with having psa, older age, being female, having a tertiary education, higher ses, having private health insurance, better self-reported function (lower haq), and 3 international journal of rheumatology the proportion of participants with cm use did not (21) chinese herbs (40) magnesium (45) flaxseed oil (19) st john's wort (23) shark fin (28) krill oil (19) kelp (24) vitamin c (28) olive oil (11) turmeric/cumin/curcumin (19) vitamin b (25) emu oil (9) pennywort (14) vitamin e (17) cod liver oil (9) garlic (15) probiotic (17) linseed oil (4) rosehip (10) zinc (16) hemp oil (1) cranberry (1)/fruit juice (7) chondroitin (11) gingko (7) coenzyme q10 (5) milk thistle (6) silver (2) international journal of rheumatology appreciably change over the 6 and 12-month follow-up from baseline. compared to baseline (38.3%), at the six-month follow-up questionnaire, out of 4,077/5630 (72.4%) participants with data, 37.9% were taking at least one cm. this was similar to the 38.2% taking at least one cm at 12 months (data for 3,901/5630 (69.3%) participants) (data not shown). figure 1 shows cm use over time for the most common cms recorded at any questionnaire between 2006 and 2016. fish oil, while still the most common form of cm used in this cohort, declined in use over time from 27.5% in 2006 to 21.4% in 2016 although the trend was nonsignificant (p = 0:85). glucosamine use also declined over this period (15.5% to 6.4%, trend p < 0:01). ginger remained stable over time (2.9% to 2.6%, trend p = 0:37), calcium decreased (2.1% to 1.0%, trend p = 0:71), while magnesium (0.3% to 2.7%, trend p = 0:02) and multivitamins (0.5% to 0.9%, trend p = 0:97) both increased. krill oil started to be used in 2009, peaked in 2013 (3.0%), and has been decreasing since then (0.8% in 2016). other cms listed in table 1 were used by a lower proportion of participants but those with significant changes, in the test for trend, between 2006 and 2016 were celery seed (0.9% to 0.4%), shark fin (1.3% to 0.3%), pennywort (0.4% to 0.04%), evening primrose oil (0.8% to 0.2%), turmeric (0.0% to 1.5%), rosehip (0.2% to 0.1%, but peaked in 2014 at 0.5%), and probiotic (0.0% to 0.9%). cm use is common among people with inflammatory arthritis in australia as evident by the 38.3% of arad participants taking at least one cm at entry into the database. this is consistent with the prevalence rates reported in the 2004-05 australian national health survey (nhs) [9] and the north west adelaide health study (nwahs) cohort in 2004-05 [10] . use among jia participants was much lower compared to the other disease groups. of those taking at least one cm, just under half were taking two or more. the most common cms were fatty acids, predominantly fish oil, and supplements, most commonly glucosamine. however, the use of both of these cms declined over the decade from 2006 to 2016. similar to other studies, we found that cm use was associated with being female and being tertiary educated [26] [27] [28] . other associations which remained in the multivariable analysis included having private health insurance, not currently smoking, consuming alcohol sometimes (rather than never), having poorer function, and current low potency opioid and nsaid use. we did not investigate the association with place of residence although another australian study found an association with living in rural and remote areas [29] . our results indicate that the use of particular types of cm fluctuates over time. the two most popular cms, fish oils, and glucosamine have been declining in use over time, while supplements (mainly vitamins) have been increasing over time. an australian study of oral cm use in the general population of south australia at three time points (1993, 2000 and 2004) reported that oral cm use rose from 48.5% to 52.2% between 1993 and 2004 [27] . reasons for use were for general health (70%) followed by for muscle, bones, or joints (21%), for the immune system (18.2%), for nerves or stress (13.0%), and for blood or circulation (9.5%). in an australian populationmatched cross-sectional survey of 2,025 australian adults in 2019 the use of cm was found to be 50.3% [30] . high quality and robust evidence to support the use of various oral cms for ra, psa, as, and jia is lacking. a 2017 systematic review based upon 22 trials found moderate-quality evidence for a small favourable effect (smd -0.21 (95% ci -0.42 to -0.00) translating to <8% improvement) for marine oil supplements in ra [31] . however, the effects varied depending upon the type of marine oil, dose, and ratio of eicosapentaenoic acid (epa) and docosahexaenoic acid (dha). a cochrane review of herbal therapy 7 international journal of rheumatology for ra, last updated in 2011, found moderate-quality evidence that oils containing gamma linolenic acid (gla) (evening primrose, borage, or blackcurrant seed oil) may also provide symptom benefit in ra [32] . a strength of our study is that arad collects longitudinal data enabling us to determine whether cm use has changed over time. our study is also the first to provide a population-based estimate of the prevalence of cm use among people with psa as well as add to the limited data available for cm use in as [15] and jia [16, 17] . a generally high number of arad participants complete questionnaires each year, 12.7% only complete one questionnaire, 87.3% complete 2 questionnaires, and 78.9% complete 3 questionnaires. loss to follow-up may be a potential source of bias for our trend analyses although those that filled in 6 and 12-month questionnaires had similar rates of cm use (37.9% and 38.2%, respectively) compared to baseline (38.3%). another strength was the size of our cohort allowing us to investigate the associations of cm use across a large number of participants with diverse demographic, disease, and treatment characteristics. potential limitations include the fact that participation in arad is voluntary and therefore our results may not be generalisable to the australian population overall or by inflammatory arthritis type. however, overall cm use in arad was similar to use in people with arthritis found in two australian population-based studies, which is reassuring and strengthens the case for validity. although we specifically ask arad participants to specify cms taken for arthritis, we cannot be certain that this is the case. we also have no data concerning the amount or dose of the cms used. our study only investigated oral forms of cm and did not consider other cm categories such as alternative medical systems, energy therapies, manipulative and body-based methods, mindbody interventions, or other natural product-based therapies. based upon the findings from arad, a large national prospective registry, oral cm use is common among australians with all types of inflammatory arthritis including jia. over a third of people with inflammatory arthritis were taking at least one cm at the time they entered arad and this was most common in people with psa. the observation that many patients with rheumatic diseases use cm in australia as a complement to their prescribed medications is important information for clinicians. obtaining details of patients' cm use is important particularly in considering adverse events and possible interactions with prescribed medicines. cm use was greater among women and those who are better educated. trends in which types of cm are used changes over time. fish oil and glucosamine were the most commonly used cm but they both declined in use over the period of study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. the global burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study 18 national health survey -first results epidemiology of the rheumatic diseases of childhood global prevalence of spondyloarthritis: a systematic review and meta-regression analysis clinical utility of therapeutic drug monitoring in biological disease modifying anti-rheumatic drug treatment of rheumatic disorders: a systematic narrative review non-steroidal anti-inflammatory drugs (nsaids) for axial spondyloarthritis (ankylosing spondylitis and non-radiographic axial spondyloarthritis) biologics or tofacitinib for people with rheumatoid arthritis naive to methotrexate: a systematic review and network meta-analysis development and classification of an operational definition of complementary and alternative medicine for the cochrane 8 australian adults use complementary and alternative medicine in the treatment of chronic illness: a national study oral complementary medicine and alternative practitioner use varies across chronic conditions and attitudes to risk use of complementary and alternative medicines is associated with delay to initiation of disease-modifying anti-rheumatic drug therapy in early inflammatory arthritis sixty percent of patients with rheumatoid arthritis in japan have used dietary supplements or health foods non-prescription complementary treatments used by rheumatoid arthritis patients attending a community-based rheumatology practice use of complementary therapies among primary care clinic patients with arthritis complementary and alternative medicines in ankylosing spondylitis: a cross-sectional study longitudinal analysis of complementary and alternative health care use in children with juvenile idiopathic arthritis complementary and alternative medicine use among youth with juvenile arthritis: are youth using cam, but not talking about it? effect of treatment with biological agents for arthritis in australia: the australian rheumatology association database the australian rheumatology association database (arad) malignancy risk in australian rheumatoid arthritis patients treated with anti-tumour necrosis factor therapy: analysis of the australian rheumatology association database (arad) prospective cohort study the dimensions of health outcomes: the health assessment questionnaire, disability and pain scales the assessment of quality of life (aqol) instrument: a psychometric measure of health-related quality of life eq-5d: a measure of health status from the euroqol group sf-36 physical and mental health summary scales: a user's manual, the health institute the covid-19 outbreak is an emerging, rapidly evolving situation use of complementary and alternative therapies by patients self-reporting arthritis or rheumatism: results from a nationwide canadian survey the continuing use of complementary and alternative medicine in south australia: costs and beliefs in 2004 dietary supplementation is more prevalent among adults with arthritis in the united states population complementary medicine use by the australian population: a critical mixed studies systematic review of utilisation, perceptions and factors associated with use use of complementary medicine products: a nationally representative cross-sectional survey of 2019 australian adults marine oil supplements for arthritis pain: a systematic review and meta-analysis of randomized trials herbal therapy for treating rheumatoid arthritis the authors gratefully acknowledge and thank australian rheumatologists and patients for contributing data to arad and also margaret staples, lyndall henderson, joan mcphee, and vibhasha chand, and the arad steering committee. the paper was presented at the 2018 acr/arhp annual meeting. the australian rheumatology association database is supported by unrestricted educational grants administered through the australian rheumatology association currently from pfizer australia and previously from, abbvie pty ltd., eli lilly australia pty ltd. sanofi australia, celgene australian & nz, bristol myers squibb australia pty ltd. amgen australia pty ltd., aventis, astrazeneca, and roche. arad was previously supported by an australian national health and medical research council (nhmrc) enabling grant (id 384330). infrastructure support is from cabrini health, monash university, royal north shore hospital, and the australian rheumatology association. the authors declare that they have no competing interests. key: cord-0032528-4mqkkx0n authors: biswas, isha; lewis, sarah; chattopadhyay, kaushik title: content, structure and delivery characteristics of yoga interventions for managing rheumatoid arthritis: a systematic review protocol date: 2022-05-17 journal: int j environ res public health doi: 10.3390/ijerph19106102 sha: 458c0f96d36e642b857f7d0b36f6ebcf6857f870 doc_id: 32528 cord_uid: 4mqkkx0n the global burden of rheumatoid arthritis among adults is rising. yoga might be a potential solution for managing rheumatoid arthritis. this systematic review aims to synthesise the content, structure and delivery characteristics of effective yoga interventions for managing rheumatoid arthritis. the jbi methodology for systematic reviews of effectiveness and the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines will be followed. prisma for systematic review protocols (prisma-p) was used to write the protocol. randomised controlled trials assessing the effectiveness of yoga interventions for managing rheumatoid arthritis in adults will be included in this review. we aim to search the following databases to find published and unpublished studies: abim, amed, ayush research portal, cam-quest, cinahl, central, embase, medline, pedro, psycinfo, sportdiscus, trip, web of science, dart-europe-e-theses portal, ethos, opengrey and proquest dissertations and theses. no date or language restrictions will be applied. a narrative synthesis will be conducted. meta-regression will be conducted to explore the statistical evidence for which components (content, structure and delivery characteristics) of yoga interventions are effective. rheumatoid arthritis is a chronic inflammatory autoimmune disease in which the immune system of the body damages the joints by attacking their membrane linings (synovium) and eroding the bones, cartilages, tendons and ligaments [1] . the main symptoms include warm, swollen, painful and stiff joints (particularly morning stiffness or after a period of inactivity) and additional ones such as poor appetite, weight loss, weakness, high fever and sweating [2, 3] . it commonly occurs in the joints of the wrists, hands and feet [1, 3] . the symptoms are usually first noticed in the small joints in the middle of the fingers and at the base of the fingers and toes, and sometimes in the shoulders, elbows, knees and ankles [1, 3] . it is a symmetrical disease which means the symptoms usually occur on both sides of the body at the same time and to the same extent [1, 3] . in this disease, there are unpredictable episodes of symptoms, from not feeling any symptoms to times when symptoms worsen known as flares [3] . these episodes vary from person to person, making it difficult to define flares [3] . this makes it difficult to differentiate between early and established rheumatoid arthritis [4] . biologically, the most appropriate way of defining an established disease is when the diagnosis is confirmed [4] . diagnosis is performed using physical examination based on symptoms and signs (e.g., inflamed tear glands or rheumatoid nodules under the skin) to assess the swelling of joints and their movement [3, 5] . main blood tests to confirm the diagnosis include erythrocyte sedimentation rate (esr), c-reactive protein (crp) and full blood count [3, 5] . other blood tests include measuring rheumatoid factors and anti-cyclic citrullinated peptides (anti-ccp) [3] . these are antibodies that are produced by the immune system when it attacks healthy tissue [1, 3] . those who test positive for both these factors are at increased risk of severe disease [3] . in some cases, joint scans such as x-ray, magnetic resonance imaging (mri) and ultrasonography are also used [1, 5] . disease activity is an important outcome in rheumatoid arthritis and is used to understand the current state of the disease, get an idea of its progression over time and make treatment-related decisions [6] . it is not clear why the immune system attacks the joints; however, there are some possible causes and risk factors of the disease [3, 7] . these include sociodemographic factors such as increasing age and female sex; environmental factors such as smoking and outdoor air pollution; genetic factors such as having a family history of rheumatoid arthritis and altered genotype of the human leukocyte antigen gene complex (hla-drb1) that increases susceptibility to the disease; health conditions such as altered microbiome of the gut and obesity (body mass index (bmi) greater than 30 kg/m 2 ) and hormonal changes especially in women (e.g., the onset of menopause) [7] [8] [9] [10] . some triggers, such as a period of physical or mental trauma or a period after a long illness, are also risk factors for rheumatoid arthritis [7] . the global prevalence of rheumatoid arthritis in 2019 was 0.46% (95% confidence interval (ci): 0.39 to 0.54) [11] . it has substantial health (physical and psychological), social and economic burden [1, 12, 13] . physical health consequences in the early stages of the disease include the inflammation of tear glands and salivary glands which reduces the production of tears and saliva and weak, deformed and stiff joints of the hands [1]. major damage to the joints, carpal tunnel syndrome, inflammation of lungs and cardiovascular diseases such as myocardial infarction and stroke can occur in severe cases [1, 3] . depression is the major psychological health consequence of the disease [12] . social implications include withdrawal from social life and limitations in performing activities of daily living [13] . the economic consequences include disease-related absenteeism from work and loss of work productivity leading to a reduction in income and an increase in treatment-related costs [13] . overall, it has a negative impact on health-related quality of life [14] . in terms of numbers and percentages, rheumatoid arthritis resulted in 3.26 million global disability-adjusted life years (dalys) in 2019 and was responsible for 0.1% of total global dalys [15] . the global disability-adjusted life years (dalys) related to rheumatoid arthritis are high and rising [15] . there is no cure for rheumatoid arthritis, but the disease can be managed using pharmacological and non-pharmacological interventions [3] . the main aim is early diagnosis and treatment to prevent irreversible damage to the joints by lowering disease activity, reducing pain and inflammation and improving function [3, 16] . pharmacological interventions include the use of disease-modifying anti-rheumatic drugs (dmards) that are administered orally, subcutaneously or intravenously to suppress autoimmune activity and delay or prevent joint degeneration [16, 17] . conventional synthetic dmards (cs-dmards) such as methotrexate are used as first-line therapy due to their effectiveness and safety, flexible administration and low cost [17] . however, csdmards can have side effects such as sore mouth, loss of appetite, feeling sick and impacts on the gastrointestinal, hepatic, hematologic and pulmonary systems [18] . biologic dmards (bdmards) such as infliximab are usually prescribed when csdmards are ineffective [19] . these are highly specific in their mechanism of action and can have serious adverse effects such as increased risk of opportunistic and bacterial infections and reactivation of tuberculosis [19] . other pharmacological interventions include janus kinase (jak) inhibitors for people with severe disease and non-steroidal anti-inflammatory drugs (nsaids) (e.g., ibuprofen) to reduce pain by decreasing inflammation but can have side effects such as gastrointestinal ulceration, renal failure and heart failure [3, 16] . glucocorticoids (e.g., dexamethasone) are more effective but less safe than nsaids due to long-term side effects such as weight gain, muscle weakness and bone thinning [20] . in short, the medications used to treat rheumatoid arthritis can have strong side effects and are often not well-tolerated and ultimately can lead to high treatment costs [16] . along with pharmacological interventions, non-pharmacological interventions such as brisk walking and moderate-to-high intensity exercise are recommended to improve or maintain mobility and joint function [1, 21] . joint surgery is recommended only at the end-stage of the disease, and joint replacements might lead to systematic articular infections [1,3]. yoga is an ancient practice, with origins in the indian subcontinent, that aims to offer a holistic sense of well-being of the body and mind [22] . yoga philosophy and practice were first described by patanjali in the classic text-yoga sutras [23] . the multi-factorial approach of yoga includes components such as yogic poses (asana), breathing practices (pranayama) and dhyana (meditation) and relaxation practices along with moderation in lifestyle [23] . among the six major branches of yoga, hatha yoga is the most popular [24] . there are various styles of hatha yoga, and each has its distinct emphasis on the individual components [24] . yoga is becoming increasingly popular across the world as there are about 300 million people who practice yoga globally [25] . generally, yoga uses a gentle approach, is easy to learn and safe to practice, demands a low to moderate level of supervision, is inexpensive to maintain because of minimal equipment required and can be practised indoors or outdoors [26, 27] . yoga could be beneficial for a wide range of health conditions such as covid-19, lung diseases, type 2 diabetes, hypertension, obesity, cardiovascular diseases, cancers, musculoskeletal conditions and mental health disorders [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] . several systematic reviews have reported the beneficial effects of yoga interventions on rheumatoid arthritis outcomes, such as lower disease activity, pain relief and functional improvement [38] [39] [40] [41] [42] . these reviews have included randomised controlled trials (rcts) except two which also included other study designs [38, 41] . a recent systematic review and meta-analysis of 10 rcts on knee, hand and feet rheumatoid arthritis showed that yoga significantly lowered disease activity (4 rcts; standardised mean difference (smd) −0.38; 95% ci: −0.71 to −0.06) and improved physical function (5 rcts; smd −0.32, 95% ci −0.58 to −0.05) compared to no intervention or usual care [42] . the positive effects of yoga on rheumatoid arthritis outcomes can be explained by some potential mechanisms. the varying postures in yoga (such as flexion, extension, adduction, abduction and rotation) strengthen and stabilise muscles by engaging them in contraction [43] . strengthening of muscles leads to a reduction in pain and improvement in function [43, 44] . stress is a major psychological health factor that can lead to muscle tension and continued joint pain [45] . the continued pain reduces pain tolerance over time, disturbs emotional regulation and can further increase stress [46] . this can lead to increased activity of the sympathetic nervous system which stimulates the secretion of inflammatory biomarkers such as interleukin-6 (il-6) causing inflammation [47] . the breathing techniques in yoga strike a balance between activity of the sympathetic and parasympathetic nervous system and counter the stress response, thus, reducing inflammation [48] . none of the above-mentioned systematic reviews has synthesised the content, structure and delivery characteristics of yoga interventions for managing rheumatoid arthritis [38] [39] [40] [41] [42] . additionally, meta-regression has never been conducted to explore the statistical evidence for which components of yoga interventions (e.g., content, structure and delivery characteristics) are effective. thus, there is a need to conduct such a systematic review so that the content, structure and delivery characteristics of effective yoga interventions for managing rheumatoid arthritis can be synthesised and used in future research and practice. for example, based on the findings, a yoga intervention could be developed, evaluated and implemented for managing rheumatoid arthritis. the systematic review aims to synthesise the content, structure and delivery characteristics of effective yoga interventions for managing rheumatoid arthritis. this systematic review will be conducted by following the jbi methodology for systematic reviews of effectiveness and the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines [49, 50] . prisma for systematic review protocols (prisma-p) was used to write the protocol [51] . the protocol is registered with prospero (crd42022320337). this systematic review will include studies conducted among adults (aged ≥ 18 years) diagnosed with rheumatoid arthritis in one or more joints. no restrictions will be applied regarding the diagnostic criteria of rheumatoid arthritis, including diagnosis based on physical examination, blood tests and joint scans. if a study includes both children and adults, only the relevant information about adults will be extracted. if it is not possible to extract the relevant information about adults, the study will be excluded. studies reporting at least one of the major components of yoga, namely, asana (yogic poses), pranayama (breathing practices) and dhyana (meditation) and relaxation practices, will be included. there will be no restrictions on the type, frequency, duration or delivery mode of the yoga intervention. studies on multimodal interventions (which include yoga among other interventions) will be excluded if relevant data cannot be extracted. studies will also be excluded if they did not explicitly label the intervention as yoga. studies comparing a yoga intervention with no intervention, sham intervention, nonpharmaceutical intervention (e.g., physical activity) or pharmaceutical intervention will be included. studies having co-interventions will be included as long as all the eligible study groups were allowed to do so. studies with a head-to-head comparison of two or more yoga interventions (i.e., different in terms of content, structure or delivery characteristics) will be excluded. this systematic review will include studies that assessed the main outcomes of rheumatoid arthritis, i.e., disease activity, pain and function [52] [53] [54] . disease activity measured using any of the validated composite disease activity scores (das) such as das28-esr and das28-crp, pain assessed using any scale such as the visual analogue scale (vas) and numeric rating scale (nrs), and function assessed using any scale such as arthritis impact measurement scale (aims) and health assessment questionnaire (haq) will be eligible [54] . radiographic outcomes such as structural joint damage and residual inflammation in the synovium will not be included in this review as inadequate blinding may affect the specificity of the classification criteria and are not standardised to be considered as core outcomes [52] . considering the feasibility and practicality of the proposed work and the hierarchy of study designs, only rcts will be included in this systematic review. the following 13 databases will be searched from their inception dates to find pubthe search strategies are developed based on the following and in consultation with a research librarian at the university of nottingham: (i) the yoga component is based on a relevant systematic review [55] , (ii) the rheumatoid arthritis component is based on the search strategies reported in the uk's national institute for health and care excellence (nice) guideline on rheumatoid arthritis [56] and a cochrane systematic review on rheumatoid arthritis [57] , and (iii) the pre-designed search filters for rcts are used [58] [59] [60] . all the search strategies are detailed in the supplementary file s1. no date or language restrictions will be applied. all the identified citations will be collated and uploaded onto endnote x9 (clarivate analytics, pa, usa) [61] , and duplicates will be removed. the remaining records will be then imported into rayyan (qatar computing research institute [data analytics], doha, qatar) to facilitate the title and abstract screening process [62] . titles and abstracts will be independently screened for eligibility using the inclusion criteria by two systematic reviewers (ib and sl/kc). studies identified as potentially eligible or those without an abstract will have their full text retrieved. full texts of the studies will be assessed for eligibility by two independent reviewers. full-text studies that do not meet the inclusion criteria will be excluded, citing reasons. any disagreements that arise between the two reviewers will be resolved through discussion. if consensus is not reached, a third reviewer (sl/kc) will be consulted. translations will be sought where necessary. included studies will be critically assessed by two independent systematic reviewers (ib and sl/kc) for methodological quality using the standardised critical appraisal tool developed by jbi for rcts [49] . this tool uses a series of criteria that can be scored as being met (yes), not met (no), unclear or not applicable (n/a). the two reviewers will independently assess each criterion and comment on it. any disagreements that arise between the two reviewers will be resolved through discussion. if consensus is not reached, then a third reviewer (sl/kc) will be involved. all studies, regardless of their methodological quality, will undergo data extraction and synthesis, where possible. two systematic reviewers (ib and sl/kc) will independently extract data from the included studies using a pre-developed and pre-tested data extraction form. any disagreements that arise between the two reviewers will be resolved through discussion. if consensus is not reached, a third reviewer (sl/kc) will be consulted. the following data will be extracted: author(s), year of publication, country, participant characteristics (e.g., age, sex, ethnicity, occupation, type of joint affected by rheumatoid arthritis), sample size, intervention and comparator, outcomes, the timing of follow-up at the end of the intervention and adverse events. for all the outcomes, the authors will extract the end of intervention data [52, 57] . where this time point is not reported, data from the time point closest to the end of intervention will be extracted. intention-to-treat (itt) data will be preferred compared to per-protocol data. itt analysis is the most preferred analysis method for rcts [63] . this is because it preserves sample size by including all the participants irrespective of adherence to the study and attrition, maximises external validity and helps in understanding real-world circumstances encountered in an actual setting [63, 64] . post-intervention data will be extracted in preference to change from baseline data (i.e., post-intervention score-baseline score). percentage change from baseline will not be extracted as it is highly sensitive to change in variance and it also fails to protect from baseline imbalances, leading to non-normally distributed outcome data [65] . in addition, the content of yoga interventions will be extracted (e.g., yogic poses, breathing practices, meditation and relaxation practices) along with the structure (e.g., duration of the yoga sessions, duration and frequency of the yoga interventions) and delivery characteristics (e.g., individual or group sessions, supervised or unsupervised sessions, sessions delivered in yoga centres or at home, strategies for yoga intervention uptake and adherence, characteristics of yoga instructors). to obtain missing data on outcomes, multiple strategies will be used. the first strategy will be to contact the corresponding author of the included study by email (at least two times per author) to obtain the relevant data. if we get no response from the corresponding author, then certain assumptions will be applied. for example, where disease activity, pain and function are reported as continuous outcomes, if the standard deviation (sd) is missing, sd will be imputed from a similar study (in terms of intervention, comparator, sample size and numerical outcome data) [66] . if only a median and interquartile range (iqr) is reported, these will be extracted, the mean will be assumed to be equal to the median and the sd will be calculated using the standard formula (=iqr/1.35) [66] . a narrative synthesis will be conducted with the help of tables, focusing on the content, structure and delivery characteristics of effective yoga interventions for managing rheumatoid arthritis (i.e., for each type of joint and outcome). for example, in the case of rheumatoid arthritis of the wrist, a narrative synthesis will be performed for disease activity, pain and function of the wrist. considering the errors in how authors analyse and report yoga interventions to be effective in studies (e.g., doing pre-post analysis of outcomes within study arms but no comparative analysis between study arms), meta-analyses will be conducted for each type of joint and outcome using review manager 5.4.1 (copenhagen, the nordic cochrane centre, the cochrane collaboration) [67] to determine the true effectiveness of each included yoga intervention. meta-analyses based on random-effects models will be conducted to provide a weighted measure of treatment effect. the i 2 statistic will be used to quantify statistical heterogeneity across studies. for studies with more than one comparator group, the comparisons will be included in separate meta-analysis models to avoid the issue of double-counting of the comparator group. where disease activity, pain and function are reported as continuous outcomes, mean differences (mds) with 95% cis will be reported where the same scale is used across the studies. where different scales are used across studies, smds with 95% cis will be reported. where necessary, post-intervention data will be pooled with changes from baseline data, and this will be conducted only for mds but not smds. if reported as binary outcomes, risk ratios with 95% cis will be reported. where there is a sufficient number of studies (at least 10) included in a meta-analysis, a funnel plot will be generated to assess for publication bias. in the final step, meta-regression will be conducted to explore the statistical evidence for which components of the intervention are effective [68] . this requires a reasonable number of studies to have sufficient enough power to show differences in effectiveness between components, and so the final decision on which components will be explored and how they will be grouped will be made once we have extracted data from the included studies on the components of each intervention. however, we anticipate exploring broad categories of intervention components including content (e.g., yogic poses, breathing exercises, meditation and relaxation practices), structure (e.g., number of yoga sessions per week, length of the yoga sessions) and delivery characteristics (e.g., one-to-one or group sessions) of yoga interventions [68] . a random-effects model will be used to analyse these subgroup effects [69] . the effects of content, structure and delivery components on outcomes, i.e., disease activity, pain and function, will be investigated by looking at the amount of heterogeneity explained by these components using the reduction in the i 2 statistic and the dersimonian-laird estimation method [70] . the results will establish the statistical significance of any observed patterns in which components of yoga are associated with a greater effect on rheumatoid arthritis outcomes. supplementary materials: the following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19106102/s1, file s1: search strategies. rheumatoid arthritis: overview; institute for quality and efficiency in health care (iqwig) rheumatoid arthritis overview: rheumatoid arthritis; nhs: london, uk established rheumatoid arthritis-redefining the concept rheumatoid arthritis: early diagnosis and treatment outcomes clinical disease activity assessments in rheumatoid arthritis possible causes and risk factors the pathogenesis of rheumatoid arthritis long-term exposure to outdoor air pollution and the risk of development of rheumatoid arthritis: a systematic review and meta-analysis body mass index and the risk of rheumatoid arthritis: a systematic review and dose-response meta-analysis the global prevalence of rheumatoid arthritis: a meta-analysis based on a systematic review the prevalence of depression in rheumatoid arthritis: a systematic review and meta-analysis samel-kowalik, p. social implications of rheumatic diseases the impact of rheumatoid arthritis on quality-of-life assessed using the sf-36: a systematic review and meta-analysis global burden of disease collaborative network. rheumatoid arthritis-level 3 cause statpearls american college of rheumatology guideline for the treatment of rheumatoid arthritis side effects of methotrexate therapy for rheumatoid arthritis: a systematic review adverse effects of biologics: a network meta-analysis and cochrane overview pharmacotherapy options in rheumatoid arthritis self-management education: history, definition, outcomes, and mechanisms the yoga of healing: exploring yoga's holistic model for health and well-being the heart of patanjali hatha yoga pradipika significant yoga statistics: 2020/2021 benefits, facts and trends the metabolic syndrome and mind-body therapies: a systematic review a guide to yoga: exercise; nhs: london, uk modified bhramari pranayama in covid 19 infection yoga as a treatment for chronic low back pain: a systematic review of the literature yoga for arthritis: a scoping review a systematic review and meta-analysis on the effects of yoga on weight-related outcomes effects of yoga in adults with type 2 diabetes mellitus: a meta-analysis yoga and hypertension: a systematic review yoga for the primary prevention of cardiovascular disease breathing exercises for adults with asthma yoga as an alternative and complementary treatment for cancer: a systematic review hatha yoga for acute, chronic and/or treatment-resistant mood and anxiety disorders: a systematic review and meta-analysis yoga as an alternative and complementary approach for arthritis: a systematic review components and reporting of yoga interventions for musculoskeletal conditions: a systematic review of randomised controlled trials yoga for rheumatic diseases: a systematic review impact of yoga on inflammatory biomarkers: a systematic review yoga for treating rheumatoid arthritis: a systematic review and meta-analysis effect of yoga training on reaction time, respiratory endurance and muscle strength a systematic review of the effects of dynamic exercise in rheumatoid arthritis chronic stress, cortisol dysfunction, and pain: a psychoneuroendocrine rationale for stress management in pain rehabilitation cognitive and emotional control of pain and its disruption in chronic pain stress of different types increases the proinflammatory load in rheumatoid arthritis the physiological effects of slow breathing in the healthy human chapter 3: systematic reviews of effectiveness the joanna briggs institute the prisma 2020 statement: an updated guideline for reporting systematic reviews preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement guideline on clinical investigation of medicinal products used in the treatment of rheumatoid arthritis rheumatoid arthritis in adults: diagnosis and management: evidence review c treat-to-target; (nice guideline international consortium for health outcome measurement set of outcomes that matter to people living with inflammatory arthritis: consensus from an international working group content, structure, and delivery characteristics of yoga interventions for managing hypertension: a systematic review and meta-analysis of randomized controlled trials. front. public health risk factors: rheumatoid arthritis in adults: diagnosis and management: evidence review b; national institute for health and care excellence (nice exercise for rheumatoid arthritis of the hand cochrane handbook for systematic reviews of interventions, version available online: www.training.cochrane.org/ handbook rayyan-a web and mobile app for systematic reviews explanation and elaboration: updated guidelines for reporting parallel group randomised trials intention-to-treat and transparency of related practices in randomized, controlled trials of anti-infectives the use of percentage change from baseline as an outcome in a controlled trial is statistically inefficient: a simulation study chapter 6: choosing effect measures and computing estimates of effect. in cochrane handbook for systematic reviews of interventions version 5.4; the cochrane collaboration a review of methods for addressing components of interventions in meta-analysis a random-effects regression model for meta-analysis meta-analysis in clinical trials we would like to thank sarah beach, research librarian at the university of nottingham, for her support in developing the search strategies.conflicts of interest: k.c. is a guest editor for ijerph and was not involved in the editorial processes and decisions associated with this manuscript. the other authors declare no conflict of interest. key: cord-0020892-q93j84t6 authors: han, liang; tu, shenghao; shen, pan; yan, jiahui; huang, yao; ba, xin; li, tingting; lin, weiji; li, huihui; yu, kun; guo, jing; huang, ying; qin, kai; wang, yu; chen, zhe title: a comprehensive transcriptomic analysis of alternate interferon signaling pathways in peripheral blood mononuclear cells in rheumatoid arthritis date: 2021-08-25 journal: aging (albany ny) doi: 10.18632/aging.203432 sha: 89c93720fae642f70c5a53a6360c0a91a9fbfa4a doc_id: 20892 cord_uid: q93j84t6 interferon (ifn) signaling pathways play crucial roles in the pathogenesis of rheumatoid arthritis (ra). prior studies have mainly studied mixed alterations in the ifn signaling pathway in ra, but these studies have not been sufficient to elucidate how imbalanced ifn signaling subtly influences immune cells. single-cell rna (scrna) sequencing makes it possible to better understand the alternations in the interferon signaling pathways in ra. in the present study, we found that ifn signaling pathways were activated in natural killer (nk) cells, monocytes, t cells, b cells, and most immune cell subclasses in ra. we then explored and analyzed the connections between abnormal ifn signaling pathways and cellular functional changes in ra. single-cell regulatory network inference and clustering (scenic) analysis and gene regulatory network (grn) construction were also performed to identify key transcription factors in ra. finally, we also investigated altered ifn signaling pathways in multiple ra peripheral blood samples, which indicated that abnormal ifn signaling pathways were universally observed in ra. our study contributes to a better understanding of the delicate and precise regulation of ifn signaling in the immune system in ra. furthermore, common alternations in ifn signaling pathway-related transcription factors could help to identify novel therapeutic targets for ra treatment. rheumatoid arthritis (ra) is a systemic, chronic, incurable autoimmune inflammatory disease affecting approximately 0.5%-1% of the world population [1] . imbalanced immune responses both in circulating peripheral blood and in diseased joint cavities are closely related to the occurrence and development of ra [2] . it has been repeatedly illustrated that aberrant cd4 + helper t (th) cells produce proinflammatory cytokines, and abnormally activated b cells can differentiate into plasma cells that ultimately produce a large panel of autoantibodies [3, 4] . in addition, aberrant alterations of innate immune cells such as monocytes, macrophages, and natural killer (nk) cells, as well as dendritic cells (dcs), are also related to ra [5] [6] [7] . interferons (ifns) are a family of cytokines produced by various cells that play pivotal roles in early defense against viral infection in mammals [8, 9] . moreover, as important immunomodulators, ifns also although the etiology and pathogenesis of ra are still not fully understood, it has been demonstrated that ifnand ifn-related signaling pathways partly promote the inflammatory response in ra patients [13] . human ifn can be broadly grouped into three main classes: type i ifn, type ii ifn, and type iii ifn [14] . type i ifns consists of ifn-α, ifn-β, ifn-ε, ifn-κ, and ifn-ω, and type ii ifns consists of only ifn-γ [15] . type iii ifn was discovered approximately two decades ago and includes ifn-λ1, ifn-λ2, ifn-λ3 as well as ifn-λ4 [16] . in our study, we primarily discuss type i ifn (ifn-α and ifn-β) and type ii ifn due to their important roles in ra. individual case reports showed that patients were diagnosed with ra after using ifn-α to treat other diseases [17, 18] . previous studies have also reported that type i ifn response genes are potential biomarkers for predicting the development of ra [11, 19] . the relationship between ifns and ra is not only restricted to high clinical relevance but is also reflected in many molecular biology studies. ifn-α increases the production of proinflammatory cytokines such as interleukin (il)-1β and il-18 in synovial cells [20] . in contrast, ifn-β inhibits expression of il-1β and tumor necrosis factor (tnf) in peripheral blood mononuclear cells (pbmcs) [21] . moreover, ifn-β-treated collagen-induced arthritis (cia) mice exhibited relieved arthritis, suggesting that ifn-β exerts potential therapeutic effects [22] . beyond expectation, randomized clinical research on the treatment of ra using recombinant ifn-β negated the therapeutic effects of ifn-β in ra [23] . thus, the effects of ifns and their response genes and pathways in ra deserve further investigation. in the past decade, the evolution of high-throughput technologies have made it possible to detect the expression of tens of thousands of genes in one sample at the same time, giving us novel insights into the pathogenesis of ra [24] . nevertheless, the technologies and analytic tools of single-cell rna (scrna) sequencing are rapidly expanding and maturing [25, 26] . researchers are able to understand the mechanisms of ra in parallel to the dimensions of cell and gene expression with the development of scrna sequencing [26, 27] . in the present study, we comprehensively analyzed the discrepancies between ra and healthy controls in the terms of both scrna transcriptomics and conventional rna transcriptomics of pbmcs. alterations in type i ifn and ifns and their response genes and pathways, as well as the resulting potential impacts on the function of immune cells are discussed in detail. to comprehensively explore the disorders in pbmcs from ra, scrna sequencing data were preprocessed, and batch effects were removed (supplementary figure 1a-1d ). all cells were grouped to produce 27 clusters first, and all cells were visualized using the principal component analysis (pca)-based uniform manifold approximation and projection (umap) method ( figure 1b ). highly expressed marker genes in each cluster were calculated, and violin plots were used to illustrate the expression of several widely used marker genes in each cluster ( figure 1c ). in the present study, nk cells, monocytes, and t and b cells were identified ( figure 1d ), and these cells were conserved for further analysis. although plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) were also distinguished ( figure 1d ), the number of cells was too small to analyze (466 mdcs, 72 pdcs, and 42 megakaryocytes), so those cells were filtered out in subsequent experiments. moreover, the marker genes of cluster 18 and cluster 19 did not support them as any common or specific cell types in pbmcs, so they were considered as mixed cells ( figure 1d ). considering that clusters 18 and 19 had few cells (the number of all cells in the two clusters was no more than 300), these two clusters were also abandoned. gene set enrichment analysis (gsea) of nk cells, monocytes, t cells, and b cells was performed to reveal the differences between ra and healthy controls (hcs), and the gsea results indicated the widespread dysregulation of immune cells in peripheral blood from the ra patient (figure 2a-2d ). among all significant gene sets, it was not difficult to observe that the gene ontology (go) biological process (bp) pathway named "response_to_type_i_interferon" was upregulated in nk cells, monocytes, t and b cells from ra sample ( figure 2e-2h) . moreover, three other gene sets named "response_to_interferon_ alpha", "response_to_interferon_beta", and "response_to_interferon_gamma" were aging figure 1 . study design and preliminary analysis. (a) workflow of scrna sequencing and microarray data analysis. step 1: we first downloaded individual rheumatoid arthritis (ra) and healthy control (hc) peripheral blood mononuclear cell (pbmc) scrna sequencing data (gse159117 and gse149689) from the gene expression omnibus (geo) database (https://www.ncbi.nlm.nih.gov/geo/). to eliminate the potential batch effects, canonical correlation analysis (cca) was performed to integrate the two datasets. afterwards, four main immune cell types (t cells, b cells, nk cells and monocytes) in pbmcs were identified. then, we explored the up-and downregulated genes and gene sets using differential gene expression analysis and gene set enrichment analysis (gsea). subsequently, four main immune cell subtypes were identified using monocle2, and up-and downregulated interferon (ifn)-related genes and gene sets in different immune cell subtypes were also identified. step 2: key transcription factors were identified, and gene regulatory networks (grns) were constructed using single-cell regulatory network inference and clustering (scenic) analysis. step 3: we downloaded microarray datasets including multiple ra and hc pbmc samples and explored the up-and downregulated ifn-related genes and gene sets. aging also activated in nk cells from the ra patient ( figure 2a ). the above results indicate that ifn-α-, and ifn-βstimulated genes are comprehensively activated in ra peripheral blood. ifn-γ-stimulated genes are also upregulated in ra nk cells. moreover, there were differentially upregulated ifn-stimulated genes in nk cells, monocytes, t cells, and b cells from ra ( figure 2i ), indicating that there were different alterations influenced by ifn in distinct immune cell types. therefore, we further analyzed the alterations of ifn-α-, ifn-β-, and ifn-γ-stimulated genes in nk cells, monocytes, t cells, and b cells from the ra patient. to better explore the impacts of ifn-stimulated genes and signaling pathways in nks from ra, we reclustered nk cells and divided them into three subclusters: activated cd56 dim nk cells, inactivated cd56 dim nk cells and cd56 bright nk cells ( figure 3a , 3b). we then examined the transcriptional changes associated with ra in these three nk subclusters using gsea. the gsea results showed that ifn-γ stimulated signaling pathways in the three nk subclusters were all activated ( figure 3c ). moreover, type i ifn stimulated signaling pathways were activated in activated cd56 bright nk cells and activated cd56 dim nk cells ( figure 3c ). we also calculated differentially expressed genes (degs) in the three nk cell subclusters between the ra patient and hcs, and the results showed that some type i ifn-and ifn-γstimulated genes, such as stat1 and socs1, were significantly upregulated in nk cell subclusters ( figure 3d -3g). the above results all indicate that ifns are partially implicated in nk cell regulation, which eventually causes abnormal changes in the immune environment in peripheral blood. in addition, it is interesting that cd56 dim nk cells might produce more ifn-γ according to the results of differential gene expression analysis ( figure 3f ), even though cd56 dim nk cells primarily exert cytotoxic effects in pbmcs. we also examined functional changes in nk subclusters in ra, and the inactivated cd56 dim nk cells in ra exhibited lower activity in the kyoto encyclopedia of genes and genomes (kegg) pathway of "kegg_natural_killer_cell_mediated_c ytotoxicity" ( figure 3h ). in activated cd56 dim nk cells, expression of the cytotoxic effector molecules prf1 and gzmb was also significantly depressed ( figure 3i ). these results indicate that the cytotoxic capacity of cd56 dim nk cells is reduced in ra. importantly, it is reasonable to believe that those functional changes in ra nk cells are partly associated with the activation of ifn-stimulated genes and pathways based on previous studies [28] . in pbmcs, monocytes are commonly divided into cd14 ++ cd16 − (classical), cd14 ++ cd16 + (intermediate), and cd14 − cd16 ++ (nonclassical) populations according to their developmental processes [29] . here, we followed a similar classification and monocytes were split into cd14 + and cd16 + monocytes using single-cell trajectory analysis ( figure 4a , 4b). we then analyzed the activity of ifn-stimulated pathways by performing gsea. cd14 + and cd16 + monocytes were all activated by type i ifn ( figure 4c , 4d), and cd14 + monocytes were activated by ifn-γ simultaneously ( figure 4c , 4e). cd14 + monocytes are more inclined to migrate to local tissues with high levels of cd62l, and chemokines (cxcr2, ccr2), and cd14 + monocytes are also considered osteoclast precursors [29] [30] [31] . in our study, high hla-drb5 expression was also observed in cd14 + monocytes from the ra patient ( figure 4e ), indicating an inflamed and erosive osteoclast phenotype in ra joint cavities. in contrast, cd16 + monocytes primarily secreted inflammatory factors in pbmcs [5] . we also found that cd16 + monocytes from ra patients expressed significantly more ch25h ( figure 4d ), which could promote the inflammatory response in ra peripheral blood [32] . we propose that these inflammatory changes are caused by ifn's influence according to prior studies [33] . the heterogeneous nature of t cells makes investigation of t cells complex. for convenience, t cells were split into three subgroups: cd4 + t cells, cd8 + t cells, and naïve t cells via single-cell trajectory analysis ( figure 5a , 5b). gsea indicated that type i ifn-and ifn-γstimulated pathways were activated in cd4 + t cells and cd8 + t cells but not in naïve t cells ( figure 5c ). meanwhile, expression of ifn-stimulated genes such as irf7 was significantly increased in cd4 + t cells and cd8 + t cells from the ra patient ( figure 5d , 5e). in addition, levels of gzmh were increased in ra cd8 + t cells ( figure 5e ), which might also be caused by type i ifn activation [34] . for b cells, cell trajectory analysis was performed, and the track plot of b cell trajectories is shown in aging aging figure 6a . three b cell clusters included naïve b cells, plasma cells, and memory b cells according to their marker genes ( figure 6b ). gsea results showed that type i ifn and ifn-γ primarily affected naïve b cells ( figure 6c ), and upregulated type i ifnstimulated genes were also observed in ra naïve b cells ( figure 6d ). moreover, gsea results indicated that naïve b cells were one of the sources of type i ifn ( figure 6c ). remarkably, the gsea results showed that naïve b cells from ra were activated and tended to proliferate faster ( figure 6e ). egr1 has been demonstrated to promote b cell differentiation into plasma cells and support antibody secretion, and higher expression of egr1 was identified in naïve b cells from ra ( figure 6d ) [35] . the pseudotimes of b cells were also significantly different between the two groups, and ra plasma cells exhibited older pseudotimes ( figure 6f ), indicating that ra b cells are primarily concentrated at the end of the differentiation trajectory. moreover, the proportions of three b cell types in ra and hc were compared, and the results revealed that the number of plasma cells in ra was increased with the same proportion of naïve b cells ( figure 6g ). in addition, gsea results demonstrated that naïve b cells from ra tended to produce more of the proinflammatory factors il-1β and il-12 ( figure 6e ). interestingly, plasma cells in ra exhibited lower activity in the "response_to_interferon_gamma" pathway ( figure 6h ), and the expression of ifn-γstimulated genes, such as ifitm3 and ifi30, were downregulated in ra plasma cells ( figure 6i ). in accordance with previous studies, this might affect antibody class switching in ra plasma cells [36] . the effects of ifns on immune cells are complex. here, we focused on the regulation of downstream transcription factors and their target genes by ifns. we first calculated and compared the auc values of the regulon between ra and hc, and significantly differential regulons are illustrated in figure 7a . regulons of irf7_127g, stat1_115g, stat2_49g and stat2_extended_101g were significantly upregulated in most cell types. instead, the regulons of irf1_14g and uqcrb_19g were significantly decreased in most cell types ( figure 7a ). subsequently, we constructed grns in different immune cell types. many ifn-stimulated genes were included in the grns ( figure 7b ), and different cell types exhibited diverse ifn-stimulated genes, indicating different and fine regulation of gene expression in distinct immune cells. considering that the above results came from a single ra patient, we used external transcriptomic data from multiple individuals to explore the expression of ifnaging 2d). furthermore, we investigated alterations in ifnstimulated pathways and genes in different cell types in ra pbmcs. the results indicated that the effects of type i ifn were primarily concentrated in cd14 + monocytes (supplementary figure 3) , while the effects of ifn-γ were primarily concentrated in monocytes, effector memory cd4 + t cells and nk cells (supplementary figure 3) . in addition, the gsea results also suggested that nk cells in ra produced more ifn-γ (supplementary figure 3) , which is consistent with the results of scrna sequencing analysis. ifns are a series of natural cytokines that exert pivotal immunomodulatory activities [37] [38] [39] . both type i and ii ifn are presumed to be the bridge between innate immune and adaptive immune responses due to their functions in enhanced antigen presentation. from this point, the intensities of type i and ii ifn signaling pathways are crucial for the balance between selfreactivity and autoimmunity, illustrating that ifns play important roles in autoimmune diseases. type i ifn can be produced by many immune cell types, including dcs, monocytes, and macrophages [13] . t cells and nk cells are the major producers of ifn-γ [40] . type i and ii ifn can also act on immune cells in an autocrine fashion. the influences of ifn signaling on immune cells are comprehensive and involve multiple signaling pathways. the type i ifn receptor (ifnar) activates its downstream target kinases, including jak1 and tyk2, which promotes phosphorylation, dimerization, and nuclear translocation of stat1 and stat2 [41] . finally, the expression of a series of genes called ifn-stimulated genes is facilitated. ifn-γ also regulates gene expression by activating the jak-stat1 signaling pathway and other factors, including the ap-1, stat3, stat5, the map kinase signaling pathway, the pi3k signaling pathway and the nf-κb signaling pathway [42] . nk cells, t and b cells and monocytes are the crucial proportions of pbmcs, which are also involved in innate and acquired immune responses and are related to a series of autoimmune diseases. gsea is a popular and useful tool to identify specific up-or downregulated pathways. in the present study, we determined that ifnrelated signaling pathways were activated in pbmcs from ra microarray data using gsea and go and kegg pathways from the molecular signatures database (msigdb). then we focused on the abnormal expression of type i and ii ifn-related genes and gene sets, and consequent alterations in nk cells, monocytes, and t and b cells from ra peripheral blood. cd56 dim nk cells exert roles in the immune response primarily through there cytotoxic effects on abnormally activated t cells and macrophages, and cd56 bright nk cells primarily regulate the immune response by producing cytokines [43] . aberrant alterations of nk cells partially contribute to the progression of ra. it has been reported that there are increased cd56 bright nk cells in inflamed synovial joints, which also express more ifn-γ than nk cells in the peripheral blood [44] . previous research also demonstrated that nk cell activity was impaired in ra [45] . nk cell activation relies on the stimulation of type i ifn [28] . in our study, we found that both type i ifnand ifn-γ-stimulated signaling pathways were activated in activated cd56 dim nk cells and cd56 bright nk cells. notably, in agreement with prior studies, our study also suggested decreased cytotoxic effects of ra activated and inactivated cd56 dim nk cells [45] . it has been reported that nk cells stimulated with ifn-γ exhibit increased phosphorylation of stat1 or stat4, and different activation of stats leads to distinct nk cell phenotypes [28] . specifically, stat1 phosphorylation promotes the cytotoxic activity of nk cells, while stat4 phosphorylation promotes nk cell cytokine secretion [46, 47] . although a higher level of stat1 in ra-activated cd56 dim was detected, we speculated that the impaired nk cell activity in ra cd56 dim nk cells was related to ifn-γ stimulation and downstream stat4 phosphorylation. aberrant activation of monocytes also contributes to ra. cd14 + monocytes are more inclined to migrate to local tissues including joint cavities [29, 30] . a large number of peripheral cd14 + monocytes differentiate into osteoclasts in ra, ultimately leading to bone erosion. herein, our study indicated that cd14 + monocytes from ra exhibited increased hla-drb5 levels and ifn-γ signaling pathway activation. cd14 + cd16 + hla-dr + monocytes secrete high levels of tnf, which indicates that hla-dr + monocytes exhibit a potential proinflammatory phenotype [48] . ifn-γ has been considered an effective inducer of hla-dr expression on synovial monocytes by in vitro cell experiments [5] . taken together with the above results, we speculate that higher expression of hla-dr in cd14 + monocytes indicates stronger immune responses in ra peripheral blood. cd16 + monocytes in ra also exert momentous effects on ra by producing proinflammatory cytokines, such as il-1β, il-6, and tnf-α [5] . in our study, we found that ra cd16 + monocytes expressed more ch25h than hcs, accompanied by activation of the type i ifn signaling pathway. ch25h is an enzyme that catalyzes aging the formation of 25-hydroxycholesterol (25hc) from cholesterol, and it has been demonstrated that ch25h acts as an inflammatory signaling amplifier in macrophages [32, 49] . moreover, individuals with higher ch25h expression in synovial membranes are more likely to develop ra [50] . previous research has demonstrated that ch25h is an ifn-stimulated gene and that expression of ch25h is induced by ifn-α [33] . thus, we hypothesized that type i ifn promotes the expression of ch25h in ra cd16 + monocytes, which aggravated the inflammatory response in ra cd16 + monocytes. in addition, although expression of ch25h represents an inflammatory signal, the relationship between ch25h and ra needs further investigation because there are few relevant studies. consistently, cd4 + t cells occupy a core status in ra [51] . cd4 + t cells contain a cd4 + t cell subtype mixture of th1, th2, th17, and regulatory t cells (tregs). among them, enhanced th1 and th17 activities, as well as elevated ifn-γ and il-17, promote inflammatory responses both in synovial membranes and in pbmcs [52] . our study demonstrated that the ifn-γ stimulated signaling pathway was activated in ra cd4 + t cells, indicating that it enhanced cd4 + th1 polarization in ra. these results are consistent with previous studies [52] . cd8 + t cells also function in ra, but their precise roles in ra pathogenesis are still unclear [53] . some animal studies have demonstrated that cd8 + t cells have proinflammatory effects via cytotoxicity [54] . however, other studies have indicated that cd8 + t cells play a regulatory role in inflamed joints [55] . in the present study, we found that gzmh, a cytotoxic gene, was upregulated in ra cd8 + t cells, supporting the view that the cytotoxicity of cd8 + t cells is enhanced in ra. it is also known that ifn-γ can enhance the cytotoxicity of cd8 + t cells. therefore, we hypothesized that the cytotoxicity of cd8 + t cells was elevated and was associated with the activation of ifnγ-stimulated pathways [34] . in ra, unnatural b cell activation leads to the production of autoantibodies, including anti-cyclic citrullinated peptide (anti-ccp) and rheumatoid factor (rf). unsurprisingly, we found that naïve b cells in ra exhibited stronger proliferation and activation, accompanied by the activation of activation of type i ifn. it has been previously demonstrated that type i ifn boosts b cell proliferation and differentiation to plasma cells [56] . egr1, a type i ifn-stimulated gene, was also upregulated in ra naïve b cells [35] . thus, we hypothesized that the activation of type i ifn partially enhanced b cell differentiation into plasma cells. activated b cells also regulate immune responses by secreting cytokines [36] . both proinflammatory cytokines and anti-inflammatory cytokines can be produced by b cells, depending on the stimulus [57] . it has been reported that b cells from the autoimmune disease multiple sclerosis produce less of the antiinflammatory cytokine il-10 [58] . here, our study indicated that naïve b cells in ra had the potential to produce the proinflammatory cytokines il-1β and il-12, which might aggravate the inflammatory status in ra peripheral blood. whether the cytokines from ra b cells are related to type i ifn and ifn-γ activation remains to be further investigated. inhibition of the ifn-γ signaling pathway in ra plasma cells is another interesting phenomenon observed in the present study. previous studies have demonstrated that ifn-γ intervenes in plasma cell isotype switch recombination by promoting igg2a production and inhibiting igg1 production [59, 60] . coincidentally, igg1 and igg4 are the primary subtypes of anti-ccps in ra [61, 62] . as a result, we conjectured that the inhibited ifn-γ signaling pathway in plasma cells might be associated with plasma cell isotype switch recombination in ra, which eventually promotes the production of autoantibodies. finally, ifn-regulated pathways and genes are complex and numerous. therefore, we attempted to use scenic to evaluate the activity of each transcription factor and their target genes. we found that the activities of the regulons irf7_127g, stat1_115g, stat2_49g, stat2_extended_101g, irf1_14g and uqcrb_19g were significantly increased or decreased in most cell types. there were several imbalanced transcription factors in ra, such as irf1, irf7, stat1, and stat2. they could be regulated by ifn, addressing the important ifn effects in ra [63] [64] [65] . furthermore, we constructed grns by integrating the results of scenic analysis and deg analysis in each cell type. our grns might not be comprehensive due to the strict calculations of degs. moreover, only a single ra sample with missing clinical characteristics was used in the main analysis of the present study, which limited the generalizability of the findings. further research is required to explain the significance of these transcription factors in ra. once their expression and functions are identified, these transcription factors could be used as potential targets in the treatment of ra due to their extensive influence instead of partial alterations in ra. knowledge of the relationship between ifn and ra is tortuous because ifn participates in multiple regulatory effects on the immune system [66] . initially, type i ifn was used as a treatment option [67] . subsequently, the aging promoting effects of ifn-stimulated signaling pathways on ra have been gradually recognized [68] . the type i ifn signature represents type i ifn response genes and pathways, that are activated in ra patients, which may play a role in the potential development of ra [11] . meanwhile, contradictory findings indicated the dual effects of ifn-γ on ra, and our findings demonstrated that ifn-γ-stimulated pathways were not always activated in all cell types in ra [69] . in the present study, we comprehensively analyzed the alterations of type i ifn-and ifn-γ-stimulated pathways in ra. parts of the findings from the transcriptome analysis corroborate previous studies. the present study indicated that ifn signaling pathways are activated in ra patients, in agreement with prior investigations. a previous study showed that cd56 dim nk cells from ra patients are impaired, and our study supported that conclusion [45] . enhanced th1 activity and imbalanced th1/th2 cells in ra were detected very early [51] . our research also found enhanced ifn-γ signaling pathway in ra cd4 + t cells, indicating that th1 polarization is enhanced in ra cd4 + helper t cells. however, the subtypes of cd4 + helper t cells are diverse, and the relationship between the th17/treg balance and ra has been observed in prior research [51] . how ifn signaling pathways and cd4 + helper t cell differentiation influence each other needs to be clearly demonstrated in future studies. our study also delivers some novel findings. we linked disease promotion of cd56 bright and cd56 dim nk cells in ra with activated ifn signaling pathways. our research also indicated that the activated type i ifn signaling pathway might promote production of the inflammatory signaling amplifier ch25h. in addition, ra patient primary b cells tended to differentiate into plasma cells, and our investigation demonstrated that the type i ifn signaling pathway might be one of the factors causing this differentiation. our analysis also demonstrated that the ifn-γ signaling pathway in ra was activated, which might influence antibody class switching in ra plasma cells and autoantibody production. finally, scenic was used to identify key transcription factors in different immune cells in ra, and grns were also constructed to reveal the mechanism of transcription regulation, which provides directions for future research on ifn signaling pathways in ra. although this study comprehensively explored the effects of abnormal type i and ii ifn signaling pathways in ra pbmc immune cell subtypes, only one ra scrna sample was used in our study, and the clinical characteristics of the ra patient were unknown. thus, we suggest that further studies should focus on ifn-stimulated signaling pathways in ra using multiple samples and corresponding clinical information. additionally, it is crucial to distinguish different inf signal alterations in distinct cell types to obtain a better cell classification performance, cell sorting is worth using before performing scrna sequencing. regardless, it is questionable whether ifn signaling pathways directly and/or indirectly promote the occurrence and development of ra according to previous studies. the relationship between ifn signaling pathways and ra immune cells should be carefully evaluated with a series of in vitro and in vivo experiments using a combination of novel sequencing technologies and traditional molecular biology techniques. both pbmc scrna sequencing data and mrna expression microarray data were downloaded from the gene expression omnibus (geo) database (https://www.ncbi.nlm.nih.gov/geo/). pbmc scrna sequencing data of one ra patient and two hcs were obtained from gse159117 and gse149689, respectively [70, 71] . the mrna expression data of 232 ra and 43 hc pbmc samples, which were preprocessed by frozen robust multi-array analysis (frma) with batch effects corrected, were obtained from gse93272 [72] . microarray data preprocessed by frma of several immune cell subtypes in pbmcs were obtained from gse93776 [72] . the design of this research is shown in figure 1a . for scrna sequencing data, the r package seurat (version 4.0.0) was used to preprocess the scrna sequencing data [73] [74] [75] [76] . first, cells were included if they met all three of the following parameters: (1) the number of genes in each cell was greater than 500; (2) the total number of molecules in a cell was greater than 1000 and less than 15000; and (3) the mitochondrial gene expression ratio was less than 5% and 15% for gse159117 and gse149689, respectively. two geo series scrna data were normalized using "normalizedata" function, and 3000 highly variable genes were identified using "findvariablefeatures". second, two geo series were integrated in seurat using the canonical correlation analysis (cca) method with the "findintegrationanchors" and "integratedata" functions. subsequently, the pca method was performed for dimension reduction after data scaling, and the top 30 principal components were selected to perform the downstream analysis. the umap algorithm was used to visualize and explore the data. cell clusters were identified by the function "findneighbors" using the knearest neighbors (knn) algorithm and the function "findclusters" with a resolution of 1.25. we calculated marker genes to annotate cell clusters to specific immune cell types. the following marker genes were used for cell type annotation: cd3d, klrf1, cd79a, cd68, lilra4, cd1c, and tubb1 [77] [78] [79] [80] . correspondence between marker genes and cell types is shown in figure 1c . considering that the numbers of pdcs, mdcs, and megakaryocytes were too small, they were excluded from all subsequent analyses. to estimate the dissimilar functions of the different immune cell types in detail, the four main classes of immune cells (nk cells, monocytes, t cells, and b cells) were further divided according to the general classification criterion and certain marker genes. nk cells identified above were chosen, and reclustering was performed in seurat using the same pipeline as previously described. nk cells were then subdivided into three clusters, and the following genes were used for nk cell subtype annotation: prf1, gzma, gzmb, gnly (activated cd56 dim nk cells), gzmh (inactivated cd56 dim nk cells), and gzmk and sell (cd56 bright nk cells) [81, 82] . for monocytes, t cells and b cells, the r package monocle (version 2.18.0) was used to perform single-cell trajectory analysis, and pseudotime trajectories were constructed using the ddrtree algorithm [83] . subsequent genes were used for cell subtype identification: cd14 (cd14 + monocytes), fcgr3a (cd16 + monocytes), ccr7, sell (naïve t cells), cd4 (cd4 + t cells), cd8a (cd8 + t cells), tcl1a (naïve b cells), prdm1 (plasma cells), fas, cd80 and cd27(memory b cells) [78, [84] [85] [86] . degs between cells from two individuals are important for determining their potential distinct biological functions. to identify degs between ra and hc in different immune cell subtypes, we used the findmarkers function in seurat to evaluate them and set min.pct = 0.1, logfc.threshold = 0.25, only.pos = false, and only genes with adjusted p-value < 0.05 were retained. gsea was performed using the r package clusterprofiler (version: 3.18.1) [87] . gene set files were downloaded from http://www.gsea-msigdb.org/gsea/ downloads.jsp, and all go gene sets and kegg gene sets were used for enrichment analysis. for scrna sequencing data, genes were ranked using the "findmarkers" function in seurat. for microarray data, gene log2 (fold-change) values were calculated using the r package limma (version: 3.46.0), and all genes were decreasingly ranked by their log2 (fold-change) values [88] . the normalized enrichment score (nes) was used to assess the results of gene set enrichment. pathways with an adjusted p-value<0.05 and |nes|>1 were considered significant. gsea plots were created using the r package enrichplot (https://yulabsmu.top/biomedical-knowledge-mining-book/, version: 1.10.2). bar plots were generated using the r package ggpubr (https://cran.r-project.org/package=ggpubr, version: 0.4.0). scenic is a computational method to infer grns from single-cell rna-seq data [89] . scenic analysis was performed according to the official workflow. we used the r package scenic (version: 1.2.4) and the grnboost2 algorithm in the python package arboreto (version: 0.1.5) to assess the gene regulatory relationships in subclasses of pbmcs [90] . genes that were expressed at either very low levels or in too few cells were removed first; subsequently, we split the targets into positive-and negative-correlated targets by calculating the correlation in r. gene coexpression networks were then constructed using the grnboost2 algorithm in python and scenic in r. auc values of the regulon were calculated to measure the activity of the regulon. a heat map of regulon auc values in each cell type was illustrated and clustered using r the package pheatmap (version: 1.0.12). finally, the auc values of the regulon in each subclass cell were compared using the limma package between the ra patients and hcs. only a regulon with a logfc≥0.05 and adjusted p-value < 0.05 was considered significantly different between ra and hc groups. finally, genes belonging to both different regulons between ra and hc and degs identified using the findmarkers function were used for grn construction. we compared b cell pseudotime between ra and hc by using mann-whitney u test by function aging "wilcox.test" in r. differences in b cell subtype proportions were compared using fisher's exact test using the function "pairwise.fisher.test" in the r package fmsb (https://cran.r-project.org/package= fmsb, version: 0.7.0), and p-values were adjusted using a benjamini-hochberg (bh) method. a p-value < 0.05 was considered significant. publicly available datasets were analyzed in this study. this data can be found here: https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=gse159117; https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse149689; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=g se93272; https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=gse93776. rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies pathogenic role of immune cells in rheumatoid arthritis: implications in clinical treatment and biomarker development. cells understanding the role of cytokines in the pathogenesis of rheumatoid arthritis b cells in rheumatoid arthritis: from pathogenic players to disease biomarkers monocytes in rheumatoid arthritis: circulating precursors of macrophages and osteoclasts and, their heterogeneity and plasticity role in ra pathogenesis natural killer cells and their role in rheumatoid arthritis: friend or foe? the function of myeloid dendritic cells in rheumatoid arthritis role of ifn and complements system: innate immunity in sars-cov-2 the interferon (ifn) class of cytokines and the ifn regulatory factor (irf) transcription factor family type-i interferon responses: from friend to foe in the battle against chronic viral infection the type i ifn signature as a biomarker of preclinical rheumatoid arthritis increased expression of interferon (ifn)-gamma together with ifn-gamma receptor in the rheumatoid synovial membrane compared with synovium of patients with osteoarthritis type i interferon in rheumatic diseases regulating immunity to mycobacterium tuberculosis infection the interferon system: an overview type iii interferons in viral infection and antiviral immunity rheumatoid arthritis induced by alpha-interferon therapy interferon-alpha-associated polyarthritis. possible induction of seropositive rheumatoid arthritis by interferon-alpha: two case reports and review of the literature patients with systemic lupus erythematosus, myositis, rheumatoid arthritis and scleroderma share activation of a common type i interferon pathway type i interferons might form the link between toll-like receptor (tlr) 3/7 and tlr4-mediated synovial inflammation in rheumatoid arthritis (ra) interferon-beta not only inhibits interleukin-1beta and tumor necrosis factor-alpha but stimulates interleukin-1 receptor antagonist production in human peripheral blood mononuclear cells amelioration of collagen-induced arthritis and suppression of interferon-gamma, interleukin-12, and tumor necrosis factor alpha production by interferon-beta gene therapy a multicentre, randomised, double blind, placebo controlled phase ii study of subcutaneous interferon beta-1a in the treatment of patients with active rheumatoid arthritis rna sequencing: the teenage years exponential scaling of single-cell rna-seq in the past decade singlecell rna-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation accelerating medicines partnership rheumatoid arthritis and systemic lupus erythematosus (amp ra/sle) consortium. defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry natural killer cells and interferon gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets intermediate monocytes contribute to pathologic immune response in leishmania braziliensis infections the proliferative human monocyte subpopulation contains osteoclast precursors 25-hydroxycholesterol acts as an amplifier of inflammatory signaling interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol interferonγ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity transcription of prdm1, the master regulator for plasma cell differentiation, depends on an sp1/sp3/egr-1 gc-box b cells responses and cytokine production are regulated by their immune microenvironment immunomodulatory functions of type i interferons type i interferons: distinct biological activities and current applications for viral infection the dual nature of type i and type ii interferons innate ifn-γ-producing cells developing in the absence of il-2 receptor common γ-chain regulation of type i interferon responses expression patterns of nkg2a, kir, and cd57 define a process of cd56dim nk-cell differentiation uncoupled from nk-cell education a subset of natural killer cells is greatly expanded within inflamed joints a significantly impaired natural killer cell activity due to a low activity on a per-cell basis in rheumatoid arthritis interferon alpha/beta-mediated inhibition and promotion of interferon gamma: stat1 resolves a paradox critical role for stat4 activation by type 1 interferons in the interferon-gamma response to viral infection the proinflammatory cd14+cd16+dr++ monocytes are a major source of tnf cdna cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism the cholesterol biosynthesis pathway regulates il-10 expression in human th1 cells the central role of t cells in rheumatoid arthritis reduction of peripheral blood t cells producing ifn-γ and il-17 after therapy with abatacept for rheumatoid arthritis potential roles for cd8(+) t cells in rheumatoid arthritis inflammatory polyarthritis induced by mercuric chloride in the brown norway rat collagen-induced arthritis in cd4-or cd8-deficient mice: cd8+ t cells play a role in initiation and regulate recovery phase of collagen-induced arthritis role of production of type i interferons by b cells in the mechanisms and pathogenesis of systemic lupus erythematosus distinct profiles of human b cell effector cytokines: a role in immune regulation? bar-or a. distinct effector cytokine profiles of memory and naive human b cell subsets and implication in multiple sclerosis regulation of human igg subclass production by cytokines. ifn-gamma and il-6 immunol ifn-gamma regulates the isotypes of ig secreted during in vivo humoral immune responses igg1 and igg4 are the predominant subclasses among auto-antibodies against two citrullinated antigens in ra igg subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin irf1 is critical for the tnf-driven interferon response in rheumatoid fibroblast-like synoviocytes : jakinibs suppress the interferon response in ra-flss activation of the stat1 pathway in rheumatoid arthritis identification of molecules associated with response to abatacept in patients with rheumatoid arthritis heterogeneity of the type i interferon signature in rheumatoid arthritis: a potential limitation for its use as a clinical biomarker type i interferon gene response is increased in early and established rheumatoid arthritis and correlates with autoantibody production new insights into ifn-γ in rheumatoid arthritis: role in the era of jak inhibitors cd127 imprints functional heterogeneity to diversify monocyte responses in human inflammatory diseases immunophenotyping of covid-19 and influenza highlights the role of type i interferons in development of severe covid-19 multiomics monitoring of drug response in rheumatoid arthritis in pursuit of molecular remission integrated analysis of multimodal single-cell data comprehensive integration of single-cell data integrating single-cell transcriptomic data across different conditions, technologies, and species spatial reconstruction of single-cell gene expression data metacell: analysis of single-cell rna-seq data using knn graph partitions single-cell landscape of immunological responses in patients with covid-19 cd68/macrosialin: not just a histochemical marker genomics and transcriptomics of megakaryocytes and platelets: implications for health and disease discordant regulation of granzyme h and granzyme b expression in human lymphocytes differential expression of human granzymes a, b, and k in natural killer cells and during cd8+ t cell differentiation in peripheral blood the dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells immune cell profiling of covid-19 patients in the recovery stage by single-cell sequencing prdm1/blimp-1 is expressed in human b-lymphocytes committed to the plasma cell lineage memory b cells clusterprofiler: an r package for comparing biological themes among gene clusters limma powers differential expression analyses for rna-sequencing and microarray studies scenic: single-cell regulatory network inference and clustering pmid:30445495 aging supplementary figure 2. upregulated interferon (ifn)-stimulated signaling pathways and genes in rheumatoid arthritis (ra) peripheral blood. (a, b) bar plots of selected results of gse93272 gene set enrichment analysis (gsea) indicated upregulated type i ifn (a) and ifn-γ (b) stimulated signaling pathways in ra. (c) violin plots of the top 10 upregulated genes in the type i ifn-stimulated signaling pathway. (d) violin plots of the top 10 upregulated genes in the ifn-γ-stimulated signaling pathway upregulated interferon (ifn)-stimulated signaling pathways in immune cells from rheumatoid arthritis (ra) peripheral blood. bar plots of selected results of gse93776 gene set enrichment analysis (gsea) showing activated ifnstimulated signaling pathways in ra peripheral blood. the horizontal axis represents the normalized enrichment score the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this study was supported by the national natural science foundation of china (81874383, 82074267) and the chinese medicine scientific research project of the health commission of hubei province (zy2021q024). key: cord-0021971-aneqe5h1 authors: kwon, eui-jong; ju, ji hyeon title: impact of posttranslational modification in pathogenesis of rheumatoid arthritis: focusing on citrullination, carbamylation, and acetylation date: 2021-09-30 journal: int j mol sci doi: 10.3390/ijms221910576 sha: c764622c7b8a332da036369a0e5e1358b8c18d8f doc_id: 21971 cord_uid: aneqe5h1 rheumatoid arthritis (ra) is caused by prolonged periodic interactions between genetic, environmental, and immunologic factors. posttranslational modifications (ptms) such as citrullination, carbamylation, and acetylation are correlated with the pathogenesis of ra. ptm and cell death mechanisms such as apoptosis, autophagy, netosis, leukotoxic hypercitrullination (lth), and necrosis are related to each other and induce autoantigenicity. certain microbial infections, such as those caused by porphyromonas gingivalis, aggregatibacter actinomycetemcomitans, and prevotella copri, can induce autoantigens in ra. anti-modified protein antibodies (ampa) containing anti-citrullinated protein/peptide antibodies (acpas), anti-carbamylated protein (anti-carp) antibodies, and anti-acetylated protein antibodies (aapas) play a role in pathogenesis as well as in prediction, diagnosis, and prognosis. interestingly, smoking is correlated with both ptms and ampas in the development of ra. however, there is lack of evidence that smoking induces the generation of ampas. rheumatoid arthritis (ra), the most common form of chronic inflammatory arthritis, is mainly targeting synovial joints [1] [2] [3] . however, ra is also a systemic autoimmune disease that involves not only joints but other organs such as the lungs, pericardium, sclera, peripheral nerves, skin, and vessels [4] [5] [6] . untreated ra destroys the articular cartilage and nearby bones, resulting in functional disability [7, 8] . the current strategy for ra treatment focuses on early and aggressive management before irreversible articular damage [7, 9] . thus, recent research has focused on events occurring before the presentation of ra; specifically, the pathogenesis and preclinical stage. the 2010 american college of rheumatology (acr)-european league against rheumatism (eular) criteria are often used as the basis for a diagnosis of ra (table 1) . the new scoring system results in a score of 0-10, and a score ≥ 6 is considered satisfactory for the diagnosis of ra. the 2010 acr-eular criteria include anti-citrullinated table 1 . scoring for rheumatoid arthritis (ra) diagnosis. joint involvement 1 large joint (shoulder, elbow, hip, knee, ankle) 0 2-10 large joints 1 1-3 small joints (mcp, pip, thumb ip, mtp, wrists) 2 4-10 small joints 3 more than 10 joints (including at least 1 small joint) 5 serologic study negative rf and negative acpas (under uln) 0 low-positive 1 rf or low-positive acpas 2 high-positive 2 rf or high positive acpas 3 acute phase reactants normal crp and normal esr 0 abnormal crp or abnormal esr 1 duration of symptoms <6 weeks 0 ≥6 weeks 1 ing b cell differentiation into plasma cells, which secrete autoantibodies such as rf and acpas [74] . autoantibodies bind to autoantigens to form immune complexes, which activate the complement system. furthermore, t h cells produce numerous cytokines such as interferon (ifn)-γ, tnf-α, lymphotoxin-β, ils (especially il-6 and il-17), and granulocyte-macrophage colony-stimulating factor (gm-csf). rituximab targets cd20, sometimes called "medical splenectomy" and is used for ra treatment because it depletes b cells [75] . several groups of t cells, called effector t cells (t eff cells), rapidly respond to these stimuli and activate macrophage-like synoviocytes and fls [76] . activated macrophagelike synoviocytes release proinflammatory cytokines including tnf-α and ils (il-1, il-6, il-12, il-15, il-18 and il-23), whereas activated fls produce il-1, il-6, and tnf-α [77] . recent theory suggests that t h17 cells are more important than t h1 cells are in ra pathophysiology [78, 79] . t h17 cells secrete il-17, which enhances the upregulation of inflammatory cytokine production in synoviocytes, including tnf-α, il-1 and il-6. it also mediates neutrophil, granulopoiesis, and osteoclast differentiation. il-23 from t h17 cells affects the activity and glycosylation of autoantibodies [80] . the t h1 /t h17 ratio in patients with ra, is inversely proportional to disease activity [81] . the janus kinase (jak)/stat signaling pathway in these inflammatory cells amplifies the immune response [82] . other intracellular signaling pathways are also involved, such as spleen tyrosine kinase (syk), mitogen-activated protein kinases (mapks), and nf-κb [83] . inhibition of these pathway is one of the mechanisms of action of small-molecule drugs. jak inhibitors such as tofacitinib, baricitinib, and upadacitinib have been approved for the treatment of ra by the us food and drug administration (fda) [84] . fostamatinib, which blocks syk, usually used in chronic immune thrombocytopenia (itp), has been in trials for ra treatment [85] [86] [87] . regulatory t (t reg ) cells expressing cd25 and the transcription factor forkhead box p3 (foxp3) regulate other immune cells to maintain tolerance [88] . some studies suggest that the loss of cytotoxic t lymphocyte antigen 4 (ctla-4) expression interferes with the suppressive role of t reg cells [89] . abatacept, a fusion protein including the constant fragment (fc) region of immunoglobulin (ig) g1 and the extracellular part of ctla-4, competes with cd28 of t cells such as t reg cells. because the binding affinity of abatacept to cd80/86 of apc is higher than that to cd28, abatacept modulates excessive immune responses in ra [72] . tnf-α stimulates the proliferation of t cells and b cells and activates fls to produce matrix metalloproteinases (mmps) [1, 90] . autophagy is upregulated in fls after tnf-α exposure, and ra patients show higher levels of autophagy than normal persons do [91] . tnf-α also upregulates adhesion molecules on endothelial cells and their pathological neovascularization [1] . production of il-1, il-6, and gm-csf is accelerated by tnf-α [92] , whereas il-6 interacts with tnf-α to promote the cell cycle for the proliferation of fls, leading to ra induction [93, 94] . tnf-α induces the expression of dickkopf-1 (dkk-1), which downregulates the wnt receptors of osteoblast precursors [94] . tnf-α also interacts with osteoclast precursors and osteoblasts (obs), leading to pathological bone destruction in ra by fusing osteoclast precursors to form activated osteoclasts (ocs), stabilizing ob, inducing osteocyte apoptosis, and enhancing bone absorption [95] [96] [97] . figure 1 shows a schematic illustration of ra pathogenesis. figure 1 . schema of rheumatoid arthritis (ra) pathogenesis. black, blue, and red arrows indicate differentiation, secretion of proinflammatory molecules, and stimulation, respectively. broken red arrow indicates stimuli mediated by specific molecules. abbreviations: tlr, toll-like receptor; cd, cluster of differentiation; ctla-4, cytotoxic t lymphocyte antigen-4; mhc, major histocompatibility complex; tcr, t cell receptor; th cell, helper t cell; th1 cell, helper th1 cell; th17 cell, helper th17 cell; treg cell, regulatory t cell; rf, rheumatoid factor; acpas, anticitrullinated protein/peptide antibodies; ic, immune complex; autoag, autoantigen; egf, epidermal growth factor; tgf-β, transforming growth factor-β; ifn-γ, interferon-γ; il, interleukin; mmp, matrix metalloproteinase; fls, fibroblast-like synoviocytes; oc, osteoclast; ob, osteoblasts [3] . created with biorender.com. ai, r. et al. [98] found that there are epigenetically similar regions in the fls of ra patients, including a study that found that huntingtin-interacting protein-1 (hip-1) in the huntington's disease signaling pathway is correlated with fls in ra patients. the action of hip-1 is related to fls invasion of the matrix, which regulates the severity of ra [98, 99] . air pollutants appear to play a role in ra pathogenesis, and the acpas titer could be predicted by exposure to particulate matter with a diameter ≤ 2.5 μm (pm 2.5) [11] . in addition, ozone exposure and living near high-traffic roads were recognized as risk factors for ra in a meta-analysis [100] . a body mass index (bmi) indicating adiposity is linked to the risk of ra development, and this is more significant in women [13] . some studies have shown that the gut microbiome and its metabolites may induce ra by stimulating th17 cells of mucosal immune tissue that control the production of autoantibodies [101] [102] [103] [104] . disease-modifying antirheumatic drugs (dmards) are a group of drugs that regulate the activity of ra. in recent decades, biological dmards have been developed based on the pathophysiology of ra, particularly targeting certain molecules or pathways. table 2 shows a list of biologic dmards targeting cytokines and cell-surface molecules approved by the fda [84] . schema of rheumatoid arthritis (ra) pathogenesis. black, blue, and red arrows indicate differentiation, secretion of proinflammatory molecules, and stimulation, respectively. broken red arrow indicates stimuli mediated by specific molecules. abbreviations: tlr, toll-like receptor; cd, cluster of differentiation; ctla-4, cytotoxic t lymphocyte antigen-4; mhc, major histocompatibility complex; tcr, t cell receptor; t h cell, helper t cell; t h1 cell, helper th1 cell; t h17 cell, helper th17 cell; t reg cell, regulatory t cell; rf, rheumatoid factor; acpas, anticitrullinated protein/peptide antibodies; ic, immune complex; autoag, autoantigen; egf, epidermal growth factor; tgf-β, transforming growth factor-β; ifnγ, interferon-γ; il, interleukin; mmp, matrix metalloproteinase; fls, fibroblast-like synoviocytes; oc, osteoclast; ob, osteoblasts [3] . created with biorender.com. ai, r. et al. [98] found that there are epigenetically similar regions in the fls of ra patients, including a study that found that huntingtin-interacting protein-1 (hip-1) in the huntington's disease signaling pathway is correlated with fls in ra patients. the action of hip-1 is related to fls invasion of the matrix, which regulates the severity of ra [98, 99] . air pollutants appear to play a role in ra pathogenesis, and the acpas titer could be predicted by exposure to particulate matter with a diameter ≤2.5 µm (pm 2.5) [11] . in addition, ozone exposure and living near high-traffic roads were recognized as risk factors for ra in a meta-analysis [100] . a body mass index (bmi) indicating adiposity is linked to the risk of ra development, and this is more significant in women [13] . some studies have shown that the gut microbiome and its metabolites may induce ra by stimulating t h17 cells of mucosal immune tissue that control the production of autoantibodies [101] [102] [103] [104] . disease-modifying antirheumatic drugs (dmards) are a group of drugs that regulate the activity of ra. in recent decades, biological dmards have been developed based on the pathophysiology of ra, particularly targeting certain molecules or pathways. table 2 shows a list of biologic dmards targeting cytokines and cell-surface molecules approved by the fda [84] . citrullination is a process that converts the amino acid arginine to citrulline [46] , and it is catalyzed by a ca 2+ -dependent enzyme, pad [105, 106] . every event where citrulline is converted to arginine increases the mass by 0.984 da and the loss of one positive charge [105] . this posttranslational modification (ptm) alters acidity, which affects the isoelectric point (pi), ability to form hydrogen bonds, interaction with other amino acid residues, and protein unfolding [107, 108] . furthermore, these changes can influence the function and half-life of the associated proteins [109] , suggesting that citrullination may create a new protein [106, 110] . the formation of a citrullinated protein also suggests the possibility of generating new epitopes that could act as a new autoantigen that escapes self-immune tolerance [111] . citrullinated peptides have a higher binding affinity to the hla-drb1 (drb1*0401 or *0404) antigen-binding groove than to the corresponding arginine-containing peptide [112] . the ca 2+ dependency of pad, which catalyzes citrullination, may be the main switch for the regulation of citrullination in the body [68] . pad requires not only ca 2+ but also a reducing environment to maintain free thiol cystine activity [113] . the oxidative environment of the extracellular space inhibits citrullination [114] . furthermore, the body regulates the concentration of ca 2+ using numerous channels and hormones by investing energy. citrulline-specific cd4+ t cells have been found in both human and mouse models [115, 116] and citrulline-specific t h1 and t h17 cells are increased in ra patients [117, 118] . the sequence of human citrullinated enolase peptide-1 (cep-1) is similar to that of the α-enolase of p. gingivalis; the anti-cep-1 antibodies and the enolase of p. gingivalis also have cross-reactivity with equivalent epitopes [119] . administration of glucocorticoids as an ra treatment may reduce the level of citrullination [120, 121] . a significantly higher proportion of citrullinated protein has been detected in synovial biopsy samples from ra patients than in the synovium of healthy individuals [120] . in chronic obstructive pulmonary disease (copd) patients and smokers, vimentin levels are increased [122] . copd patients without ra sometimes show positive test results for anti-mutated citrullinated vimentin antibodies (anti-mcv), one of the acpa tests, and anti-mcv positivity is correlated with the manifestation of a severe form of extra-articular ra [123] . additionally, pad2 expression accelerates citrullination in the lungs, which is enhanced by smoking [124, 125] . in a rat model of autoimmune encephalitis, odoardi et al. [126] demonstrated that the autoimmune cells acquire the capacity to enter the cns only after residing within the lung tissue; first the autoimmune cells drained to lymphatics of airways, then entered to blood circulation to reach for the cns. in conclusion, valesini et al. [46] hypothesized that citrullination in the lung could be an extra-articular factor in the origin of ra autoimmunogenicity that generates lung-resident autoreactive t cells, which migrate to other target organs by the upregulation of chemokine receptors-as in case of a rat model of autoimmune encephalitis [126] . pad, which hydrolyzes guanidinium side chains in peptidylarginine to peptidylcitrulline and ammonia [30], belongs to another larger group of enzymes called the amidinotransferase superfamily. isoforms of pad share approximately 50% sequence similarity [113] . pad5, a designation not currently used, was once considered to be a human homolog of rodent pad4; however, it was proved that the pad5 is identical to pad4, as indicated by expression, sequence data, and genomic organization [30] . another notable type of pad is found in eukaryotes. the pad of p. gingivalis (ppad), the major pathogen of periodontitis, is independent of the ca 2+ concentration [127, 128] , which makes it active at higher ph, and it has a preference for c-terminal arginine citrullination, regardless of whether it is the peptide-bound or free form [128, 129] . when arginine gingipains get activated, bacterial enzymes similar to human trypsin, they cut polypeptides into short peptide fragments with a c-terminal arginine [46] . then, ppad rapidly citrullinates c-terminal arginine in the fragment [130] . table 3 shows the site of expression and substrate of the five isoforms of pad in addition to those of ppad. there are five pad isoenzymes. pad2 stabilized by the ntz, substrates β-catenin. trichohyalin is a major structural protein in hair follicles. note that pad4 has a homodimer structure, the only type of pad localized to the cell nucleus. pad4 and ppad can autocitrulate to generate antibodies. pad6 is uniquely expressed in germ cells, and its precise role is unclear; it is thought to be essential for germ cell-specific structures in zygote/embryo development. ppad is independent of ca 2+ concentration and active at higher ph; it prefers c-terminal arginine citrullination. autocitrullination is occasionally considered to mediate the inactivation and regulation of enzymes, but its definite role is still controversial [48, 146, 147] . pad4 has multiple citrullination sites including the arg-372 and arg-374 of pad, which are considered potential autocitrullination targets. autocitrullination modifies the structure of pad4 and may increase its recognition by autoantibodies [48] . activity of pad and ppad is elevated in patients with both ra and periodontitis [147] . citrullination by ppad may induce an immunologic response against citrullinated proteins in ra patients with periodontitis and se [46] . a higher igg anti-p. gingivalis antibody titer is associated with hla-drb1 neutral alleles [148] . the roles of pad2 and pad4 have been identified in ra, and they have been detected in macrophages of the sf of ra patients (ra-sf) and granulocytes isolated from the synovium of a mouse arthritis model, respectively [149, 150] . autocitrullination of pad4 enhances the chance of its recognition by autoantibodies, and anti-pad4 antibodies have predictive and prognostic value in ra [48, 151] . ppad also undergoes autocitrullination and generates antibodies that are cross-reactive with other citrullinated proteins of the human body [128] . however, in contrast with anti-pad4 antibodies, anti-ppad antibodies have no correlation with disease activity or acpas levels [142] . acpas are a group of antibodies that sense citrulline-containing proteins/peptides and share partial cross-reactivity [152, 153] . an in vitro study showed that 66% of acpas showed cross-reactivity with different epitopes, whereas 33% were mono-active [154] . acpa is a well-established diagnostic serology test molecule for ra, with a specificity of 85-95% and a sensitivity of 67% [155] [156] [157] [158] . the specificity and sensitivity of the acpa test has improved gradually from the first generation (anti-ccp1 test) to the third generation (anti-ccp3 test) [46, 159] . acpa is useful for the prediction of ra because it already exists before the onset of ra [160] , and its positivity indicates a more erosive disease course and severity [161] [162] [163] . although there are various isotypes of acpas, such as igg, iga and igm, the igg isotype is the most dominant form in ra patients [164] . the fc region of acpa undergoes remodeling, which causes its fc fragment to show two characteristics: decreased galactosylation and sialylation and increased core fucosylation, which differs from those of other serum antibodies [165] [166] [167] [168] [169] . sialylation has a protective effect against the autoimmunogenicity of anti-type ii collagen antibodies [165] . therefore, remodeling of the fc region could indicate the alteration of the functional activities of acpa [165, 168] , and this remodeling of acpa occurs before the change in total serum ig [167, 170] . in vitro, acpa binds to the fc receptors of myeloid lineage immune cells, activating the component system through both classical and alternative pathways [171, 172] . citrullinated fibrinogen-acpa complexes in ra patients could activate macrophages to release tnf-α [173] . igm-rf enhances this cascade and extends the spectrum by inducing the secretion of other cytokines (il-1β, il-6 and il-8) [171] . the fc-glycan profile showed different fc receptor and complement binding affinities [168] . the agalatosylated profile of the fc of acpa facilitates the production of high-affinity rf for agalactosylated igg [166] . acpa also undergoes extensive variable domain glycosylation associated with the se allele, except for igm acpa [174, 175] . the incorporation of n-linked glycosylation sites modulates the affinity of acpa [166, 168, 176] . because acpas are a collection of heterogeneous antibodies, the specificity of acpas differs even in individual patients [177, 178] . candidate citrullinated autoantigens are listed in table 4 . [190, 191] bip § er [170, 192] tenascin-c ecm of joint [193, 194] filaggrin epithelium [195, 196] apo e plasma, cns, ra-sf [197, 198] mnda inflammatory cells ¶ [197, 199] inflammatory cells ¶ [197, 199] exogenous viral citrullinated peptides from ebv antigen [190] α-enolase from p. gingivalis [190] gns sequence homologue of surface proteins of the p. copri the concentration of igm-acpa in the ra-sf, corrected for the total amount of igg, was higher than that in the serum of the same patient, suggesting that there is local production of acpas [202] . following binding to peripheral blood mononuclear cells, acpa activates extracellular signal-regulated kinase (erk) 1/2 and c-jun n-terminal kinase (jnk) signaling pathways. this effect further enhances inhibitor of nf-κb kinase (ikk)-α phosphorylation, followed by activation of nf-κb and production of tnf-α [203] . certain acpas, such as anti-citrullinated vimentin antibodies, induce net formation, and netosis provides the citrullinated autoantigen and pad enzymes, which provides a positive feedback loop, further enhancing the formation of acpas [183] . in summary, acpa has a pathogenic effect on ra, both in vitro and in vivo. first, acpa induces netosis, creating positive feedback. second, acpa stimulates macrophages to induce tnf-α and activate the complement of both classical and alternative pathways. third, acpa interacts with other autoantibodies associated with ra to initiate disease cascades. fourth, these substances pre-existed before the onset of ra, called the pre-clinical stage, and their titer increases with a widening spectrum and stronger affinity during the development of ra. fifth, the characteristics of acpa could be altered through remodeling by ptm. finally, these mechanisms mediate the applicability and usefulness of acpa not only in the diagnosis of ra but also as an indicator of a more severe clinical course and structural damage. a recent study by chirivi et al. [204] showed that therapeutically inhibiting net formation with acpa, especially targeting the n-termini of histones 2a and 4, suppressed net release or uptake by macrophages in various mouse models. won et al. [205] detected circulating citrullinated antigens, such as type ii collagen and filaggrin, in the sera of ra patients, including seronegative ra, using a monoclonal 12g1 antibody and proposed the possibility of it serving as a diagnostic tool for seronegative ra. citrullination is the process of preparing for apoptosis, and it involves the induction of a functional loss of filament proteins [106] . in apoptosis, unlimited ca 2+ influx into the intracellular space activates the pad, leading to citrullination [46] . in the normal apoptosis process, phagocytosis rapidly eliminates apoptotic bodies [206] . however, once dysregulation occurs during apoptosis or phagocytosis to eliminate apoptotic bodies, citrullinated intracellular antigens are exposed [207] . similarly, the necrosis of neutrophils releases pad into the extracellular space and leads to citrullination [208] . compared with the sf of osteoarthritis (oa) patients, that of ra patients showed an elevation in the concentration of intranuclear materials and the activity of pad4 [151] . pad4 activity in vitro, released to the extracellular space, is higher in necrosis than it is in netosis [209] . the names of newly identified cell death mechanisms, lth and defective mitophagy, were formulated to distinguish them from other forms of neutrophil death that do not involve hypercitrullination, even if they form net-like structures [68] . lth releases citrullinated intranuclear molecules [210] . in lth, perforin and the membrane attack complex (mac), the pore-forming cytolytic proteins, induce the influx of ca 2+ and other ions, leading to osmotic lysis [211] [212] [213] . lth is independent of erk and nicotinamide adenine dinucleotide phosphate (nadph) oxidase activity, a hallmark of netosis [214, 215] . initiation of lth originates not only endogenously but also exogenously. streptomyces sp. have bacterial calcium ionophores, such as ionomycin and calcimycin [67] . other bacterial pore-forming toxins also trigger lth. the periodontal pathogen aggregatibacter actinomycetemcomitans secretes the pore-forming protein leukotoxin a (ltxa) to activate citrullination in neutrophils [216] [217] [218] . p. gingivalis induces citrullination using ppad independent of ca 2+ and ph level [43]. as described above, a. actinomycetemcomitans and streptomyces sp. induce lth that involves citrullination; p. copri and ebv have homologous epitopes that could trigger the production of acpas and autoimmunogenicity. some other bacterial pathogens also induce membranolytic damage, which may induce citrullination in neutrophils and acpa production via cell death mechanisms. certain microbiomes in the lung, gut, and urothelium also produce pore-forming toxins that target neutrophils [219] . staphylococcus aureus and streptococcus pyogenes are microorganisms that colonize the extra-articular mucosa and produce panton-valentine leukocidin (pvl) and streptolysin o (slo), respectively, which could induce the citrullination of neutrophils [67, [220] [221] [222] . carbamylation occurs in the human body under uremic or inflammatory conditions [223, 224] . carbamylation is both an enzymatic and nonenzymatic process of adding the "carbamoyl" part (−conh 2 −), which is related to cyanate ( − n=c=o), to proteins, or to peptides [225] [226] [227] . this ptm process leads to the production of homocitrulline or α-carbamyl-protein [226] . carbamylation is thought to be an enzyme-independent process that typically occurs under uremic conditions [228, 229] . urea dissolved in water spontaneously generates cyanate, which is a carbamylate protein or peptide [230] . however, any inflammation causes oxidation of thiocyanate to produce cyanate, catalyzed by mpo and peroxide, regardless of whether the conditions are uremic or not [231] . smoking may also induce carbamylation [232] , and mononuclear cells of treatmentnaïve ra patients show a correlation between autophagy and the level of carbamylation [233] . the carbamylated form of hemoglobin and low-density lipoprotein (ldl), detectable by laboratory tests, is correlated with acute or chronic renal failure and atherosclerosis, respectively [234] [235] [236] [237] [238] . despite these findings, the precise role and effect of carbamylation in ra have not yet been definitively established. homocitrulline, which is generated by carbamylation, shows immunogenicity in ra, producing anti-carbamylated protein (anti-carp) antibodies [239] . anti-carp antibodies are detected in ra patients, regardless of acpa-positivity or acpa-negativity [240] . interestingly, anti-carp antibodies and acpa demonstrated definitive cross-reactivity in vitro [240] . the sensitivity and specificity of anti-carp antibodies for ra diagnosis are 44% and 89%, respectively [241] . hla-dr3 alleles, found at higher levels in acpa-negative ra patients than in controls, are related to anti-carp antibody-positive ra without acpas [242] [243] [244] [245] . although igg levels of anti-carp antibodies were shown to increase under uremic conditions and smoking in a mouse model, the levels did not increase in heavy smokers or show any association with smoking, or the association became insignificant after correcting for acpas in humans [244, [246] [247] [248] . in acpa-negative ra patients, positivity of anti-carp antibodies is associated with more erosive manifestation of ra than negativity, independent of rf or acpa [249] [250] [251] . anti-carp antibodies are also used to screen for those at risk, and the odds ratio is highest when all three autoantibodies (rf, acpas, and anti-carp antibodies) are co-analyzed [252] . the combined presence of acpas and anti-carp antibodies could strongly indicate ra [253] . studies have shown that carbamylation plays a role that is like citrullination in ra [249, 254, 255] . similar to rf and acpas, anti-carp antibodies show an association with risk when combined with smoking [256] . the presence of rf is associated with positivity for both acpas and anti-carp antibodies [257] . smoking induces both citrullination and carbamylation. however, there is a lack of evidence that smoking induces the production of autoantibodies; conversely, it correlates with the initiation of intolerance to multiple autoantigens, which may correlate with the overlapping of rf, acpas, and anti-carp antibodies [258] . specifically, cigarette smoking only induces autoimmunity, which is affected by hla-drb1 se, and evidence does not support the notion that it induces de novo intolerance to citrullinated or carbamylated proteins or peptides [39] . like acpas, anti-carp antibodies could exist for several years before the onset of ra, and they could increase gradually in quantity just before disease presentation [254, 259] . notably, the presence of both acpas and anti-carp antibodies strongly suggests ra, even if they are not only specific for ra [248] . the presence of each autoantibody is a marker of aggressive joint destruction in ra patients, making it possible to predict progression to ra in arthralgia patients [49, 260] . avidity of acpas and anti-carp antibodies are lower than other antibodies to usual recall antigen; nevertheless, acpas and anti-carp antibodies, composed of broad isotypes and subclasses (such as igm, iga and igg subclasses), undergo isotype-switching to diminish their avidity more [261] . published data suggest that smoking and periodontitis due to p. gingivalis are risk factors for cardiovascular disease [262] . citrullinated proteins and pad4 were detected in the atherosclerotic plaques in individuals without ra [263] , and the acpas in ra patients can target these proteins from atherosclerotic plaques [264] . according to hermans et al. [265] , st-segment elevation myocardial infarction (stemi) is associated with acpa. citrullination is also associated with neurodegenerative diseases, cancers [105, 144] , and type 1 diabetes [266] . citrullination of mbp in the cns is correlated with the onset of ms [267, 268] , and it causes other autoimmune diseases, such as systemic lupus erythematosus (sle) and autoimmune encephalomyelitis [268, 269] . because pad and citrullination are involved in transcription, net formation, and cell signaling, the connection between malignancy and citrullination has also been suggested [134, 144] . carbamylation is related to cardiovascular diseases [264] and plays a role as a proatherosclerotic activator by accumulating cholesterol in macrophages, inducing endothelial apoptosis, and increasing scavenger receptor recognition [231] . cataracts are induced by carbamylation of α-crystallin [227] . usually, the carbamylation of protein hormones downregulates the function of hormones; however, carbamylation of uncommon residues occasionally exhibits different effects on the intensity of downregulation, such as the a-chain of insulin, oxytocin, and arginine-vasopressin [270] [271] [272] . in the uremic status of end-stage renal disease (esrd), the lysine residue of erythropoietin (epo) is carbamylated and decreases in activity, resulting in the production of non-functional epo, which leads to hypoxemia [273, 274] . patients with inflammatory bowel disease (ibd) showed significant differences in the proportion that presented with serum anti-carp antibodies compared with normal controls [248] . carbamylated ldl (c-ldl) shows low affinity for its receptor, which decreases the clearance rate in rabbits [275, 276] . c-ldl induces the accumulation of cholesterol and the formation of foam cells and signals for inflammation [277] , and it facilitates monocyte adhesion to the endothelium and vascular smooth muscle to enhance their proliferation [278] . acetylation, which normally occurs in the human body through both co-translation and post-translation [279, 280] , has two distinct pathways through which an acetyl group donated by acetyl-coenzyme a is attached to either the n-terminus of proteins or lysine residues [281] . acetylation is a reversible enzymatic process catalyzed by various nterminal and lysine acetyltransferases [281, 282] . similar to carbamylation, acetylation can modify lysine residues, change the molecular features of proteins or peptides, and induce the production of modified epitopes [160] . in addition, acetylated proteins can induce the production of anti-acetylated protein antibodies (aapas) [160] . in seronegative ra patients, acetylation of histones, a padindependent process, showed cross-reactivity with acpa [283] . kampstra et al. [284] showed that autoantibodies to ptm molecules, called anti-modified protein antibodies (ampas), which include acpas, anti-carp antibodies, and aapas, could be part of a single concept because they share partial cross-reactivity with each post-translated autoantigen. su et al. [285] showed that the dysregulation of acetylation attenuates the development of ra by downregulating foxp3 expression. figure 2 shows a schema that illustrates posttranslational modifications (ptms) and anti-modified protein antibodies (ampas) in ra pathogenesis. citrullination and carbamylation are reported in a wide range of inflammatory tissues and, therefore, are considered as markers of inflammation rather than specific disease-dependent processes. pad, which mediates citrullination, has five isoenzymes, including some with the potential to autocitrullinate. ppad is an exogenous pad independent of ca 2+ and ph. acetylation is also involved in autoimmunogenicity and the production of autoantibodies in ra. both carbamylation and acetylation have a common target of lysine residues, but the results of each ptm have distinguishable characteristics. carbamylation are reported in a wide range of inflammatory tissues and, therefore, are considered as markers of inflammation rather than specific disease-dependent processes. pad, which mediates citrullination, has five isoenzymes, including some with the potential to autocitrullinate. ppad is an exogenous pad independent of ca 2+ and ph. acetylation is also involved in autoimmunogenicity and the production of autoantibodies in ra. both carbamylation and acetylation have a common target of lysine residues, but the results of each ptm have distinguishable characteristics. ptms, including citrullination, carbamylation, and acetylation alone, are not sufficient to induce intolerance and autoimmunity, but this does not negate their importance. because ra is caused by complex interactions between multiple pathogenic factors, the role of these ptms is as significant as that of other pathogenic factors. ptms are also correlated with one or more pulmonary, cardiovascular, neurodegenerative, malignant, or other autoimmune diseases, other than ra. ampa is closely related to the pathogenesis, prediction, diagnosis, and prognosis of ra, whereas smoking induces ptms and interacts with ampa to enhance the risk of ra development. cell death mechanisms, including apoptosis, autophagy, netosis, lth, and necrosis induce or present ptm autoantigens, generating ampas. some microbes ptms, including citrullination, carbamylation, and acetylation alone, are not sufficient to induce intolerance and autoimmunity, but this does not negate their importance. because ra is caused by complex interactions between multiple pathogenic factors, the role of these ptms is as significant as that of other pathogenic factors. ptms are also correlated with one or more pulmonary, cardiovascular, neurodegenerative, malignant, or other autoimmune diseases, other than ra. ampa is closely related to the pathogenesis, prediction, diagnosis, and prognosis of ra, whereas smoking induces ptms and interacts with ampa to enhance the risk of ra development. cell death mechanisms, including apoptosis, autophagy, netosis, lth, and necrosis induce or present ptm autoantigens, generating ampas. some microbes such as p. gingivalis can cause self-citrullination of molecules to produce de novo epitopes, and a. actinomycetemcomitans uses ltxa to induce lth in citrullinated neutrophils. tcr t cell receptor t eff cell effector t cells tgf-β transforming growth factor-β t h1 cell helper t1 cell t h17 cell helper t17 cell tlr toll-like receptor tnf tumor necrosis factor traf1-c5 tnf receptor associated factor 1-c5 t reg cell regulatory t cell tyk tyrosine autoimmun. rev. 2015, 14, 490-497. [crossref] the pathogenesis of rheumatoid arthritis puxeddu, i. one year in review 2021: pathogenesis of rheumatoid arthritis rheumatoid arthritis. in harrison's principles of internal medicine, 20e management issues in rheumatoid arthritis-associated interstitial lung disease beyond the joints, the extra-articular manifestations in rheumatoid arthritis the clinical features of rheumatoid arthritis update on the pathomechanism, diagnosis, and treatment options for rheumatoid arthritis weight bearing joints destruction in rheumatoid arthritis benefit of very early referral and very early therapy with disease-modifying anti-rheumatic drugs in patients with early rheumatoid arthritis exposure to ambient air pollution and autoantibody status in rheumatoid arthritis epigenetic changes in the pathogenesis of rheumatoid arthritis adiposity and the risk of rheumatoid arthritis: a systematic review and meta-analysis of cohort studies rheumatoid arthritis incidence and prevalence of rheumatoid arthritis racial and ethnic disparities in disease activity in patients with rheumatoid arthritis the environment, geo-epidemiology, and autoimmune disease: rheumatoid arthritis racial or ethnic differences in allele frequencies of single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene and their influence on response to methotrexate in rheumatoid arthritis ethnic variation in the clinical manifestations of rheumatoid arthritis: role of hla-drb1 alleles genetics and epigenetics of rheumatoid arthritis systematic approach demonstrates enrichment of multiple interactions between non-hla risk variants and hla-drb1 risk alleles in rheumatoid arthritis citrullination and rheumatoid arthritis autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell activation the role of anti-citrullinated protein antibodies (acpa) in the pathogenesis of rheumatoid arthritis. cent temporomandibular joint biomechanical restrictions: the fluid and synovial membrane electron microscopy of the human synovial membrane nomenclature clarification: synovial fibroblasts and synovial mesenchymal stem cells review: synovial cell metabolism and chronic inflammation in rheumatoid arthritis fibroblast-like synoviocyte metabolism in the pathogenesis of rheumatoid arthritis nr1d1 modulates synovial inflammation and bone destruction in rheumatoid arthritis monocytes in rheumatoid arthritis: circulating precursors of macrophages and osteoclasts and, their heterogeneity and plasticity role in ra pathogenesis role of endoplasmic reticulum stress in rheumatoid arthritis pathogenesis somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium analysis of the p53 tumor suppressor gene in rheumatoid arthritis synovial fibroblasts p53 tumor suppressor gene mutations in fibroblast-like synoviocytes from erosion synovium and non-erosion synovium in rheumatoid arthritis apoptosis in rheumatoid arthritis cell death in rheumatoid arthritis molecular mechanisms, role in physiology and pathology molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death neutrophil extracellular traps kill bacteria the role of neutrophil netosis in organ injury: novel inflammatory cell death mechanisms a critical reappraisal of neutrophil extracellular traps and netosis mimics based on differential requirements for protein citrullination rheumatoid arthritis and citrullination the ins and outs of mhc class ii-mediated antigen processing and presentation co-stimulatory blockade of the cd28/cd80-86/ctla-4 abatacept, a novel cd80/86-cd28 t cell co-stimulation modulator, in the treatment of rheumatoid arthritis a role for th1-like th17 cells in the pathogenesis of inflammatory and autoimmune disorders human b cell tolerance and its failure in rheumatoid arthritis sanz, i. b cell therapies for rheumatoid arthritis: beyond b cell depletion the immunology of rheumatoid arthritis fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis il-17-mediated mitochondrial dysfunction impairs apoptosis in rheumatoid arthritis synovial fibroblasts through activation of autophagy the il-23-il-17 axis in inflammatory arthritis regulation of autoantibody activity by the il-23-t(h)17 axis determines the onset of autoimmune disease th1-th17 ratio as a new insight in rheumatoid arthritis disease. iran the role of the jak/stat signal pathway in rheumatoid arthritis small molecule inhibitors in the treatment of rheumatoid arthritis and beyond: latest updates and potential strategy for fighting covid-19 american college of rheumatology guideline for the treatment of rheumatoid arthritis fostamatinib, an oral spleen tyrosine kinase inhibitor, in the treatment of rheumatoid arthritis: a meta-analysis of randomized controlled trials efficacy and safety of multiple dosages of fostamatinib in adult patients with rheumatoid arthritis: a systematic review and meta-analysis safety and efficacy of fostamatinib in rheumatoid arthritis patients with an inadequate response to methotrexate in phase ii oskira-asia-1 and oskira-asia-1x study defective t-cell apoptosis and t-regulatory cell dysfunction in rheumatoid arthritis defects in ctla-4 are associated with abnormal regulatory t cell function in rheumatoid arthritis anti-tnf in rheumatoid arthritis: an overview autophagy enhances the resistance to tumor necrosis factor-alpha treatment in rheumatoid arthritis human fibroblast-like synovial cell selected cytokine pathways in rheumatoid arthritis interleukin-6 and tumour necrosis factor-α cooperatively promote cell cycle regulators and proliferate rheumatoid arthritis fibroblast-like synovial cells wnt signaling and biological therapy in rheumatoid arthritis and spondyloarthritis tnf-α directly enhances osteocyte rankl expression and promotes osteoclast formation osteocyte-related cytokines regulate osteoclast formation and bone resorption tnf-α and il-6: the link between immune and bone system comprehensive epigenetic landscape of rheumatoid arthritis fibroblast-like synoviocytes huntingtin-interacting protein 1 (hip1) regulates arthritis severity and synovial fibroblast invasiveness by altering pdgfr and rac1 signalling long-term exposure to outdoor air pollution and the risk of development of rheumatoid arthritis: a systematic review and meta-analysis the oral and gut microbiome in rheumatoid arthritis patients: a systematic review probiotic supplementation for rheumatoid arthritis: a promising adjuvant therapy in the gut microbiome era metagenome-wide association study of gut microbiome revealed novel aetiology of rheumatoid arthritis in the japanese population regulating gut microbiome: therapeutic strategy for rheumatoid arthritis during pregnancy and lactation citrullination: a posttranslational modification in health and disease ca2+-dependent deimination-induced disassembly of intermediate filaments involves specific modification of the amino-terminal head domain the complete microspeciation of arginine and citrulline oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific t cells citrullination of cxcl8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation protein unfolding by peptidylarginine deiminase. substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis the interplay between citrullination and hla-drb1 polymorphism in shaping peptide binding hierarchies in rheumatoid arthritis the protein arginine deiminases: structure, function, inhibition, and disease redox regulation in the extracellular environment a citrullinated fibrinogen-specific t cell line enhances autoimmune arthritis in a mouse model of rheumatoid arthritis post-translational modifications such as citrullination are excellent targets for cancer therapy citrulline-specific th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy cd4+ memory stem t cells recognizing citrullinated epitopes are expanded in patients with rheumatoid arthritis and sensitive to tumor necrosis factor blockade antibodies to citrullinated α-enolase peptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase local administration of glucocorticoids decreases synovial citrullination in rheumatoid arthritis inhibiting citrullination in rheumatoid arthritis: taking fuel from the fire epithelial to mesenchymal transition is increased in patients with copd and induced by cigarette smoke frequency of antibodies to mutated citrullinated vimentin in chronic obstructive pulmonary disease: comment on the article by demoruelle et al. arthritis rheum smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in bal cells smoking is associated with increased levels of extracellular peptidylarginine deiminase 2 (pad2) in the lungs t cells become licensed in the lung to enter the central nervous system purification, characterization, and sequence analysis of a potential virulence factor from porphyromonas gingivalis, peptidylarginine deiminase heightened immune response to autocitrullinated porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis porphyromonas gingivalis peptidylarginine deiminase substrate specificity citrullination as a plausible link to periodontitis, rheumatoid arthritis, atherosclerosis and alzheimer's disease sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and keratin 1 deimination pad1 promotes epithelialmesenchymal transition and metastasis in triple-negative breast cancer cells by regulating mek1-erk1/2-mmp2 signaling small molecule promotes β-catenin citrullination and inhibits wnt signaling in cancer mining the human tissue proteome for protein citrullination relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone h3 peptidylarginine deiminase of the hair follicle: characterization, localization, and function in keratinizing tissues citrullination of inhibitor of growth 4 (ing4) by peptidylarginine deminase 4 (pad4) disrupts the interaction between ing4 and p53 dysregulation of pad4-mediated citrullination of nuclear gsk3β activates tgf-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells peptidylarginine deiminase (pad) 6 is essential for oocyte cytoskeletal sheet formation and female fertility peptidylarginine deiminase from porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis defining the role of porphyromonas gingivalis peptidylarginine deiminase (ppad) in rheumatoid arthritis through the study of ppad biology protein arginine deiminases and associated citrullination: physiological functions and diseases associated with dysregulation peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis protein arginine deiminase 4: a target for an epigenetic cancer therapy autocitrullination of pad4 does not alter its enzymatic activity: in vitro and in silico studies citrullination in the periodontium-a possible link between periodontitis and rheumatoid arthritis relation between anti-porphyromonas gingivalis antibody titers and hla-drb1 neutral alleles in individuals with rheumatoid arthritis expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages citrullination of synovial proteins in murine models of rheumatoid arthritis prevalence and significance of anti-peptidylarginine deiminase 4 antibodies in rheumatoid arthritis autoimmunity in rheumatoid arthritis: different antigens-common principles structural basis of cross-reactivity of anti-citrullinated protein antibodies. arthritis rheumatol autoantibodies from single circulating plasmablasts react with citrullinated antigens and porphyromonas gingivalis in rheumatoid arthritis anti-citrullinated peptide antibody assays and their role in the diagnosis of rheumatoid arthritis anti-citrullinated peptide antibody: death of the rheumatoid factor? anticitrullinated protein/peptide antibody multiplexing defines an extended group of acpa-positive rheumatoid arthritis patients with distinct genetic and environmental determinants meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis superior performance of the ccp3.1 test compared to ccp2 and mcv in the rheumatoid factor-negative ra population an overview of autoantibodies in rheumatoid arthritis antibodies to citrullinated proteins (acpa) associate with markers of osteoclast activation and bone destruction in the bone marrow of patients with rheumatoid arthritis the acpa isotype profile reflects long-term radiographic progression in rheumatoid arthritis anti-citrullinated peptide antibodies are the strongest predictor of clinically relevant radiographic progression in rheumatoid arthritis patients achieving remission or low disease activity: a post hoc analysis of a nationwide cohort in japan antibodies against cyclic citrullinated peptides of igg, iga and igm isotype and rheumatoid factor of igm and iga isotype are increased in unaffected members of multicase rheumatoid arthritis families from northern sweden sialylation converts arthritogenic igg into inhibitors of collagen-induced arthritis glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid anti-citrullinated protein antibodies acquire a pro-inflammatory fc glycosylation phenotype prior to the onset of rheumatoid arthritis igg antibodies to cyclic citrullinated peptides exhibit profiles specific in terms of igg subclasses, fc-glycans and a fab-peptide sequence structural analysis of variable domain glycosylation of anti-citrullinated protein antibodies in rheumatoid arthritis reveals the presence of highly sialylated glycans detection of autoantibodies to citrullinated bip in rheumatoid arthritis patients and pro-inflammatory role of citrullinated bip in collagen-induced arthritis igm and iga rheumatoid factors purified from rheumatoid arthritis sera boost the fc receptor-and complement-dependent effector functions of the disease-specific anti-citrullinated protein autoantibodies fc gamma receptor binding profile of anti-citrullinated protein antibodies in immune complexes suggests a role for fcγri in the pathogenesis of synovial inflammation immune complexes containing citrullinated fibrinogen costimulate macrophages via toll-like receptor 4 and fcγ receptor the extensive glycosylation of the acpa variable domain observed for acpa-igg is absent from acpa-igm extensive glycosylation of acpa-igg variable domains modulates binding to citrullinated antigens in rheumatoid arthritis n-linked glycans in the variable domain of igg anti-citrullinated protein antibodies predict the development of rheumatoid arthritis differences in the spectrum of anti-citrullinated protein antibody fine specificities between malaysian and swedish patients with rheumatoid arthritis: implications for disease pathogenesis acpa fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression the major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha-and beta-chains of fibrin marked differences in fine specificity and isotype usage of the anti-citrullinated protein antibody in health and disease multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with hla-drb1 alleles rheumatoid arthritis specific anti-sa antibodies target citrullinated vimentin nets are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis antibodies to mutated citrullinated vimentin for diagnosing rheumatoid arthritis in anti-ccp-negative patients and for monitoring infliximab therapy identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis apolipoprotein b binds to enolase-1 and aggravates inflammation in rheumatoid arthritis interaction between extracellular matrix molecules and microbial pathogens: evidence for the missing link in autoimmunity with rheumatoid arthritis as a disease model anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-drb1 shared epitope alleles immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin) antibodies directed against endogenous and exogenous citrullinated antigens pre-date the onset of rheumatoid arthritis characterization of autoantigens targeted by anti-citrullinated protein antibodies in vivo: prominent role for epitopes derived from histone 4 proteins immunoglobulin heavy-chain-binding protein (bip): a stress protein that has the potential to be a novel therapy for rheumatoid arthritis identification of an immunodominant peptide from citrullinated tenascin-c as a major target for autoantibodies in rheumatoid arthritis potential roles for tenascin in (very) early diagnosis and treatment of rheumatoid arthritis identification of citrullinated rheumatoid arthritis-specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection by line immunoassay citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies the rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein e, myeloid nuclear differentiation antigen, and β-actin apolipoprotein e triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding c1q neutrophil proteases degrade autoepitopes of net-associated proteins candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes antibodies to native and citrullinated ra33 (hnrnp a2/b1) challenge citrullination as the inciting principle underlying loss of tolerance in rheumatoid arthritis the concentration of anticitrullinated protein antibodies in serum and synovial fluid in relation to total immunoglobulin concentrations anti-citrullinated protein antibodies activated erk1/2 and jnk mitogen-activated protein kinases via binding to surface-expressed citrullinated grp78 on mononuclear cells therapeutic acpa inhibits net formation: a potential therapy for neutrophil-mediated inflammatory diseases pathogenic role of circulating citrullinated antigens and anti-cyclic monoclonal citrullinated peptide antibodies in rheumatoid arthritis apoptosis: controlled demolition at the cellular level an overview of the intrinsic role of citrullination in autoimmune disorders inflammatory but not apoptotic death of granulocytes citrullinates fibrinogen release of active peptidyl arginine deiminases by neutrophils can explain production of extracellular citrullinated autoantigens in rheumatoid arthritis synovial fluid antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps membrane attack by complement: the assembly and biology of terminal complement complexes a central role of perforin in cytolysis? intracellular ca2+ and cell injury: a paradoxical role of ca2+ in complement membrane attack sk3 channel and mitochondrial ros mediate nadph oxidaseindependent netosis induced by calcium influx requirements for nadph oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus aggregatibacter actinomycetemcomitans-induced hypercitrullination links periodontal infection to autoimmunity in rheumatoid arthritis evaluation of the virulence of aggregatibacter actinomycetemcomitans through the analysis of leukotoxin aggregatibacter actinomycetemcomitans leukotoxin (ltxa) requires death receptor fas, in addition to lfa-1, to trigger cell death in t lymphocytes microbiome and mucosal inflammation as extra-articular triggers for rheumatoid arthritis and autoimmunity pore-forming toxins: ancient, but never really out of fashion role of pore-forming toxins in bacterial infectious diseases. microbiol ab0097 the presence of staphylococcal toxins in the urine of patients with rheumatoid arthritis plasma protein carbonylation in chronic uremia protein carbamylation in kidney disease: pathogenesis and clinical implications protein carbamylation in chronic kidney disease and dialysis carbamylation-derived products: bioactive compounds and potential biomarkers in chronic renal failure and atherosclerosis protein carbamylation and cardiovascular disease hemoglobin carbamylation in uremia role of cyanate in the induction of vascular dysfunction during uremia: more than protein carbamylation? kidney int reactions of the cyanate present in aqueous urea with amino acids and proteins protein carbamylation links inflammation, smoking, uremia and atherogenesis carbamylation of vimentin is inducible by smoking and represents an independent autoantigen in rheumatoid arthritis autophagy induces protein carbamylation in fibroblast-like synoviocytes from patients with rheumatoid arthritis quantification of carbamylated ldl in human sera by a new sandwich elisa factors determining hemoglobin carbamylation in renal failure differentiation of acute from chronic renal impairment by detection of carbamylated haemoglobin kinetics of carbamylated haemoglobin in acute renal failure carbamylated low-density lipoprotein induces death of endothelial cells: a link to atherosclerosis in patients with kidney disease is there a link between carbamylation and citrullination in periodontal disease and rheumatoid arthritis? antibodies to carbamylated α-enolase epitopes in rheumatoid arthritis also bind citrullinated epitopes and are largely indistinct from anti-citrullinated protein antibodies the specificity of anti-carbamylated protein antibodies for rheumatoid arthritis in a setting of early arthritis association of hla-dr3 with anti-cyclic citrullinated peptide antibody-negative rheumatoid arthritis regulation of anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis: contrasting effects of hla-dr3 and the shared epitope alleles anti-carp antibodies in two large cohorts of patients with rheumatoid arthritis and their relationship to genetic risk factors, cigarette smoking and other autoantibodies specific association of hla-drb 1*03 with anti-carbamylated protein antibodies in patients with rheumatoid arthritis carbamyl adducts on low-density lipoprotein induce igg response in ldlr-/-mice and bind plasma autoantibodies in humans under enhanced carbamylation the isotype and igg subclass distribution of anti-carbamylated protein antibodies in rheumatoid arthritis patients anti-carbamylated protein antibodies: a specific hallmark for rheumatoid arthritis. comparison to conditions known for enhanced carbamylation; renal failure, smoking and chronic inflammation autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage the association between anti-carbamylated protein (anti-carp) antibodies and radiographic progression in early rheumatoid arthritis: a study exploring replication and the added value to acpa and rheumatoid factor association of the presence of anti-carbamylated protein antibodies in early arthritis with a poorer clinical and radiologic outcome: data from the french espoir cohort. arthritis rheumatol triple positivity for anti-citrullinated protein autoantibodies, rheumatoid factor, and anti-carbamylated protein antibodies conferring high specificity for rheumatoid arthritis: implications for very early identification of at-risk individuals the combination of three autoantibodies, acpa, rf and anti-carp antibodies is highly specific for rheumatoid arthritis: implications for very early identification of individuals at risk to develop rheumatoid arthritis anti-carbamylated protein antibodies are present prior to rheumatoid arthritis and are associated with its future diagnosis citrullination and carbamylation in the pathophysiology of rheumatoid arthritis a predominant involvement of the triple seropositive patients and others with rheumatoid factor in the association of smoking with rheumatoid arthritis rheumatoid factor isotypes in relation to antibodies against citrullinated peptides and carbamylated proteins before the onset of rheumatoid arthritis smoking is associated with the concurrent presence of multiple autoantibodies in rheumatoid arthritis rather than with anti-citrullinated protein antibodies per se: a multicenter cohort study anti-carbamylated protein antibodies in the pre-symptomatic phase of rheumatoid arthritis, their relationship with multiple anti-citrulline peptide antibodies and association with radiological damage arthritis development in patients with arthralgia is strongly associated with anti-citrullinated protein antibody status: a prospective cohort study the anti-carbamylated protein antibody response is of overall low avidity despite extensive isotype switching the relationship of diabetes, periodontitis and cardiovascular disease report: citrullination within the atherosclerotic plaque: a potential target for the anti-citrullinated protein antibody response in rheumatoid arthritis post-translational modifications in rheumatoid arthritis and atherosclerosis: focus on citrullination and carbamylation long-term mortality in patients with st-segment elevation myocardial infarction is associated with anti-citrullinated protein antibodies citrullination and pad enzyme biology in type 1 diabetes-regulators of inflammation myelin basic protein citrullination in multiple sclerosis: a potential therapeutic target for the pathology protein arginine deiminases (pads): biochemistry and chemical biology of protein citrullination biochemically altered myelin triggers autoimmune demyelination carbamylation of oxytocin and arginine-vasopressin uremia and insulin resistance: n-carbamoyl-asparagine decreases insulin-sensitive glucose uptake in rat adipocytes inhibition of erythropoietin activity by cyanate. scand chemical modification of erythropoietin: an increase in in vitro activity by guanidination decreased clearance of uraemic and mildly carbamylated low-density lipoprotein role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts how do macrophages sense modified low-density lipoproteins? carbamylated low-density lipoprotein induces proliferation and increases adhesion molecule expression of human coronary artery smooth muscle cells 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond functions and mechanisms of non-histone protein acetylation developmental roles of protein n-terminal acetylation proteomics analyses reveal the evolutionary conservation and divergence of n-terminal acetyltransferases from yeast and humans differential acpa binding to nuclear antigens reveals a pad-independent pathway and a distinct subset of acetylation cross-reactive autoantibodies in rheumatoid arthritis different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modification impaired tip60-mediated foxp3 acetylation attenuates regulatory t cell development in rheumatoid arthritis key: cord-0031693-frnv0ta8 authors: miše, joško; jukić, ines lakoš; marinović, branka title: rituximab progress but still not a final resolution for pemphigus patients: clinical report from a single center study date: 2022-05-03 journal: front immunol doi: 10.3389/fimmu.2022.884931 sha: 087c838090219999150a293daeeb9a95c673a103 doc_id: 31693 cord_uid: frnv0ta8 pemphigus is a rare autoimmune disease characterized by the production of pathogenic autoantibodies against desmosomal adhesion proteins, desmoglein 1 and 3. the pathophysiological process leads to the development of blisters and erosions on mucosal and/or skin surfaces as the main clinical manifestation of the disease. rituximab emerged as the first-line therapeutic option for pemphigus due to its ability to induce remission by depleting peripheral b lymphocytes. our aim was to assess the efficacy of rituximab in the treatment of patients in croatia. a single-center, retrospective study was conducted on 19 patients treated with rituximab following a rheumatoid arthritis dosing protocol between october 2015 and march 2021, with a mean follow-up of 24.1 months. after the first rituximab cycle, two patients achieved complete remission off therapy (10.5%), and six patients achieved complete remission on minimal therapy (31.6%). partial remission was observed among ten patients (52.6%). eight patients (44.4%) relapsed after the first rituximab cycle. the mean relapse time was 21 months. seven patients received two rituximab cycles, and three patients received three cycles. overall, 13 out of 19 patients experienced complete remission at some point during the study, while there were no non-responders after the rituximab treatment. no statistically significant associations were observed between age, sex, type of disease involvement and clinical remission, either on or off therapy. a steady decrease in anti-desmoglein 1 and anti-desmoglein 3 levels was measured among all patients following rituximab treatment. one patient experienced a treatment-related adverse event of infectious etiology (cellulitis). one patient died following the first rituximab cycle, with the cause of death likely not to be associated with the treatment. rituximab is an effective disease-modifying agent in the treatment of pemphigus with the main benefit of reducing corticosteroid exposure and steroid-related side effects among pemphigus patients. however, a feature of rituximab therapy is high relapse rates and the need for repeated treatment cycles to achieve complete remission. developing an optimal protocol for rituximab treatment and finding suitable markers for predicting relapse will improve the management of pemphigus patients. pemphigus diseases are a group of autoimmune bullous disorders characterized by the formation of autoantibodies against the desmosomal adhesion proteins, desmoglein 1 (dsg1) and/or desmoglein 3 (dsg3), leading to the formation of blisters and erosions of skin and mucosa. pemphigus is a rare disease with an incidence in croatia of 3.7 new patients per 1 million inhabitants per year (1) . before the introduction of systemic corticosteroids, the diagnosis of pemphigus was almost always a fatal one. systemic corticosteroids and immunosuppressive drugs have drastically reduced pemphigus mortality from 75% to less than 10% in severe cases (2) . the combination of prednisone/prednisolone (1.0-1.5 mg/ kg/day) and corticosteroid-sparing immunosuppressive agents, mostly azathioprine and mycophenolate mofetil was regarded as a standard first-line therapy by most clinicians (3) . however, severe and sometimes even life-threatening side effects related to chronic use of thesedrugswerestillasignificantissue.theincreasingevidenceforthe successful use of rituximab was a breakthrough in the treatment of pemphigus. rituximab, a monoclonal antibody directed against the cd20 antigen on b-lymphocytes, depletes cd20 b cells from the circulation and has been used in b-cell lymphoma, rheumatoid arthritis, vasculitides and off-label in autoimmune dermatologic conditions (4) . the first case of a pemphigus patient successfully treated with rituximab was published twenty years ago (5) . in a randomized controlled trial published in 2017, joly et al. showed that 89% of patients with pemphigus vulgaris (pv) and pemphigus foliaceus (pf) assigned to the rituximab group achieved complete remission off therapy compared to 34% of patients assigned to the treatment with prednisone alone (6) . in 2018 rituximab was licensed forthetreatmentofmoderatetoseverepemphigusintheunitedstates and the european union. more recently, the consensus statement on management of pemphigus by the international panel of experts and the guidelines by the european academy of dermatology and venereology (eadv) recommended intravenous cd20 inhibitors as a first-line therapy option for mild and moderate-to-severe pemphigus (7, 8) . in the years following, a number of studies evaluating the efficacy and outcomes of rituximab therapy for pemphigus were published. this study aimed to assess the efficacy of rituximabintreatingpatientswithpemphigusincroatiaandcompare our results with the published studies on rituximab effectiveness from other centers. we identified eligible patients from treatment logs at the university hospital center zagreb, the croatian referral center for bullous dermatoses. the study included all patients with pv and pf treated with rituximab from october 2015 to march 2021. nineteen patients were identified, out of which 16 were diagnosed with pv and 3 with pf. patient characteristics are summarized in table 1 . a diagnosis of pv and pf was based on the clinical appearance of mucosal and/or cutaneous lesions and confirmed by the histopathological finding of suprabasal (pv) or subcorneal (pf) acantholysis and direct immunofluorescence results of intercellular immunoglobulin g, with or without c3 deposits, in the epidermis/epithelium. patients received an initial intravenous (iv) infusion of 1000 mg rituximab on day 1 and a second iv infusion of 1000 mg rituximab on day 15 (rheumatoid arthritis protocol). each patient received rituximab while being hospitalized at our department. before starting rituximab treatment, all patients underwent general, and laboratory examination and vaccinations were administered as indicated (8) . patients received premedication 30 minutes prior to rituximab infusion with paracetamol 1g iv, loratadine 10 mg per os and methylprednisolone 125 mg iv. vital parameters of each patient were monitored during and after rituximab infusion. patients who relapsed were treated with an additional cycle of 2g of rituximab combined with reintroduced or escalated prednisone dose. first-line treatment in all patients included oral prednisone at an initial dosage of up to 1 mg/kg/day tapered off over 6-12 months. in 16 out of 19 patients, a steroid-sparing drug (azathioprine) was also introduced at the dosage between 0.5 mg/kg/day and 2.5 mg/ kg/day depending on the activity level of the thiopurine methyltransferase (tpmt) enzyme. clinical response was defined by the criteria outlined in the consensus statement on definitions of endpoints and therapeutic response for pemphigus (9) . complete remission off therapy (crot) was defined as complete epithelialization and absence of new or established lesions while the patient is off all systemic therapy for at least 2 months while complete remission on minimal therapy (crmt) was defined as the absence of new lesions while the patient is receiving minimal doses of systemic therapy. partial remission off therapy (prot) was defined as the presence of transient new lesions that healed within 1 week without treatment and while off all systemic therapy. minimal therapy was defined as prednisone up to 10 mg/day or azathioprine up to 1.25 mg/kg/day. relapse was defined as the appearance of at least three new lesions in 1 month that did not heal spontaneously within 1 week or the extension of established lesions in a patient who had previously achieved disease control. anti-desmoglein 1 (anti-dsg 1) and anti-desmoglein 3 (anti-dsg 3) antibody titers were measured at the time of pemphigus diagnosis, before the start of rituximab treatment (baseline) and at months 3, 6 and 12 after receiving rituximab. the primary study outcome was crot six months after one rituximab cycle. secondary study outcomes included crot six months after additional rituximab cycles, levels of anti-dsg 1 and anti-dsg 3 titers after one rituximab cycle, relapse rates, the median time to relapse and incidence of treatment-related serious adverse events. serious adverse events were defined according to the food and drug administration (fda) definition as any event that is fatal or life-threatening, requires hospitalization or causes disability or permanent damage. we conducted a single-center retrospective study of 19 patients with pv and pf treated with rituximab in our center from october 2015 and march 2021 with at least 6 months of followup. this study was conducted in accordance with the declaration of helsinki and was approved by the committee of ethics of the university hospital center zagreb. the requirement for the acquisition of informed consent was waived due to the study's retrospective design. data were analyzed using statistical packages statistica ver. 12 (statsoft, inc., tulsa, ok, usa) and medcalc ® statistical software ver. 20.015 (medcalc software ltd, ostend, belgium). categorical variables were presented as numbers and proportions (%) and continuous variables as mean with standard deviation (sd) or as median with interquartile range (iqr) depending on the distribution. categorical data were tested using a chi-square test for the differences between groups. repeated measures anova was used to test for differences in time dynamics of outcome measures between groups. kaplan-meier survival analysis was used to test for differences in time to the first event between groups. logistic regression analysis was used to assess associations with different outcomes. roc analysis was used to assess the pretreatment level of anti-dsg with outcomes. p<0.05 was considered statistically significant for all tests, corrected for multiple comparisons. a total of 19 patients, 16 of them with pv and 3 with pf, received the first cycle of rituximab. there were 14 female and 5 male patients. the median age was 55.3 (m: 55.2,f: 55.4) and the mean disease duration before administration of rituximab was 80.57 months with range from 9 to 221 ( table 1) . two patients achieved crot (10.5%), six patients achieved crmt (31.6%), and pr was observed in 10 out of 19 patients (52.6%). one patient died during the follow-up after the first rituximab cycle. overall, significant improvement was observed in 8 of 19 patients (42.1%). eight patients (44.4%) relapsed after the first cycle (p=0.0395); 4 patients who were in pr and 4 patients who were in cr. the mean relapse time was 21 months (range: 6-50). seven of these eight patients went through the second cycle of rituximab. six achieved crmt (85.7%), with one patient achieving crot (14.3%). among these seven patients, three relapses were observed after the second cycle (42.8%). they were retreated with a third cycle of rituximab, and all of them achieved crmt. there were no relapses observed among patients who received the third rituximab cycle by the end of the inclusion period of this study. figure 1 represents a flowchart describing patients' clinical response following the administration of rituximab treatments. the mean remission length after the first rituximab cycle was 20 months. overall, 13 out of 19 patients experienced complete remission at some point during the study, while there were no non-responders after the rituximab treatment. patients achieving complete remission had a mean disease duration prior to rituximab of 67 months, against 82 months in those not achieving complete remission. however, the difference was not statistically significant (p=0.40). there were no other statistically significant associations between age, sex, type of disease and clinical remission, either on or off therapy. patient demographics, including sex and type of disease were not significant predictors of relapse. all patients in every rituximab cycle were concomitantly receiving corticosteroids with the dose gradually tapered depending on the clinical status. the mean corticosteroid dose per day decreased progressively with each new cycle (31 mg in the first cycle, 21 mg in the second cycle, and 16 mg in the third cycle). sixteen patients received adjuvant immunosuppressive (azathioprine) during the first cycle. four patients continued the medication throughout the second, and only one patient received the adjuvant in the third cycle with achieved complete remission on minimal therapy ( table 2) . we recorded a steady decrease in anti-dsg 1 and anti-dsg 3 levels after rituximab treatment among all patients ( table 3) . mean anti-dsg 1 and anti-dsg 3 values dropped at months 3, 6 and 12 following rituximab infusion ( figure 2 ). there were no statistically significant associations observed between the baseline levels of anti-dsg 1 or 3 antibodies and crot, crmt or both (p=0.44, p=0.28, p=0.64). no statistically significant association was observed between anti-dsg 1 or 3 levels and relapse after rituximab treatment (p=0.38). one adverse event (5.2%) was observed that we attribute to rituximab treatment. a female patient developed cellulitis that was treated with systemic antibiotics. one patient died in the first year of completing a rituximab cycle. however, the cause of death (acute myocardial infarction) is most likely not attributable to rituximab treatment. we did not record any adverse events related to the infusion of rituximab. no serious adverse events, as defined by the fda, were observed. however, there could be more adverse events that we failed to capture because of the retrospective design and less reporting of side effects as many get treated in the primary or secondary care units. furthermore, it is difficult to attribute adverse events to rituximab, given that patients were concomitantly receiving other immunosuppressive therapies. our study showed that rituximab is effective in inducing remission as 68% of pemphigus patients who received rituximab achieved a complete remission at some point during the follow-up period. however, more than one cycle of rituximab therapy is needed to achieve the desired treatment outcome. after the first cycle, complete remission, off or on minimal therapy, was achieved in 31.5% of patients, and after the second rituximab cycle, all patients achieved complete remission, either off or on minimal therapy. this finding is consistent with almost all studies involving the assessment of rituximab efficacy, showing that it effectively induces remission, but with more than one administered cycle, ranging from two to as many as seven in some patients (10) . rituximab has, until 2020, largely been used as a second or thirdline treatment option for pv, and we have used it as such at our department for the patients involved in this study. however, it has recently been recommended by the international panel of experts, as well as by european guidelines, as a first-line treatment for pv. among a number of studies related to the use of rituximab as the first vs second-line treatment, several studies reported a higher probability of achieving complete remission when rituximab is used as a first-line agent (11) (12) (13) (14) . however, contesting these findings are studies that observed no statistically significant difference in achieving complete remission between the two groups (15) (16) (17) (18) . among them is a systematic review by amber et al. reporting no association between the number of previous treatments and clinical outcomes (19) . it needs to be highlighted that the findings of the superior effect of rituximab when administered as the first-line therapy may be influenced by the possible presence of more recalcitrant patients who previously relapsed and therefore received rituximab as the second or third-line agent. the absence of pemphigus disease area index (pdai) score in the reviewed studies makes it difficult to assess disease severity among patient cohorts who received rituximab as first or second-line treatment impeding us to provide a definite answer to this question. however, studies are showing that patients who received rituximab earlier during the course of their disease had a higher chance of achieving complete remission. lunardon et al. reported that patients in complete remission had a median disease duration of 19 months compared to 86 months in those not achieving complete remission (20) . furthermore, balighi et al. found better outcomes in patients treated with rituximab within 6 months of diagnosis (12) . in our study, patients achieving complete remission had a mean disease duration of 67 months prior to rituximab therapy, against 82 months in those not achieving complete remission. however, the (21) . conducting the literature review, we found that the relapse rate after the first rituximab cycle ranged from 24% to 65%. curimbhoy et al. reported that 65% of patients relapsed after the first rituximab cycle, 18% relapsed after the second cycle and 20% after the third administered cycle (22) . a similar "crescendo effect" was observed in the study by shimanovich et al. with a 63% relapse rate after the first rituximab cycle and 41% and 43% after the second and third cycle, respectively (15) . we found that 44.4% of our patients relapsed after the first rituximab cycle, 42% after the second, and none after the third cycle supporting the observation that repeated rituximab cycles lead to progressively decreasing relapse rates. the overall relapse rate is difficult to narrow down because of the differences in the follow-up time between the studies. the mean relapse time following rituximab treatment among our patients was 21 months, with one patient relapsing at month 50 after a rituximab cycle. in the literature, the mean relapse time following rituximab therapy was reported between 8 and 24 months (21) . recent research has also focused on finding relevant prognostic factors for predicting clinical remission and relapse. assessing the correlation between age, sex, anti-dsg titer levels, disease duration, and the outcomes of rituximab therapy is of practical interest. with the collected data from our study, we analyzed anti-dsg 1 and 3 values and tried to see whether the titer levels could serve as an indicator for predicting the clinical outcome. even though anti-dsg 1 and 3 levels progressively decreased following each rituximab cycle, we could not establish a statistically significant association between anti-desmoglein titer levels and remission or relapse. the existing data from different studies is somewhat conflicting regarding the role of anti-dsg 1 and anti-dsg 3 in predicting treatment outcomes and relapse. in recent years, some studies suggested anti-dsg 1 as a more valuable marker of clinical outcome than anti-dsg 3 in pemphigus patients (12, 23) . however, the consensus is lacking, as there are studies that find no statistically significant difference between anti-dsg 1 and 3 in predicting a favorable clinical outcome and relapse (11) . the study by albers et al., which focused on identifying biomarkers predictive of relapse, found that anti-dsg 3 level had a strong predictive value for relapse among all patients and that positive anti-dsg 1 level had significant predictive value among patients with the mucocutaneous disease, which is contrary to the previous findings of the role of antidsg1 in pemphigus phenotype (24) . this should come as no surprise as there is increasing evidence that antibody specificities and titers do not always correlate to the disease activity and clinical features of pemphigus. some studies suggest that these discrepancies account for between 36% and 48% of all pemphigus cases (25) . the reason for these cases that challenge "desmoglein compensation theory" could be in a distinct set of antibodies to desmoglein and various non-desmoglein antigens (desmocollin, plakins, armadillo proteins, cholinergic receptors, hspca1 and antimitochondrial proteins) that each patient develops during the course of the disease (26, 27) . antibodies against these non-desmoglein antigens could maintain disease activity, casting doubts at the attempts to use anti-dsg 1 and 3 titer levels to predict relapse and remission successfully. kushner et al., when controlling for age and dosing protocol, revealed that older age (65 and older) was significantly associated with achieving complete remission after rituximab therapy (10) . the explanation given for this finding could be in the weakened immune system in elderly patients, making remissions of autoimmune diseases easier to achieve. toosi et al. analyzed the differences between pdai scores in patients with or without relapse and found that patients with higher pdai scores, especially higher mucosal pdai scores at baseline, may have a higher risk of relapse in the future (11) . it is evident that relapse poses a problem after treatment with rituximab. a plausible explanation for high relapse rates could be in the existence of ectopic lymphoid structures within pemphigus lesions that consist of t and b lymphocytes in various stages of differentiation. the question remains if anti-cd20 treatment substantially depletes b-cells in the pemphigus lesions as the depth of b cell depletion depends on the target tissue (28) . recent findings suggest that locally present ectopic lymphoid structures evade the systemic depletion induced by rituximab and facilitate the resistance of lesions even in the absence of circulating dsgautoantibodies. furthermore, the study by zhou et al. detected a much higher fraction of dsg-specific b cells in pemphigus lesions than in peripheral blood, indicating that pemphigus lesions could be a reservoir of b-cells maintaining the disease activity (29) . these findings could provide an explanation for relapses after rituximab treatment and a possible future therapeutic target -chemokines that facilitate the migration of b lymphocytes into the skin, or a different treatment modality -intralesional rituximab. furthermore, hematological dosing protocol for rituximab administration shows a deeper b-cell depletion in the secondary lymphoid tissues than the rheumatoid arthritis dosing protocol (30) . several studies confirm this finding by reporting lower relapse rates among patients receiving hematological dosing protocol (375 mg/m2 body surface area, four infusions one week apart) (22, 31, 32) . however, concerns remain regarding the safety and consequences of a complete b-cell clone eradication (19) . identifying the patients who are more likely to relapse or have a poor response to therapy is beneficial for determining the optimal dosage and timing of maintenance therapy. eight of our patients who relapsed after the first cycle relapsed between month 6 and month 50 (median: 16 months), which gives clues on the best timing of maintenance rituximab infusion. considering that half of our patients relapsed in the second year of follow-up, it seems reasonable to give patients a maintenance dose of rituximab at month 12 to prevent these relapses. however, results are lacking on the treatment outcomes of patients who received maintenance therapy making valid counterfactual reasoning if patients who received maintenance therapy would have achieved the same relapse rates. nevertheless, eadv guidelines on pemphigus management from 2020 recommend maintenance infusion of rituximab at month 6 for patients with severe pemphigus and/or who still have high titers of anti-dsg antibodies, whereas maintenance infusion at month 12 is considered for all patients in complete remission and in particular for those who have positive anti-dsg antibodies (7) . a challenge in assessing the treatment outcomes of rituximab is the heterogeneity of definitions, follow-up duration and patient data available among different studies. in order to streamline the reports on rituximab efficacy which would allow for a more robust and reliable comparison between various studies, we recommend following the consensus statement on definitions of disease and endpoints (9) . collecting patient data, such as pdai scores, and measuring anti-dsg 1 and 3 levels at 3month intervals, could provide valuable information in predicting and preventing a relapse, which still comprises a significant problem in the management of pemphigus patients. further research is needed to establish the optimal rituximab dosing protocol taking into account clinical outcomes, safety and cost-effectiveness of anti-cd20 therapy. limitations of our study are the absence of pdai and/or absis scores and small sample size. due to the study's retrospective nature, pdai or absis scores were not available for all patients, so we decided not to include them in the study. the number of patients is small, but in accordance with the number of newly diagnosed pemphigus patients in croatia and the number of patients with severe pemphigus referred to our center. despite these limitations, our study provides important information on the long-term effects of rituximab in a welldefined group of patients treated in a single center with the same treatment regimens. the results of our study show that rituximab is generally well tolerated and effective in inducing remission among pemphigus patients. the indispensability of rituximab lies in its ability to significantly decrease corticosteroid exposure and corticosteroidrelated side effects, making it major progress for pemphigus patients. however, a feature of rituximab therapy is high relapse rates and the need for repeated treatment cycles. the presence of ectopic lymphoid structures in pemphigus lesions and the variety of antibodies involved in the pathophysiology of pemphigus make it difficult to establish a one-size-fits-all approach in the management of pemphigus. furthering the research into understanding the complete autoantibody profile, not only antidesmosomal, will help establish a personalized pemphigus subtype for each patient, which will open new therapeutic opportunities. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by committee of ethics of the medical school university of zagreb. written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. autoimmune blistering diseases: incidence and treatment in croatia pemphigus: current and future therapeutic strategies anti-cd 20 chimeric monoclonal antibody (rituximab) for the treatment of recalcitrant, life-threatening pemphigus vulgaris with implications in the pathogenesis of the disorder first-line rituximab combined with short-term prednisone versus prednisone alone for the treatment of pemphigus (ritux 3): a prospective, multicentre, parallel-group, open-label randomised trial updated s2k guidelines on the management of pemphigus vulgaris and foliaceus initiated by the european academy of dermatology and venereology (eadv) diagnosis and management of pemphigus: recommendations of an international panel of experts consensus statement on definitions of disease, end points and therapeutic response for pemphigus factors associated with complete remission after rituximab therapy for pemphigus efficacy and safety of biosimilar rituximab in patients with pemphigus vulgaris: a prospective observational study anti-desmoglein-1 levels as predictor of prednisolone tapering in pemphigus vulgaris patients treated with rituximab efficacy of repeated courses of rituximab as treatment for pemphigus vulgaris long-term remissions of severe pemphigus after rituximab therapy are associated with prolonged failure of desmoglein b cell response long-term outcomes of rituximab therapy in pemphigus rituximab as first-line adjuvant therapy for pemphigus: retrospective analysis of long-term outcomes at a single-center rituximab as an adjuvant therapy for pemphigus: experience in 61 patients from a single center with long-term follow-up effectiveness and safety analysis of rituximab in 146 indian pemphigus patients: a retrospective single-center review of up to 68 months follow-up an assessment of treatment history and its association with clinical outcomes and relapse in 155 pemphigus patients with response to a single cycle of rituximab adjuvant rituximab therapy of pemphigus: a single-center experience with 31 patients efficacy of rituximab for pemphigus: a systematic review and meta-analysis of different regimens rituximab in the treatment of 38 patients with pemphigus with long-term follow elisa testing of anti-desmoglein 1 and 3 antibodies in the management of pemphigus developing biomarkers for predicting clinical relapse in pemphigus patients treated with rituximab bukvic mokos z. pemphigus-the crux of clinics, research and treatment during the covid-19 pandemic the evolving story of autoantibodies in pemphigus vulgaris: development of the "super compensation hypothesis" non-desmoglein antibodies in patients with pemphigus vulgaris b cell depletion therapies in autoimmune diseases: advances and mechanistic insights autoreactive b cell differentiation in diffuse ectopic lymphoid-like structures of inflamed pemphigus lesions b-cell subpopulations in humans and their differential susceptibility to depletion with anti-cd20 monoclonal antibodies adjuvant rituximab treatment for pemphigus: a retrospective study of 45 patients at a single center with long-term follow up rituximab therapy in pemphigus: a long-term follow-up conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.copyright © 2022 miše, jukićand marinovic. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-0035199-jwv0g2ey authors: ueda, shiro; porter, george a. title: gold salts, d-penicillamine and allopurinol date: 2008 journal: clinical nephrotoxins doi: 10.1007/978-0-387-84843-3_19 sha: a74a822dd8930ada5de70d41863b20062b0e3d1d doc_id: 35199 cord_uid: jwv0g2ey gold salts have been used in the treatment of patients with rheumatoid arthritis since 1927 [1]. after a controlled study, the empire rheumatism council [2], confirmed the effectiveness of gold salts for the treatment of rheumatoid arthritis. even today, chrysotherapy has remained one of the major therapeutic modalities in the second line treatment of progressive rheumatoid arthritis. gold salts are also used in the treatment of pemphigus vulgaris [3] and bronchial asthma [4]. before the introduction of an orally administered gold compound, auranofin (triethylphosphine gold tetra-acetyl glycopyranoside), to clinical use [5-7], parenterally administered gold salts, such as sodium aurothiomalate and gold thioglucose comprised chrysotherapy. the frequency and severity of the side effects for patients treated with parenteral gold versus those given oral gold preparations are significantly different [8-10]. with introduction of newer parental dmards, toxicity has been reduced using combination therapy [10a, 10b]. introduction g old salts have been used in the treatment of patients with rheumatoid arthritis since 1927 [1] . after a controlled study, the empire rheumatism council [2] , confirmed the effectiveness of gold salts for the treatment of rheumatoid arthritis. even today, chrysotherapy has remained one of the major therapeutic modalities in the second line treatment of progressive rheumatoid arthritis. gold salts are also used in the treatment of pemphigus vulgaris [3] and bronchial asthma [4] . before the introduction of an orally administered gold compound, auranofin (triethylphosphine gold tetra-acetyl glycopyranoside), to clinical use [5] [6] [7] , parenterally administered gold salts, such as sodium aurothiomalate and gold thioglucose comprised chrysotherapy. the frequency and severity of the side effects for patients treated with parenteral gold versus those given oral gold preparations are significantly different [8] [9] [10] . with introduction of newer parental dmards, toxicity has been reduced using combination therapy [10a, 10b] . despite the efficiency of injectable gold salts in the treatment of rheumatoid arthritis, they are associated with a variety of adverse effects, such as skin rashes [11] [12] [13] , thrombocytopenia [14, 15] , granulocytopenia [11, 16] , aplastic anemia [17, 18] , interstitial pneumonitis [19, 20] , gastrointestinal side effects [11, 21] , chrysiasis of cornea and lens [12] , and proteinuria and nephrotic syndrome [11, 22, 23] . one or more of these adverse reactions have been reported in approximately onethird of patients treated with gold salts [12] . proteinuria, including nephrotic syndrome, is the commonest manifestation of gold-induced nephropathy, occurring in 2% to 10% in patients receiving chrysotherapy [10, [22] [23] [24] . however, the decreased frequency of proteinuria has paralleled the reduction in dosage of injectable gold salts, prolonging the interval between injections and the introduction of several new disease modifying agents. the risk of proteinuria is increased at higher doses [26] and in the patients with hla dr3 [27] [28] [29] [30] . in one-third to half of the patients, the proteinuria is accompanied by microscopic hematuria [31, 32] . the severity of the proteinuria varies greatly and does not correlate with the duration of treatment or the total dose of gold received [31, 33] . the peak incidence of proteinuria occurs after four to six months of treatment [33] , but it may develop at any time from 1 week to 39 months after the start of treatment [33, 34] . complete resolution of gold-induced proteinuria occurs in all patients 3 years after cessation of therapy, however, one-third of patients had resolved their proteinuria in 6 months after stopping therapy [34a] . progressive loss of renal function following withdrawal of gold therapy is rare [34b] . furthermore, reinstitution of gold therapy at a lower dose in patients with prior history of goldinduced proteinuria without recurrence suggests that the proteinuria may have a dose dependency [34c] . renal function is usually normal to minimal impairment in these proteinuric patients. histopathological examinations of the renal biopsy specimens from patients with proteinuria show predominantly membranous glomerulopathy [10, 22, [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . electron microscopy of renal tissue usually demonstrates subepithelial electron dense deposits (especially when the disease is of short duration) [22, [32] [33] [34] [35] [36] [37] [38] [39] [40] , intramembranous electron dense deposits [32, 34, 40] , and fusion and increased density of foot processes of epithelial cells [22, 32, [35] [36] [37] [38] [39] [40] . light microscopy occasionally discloses varying degrees of uniform thickening of the glomerular basement membrane. small, fuchsinophilic deposits with associated spike like extensions of the basement membrane may be identified on trichrome-stained sections. immunofluorescent study of the renal tissues with subepithelial electron dense deposits reveals granular deposition of igg, igm and/or complements [10, 33, 34, 37, 39] . in addition to membranous glomerulonephritis, there are reports of minimal change glomerulonephritis [32, 41] , focal segmental glomerulonephritis [32] , and mesangioproliferative glomerulonephritis with immune complex deposition in mesangial areas [10, 31, 40] . skrifvars et al. [42] reported a highly unusual fatal renal complication induced by sodium aurothiomalate. this complication was characterized by microhematuria, impaired renal function and by a granulomatous glomerulonephritis. in addition to the glomerular lesions mentioned above, focal tubular atrophy of variable severity is a feature of the majority of biopsy specimens of gold induced nephropathy [32, 37, 39, 43] . interstitial fibrosis can be recognized in many of the specimens (figure 1) , and the degree of fibrosis tends to parallel the severity and extent of the tubular atrophy. however, interstitial inflammation is not usually prominent [32] . electron microscopy reveals the existence of characteristic filamentous, electron dense cytoplasmic inclusions in various renal cells at high frequency [22, 37-39, 44, 45] . these filamentous inclusions may be complexes containing gold and other molecules [52, 50, 53] . the inclusions are concentrated in proximal tubular epithelial cells, interstitial macrophages, but rarely occur in mesangial cells and visceral epithelial cells, and spare the basement membrane or subepithelial space. they are much more prominent in patients who have received large doses of gold [22] . there may be a significant association between the degree of histological interstitial changes and the number of gold inclusions. cramer et al. [43] reported a patient who suffered from chronic interstitial nephritis after receiving large quantities of aurothioglucose for rheumatoid arthritis. gold deposition was seen by electron microscopy and confirmed by microprobe x-ray analysis within both tubular epithelial cells and interstitial macrophages but not the interstitium. they hypothesized that the administration of massive amounts of gold salts resulted in these depositions and the subsequent interstitial nephritis [43] . lesato et al. [46] reported a high incidence of subtle renal tubular dysfunction in rheumatoid arthritis patients receiving gold treatment, demonstrating tubular proteinuria and the urinary excretion of large amounts of renal tubular epithelial antigen, tubular basement membrane (tbm) antigen, and β2-microglobulin. however, the amounts of these proteins in urine did not correlate with the total dose of gold [46] . renal tubular dysfunction has been induced in hartley guinea pigs by the injection of sodium aurothiomalate, as manifested by the urinary excretion of tubular basement membrane and renal tubular epithelial antigens and tubular proteinuria. excretion of these proteins tended to be dose dependent [47] . following the tubular dysfunction, autoimmune tubulointerstitial nephritis with anti-tbm antibodies developed in the animals [47] . there are mainly two types of gold-induced nephropathy, one being immune complex type glomerulonephritis and the other limited to tubular lesions. the latter may be induced by the direct toxic action of gold, and this toxicity seems to be dose dependent. the morphological changes in the tubules usually involve gold inclusions [22, 37, 39, [44] [45] [46] . nagi et al. [48] using large doses of sodium aurothiomalate (1 mg/week) produced renal tubular necrosis in rats, characterized by degenerative changes of the cytoplasmic contents of epithelial cells of proximal convoluted tubules. the ultracellular structure changes involved swollen mitochondria that had lost their shape. eiseman et al. [49] figure 1. photomicrographs of the kidney from a rheumatoid arthritis patient with gold nephropathy, demonstrating prominent interstitial fibrosis and tubular cell degeneration (magn. x340). above: masson's trichrome staining; below: pam staining. reported morphofunctional and biochemical changes in rat kidneys following a single ip injection of a high dose (75 mg/kg) of gold sodium thiomalate. this included severe coagulative necrosis of the proximal tubular epithelium at one day, followed by epithelial regeneration by day 4 and nearly complete resolution by day 8. alternations in renal heme biosynthesis and drug metabolism paralleled the morphological changes [49] . tubular dysfunction has also been reported in rheumatoid arthritis patients receiving gold treatment [46] and in animals being treated with low doses of gold salts [47, 48] . the pathogenesis of immune complex type glomerular lesions associated with chrysotherapy remains unclear. to clarify the pathogenesis of this nephropathy, it is necessary to confirm the specificity of the antigens and antibodies responsible for the immune complex of the glomerular lesions. gold salts may act as a hapten, and specific ige antibodies against gold salts have been detected in the sera of rheumatoid arthritis patients with mucocutaneous and hematologic adverse reaction to gold salts [50, 51] . a positive lymphocyte transformation test to gold salts has been reported in some rheumatoid arthritis patients with hematologic side effects after chrysotherapy [52] . derot et al. [53] reported a rare case of fatal acute tubular necrosis due to gold induced nephropathy. allergic reaction to gold salts might have been responsible for the development of this nephritis; however, such immunological phenomena are rarely seen in the patients with gold-induced nephropathy [50, 51] . to date, no evidence for the presence of gold in renal immune deposits has been reported. it is difficult to confirm that gold is the causal antigen or hapten in gold-induced immune complex nephropathy. palosuo et al. [54] demonstrated a circulating antigen in a patient with gold-induced nephropathy before and after the development of nephropathy, which shared immunological determinants with tissue antigens extracted with deoxycholate from microsomal fractions of various organs including human liver, human kidney, and rat liver. precipitating antibodies against this circulating antigen were found in the serum sample pre-dating diagnosis. this serum reacted with various tissue antigens extracted from human organs, but not with kidney specific antigen [54] . in an experimental rat model, nagi et al. [48] reported the successful induction of slowly progressive immune complex nephropathy by weekly injections of small doses of sodium aurothiomalate (0.0025 mg/week), suggesting the important pathogenetic role of renal tubular antigen released from damaged tubular epithelial cells ( figure 2 ). skrifvars [55] also emphasized the possible role of autoimmunization secondary to released tubular antigens in the pathogenesis of gold-induced glomerular lesions. in the guinea pig model, renal dysfunction was also induced by injections of sodium aurothiomalate, as manifested by the urinary excretion of renal tubular antigens including renal tubular epithelial and tubular basement membrane antigens. following the tubular dysfunction, immune complex nephropathy with circulating anti-renal tubular epithelial antibody, including deposition of renal tubular epithelial antigen in the glomerular immune complexes, developed in the animals [47] . thus, shed renal tubular antigens from damaged tubular epithelium may play an important role in the pathogenesis of gold-induced immune complex nephropathy. there are many drugs that injure the renal tubular epithelium, but rarely induce immune complex nephropathy. thus, in addition to tubular damage, there must be other factors that promote the development of gold nephropathy. other tissue autoantigens released and/or altered by the effect of gold and heterogeneous antigens may also participate in the pathogenetic mechanisms. that gold salts possess immunosuppressive effects has been demonstrated by both in vivo and in vitro studies [56] [57] [58] [59] . in addition, they also have an immunoenhancing effect on the immune response of mice, depending on dosage [60] . balb/c mice are highly susceptible to autoimmune interstitial nephritis, while c57bl/6 mice are genetically resistant to this nephritis when immunized with tubular basement membrane antigen with adjuvant [61] . when both strains of mice are following pretreated with appropriate doses of sodium aurothiomalate immunization with tubular basement membrane antigen with adjuvant, balb/c mice become resistent to the development of nephritis, but nephritis is induced in the genetically resistant c57bl/6 mice. thus, gold salts may depress the activity of all t cells, and the phenotypical effect of gold salts on the immune response to some antigens may depend on the character of the dominant t cells [62] . selective in vitro inhibition of t cells has also been shown in patients receiving chrysotherapy [63] . there must be other, as yet defined factors that are involved in the development of gold nephropathy. proteinuria is usually slow to resolves after withdrawal of the drug. in 1970, vaamonde et al. [31] reviewed 19 case reports of nephrotic syndrome associated with chrysotherapy. in 17 patients whose outcomes were known, 13 recovered in 3 months to 7 years. hall et al. [33] reported a long-term study of 21 patients with rheumatoid arthritis who developed proteinuria during treatment with sodium aurothiomalate. ten patients developed proteinuria after 6 months' of treatment, 15 after 12 months, and 18 after 24 months. when chrysotherapy was stopped the proteinuria had reached a median peak of 2.1 g/day (range 0.7-30.7 g/day) at two months (range 1-13 months) before resolving spontaneously, in 8 patients by 6 months, in 13 by 12 months, and in 18 by 24 months. all patients were free of proteinuria after 39 months, the median duration being 11 months after withdrawal. renal function did not deteriorate, and no patient died from or needed treatment for renal failure. hla-b8 and/or dr3 alloantigens were identified in seven of the patients [33] . newton et al. [64] studied 27 patients with goldinduced proteinuria, and provided guidelines as to when gold should be permanently stopped in these patients. they demonstrated that proteinuria of up to 2 g/l is compatible with continued gold therapy, since the low risk of more serious nephropathy developing was low. they concluded: 1) mild proteinuria (less than 0.4 g/l) is common in rheumatoid arthritis patients on gold, and such a level may not even be re-lated to this drug. it usually disappears spontaneously without alteration of therapy, but rarely can proceed to more serious problems. 2) moderate proteinuria (0.4-2.0 g/l) should be treated more seriously. gold injections should be stopped. if the urine clears within three months, then further treatment with gold may be given without precipitating heavy proteinuria. 3) none of their subjects have sustained permanent renal impairment [66] . the advice of howard-lock et al about d-penicillamine therapy may also be suitable for gold therapy. they advocate withholding the drug if there is (1) proteinuria of 2 + on the dipstick, (2) persistent (longer than 3 weeks) proteinuria of 1 + , (3) if there are red cell casts, white cell casts, or hyaline casts present, or (4) if red cells >10 per high power field are present. for patients whose disease has improved but who developed proteinuria of between 300 to 1, 000 mg/day but without other renal abnormality, they suggest continuing the drug cautiously at a reduced dose with close monitoring. if the proteinuria exceeds 2 g/day or the glomerular filtration rate falls, the drug should be discontinued immediately [65] . manthorpe et al. reported a successful one year treatment with auranofin (6 mg/day) in 7 rheumatoid arthritis patients with previous proteinuria associated with parenterally injected gold salts [66] . to predict the adverse effects of gold, the association with hla antigen has been studied [27, 28, [67] [68] [69] . a genetic predisposition to gold toxicity was first suggested by panayi et al. [67] . wooley et al. [68] investigated the possible relation between hla antigens and toxicity of d-penicillamine and sodium aurothiomalate in rheumatoid arthritis patients. nineteen of 24 patients in whom proteinuria developed were positive for hla-b8 and drw3 antigens. furthermore, all 13 episodes of proteinuria exceeding 2 g/day occurred in patients with drw3. several investigators confirmed the association between gold-induced proteinuria and dr3 [27] [28] [29] [30] and b8 [30] , but others were unable to confirm it [70] . conversely, dr3 patients tended to exhibit a better therapeutic response to sodium aurothiomalate than patients with dr4 [28] . dr4 and/or dr2 positive patients may have some degree of protection against gold toxicity [28, 29] . given the uncertainty about hla types and toxic reactions, together with the suggestion that patients with dr3 respond better than the more numerous dr4, and taking into account the cost involved, any suggestion of using hla typing as a guide to therapy seems premature [71] . while van riel et al. [72] reported the predictive value of serum iga for gold toxicity, the study of ostuni et al., involving a larger population, concluded that the monitoring of serum iga was not useful in predicting gold toxicity [73] . recently, ayesh et al. [74] reported the predictive efficacy of the prior measurement of sulphoxidation capacity. a patient with poor sulphoxidation capacity had a nine-fold greater risk of developing gold-induced adverse reactions including nephropathy. hopefully this will be confirmed by prospective studies involving various races and a large population. to date, there is no confirmed method for predicting gold toxicity including nephropathy, thus it is essential to monitor patients closely for any appearance of nephropathy. however, shah et al [74a] have evaluated the association between gold adr's (thrombocytopenia or proteinuria) and hla-dr3 status. based on a cohort of 41 patients they concluded that patients with nodular disease were more likely to develop ards (51.3% vs. 25.6%, or= 3.0, p=0.02 and also more likely to be hla-dr3 positive (41.2% vs. 17.6%, or= 3.0, p= 0.045. the authors suggest that nodular patients with hla-dr3 should not receive parenteral gold as their primary treatment for ra. the decline in the number of reports of parenterally administered gold-induced nephropathy may indicate that the dose of gold salts used per injection is decreased and intervals between injections are being extended to prevent adverse reactions. furthermore, introduction of methotrexate therapy, along with several biological agents, for rheumatoid arthritis, has contributed to decreased reliance on gold salts. however, intriguing reports using nanotechnology gold in treatment of malignancies has renewed interest in gold as a therapeutic agent [74b, c] . auranofin, a unique gold compound, has been available for clinical use for 25 years after it proved to be one of the most potent oral antiarthritic com-pounds among alkylphosphine gold coordination complexes [75] . initial clinical studies suggested that this compound was therapeutically active when taken by mouth, with no renal adverse effects in any of the 32 patients studied [5] [6] [7] . subsequently, the therapeutic benefits and toxicity of auranofin have been evaluated [24, 76] , compared with placebo [9, 77, 78] , sodium aurothiomalate [8] [9] [10] 79] , and d-penicillamine [80] [81] [82] . the incidence of proteinuria in a world-wide trial was 3% for auranofin [10, 24] . the risk of developing proteinuria with auranofin therapy is significantly less than with parenteral gold [9, 24] , or d-penicillamine [82] . histopathological findings in renal biopsy specimens from patients with moderate to heavy proteinuria are consistent with the membranous nephropathy similar to injectable gold nephropathy [33, 83, 84] . heuer et al. [10] reported a total of 3, 475 rheumatoid arthritis patients receiving auranofin therapy in 27 countries. proteinuria developed in 3% of the patients, resulting in drug withdrawal in 0.9%, compared with 4% proteinuria in patients receiving injectable gold, with 0.8% being withdrawn. katz et al. [24] evaluated proteinuria in 1800 rheumatoid arthritis patients given chrysotherapy. three percent (41 cases) of 1283 auranofin-treated patients had an abnormal 24-hour urine protein level: 15 had mild (0.15 to 1 g/day), 17 had moderate (1 to 3.5 g/day), and 9 had heavy (>3.5 g/day) proteinuria. permanent renal impairment did not occur in any patient. in 36 patients with long-term follow-up after drug withdrawal, proteinuria cleared in 31 patients within 1 week to 24 months. seven of 8 patients who were rechallenged once the proteinuria had cleared were able to continue treatment without recurrent episodes [24] . pathogenic mechanism of auranofin-induced nephropathy resemble those of parenteral gold-induced nephropathy. the reason for the reduced risk of proteinuria with auranofin compared to parenteral gold salts is not known. however, differences in the pharmacokinetics of the two types of gold preparations may be important. in rats treated with auranofin or sodium aurothiomalate for one year, renal gold concentrations were 33 times higher with the latter formulation [85] . renal elimination of an orally administered dose of auranofin in human is less than 15%, compared with greater than 70% for parenterally administered sodium aurothiomalate [86] . introduction d-penicillamine is so named because it was first isolated as an amine, from the degradation products of penicillin by abraham et al [87] . later studies showed the characteristic chemical behavior of d-penicillamine which involves three types of reactions, formation of disulphide links, formation of thiazolidine rings, and formation of metal complexes and chelates [67] . it was first used in 1956 in the treatment of wilson's disease [88] . d-penicillamine has since been used in the treatment of many diseases, such as cystinuria [89] , rheumatoid arthritis [90] [91] [92] , systemic sclerosis [93] , primary biliary cirrhosis [94] , heavy metal poisoning due to lead [95] , cadmium [96] , and mercury [97] , and hyperviscosity syndrome [99] . in rheumatoid arthritis, d-penicillamine has been widely accepted as an effective second line treatment. despite of its effectiveness, it causes many adverse effects, such as skin rashes [99, 100] , taste abnormalities [100, 101] , hepatic dysfunction [102] [103] [104] , gastrointestinal toxicity [99, 105] , proteinuria [100, 106] , hematuria [107, 108] , thrombocytopenia [92, 109] , aplastic anemia [110] , lupus-like syndrome [111, 112] , goodpasture's-like pulmonary renal syndrome [113] [114] [115] , vasculitis [116, 117] , myasthenia gravis [118] [119] [120] [121] [122] , polymyositis [123, 124] , and dermatomyositis [125] . one or more of these adverse reactions was recorded in nearly 60% of patients treated with d-penicillamine [100, [126] [127] [128] [129] . among these adverse reactions, nephropathy developed in patients with proteinuria, hematuria, lupus-like syndrome, goodpasture's-like pulmonary renal syndrome, and vasculitis. proteinuria, including nephritic syndrome, is the commonest manifestation of nephropathy, reported as occurring in between 2 and 32% of patients [100, 101, 109, 124, [126] [127] [128] [129] [130] . the risk of proteinuria is increased at higher doses [100, [131] [132] [133] , in patients with hla b8 and/or drw3 antigens [68] , and in patients with previous gold toxicity [134, 135] . however, others have not confirmed the relationship to the drug dosage [136] , duration of therapy [137] , or hla antigens [70] . in the majority of patients, proteinuria is accompanied by microscopic hematuria [100, 127] . the peak incidence of proteinuria occurs in the second six months of treatment, but it may develop at any time from 6 weeks to 74 months [107, 101, 138] . proteinuria may be persistent or may slowly progress to nephrotic syndrome if therapy is continued. up to 1/3 of the patients with significant proteinuria progress to nephrotic syndrome if therapy is continued [106] . renal function is normal to minimal impairment in patients with isolated proteinuria. histopathological examination of renal biopsy specimens from the patients with isolated proteinuria due to d-penicillamine shows predominant membranous glomerulopathy [139] [140] [141] . electron microscopy of renal tissue usually demonstrates subepithelial electron dense deposits and fusion of epithelial foot processes [139] [140] [141] . the deposits on the epithelial side of the glomerular basement membrane appear to be slowly covered and later incorporated into the basement membrane. with time the deposits become fainter and move towards the endothelial side of the basement membrane [142] . immunofluorescent study may demonstrate granular deposits of igg and c3 in the capillary wall. these changes in glomerular histology can persist for at least a year after the withdrawal of the drug [139] . sellars et al. [143] reviewed the renal biopsies of 30 patients with rheumatoid arthritis and clinical evidence of renal disease. they reported all 9 patients with membranous glomerulonephritis but only 6 of 13 with mesangial change had received dpenicillamine or gold. besides membranous glomerulonephritis, there are reports of minimal change glomerulonephritis [144, 145] , mild mesangioproliferative glomerulonephritis without crescent [110, 142, 146] , or igm nephropathy [147, 148] associated with d-penicillamine induced proteinuria. proteinuria usually resolves slowly after withdrawal of the drug. hall et al. [149] reported a long-term study of 33 patients with rheumatoid arthritis who developed proteinuria during treatment with d-penicillamine. of these, fourteen patients developed proteinuria within 6 months after the start of treatment and 27 within 12 months. when treatment was stopped, the proteinuria reached a median peak of 4.2 g/day (range 0.3-15 g/ day) at one month (range 0-7 months) before resolving spontaneously by six months in 12 patients, 12 months in 21, and 21 months in all. in all their patients whose nephropathy was due to d-penicillamine the proteinuria resolved completely when the drug was withdrawn; renal function did not deteriorate, and corticosteroids were unnecessary [149] . jaffe [150] reported that reintroduction of d-penicillamine in patients with drug induced proteinuria, starting with a daily dose of 250 mg, was usually followed by a return of proteinuria at about the same time and at about the same cumulative dose as on the first occasion. however, hill et al. [133] reported successful reintroduction and continuation for a minimum of 13 months in 5 rheumatoid arthritis patients who developed proteinuria during the first course of the drug. they instituted the "go slow, go low" method of jaffe [151] , starting with a daily dose of 50 mg and increasing by monthly increment of 50 mg to a maintenance dose of 150 mg daily. the dose was held at 150 mg/day for 4 months and thereafter increased by 50 mg at 3-months intervals if disease remained active. proteinuria did not recur, and improvement of disease was shown in all 5 patients [133] . howard-lock et al. [65] advocated withholding d-penicillamine if there is (1) proteinuria of 2+ on the dipstick, (2) persistent (longer than 3 weeks) proteinuria of 1+ (3) if there are red cell casts, white cell casts, or hyaline casts present, or (4) if red cells > 10 per high power field are present. for patients whose disease has improved but who developed proteinuria between 300 to 1, 000 mg/day, but without other renal abnormality, they suggest the continued use of the drug cautiously at a reduced dose with close monitoring. if proteinuria exceeds 2 g/day or the glomerular filtration rate falls, the drug should be discontinued immediately. besides the benign proteinuria mentioned above, proliferative glomerulonephritis with fulminant renal failure has also occurred with d-penicillamine therapy. one is goodpasture's-like syndrome, which is characterized by pulmonary hemorrhage and rapidly progressive glomerulonephritis. goodpasture's -like syndrome associated d-penicillamine treatment has been reported in patients with wilson's disease [113] , rheumatoid arthritis [114, 115, 152, 153] , primary bil-iary cirrhosis [154] , and progressive systemic sclerosis [155] . d-penicillamine was given for at least 7 months (range: 7-84 months), and at a daily dose higher than 750 mg (range: 750-2, 000 mg) preceding the onset of symptoms. pulmonary x-rays showed bilateral extensive infiltrates in all 10 cases. lung hemorrhage was the principle cause of death in 3 cases [113] . the histopathology of renal specimens usually showed proliferative glomerulonephritis with crescent formation in 30 to 100% of the glomeruli. direct immunofluorescent study failed to show linear igg deposition along the glomerular basement membrane, but granular deposition of igg and/or c3 were present along the glomerular capillary walls in 5 of 6 patients. subepithelial electron dense deposits were observed in 3 of 4 patients tested. circulating anti-glomerular basement membrane antibody was not detected in any of the cases tested. in brown norway rats, the administration of d-penicillamine induced antinuclear antibodies and significantly high concentrations of immune complexes. iin these animals there was no granular deposition of igg, but linear deposition of igg along the glomerular basement membrane. igg eluted from diseased kidneys bound both in vitro and in vivo to the kidney basement membrane [156] . hla-dr2 antigen was absent in the 2 cases where hla phenotype was determined, whereas there is a strong association between hla-dr2 and antibody-mediated good-pasture's syndrome [157] . anti-nuclear antibodies have been detected both before [115, 156] and after initiation of the drug [152, 115] . although this syndrome is potentially life-threatening, aggressive treatment with plasmapheresis, steroids, immunosuppressive drugs such as azathioprine and cyclophosphamide, and mechanical ventilation with peep may be life saving [113, [152] [153] [154] [155] . derk and jimenez [155a] recently reviewed the case for goodpasture-like syndrome occurring in systemic sclerosis patients treated with penicillamine. basically they describe rapidly progressive glomerulonephritis without anti-gbm antibiodies, but with linear or granular glomerular deposits. while they raise the possibility of pausi-immune gn this could not be confirmed since anca was not tested in their patient. despite their conclusions, the case report by bienaime et al ( [155b] makes a compelling case for penicillamine induced anca associated rpgn in a patient with wilson disease. since wilson disease has never been associated with pauci-immune gn, the authors are confident that penicillamine is capable of inducing anca associated rpgn, to date all patients with suspected pauci-immune gn have tested positive for anti-mpo antibodies. since this observation was limited to a patient with wilson disease, it remains to be confirmed that patients with systemic sclerosis or rheumatoid arthritis receiving penicillamine who develop the goodpasture-like syndrome described as a complication of penicillamine treatment should be classified as pauci-immune gn with anti-mpo antibodies. extracapillary glomerulonephritis with renal vasculitis is also been reported as a rare complication of d-penicillamine therapy [117, 126, 156] . necrosis of interlobular arteries with glomerular crescent [117] and necrotic and occluded periglomerular arterioles [156] have been reported. aggressive treatment with pulse steroid, anticoagulants, and antiplatelet agents may be beneficial. the two patients with renal vasculitis, whose outcome was known, died from bacterial infection within ten months after the onset of the disease [117, 156] . these cases mostly likely represent pauci-immune gn as reviewed in the preceding paragraph. a drug-induced systemic lupus erythematosus (sle) with proliferative glomerulonephritis has also been described in patients treated with d-penicillamine [111, 157] . systemic lupus erythematosus syndrome is induced in approximately 2% of patients treated with d-penicillamine [112, 158] . unlike other forms of druginduced systemic lupus erythematosus, anti-doublestrand dna antibodies and/or hypocomplementemia are seen in d-penicillamine-induced systemic lupus erythematosus syndrome [111, 156] . nephropathy is rare in d-penicillamine-induced systemic lupus erythematosus syndrome [111] . walshe [112] reported that 8 patients developed the serological change of systemic lupus erythematosus of 120 patients with wilson's disease treated with d-penicillamine, but none of them showed nephropathy. chalmers [111] reported 6 rheumatoid arthritis patients with d-penicillamine-induced systemic lupus erythematosus syndrome. all patients had previous mucocutaneous reactions to chrysotherapy. manifestations included pleurisy in 5 of 6 patients, rashes in 3, and nephritis in 2. le cells were present in 5 patients, anti nuclear antibodies in all 6, anti-double-strand dna in 3, 3 were coomb's test positive, and low c4 complement in 5 of the 6 [111] . results of a renal biopsy from a patient with nephritis showed diffuse endocapillary proliferative glomerulonephritis with focal crescent formation and vasculitis. electron microscopy showed scattered subendothelial deposits, and immunofluorescent study revealed granular deposition of igg, igm, c3 complement and c1q. the patient was successfully treated with prednisolone and azathioprine [112] . ntoso et al. [156] reported penicillamine-induced rapidly progressive glomerulonephritis in two patients with progressive systemic sclerosis. anti nuclear antibodies, anti-sm antibody, and coomb's antibodies were positive in both patients. renal biopsies from the two patients demonstrated a diffuse, predominantly extracapillary, proliferative glomerulonephritis with crescents and focal necrosis, and by immunofluorescence, focal areas of igg, c3, and fibrinogen were observed in areas of glomerular necrosis. subendothelial and mesangial deposits were observed by electron microscopy. both patients responded to pulse methylprednisolone and subsequent daily steroids [156] . deposition of immune complexes in the glomerular basement membrane may play an important role in the pathogenesis of d-penicillamine-induced nephropathy, such as isolated proteinuria, goodpasture's-like syndrome, and nephritis associated with d-penicillamineinduced systemic lupus erythematosus rheumatoid arthritis syndrome. immunofluorescent study show predominantly granular deposition of igg and/or c3, and electron microscopy revealed subepithelial or subendothelial electron dense deposits. in rheumatoid arthritis patients, d-penicillamine alters the circulating immune complexes [159] . d-penicillamine has the capacity to convert large complexes into small ones in vitro and there has been speculation that similar mechanisms in vivo could explain the deposition of complexes and renal damage [160] . small immune complexes deposit in the glomeruli easier than big ones. in addition to penicillamine nephropathy, other side effects of the drug may be related to the widespread deposition of immune complexes (figure 3) . dense, granular immunoglobulin deposits have been identified at the epidermodermal junction in 4 rheumatoid arthritis patients who developed toxic reactions, such as severe rashes, thrombocytopenia, aplastic anemia, and proteinuria. three of 4 penicillamine-induced systemic lupus erythematosus syndrome patients had similar findings on skin biopsy [161] . besides immune complex deposition, autoantibodies against several autoantigens are frequently detected in patients treated with d-penicillamine, leading to autoimmune diseases. the exact mechanism by which this drug induces autoimmunity remains to be investigated. it may directly stimulate oligoclonal b cell activity, upset the balance between t cell subsets, or alter antigens by hapten formation. d-penicillamine can bind with various proteins, and may change the antigenicity of these proteins as a hapten. however, to date, no evidence for the presence of penicillamine in renal immune deposits has been reported. nagata et al. [162] reported that d-penicillamine can act as a hapten for specific t cells when presented on the surface of appropriate stimulator cells, and suggested that the adverse immunological side effects of this drug in patients may have a pathogenesis similar to graft-versus-host reaction. the possibility of anca associated vasculitis, as described in the case report of bienaime et al [155b] , raises an alternate explanation for the pathogenesis of penicillamine-induced vasculitis. since all of the anca associated gn due to penicillamine have had anti-mpo antibodies, this suggests that an interaction with mpo is critical in triggering the anca induced vasculitis. however, it is not clear that penicillamine induces autoimmunity, thus the exact mechanism remains to be elucidated, although both humoral and cellular immunity are thought to play significant roles [162a] . to predict d-penicillamine side effects, the association between side effects and various factors, such as hla antigens [68, 70, 128, 130, 163, 164] , autoantibodies [165, 166] , and previous gold toxicity [101, 138, 167, 168] has been studied. wooley et al. [68] investigated the possible interaction between hla antigens and toxicity of d-penicillamine and sodium aurothiomalate in rheumatoid arthritis patients. nineteen of 24 patients in whom proteinuria developed were positive for hla-b8 and drw3 antigens. furthermore, all 13 episodes of proteinuria exceeding 2 g/day occurred in patients with drw3 [68] . there is also a strong association between idiopathic membrane nephropathy and hla-drw3, b8 and b18 [169] . other investigators have confirmed the association between d-penicillamineinduced proteinuria and dr3 [128, 130, 164] and b8 [70, 128, 130] . however, other investigators could not confirm a significant association between d-penicillamine proteinuria and hla-dr3 [70, 170] . in addition to hla antigens, emery et al. [163] emphasized the sulphoxidation status of patients as a new predictor of outcome of drug toxicity. moutsopoulos et al. [165, 166] reported that anti-ro (ssa) positive greek rheumatoid arthritis patients experienced a significantly high frequency of side effects from d-penicillamine. despite their dissimilar chemical structures, the thiol compounds, sodium aurothiomalate and d-penicillamine, have remarkably similar clinical effects, and this similarity extends to the incidence and type of adverse effects [138, 167] . several investigators have noted the association between prior gold nephropathy and d-penicillamine. billingsley and stevens reported the significant correlation of d-penicillamine-induced proteinuria to a previous history of gold nephropathy [134] . patients with gold-induced proteinuria are at a higher risk for the development of proteinuria during d-penicillamine therapy (p<0.001), and this occurs within the first six months of treatment [138] . all six patients who developed systemic lupus erythematosus syndrome while being treated with dpenicillamine had previous mucocutaneous reactions to chrysotherapy [114] . dood et al. [165] noted that all patients who took d-penicillamine within six months after an adverse reaction to gold developed side effects from d-penicillamine, and recommended an interval exceeding six months between treatment with gold and treatment with d-penicillamine in patients who have developed adverse reactions to gold, to reduce the risk of adverse reactions to d-penicillamine, kean et al. [101] analyzed the influence of previous sodium aurothiomalate therapy on the toxicity pattern of d-penicillamine, but could not confirm a synergistic effect of d-penicillamine and sodium aurothiomalate leading to increased adverse reaction in patients with rheumatoid disease [101] . although there are several predictors of adverse reactions, the most useful clinical predictor is urinalysis. patients on d-penicillamine therapy should be closely monitored for evidence of proteinuria as the first sign of penicillamine induced nephropathy [170a] . allopurinol (4-hydroxypyrazolo [3, 4-d] pyrimidine) is an inhibitor of xanthine oxidase that was successfully introduced in the treatment of primary gout about 45 years ago [171] . allopurinol continues to be accepted as standard therapy in the treatment of primary and secondary hyperuricemia. adverse reactions occur in about 10% of patients treated with allopurinol and are relatively mild and self-limited [171, 172] . a mild maculopapular eruption or gastrointestinal disorders are usually noted, which promptly regress with cessation of therapy. isolated instances of allopecia [173] , bone marrow depression [174] , ocular lesions [175] , acute cholangitis [176] , various types of hepatic injuries [177, 178] temporal arthritis [179] , and xanthine stones [180] have been reported. recently, larosa et al [180a] have reported a case of xanthine nephropathy during treatment of childhood t-cell all. xanthine nephropathy has been reported in tumor lysis syndrome (tls) in patients with hypoxanthineguanine phosphoribosyl transferase (hgprt) enzyme deficiency [180b] , however, this patients' cultured fibroblasts yielded normal levels of hgprt enzyme. allopurinol pretreatment allows the build up of both xanthine and hypoxanthine which, in the absence of hgprt, cannot be recycled and thus xanthine supersaturation in the urine resulting in xanthine stones with subsequent obstructive renal failure. in 1970, reports began to appear of systemic, severe, prolonged hypersensitivity reactions occurring in patients during treatment with allopurinol, now known as allopurinol hypersensitivity syndrome (ahs) [182] . these reactions are characterized by fever, chills, malaise, generalized dermatitis, eosinophilia, abnormalities of liver function tests, and rapidly progressive renal failure [181] [182] [183] [184] [185] [186] [187] [188] . allopurinol-induced nephropathy is usually reported as a part of these reactions. in 1979, gorge et al. [186] reported 3 cases of such reactions and reviewed 38 patients including their 7 patients. the average dose of the drug in these patients was 300 mg/day. the average time from initiation of the therapy to onset of the reaction was 3.8 weeks. the most common type of dermatitis was a pruritic, diffuse, erythematosus, maculopapular eruption noted in over 60% of the patients. toxic epidermal necrosis, stevens-johnson syndrome, and exfoliative dermatitis were also noted in some patients. exfoliative dermatitis has also been reported in a patient with metabolic syndrome as a delayed skin reaction heralding the onset of ahs [188a] . the presence of eosinophilia (4-53%) was noted in all but two patients. thirty-one of 32 patients (97%) had documented impaired renal function prior to allopurinol therapy. following the onset of the hypersensitivity reaction, further deterioration of renal function occurred in 30 of 32 patients [186] . in 1986, singer et al. [188] reported 8 additional patients with such reactions and reviewed an additional 72 patients described in the literature. forty of 80 patients (50%) had impaired renal function prior to allopurinol therapy. further deterioration of renal function was found in 48 of 80 patients. underestimation of the presence of impaired renal function in hospitalized patients with gout was recently highlighted by the findings of petersel and schlesinger [188b] . based on a two year retrospective review of records of hospitalized patients with acute gout they found renal failure (serum creatinine > 1.5 mg/dl) in 65% and a decreased egrf in 73%. ckd iii was present in 47%, ckd iv in 20% and ckd v in 5%. combination therapy with colchicine and nsaids was used in over 80% of the patients with renal failure and gout. only 27% of the patients admitted with gouty arthritis were receiving allopurinol prophylaxis. in patients receiving allopurinol prophylaxis as outpatient treatment, one quarter do not have their serum creatinine monitored [188c] . histopathological examination of renal biopsy or autopsy specimens revealed renal vasculitis [181] , focal segmental glomerulonephritis [184] , and acute interstitial nephritis [185, 187, 189, 190] . jarzobski et al. [181] reported a case of the hypersensitivity type of vasculitis with fibrinoid necrosis and eosinophilic reaction, involving multiple organs, especially the kidney, resulting in uremia and death. boyer et al. [191] also reported 3 cases of the same type including the efficacy of prednisolone in treating this type of disease. kantor et al. [182] reported a case of glomerulonephritis associated with allopurinol-hypersensitivity. linear deposition of igg and complement along the glomerular basement membrane were demonstrated, and a necrotizing, hemorrhagic pneumonitis was also reported. however, no circulating anti-glomerular basement membrane antibody was detected. acute interstitial nephritis has also been reported associated with by the administration of allopurinol [185, 187, 189, 190] . gelbart et al. [185] reported a case of allopurinolinduced interstitial nephritis with extensive infiltration of lymphocytes, plasma cells and tubular damage. no immunoglobulins, complement, or fibrin were evident in the tubular basement membrane. this patient also had other typical symptoms of hypersensitivity reactions. grussendrof et al. [187] also reported a case of acute interstitial nephritis with circulating anti-tubular basement membrane antibody and granular c3 deposition on the tubular basement membrane. the interstitium was diffusely widened, edematous and infiltrated with lymphocytes, plasma cells, histiocytes and numerous eosinophils. the nephritis was induced by controlled re-exposure to allopurinol in a patient who had two successive severe hypersensitivity reactions to this drug. more recently, morel et al [191a] reported a case of allopurinol hypersensitivity reac-tion with renal failure on admission, in which skin manifestations and renal failure recurred after initial recovery. the case was considered unique due to the presence of an ana titer or 1:2000 on admission. treatment consisted of intravenous and oral steroids with residual renal impairment after 7 months of therapy. a renal biopsy, at the time of recurrence, yielded deposits of c3 complement in the vessel walls. a previous skin biopsy on admission yielded leukocytoclastic, non-specific vasculitis. the authors concluded that the "findings suggested the participation of ribonucleotide alterations in the pathophysiology of allopurinol hypersensitivity syndrome". the pathogenesis of nephropathy associated with allopurinol-induced hypersensitivity reactions is unclear. however, pathogenic role of the immune reactions against allopurinol or its metabolites has not been excluded. emmerson et al. [192] studied the lymphocyte reactivities to allopurinol and its active metabolite, oxypurinol, in 9 patients with previous documented adverse reactions to allopurinol. they suggested that some adverse reactions to allopurinol represented delayed type hypersensitivity to oxypurinol, but not to allopurinol. allopurinol is oxidized by xanthine oxidase to oxypurinol, which is also an inhibitor of the enzyme (figure 4 ). allopurinol plasma half life is less than 2 hours due to rapid renal clearance and oxidation to oxypurinol [193] . oxypurinol, because of its reabsorbance by the renal tubules, has a plasma half-life of 18 to 30 hours. the clearance of oxypurinol is diminished in renal insufficiency [194] . in addition, thiazide diuretics might be expected to cause accumulation of oxypurinol since its renal handling is similar to that of uric acid [195] . hypersensitivity syndrome has been found to occur most frequently when allopurinol is given with thiazides or in patients with renal insufficiency [184, 188] . the immune reactions to oxypurinol may play an important role in the pathogenesis of the syndrome, including being dose dependent. the serum concentration of oxypurinol has been monitored to prevent adverse reactions [195, 196] . recommended plasma oxypurinol concentrations are below 100 μmol/l [196] . several authors [195, 196] reported that no adverse reactions have occurred in patients with lower plasma oxypurinol levels; how-ever, hypersensitivity syndrome occasionally develops in patients with a therapeutic plasma oxypurinol concentration [197] . in addition to plasma oxypurinol concentration, other factors probably contribute to the development of the syndrome. human herpes virus 6 (hhv 6) infection is recently attracted a great deal of attention as a possible cause of drug-induced hypersensitivity. suzuki et al reported a case of allopurinol-induced hypersensitivity syndrome with dramatically increased anti-hhv 6 igg antibodies. they also demonstrated the presence of hhv 6 in the skin of this patient using a polymerase chain reaction and in situ hybridization [198] . thus, drug-induced hypersensitivity syndrome may not be a simple allergic reaction to drug. further investigations regarding the relation of hhv 6 infection and drug-induced hypersensitivity syndrome may provide insight to the pathogenesis of allopurinol-induced hypersensitivity syndrome. withdrawal of the drug and the prolonged administration of systemic steroids are beneficial for the hypersensitivity syndrome with renal involvement. initial dose of steroid should be 1 to 2 mg/kg/day of methylprednisolone, with careful gradual tapering of steroids required in the majority of patients. the recovery time ranged from 1 week to 11 months. mortality from this syndrome is high, with twenty-one of 80 patients died as a result of the syndrome [188] . in fulminant cases, such as acute renal failure complicating toxic epidermal necrosis or stevens-johnson syndrome, methylprednisolone 'pulse' therapy might be beneficial. patients with hhv 6 infection also require prednisolone therapy. to prevent unnecessary morbidity and mortality due to the allopurinol hypersensitivity, singer et al. [188] recommended the indications for allopurinol as follow: 1) tophaceous gout; 2) major uric acid overproduction (urinary excretion of more than 900 mg of uric acid/day on a diet with rigid purine restriction); 3) frequent gouty attacks unresponsive to prophylactic colchicines, when uricosuric agents cannot be used due to intolerance, lack of efficacy, renal insufficiency, or poor patient compliance; 4) recurrent uric acid renal calculi; 5) recurrent calcium oxalate renal calculi when associated with hyperuricosuria; or 6) prevention of acute urate nephropathy in patients receiving cytotoxic therapy for malignancies. the tumor lysis syndrome (tls) has come under increased scrutiny with the more aggressive chemotherapeutic management of both hemopoeitic and solid tumor malignancies. because of the massive release of purine nuceloties, pretreatment with allopurinol often is inadequate to control the hyperuricemia and acute uric acid nephropathy develops [198a] . to overcome this deficiency of allopurinol protection, febuxostat, a more prowerful xanthine oxidase inhibitor has been developed. however, in criticizing a recent clinical trial comparing febuxostat with allopurinol [198b] , gelber [198c] wrote "caution, however, needs to be exercise in as much as the reported frequency of adverse events leading to discontinuation of the drug occurred two and three times as often in the low-dose and high-dose febuxostat group, respectively, as in the allopurinol group". rasburicase is a urate oxidase that converts uric acid to allantoin which is much more soluble thus precluding acute urate nephropathy in tls [198d, h] . a combination of rasburicase and allopurinol has been successfully used in preventing hyperuricemia of tls [198i] . while rasburicase has been shown to be successful in preventing the hyperuricemia of tls [198e, f, g] it should not be given to patients with g6pd deficiency, methemaglobinemia and history of anaphalaxis [198e] . also, concern has been raised about the high immunogenicity of rasburicase and native uricase since antibodies to the drug occurred in 14% of patient in a clinical trial of tls [198a] . there is disagreement regarding the value of allopurinol treatment for asymptomatic hyperuricemia, uncomplicated gout, and acute gouty attacks which singer et al [188] consider counter-indicated while kelley [199] advised allopurinol therapy for asymptomatic hyperuricemia, but only when it is truly severe (serum uric acid level > 13 mg/dl and 24-hour urine excretion > 1, 100 mg). for the treatment of acute hyperuricemia with renal insufficiency ronco and coworkers [198g] present compelling data supporting the use of rasburicase. the allopurinol hypersensitivity syndrome occurs most frequently when the drug is given with diuretics or in patients with renal insufficiency. ckd patients on allopurinol therapy should be closely monitored especially within the first several weeks after initiating administration of the drug. if the ahs develops, allopurinol should be withdrawn but may be reintroduced using a gradually increasing dosage schedule [200] . finally, patients at high risk for developing hyperuricemia should start the therapy with lower dose of allopurinol. indications for chronic treatment of symptomatic gout in high risk patients using either rasburicase or febuxostat needs to be confirmed by clinical trials. die gunstige beeinflussung schleichender dauerinfekte durch solganal the research sub-committee of the empire rheumatism council. gold therapy in rheumatoid arthritis. final report of a multicenter controlled trial management of pemphigus with gold compounds gold salt in the treatment of bronchial asthma -a double blind study auranofin -new oral gold compound for treatment of rheumatoid arthritis auranofin: an oral chrysotherapeutic agent for the treatment of rheumatoid arthritis management of rheumatoid arthritis with oral gold one-year comparative study of gold sodium thiomalate and auranofin in the treatment of rheumatoid arthritis comparison of auranofin, gold sodium thiomalate and placebo in the treatment of rheumatoid arthritis: a controlled clinical trial an analysis of worldwide safety experience with auranofin etanercept in combination with sulfasalaazine, hydroxychloroquine, or gold in the treatment of rheumatoid arthritis longterm combination therapy of refractory and destructive rheumatoid arthritis with methotrexate (mtx) and intramuscular gold or other disease modifying antirheumatic drugs compare to mtx monotherapy gold treatment of arthritis. a review of 900 cases complication of chrysotherapy. a review of recent studies gold dermatitis hla antigens and toxic reactions to sodium aurothiomalate in patients with rheumatoid arthritis gold-induced thrombocytopenia: platelet associated igg and hla typing in three patients diagnosis and management of adverse reaction from gold compounds bone marrow transplantation in patients with gold-induced marrow aplasia aplastic anemia in association with gold therapy for rheumatoid arthritis diffuse pulmonary injury associated with gold treatment patients with rheumatoid arthritis and fold-induced pneumonitis express two high-risk major histocompatibility complex patterns fulminant colitis complicating gold therapy gold nephropathy. a clinical and pathological study long-term chrysotherapy. incidence of toxicity and efficacy during sequential time periods proteinuria in gold treated rheumatoid arthritis gold nephrosis a double-blind trial of high versus conventional dosages of gold salts for rheumatoid arthritis hla dr antigens and gold toxicity hla antigens and toxic reactions to sodium aurothiomalate in patients with rheumatoid arthritis dr antigens and gold toxicity in white rheumatoid patients association of different hla antigens with various toxic effects of gold salts in rheumatoid arthritis the nephritic syndrome as a complication of gold therapy god-associated nephropathy the natural course of gold nephropathy: long term study of 21 patients prototype of membranous glomerulonephritis the natural course of gold and penicillamine nephropathy. a longterm study of 54 patients reinstitution of gold after gold induced proteinuria renal lesions associated with gold therapy. light and electron microscopic studies glomerular diseases as a complication of gold therapy gold nephropathy. an immunopathologic study ultrastructural fluorescent and microanalytic study of two human cases gold-induced immune complex nephritis in seronegative rheumatoid arthritis minimal change nephropathy during gold treatment. a case with unusual histopathological and immunopathological features granulomatous glomerulonephritis in a patient with rheumatoid arthritis treated with gold salts chronic interstitial nephritis associated with gold nephropathy ultrastructural and electron microprobe examination of clinical and experimental materials renal tubular dysfunction as a complication of gold therapy in patients with rheumatoid arthritis experimental gold nephropathy in guinea pigs: detection of autoantibodies to renal tubular antigens gold nephropathy in rats. light and electron microscopic studies acute nephropathy induced by gold sodium thiomalate: alterations in renal heme metabolism and morphology association of ige antibodies to sodium aurothiomalate and adverse reactions to chrysotherapy for rheumatoid arthritis immunological studies on the mechanism of gold hypersensitivity reactions the lymphocyte transformation test and gold hypersensitivity fatal anuric nephritis with associated chrysocyanosis following gold therapy serologic data suggesting an immune complex disease hypothesis for the pathogenesis of sodium aurothiomalate induced immune complex nephritis effects of gold salts and prednisolone on inflammatory cells. i. phagocytic activity of macrophages and polymorphs in inflammatory exudates studied by a 'skin-window' technique in rheumatoid and control patients effects of gold salts and prednisolone on inflammatory cells. ii. suppression of inflammation and phagocytosis in the rat effects of certain antirheumatic drugs on normal human peripheral blood lymphocytes. inhibition of mitogen-and antigen-stimulated incorporation of tritiated thymidine effects of a gold salt on lymphocyte responses effects of gold on the immune response of mice autoimmune interstitial nephritis in inbred mice. analysis of mouse tubular basement membrane antigen and genetic control of immune response to it gold nephropathy -effect of gold on immune response to renal tubular basement membrane (tbm) antigen in mice. in: nephrotoxicity. in vitro to in vivo, animals to man selective inhibition of t suppressor lymphocytes during chrysotherapy proteinuria with gold nephropathy: when should gold be permanently stopped? d-penicillamine; chemistry and clinical use in rheumatic disease auranofin in rheumatoid arthritis: use in patients with side-effect or lack of effect to gold sodium thiomalate or gold thioglucose and/or d-penicillamine genetic basis of r.a. hla antigen, disease manifestations and toxic reactions to drugs hla-dr antigens and toxic reaction to sodium aurothiomalate and d-penicillamine in patients with rheumatoid arthritis hla antigens and seronegative rheumatoid arthritis a systematic survey of hla-a, b, c, and d antigens and drug toxicity in rheumatoid arthritis hla antigens and drug toxicity in rheumatoid arthritis serum iga and gold induced toxic effects in patients with rheumatoid arthritis serum iga and gold toxicity in rheumatoid arthritis: lack of predicting value sodium aurothiomalate toxicity and sulphoxidation capacity in rheumatoid arthritis patients can gold ltherapy be used more safely in rheumatoid arthritis? adverse drug reactions are more likely in patients with nodular disease, independent of hla-dr3 status therapeutic possibilities of plasmonicall heated gold nanoparticles synergistic enhancement of selective nanophotothermolysis with gold nanoclusters: potential for cancer therapy auranofin: a unique oral chrysotherapeutic agent oral gold therapy with auranofin (sk&f39162) the efficacy and safety of auranofin compared to placebo in rheumatoid arthritis auranofin versus placebo in the treatment of rheumatoid arthritis gold therapy in rheumatoid arthritis. interim report of the canadian multicentre prospective trial comparing sodium aurothiomalate and auranofin a 12-month comparative trial of auranofin and d-penicillamine in rheumatoid arthritis auranofin versus penicillamine in rheumatoid arthritis. one-year results from a prospective clinical investigation auranofin or d-penicillamine in the treatment of rheumatoid arthritis reversible proteinuria as a complication of oral gold therapy membranous glomerulonephritis as a complication of oral gold therapy comparative pharmacokinetics of parenteral and oral gold compounds auranofin versus injectable gold: comparison of pharmacokinetic properties penicillamine, a characteristic degradiation product of penicillin a new oral therapy for wilson's disease effects of penicillamine on cystinuria intra-articular dissociation of the rheumatoid factor the effect of penicillamine in the laboratory parameters in rheumatoid arthritis controlled trial of d-penicillamine in severe rheumatoid arthritis d-penicillamine therapy in progressive systemic sclerosis (scleroderma). a retrospective analysis a controlled trial of d-penicillamine therapy in primary biliary cirrhosis treatment of lead poisoning with oral penicillamine metal binding by d-penicillamine: crystal structure of d-penicillamine cadmium (ii) hydrate mercury poisoning from an unsuspected source dissociation of human serum macroglobulins the toxicity pattern of d-penicillamine therapy. a guide to the use in ra adverse effects of d-penicillamine in rheumatoid arthritis prior gold therapy does not influence the adverse effects of d-penicillamine in rheumatoid arthritis liver abnormalities in penicillamine treated patients with rheumatoid arthritis cholestatic jaundice caused by d-penicillamine cholestatic hepatitis caused by penicillamine penicillamine causing acute colitis proteinuria in d-penicillamine-treated rheumatoid arthritis effect of penicillamine on the kidney microscopic haematuria in patients with rheumatoid arthritis on d-penicillamine toxicity profiles of disease modifying antirheumatic drugs in rheumatoid arthritis toxicity of d-penicillamine in rheumatoid arthritis. a report of 63 patients including two with aplastic anemia and one with the nephritic syndrome systemic lupus erythematosus during penicillamine therapy for rheumatoid arthritis penicillamine and the sle syndrome d-penicillamine induced goodpasture's syndrome in wilson's disease goodpastures' syndrome and d-penicillamine penicillamine-induced 'goodpasture's syndrome'; successful treatment of a fulminant case the treatment of rheumatoid arthritis and necrotizing vasculitis with penicillamine extracapillary glomerulonephritis with necrotizing vasculitis in d-penicillamine treated patients with rheumatoid arthritis penicillamine induced myasthenia gravis in progressive sclerosis myasthenia gravis and d-penicillamine acetylcholine receptor antibody characteristic in myasthenia gravis. patients with penicillamineinduced myasthenia or idiopathic myasthenia of recent onset penicillamine induced myasthenia gravis. effects of penicillamine on acetylcholine receptor myasthenia gravis in penicillamine treatment of ra d-penicillamine induced polymyositis in ra polymyositis and penicillamine dermatomyositis induced by penicillamine toxicity of penicillamine. a serious limitation to therapy in rheumatoid arthritis the toxicity of d-penicillamine in systemic sclerosis longterm followup of treatment with d-penicillamine to rheumatoid arthritis: effectivity and toxicity in relation to hla antigens clinical evaluation of d-penicillamine by multicentric double blind comparative study in chronic rheumatoid arthritis genetic markers in rheumatoid arthritis-relationship of toxicity from d-penicillamine maintenance dose of penicillamine in rheumatoid arthritis: a comparison between standard and a response-related flexible regimen low dose d-penicillamine treatment of r.a. a controlled double blind clinical trial resumption of treatment with penicillamine after proteinuria the relationship between d-penicillamine-induced proteinuria and prior gold nephropathy sequential gold and penicillamine therapy in rheumatoid arthritis penicillamine nephropathy immunopathology of penicillamine-induced glomerular disease sequential gold and penicillamine therapy in rheumatoid arthritis penicillamine nephropathy in rheumatoid arthritis nephropathy induced by d-penicillamine penicillamine induced membranous nephritis renal biopsy appearances in rheumatoid disease nephrotic syndrome with minimal glomerular lesions during treatment with d-penicillamine d-penicillamine induced lipoid nephrosis in a patient with scleroderma focal glomerulitis due to penicillamine igm nephropathy associated with penicillamine igm nephropathy after d-penicillamine therapy: report of a case natural course of penicillamine nephropathy: a long-term study of 33 patients penicillamine in rheumatoid disease with particular reference to the rheumatic factor plasma exchange in the successful treatment of drug-induced renal disease goodpasture's syndrome in a patient receiving penicillamine and carbimazole d-penicillamine-induced goodpasture's-like syndrome in primary biliary cirrhosis: successful treatment with plasmapheresis and immunosuppressives d-penicillamine induced crescentic glomerulonephritis: report and review of the literature goodpasture-like syndrome induced by d-penicillamine in a patient with systemic sclerosis: report and review of the literature d-penicillamine-induced anca-associated crescentic glomerulonephritis in wilson disease fatal renal vasculitis with minimal change glomerulonephritis complicating treatment with penicillamine penicillamine-induced rapidly progressive glomerulonephritis in patients with progressive systemic sclerosis: successful treatment of two patients and review of the literature autoantibody formation in d-penicillamine treated rheumatoid arthritis effect of d-penicillamine therapy on circulating immune complexes in rheumatoid arthritis pathogencity of immune complexes d-penicillamine and immune complex deposition specific sensitization of lyt-1 + 2 -stereoisomer drug-induced vasculitis e-penicillamine induced toxicity in rheumatoid arthritis: the role of sulphoxidation status and hla-dr3 hla-dr antigens and proteinuria induced by aurothioglucose and d-penicillamine in patients with rheumatoid arthritis anti-ro(ssa) positive rheumatoid arthritis: a clinicoserological group of patients with high incidence of d-penicillamine side effects antibodies to cellular antigens in greek patients with autoimmune diseases: anti-ro(ssa) antibody a possible marker of d-penicillamine intolerance adverse reaction to d-penicillamine after gold toxicity is penicillamine therapy in ra. influenced by previous gold ? strong association between idiopathic membranous nephropathy and hla-drw3 swiss federal commission for the rheumatic diseases, subcommission for research. hla-dr antigens in rheumatoid arthritis -a swiss collaborative study; final report renal toxicity associated with disease-modifying antirheumatic drugs used for the treatment of rheumatoid arthritis effects of a xanthine oxidase inhibitor on thiopurine metabolism, hyperuricemia, and gout allopurinol in the treatment of gout alopecia and ichthyosis secondary to allopurinol boston collaborative drug surveillance program. allopurinol and cytotoxic drugs possible association between macular lesions and allopurinol acute cholangitis after allopurinol treatment allopurinol-hypersensitivity. vasculitis and liver damage fulminant hepatic failure due to allopurinol allopurinol-associated arteritis urinary xanthine stones, a rare complication of allopurinol therapy acute renal failure from xanthine nephropathy during management of acute leukemia xanthinuria with xantine lithiasis in a patient with lesch-nyhan syndrome under allopurinol therapy vasculitis with allopurinol therapy toxic epidermal necrolysis, azotemia, and death after allopurinol therapy severe hypersensitivity reactions associated with allopurinol severe allopurinol hypersensitivity allopurinol-induced interstitial nephritis the allopurinol hypersensitivity syndrome systemic hypersensitivity to allopurinol with acute interstitial nephritis the allopurinol hypersensitivity syndrome. unnecessary morbidity and mortality the janus faces of allopurinol -allopurinol hypersensitivity syndrome treatment of acute gout in hospitalized patients frequency of serum creatinine monitoring during allopurinol therapy in ambulatory patients drug induced interstitial nephritis, hepatitis and exfoliative dermatitis allopurinol hypersensitivity allopurinol-hypersensitivity. vasculitis and liver damage recurrent renal failure associated with hypersensitivity to allopurinol some adverse reactions to allopurinol may be mediated by lymphocyte reactivity to oxypurinol metabolic studies of allopurinol, an inhibitory of xanthine oxidase renal clearance of oxypurinol, the chief metabolite of allopurinol severe allopurinol toxicity. description and guidelines for prevention in patients with renal insufficiency allopurinol in renal failure and the tumor lysis syndrome plasma oxypurinol concentration in a patient with allopurinol hypersensitivity human herpesvirus 6 infrction a risk factor for the development of severe druginduced hypersensitivity syndrome tumor lysis syndrome febuxostat compared with allopurinol in patients with hyperuricemia and gout febuxostat versus allopurinol for gout gout in solid organ transplantation; a challenging clinical problem clarifying the role of rasburicase in tumor lysis syndrome rasburicase: a new approach for preventing and/or treating tumor lysis syndrome. current pharmaceutical design rasburicase therapy in acute hyperuricemia and renal dysfunction acute lymphoblastic leukemia presenting as acute renal failure is rasburicase an effective alternative to allopurinol for the management of hyperuricemia in renal failure patients/ a double -blind randomized study gout and related disorders of purine metabolism gout in the elderly key: cord-0015089-wjctpqu8 authors: ledón-llanes, loraine; contreras-yáñez, irazú; guaracha-basáñez, guillermo; valverde-hernández, salvador saúl; gonzález-marín, anayanci; ballinas-sánchez, ángel de jesús; durand, marta; pascual-ramos, virginia title: views of mexican outpatients with rheumatoid arthritis on sexual and reproductive health: a cross-sectional study date: 2021-01-28 journal: plos one doi: 10.1371/journal.pone.0245538 sha: bb3d4d74b76dad26def4c5df25b4fad9a6b4482b doc_id: 15089 cord_uid: wjctpqu8 background: rheumatoid arthritis (ra) impacts sexual and reproductive health (srh), which is a prominent component of a patient´s quality of life and highly influenced by the cultural background. the aim of the study was to explore the interest of mexican outpatients with ra in srh and to examine patient view on srh. methods: this cross-sectional study surveyed 303 consecutive outpatients with ra on their perceptions of srh importance, srh satisfaction, access to srh information, preferences regarding srh communication with healthcare professionals, and understanding of srh (qualitative open-ended descriptions). descriptive statistics and inferential analysis were used. patient knowledge of each dimension of srh was rated based on pre-specified criteria. two assessors assigned ten major themes to each patient´s description of both dimensions of srh. results: patients perceived their srh as an important component of their general health and wished to address the topic, although few had access to such communication. female patients assigned lesser importance to srh, showed lesser degree of satisfaction with srh, and expressed preference for a truthful physician. age showed a linear association with individual survey responses, except for satisfaction with reproductive health dimension. there was a linear association between increased age and decreased years of formal education with a lower level of srh knowledge. ten major themes emerged for each of the two dimensions of the srh construct, although most individual descriptions were assigned to one or two major themes. conclusions: further education and assessment of srh in mexican patients with ra is warranted. this cross-sectional study surveyed 303 consecutive outpatients with ra on their perceptions of srh importance, srh satisfaction, access to srh information, preferences regarding srh communication with healthcare professionals, and understanding of srh (qualitative open-ended descriptions). descriptive statistics and inferential analysis were used. patient knowledge of each dimension of srh was rated based on pre-specified criteria. two assessors assigned ten major themes to each patient´s description of both dimensions of srh. patients perceived their srh as an important component of their general health and wished to address the topic, although few had access to such communication. female patients assigned lesser importance to srh, showed lesser degree of satisfaction with srh, and expressed preference for a truthful physician. age showed a linear association with individual survey responses, except for satisfaction with reproductive health dimension. there was a linear association between increased age and decreased years of formal education with a lower level of srh knowledge. ten major themes emerged for each of the two dimensions of the srh construct, although most individual descriptions were assigned to one or two major themes. in recent decades, the concept of reproductive health has evolved to offer a comprehensive and integrated approach to individual health needs related to sexuality and reproduction [1] . the program of action of the united nations international conference on population and development held in cairo in 1994 [2] and the fourth world conference on women sponsored by the united nations held in beijing in 1995 [3] , adopted the following definition for reproductive health: "reproductive health is a state of complete physical, mental, and social well-being, and not merely the absence of disease or infirmity, in all matters relating to reproductive system and to its functions and processes. . . it also includes sexual health, the purpose of which is the enhancement of life and personal relations, and not merely counselling and care related to reproduction and sexually transmitted diseases". more recently, the world health organization (who) recognized that sexual health is an umbrella term that includes reproductive health [4] . in both definitions, a dissociation of the act of sex from reproduction is recognized, and sex is elevated from an act of reproductive instinct to an expression of love and confirmation of human bonding [1] . accordingly, sexual and reproductive health (srh) is a complex construct, integrated by two dimensions that need to be addressed separately. however, both srh dimensions are important for quality of life at a personal level and for the individual's integration into society [5] . similar to health in general, srh is promoted or undermined by lifestyle-related behaviors and simultaneously greatly influenced by the social, cultural, educational, and economic conditions of the community in which people are born and live [1] . moreover, the relative importance of these determinants varies among countries, and their relevance varies for the two dimensions of the srh construct, which limits the generalization of the results obtained from studies performed with populations with a different anthropologic framework. rheumatoid arthritis (ra) is a chronic inflammatory condition with worldwide distribution and female preponderance. the disease presents particular characteristics in the latin american region, where it is differentiated by a younger age at presentation and extreme female preponderance compared to that in caucasian populations [6] [7] [8] . ra can potentially impact every domain of a patient´s life [9] , extending to srh [10] . two recent systematic literature reviews revealed an association between ra and sexual dysfunction among women and men [11, 12] . impaired sexual health has been attributed to ra-related symptoms and patient reduced function [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] , the long and chronic course of the disease [23, 24] , associations with comorbid conditions, primarily psychological [22, [25] [26] [27] , the medications used [28, 29] , and hormone imbalance [30, 31] . importantly, chronic conditions, socio-demographic factors, and medications have also been considered risk factors associated with sexual dysfunction in the general population [32] . meanwhile, most studies on reproductive health in rheumatic diseases have focused on fertility [33] . reduced fertility has been described in women with ra by several studies [33] [34] [35] [36] [37] [38] [39] , as a result of the disease process itself [35] or related to therapy [33] , gonadal dysfunction [40] , and due to medical advice and individual decisions [35] , which are sometimes driven by fear of transmitting the disease to children or inability to care for small children [34] . in addition, lower parity [36] and subfertility [37] [38] [39] have also been associated with disease-and treatment-related variables in patients with ra. finally, ra is a prevalent disease among young latin american women who are expected to be sexually active. srh remains an area that patients and healthcare providers are reluctant to discuss in person [20, 33, 41] . frequent reasons provided by healthcare professionals are poor training or education in sexual health, lack of relevant experience, religious or personal views, the belief that the topic is not important or appropriate, and embarrassment [42] . josefsson et al. [14] confirmed that communication regarding sexual health between 63 swedish patients with ra and their physicians was uncommon, whereas a considerable proportion of the patients expressed a desire for communication. in accordance, in a study with 74 ra patients from the uk, there was an 80% response rate (returned by pre-paid post) to a questionnaire concerning patient sexuality [20] . with the above considerations in mind, and given that srh had strong cultural determinants, the aim of the study was to explore the interest of mexican outpatients with ra in addressing srh as part of their rheumatologic evaluation and to examine patient view on srh, which has not been previously performed. the study was performed in compliance with the helsinki declaration [43] . the research ethics committee of the instituto nacional de ciencias médicas y nutrición salvador zubirán (incmyn-sz) approved the study (reference number: ire-1909). written informed consent was waived due to the study characteristics (survey application). the research ethics committee required anonymous application of the survey, due to sensitivity associated with the topic. consecutive patients from the outpatient clinic of the department of immunology and rheumatology were invited to answer the survey, by trained personnel not involved in patient s care, while waiting to receive their schedule consultation. informed consent process was performed in all the patients who gave verbal consent and agreed to respond the survey. verbal informed consent process was confirmed by the primary rheumatologist in charge of the patient, at the end of the consultation. the study was cross-sectional and distributed a survey between january 20 th and february 29 th 2020 at the outpatient clinic of the department of immunology and rheumatology of the incmyn-sz, a national referral and academic center for rheumatic diseases. the survey was distributed to all the consecutive patients with ra (according to their primary rheumatologist criteria) who arrived at the outpatient clinic to attend a schedule visit (inclusion criteria). exclusion criteria considered were additional (to ra diagnosis) rheumatic diagnosis, but secondary sjögren syndrome, and patients who disagreed to participate. during the study period, the outpatient clinic identified 436 patients who met inclusion criteria and were considered potential candidates to survey application, although 330 patients arrived to their schedule appointment. the characteristics of these patients, are summarized in s1 table (please refer to the s1 "target population characteristics", s1 table) . briefly, patients were primarily middle-aged females, with substantial disease duration. almost two-third of the patients had adequate control of the rheumatic disease according to the primary rheumatologist, meanwhile up to 46% of the patients referred joint pain, 24.4% morning stiffness and 9.8% substantial fatigue. there were 204 patients (61.8%) with at least one comorbid condition; among them, 84 patients (4.9%) had depression. finally, the majority of the patients were on disease modifying-anti-rheumatic drugs (dmards) and 28.2% on low doses of oral prednisone. finally, patients who completed the survey, corresponded to 91.8% of potential candidates and they were considered representative of the target population. because of the anonymous application of the survey, their characteristics are not provided. strobe (strengthening the reporting of observational studies in epidemiology) statement was followed [44] and the checklist of items that should be included is summarized in s2 table (please refer to the s2 "strobe checklist for cross-sectional studies", s2 table) . good practice in the conduct and reporting of survey research was followed [45] . the aim of the study was to explore the interest of mexican outpatients with ra in addressing srh as part of their rheumatologic evaluation and to examine patient view on srh. a literature search failed to identify validated tools in spanish, suitable to address the primary objective in our population. survey conceptualization was driven by clinical experience of a senior rheumatologist and of a phd psychologist, previous clinical research in mexican male patients with systemic lupus erythematosus (sle) from the same institution, where patient's interest in addressing the topic was identified [46] , and the theoretical conceptualization of srh [1] . the survey content was proposed by a committee consisting of one general practitioner, one senior rheumatologist in charge of an early ra clinic, and one phd psychologist with experience in srh. the committee agreed on five components to be included in the survey and suggested a reduced number of items to be included in the survey, due to the limited education of the target population. subsequently, ten individual items/questions, the scale responses to the ten items, and their distribution into the five components of the survey, were constructed by one coauthor. they were independently reviewed by the remaining two members of the committee who suggested splitting srh in to sexual health and reproductive health, which were presented into one single item (n˚10). for this particular item that was intended to recall patient view of srh, the expert committee considered it was convenient to direct patients to recognize both dimensions of the srh construct. in addition, four demographic variables were included to be optionally filled by the patients: age, sex, level of formal education, and civil status (available in the last 130 surveys). the first version of the survey (sv 1 ) was integrated and included 11 items. (please refer to the s3 "survey components, items and scale´s response", s3 table) . judgment experts determined the face and content validity of sv 1 . the expert committee consisting of a rheumatologist, reproductive health biologist, social worker, gynecologist, and bioethicist, who were blinded to each other's evaluation, scored the following characteristics on standardized formats: adequate wording and appropriate language and meaning (for the target population) regarding individual items and instructions, adequacy of the item´s scale response, and relevance and pertinence of individual items to the survey purpose were also explored. as a result, item 3 was modified, and additional options for the item´s scale response were included for items 1-5 and item 9. in all cases, at least 80% agreement among experts was deemed necessary to approve modifications, and sv 2 was integrated. fifty consecutive outpatients with ra were interviewed by two coauthors to assess their perception of instruction clarity, item adequate wording and meaning, and scale responses; standardized formats were used. patients agreed on instruction clarity (85%), item adequate wording and meaning (90%), and adequacy of the scale response (95%). in addition, patients were directed to identify items they considered should be deleted, and to suggest items they considered should be added. there were no suggestions from patients but a blank section for additional comments that was added, and survey version 3 (sv 3 ) was integrated and was considered suitable for final application (please refer to the s4 "the sexual and reproductive health survey", spanish and english versions, s1 appendix). first, all patients with a scheduled visit were identified on the day prior to their scheduled visit to the outpatient clinic of the department of immunology and rheumatology. on the day of the scheduled visit, all patients who attended the outpatient clinic were invited to participate. patients were instructed to read the survey, answer or not, and leave it at a designated area after their consultation. every day, all surveys were collected at the end of the day, and answered surveys were identified. this procedure was repeated until the target sample of at least 248 completed surveys was achieved (fig 1) . the sample size for pilot testing was 50 patients, and the number of patients included followed recommendations for pilot testing [47] . for survey application, we estimated a sample size of at least 248 patients. we considered that 82% of the outpatients with ra would be interested in discussing srh, based on local data obtained from men with sle [46] . the outpatient clinic of the department of immunology and rheumatology registered 1578 outpatients coded with ra during 2018 and a similar number was expected during 2019. descriptive statistics included frequencies and percentages, mean (± standard deviation [sd]) for normally distributed variables, and median (interquartile range [iqr]) for non-normally distributed variables. descriptive statistics were also used to describe the distribution of major themes in the study population (see qualitative analysis below). face validity and content validity were examined by experts with agreement percentages. differences in the sex distribution of patient responses to the survey was compared with the x 2 test. linear regression analysis was used to examine age as a predictor of response selection to individual items from the survey. kruskal-wallis and x 2 tests were used to compare the characteristics among patients classified according to their srh knowledge (insufficient, borderline and sufficient knowledge). based on the distribution of the patients´responses, scale responses were further grouped into five categories, instead of nine, as follows: (1) "very important/important," (2) "somewhat important," (3) "of minor importance/unimportant," (4) "i don´t know/i haven´t thought about it," and (5) "i don´t want to answer" for questions 1, 4, 5, and 9; (1)"very satisfied/satisfied," (2)"somewhat satisfied," (3)"mostly dissatisfied/dissatisfied," (4)"i don´t know/i haven´t thought about it," and (5)"i don´t want to answer" for questions 2 and 3; and finally, (1)"very frequent/frequent," (2)"occasionally," (3)"rarely/never," (4)"i don´t know/i haven´t thought about it," and (5)"i don´t want to answer" for questions 6 and 7. a phd psychologist rated patient knowledge of sexual health (item 10) and of reproductive health (item 11), according to the patient answers expressed in free-text, into three categories: "insufficient knowledge," "borderline knowledge," and "sufficient knowledge" based on the who definition and pre-specified criteria (please refer to the s5 "criteria for sexual and reproductive health knowledge classification", s4 table) . missing survey data (except for items 10 and 11) varied from 0% (for question 2) to 6.6% (for question 3) . no imputation was performed. all statistical analyses were performed using the statistical package for the social sciences version 21.0 (ibm corp., armonk, ny). a value of p < 0.05 was considered statistically significant. qualitative analysis was conducted on free-text comments related to patients understanding of srh, using a brief thematic analysis approach [48] . first, three assessors read the free-text comments for item 10 (patient understanding of sexual health) and item 11 (patient understanding of reproductive health) and identified a list of categories for each dimension of the srh construct, which was further organized into major themes. then, the assessors met to discuss and agree on a list of ten major themes and their corresponding categories, which allowed an accurate description of the relevant characteristics of the content of each srh dimension. in the second step, two assessors independently assigned each patient´s answers regarding both dimensions of the srh construct, to the categories and to the ten major themes from the list; when differences were present, they were resolved by consensus. finally, based on the content identified through the coding system of major themes and their corresponding categories, a brief qualitative analysis was carried out based on a general interpretation of the main subjective and social representations, which may support the expression of major themes related to their corresponding categories [48] . during the study period, there were 303 filled surveys (including two surveys with missing answers to item 8), and overall, most patients rated questions 1 (63.9%), 4 (79.8%), 5 (84.8%) and 9 (51%) "very important/important." additionally, patients frequently rated questions 2 (48.8%) and 3 (60.1%) "very satisfied/satisfied". finally, most patients rated questions 6 (60.9%) and 7 (77.3%) as "never/rarely." the sex distribution of patient responses was then compared; there were differences for questions 1 and 2, as depicted in figs 2(a) and 3(a), respectively. regarding question 1, female patients selected more frequently the scale response "i don't know/i haven´t thought about it" and "of minor importance/not important"; meanwhile, the opposite was true for the scale response "somewhat important," which was more frequently selected by male patients. similarly, female patients selected more frequently response options "i don´t know/i haven´t thought about it" and "i don't want to answer" for question 2, meanwhile, the opposite was true for the response options "moderately satisfied" and "mostly dissatisfied/dissatisfied," which were more frequently selected by male patients. two hundred and seventy female patients and 31 male patients answered question 8 ("which healthcare provider, among those described below, would you like to talk with about srh?"). the results of the patients' responses are summarized in table 1 . overall, most patients selected the "srh expert" option (53.8%), without differences between male and female patients. more female patients selected a "trustful physician" as the second option (24.1% women vs. 9.7% men, p = 0.073); meanwhile, male patients selected the "any physician" option more frequently than female patients did (8 [25.8%] vs. 19 [7%], p = 0.003). finally, a similar proportion of male and female patients selected the "rheumatologist" option. age was examined as a predictor of response selection, for each individual item/question from the survey. results are summarized in table 2 and highlight that age predicted option response for every question except question 3 ("how do you rate your reproductive health satisfaction?"). magnitudes of the relationship were similar across the questions but the direction differed, as negative correlations were found for questions 1-7, whereas a positive correlation was found for question 9. overall, patients rated survey questions as easy to understand (75.4%), easy to answer (72.9%), and with adequate format (80.7%). there were 71 (23.4%) and 72 (23.7%) missing data points, respectively, regarding the patient s description of sexual health (item 10, open format) and of reproductive health (item 11, open format); among them, 58.7% coincided. there were 232 patient descriptions of sexual health dimension, and 20 (8.6%) were classified as "insufficient knowledge," 171 (73.7%) as "borderline knowledge," and 41 (17.7%) as "sufficient knowledge" by the psychologist. similarly, there were 231 patient descriptions of reproductive health dimension, and 16 (6.9%) were classified as "insufficient knowledge," 186 (80.5%) as "borderline knowledge," and 29 (12.6%) as "sufficient knowledge." we further explored differences among patients with insufficient, borderline, and sufficient knowledge regarding age, sex distribution, level of formal education, and civil status. the results are summarized in table 3 for sexual health dimension and table 4 for reproductive health dimension, and showed a linear association between increased age and decreased years of formal education with lower levels of knowledge for both dimensions of the srh construct. there were 219 surveys (72.3%) with a description of sexual health (one description per survey), 73 surveys (24.1%) with missing information, and 11 surveys (3.6%) where patients stated that they could not provide a description. among the former, the assessors first agreed on major theme assignment in 184 sexual health descriptions (84%) and reached a consensus on the remaining sexual health descriptions. most of the sexual health descriptions (138 [63%]) were assigned to only one major theme, while the remaining descriptions (81 [37%] ) were assigned to a combination of at least two major themes; in accordance, the median (iqr) of major themes/ for each srh construct, assessors agreed on the same 10 major themes: 1. table 5 . tables 6 (sexual health) and 7 (reproductive health) present illustrative quotes for main categories and each major theme identified in free-text comments. finally, major themes distribution among the 219 and 223 surveys where the patients provided a description of sexual and of reproductive health, respectively, is summarized in table 8 . overall prevention-oriented patient care, overall health and sexuality expression were the most frequently assigned themes to sexual health, while biological aspects, overall health, and overall preventionoriented patient care were the most frequently assigned themes to reproductive health. the same ten major themes were described for each patient definition of sexual health and of reproductive health, which could be considered an expression of the shared boundaries between the two dimension definitions from the patient perspective. these shared boundaries were specifically expressed in the sexual health definitions, which showed several major themes that included categories related to reproductive health (for example: references to reproduction, reproductive organs and their functioning, family planning issues, contraception, unwanted pregnancy prevention). furthermore, the overlap between the major themes represented in the sexual health and reproductive health definitions could be considered either an expression of an integrative view from the patient perspective, or may be related to patient difficulty in effectively identifying both dimensions of the srh construct. in general, the patients used more categories to define sexual health than reproductive health, except for the major themes "family planning," "biological aspects (primarily)," and "psychological aspects being knowledgeable or informed regarding reproduction, family planning, and pregnancy. overall preventionoriented patient care 18 categories 14 categories (self) care related to overall heath and sexuality, prevention of sexually transmitted infections and sexual diseases, and prevention of infections and diseases (in general). general (self) care related to reproduction, receiving systematic prevention-oriented care related to the reproductive system organs and function, and healthcare related to pregnancy. family planning 5 categories 14 categories contraception and contraceptive methods used, family planning issues, and unwanted pregnancy prevention. family planning issues (for example, making decision process about if, when and how to reproduce, planning the extension of the family), and reproductive desires. overall health 16 categories 11 categories sexuality-related health and healthy sexual organs, the state of health need to optimal sexuality, overall health, and disease, comorbidities and age-related changes. the (optimal) state of health for achieving reproduction, healthy reproductive organs, system and function, having a good overall health, and gynecological, obstetric and perinatal health. sexual organs and its functioning, as part or function body-related, and reproductive organs and its functioning. conceiving or having children, fertility, the life stage associated to biological changes related to reproduction, and pregnancy. psychological aspects (primarily) attitudes regarding sexuality (for example: responsibility, autonomy, ethics, freedom), and reference to different expressions of identity (gender-related, sex-related, and sexual orientation-related). attitudes regarding reproduction (for example: responsibility, autonomy, awareness the reproductive decision-making process), reference to gender identity (female or male), and the assessment of reproductive health relevance. optimal expression of sexuality, enjoying intercourse and sexual satisfaction, sexual activity and/or intercourse and their wellfunctioning, and references to sexual discomfort, disorders, or changes. sexual activity and/or intercourse and well-functioning of sexual activity. holistic concept 2 categories 1 category balanced integration of physical, psychological, and social aspects in general and related to sexuality in particular. balanced integration of physical, psychological, and social aspects related to reproduction. https://doi.org/10.1371/journal.pone.0245538.t005 having information or knowledge about overall health "to be aware of how to be well". 1 "estar enterada de como estar bien". overall patient care prevention-oriented (self) care related to the overall health "related to the means of prevention and care". 2 "relacionado a los medios de prevención y cuidados". "taking care in all aspects to prevent disease". 1 "cuidarse en todos los aspectos para prevenir enfermedad". contraception and contraceptive methods' use "to use family planning methods". 1 "utilizar métodos de planificación familiar". sexuality-related health and healthy sexual organs "being healthy in the intimacy, without discharge or pain or secretions or odors". 1 the state of health needed to optimal sexuality "i have to be healthy to have good sexual health". (180) "tengo que estar bien de salud para tener salud sexual bien". sexual satisfaction in couple "having sex with your partner (. . .) and having it satisfying". (86) "el tener relaciones sexuales con tu pareja (. . .) y que sea satisfactorio". sexual organs and its functioning "everything related to the genital apparatus regarding its functioning and state". (246) "todo lo relacionado con el aparato genital respecto a su funcionamiento y estado". a part or function body-related "i don't know how to explain it but i think it is the function of the body that requires it so". (295) "no sé cómo explicarlo pero pienso que es la función del cuerpo que lo requiere así". references to sexual discomfort, disorders or changes "(. . .) any discomfort, (. . .) the changes that one presents with age and medications". 1 "(. . .) cualquier molestia, (. . .) los cambios que uno va presentando con la edad y medicamentos". the balanced integration among physical, psychological and social aspects in general "physical, mental and emotional issue of a person". 1 the balanced integration among physical, psychological and social aspects related to sexuality "the union of a mental, physical and social state related to sexuality and healthy relationships". 1 "la unión de un estado mental, físico y social relacionado con la sexualidad y relaciones saludables". having information or knowledge about family planning "learning about the methods of reproduction, planning and monitoring". 1 "conocer sobre los métodos de reproducción, planificación y seguimiento". having information or knowledge about pregnancy "to know (. . .) how to take care of yourself when you want to get pregnant, and how to proceed in case of pregnancy". 1 "saber (. . .) cómo cuidarse para cuando así se desee embarazarse, y cómo proceder en caso de embarazo". general (self) care related to reproduction "care and prevention, so that the reproduction would be satisfactory at any time". 1 "cuidados y prevención para que la reproducción sea satisfactoria en cualquier punto". receiving systematic prevented-oriented care related to the reproductive system, organs and function "to follow a good control with your gynecologist regarding ovaries and womb". 1 "llevar un buen control con tu ginecólogo respecto a ovarios y matriz". health care related to pregnancy "care and attendance during pregnancy for avoiding problems with the baby". 1 "cuidados y atención durante el embarazo para evitar problemas con el bebé". the (optimal) state of health for achieving reproduction "to be in optimal physical and mental conditions if it is planned to conceive". 1 healthy reproductive' organs, system and function "optimal functioning for reproduction, healthy spermatozoids and eggs". 2 "funcionamiento óptimo para reproducirse, salud de espermatozoides y óvulos". having a good overall health "be in good health and be productive in the things i do". 1 "tener buen estado de salud y ser productivo en las cosas que hago". gynecological, obstetric and perinatal health "being able to have a pregnancy without any risk and without complication for the baby". 1 "poder tener un embarazo sin ningún riesgo y sin complicación para el bebé". well-being reproduction-related "it implies physical well-being with the reproductive system". 1 "implica bienestar físico con el sistema reproductor". overall well-being covering biological, psychological and social dimensions "it is a general state of physical, mental and social well-being, and absence of pain and disease". 1 "es un estado general de bienestar físico, mental y social, y ausencia de dolor y enfermedad." (continued ) (primarily)". this could be considered an indicator of being more informed regarding sexual health as well as an indicator that the sexual health dimension elicits a greater diversity of meanings for the patient, compared with the reproductive health dimension. finally, it must be considered that in almost all major themes, patients combined the biomedical and psychosocial categories expressing a holistic perspective of sexual and reproductive health, framed in the context of overall health, which influences psychosocial (for example, relationships, values, communication, information, and satisfaction) and biomedical (for example, healthcare assistance, organs, systems and functions, and biological stages) domains to achieve a state of wellbeing. main categories illustrative quote the couple relationship as the basis for reproduction "when it is wanted to have a child, the couple is important." 2 "cuando se quiere tener un hijo es importante la pareja." talking about reproduction "communication with the couple, knowing how many children for a better coexistence". 1 "la comunicación con la pareja, saber cuántos hijos para una mejor convivencia". conceiving or having children "to be able to conceive or to have a child or several". 1 fertility "ability to be able to get pregnant without any risk". 1 "capacidad de poderse embarazar sin ningún riesgo". the life stage associated to biological changes related to reproduction "stage in which your body begins to have hormonal changes and you become reproductive". 1 "etapa en la que tu cuerpo empieza a tener cambios hormonales y te vuelves reproductiva". pregnancy "everything related to (. . .) pregnancy". 1 "todo lo relacionado con (. . .) el embarazo". attitudes regarding reproduction (for example: responsibility, autonomy, to be aware about the reproductive making decision process) "to be prepared to conceive, to be aware, to do it with love, and not only for need it". 2 "prepararse para engendrar, estar consciente, hacerlo con amor, y no solo por necesidad". reference to gender identity (female or male) "when the reproductive age' woman has her family without any problem". 1 "cuando la mujer en edad reproductiva tiene a su familia sin ningún problema". the balanced integration among physical, psychological and social aspects related to reproduction "the healthy way, mental and physical, with planning to create life". 1 "la forma sana, mental y física con planeación para crear vida". the present study distributed an srh-related-survey to mexican outpatients with ra attending a tertiary care level center at the time of the study. the survey was locally developed; face and content validity were determined by an expert committee, and additionally confirmed with 50 patients who participated in a pilot test. the study revealed that mexican patients with ra perceived their srh as an important component of their general health and wished to address the topic with healthcare professionals, primarily with an srh expert, although few had access to such communication. meanwhile, most patients reported some degree of satisfaction with their srh. nationality and ethnicity determine the particular cultural background that influences patient perceptions and views regarding the ra priority domains [49] [50] [51] [52] , trust in the physicians [53] , and sexuality dimension and ways in which sexuality is conceptualized and explored [54] . additionally, patients from our region expressed particular concerns and interests regarding their disease [55] and did not object to physician-centered/paternalistic patientdoctor relationships [56] [57] [58] [59] . ultimately, all conditions mentioned above integrate in a regional-cultural framework that shapes the results obtained herein. accordingly, we consider that the study adds relevant information to the current knowledge on srh in patients with ra, which has been conceived based on results from studies primarily performed in developed countries and with caucasian populations [14, 20, 21, 29, 60, 61] . to the best of our knowledge, only two studies have addressed this topic with mexican patients [62, 63] , although one was limited to comparing obstetric prognosis before and after ra onset [62] . skinner-taylor et al. [63] described the srh characteristics and contraceptive use of 135 mexican women in childbearing age with a wide range of rheumatic diseases, among whom 35 had ra; authors found that 29% of their patients did not receive contraceptive counseling by any healthcare specialist. lower rates of counseling regarding pregnancy and contraception have also been described in usa patients with rheumatic diseases [64] [65] [66] . andreoli et al. [67] confirmed these results in 249 italian patients with connective tissue diseases and 149 patients with chronic arthritis and additionally found that 34.5% of the patients with chronic arthritis, among whom 100 patients had ra, had never been asked by their rheumatologist regarding the desire to have children, while counseling was shown to play a role in increasing knowledge and had a positive impact on family planning. we found that a significant number of the patients surveyed reported that they "never/ rarely" talk about srh with their rheumatologists; our approach to srh was holistic, not limited to counseling for specific concerns, and targeted female and male patients of varying ages and with a wide range of disease durations. similar percentages have been described for european patients with ra [14, 20, 68] . more recently, twisttman et al. [69] found that up to 92% of 329 danes ra patients, had never discussed sexual issues with a health care professional during the last 5 years. chakravarty et al. [70] conducted a multinational survey with 969 women aged 25-45 years with chronic inflammatory conditions from six countries (usa, uk, germany, france, italy, and spain), 50 rheumatologists each from germany, france, italy, and the usa, and 100 gastroenterologists from the usa. the authors found that family planning and pregnancy were considered extremely important issues to be addressed by their patients and confirmed our results and those from other studies regarding patient-assigned importance to srh [60, 71] . meanwhile, general practitioner (gps)/primary care physicians and gynecologists were identified by the patients as frequently central to their discussions; it should be highlighted that in the uk, the patient's gp and local midwife may oversee most of the pregnancy planning and management and could be considered surrogates of an "expert". in addition, patients did also report that they wished to discuss family planning and pregnancy-related issues with their specialists (either rheumatologists or gastroenterologists). these results differed from those found in danes ra patients were half of them stated that they did not want health care professionals to address sexual themes (occasionally) [69] , and in uk ra patients with long-standing disease, where nurses were the patient´s preferred confidants to talk about sexual issues, followed by physicians [20] . in swedish patients, the rheumatologist was the most frequent healthcare professional selected with whom to address sexual concerns, while french patients selected psychologists [68] . our patients preferred an srh expert and a trustful physician even more frequently than a rheumatologist [14] . the differences observed among populations regarding these preferences may be related to variation across countries in the healthcare system-related-working models, the (patient´s perception of the) training and communication skills of the healthcare providers involved in patient care, and the patient´s preferred communication pattern, which may be highly nuanced by the cultural background [42, 53, 72] . importantly, high-power-distance cultural communities may assign higher value on healthcare professionals' training and expertise. finally, trust in physicians is a distinct and complex construct in itself [72] , which has been associated with racial, cultural, socio-demographic, and disease-related variables [53, 72, 73] all of which may differ in our patients. a second important finding was the differences between male and female patients in the answers provided to some components of the survey; female patients assigned lesser importance to srh, showed lesser degree of satisfaction with srh, and reported preference for a truthful physician to a greater extent than male patients. sexual problems have been reported more frequently by women either in ra populations [21, 23, 60, 74] or in the general population [75] [76] [77] ; meanwhile, the male sex has been associated with desire for sexual intercourse and frequency of sexual contact [74] , which could be considered a surrogate for "importance assigned to srh." however, there is a hegemonic masculinity that requires confirmation and legitimization through active male involvement in sexuality [78] . in addition, men with ra reported a large impact on sexuality compared to women [21] . in a recent survey [68] , french women with ra were reported to be more comfortable addressing sexual health with nurses and psychologists, while male patients selected rheumatologists or gps; nurses and psychologists may represent trustful healthcare providers from the female perspective suggesting that women's sexual concerns (extended to female patients with ra concerns) might be more psycho-affective than biologically driven [21] and should be addressed in an intimate environment. the results discussed above highlight that there are culturally driven differences between men and women in the value and concerns assigned to sexuality, the differential impact of disease-related body damage on one´s body satisfaction and intimate relationships, and the social roles and expectations. consequently, srh-related conceptualizations and behaviors are expected to be different for women and men. third, age showed a linear association with responses to single items of the survey, except for satisfaction with reproductive health. the results could be aligned with the general conception that sexual activity decreases later in life, although it remains an important part of many relationships [79] , and confirm results from previous studies where aging appeared to have an impact on the importance a patient places on sexual ability [20] , although another study limited the (negative) association between age and sexual motivation/sexual activity and fantasies, to female patients [16] . finally, the lack of association between age and satisfaction with reproductive health may be related to the population studied, primarily women in their 50s, who might have completed their family planning when the survey was conducted. fourth, in most patients, knowledge on srh was rated as borderline; in addition, there was a linear association between increased age and decreased years of formal education with a lower level of knowledge. finally, the qualitative analysis revealed ten major themes for each domain of the srh construct, each with an integrative (biomedical, emotional, and social) approach, while most individual descriptions were limited to one, followed by two major themes. when searching for additional information regarding srh knowledge, studies with mexican populations have targeted healthy adolescents and young individuals; in agreement with our results, the literature highlights a low to moderate level of general knowledge on sexuality and srh [80] , which might be partially explained by the limited perspective of the srh-related topics taught in mexican schools [80] , in addition to biases integrated into national srh programs [80] . furthermore, advanced age might be an indicator of a subpopulation of patients with fewer years of education in our country; in fact, we found a low but significant inverse correlation between age and education level. srh knowledge might be considered part of health literacy [81] . the relevance of achieving (health) literacy relies on literacy to impact health knowledge, health status, and access to health services, in addition to income level, occupation, education, housing, and access to medical care [81, 82] ; all of them are "red spots" in the latin-american region and contribute to the current humanitarian crisis. meanwhile, health literacy has been shown to positively impact different health-related outcomes [83, 84] , and should be integrated in to national education initiatives. the first pre-specified criteria used to rate srh knowledge adopted an integrative approach to srh (biomedical, emotional, and social), which was recognized in the major themes from each dimension of the srh construct emerging from the assigned categories. meanwhile, most individual descriptions to each dimension were assigned to one or two major themes. as such, it appears that at the population level, srh knowledge was more broadly conceptualized, as a reflection of the diversity of the patients included; meanwhile, at the individual level, srh conceptualization was more limited, in accordance with srh patient descriptions being primarily rate as reflective of borderline knowledge. the widely varied content included in each definition of the srh construct (ten major themes with a minimum of one to a maximum of 18 categories) might be consistent with the advantage of using open-ended questions to provide respondents with the freedom to express their opinion on the topic of research [85] , which allows a better comprehension of the diversity of individual approaches. moreover, and according to the major theme distribution, patients described each dimension of the srh construct with a predominant content, they tended to define both dimensions through the major themes "overall prevented-oriented patient care" and "overall health", indicating a strong representational association with health prevention and care, and with the health status as a preeminent condition for sexual health and reproductive health. the link between sexual health and reproductive health with different aspects of health and healthcare is probably related to the most widespread updated information on srh, which is usually framed from a risk perspective, aimed to prevent sexually transmitted infections and unplanned pregnancies and is usually offered from healthcare services [80] . patients' definitions of sexual health included a group of categories related to the major theme "sexuality expression," involving content related to sexual activity and intercourse, their well-functioning, sexual satisfaction and pleasure, and references to sexual disorders. it could express a representational separation between sexuality and reproduction, even when both appeared also to be mixed. the inclusion of the concept of sexual pleasure and satisfaction could indicate an investment from the human rights approach as well as the deeper significance of sexuality in patient lives and identities [86] . this appeared to be even more significant, considering that higher importance is attributed to reproduction than to sexuality, especially among mexican women [78] . nevertheless, it is quite possible that this approach (sexuality separated from reproduction) could coexist with the (inadequate) identification between sexuality and reproduction, which has been described in the mexican population [78] . finally, patient references to sexual disorders could be related to their personal experiences and to the previously described increased prevalence of sexual dysfunction in patients with ra [10] [11] [12] 33] ; it needs to be emphasized that ra patients from latin america present distinct epidemiological characteristics (female preponderance and a younger age at presentation), which have the potential to shape personal srh experience. conversely, patient definitions of reproductive health included a group of categories related to the major theme "biological aspects (primarily)," especially represented by categories related to fertility, pregnancy, and having children. considering that the study population primarily comprised female patients, this approach could be related to the preponderant role of motherhood in shaping female identity previously identified in mexican women [78] . some limitations of the study need to be addressed. first, the study was conducted at a single academic center located in a metropolitan area, and referred patients with ra may present unique characteristics. additionally, the number of included male patients was limited, reflecting the worldwide female preponderance of ra; accordingly, the results might not be generalizable. second, we used a locally developed survey, which lacked formal validation, although face and content validity were determined by an expert committee and confirmed in a pilot test with 50 outpatients with ra. third, numerous factors may affect patient perceptions of srh, and the survey content was certainly limited to assess all of them; in addition, some variables (marital status) were missing in a large number of patients. fourth, the survey took place from january 20 th to february 29 th 2020 just a few weeks before the who declared the covid-19 pandemic [87] , which might have influenced the results. finally, some patients with a scheduled appointment could not be invited to fill the survey, while few denied participation; their characteristics were not compared to those of the included patients, which could have biased results; nonetheless, the percentage of patients within each category was low (below 10%). current knowledge regarding srh in ra lacks the perspective of latin american patients. our study confirmed that srh is a relevant outcome for mexican outpatients with ra, although it is infrequently addressed by healthcare professionals. age, sex, and literacy appear to shape mexican patients' srh views and preferences for healthcare professionals with whom to address the topic; female mexican patients valued trust. at the population level, mexican patients with ra present a broader and integrative conceptualization (biomedical, emotional, and social) of each dimension of the srh construct, framed from a preventive and assistant health approach. at the individual level, the integrative conceptualization remains, but it is more limited and translates into borderline srh knowledge. the study findings support the relevance of assessing patients with ra from a patient-centered approach, which might begin with the consideration that patients with ra could have srh concerns. the patient-centered approach should be sensitive to the patient´s sex, lifecourse, and sociocultural particularities, to name the most relevant. there is need to provide patients with ra with srh counseling, assistance, and rehabilitation interventions. it remains to be determined in our public health and academic environments, which healthcare professionals should be involved and how they might operationalize the provision of srh care. a multidisciplinary approach should ideally be considered. supporting information s1 sexual and reproductive health: overview united nations fourth world conference on women platform for action and the beijing declaration report of technical consultation on sexual health determinants of satisfaction with individual health in male and female patients with chronic low back pain medication persistence over 2 years of follow-up in a cohort of early rheumatoid arthritis patients: associated factors and relationship with disease activity and with disability treatment of early rheumatoid arthritis in a multinational inception cohort of latin american patients: the gladar experience challenges in the management of rheumatoid arthritis in developing countries consequences of rheumatoid arthritis for performance of social roles-a literature review rheumatoid arthritis is associated with negatively variable impacts on domains of female sexual function: evidence from a systematic review and metaanalysis rheumatoid arthritis and risk of sexual dysfunction: a systematic review and metaanalysis sexual function and reproduction can be impaired in men with rheumatic diseases: a systematic review sexual difficulties and total hip replacement in rheumatoid arthritis sexual health in patients with rheumatoid arthritis: experiences, needs and communication with health care professionals mohamed said ms. sexual dysfunction and its determinants in malaysian women with rheumatoid arthritis sexual functioning of people with rheumatoid arthritis: a multicenter study determinants of sexual disability and dissatisfaction in female patients with rheumatoid arthritis sexual self-concept and general health in rheumatoid arthritis patients women's experiences of sexual health when living with rheumatoid arthritis-an explorative qualitative study effects of rheumatoid arthritis on sexual activity and relationships perceived influence of health status on sexual activity in ra patients: associations with demographic and disease-related variables sexual dysfunction in rheumatoid arthritis patients: arthritis and beyond life satisfaction in early rheumatoid arthritis: a prospective study factors associated with sexual dysfunction in taiwanese females with rheumatoid arthritis body image of women with rheumatoid arthritis evaluation of sexual dysfunction in women with rheumatoid arthritis: a controlled study the impact of rheumatic diseases on sexual function sexual function in moroccan women with rheumatoid arthritis and its relationship with disease activity assessment of objective and subjective quality of life in people with rheumatoid arthritis-preliminary study rheumatoid arthritis: a female challenge efficacy of intravaginal dehydroepiandrosterone (dhea) on moderate to severe dyspareunia and vaginal dryness, symptoms of vulvovaginal atrophy, and of the genitourinary syndrome of menopause definitions/epidemiology/risk factors for sexual dysfunction sexual and reproductive health in rheumatic disease effects of infertility, pregnancy loss, and patient concerns on family size of women with rheumatoid arthritis and systemic lupus erythematosus fertility in women with chronic inflammatory arthritides parity in patients with chronic inflammatory arthritides childless at time of diagnosis time to pregnancy among women with rheumatoid arthritis fertility in women with rheumatoid arthritis: influence of disease activity and medication subfertility in rheumatoid arthritis is often unexplained or caused by anovulation ovarian reserve alterations in premenopausal women with chronic inflammatory rheumatic diseases: impact of rheumatoid arthritis, behçet's disease and spondyloarthritis on anti-mü llerian hormone levels sexual health promotion and nursing sexual and reproductive health rcn report on the impact of funding and service changes in england. royal college of nursing ethical principles for medical research involving human subjects the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies good practice in the conduct and reporting of survey research epidemiologic profile of erectile dysfunction in patients with systemic lupus erythematosus: the latin american landscape health measurement scales. a practical guide to their development and use content analysis and thematic analysis: implications for conducting a qualitative descriptive study fears and beliefs of people living with rheumatoid arthritis: a systematic literature review illness beliefs predict disability in rheumatoid arthritis beliefs about medicines in patients with rheumatoid arthritis and systemic lupus erythematosus: a comparison between patients of south asian and white british origin in wealthier countries, patients perceive worse impact of the disease although they have lower objectively assessed disease activity: results from the cross-sectional comora study trust in physicians and elements of the medical interaction in patients with rheumatoid arthritis and systemic lupus erythematosus culture and society: shifting paradigms in sexuality research educational website incorporating rheumatoid arthritis patient needs for latin american and caribbean countries attitudes about principle of autonomy in hispanic patients from a dynamic early rheumatoid arthritis cohort ethics and communication between physicians and their patients with cancer, hiv/aids, and rheumatoid arthritis in mexico doctor-patient relationship between individuals with fibromyalgia and rheumatologists in public and private health care in mexico can physicians demonstrate high quality care using paternalistic practices? a case study of paternalism in latino physician-patient interactions factors affecting the sexual satisfaction of patients with rheumatoid arthritis and ankylosing spondylitis rheumatology outcomes: the patient's perspective. a multicentre focus group interview study of swedish rheumatoid arthritis patients desenlace obstétrico antes y después del inicio de la artritis reumatoide reproductive health counseling and contraceptive use in mexican women with rheumatic diseases: a cross-sectional study contraception use among reproductive-age women with rheumatic diseases contraceptive use in women of childbearing ability with rheumatoid arthritis contraceptive counseling and use among women with systemic lupus erythematosus: a gap in health care quality? disease knowledge index" and perspectives on reproductive issues: a nationwide study on 398 women with autoimmune rheumatic diseases french survey on the crossed needs on sexual health for chronic inflammatory rheumatism patients and healthcare professionals sexual health and dysfunction in patients with rheumatoid arthritis: a cross-sectional single-center study family planning and pregnancy issues for women with systemic inflammatory diseases: patient and physician perspectives an exploratory survey of the practice of rheumatology nurses addressing the sexuality of patients with rheumatoid arthritis use of the trust in physician scale in patients with rheumatic disease: psychometric properties and correlates of trust in the rheumatologist racial and ethnic disparities in perceptions of physician style and trust sexual health in patients with rheumatoid arthritis and the association between physical fitness and sexual function: a cross-sectional study physical fitness, aging, and sexuality sexual dysfunction: overview of prevalence, etiological factors, and treatments sexual function problems and help seeking behaviour in britain: national probability sample survey the hispanic family and male-female relationships. an overview how important is sex in later life? the views of older people sexual and reproductive health outcomes are positively associated with comprehensive sexual education exposure in mexican high-school students health literacy definition. network of the national library of medicine web site nnlm web site e-health literacy and associated factors among chronic patients in a low-income country: a cross-sectional survey health literacy: a prescription to end confusion health literacy and mortality among elderly persons surveys and questionnaires global survey of national constitutions: mapping constitutional commitments to sexual and reproductive health and rights world health organization. coronavirus disease 2019 (covid-19): situation report, 51. world health organization; 2020 we acknowledge all the clinicians from the department of immunology and rheumatology of the incmyn-sz. key: cord-0034062-3hl8scyi authors: parke, a.; parke, d. v. title: the pathogenesis of inflammatory disease: surgical shock and multiple system organ failure date: 1995 journal: inflammopharmacology doi: 10.1007/bf02674919 sha: 6d1d15fd6108b380cd13259c64949ab5e801562b doc_id: 34062 cord_uid: 3hl8scyi chronic inflammatory disease, embracing rheumatoid arthritis (ra), inflammatory bowel disease (ibd), hepatitis, asthma, atherosclerosis, multiple system organ failure (msof), etc., is mediated by reactive oxygen species (ros). these ros originate from activated neutrophils in infections and in immune and autoimmune reactions, from tissue deposits of ferritin, and from futile cycling of cytochrome p450 (cyp) following exposure to persistent chemicals, and may be perpetuated by the actions of complement, cytokines and eicosanoids. acute inflammation is normally arrested by removal of ros by tissue glutathione (gsh) and the antioxidant vitamins, a, c and e, all of which are regenerated by nadh and nadph. failure of this antioxidant defence system can lead to oxidative stress and to chronic inflammatory disease, including surgical shock and msof. the roles of oxidative stress and microcirculatory arrest in promoting msof, and of gsh, the antioxidant defence system, and fibronectin in preventing this, are reviewed in the light of recent experimental studies of surgical shock, including fasting, anaesthesia, hepatic ischaemia and reperfusion. inflammation is a valuable defence mechanism, an aspect of the immune cascade which protects living animals against infections from invading organisms, antigens and foreign bodies. it involves vasodilatation, increased vascular permeability, tissue oedema, lcucocyte migration and activation, leucocyte production of reactive oxygen species (ros) [1] and release of lysosomal enzymes, the release of cytokines, and the formation of eicosanoids. inflammation can progress from an acute, episodic phenomenon to a self-perpetuating chronic condition. the ultimate products of inflammation are ros, which can have a cascade effect on the process, thereby augmenting and extending the phenomenon ( figure 1) . hence, the production of ros by other mechanisms, e.g. redox cycling, futile cycling by the cytochromes p450 (figure 2 ), prostanoid biosynthesis, and iron-promoted ros damage [1] , may similarly augment, or even initiate, inflammation. ros-mediated inflammation is well-known to be involved in the pathogenesis of infectious disease, including pneumonic plague and septic shock [2] , in immune and autoimmune diseases, such as rheumatoid arthritis (ra) [1, 3] and inflammatory parkeand parke bowel disease (ibd) [4] , and although its role is less well established, in diseases such as cancer [5-71, atherosclerosis [6, 8, 9] , hepatitis [3, 10] , alzheimer's dementia [11] , aids [12] , multiple system organ failure (msof) [13,j4] , and asthma and adult respiratory distress syndrome (ards) [15] . give the oxygen-enzyme-substrate complex. if the substrate is readily oxygenated, this complex breaks down by pathway (b) to yield the oxygenated substrate and water, and regenerates the enzyme (mixed-function oxidation). if, however, the substrate is not readily oxygenated, the complex breaks down by pathway (a) to yield superoxide radical and the enzyme-substrate complex, and the process of reduction of 0 2 to 02 ° continues cyclically via the oxygen-enzyme-substrate complex (futile cycling). from reference 179] modified over the past century, the complex mechanisms of the interactive multicomponent defence system, which initiates and maintains inflammation, have been the subject of numerous research investigations. presumably, a primary objective has been to identify a critical mechanism that might be exploited to provide a treatment or prophylaxis. one component after another has been considered to be the critical self-promoting factor that prolongs the protective benefits of acute inflammation into the degenerative, chronic inflammatory disease states, such as ra, ibd, msof, etc. these have ranged from infecting micro-organisms [16] to complement [17] , prostanoids [18] , iron complexes [1] , loss of glutathione [19] , or energy (nadph) to recharge the antioxidant defence mechanism [20] . this present review aspires to update this perplexing problem, and introduces additional new factors, namely the specific oxygen-activating enzymes, the cytochromes p450, which have been shown to be involved in oxidative stress, inflammation, tissue necrosis, surgical shock and msof [20] . these comprise 02" ~ the superoxide ion radical; o2-, the peroxide anion; 6h, the hydroxyl radical; 02, singlet oxygen; and ocl-, the hypochlorite ion, all of which are cytotoxic and are involved in the destruction of invading micro-organisms [9] . they are formed by one-electron reduction of molecular oxygen, and are produced by neutrophils and macrophages for the specific defensive role of microbial destruction, but are formed continuously in other tissues by a variety of metabolic processes, including electron leakage from mitochondrial membranes, transoxygenation in the biosynthesis of the eicosanoids, oxygen activation by the cytochromes p450 in anoxia, the action of xanthine oxidase in anoxia, futile cycling of cytochrome p4502e1, and the redox cycling of quinones and carbon-centred radicals [21] [22] [23] (figure 3 ). ros have also been shown to be involved in the activation of carcinogens, the formation of neoantigens [3] , dna strand cleavage [24] , and the initiation and promotion of carcinogenesis [25] . ros may promote and extend inflammation by chemotactic attraction of neutrophils, and their activation to generate ros, and by contributing to eicosanoid formation [22] ; ros also contribute to the regulation of prostacyclin (pgi2) synthesis and release during acute haemorrhage [26] . biological systems are protected from the damaging effects of ros by the antioxidant defence system (figure 3 ), a complex integrated array of enzymes, antioxidants and radical scavengers, dependent largely on the intracellular redox buffer, glutathione (gsh), and involving gsh reductase, gsh peroxidase (gpx), phospholipid hydroperoxide gsh peroxidase (phgpx) [27] , superoxide dismutase (sod), and catalase, the tocopherols (vitamin e), ascorbic acid (vitamin c), and retinoids (vitamin a) [27] . b-carotene and a-toeopherol act synergistically, thereby enhancing the radicaltrapping activity of vitamin e [28] . retinoids are involved in limiting inflammation, inhibiting neutrophil generation of ros, but with little effect on the xanthine oxidase system, and have been used in the treatment of inflammatory skin disease [29] . figure 3 . oxygen radical toxicity and the antioxidant defence system. ros are formed by the reduction of molecular oxygen, by radiation, cyp2e1, or cytochrome p450 futile cycling. antioxidant defence depends largely on radical scavengers, including gsh, tocopherols (vit. e), ascorbate (vit. c), retinoids (vit. a), and on antioxidant enzymes, such as sod, gpx, etc. chemical defence depends largely on uridine diphosphate glucuronyl (udpga) and phosphoadenosine phosphosulphate (paps) transferases. chemicals may also be metabolized to give rise to reactive intermediates, which bind to proteins to give neoantigens, or to dna to initiate malignancy haem iron may also generate ros, and may amplify and prolong the inflammatory reaction [30] . it has been postulated that, in ra, iron is released from accumulated ferritin deposits in inflamed joints, leading to the iron-catalysed production of ros, and the consequent processes of chronic inflammation [1] . the free radical nitric oxide (no) is involved in a wide range of physiological processes including vasodilatation; it is identical to endothelium-derived relaxing factor (edrf) and contributes to the cardinal signs of inflammation [31] . it is an important cytotoxic molecule in the defenee against malignant cells, fungi, protozoa, helminths and mycobacteria, but not against extracellular bacteria [32] . it is a neurotransrnitter and neuromodulator in the central and peripheral nervous systems, and has a regulatory function on the immune system [31] . no is generated from l-arginine by no synthase; it inhibits osteoclast activity and no synthase activity is decreased in old age and osteoporosis [1] . inflammation is characterized by migration of erythrocytes, platelets, neutrophils, and then macrophages and lymphocytes, from the vascular compartment into tissues, in response to the inflammatory stimulus of bacteria, endotoxin, necrotic tissue, etc. the leucocytes interact with target cells dependent on regulated adhesion molecules (integrins and selectins -glycoproteins and lectins from the immunoglobulin and other supergene families) which are essential for most aspects of cellular immunity and inflammation, including antigen presentation, leucocyte activation, and leucocyte migration to sites of inflammation [33] . leucocytes possess an array of cell-surface receptors, mediating varying degrees of adhesion to a miscellany of adhesion molecules specific for lymphocytes, monocytes, platelets and endothelial cells, and for fibrinogen, collagen, fibronectin, complement, etc., facilitating chemotaxis, leucocyte migration, leucocyte adhesion and activation [33] . figure 4 . role of complement in perpetuating inflammation. the activation of complement by endotoxin, ros, necrotic tissue, and other inflammatory stimuli, in turn activates neutrophils, which release ros and lysosomal enzymes, damaging the vascular endothelium. this activates platelets, liberating txa2, resulting in platelet aggregation, local vasoconstriction and ischaemia, leading to microcirculatory arrest and tissue necrosis. in turn, the necrotic tissue again activates complement, thereby perpetuating this cycle of the inflammatory process. from reference [35] modified complement is a cascade system of more than 20 proteins, which is concerned in the amplification of antigen-antibody reactions, and is also a mediator of the inflammatory response [17] . complement activation markedly decreases liver perfusion, and the hepatic ischaemia associated with trauma and sepsis is probably mediated by complement activation. complement is also a major determinant of eicosanoid release, eicosanoids acting as intermediate messengers mediating the response to bacteria, endotoxin, etc. [17, 34] . it has been suggested by schirmer and fry [35] that complement may be a causative factor in the continuing activation of inflammation; bacteria, endotoxin and other inflammatory stimuli activate complement, which in turn activates neutrophils, releasing ros and initiating a continuous cycle of inflammatory response [35] (figure 4 ). cytokines are regulatory proteins secreted by macrophages and other cells, which provoke numerous effects on cells of the immune system, and modulate the inflammatory response. cytokines have high affinities for leucocytes and other cells, effecting immunoregulation and cell proliferation, and include the interleukins (il-1 to il-12), tumour necrosis factors (tnfa and t) and the interferons. il-1 (a and//) are related interleukins with similar biological activity, produced mainly by activated mononuclear phagocytes. il-1 is a major inflammatory hormone, causing leucocyte accumulation and activation, inducing fever and the acute phase response, and is also implicated in shock-induced atp depletion [36] . il-1 results in hypotension and tachycardia, mediated by increased pgi 2 and pge 2 synthesis from several cell types, increased txb 2 from macrophages and neutrophils, and increased ltb 4 from neutrophils. il-1 induces many genes (including some oncogenes), activates regulatory g proteins and protein kinase c, and increases phosphorylation of certain cellular proteins [37] ; it also increases muscle pge 2 and muscle proteolysis. interleukin 6 (il-6), synthesized by monocytes/macrophages, endothelial cells, fibroblasts, and other cells, regulates the induction of fever, the synthesis of acute phase proteins, t-cell proliferation, and induction of immunoglobulin production by activated b cells. the synthesis of il-1 and il-6 are promoted by ros [38] . it has been suggested that dysregulation of il-6, by chemical damage or viral infection, may play a role in the pathogenesis of autoimmune disease, leading to a self-perpetuating inflammatory process [39] . il-6 is the main cytokine released after elective surgery, and tissue damage is a major determinant of the circulating levels of this cytokine [40] . interleukin-2 (il-2) is a t-cell growth factor and is a pivotal cytokine for generating effective immune response. it is associated with increased vascular permeability due to its increasing the adhesion of platelets and neutrophils to the endothelium [41] . whereas the inflammatory cytokines, tl-1 and il-6, decreased the activities of rat liver cytochrome p450-1inked mono-oxygenase activities, il-2 results in the induction of rat liver cytochrome p4502d (cyp2d) [42] . the cytokines, il-3, il-5 and granulocyte macrophage colony-stimulating factor (gm-csf), synthesized and released from specific t-cells, have relatively selective activities in activating eosinophils, and are involved in the inflammatory process of asthma [43] . tnf, another cytokine elaborated by macrophages and other cell types, is one of the major endogenous mediators of endotoxic shock, resulting in increased eicosanoid production, hypotension and ischaemic necrosis of liver, lungs and bowel. it stimulates collagenase activity, pge 2 formation in macrophages and fibroblasts, and increases endothelial pgi 2 synthesis mediating the hypotension. the eicosanoids (prostanoids) are oxidative metabolites of arachidonic acid, a family of vasoactive mediators of major importance in inflammation, regulation of the immune response, and circulatory shock [3, 18, 34, 44] . arachidonic acid, released from membrane phospholipids by phospholipase, is metabolized by the cyclo-oxygenase arachidonic acid released from membrane phospholipids is metabolized by cyclo-oxygenase to prostaglandins (pgh2, pge2), prostacyclins (pgi2) and thromboxanes (txa2, txb2) , or by lipoxygenases to hydroxyeicosatetraenoic acids (hete) and leukotrienes (ltb4). note the ros released in the conversion of pgg 2 to pgh 2 and in the conversion of 5-and 12-hpete to 5-and 12-hete pathway (platelets, endothelial cells) to form the prostaglandins, thromboxanes and prostacyclins, and by the lipoxygenase pathway (neutrophils) to yield the leukotrienes ( figure 5 ). platelets synthesize large amounts of thromboxane (txaz) but not prostacyclins (pgi2), whereas endothelial cells synthesize mainly pgi 2. the leukotriene, ltb4, and to a lesser extent other leukotrienes, is formed in neutrophils and macrophages but not lymphocytes. eicosanoids are both pro-and anti-inflammatory. prostaglandin e (pgez) and prostacyclin pgi 2 are potent vasodilating and hyperalgesic agents mediating the acute inflammatory response; they suppress the functions of inflammatory cells (mast cells, neutrophils, lymphocytes, macrophages) and suppress the production of ltb 4 and the cytokines, il-t and il-2. pge 2 inhibits neutrophil ros production, suppresses b lymphocyte activity (antibody response) but enhances t-cell activity (cell-mediated immunity); txb 2 antagonizes the pge compounds. txa 2 induces platelet aggregation; pgi 2 inhibits platelet aggregation and is a vasodilator. the leukotrienes are potent mediators of inflammation; they increase vascular permeability, and endotoxin produces a marked increase in lte 4 metabolites in the bile. ltb4, increased in rheumatoid synovial exudates, in psoriatic skin lesions, and in ibd, inhibits the leucocyte proliferative response, activates t lymphocytes to secrete interferon (ifn-~,), and induces neutrophil ros formation and monocyte-mediated cytotoxicity. tnfc~ may initiate the eicosanoid cascade in endotoxaemia, and increased levels of txb 2 and its metabolites are seen in patients with advanced sepsis, severe burns, hepatic failure and msof. renal failure is a prominent feature of the later stages of msof, and ltd 4 and ltc 4 have been implicated in acute renal injury. ltd 4 causes marked splanchnic vasoconstriction and may be involved in stress-induced gastrointestinal ulceration. platelet-activating factor (paf) is a phospholipid with diverse inflammatory actions, and, given intravenously, produces severe hypotension and shock. eicosanoids, particularly ltc 4 and ltd4, and txb 2 are probably mediators of paf action. the eytochromes p450 (cyp), a superfamily of mixed-function oxygenases [45] , concerned in the oxygenation and metabolic detoxication of toxic chemicals and other xenobiotics [46] , and in the elaboration of the steroid hormones, are also able to generate ros, either directly (cytochrome p4502e1; cyp2e1) or by futile cycling (table 1, figure 2 ). in futile cycling, xenobiotic substrates (e.g. chlorodioxins) activate the p450-oxygen complex, but, because of the conformational difficulty of inserting oxygen into the substrate, oxidation does not occur; instead, superoxide anions are released and ros generated (figure 3 ). many polyhalogenated, and other complex, molecules (e.g. tcdd; 2,3,7,8-tetrachlorodibenzo-p-dioxin) are known to initiate chronic inflammatory conditions because they: two families of the cytochromes p450, namely cyp1a and cyp2e, metabolize xenobiotics to products with increased toxicity (reactive intermediates from cyp1a, and carbon-centred radicals plus ros from cyp2e), and hence are involved in the activation of carcinogens and mutagens, and in the elaboration of immunotoxins and neoantigens [3, 46, 47] (figure 3) . classic examples of the latter are the cyp autoantibodies of hepatitis [48] , the endoplasmic reticulum autoantibodies of systemic lupus erythematosus [49] , and the fatal hepatitis of tienilic acid antigens [50] . the cyp enzymes are particularly active in the liver, metabolizing nutrients and xenobiotics present in food; they are also present in most other tissues, including the leucocytes, where their function is as yet unknown [3] . high rates of exposure to toxic chemicals and consequent heavy dependence on the chemical detoxication system, make large demands on the endogenous redox buffer, glutathione (gsh), both for the conjugation/detoxication of epoxide metabolites (potential carcinogens, immunotoxins) and for the reduction of quinone metabolites [51] , thus depleting the gsh available for antioxidant defence and so enhancing the potential for oxidative stress (figure 3) . furthermore, depletion of gsh decreases the integrity of lymphocytes and other cells of the immune system, resulting in impairment of t-cell and macrophage immune function, loss of immunocompetence [52] and inhibition of leucocyte differentiation [53] . the past few decades have seen the emergence of several widespread fatal chronic inflammatory syndromes associated with exposure to specific chemicals, namely, the toxic oil syndrome (tos) associated with the ingestion of reprocessed adulterated rapeseed oil, the eosinophilia-myalgia syndrome (ems) associated with the amino acid, tryptophan, and the acquired immunodeficiency syndrome (aids) associated with the use of nitrate vasodilators and other drugs. the toxic oil syndrome (tos) that developed in spain in 1981 followed the ingestion of fraudulently-marketed illicitly-recovered denatured rapeseed oil [54] [55] [56] [57] . over 20 000 cases were recorded, with symptoms of fever, rash, gastrointestinal pain, hepatomegaly, pulmonary and neurological disturbances, arthralgia and myalgia; over 300 died and many had persistent scleroderma-like symptoms for a decade or so [54] . rapeseed oil, long known to be atherogenic due to its content of erucic acid, a natural mono-unsaturated fatty acid, is metabolized only with great difficulty. it results in hepatic peroxisomal proliferation, was associated with atherosclerosis, and was marketed as an industrial lubricant after denaturation by addition of aniline/pyridine and a brown dye to prevent human consumption. illicit treatment to remove the aniline by steam distillation is thought to have resulted in the production of toxic acyl anilides [58] . erucic acid is resistant to/~-oxidation and is therefore metabolized by cyp4 and peroxisomal enzymes which are associated with peroxide formation and hepatotoxicity; anilides of fatty acids, which are even more resistant to /~-oxidation, would accumulate, and could manifest immunotoxicity. although not normally antigenic, fatty acids can be made immunogenic by chemical modification, and antibodies to several common fatty acids have been synthesized [59] . in 1989, a second such epidemic of chronic inflammatory disease, namely, eosinophilia-myalgia syndrome (ems), associated with the ingestion of l-tryptophan, occurred in the usa and canada [60, 61] . the disease was marked by progressive neuromuscular, pulmonary and skin manifestations, resembling the symptoms of toxic oil syndrome, and although quinolinic acid and other metabolites of tryptophan were first indicted as possible causes, an impurity with the chemical structure of two tryptophan molecules joined by an ethylidine bridge -a manufactured impurity -was finally identified as the toxic entity [62] . the third example of these recent disastrous inflammatory diseases, namely, acquired immunodeficiency syndrome (aids), a disease of systemic inflammation and changed lymphocyte function, and generally believed to result from hiv infection, is now often considered to be the consequence of several interacting factors, including a viral-induced cysteine/glutathione deficiency [63] and the cytotoxicity of vasodilatory alkyl nitrites [64] . these chemicals are used as recreational drugs, and form s-nitrosoglutathione, depleting lymphocyte gsh and atp, resulting in oxidative stress, dna damage and cell death [64] [65] [66] . the initial phase of critical illness (trauma, surgery, burns, infection), namely shock and hypovolaemia, is accompanied by decreased metabolic activity (raised blood glucose, decreased oxygen consumption, raised levels of adrenaline and other stress hormones), which is followed by a prolonged phase of hypermetabolism (increased oxygen consumption, lowered blood glucose, nitrogen loss, cytokine production). the degree of hypermetabolism is related to wound severity and the extent of associated infection. in the absence of continuing external inflammatory stimuli (wounding, infection, antigens), ros from activated neutrophils and other products of the primary inflammatory response are the major cause of progression and perpetuation of the acute inflammation to a chronic condition. normally, the lipid-soluble a-tocopherol, and the water-soluble ascorbic acid and glutathione, scavenge ros and other radicals, limiting the oxidative stress and inflammatory response, a-tocopherol is the primary radical scavenger in biological membranes, and is continuously regenerated at the expense of ascorbate and glutathione oxidation ( figure 6 ), with the ascorbate and glutathione being continuously regenerated by nadh and nadph in the presence of the relevant reductases [67, 68] . hence, the prevention of oxidative stress and progressive inflammation is highly dependent on sufficient provision of energy to regenerate glutathione and other antioxidants, and on adequate dietary provision of a-tocopherol, ascorbic acid, sulphur amino acids, selenium, etc. to repair the daily figure 6 . antioxidant regeneration systems. biological oxidants and ros (represented by x') are reduced directly by the tocopherols. the oxidized tocopheryl chromanoxy radical is cyclically reduced to the tocopherols again by ascorbic acid or gsh. the dehydro-ascorbic acid is cyclically reduced back to ascorbic acid at the expense of gsh or nadh, and oxidized glutathione (gssg) is reduced back to gsh by nadph in the presence of glutathione reductase losses of these vital chemicals and enzymes of the antioxidant defence system [19] . chain oxidation of intraceuular gsh to gssg by superoxide-dependent production of h20 2 is precluded by the presence of superoxide dismutase, so that the combination of gsh and sod constitutes an integral component of cellular antioxidant defence [69] . glutathione peroxidase (gpx), a selenoenzyme, is involved in protection against h20 2 and other peroxides; hence the dietary need for selenium, deficiencies of which result in keshan disease, a cardiomyopathy widespread in china [70] . critical illness is associated with both immunosuppression and glntathione deficiency [52] ; gsh deficiency decreases lymphocyte proliferation and production of turnout necrosis factor. therapeutic or nutritional intervention to maintain gsh levels may enhance immunocompetenee and improve the ability of patients to combat infection and recover from critical illness [52, 63] . surgical shock, for long a major cause of death following emergency amputation and similar elective surgery, was considered by the eminent 19th century surgeon, joseph lister, to be due to microbial infection, which was prevented by the use of antiseptic phenol sprays in the theatre. subsequent surgeons considered that blood loss was the major cause of postsurgical fatality, and this led to fluid replacement by saline or blood transfusions. later still, anaesthetics were believed to interfere with hepatic function, and postmortems on surgical fatalities revealed that hepatic necrosis and renal necrosis were frequent causes of death. surgical shock is a major cause of death of critically ill and injured patients, particularly.military casualties in time of war and road traffic accidents in peace time. resuscitation of casualties to normal blood pressure proved to be inadequate, with many patients dying of renal failure, so that urine output became the factor to monitor for successful shock resuscitation in the 1950s. in world war 2, gastrointestinal haemorrhage, often fatal, was a major feature of shock in flying personnel suffering severe burns and shot down in combat. following the correction of blood volume and renal function, pulmonary dysfunction became the next weak link, and, in the vietnam war, 'shock lung' or da nang lung (acute respiratory distress syndrome; ards) was a major cause of fatalities. in the 1980s, medical technology had advanced to correct the various single organ dysfunctions, and multiple system organ failure (msof) emerged as a consequence [14] . four major organ systems at risk have been identified, namely pulmonary failure, hepatic failure, renal failure and stress gastrointestinal bleeding. ards has been associated with massive effort at resuscitation, hepatic failure is associated with anaesthetic and drug toxicities, renal failure with drug toxicity and transfusion reactions, and hypovolaemic shock and uncontrolled infections have both been associated with all four organ failures. in those instances where any of these organ failures were studied in detail, inflammation with nentrophil invasion, ros production, oxidative stress and eventual tissue necrosis, were characteristic features. hence, msof may be considered to be a form of systemic inflammation, progressing from one organ to another, as modern medical technology is applied to rescue each individual system in the resuscitation of the patient. this is a high-molecular-weight glycoprotein, found in a soluble form in blood and lymph, and in an insoluble form in cells and cellular matrix; it has high affinity for cell surfaces, fibrin, collagen, bacteria, etc. incorporation of fibronectin into tissues augments macrophage clearance of microaggregates of tissue debris, thus preventing microvascular injury from microemboli [71] . fibronectin is synthesized in the liver, vascular endothelium, macrophages and fibroblasts; it has a fast turnover (plasma half-life of 30 h) and is increased in injury and shock. plasma fibronectin is low in burn cases, infections and msof, and deficiency develops rapidly in starvation and trauma. microaggregates of fibrin, fibrin degradation products, platelets, etc., circulating in the blood, are removed by the reticuloendothelial kupffer cells in the liver, thereby preventing the formulation of microemboli, protecting the lungs and other organs from microcirculatory arrest. blood-borne microaggregate debris in association with fibronectin deficiency may lead to leucocyte activation and sequestration, releasing ros and proteases, resulting in endothelial injury, increased vascular permeability, oedema, tissue necrosis and organ failure [71] . in various forms of trauma, including crush injury, fractures and burns, necrotic tissue, fracture fragments and haematoma fragments serve as stimuli for continuing activation of inflammation. in instances of exposure to toxic chemicals activating the cytochromes p450 to give rise to futile cycling or neoantigen formation, numerous pathogenic mediators of inflammation are activated; these include cellular mediators (cytokines) and humoral mediators (complement, ros, eicosanoids, kinins, etc.). this systemic inflammation is likely to be the primary trigger for widespread microvascular injury, microaggregation of tissue debris, and microcirculatory arrest, ischaemia and organ failure. msof is thus the likely consequence of generalized autodestructive systemic inflammation 135] (see figure 4 ). organs fail sequentially, rather than simultaneously. the sequence of organ failure is the clinical manifestation of the organ-specific responses to systemic inflammation, microcirculatory arrest, ischaemia, and tissue necrosis. hepatic and pulmonary failure are the earlier manifestations of msof, probably because these tissues, unlike the kidney, are rich in tissue-fixed macrophages which release numerous inflammation-mediating factors, including il-1, tnfa, paf, eicosanoids, etc. renal failure is usually terminal, since in the ultimate oxidative stress, ros, lipid peroxides, and other toxic products of the ros-mediated tissue lipid peroxidation, are excreted via the kidney, resulting in fatal damage, as has been reproduced in numerous animal models i72]. because of the self-promoting nature of the systemic inflammation, removal of the original, initiating stimulus may do little to prevent the propagation of the inflammation, the organ ischaemia and organ failure, and eventual death. in an endeavour to elucidate in more detail the pathogenesis of surgical shock, studies in experimental animals were conducted to examine the roles of possible contributory factors, including (a) preoperation fasting, (b) anaesthesia, (c) duration of surgery, (d) blood loss and hepatic ischaemia, and (e) reperfusion [19, 20, [73] [74] [75] [76] . american surgeons in the first world war considered that anaesthetic toxicity and consequent liver damage were primary causes of battle fatalities and found that transfusion with 5% glucose/saline for 24 h greatly decreased the mortality rate [74, 77] . diethyl ether was the major anaesthetic in use at the time, and was known to cause damage to the liver and kidneys, especially after fasting and fluid loss [74, 78] . indeed, from these early observations by philadelphian surgeons nearly eighty years ago, came the first understandings of chemical detoxication and of the susceptibility of the liver to chemical toxicity, the protective effects of good nutrition, especially sulphur amino acids, and the role of hepatic glutathione [78] . in recent studies, rats exposed to fasting (20 h) and light ether anaesthesia showed marked evidence of oxidative stress including: a) increased exhalation of alkanes in the expired air, b) 4-fold increases in the chemiluminescence and thiobarbituric acid-reacting (tbar) material of liver and kidney cytosols, c) a 70% loss in the total cytochromes of the rat liver and kidney, and d) a 140% increase in cytochrome p4502e1 (cyp2e1) activity [74] . the effects of the pre-anaesthetic fasting, and the effects of the fight ether anaesthesia, were about equal, and were additive [19, 74] . hence, it was concluded that both food deprivation and ether anaesthesia result in ros generation, lipid peroxidation and oxidative stress, probably by the induction of cyp2e1, a cytochrome p450 which is known to be highly active in futile cycling and the generation of ros [75] . thus, in addition to the generation of ros by infections and by immune reactions, we now have the possibility of ros generation from the eytochromes p450 following exposure to certain chemicals. like microbial infections and immune reactions, the cyp2e1 generation of ros may also lead to inflammation. in subsequent studies, tissue glutathione (gsh) and total radical-trapping activity (trap) (ascorbic acid, tocopherols, retinoids, glutathione, etc.) were determined, together with specific cytochrome p450 (cyp) activities; these showed that gsh, trap, total cyp, cyp1 and cyp2b decreased progressively with increasing exposure to ether, in both liver and kidney, while tbar and cyp2e progressively increased [76] . this is considered to provide quantitative evidence of the effects of the duration of ether anaesthesia on cyp2e induction, its generation of ros, tissue lipid peroxidation, and organ necrosis. although diethyl ether was the anaesthetic used in all these experiments, other volatile anaesthetics, particularly the halogenated anaesthetics, e.g. halothane, enflurane, and other substrates/inducers of cyp2e, are likely to exhibit the same ros-generating effects. in parallel experiments, in a rat liver ischaemia-reperfusion model to examine the molecular pathology of hypovolaemic shock [20] , it was found that liver ischaemia alone resulted in slight oedema, a slight increase in tbar substances (lipid peroxidation), neutrophil infiltration, and decreases in liver gsh and trap, all indices figure 7 . possible mechanisms of ros-mediated toxicity in surgical shock and msof. ros are generated by cytochrome p4502e1 (cyp2e), from hepatic ischaemia (hypovolaemic shock) or in reperfusion injury. these deplete tissue gsh and total radical trapping activity (trap = vitamins c, e, a and gsh), and also release interleukins (il-1, il-6), leukotrienes (ltb4) and paf. this in turn activates leucocytes and results in oxidative stress, and migrating leucocytes follow the ros into other organs, resulting in msof, ards, etc. liver reduced gluathione (gsh) is expressed as nmol/mg tissue; oxidized glutathione (gssg) is expressed as nmol gsh equiv/mg tissue; total radical trapping activity (trap) is nmol 0 2 uptake per min per mg tissue; thiobarbituric acid-reactive substance (tbar) is expressed as nmol tetraethoxypropane equiv/mg tissue protein. six rats per group were studied and mean values are given; sem values were 20% of the mean values or less. data is taken from references [19, 20] of hepatic inflammation. after 60 minutes of liver reperfusion, liver oedema was increased by 40%, neutrophil infiltration into the liver was increased 40-fold, tbar substances were increased 20-fold and were maximal; liver gsh was 95% decreased, trap was 90% decreased, and both these were at a minimum [20] . all parameters of hepatic oxidative stress (inflammation) were exacerbated by 24-h starvation; liver gsh closely paralleled the trap, and ischaemia alone depleted both by 30% in fed rats and 50% in fasted rats. the oxidative stress and lipid peroxidation were associated more with the period of reperfusion than with the initial ischaemia and more with the later neutrophil infiltration than with the initial ros generation by the xanthine oxidase system [20[. after 90 rain of liver reperfusion, neutrophil infiltration was observed also in the rat lung, indicating that inflammation of the liver could generate circulating mediators, possibly interleukins (il-1, il-6), platelet-activating factor (paf), and leukotrienes (ltb4) , that provoked sympathetic inflammation in the lungs. hence, the phenomenon of ards following surgical shock or msof is explained as a manifestation of systemic inflammation, and possible mechanisms are illustrated in figure 7 . in a study of the role of tissue (liver/kidney) gsh in the prevention of surgical shock, it was found that fasting of rats alone decreased tissue gsh by 50% and increased tbar by 100%; fasting plus 30 rain ether anaesthesia decreased tissue gsh by 80% and increased tbar by 600% [19] . the oxidation of tissue glutathione increased progressively with fasting, anaesthesia, ischaemia, and ischaemia followed by reperfusion (table 2 ) [20] . a complex series of molecular mechanisms has been advanced to account for the pathogenesis of surgical shock and msof, and these include: a) energy and gsh depletion, b) induction of cyp2e1 activity by fasting and anaesthesia, c) generation of ros, d) cytokine release, e) oxidative stress and lipid peroxidation, f) neutrophil activation and sequestration, and g) oxidative stress, cell death and tissue necrosis [19] . hence, it would appear that the protective acute inflammatory response may progress to destructive chronic inflammatory disease states, such as rheumatoid arthritis, peptic ulceration, hepatitis and msof, by progressive systemic oxidative stress associated with a variety of factors, including: tissue iron [30] , infection [2] , starvation [27] , cytochrome p4502e1 [10] , complement [17] , or loss of tissue gsh [19, 20] . it is known that loss of gsh from increased ros formation, and greater demands for chemical detoxication [51] , also has adverse effects on lymphocyte function and immune competence [52, 53, 66] . thus, high levels of exposure to environmental chemicals may constitute yet another causative mechanism of inflammatory disease; and epidemiology confirms this. chemically-degraded rapeseed oil in tos, a manufactured aberrant of tryptophan as the causative agent in ems, alkyl nitrites and aids, anaesthetics in surgical shock, and drug-associated lupus and similar autoimmune diseases, are some of the many examples of inflammatory disease now recognized as being chemically-induced [3, 49, 50] . this may indeed prove an explanation of why, in this age of chemical industry and environmental pollution, inflammatory disease is now most prevalent. free radicals in inflammation: second messengers and mediators of tissue destruction endotoxin, septic shock and acute lung injury:, neutrophils, macrophages and inflammatory mediators molecular pathology of drug-disease interactions in chronic autoimmune inflammatory diseases oxygen radicals in ulcerative colitis an overview of the relationship between oxidative stress and chemical carcinogenesis oxidants, antioxidants and the degenerative diseases of aging active oxygen species as factors in multistage carcinogenesis evidence for lipid peroxidation in atherosclerosis free radicals and antioxidants: a personal view hepatotoxicity of volatile anaesthetics free radicals in brain metabolism and pathology the role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus the gastrointestinal tract. the undrained absess of multiple organ failure multiple system organ failure. st louis: mosby year book ards and sepsis -definitions and new therapy oxygen dependent microbial killing by phagocytes multiple system organ failure. st louis: mosby year book multiple system organ failure. st louis: mosby year book role of tissue glutathione in prevention of surgical trauma studies in surgical trauma: oxidative stress in ischaemia-reperfusion of rat liver the effects of nutrition on chemical toxicity multiple system organ failure production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes from endrin-treated rats dna strand cleavage as a sensitive assay for the production of hydroxyl radicals: role of cytochrome p4502e1 in the increased activity after ethanol treatment the cytochromes p450 and mechanisms of chemical carcinogenesis role of oxygen-derived free radicals on rat splanchnic eicosanoid production during acute haemorrhage the importance of diet and nutrition in the detoxication of chemicals /~-carotene and a-tocopherol are synergistic antioxidants anti-oxidant effects of retinoids on inflammatory skin diseases iron-promoted oxidative damage in rheumatic diseases nitric oxide -from mediator to medicines nitric oxide: physiology, pathophysiology and pharmacology leucocyte adhesion molecules and alcoholic liver disease cytochrome p450 metabolism of arachidonic acid multiple system organ failure. st louis: mosby year book interleukin-1 receptor antagonist improves survival and preserves organ adenosine-5'-triphosphate after haemorrhagic shock interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide t669 hypoxia/reoxygenation stimulates endothelial cells to promote interleukin-1 and interleukin-6 production. effects of free radical scavengers interleukin-6 in autoimmune diseases interleukin-6 and the metabolic response to surgery interleukin-2 acutely induces platelet and neutrophil-endothelial adherence and macromolecular leakage interleukin 2 induction of cytochrome p450-1inked monooxygenase systems of rat liver microsomes t cells, cytokines and asthma diet, eicosanoids and chemical toxicity evolution of the p450 gene superfamily the role of cytochromes p450 in the detoxication and activation of drugs and other chemicals the cytochrome p4501 gene family of microsomal haemoproteins and their role in the metabolic activation of chemicals identification of cytochrome p4501 a2 as a human autoantigen liver abnormalities and liver membrane autoantibodies in systemic lupus erythematosus immune mechanisms in tienilic acid associated hepatotoxicity glutathione metabolism and its role in hepatotoxicity glutathione depletion in rats impairs t-cell and macrophage immune function inhibition of the differentiation of human myeloid cell lines by redox changes through glutathione depletion clinical epidemiology of toxic oil syndrome: manifestations of a new illness manufacturing processes at two french rapeseed oil companies: possible relationship to toxic oil syndrome in spain chemical correlates of pathogenicity of oils related to the toxic oil syndrome epidemic in spain toxic-allergic syndrome caused by the ingestion of rapeseed oil denatured with aniline anilides and the spanish toxic oil slnldrome fatty acids rendered immunogenic l-tryptophan-associated eosinophilia-myalgia syndrome: perspective of a new illness l-tryptophan associated eosinophilia myalgia syndrome characterisation of 'peak e' a novel amino acid with the eosinophilia-myalgia syndrome cysteine and glutathione deficiency in aids patients: a rationale for treatment with n-acetylcysteine nitrite inhalants: history, epidemiology, and possible links to aids. envir health persp toxicity, immunosuppressive effects and carcinogenic potential of volatile nitrites: possible relationship to karposi's syndrome s-nitrosylglutathione-mediated hepatocyte toxicity the pecking order of free radicals and antioxidants: lipid peroxidation, c~-tocopherol, and aseorbate vitamin e and oxidative stress reduced glutathione in combination with superoxide dismutase as an important biological antioxidant defence mechanism low dietary selenium and its relationship to human disease microaggregation hypothesis of multiple system organ failure effects of acute and sub-chronic administration of iron nitrilotriacetate in the rat autoxidative injury with loss of cytochrome p-450 following acute exposure of rats to fasting and ether anaesthesia the effects of fasting and ether anaesthesia on hepatic and renal function in surgical trauma effects of ether anaesthesia and fasting on various cytochromes p450 of rat liver and kidney the effects of ether anaesthesia on oxidative stress in rats -dose response principles and practice of surgery anaesthetics and liver function molecular orbital studies of oxygen activation and mechanisms of cytochrome p450-mediated oxidative metabolism of xenobiotics key: cord-0073836-j1mvy902 authors: molero-chamizo, andrés; nitsche, michael a.; bolz, armin; andújar barroso, rafael tomás; alameda bailén, josé r.; garcía palomeque, jesús carlos; rivera-urbina, guadalupe nathzidy title: non-invasive transcutaneous vagus nerve stimulation for the treatment of fibromyalgia symptoms: a study protocol date: 2022-01-12 journal: brain sci doi: 10.3390/brainsci12010095 sha: 4c5af0b19cd766424c98b830ca6e06dad796ac08 doc_id: 73836 cord_uid: j1mvy902 stimulation of the vagus nerve, a parasympathetic nerve that controls the neuro-digestive, vascular, and immune systems, induces pain relief, particularly in clinical conditions such as headache and rheumatoid arthritis. transmission through vagal afferents towards the nucleus of the solitary tract (nst), the central relay nucleus of the vagus nerve, has been proposed as the main physiological mechanism that reduces pain intensity after vagal stimulation. chronic pain symptoms of fibromyalgia patients might benefit from stimulation of the vagus nerve via normalization of altered autonomic and immune systems causing their respective symptoms. however, multi-session non-invasive vagal stimulation effects on fibromyalgia have not been evaluated in randomized clinical trials. we propose a parallel group, sham-controlled, randomized study to modulate the sympathetic–vagal balance and pain intensity in fibromyalgia patients by application of non-invasive transcutaneous vagus nerve stimulation (tvns) over the vagal auricular and cervical branches. we will recruit 136 fibromyalgia patients with chronic moderate to high pain intensity. the primary outcome measure will be pain intensity, and secondary measures will be fatigue, health-related quality of life, sleep disorders, and depression. heart rate variability and pro-inflammatory cytokine levels will be obtained as secondary physiological measures. we hypothesize that multiple tvns sessions (five per week, for 4 weeks) will reduce pain intensity and improve quality of life as a result of normalization of the vagal control of nociception and immune–autonomic functions. since both vagal branches project to the nst, we do not predict significantly different results between the two stimulation protocols. fibromyalgia is a disabling chronic disease. in addition to chronic and generalized pain, the syndrome includes extreme fatigue, insomnia and other sleep disorders, affective and emotional disorders, such as depression and anxiety, cognitive impairment, paresthesia, allergy symptoms, joint stiffness, tendinopathy, neuropathic and rheumatologic symptoms, chemical and skin hypersensitivity, irritable bowel syndrome, and other symptoms whose common etiopathogenesis is unknown [1] [2] [3] . because of the unknown biological mechanisms of fibromyalgia, current pharmacological therapies are aimed at alleviating its symptoms, and the efficacy of available treatments is limited [4] . moreover, generalized pain and extreme fatigue are usually refractory to any therapeutic attempt [4, 5] . the parasympathetic vagus nerve (the 10th cranial nerve) innervates multiple internal organs and integrates sensory, motor, and autonomic information by four vagal nuclei. the primary central relay of vagal afferents is the nucleus of the solitary tract (nst) in the brainstem, and several branches of this nucleus project to different brain areas [6] . the myelinated a-and b-fibers of the vagal nerve send somatosensory, motor, and autonomic signals [7] , and the complex system of vagal projections is involved in inflammatory, immune, nociceptive, and emotional processes [8, 9] . a relevant role of the vagal nerve in pain processing is the transmission of peripheral inflammation signals to the central nervous system [10] . additionally, neuroimaging studies have identified neuromodulatory activity in areas related to the sensory and emotional processing of pain, such as the insular cortex and anterior cingulate cortex, after vagal stimulation [11] , which reveals that the central system of pain processing receives vagal afferences. due to the multisystem control of the vagal nerve [12] , some clinical conditions refractory to conventional treatment, such as epilepsy, depression, and inflammatory diseases, have been shown to benefit from invasive stimulation of this nerve [13] . recent clinical and experimental studies have shown that stimulation of the vagus nerve also has therapeutic potential in other diseases, including pain syndromes [14, 15] . vagus nerve stimulation is a food and drug administration (fda)-approved somatic treatment for different pathologies. the precise mechanisms through which vagal stimulation induces clinical improvements are not well understood in detail [16, 17] , but regulation of the autonomic and immune systems and a specific effect on chemical mediators of inflammation are relevant physiological mechanisms of this intervention [18] . considering that the vagus nerve controls pain signals towards the central nervous system [14, 19, 20] , and growing evidence of autonomic dysfunction in fibromyalgia in terms of sympathetic hyperactivity [21] [22] [23] [24] [25] [26] [27] , it can be argued that rebalancing the sympatheticparasympathetic activity via vagal stimulation might reduce the intensity of pain and stabilize the autonomic symptoms of this disease. in addition, an immune system alteration [28] seems to mediate the interaction between the autonomic nervous system and the chronic inflammatory state in fibromyalgia, apparently by the influence of inflammatory cytokines and chemokines [28] [29] [30] . this well-described inflammatory state in fibromyalgia patients is critically important for treatment approaches. auricular and cervical vagus nerve stimulation might reduce such inflammatory overactivity as well via efferent modulation of the activity of the spleen and inflammatory processes [18] and hereby reduce pain symptoms [19] . in accordance, invasive vagal nerve stimulation has been shown to be effective in treatment-resistant fibromyalgia patients, with a tolerability profile similar to that described for the treatment of epilepsy and depression [31] . however, to the best of our knowledge, the potential of non-invasive auricular and cervical vagus nerve stimulation under repeated sessions to improve fibromyalgia symptoms has not been evaluated in randomized, sham-controlled clinical studies. auricular and cervical transcutaneous vagus nerve stimulation (tvns), two non-invasive low-intensity electric stimulation procedures [32, 33] , have however been explored with respect to their potential to modulate the activity of this nerve in other clinical settings [20, [34] [35] [36] [37] , and the effectiveness of these non-invasive methods might not differ significantly from those of invasive stimulation, while non-invasiveness provides an advantageous safety profile [14, 19, [38] [39] [40] . since fibromyalgia involves a dysregulation of the autonomic (high sympathetic tone) and immune (enhanced pro-inflammatory activity) systems [25, 41] , non-invasive tvns is expected to improve the symptoms of fibromyalgia, including chronic, musculoskeletal, and generalized pain [42, 43] , through modulation of the vegetative and immune systems [44] . the main objective of this intervention study is to evaluate the effects of repeated sessions of tvns on pain and other symptoms in fibromyalgia patients, comparing auricular and cervical tvns protocols which have been probed in other pathologies [45] [46] [47] [48] [49] . according to the effectivity reported in these studies, we hypothesize that a multi-session auricular and cervical tvns protocol carried out under safety standards will provide analgesic and therapeutic effects in fibromyalgia patients, in comparison with sham and axillary nerve stimulation controls, and secondarily will also normalize the autonomic (via heart rate variability) and immune (via pro-inflammatory cytokine levels) functions supposedly altered in this disease. adult fibromyalgia patients, in an age range between 18 and 69 years, recruited from the hospital juan ramón jiménez of huelva, spain, will be interviewed to voluntarily participate in this monocentric study. inclusion criteria will be the diagnosis of fibromyalgia by a physician, based on the 2010 american college rheumatology (acr) fibromyalgia diagnostic criteria, and the maintenance of characteristic symptoms for more than a year. a moderate to high pain intensity according to analog pain scales (above 4 points over 10), maintained for more than 6 months, will also serve as an inclusion criterion. the usual individual medical treatment for fibromyalgia will be maintained throughout the study, including psychopharmacological treatments. all genders will be included. exclusion criteria will include standard conditions to be considered for vns (cardiac arrhythmia, sleep apnea, pregnancy, etc.) [49] , other chronic diseases producing widespread pain not related to known symptoms of fibromyalgia, multiple pharmacological treatments for symptoms different from those of fibromyalgia, primary psychiatric syndromes (psychosis, bipolar disorder, etc.) with or without treatment via central nervous system-acting medication, drug dependence (including smoking), vagal syndrome or history of vagal symptoms, and chronic pain and fibromyalgia secondary to inflammatory rheumatic diseases. previous interventions with non-invasive brain stimulation methods or invasive vns will also constitute exclusion criteria. an electrocardiogram will be performed in all participants before the study. participants with arrhythmia and other cardiac alterations will be excluded from the study. sample size calculation will be based on the hypothesized significant differences between (auricular vs. cervical vs. sham vs. axillary) and within (baseline vs. postintervention) groups. we conducted an a priori calculation of the sample size by the gpower statistical power analysis tool (3.1.9.2) for a mixed-model repeated-measures anova (with groups and pre/post-intervention measures as levels of the between-group and within-group factors) [50] , as a primary outcome parameter conducted for the pain questionnaire (bpi). because the main parameter of interest for this calculation is the interaction between the factors group and measure, we conducted the analysis for the within-between interaction, setting the following input parameters: effect size (f) = 0.2 (medium size), α value = 0.05, power (1-ß) = 0.95, number of groups = 4 (pooled sham, axillary stimulation, cervical tvns, and auricular tvns), number of measurements = 2 (baseline as pre-intervention, and at the end of the intervention), correlation for repeated measures = 0.5, and with nonsphericity correction (1). the output parameters were: noncentrality parameter = 17.92, critical f = 2.68, actual power 0.951, and total sample size = 112. taking into account these estimates and adding 20% to the calculated sample to compensate for possible dropouts, we will include 34 patients per group (total sample size of 136). written informed consent (available in supplemental material) will be provided by all patients prior to their participation in the study. the clinical trial will be submitted for consideration and approval by the regional ethics committee for clinical studies. in accordance with national legislation (93/42/eec and rd 1090/2015), the study will be pre-registered in the spanish registry of clinical studies (https://reec.aemps.es/reec/ public/web.html#, accessed on 28 december 2021). the study complies with the code of ethics of the world medical association declaration of helsinki (amend 64th, 2013). auricular vagal stimulation will be performed by the tvns ® l device (tvns technologies gmbh, erlangen, germany, https://u.pcloud.link/publink/show?code=kz0 myfxzrxxqqurvy9sb8okpgtac55ax7nrx, accessed on 28 december 2021). tvns ® l is a battery-driven electrical stimulator connected to an ear electrode which is positioned over the skin of the cymba conchae ( figure 1 ). thus, auricular vagal stimulation will be applied using the typical ear electrodes of the tvns ® l device. a technical variant of this device, in which the original connector is modified to incorporate self-adhesive electrodes, will be used for cervical vagal stimulation and axillary nerve stimulation (the latter as an added control group). thus, stimulation over the cervical branch of the vagus nerve and axillary nerve stimulation will be applied by adapted electrodes for transcutaneous stimulation outside the ear ( figure 1 ). in order to effectively access cervical vagal projections, as reported in previous studies [36] , 2 cm 2 self-adhesive anodal and cathodal electrodes will be used for cervical vagal stimulation. these adapted electrodes will also be used in the active control group with axillary nerve stimulation ( figure 2 ). since efferent fibers of the vagus nerve controlling cardiac functions are mainly located in the right branch [51] , but no arrhythmic effects have been reported after left transcutaneous vagus nerve stimulation [52] , tvns will be applied over the left branch for safety reasons. for sham auricular tvns, the anodal electrode will be placed over the center of the left ear lobe as previously reported [53] , because there is no vagal innervation around the ear lobe [54] and stimulation of this area does not alter brain activity, including the brain stem [55] . the cathode will be placed over the antitragus. (figure 2a ). accordingly, in the sham cervical tvsn protocol, the anodal electrode will be placed 2 cm anterior to the cervical branch of the vagus nerve, and the cathode below the cheek ( figure 2b ). this method and these stimulation parameters have been previously used for reliable blinding in other clinical studies [56] . series of electrical pulses with 250 µs pulse width, 25 hz frequency, and 28 s inter-burst interval (32 s on/28 s off duty cycle) will be applied in each intervention session to induce effective vagal stimulation and avoid stimulation habituation. auricular tvns will be applied at 1ma intensity for 30 min per session (1 session per day, 5 consecutive days a week, 20 sessions in total). in order to reach equivalent current density as for the auricular electrodes, which include a maximum contact area of 1 cm 2 , the stimulation intensity for non-auricular electrodes (2 cm 2 ) will be adjusted to 2 ma, which will allow achieving comparable current densities at the electrode-skin interface for the respective electrode sizes. the other non-auricular stimulation parameters will remain the same as for the auricular stimulation protocol. auricular vagal stimulation will be performed by the tvns ® l device (tvns technologies gmbh, erlangen, germany, https://u.pcloud.link/publink/show?code=kz0myfxzrxxqqurvy9sb8okpgtac55ax7nrx, accessed on 28 december 2021). tvns ® l is a battery-driven electrical stimulator connected to an ear electrode which is positioned over the skin of the cymba conchae ( figure 1 ). thus, auricular vagal stimulation will be applied using the typical ear electrodes of the tvns ® l device. a technical variant of this device, in which the original connector is modified to incorporate selfadhesive electrodes, will be used for cervical vagal stimulation and axillary nerve stimulation (the latter as an added control group). thus, stimulation over the cervical branch of the vagus nerve and axillary nerve stimulation will be applied by adapted electrodes for transcutaneous stimulation outside the ear (figure 1 ). in order to effectively access cervical vagal projections, as reported in previous studies [36] , 2 cm 2 self-adhesive anodal and cathodal electrodes will be used for cervical vagal stimulation. these adapted electrodes will also be used in the active control group with axillary nerve stimulation ( figure 2 ). since efferent fibers of the vagus nerve controlling cardiac functions are mainly located in the right branch [51] , but no arrhythmic effects have been reported after left transcutaneous vagus nerve stimulation [52] , tvns will be applied over the left branch for safety reasons. for sham auricular tvns, the anodal electrode will be placed over the center of the left ear lobe as previously reported [53] , because there is no vagal innervation around the ear lobe [54] and stimulation of this area does not alter brain activity, including the brain stem [55] . the cathode will be placed over the antitragus. (figure 2a ). accordingly, in the sham cervical tvsn protocol, the anodal electrode will be placed 2 cm anterior to the cervical branch of the vagus nerve, and the cathode below the cheek ( figure 2b ). this method and these stimulation parameters have been previously used for reliable blinding in other clinical studies [56] . series of electrical pulses with 250 μs pulse width, 25 hz frequency, and 28 s inter-burst interval (32 s on/28 s off duty cycle) will be applied in each intervention session to induce effective vagal stimulation and avoid stimulation habituation. auricular tvns will be applied at 1ma intensity for 30 min per session (1 session per day, 5 consecutive days a week, 20 sessions in total). in order to reach equivalent current density as for the auricular electrodes, which include a maximum contact area of 1 cm 2 , the stimulation intensity for non-auricular electrodes (2 cm 2 ) will be adjusted to 2ma, which will allow achieving comparable current densities at the electrode-skin interface for the respective electrode sizes. the other non-auricular stimulation parameters will remain the same as for the auricular stimulation protocol. the intensity and magnitude of the baseline symptoms and primary (pain) and secondary (fatigue and health-related quality of life, sleep disorders, depression, physiological measures) outcome measures will be recorded by a validated spanish version of the brief pain inventory (bpi) [57] (pain intensity measure), the patients' global impression of change (pgic) [58] (restricted to pain intensity changes), a validated spanish version of the revised fibromyalgia impact questionnaire (fiqr) [59] (fatigue and health-related quality of life, as a secondary measure of general symptom improvement), the pittsburgh sleep quality index (psqi) [60, 61] (sleep measure), and the beck depression inventory (bdi) [62] [63] [64] [65] (depression measure). additionally, heart rate variability (the high/low heartbeat ratio as an index of cardiac vagal regulation) and pro-inflammatory cytokine levels (tnf α, il-1β, il-6) will be recorded as secondary physiological outcome measures. heart rate variability will serve as an indirect marker of the efficacy of vagal neuromodulation, at the level of its autonomic effects. the tvns effects on fibromyalgia symptoms will be evaluated in a randomized, prospective, double-blind, sham-controlled, and parallel group clinical trial, with a mixed between-group and within-group design. the data of the recruited participants will be included in a secure database, and a code per group for randomization will be generated. to allocate subjects to each group with complete randomness and independence regarding the intervention, simple randomization will be applied [66] , by which the participant codes will be assigned to the respective groups via a 1:1 randomization procedure (equal allocation) [67, 68] using the computer spreadsheet microsoft excel. the patients will be recruited according to the inclusion and exclusion criteria. then, only patients without alterations in the electrocardiogram will be randomly assigned to the intensity and magnitude of the baseline symptoms and primary (pain) and secondary (fatigue and health-related quality of life, sleep disorders, depression, physiological measures) outcome measures will be recorded by a validated spanish version of the brief pain inventory (bpi) [57] (pain intensity measure), the patients' global impression of change (pgic) [58] (restricted to pain intensity changes), a validated spanish version of the revised fibromyalgia impact questionnaire (fiqr) [59] (fatigue and health-related quality of life, as a secondary measure of general symptom improvement), the pittsburgh sleep quality index (psqi) [60, 61] (sleep measure), and the beck depression inventory (bdi) [62] [63] [64] [65] (depression measure). additionally, heart rate variability (the high/low heartbeat ratio as an index of cardiac vagal regulation) and pro-inflammatory cytokine levels (tnf α, il-1β, il-6) will be recorded as secondary physiological outcome measures. heart rate variability will serve as an indirect marker of the efficacy of vagal neuromodulation, at the level of its autonomic effects. the tvns effects on fibromyalgia symptoms will be evaluated in a randomized, prospective, double-blind, sham-controlled, and parallel group clinical trial, with a mixed between-group and within-group design. the data of the recruited participants will be included in a secure database, and a code per group for randomization will be generated. to allocate subjects to each group with complete randomness and independence regarding the intervention, simple randomization will be applied [66] , by which the participant codes will be assigned to the respective groups via a 1:1 randomization procedure (equal allocation) [67, 68] using the computer spreadsheet microsoft excel. the patients will be recruited according to the inclusion and exclusion criteria. then, only patients without alterations in the electrocardiogram will be randomly assigned to one of five groups: auricular vagus nerve stimulation at 25 hz frequency (atvns) (n = 34); sham auricular vagus nerve stimulation (sham atvns) (n = 17); cervical vagus nerve stimulation at 25 hz frequency (ctvns) (n = 34); sham cervical vagus nerve stimulation (sham ctvns) (n = 17); axillary nerve stimulation at 25 hz frequency (ans) (n = 34). pre-intervention baseline measures (fiqr, bpi, psqi, and bdi) will be performed once a week for 3 weeks and before the first intervention day, resulting in four baseline measures (one per week) to evaluate the stability of the symptoms. then, each participant will receive five 30 min consecutive sessions of stimulation (verum or sham) per week (one per day), for four weeks (in total, 20 sessions) over the left branch of the auricular or cervical vagal nerve or over the left axillary nerve, depending on the group. the patients will not be aware of the stimulation condition (verum vs. sham) within each group, and data analyses will be conducted by researchers not involved in the respective interventions, who will use numerical codes for each experimental condition in order to assure double blinding. once the analyses have been conducted, the codes will be revealed. in the atvns verum and sham groups, the anodal electrode will be positioned over the cymba conchae of the left auricle and the center of the left ear lobe, respectively, and the cathode (return electrode) over the antitragus. in the ctvns verum group, the anodal electrode will be positioned on the surface of the throat that corresponds to the position of the left cervical branch of the vagus nerve, adjacent to the position of the cervical carotid artery (2 cm anterior to this branch for the sham ctvns condition), and the cathode over the lower cheek. in the ans verum group, the anode will be positioned over the left axillary nerve under the shoulder, and the cathode over the lower cheek. the positions of the electrodes for the respective procedures are shown in figure 2 . the post-baseline outcome measures (fiqr, bpi, pgic, psqi, and bdi) will be recorded immediately after the fifth session of each intervention week, resulting in four intra-intervention measurements. four additional post-intervention follow-up measures will be recorded to evaluate potential short-and long-lasting treatment effects, a week, a month, 3, and 6 months after the last intervention session, resulting in four intraintervention and four post-intervention measurements. although no conclusive evidence about tvns effects on heart rate variability is available [69] , heart frequency will be recorded before and after each tvns session, the high-frequency/low-frequency heartbeat ratio will be calculated, and data will be correlated with the outcome measures. pro-inflammatory cytokine levels taken from blood samples before the start of the intervention and immediately after 10 and 20 intervention sessions, as well as 1 week, and 1 month after the end of the intervention, will be analyzed as proposed biomarkers of the anti-inflammatory effects of the intervention [70] . to measure plasma cytokine levels, the enzyme-linked immunosorbent assay (elisa) protocol will be used [71] . plasma collection will be selected to optimize the yield of cytokines of interest (tnf α, il-1β, il-6) [72] . within 1 h after collection, blood will be centrifuged for 10 min at 1300 g and 4 • c, and the supernatant will be transferred to a separate tube and then centrifuged for 10 min at 16,000 g and 4 • c. the resulting platelet-poor plasma will be stored in 0.5 ml aliquots at low temperatures (−80 • c) in order to maintain cytokine stability until the analysis. after each intervention, participants will be asked to guess the stimulation condition (real, sham, or unable to guess) for evaluation of successful blinding as previously reported [73] , and any adverse effect of the stimulation or any aspect related to tvns will be evaluated via an adapted standard questionnaire for assessment of adverse effects induced by a related non-invasive brain stimulation tool, namely, transcranial direct current stimulation (tdcs) [74] , as a measure of the safety of the intervention. all sessions will take place at the aforementioned hospital. figure 3 depicts the design of the study. figure 3 . study design. measurements refer to primary and secondary outcome measures (fiqr, bpi, pgic, psqi, and bdi scores). heart rate variability will be recorded before and after each tvns session, and pro-inflammatory cytokine levels (tnf α, il-1 β, il-6) will be analyzed before the start of the intervention and after 10 and 20 intervention sessions, as well as 1 week, and 1 month after the end of the intervention. ans, axillary nerve stimulation at 25 hz frequency; atvns, auricular vagus nerve stimulation at 25 hz frequency; ctvns, cervical vagus nerve stimulation at 25 hz frequency; sham atvns, auricular vagus nerve stimulation at 1 hz frequency; sham ctvns, cervical vagus nerve stimulation at 1 hz frequency. to exclude differences of baseline measurements (bpi scores as primary outcome measure, and fiqr, pgic, psqi, and bdi scores as secondary outcome measures) between groups, a mixed model 4 × 4 anova will be used, with one between-group factor with four levels (4 groups), and one within-subject factor (four baseline measurements). a mixed model 4 × 8 anova, with one between-group factor with four levels (four groups) and one within-subject factor (8 measurement days), will be used to analyze the scores of the outcome measures obtained in each of the questionnaires throughout the study (intra-intervention and follow-up period). in case of significant results of the ano-vas, exploratory bonferroni-corrected post-hoc t-tests will be conducted. a univariate analysis by a chi-square test will be performed to explore the success of blinding with regard to the intervention, and t-tests as well as chi-square tests will be conducted to exclude demographic differences (gender, age, and years of education) between groups. pearson's correlation coefficient (pearson's r) will be calculated for each tvns group to analyze possible correlations between vagal activity measures (heart rate variability) during the intervention and questionnaire scores (fiqr, bpi, pgic, psqi, and bdi), as well as between pre-and post-intervention pro-inflammatory cytokines levels and outcome measures. the critical level of significance for all statistical analyses will be set to p < 0.05. the analyses will be carried out using the spss statistical software package. this study aims to explore the effects of multiple tvns sessions on fibromyalgia symptoms. because the vagus nerve modulates afferent pain signals to the central nervous figure 3 . study design. measurements refer to primary and secondary outcome measures (fiqr, bpi, pgic, psqi, and bdi scores). heart rate variability will be recorded before and after each tvns session, and pro-inflammatory cytokine levels (tnf α, il-1 β, il-6) will be analyzed before the start of the intervention and after 10 and 20 intervention sessions, as well as 1 week, and 1 month after the end of the intervention. ans, axillary nerve stimulation at 25 hz frequency; atvns, auricular vagus nerve stimulation at 25 hz frequency; ctvns, cervical vagus nerve stimulation at 25 hz frequency; sham atvns, auricular vagus nerve stimulation at 1 hz frequency; sham ctvns, cervical vagus nerve stimulation at 1 hz frequency. to exclude differences of baseline measurements (bpi scores as primary outcome measure, and fiqr, pgic, psqi, and bdi scores as secondary outcome measures) between groups, a mixed model 4 × 4 anova will be used, with one between-group factor with four levels (4 groups), and one within-subject factor (four baseline measurements). a mixed model 4 × 8 anova, with one between-group factor with four levels (four groups) and one within-subject factor (8 measurement days), will be used to analyze the scores of the outcome measures obtained in each of the questionnaires throughout the study (intra-intervention and follow-up period). in case of significant results of the anovas, exploratory bonferroni-corrected post-hoc t-tests will be conducted. a univariate analysis by a chi-square test will be performed to explore the success of blinding with regard to the intervention, and t-tests as well as chi-square tests will be conducted to exclude demographic differences (gender, age, and years of education) between groups. pearson's correlation coefficient (pearson's r) will be calculated for each tvns group to analyze possible correlations between vagal activity measures (heart rate variability) during the intervention and questionnaire scores (fiqr, bpi, pgic, psqi, and bdi), as well as between pre-and post-intervention pro-inflammatory cytokines levels and outcome measures. the critical level of significance for all statistical analyses will be set to p < 0.05. the analyses will be carried out using the spss statistical software package. this study aims to explore the effects of multiple tvns sessions on fibromyalgia symptoms. because the vagus nerve modulates afferent pain signals to the central nervous system and the tone of the autonomic, immune, and emotional systems [19] , the application of tvns, a non-invasive low-intensity electric stimulation technique with potential to acti-vate a-and b-fibers [75] , might normalize the functions of vagal afferences, which seem to be pathologically altered in fibromyalgia [25] , and thus reduce the characteristic symptoms of this disease, particularly muscular and generalized pain. encouraging results have been reported for invasive vagal stimulation procedures in fibromyalgia treatment [31] , but controlled clinical trials using non-invasive vagal stimulation are missing with respect to the effectiveness and safety of this neuromodulation option. the proposed study might shed light on the involvement of vagal activity and sympathetic-vagal balance in chronic pain and other symptoms of patients with fibromyalgia and also provide direct comparisons between two tvns protocols in terms of effectiveness and safety. in particular, we hypothesize that this protocol of vagal neuromodulation is effective and safe for improving pain and other symptoms in these patients. the inclusion of a sham intervention condition is important to demonstrate the specificity of the therapeutic effectivity of this intervention. the inclusion of an active control condition, in which a nerve not related to fibromyalgia symptoms (the axillary nerve) is stimulated, will deliver further evidence about the specificity of the effects. the high number of intervention sessions (20 sessions, over 4 weeks) included in this stimulation protocol might serve to control for null effects of tvns due to under-dosing. the results of this intervention will help to elucidate if tvns is a feasible method to induce clinically relevant analgesic effects in fibromyalgia patients (primary outcome measure). in addition, considering that the vagus nerve controls the autonomic nervous system and inflammatory signals [18] [19] [20] , whose activity is apparently altered in fibromyalgia patients [25, 27] , other symptoms, including sleep and mood disorders, and quality of life (secondary outcome measures) could also benefit from this intervention. however, this study might face some limitations. first, high inter-individual variability in the fibromyalgia symptoms [76] might make it difficult to evaluate the efficacy of the treatment, despite the control of the baseline symptom state. future multi-center studies with highly representative samples might be required to support the validity of the results of the envisaged pilot study. preliminary results accomplished by this study may also be extended in future studies by the inclusion of other groups with chronic pain not primarily related to disturbances of the autonomic or immune systems, such as, for example, post-stroke pain, in which muscle pain symptoms are due to central alterations. we have described a non-invasive vagal nerve stimulation protocol aimed to ameliorate chronic pain and improve quality of life in fibromyalgia patients. the results of this study will also provide information about the effectiveness and safety of two different vagal nerve stimulation protocols. in addition, this study protocol will allow to attribute a therapeutic effect of tvns to the specific neuromodulation of the vagus nerve because of the inclusion of a control group with non-vagal stimulation. preliminary findings from this pilot study will provide valuable information to design future clinical trials and systematic studies to optimize the therapeutic outcomes of tvns. institutional review board statement: the study will be conducted in accordance with the declaration of helsinki, and submitted to approval by the regional ethics committee for clinical studies. in accordance with national legislation (93/42/eec and rd 1090/2015), the study will be pre-registered in the spanish registry of clinical studies. informed consent statement: informed consent will be obtained from all subjects involved in the study. data availability statement: not applicable. a critical and comprehensive review an update on clinical characteristics, aetiopathogenesis and treatment fibromyalgia pathogenesis and treatment options update recent advances in diagnosis, classification, pharmacotherapy and alternative remedies transcutaneous electrical nerve stimulation (tens) for fibromyalgia in adults. in cochrane database of systematic reviews; the cochrane collaboration human vagus nerve branching in the cervical region noninvasive vagus nerve stimulation alters neural response and physiological autonomic tone to noxious thermal challenge modulation of the innate immune response through the vagus nerve the vagus nerve is a causal element in controlling recognition of other's emotions the vagus nerve and the inflammatory reflex: wandering on a new treatment paradigm for systemic inflammation and sepsis vagus nerve afferent stimulation: projection into the brain, reflexive physiological, perceptual, and behavioral responses, and clinical relevance mental health during the covid-19 pandemic and beyond: the importance of the vagus nerve for biopsychosocial resilience theoretical basis of vagus nerve stimulation vagus nerve and vagus nerve stimulation, a comprehensive review: part ii spotlight on cervical vagus nerve stimulation for the treatment of primary headache disorders: a review impact of transcutaneous auricular vagus nerve stimulation on large-scale functional brain networks: from local to global transcutaneous auricular vagus nerve stimulation induces stabilizing modifications in large-scale functional brain networks: towards understanding the effects of tavns in subjects with epilepsy vagus nerve controls resolution and pro-resolving mediators of inflammation vagus nerve and vagus nerve stimulation, a comprehensive review: part i: headache vagus nerve and vagus nerve stimulation, a comprehensive review: part iii autonomic nervous system profile in fibromyalgia patients and its modulation by exercise: a mini review autonomic nervous system derangement in fibromyalgia syndrome and related disorders the plot thickens hypothalamic-pituitary-adrenal and autonomic nervous system functioning in fibromyalgia sympathetic nervous system dysfunction in fibromyalgia, chronic fatigue syndrome, irritable bowel syndrome, and interstitial cystitis: a review of case-control studies a cross-sectional study on central sensitization and autonomic changes in fibromyalgia a comprehensive study of autonomic dysfunction in the fibromyalgia patients role of inflammation in the pathogenesis and treatment of fibromyalgia neurogenic inflammation in fibromyalgia autoinflammation and immunomodulation in inflammatory fibromyalgia syndrome-a review safety and efficacy of vagus nerve stimulation in fibromyalgia: a phase i/ii proof of concept trial low intensity transcranial electric stimulation: safety, ethical, legal regulatory and application guidelines basic and functional effects of transcranial electrical stimulation (tes)-an introduction the anatomical basis for transcutaneous auricular vagus nerve stimulation mechanisms underpinning sympathetic nervous activity and its modulation using transcutaneous vagus nerve stimulation access to vagal projections via cutaneous electrical stimulation of the neck: fmri evidence in healthy humans optimization of transcutaneous vagus nerve stimulation using functional mri: transcu-taneous vns optimization using fmri auricular vagus neuromodulation-a systematic review on quality of evidence and clinical effects a review of parameter settings for invasive and non-invasive vagus nerve stimulation (vns) applied in neurological and psychiatric disorders transcutaneous vagus nerve stimulation in the treatment of drug-resistant epilepsy one year in review a review of vagus nerve stimulation as a therapeutic intervention the impact of auricular vagus nerve stimulation on pain and life quality in patients with fibromyalgia syndrome non-invasive vagus nerve stimulation in healthy humans reduces sympathetic nerve activity transcutaneous auricular vagus nerve stimulation critical review of transcutaneous vagus nerve stimulation: challenges for translation to clinical practice transcutaneous vagus nerve stimulation in patients with attention-deficit/hyperactivity disorder: a viable option? international consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (version 2020) size determination and power analysis using the g*power software vns therapy in treatment-resistant depression: clinical evidence and putative neurobiological mechanisms transcutaneous vagus nerve stimulation: retrospective assessment of cardiac safety in a pilot study bold fmri deactivation of limbic and temporal brain structures and mood enhancing effect by transcutaneous vagus nerve stimulation the nerve supply of the human auricle cns bold fmri effects of sham-controlled transcutaneous electrical nerve stimulation in the left outer auditory canal-a pilot study treatment of chronic migraine with transcutaneous stimulation of the auricular branch of the vagal nerve (auricular t-vns): a randomized, monocentric clinical trial validación española del cuestionario brief pain inventory en pacientes con dolor de causa neoplásica patients' global impression of change in the management of peripheral neuropathic pain: clinical relevance and correlations in daily practice validation of a spanish version of the revised fibromyalgia impact questionnaire (fiqr). health qual the pittsburgh sleep quality index: a new instrument for psychiatric practice and research reliability and validity of the spanish version of the pittsburgh sleep quality index (psqi) in patients with fibromyalgia on the validity of the beck depression inventory spanish adaptation of the inventory of depression and anxiety symptoms (idas-ii) and a study of its psychometric properties psychometric properties of the spanish beck depression inventory-ii in a medical sample psychometric evaluation of the beck depression inventory-ii with primary care medical patients randomization in clinical studies the questionable use of unequal allocation in confirmatory trials a roadmap to using randomization in clinical trials does transcutaneous auricular vagus nerve stimulation affect vagally mediated heart rate variability? a living and interactive bayesian meta-analysis transcutaneous cervical vagal nerve stimulation modulates cardiac vagal tone and tumor necrosis factor-alpha cytokine assays: an assessment of the preparation and treatment of blood and tissue samples comparison of multiplex cytokine assays in a pediatric cohort with epilepsy tolerability and blinding of 4x1 high-definition transcranial direct current stimulation (hd-tdcs) at two and three milliamps a systematic review on reporting and assessment of adverse effects associated with transcranial direct current stimulation high-resolution multi-scale computational model for non-invasive cervical vagus nerve stimulation: high-resolution multi-scale model for nvns conflicts of interest: michael a. nitsche is member of the scientific advisory boards of neuroelectrics and neurodevice. armin bolz is ceo and owner of tvns technologies gmbh, erlangen. the other authors declare that they have no conflict of interest. key: cord-0074606-q4kko9sm authors: soós, boglárka; fagyas, miklós; horváth, ágnes; végh, edit; pusztai, anita; czókolyová, monika; csongrádi, alexandra; hamar, attila; pethő, zsófia; bodnár, nóra; kerekes, györgy; hodosi, katalin; szekanecz, éva; szamosi, szilvia; szántó, sándor; szűcs, gabriella; papp, zoltán; szekanecz, zoltán title: angiotensin converting enzyme activity in anti-tnf-treated rheumatoid arthritis and ankylosing spondylitis patients date: 2022-01-27 journal: front med (lausanne) doi: 10.3389/fmed.2021.785744 sha: 45db4cb52c4333f82c05cde7dfbe5dd733113a02 doc_id: 74606 cord_uid: q4kko9sm introduction: angiotensin-converting enzyme (ace) and ace2 have been implicated in the regulation of vascular physiology. elevated synovial and decreased or normal ace or ace2 levels have been found in rheumatoid arthritis (ra). very little is known about the effects of tumor necrosis factor α (tnf-α) inhibition on ace or ace2 homeostasis. in this study, we assessed the effects of one-year anti-tnf therapy on ace and ace2 production in ra and ankylosing spondylitis (as) in association with other biomarkers. patients and methods: forty patients including 24 ra patients treated with either etanercept (etn) or certolizumab pegol (czp) and 16 as patients treated with etn were included in a 12-month follow-up study. serum ace levels were determined by commercial elisa, while serum ace2 activity was assessed using a specific quenched fluorescent substrate. ultrasonography was performed to determine flow-mediated vasodilation (fmd), common carotid intima-media thickness (ccimt) and arterial pulse-wave velocity (pwv) in all patients. in addition, crp, rheumatoid factor (rf) and acpa were also measured. all assessments were performed at baseline and 6 and 12 months after treatment initiation. results: anti-tnf therapy increased ace levels in the full cohort, as well as in the ra and as subsets. ace2 activity increased in the full cohort, while the ace/ace2 ratio increased in the full cohort and in the ra subset (p < 0.05). uniand multivariable regression analyses determined associations between ace or ace/ace2 ratios at different time points and disease duration, crp, rf, fmd and imt (p < 0.05). ace2 activity correlated with crp. the changes of ace or ace2 over 12 months were determined by treatment together with either rf or fmd (p < 0.05). conclusions: anti-tnf treatment may increase ace and ace2 in the sera of ra and as patients. ace and ace2 may be associated with disease duration, markers of inflammation and vascular pathophysiology. the effects of tnf inhibition on ace and ace2 may reflect, in part, the effects of these biologics on the cardiovascular system. rheumatoid arthritis (ra) and ankylosing spondylitis (as) have been associated with increased cardiovascular (cv) morbidity and mortality (1) (2) (3) (4) (5) . non-invasive ultrasound-based techniques are suitable to assess preclinical vascular pathophysiology in ra and as (1, 6) . early endothelial dysfunction of the brachial artery, carotid atherosclerosis and increased arterial stiffness are indicated by abnormal endothelium-dependent, flow-mediated vasodilation (fmd) (4, 6, 7) , common carotid intima-media thickness (imt) and carotid plaques (4) (5) (6) 8) , as well as arterial pulse-wave velocity (pwv) (5, 6, 9) , respectively. systemic inflammation and pro-inflammatory cytokines including tumor necrosis factor α (tnf-α) are involved in the pathogenesis of arthritis-associated secondary atherosclerosis and cv disease (10, 11) . anti-tnf agents are effective and safe in the therapy of ra and as (12) (13) (14) (15) (16) . tnf inhibitors also suppress synovial angiogenesis and vascular endothelial growth factor (vegf) production (17, 18) . the control of inflammation by targeted therapies including tnf-α inhibitors, may decrease cv morbidity and mortality in arthritides (1, 10, 14, 19) , especially in anti-tnf responder patients (14, 20) . anti-tnf biologics may improve or at least stabilize vascular physiology indicated by fmd, imt and pwv (14, 19, (21) (22) (23) (24) . angiotensin-converting enzyme (ace) is a member of the renin-angiotensin-aldosterone system (raas), which is an important regulator of blood pressure and salt-water homeostasis (25) . ace catalyzes the conversion of angiotensin i to angiotensin ii, and the metabolism of bradykinin (25) . ace has been implicated in cv disease, myocardial infarction, hypertension, heart failure and diabetic nephropathy (25, 26) . ace inhibitors are among the most frequently prescribed drugs with antihypertensive and cardioprotective effects (25, 26) . ace2 is an ace homolog with monocarboxypeptidase activity (27) . ace2 generates angiotensin peptides ang 1−9 and ang 1−7 from ang-i and ang-ii, respectively (27, 28) . ace2, through ang 1−7 , is capable of reducing myocardial oxidative stress and pathological remodeling (28, 29) . tnf-α converting enzyme (tace/adam17) is responsible for ace2 shedding from cardiomyocytes and endothelial cells (30) . thus, opposite to the ang-i-ace-ang-ii pathway, ace2 exerts vasculoprotective and antihypertensive mechanisms by the counter-regulation of the raas system (28, 31) . increased soluble ace2 activity has been associated with advanced heart failure (32), ventricular arrhythmias (33) and hypertension (28) . ace and ace2 concentrations and activity are readily measurable in the serum. ace activity is affected by the intake of ace inhibitors while ace2 activity is not (26, 28) . changes in soluble ace and ace2 may reflect redistribution and opposite changes of tissue activity of these enzymes (28, 34) . with respect to ace and ace2 in arthritides, ace insertiondeletion (i/d) gene polymorphism has been associated with ra and as in some populations, primarily in cohort studies carried out in arabic countries (35) (36) (37) (38) (39) (40) . when studying dd, id and ii genotypes, the frequency of the d allele was higher in ra compared to healthy controls (35) . moreover, the dd genotype may confer increased susceptibility to ra (35, 38, 40) . results in as are controversial. in one study, similarly to ra, the dd genotype has been associated with as including sacroiliac and ocular involvement (39) . moreover, in as, carrying the d allele was correlated with higher crp levels (41) . in contrast, another study reported association between the i allele and as (42) . the ii genotype was also associated with juvenile idiopathic arthritis (37) . ace polymorphisms could not be correlated with psoriatic arthritis (psa) (36) . regarding serum or plasma ace and ace2 levels, the very first study on ace serum and synovial fluid ace levels in various arthritides was published as early as in 1986. in this study, serum ace levels were similar in ra, as, psa, osteoarthritis (oa) patients and healthy controls. on the other hand, synovial fluid ace levels were increased in ra vs. oa (43) . more recently, 50 ra compared to 30 healthy women, ra patients had increased ang-ii, ang 1−7 and ace plasma levels, as well as ace/ace2 ratios vs. controls. ra was associated with lower ang-ii/ang 1−7 ratios. ace inhibitors did not significantly influence serum ang-ii, ang 1−7 , ace and ace2 levels in ra patients. ace2 levels inversely correlated with carotid imt (44) . decreased ace2 levels were also found in ra, systemic sclerosis (ssc) and systemic lupus erythematosus (sle) vs. healthy controls (45) . in contrast, other studies found similar serum ace levels in ra, oa and healthy individuals (46) . increased synovial fluid ace concentrations have been described in ra compared to oa (46, 47) . within the ra synovial tissue, ace expression is localized to endothelial cells and synovial macrophages (48) . with respect to the regulation of ace and ace2 production in arthritides, in animal models of arthritis, ang 1−7 exerted significant anti-inflammatory effects as it attenuated oxidative stress, as well as tnf-α, interleukin 1 (il-1) and il-6 production (49) . moreover, il-6 upregulated ace2 in synovial tissues (50) . anti-ace2 antibodies that inhibit the anti-inflammatory and anti-fibrotic effects of ace2 have been described in connective tissue diseases with constrictive vasculopathies, such as ssc, sle and mixed connective tissue disease (mctd) (51, 52) . therapeutically, both ace inhibitors and angiotensin receptor blockers (arb) may exert significant anti-inflammatory effects (53) . however, in the multiple regression analysis of ra patients receiving ace inhibitors or arbs in comparison to those not taking such drugs, the use of either ace inhibitors or arbs was not associated with disease activity (54) . there have been very few studies on the possible effects of tnf inhibitors on ace and ace2. in a small study, ace2 plasma levels were significantly lower in ra patients on anti-tnf treatment compared to healthy controls (55) . in the study of ra, ssc and sle patients, most antirheumatic treatments did not affect ace2 levels (45) . we have not found any studies where changes in ace or ace2 levels or activity were evaluated upon anti-tnf therapy. thus, ace and ace2 may be involved in the inflammatory processes underlying ra and as, as well as in vascular pathology associated with these arthritides. yet, very little information has become available on the effects of anti-tnf therapy on ace and ace2 production and on their correlation with disease activity, markers of inflammation, autoantibodies and vascular pathophysiology. we have recently set up a mixed cohort of ra and as patients and reported multiple effects of anti-tnf treatment over one year on vascular pathophysiology (19) and various vascular biomarkers (56, 57) . we have published data on vascular pathophysiology and bone in the very same cohort before (19, 56, (58) (59) (60) . as a novelty in comparison to the previous publications of the same cohort, now we wished to study ace and ace2 production in context with inflammation, autoantibodies and vascular pathophysiology in the very same cohort in order to obtain more information on the possible effects of biologics on the raas. fifty one patients with inflammatory arthritis (35 ra and 16 axial radiographic as) selected for the initiation of anti-tnf therapy but unselected for cv disease (any previous cv events) were enrolled in the study as described before (19, 56, 57) . these patients were consecutively selected in one tertiary rheumatology center (university of debrecen). patient characteristics in the full, ra and as cohorts are seen in table 1 . the full cohort included 33 women and 18 men with mean age of 51.4 ± 11.8 (range: 24-83) years, while mean age at diagnosis was 43.1 ± 10.8 (range: 11-71) years. the mean disease duration was 8.3 ± 7.6 (range: 1-44) years. exclusion criteria included untreated, unstable hypertension (blood pressure >140/90 mmhg), current inflammatory disease other than ra or as, infectious disease or renal failure (based on egfr and hospital records). none of patients received antiplatelet (e.g., aspirin, clopidogrel) or anticoagulant therapy (e.g., heparin, warfarin) at the time of inclusion. as antihypertensive drugs may affect the vascular status, hypertension had been stabilized for at least 6 months before the onset of this study. moreover, antihypertensive drugs remained unchanged throughout the study. some patients received ace inhibitors prior to the study. however, we have previously demonstrated that ace inhibitor treatment does not have any effects on circulating ace concentration or ace2 activity (28) . patients with active disease were recruited prior to initiating a biological therapy. at baseline ra patients had a mean das28 of 4.98 ± 10.86, while as patients exerted mean basdai of 5.94 ± 1.03. all patients started on an anti-tnf therapy at baseline and continued the same biological treatment during one year. among the 35 ra patients, 20 received etanercept (etn) 50 mg/week subcutaneous (sc) and 15 received certolizumab pegol (czp) (400 mg at 0, 2 and 4 weeks, and thereafter 200 mg every two weeks sc). altogether 12 ra patients were treated with etn and eight with czp in combination with methotrexate (mtx). the other patients received anti-tnf monotherapy. ra patients did not take dmards other than mtx. all 16 as patients received etn monotherapy 50 mg/week sc. altogether eight ra and one as patients took low-dose (<6 mg/day) methylprednisolone ( table 1) . the study was approved by the hungarian scientific research council ethical committee (approval no. 14804-2/2011/eku). written informed consent was obtained from each patient and assessments were carried out according to the declaration of helsinki. first, detailed medical history was taken. we inquired for history of cvd, as well as current smoking, experience of chest pain resembling angina pectoris, hypertension and diabetes mellitus during the last 2 years prior to the start of this study by a questionnaire ( table 1) . we also determined body mass index (bmi) and obesity ( table 1) . further clinical assessments including physical examination were performed at baseline (b), and after 6 (6 m) and 12 months (12 m) of therapy. at baseline ra patients had a mean das28 of 4.98±0.86, while as patients exerted mean basdai of 5.94 ± 1.03 ( table 1) . blood samples were collected from patients by using a standard aseptic technique. native blood was incubated for 60 min at room temperature; serum fractions (separated by centrifugation at 1,500 g for 15 min) were stored at −20 • c until further use. serum high sensitivity c reactive protein (hscrp; normal: ≤ 5 mg/l) and igm rheumatoid factor (rf; normal: ≤ 50 iu/ml) were measured by quantitative nephelometry (cobas mira plus-roche), using crp and rf reagents (both dialab). acpa (anti-ccp; accp) autoantibodies were detected in serum samples using a second generation immunoscan-ra ccp2 elisa test (euro diagnostica; normal: ≤ 25 iu/ml). the assay was performed according to the manufacturer's instructions. in order to exclude the possible interference effect of rf, we compared ace concentration and ace2 activity values in rf positive and negative patients at baseline. we did not find statistically significant differences between positive or negative patients, thus presence of rf in the sample may not interfere with the tests (data not shown in figure) . serum ace concentration was determined by a commercial human ace elisa development kit (r&d systems) according to the manufacturer's instructions, with minor modifications, as described previously (61) . enzyme-linked immunosorbent plates (greiner bio-one) were coated with 80 ng/well capture antibody, and the remaining binding sites were then blocked with reagent diluent (10 mg/ml bovine serum albumin [sigma-aldrich] in dulbecco's phosphate buffered saline solution [pbs, gibco]). diluted sera (in reagent diluent, 100-fold dilution) were added to the wells, and the antibody-antigen complexes were labeled with a biotinylated detection antibody (20 ng/well). two hundredfold-diluted streptavidin-conjugated horseradish-peroxidase (kit component) was added to the wells. finally, the amounts of complexes were detected with a substrate solution containing 0.3 mg/ml tetramethylbenzidine, 0.1 mm h 2 o 2 and 50 mm acetic acid. the reaction was terminated after 20 min by the addition of 0.5 m hcl, and the optical density was measured at 450 nm. serum ace concentration rather than activity was measured as 11 ra patients had been receiving ace inhibitor treatment ( table 1) . ace levels are expressed in ng/ml units. serum ace2 activity was determined using a specific quenched fluorescent substrate as previously described (28) one unit of fluorescence (uf) corresponds to the quantity of enzyme which can degrade 0.1 nmol mca-apk(dnp) in one h at 37 • c. the specificity of the serum ace2 enzyme activity assay was tested using the specific human ace2 inhibitor dx600 before (28) . ace2 activity is expressed in uf/ml units. ace inhibitors do not interfere with ace2 activity. the fmd, imt and pwv assessments carried out in the very same cohort were performed and published previously (19) . statistical analysis was performed using spss version 22 separate vascular imaging, as well as disease activity, inflammatory and vascular biomarker data obtained in the very same cohort have been published (19, 56, 57) . here we used those data to associate them with the ace and ace2 measurements. none of the data presented here have been published elsewhere. in (figure 1a) . ace levels in ra and as did not differ at b (p = 0.055). on the other hand, ace concentration was significantly higher in ra compared to as after 6 m (p = 0.004) and 12 m of treatment (p = 0.024) (figure 1a) . in the full ra+as cohort, ace2 activity significantly increased after 6 m (41.1 uf/ml; p = 0.044) and 12 m (figure 1b) . ace2 activity was higher in as than in ra at b (p = 0.037) and after 12 m (p = 0.019) ( figure 1b) . finally, in order to reflect the ace/ace2 balance, we calculated ratios of ace concentrations and ace2 activity (ace/ace2 ratios) (figure 1c) . in the mixed cohort, ace/ace2 ratio did not change after 6 m (4. (figure 1c) . ace/ace2 ratio was higher in ra than in as at b (p = 0.004), as well as after 6 m (p = 0.001) and 12 m (p = 0.003) (figure 1c ). all results of the simple correlation analysis are seen in supplementary table s1 . this table indicates the full cohort, as well as the ra and as subsets. in the univariable regression analysis of the ra+as cohort, ace levels at various time points were independently and positively associated with rf and fmd and inversely with age, disease duration, crp, rf, imt and fmd (p < 0.05) ( table 2) . ace2 activity was independently determined by crp (p < 0.05) ( table 2) . the ace/ace2 ratio independently correlated with age, disease duration, crp, rf, imt and fmd ( table 2 ). in ra, ace concentrations were only associated with crp, rf and fmd, while ace2 activity did not show any correlations. in ra, ace/ace2 ratio correlated with disease duration, crp, rf and fmd (p < 0.05) ( table 2 ). in as, ace level was independently associated with basdai, while ace2 activity did not show any correlations. ace/ace2 ratio positively associated only with disease duration (p < 0.05) ( table 2) . the multivariable regression analysis confirmed the positive correlation among ace levels and age, crp and imt in the mixed cohort. similarly, ace/ace2 ratios also correlated with age and disease duration (p < 0.05) ( table 2 ). in ra, the only correlation revealed was that between ace levels and fmd (p < 0.05) ( table 2) . no such correlations were observed in as (table 2) . finally, rm-anova analysis was performed in order to assess determinants of ace or ace2 changes over time. in the full cohort, one-year change in ace concentration or in ace/ace2 ratio was determined by anti-tnf treatment together with higher rf or fmd at b (p < 0.05) ( table 3 ). in ra, ace level or ace/ace2 ratio changes were associated with treatment along with higher rf (p < 0.005) ( table 3) . no such associations were observed in as ( table 3) . to our best knowledge, these may be the first data on the effects of one-year anti-tnf therapy on ace level and ace2 activity in arthritis patients. we found that one-year anti-tnf treatment significantly increased ace concentration in the mixed cohort, as well as in the ra and as subset. tnf inhibition also stimulated ace2 activity in the ra+as cohort but not in ra or as. ace/ace2 ratios significantly increased in the mixed cohort and in ra, but not in as. interestingly, ace levels and ace/ace2 ratios were higher in ra vs. as, while ace2 activity values were higher in as vs. ra at most time points. moreover, baseline, 6-and 12-month ace levels, as well as ace/ace2 ratios variably correlated with disease duration, crp, rf and various parameters of vascular pathophysiology. ace2 activity only correlated with crp. we assessed ace levels and ace2 activity. ace activity could not be included in this study as some patients received ace inhibitors ( table 1) , which could interfere with this parameter (26, 28) . ace2 activity is not affected by the use of ace inhibitor treatment (28) . upon tnf-α inhibition, ace concentration increased in the mixed patient cohort throughout the treatment period. ace levels also increased in ra and exerted a late, 12 m increase in as. similarly, anti-tnf therapy increased ace2 activity in the mixed cohort, while ace/ace2 ratios showed late, 12 m increases in the full cohort and in ra. we could not compare our data from those published by others as, to our best knowledge, there have not been any studies evaluating the longitudinal effects of tnf-α inhibition on ace or ace2 levels or activity. in ra, increased synovial fluid ace concentrations (46, 47) and decreased serum ace2 (45) or unchanged serum ace levels have been reported (46) . thus, in ra, there may be a redistribution of ace and ace2 from the serum to the synovium. this may be reversed by anti-tnf treatment, however, this was hypothetical as no prospective studies assessing the effects of tnf inhibition of ace and ace2 redistribution have been conducted. similarly to ra, potdar et al. (34) reported increased expression of colonic ace2 in active ulcerative colitis (uc) and low expression of small bowel ace in crohn's disease (cd) vs. controls. anti-tnf therapy restored ace2 tissue expression by decreasing colonic ace2 in uc and stimulating small bowel ace2 in cd (34) . the redistribution of ace and ace2 between tissue and serum was discussed above (28) . thus, although we did not conduct tissue expression studies, elevated serum ace concentrations and ace2 activity upon one-year anti-tnf treatment may reflect redistribution between tissue and serum of ace and ace2 (28, (45) (46) (47) . the pattern of ace2 changes in our cohort is similar to the uc results reported by potdar et al., however, they only assessed tissue ace2 levels and not serum ace2 activity (34) . in addition, increased synovial fluid ra ace levels were reported in ra compared to controls (43, 46, 47) . the synovial fluid compartment reflects the characteristics of the tissue better than the serum. again, considering the redistribution patterns of ace and ace2 between serum and tissue (28), our results indicating increasing ace levels and ace2 activity upon treatment may reflect decreasing synovial ace and ace2 expression. when comparing ra and as, there was no difference in ace level between the two groups at baseline. on the other hand, after 6 m and 12 m, ace concentrations were higher in ra compared to as suggesting that tnf inhibition might have a more pronounced effect on ace in ra vs. as. with respect to ace2 activity, it was higher in as compared to ra both at b and after 12 m. yet, the difference was greater between as and ra after one-year therapy again suggesting a stronger stimulating effect of anti-tnf agents on ace2 activity in as vs. ra. moreover, one-year anti-tnf treatment resulted in the increase of ace levels in both ra and as. in contrast, ace2 activity was only increased in the full cohort, but not in the ra and as subsets. ace/ace2 ratios were higher in ra compared to as at all time points. there have been no studies on the effects of biologics on ace and ace2 in ra or as, therefore, we cannot compare our data to other reports. the baseline time point represents a pre-treatment status when both ra and as patients had higher inflammatory state and disease activity. after 12 m, as anti-tnf therapy was proven to be clinically effective, most ra and as patients had remission or at least low disease activity (lda). that is why it is important to compare correlations between ace, ace2 or ace/ace2 and other parameters at baseline and after 12 m. in the regression analyses, at baseline ace concentration only correlated with age, rf and imt. however, after one-year treatment it also correlated with crp and fmd. the correlation between 6 and 12 m ace and crp or disease activity was observed in the full cohort (crp), as well as in the ra (crp) and as subsets (basdai). thus, in treated arthritis patients with lower degree of inflammation, ace levels may become a marker of remaining systemic inflammation or disease activity. moreover, high baseline crp and rf levels may predict ace levels after 12 m. increased plasma ace levels were found in some cohorts (44) , while others reported similar ace levels in ra and controls (46) . our study was an uncontrolled, longitudinal therapeutic study comparing postand pre-treatment ace levels. ace2 activity after 6 m positively correlated with baseline crp suggesting that more extensive baseline inflammation may predict higher ace2 activity after 6 m, when inflammation is already dampened. in another study of tang et al. (39) , decreased ace2 levels were found in ra vs. healthy controls. yet, that study included ra patients with mixed characteristics and the study did not assess longitudinal treatment effects (39) . ace/ace2 ratios at baseline exerted correlations with disease duration, rf and imt. again, after 6 and 12 m, ace/ace2 ratios also correlated with crp and fmd, which pattern is similar to that observed with ace discussed above. in the rm-anova analysis, changes of ace levels or ace/ace2 ratios over 12 m were positively associated with baseline rf and fmd in the full cohort and with baseline rf in the ra subset. thus, in ra, high baseline rf may be the most important denominator of ace concentration changes among the disease-related parameters included in this study. our study certainly has strengths and limitations. the strength of this study is that, for the first time, we assessed the effects of biologics on ace and ace2 in arthritides in a prospective manner. in addition, ours is the first study to evaluate ace and ace2 in association with multiple disease-related laboratory markers and those of vascular pathophysiology. possible limitations may include the relatively low patient number and the lack of control groups. in conclusion, anti-tnf treatment may increase ace and ace2 in the sera of ra and as patients, which may reflect the shedding and redistribution of ace and ace2 from the tissue to the blood. baseline ace and ace2 may be associated with disease duration, markers of inflammation (crp), autoimmunity (rf) and vascular pathophysiology (fmd, imt). the effects of tnf inhibition on ace and ace2 release may reflect, in part, the beneficial effects of biologics on vascular pathology. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the study was approved by the hungarian scientific research council ethical committee (approval no. 14804-2/2011/eku). written informed consent was obtained from each patient and assessments were carried out according to the declaration of helsinki. the patients/participants provided their written informed consent to participate in this study. bs: study conceptualization, patient recruitment, data collection, and draft writing. mf, zpa, and ac: ace and ace2 measurements, data analysis, and manuscript drafting. áho, ev, aha, zpe, nb, sszam, and sszán: patient recruitment and examination and data collection. ap and mc: laboratory assessments and data analysis. gk: vascular ultrasound examination and data collection. kh: statistical analysis and interpretation. és: study concept, data interpretation, and draft writing. gs: study concept, draft writing, manuscript finalization, and senior coordinator. zs: study concept, principal investigator, senior coordinator, and manuscript finalization. all authors contributed to the article and approved the submitted version. were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. eular recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update cardiovascular disease in inflammatory rheumatic diseases endothelial dysfunction and atherosclerosis in rheumatoid arthritis: a multiparametric analysis using imaging techniques and laboratory markers of inflammation and autoimmunity assessment of subclinical vascular disease associated with ankylosing spondylitis validated methods for assessment of subclinical atherosclerosis in rheumatology endothelial dysfunction, carotid intima-media thickness, and accelerated atherosclerosis in rheumatoid arthritis carotid intimamedia thickness predicts the development of cardiovascular events in patients with rheumatoid arthritis a comparative study of arterial stiffness, flow-mediated vasodilation of the brachial artery, and the thickness of the carotid artery intima-media in patients with systemic autoimmune diseases tumour necrosis factor antagonist use and associated risk reduction of cardiovascular events among patients with rheumatoid arthritis eular recommendations for the management of rheumatoid arthritis with synthetic and biological diseasemodifying antirheumatic drugs: 2019 update biologics, cardiovascular effects and cancer vascular effects of biologic agents in ra and spondyloarthropathies anti-tnf-alpha inhibitors: a new therapeutic approach for inflammatory immune-mediated diseases: an update upon efficacy and adverse events update upon the infection risk in patients receiving tnf alpha inhibitors effects of tnf-alpha inhibitors upon the mechanisms of action of vegf targeting angiogenesis in rheumatoid arthritis effects of 1-year anti-tnf-alpha therapy on vascular function in rheumatoid arthritis and ankylosing spondylitis reduction in the incidence of myocardial infarction in patients with rheumatoid arthritis who respond to anti-tumor necrosis factor alpha therapy: results from the british society for rheumatology biologics register changes in arterial stiffness during continued infliximab treatment in patients with inflammatory arthropathies response to anti-tumour necrosis factor alpha blockade is associated with reduction of carotid intima-media thickness in patients with active rheumatoid arthritis the effect of treatments for rheumatoid arthritis on endothelial dysfunction evaluated by flow-mediated vasodilation in patients with rheumatoid arthritis tumour necrosis factor blocking agents and progression of subclinical atherosclerosis in patients with ankylosing spondylitis recent advances in knowledge of the structure and function of the angiotensin i converting enzyme new perspectives in the renin-angiotensin-aldosterone system (raas) i: endogenous angiotensin converting enzyme (ace) inhibition a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 new perspectives in the renin-angiotensin-aldosterone system (raas) iv: circulating ace2 as a biomarker of systolic dysfunction in human hypertension and heart failure angiotensin-converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) role of ace2 in diastolic and systolic heart failure soluble angiotensin-converting enzyme 2 in human heart failure: relation with myocardial function and clinical outcomes enzymatic activity of dpiv and renin-angiotensin system (ras) proteases in patients with left ventricular dysfunction and primary prevention implantable cardioverter/defibrillator (icd) altered intestinal ace2 levels are associated with inflammation, severe disease, and response to anti-cytokine therapy in inflammatory bowel disease patterns of angiotensin converting enzyme insertion/deletion gene polymorphism among an egyptian cohort of patients with rheumatoid arthritis angiotensinconverting enzyme gene polymorphism in patients with psoriatic arthritis angiotensin converting enzyme gene insertion-deletion polymorphism is associated with juvenile rheumatoid arthritis genetic polymorphisms of ace i/d, il-1beta g > a and il-4 vntr among egyptian subjects with rheumatoid arthritis significant association between insertion/deletion polymorphism of the angiotensin-convertig enzyme gene and ankylosing spondylitis the angiotensin-converting enzyme insertion/deletion polymorphism and susceptibility to rheumatoid arthritis, vitiligo and psoriasis: a meta-analysis association of gstm1, gstt1, gstp1-ile105val and ace i/d polymorphisms with ankylosing spondylitis association of angiotensin-converting enzyme (ace) gene insertiondeletion polymorphism with spondylarthropathies serum and synovial fluid levels of angiotensin converting enzyme in polyarthritis renin-angiotensin system molecules are associated with subclinical atherosclerosis and disease activity in rheumatoid arthritis decreased serum ace2 levels in patients with connective tissue diseases renin and angiotensin-converting enzyme (ace) as active components of the local synovial renin-angiotensin system in rheumatoid arthritis differential expression of renin-angiotensin system-related components in patients with rheumatoid arthritis and osteoarthritis production of angiotensin converting enzyme by rheumatoid synovial membrane angiotensin-(1-7) attenuates collagen-induced arthritis via inhibiting oxidative stress in rats angiotensin-converting enzyme 2, a sars-cov-2 receptor, is upregulated by interleukin 6 through stat3 signaling in synovial tissues angiotensin-converting enzyme 2 autoantibodies: further evidence for a role of the renin-angiotensin system in inflammation autoantibodies to angiotensinconverting enzyme 2 in patients with connective tissue diseases the potential therapeutic use of renin-angiotensin system inhibitors in the treatment of inflammatory diseases angiotensininhibiting drugs do not impact disease activity in patients with rheumatoid arthritis: a retrospective cross-sectional study anti-tnf-alpha agents modulate sars-cov-2 receptors and increase the risk of infection through notch-1 signaling effects of one-year anti-tnf-α therapy on biomarkers of angiogenesis and functional vascular parameters in arthritides soluble vascular biomarkers in rheumatoid arthritis and ankylosing spondylitis: effects of one-year anti-tnf-alpha therapy peripheral quantitative computed tomography in the assessment of bone mineral density in anti-tnf-treated rheumatoid arthritis and ankylosing spondylitis patients associations of vascular and bone status in arthritis patients effects of 1-year anti-tnf-alpha therapies on bone mineral density and bone biomarkers in rheumatoid arthritis and ankylosing spondylitis new perspectives in the renin-angiotensin-aldosterone system (raas) iii: endogenous inhibition of angiotensin converting enzyme (ace) provides protection against cardiovascular diseases the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.copyright © 2022 soós, fagyas, horváth, végh, pusztai, czókolyová, csongrádi, hamar, pethő, bodnár, kerekes, hodosi, szekanecz, szamosi, szántó, szűcs, papp and szekanecz. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. original article j fac med baghdad vol.. 48 , no. 4 , 2006 442 rheumatiod factor isotype in rheumatiod arthritis patients . nahida r. abbas msc. khalidh m. mousawy ph.d jassim t. khafaji sami salman. summary background: rheumatiod factor in patients with rheumatoid arthritis (ra) could be of igm, igg, or igg isotypes. aim: to study the occurance of each or all these isotypes in ra patients . methods: enzyme linked immune sorbent assay (elisa) & latex agglutination test were used to asses rf in 74 patients with ra. results: using elisa, 58 (78.4%) patients had positive rf (igm 48.6%, igg in 47.3% & iga in 54.1% ), while only 43 (58.1) patients were positive by ag . conclusion all rf isotypes should be assessed in patients with ra. key words : ra, rf, igg, igm, iga, elisa, ag. __________________________________________________________________________________________ introduction :______________________________ ra is a systemic inflammatory disease that predominantly affects synovial membrane of diarthridal joints (1). one of the characteristic features of this disease is the production of autoantibodies against fc portion of immunoglobulin g. these autoantibodies are generally known as rf and may be iga, igg, or igm isotypes and it might be present all three together or only two types or just one. patients and methods patients: seventy four patients (19 male, 55 female) with ra who met the american college of rheumatology (acr)1987revised criteria (2) attending the rheumatology consultation clinic or admitted to baghdad teaching hospital in a period between november 2001 and february 2002. laboratory investigation: rf was detected using the latex test which has been supplied by biokit company, spain, and results were expressed in international unit / ml (iu / ml). the elisa technique used human igg fc as the antigen (sigma, st. louis, mo.) coated the microwells plate and isotype-specific horse antibodies coupled to radish peroxidase; result were expressed as the optical density. a level>20 iu/ ml was considered positive . results the male to female ratio was 1:2.9 with the mean age 42.111.3 as shown in table 1&2. rheumatoid factor fourty three patients were rf positive (58.1%), while the rest (41.9%) were negative these detected by latex agglutination test where 10 iu/ml was the best cutoff with sensitivity 58.1%, while by elisa technique 25 iu/ml was the best cutoff with sensitivity of 78.4% as shown in table 3. in relation to rf isotypes as expressed in the same table where iga, igg & igm rf was 20 iu/ml the best cutoff with sensitivity 54.1, 47.3 & 48.6% respectively. considering rf concentration for positive cases that the median was 63 iu/ml; range 25-600 iu/ml as shown in table 4. table 5 shows that iga rf concentration that median was 96 iu/ml; range 27-600 iu/ml. it has been noticed that the median concentration of igg rf was 160 iu/ml; range 27-600 iu/ml. considering igm rf concentration the median was 310 iu/ml; range 21-600 iu/ml. table 1 : age distribution of ra patients table 2: distribution of patients by gender ra n % gender female 55 74.3 male 19 25.7 total 74 100 age in years ra n % <20 1 1.4 20-29 9 12.2 30-39 23 31.0 40-49 19 25.7 50-59 16 21.6 60+ 6 8.1 total 74 100 range 18-67 mean 42.1 sd 11.3 rheumatiod factor isotype in rheumatiod arthritis patients . nahida khalidh jassim sami j fac med baghdad 443 vol. 48 , no. 4 , 2006 table 3 : detection of rf and rf isotypes test ra sensitivity rf by latex negative 31 positive >10 43 58.1% rf by elisa negative (<25) 16 positive (>25) 58 78.4% rfiga type by elisa negative (<20) 34 positive (>20) 40 54.1% rfigg type by elisa negative (<20) 39 positive (>20) 35 47.3% rfigm type by elisa negative (<20) 38 positive (>20) 36 48.6% table 4 : median serum rf concentration (iu/ml) in ra patients. * rheumatoid factor ra rf by elisa range 25-600 mean 194.9 median 63 sd 233.9 n 58 *detect by elisa positive > 24 iu/m according to the frequency distribution of rf isotypes number table 6 revealed that 10.8% has one type either iga or igm rf in 6 (8.1%) and 2 (2.7%) patients respectively . presence of two types of rf together were 23% either (iga+igm) , (iga+igg) or ( igg+igm) were found in 5 (6.8%) , 6(8.1%) and 6(8.1%) respectively . finally, the combination of three rf isotypes was found in 23 (31.1%). * rf isotypes ra iga range 27-600 mean 171.3 median 96 sd 188 n 40 igg range 27-600 mean 309.8 median 160 sd 261.5 n 35 igm range 21-600 mean 379.6 median 310 sd 223.8 n 36 * detect by elisa table 6: frequency distribution of rf and rf isotypes among ra patients rf isotypes ra n % negative for all ig types 26 35.1 iga + igg + igm 23 31.1 iga + igm 5 6.8 iga + igg 6 8.1 igg + igm 6 8.1 iga 6 8.1 igm 2 2.7 total 74 100 discussion the male to female ratio in this study was 1:2.9 which is somewhat comparable to 1:2.7 reported by ubaid (3) locally & canstantine (1:3.4) abroad (4), and higher than farhat (1:2.1) & saraux (1:2.1) (5,6),this is well accepted that females are more prone to have autoimmune disease than males & that simply might be related to sex hormone e.g. estrogen. it is generally accepted that rf have been widely used as an immune marker, hence latex agglutination test was 58.1% shown to be less than other iraqi studies where 72.6%, 75.3% as had been reported by ubaid, & al-rawi, respectively (3,7) while abroad studies showed 56.6% and 70% which were reported by adhya, & tighe and carson, respectively (8,9). unlike agglutination tests which failed to detect other rf isotypes rather than igm, the other technique, elisa did this job quite well. moreover this technique usually discriminates rf isotypes where (45.1,47.3, & 48.6%) showed positivity for iga, igg & igm respectively compared with other studies abroad where (44,59, & 65%) and other study (44,72,& 69%) for iga, igg & igm respectively (5,10), these differences may be related to the source of the kit or some vague other reasons which need further studies. in spite of igm rf which was regarded as the classical one that detected by latex and found in higher rate than the other two rf types in many abroad studies (10). yet other studies suggested that igg and iga rf may be more specific than igm rf and may assist in the diagnosis (11), in this study presence of these rf isotypes where nearly to be equal to each other. rheumatiod factor isotype in rheumatiod arthritis patients . nahida khalidh jassim sami j fac med baghdad 444 vol. 48 , no. 4 , 2006 in relation to frequency distribution of rf isotypes number was showed that the combination of (iga+igg+igm) with 31.1% higher than 23% and 10.8% for two and one type of rf isotypes. references: 1.ope rm. rheumatoid arthritis: pathogenesis and early recognition . am j med 100 suppl+ 2a.:35-95,1996. 2arnett fc, edworthy sm, bloch da, mc.shane dj, fries jf, cooper ns. et al . the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis . arthritis rheum 1988;31:315-24. 3ubaid ah: formal education level as a marker of clinical status in ra among iraqis. a thesis submitted to the college of medicine, university of baghdad for the partial fulfillment of the master degree in community medicine (1996). 4farhat hf: antiphospholipid antibodies in rheumatoid arthritis. clinical and laboratory correlations. a thesis submitted to the college of medicine, university of baghdad in partial fulfillment of the requirements for the degree of master of science in pathology. (2000). 5saraux a, berthelot jm, charles g, et al. value of laboratory tests in early prediction of rheumatoid arthritis. j, arthi & rheum. 2002; 47(2):155-65. 6constantin a, cances vl, navaux f, et al. stromelysin 1 (mafrix metalloproteinase 3) and hla-drb1 gene polymorphismsj. arth. & rheum. 2002; 46(7):1754-62. 7al-rawi zs, al azzawi aj, al ajili fm, al wakil, r. “rheumatoid arthritis in population samples in iraq “ann rheum. dis. 1978;37:73-75. 8adhya s, chakraborty g, hajra b, bhattacharya s, sikdar pk, sinha s, banerjee pp, ghosh e. serology and immunoglobulin profile in rheumatoid arthritis. indian -jpathol-microbid. 1998; 41(1):43-7. 9tighe h, carson da: rheumatoid factors in: kelly w.n; ruddy s: harris ed, and sledge, cb, textbook of rheumatology (5th ed). sanders, w.b. company. 1997: 241-49. 10visser h, gelink lbs, kaamphraath ah, breedvald fg, hazes jmw. diagnostic and prognostic characteristics of the enzyme linked immunosorbent rheumatoid factors assays in rheumatoid arthritis. ann rheum dis. 1999; 55:157-61. 11jonsson t, steinsson k, et al. combined elevation of igm and iga rheumatoid factor has high diagnostic specificity for rheumatoid arthritis, rheumatoid. int. 1998; 18(3):119-22. key: cord-0063314-txvr42cu authors: shipa, muhammad r; yeoh, su-ann; embleton-thirsk, andrew; mukerjee, dev; ehrenstein, michael r title: o07 the increase in erythrocyte mean corpuscular volume by methotrexate is potentiated by hydroxychloroquine and is an early indicator of clinical response in rheumatoid arthritis date: 2021-04-26 journal: rheumatology (oxford) doi: 10.1093/rheumatology/keab246.006 sha: 696800b7e2c654d97e1c94a663c71348a608b947 doc_id: 63314 cord_uid: txvr42cu background/aims rheumatologists are facing a significant challenge in the management of early rheumatoid arthritis (ra) due to limitations placed on outpatient visits during the covid-19 pandemic. frequent clinical assessments of disease activity are recommended during implementation of the treat to target strategy to achieve remission. a biomarker indicating response to methotrexate during the early phase of therapy could complement clinical examination. methotrexate increases erythrocyte mean corpuscular volume (mcv), which is measured routinely, prompting us to investigate whether changes in mcv could act as an early indicator of response. methods patients with early ra who were started on methotrexate therapy were included from two independent cohorts. the larger cohort (discovery cohort, n = 655) was used to build the model and the second cohort (validation cohort, n = 225) was applied to test the prediction of the model. conventional statistical, and machine learning approaches were adopted to identify key determinants that influence the potential relationship between mcv and clinical response, defined as attainment of remission or low disease activity, at six months after starting methotrexate. results a lasso penalised logistic regression model was built with the discovery cohort [area under the receiver operating characteristics (auroc) curve = 0.76], where change of mcv from three months [odds ratio (or) 1.53 (95% ci 1.38-1.70)], concomitant use of hydroxychloroquine [or 2.16 (95% ci 1.52 3.07, p < 0.001)], and seropositivity [or 1.83 (95% ci 1.12 3.03, p = 0.02)] were associated with favourable methotrexate response [accuracy 81% (95% ci 76%-86%) of the model testing against discovery model]. different machine learning classification methods were applied. random forest exhibited the maximum accuracy and auroc (89%, and 86%, respectively), confirming the above three predictors as the most significant. two latent classes (class 1: smaller mcv increase and class 2: greater mcv increase) were identified based on the mcv changes over six months. class 1 had fewer responders and a lower number of patients on hydroxychloroquine compared to class 2. the earliest time point of significant difference of mcv between responders and non-responders was three months [mean difference 1.43 (95% ci 0.57-2.3)]. combination hydroxychloroquine and methotrexate caused the greatest increase in mcv with a difference between responders and non-responders at 2 months. change of mcv at three months showed auroc of 0.75 to predict treatment response to the combination of methotrexate and hydroxychloroquine at six months with an optimal cut-point of mcv 3.5 fl (95% ci 3.5-3.6) with 71% sensitivity and 75%, specificity. conclusion our data provides mechanistic insight into the synergistic clinical benefit of concomitant hydroxychloroquine with methotrexate, boosting the rise in erythrocyte mcv which could serve as an early biomarker of treatment response. disclosure m.r. shipa: grants/research support; versus arthritis. s. yeoh: grants/research support; royal college of physicians, rosetrees trust, nihr university college london hospitals biomedical research centre, uclh charities. a. embleton-thirsk: none. d. mukerjee: none. m.r. ehrenstein: grants/research support; university college london hospital biomedical research centre. methotrexate (mtx) is the most common treatment for rheumatoid arthritis (ra). the prevalence of adverse events (aes) associated with mtx treatment for ra have been studied extensively, but there are limited data on the predictors of these aes. this study aims to summarise the prevalence rates of mtx aes, including gastrointestinal (gi), neurological, mucocutaneous, and elevated alanine transaminase (alt) enzyme, and to identify baseline demographic and clinical predictors of these aes. the rheumatoid arthritis medication study (rams) is a uk multicentre prospective cohort study of patients with ra starting mtx for the first time. relevant demographic, medication, clinical and disease related data were collected at baseline. aes were reported at six and twelve months follow-ups. the prevalence rates of aes were calculated based on the proportions of patients who reported having had an ae within one year of follow-up. the associations between candidate baseline predictors and aes were assessed using multivariable logistic regression. a total of 2,089 patients were included with a mean age of 58.4 (standard deviation: 13.5) years, 1390 (66.5%) were women. 1,814 and 1,579 patients completed the 6 and 12 months follow-up visits, respectively. the prevalence rates of the aes within one year of followup were: gi ¼ 777 (40.6%), mucocutaneous ¼ 441 (23.1%), neurological ¼ 487 (25.5%), elevated alt (> upper limit of normal [uln]) ¼ 286 (15.5%). younger age and being a woman were associated with increased risk of gi aes, (age: or 0.97 per year increase in age, 95% ci 0.98, 1.00; male sex: or 0.58 vs female, 95% ci 0.46, 0.74) ( table 1) . higher baseline health assessment questionnaire (haq) score was an independent predictor of gi, mucocutaneous, and neurological aes. furthermore, having alt >1xuln at baseline or history of diabetes was associated with increased risk of subsequent alt elevation during the study follow-up. in patients with ra starting mtx, gi aes were the most commonly reported aes during the first year of follow-up. the identified predictors of aes may facilitate discussions between clinicians and patients prior to commencing mtx, and may lead to increased adherence and consequently improved effectiveness. rheumatologists are facing a significant challenge in the management of early rheumatoid arthritis (ra) due to limitations placed on outpatient visits during the covid-19 pandemic. frequent clinical assessments of disease activity are recommended during implementation of the treat to target strategy to achieve remission. a biomarker indicating response to methotrexate during the early phase of therapy could complement clinical examination. methotrexate increases erythrocyte mean corpuscular volume (mcv), which is measured routinely, prompting us to investigate whether changes in mcv could act as an early indicator of response. methods patients with early ra who were started on methotrexate therapy were included from two independent cohorts. the larger cohort (discovery cohort, n ¼ 655) was used to build the model and the second cohort (validation cohort, n ¼ 225) was applied to test the prediction of the model. conventional statistical, and machine learning approaches were adopted to identify key determinants that influence the potential relationship between mcv and clinical response, defined as attainment of remission or low disease activity, at six months after starting methotrexate. . different machine learning classification methods were applied. random forest exhibited the maximum accuracy and auroc (89%, and 86%, respectively), confirming the above three predictors as the most significant. two latent classes (class 1: smaller mcv increase and class 2: greater mcv increase) were identified based on the mcv changes over six months. class 1 had fewer responders and a lower number of patients on hydroxychloroquine compared to class 2. the earliest time point of significant difference of mcv between responders and non-responders was three months [mean difference 1.43 (95% ci 0.57-2.3)]. combination hydroxychloroquine and methotrexate caused the greatest increase in mcv with a difference between responders and nonresponders at 2 months. change of mcv at three months showed auroc of 0.75 to predict treatment response to the combination of methotrexate and hydroxychloroquine at six months with an optimal cut-point of mcv 3.5 fl (95% ci 3.5-3.6) with 71% sensitivity and 75%, specificity. early diagnosis and intervention improves outcomes of immune mediated rheumatic and musculoskeletal diseases (rmds) but may be hampered by diagnostic uncertainty. the extent to which rationally selected molecular parameters add value to clinical characteristics for diagnostic prediction in undifferentiated disease states warrants investigation. b lymphocytes play an increasingly recognised role in rheumatoid arthritis (ra) pathogenesis, and cell-specific methylation patterns link environmental exposures to genetic risk. we derived and tested the practical utility of a b lymphocyte-derived dna methylation signature for predicting ra in an early arthritis clinic cohort. methods cd19þ b cell and peripheral blood mononuclear cell (pbmc) whole genome dna methylation array data were available, respectively, from 109 inflammatory arthritis patients naïve to immunomodulatory drugs (newcastle, uk; 38% confirmed to have a diagnosis of ra within 1 year) and 50 untreated undifferentiated arthritis (ua) patients (leiden, the netherlands; 68% classifiable ra within 1 year by 1987 acr criteria versus alternate diagnoses). a bespoke machine learning pipeline employed a sequential model-based optimisation (smbo) procedure for selecting, tuning and applying methods amongst ten feature-selection, six data-sampling and two classification algorithms in the newcastle ''training cohort.'' the predictive performance of the resultant optimised molecular classifier was assessed in the independent leiden ''test cohort'' alongside a previously described clinical prediction rule, using comparative area under receiver operating characteristic (auroc) curves. a modification to the clinical prediction rule that incorporated a single parameter to reflect molecular classification was also assessed. the pipeline was implemented using the r machine learning package mlr. using the smbo approach, 27 cpgs maximally discriminatory for ra were selected from b lymphocyte dna methylome training data, and a molecular classifier was derived using the random forest algorithm. we provide a proof of principle for the application of a b lymphocytederived epigenetic signature to enhance prediction of ra in ua patients using stored pbmcs. further refinement of our pipeline represents a plausible means to expedite the diagnosis in undifferentiated rmds and could offer pathophysiological insight. irs for deep vein thrombosis(dvt), pulmonary embolism(pe), and dvt and/or pe(dvt/pe) were also calculated for groups of patients while receiving bari 2mg/4mg within all-bari-ra. major adverse cardiovascular events(mace) were adjudicated in 5 ph3 studies and the lte. results 3770 pts received bari for 13,148 py, with median and maximum exposure: 4.2 and 8.4 years, respectively. overall irs per 100 py were: for any treatment-emergent adverse event (ae)(25.8); serious ae hz (3.0) 46); malignancies excluding non-melanoma skin cancer (nmsc)(0.91) 07); and gastrointestinal perforation (0.04). (irs)[95% confidence intervals] for patients while receiving bari 2mg (n ¼ 1077) and bari 4mg (n ¼ 3400) were dvt irs for death tended to increase in later time intervals (beyond 192 weeks). no particular cause of death contributed to this increase key: cord-0028740-3gf4cbky authors: pan, zijian; zhu, tong; liu, yanjun; zhang, nannan title: role of the cxcl13/cxcr5 axis in autoimmune diseases date: 2022-03-04 journal: front immunol doi: 10.3389/fimmu.2022.850998 sha: 3ee8fa83741f883a3b082bc407b1f171ffc8ae3f doc_id: 28740 cord_uid: 3gf4cbky cxcl13 is a b-cell chemokine produced mainly by mesenchymal lymphoid tissue organizer cells, follicular dendritic cells, and human t follicular helper cells. by binding to its receptor, cxcr5, cxcl13 plays an important role in lymphoid neogenesis, lymphoid organization, and immune responses. recent studies have found that cxcl13 and its receptor cxcr5 are implicated in the pathogenesis of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, primary sjögren’s syndrome, myasthenia gravis, and inflammatory bowel disease. in this review, we discuss the biological features of cxcl13 and cxcr5 and the recent findings on the pathogenic roles of the cxcl13/cxcr5 axis in autoimmune diseases. furthermore, we discuss the potential role of cxcl13 as a disease biomarker and therapeutic target in autoimmune diseases. chemokines are a family of small chemotactic cytokines that play roles in immune cell trafficking (1) . chemokines can be divided into four subfamilies depending on the position of the first two conserved n-terminal cysteine residues: xc, cc, cxc, and cx3c chemokines (2) . by interacting with their corresponding chemokine receptors, seven transmembrane g protein-coupled receptors (gpcrs), chemokines play multiple roles in immune response, embryonic development, and angiogenesis (3) . in addition, aberrant activation of chemokine signaling pathways is implicated in the development of many human diseases, such as autoimmune diseases, chronic inflammatory diseases and cancers (2) . cxcl13 is initially known as b cell-attracting chemokine 1 (bca-1) and b-lymphocyte chemoattractant (blc), for its strong chemotaxis to b cells (4, 5) . cxcl13 is constitutively expressed in the b-cell follicles of secondary lymphoid organs (5, 6) . follicular dendritic cells (fdcs) have been proved to be an important source of cxcl13 (7) . in addition, cxcl13 is also produced by t follicular helper (tfh) cells in humans, but not by murine tfh cells (8) . tfh cells, with the expression of cxcr5, b cell lymphoma 6 (bcl-6), interleukin-21 (il-21), programmed death-1 (pd-1), and inducible t cell co-stimulator (icos), dwell in the germinal centers (gcs) of secondary lymphoid organs where these cells can support b-cell survival and differentiation with the help of cd40l/cd40 interactions and cytokines such as interleukin-4 (il-4) and il-21 (9) . when interacting with b cells, tfh cells can release dopamine, which can promote the rapid translocation of intracellular icosl to cell surface of b cells (10) . the icosl/icos interaction can further augment the accumulation of cd40l and chromogranin b granules at the synapse of tfh cells, facilitating t-b interactions and gcs development (10) . besides, expression of cxcl13 is also detected in cd4 + pd-1 hi cxcr5 -t peripheral helper (tph) cells in ra synovial tissue specimens (11) . the production of cxcl13 by tph cells could exert an indispensable role in recruiting cxcr5 + b cells (11, 12) . due to the lack of expression of th-related cytokines such as interferon-g (ifn-g), il-4, and il-17, the tph cells are recognized as a distinct th-cell subpopulation from th1 cells, th2 cells, and th17 cells (13) . besides cxcl13 secretion, these cells also express b lymphocyte-induced maturation protein-1 (blimp-1), il-21, icos, and maf to further promote b-cell activation and differentiation (11, 14) . and the transcription factor sox-4 is responsible for cxcl13 production in tph cells (15) . compared with tfh cells, tph cells do not express cxcr5, however, these cells can express other chemokine receptors such as ccr2, ccr5, and cx3cr1, which may determine their peripheral localization (11) . although tfh cells and tph cells can both exert b-cell helper activities, the b-cell helper abilities between the two cells to different b-cell subpopulations are distinct (16) . for instance, tfh cells could help both naïve b cells and memory b cells, while the targets of tph cells are mainly limited to memory b cells (16) . cxcr5, formerly named burkitt's lymphoma receptor 1 (blr1), is the sole receptor of cxcl13 (4, 5) . physically, cxcr5 is highly expressed on mature b cells, subpopulations of cd4 + t cells [i.e. tfh and t follicular regulatory (tfr) cells], and skin-derived migratory dendritic cells (dcs) (17) (18) (19) (20) . by attracting these cells in a cxcr5-dependent manner, cxcl13 is essential in the development, organization, and immune activation of secondary lymphoid organs including lymph nodes (lns), peyer's patches, and spleen (21) . additionally, cxcl13/cxcr5 axis is also required for b1 cells homeostasis (22) and b-cell receptor (bcr)-induced b-cell activation (23) . overall, the cxcl13/cxcr5 axis can affect the b-cell and tfh cells' functions, and plays important roles in immunity. dysregulated cxcl13 expression has been associated with various human diseases, such as cancers (24) , infectious diseases (25, 26) , idiopathic pulmonary fibrosis (ipf) (27, 28) , transplantation rejection (29) , and neuropathic pain (30) (figure 1) . moreover, cxcl13 is also related to many autoimmune diseases, such as rheumatoid arthritis (ra), multiple sclerosis (ms), systematic lupus erythematosis (sle), primary sjögren's syndrome (pss), myasthenia gravis (mg), and inflammatory bowel diseases (ibd). in this review, we provide a comprehensive overview of the pathogenic role of cxcl13/ cxcr5 axis in autoimmune diseases, while also discussing the potential usage of cxcl13 as a novel clinical biomarker and treatment target. the cxcl13/cxcr5 axis plays an essential role in the neogenesis and organization of secondary lymphoid tissues, and in immune responses (31) . the role of the cxcl13/cxcr5 axis in the development of secondary lymphoid organs has been documented in cxcl13-and cxcr5-deficient mice, whose lymphoid organs exhibit impaired development both in size and organization (32, 33) . in cxcl13 -/and cxcr5 -/mice, most of the lns (e.g., inguinal, iliac, sacral, brachial, and axillary lns) are absent, consistent with impaired numbers of peyer's patches (33) . however, facial, superficial cervical, and mesenteric lns have been confirmed to be retained in these mice (33) . in the fetal stage, cxcl13 produced by mesenchymal lymphoid tissue organizer (mlto) cells attracts lymphoid tissue inducer (lti) cells towards the parenchyma of the lns anlagen (34, 35) . recruited lti cells subsequently upregulate lymphotoxin-a1b2 (lta1b2) expression and activate mlto cells through lta1b2-lymphotoxin b receptor (ltbr) interactions (36) . then, activated mlto cells enhance the expression of cxcl13 together with other homeostatic chemokines (e.g. ccl19 and ccl21), cytokines [e.g. il-7 and receptor activator of nuclear factor-kb ligand (rankl)], and adhesion molecules [e.g. vascular cell adhesin molecule 1 (vcam-1) and intercellular adhesion molecule 1 (icam-1)] to further recruit and retain more lti cells, thus forming a positive feed-forward loop that contributes to lns formation (37) . in addition, cxcl13 and cxcr5 are required for b-cell homing in secondary lymphoid organs (33) . b cells can migrate toward b cell follicles following the cxcl13 gradients produced by fdcs, the constitutive stromal cells in the follicles (38) , while in the cxcr5 -/mice, b cells could not migrate toward the follicles of secondary lymphoid organs (33) . furthermore, cxcl13 induces recruited b cells to upregulate lta1b2, which can promote fdcs development and increase the expression of cxcl13, leading to a positive feedback loop which is crucial for the development of b cell follicles (33) . cxcl13 is also involved in gcs compartmentalization, an essential biological process for adaptive immune responses (23, 39) . cxcl13 produced by fdcs also attracts b cells toward the light zones within gcs, where b cells undergo affinity selection and become long-lived plasma cells or memory b cells (37, 39) . the cxcl13/cxcr5 axis also regulates t-cell positioning, and thus facilitates gcs response (40) . cxcl13 can attract the tfh cells toward the boundary between t cell zones and b cell follicles (t-b border), where tfh cells can interact with activated b cells and further promote the b-cell activation and proliferation (41) . indeed, in mice with t cells lacking cxcr5, the gcs response is impaired, as demonstrated by the fewer and smaller gcs and reduced b-cell frequency found within gcs (42) . cxcl13/cxcr5 axis also regulates the migration of tfr cells, which can suppress excessive humoral responses by acting on mature b cells and tfh cells within gcs (19) . the cxcl13/cxcr5 axis is also implicated in b1 cell response (22) . cxcl13 is constitutively produced by peritoneal macrophages, and attracts b1 cell toward the peritoneal cavity, which is important in maintaining the body cavity innate immunity (22) . bröker et al. found that toll-like receptor 2 (tlr2) activation consistent with tlr2-triggered il-10 could induce the production of cxcl13 and c5a in peritoneal macrophages (43) . they further found that the c5a could subsequently enhance cxcl13 secretion of peritoneal macrophages by interacting with c5a receptor (c5ar) (43) . cxcl13/cxcr5 axis is also involved in bcr-triggered b-cell activation by shaping cell dynamics, which enhances antigen gathering at the immune synapse (23) . the possible mechanisms are that cxcl13/cxcr5 axis promotes the membrane ruffling and adhesion supported by lymphocyte function associated antigen 1 (lfa-1) and integrates the bcr signaling activation by the migratory junction supported by lfa-1 (23) . in a word, cxcl13/cxcr5 axis is important in regulating b-cell homeostasis ( figure 2 ). cxcl13 protein contains 109 amino acids, including four cysteine residues showing a c-x-c chemokine pattern ( figure 3a ) (4). typically, the tertiary structure of chemokines is relatively conserved, consisting of a disordered n-terminal 'signaling domain', followed by a 'core domain', which includes an 'n-loop', a 3 10 -helix, a three-stranded b-sheet, and a c-terminal a-helix (44) . however, in the human cxcl13 protein, the n-terminus forms an additional parallel b-strand (b0), which interacts with the core bsheet, thus forming a four-stranded b-sheet ( figure 3b ) (45) . in the crystal structure of human cxcl13, the initiating methionine was found to interact with the 'core domain' of cxcl13, which may stabilize the n-terminal structure (46) . in addition, an unusual long and disordered c-terminal domain was also observed in the cxcl13 protein (46) . the human cxcr5 is a seven transmembrane gpcr, and it contains two isoforms due to the alternatively spliced transcript variants (47) . compared with the predominant transcript variant 1, the transcript variant 2 is different in the 5' end, resulting in translation starting from the downstream in-frame aug, leading to an isoform with a shorter n-terminus ( figure 3a ) (47) . however, the crystal structure of cxcr5 is largely unknown. rosenberg et al. explored the effects of n-terminal length variation and side-chain composition of cxcl13 on cxcr5 activity (45) . they found that the orthosteric pocket of cxcr5 can tolerate minor variations in the length and side-chain of the cxcl13 n-terminus, without severely impairing the activity (45) . and the enlarged bulk in the cxcl13 orthosteric site was well tolerated by cxcr5, while a loss of contacts between cxcl13 and cxcr5 was less tolerated (45) . however, the structural basis for cxcl13-cxcr5 interactions is also less studied. cxcl13 binding to cxcr5, the seven-transmembrane gpcr, induces a variety of downstream signaling pathways, leading to various cellular events such as migration, survival, proliferation, and modulation of gene transcription (24) . the signal transduction pathways mediated by cxcr5 follow the classical gpcr activation pattern (24) . g proteins are heterotrimers that comprise three subunits: ga, gb, and gg (48) . ligand binding leads to a three-dimensional conformation change of gpcrs, and results in the dissociation of ga subunit from gbg dimer, which then regulates the downstream effectors (48) . however, furthermore, cxcl13-mediated tfh cells migration is essential in facilitating gcs response. in addition, cxcl13/cxcr5 axis is also implicated in b1 cell response through attracting b1 cell toward the body cavity. in autoimmune diseases, ectopic cxcl13 expression promotes ectopic lymphoid neogenesis and the production of disease-specific autoantibodies. in ra, cxcl13 drives epc homing and vegf expression, thus inducing angiogenesis in synovial tissue. in ln, cxcl13 promotes proliferation and tgf-b1 production of mesangial cells and induces podocyte secretion of proinflammatory cytokines/chemokines such as cxcl1, cxcl12, mif, lif, and soluble icam-1 and vcam-1. dcs, dendritic cells; epc, endothelial progenitor cells; fdc, follicular dendritic cells; gcs, germinal centers; ibd, inflammatory bowel disease; icam-1, intercellular adhesion molecule 1; ln, lupus nephritis; lif, leucocyte inhibitory factor; lti, lymphoid tissue inducer; mg, myasthenia gravis; mif, macrophage inhibitory factor; mlto, mesenchymal lymphoid tissue organizer; ms, multiple sclerosis; pss, primary sjögren's syndrome; ra, rheumatoid arthritis; sle, systemic lupus erythematosus; tfh, t follicular helper; tgf-b1, transforming growth factor b1; tph, t peripheral helper; vcam-1, vascular cell adhesion molecule 1; vegf, vascular endothelial growth factor. the specific transduction mechanisms involved in cxcl13-cxcr5 signaling may vary in a cell-and stimulus-dependent manner (24) . early studies demonstrated that cxcr5 induced ca 2+ influx and chemotaxis independently of inhibitory g proteins, whereas cxcr5-mediated mitogen-activated protein kinase (mapk) signaling via extracellular signal-regulated kinase 1 and 2 (erk1/2) was found to occur in an inhibitory g proteins dependent manner (49) . structurally, these signaling pathways induced by cxcr5 are dependent on the second intracellular domain of cxcr5 (49) . moreover, barroso et al. found that these pathways also depended on the regulatory role of epstein-barr virus-induced g-protein coupled receptor 2 (ebi2) on cxcr5 (50) . through forming a heterodimer with cxcr5, ebi2 modulates cxcl13/cxcr5-induced cell responses (50) . emerging studies have also shown that dysregulated cxcr5mediated pathways are associated with human diseases, especially with tumors. for instance, in human osteosarcoma cell lines, it is reported that cxcl13/cxcr5 signaling relies on phospholipase cb (plcb) and protein kinase c-a (pkca) to activate downstream nuclear factor-kb (nf-kb) signals, thereby promoting cell migration (51) . in return, pkcϵ overexpression and phosphatase and tensin homologue (pten) inactivation were found to upregulate cxcl13 expression through the non-canonical nf-kb the amino acid sequence of human cxcl13 and two isoforms of human cxcr5. the human cxcl13 protein consists of 109 amino acids, including four conserved cysteine residues (marked in yellow), and the first two of the cysteine residues show a typical c-x-c chemokine pattern. the human cxcr5 has two isoforms due to the alternatively spliced transcript variants. the difference between the two isoform of cxcr5 is additionally marked in bold. (b) the tertiary structure of human cxcl13 [protein data bank (pdb) id: 7jny]. the human cxcl13 protein has a flexible n-terminus, followed by the b0-sheet, a long n-loop ending in a short 3 10 -helix, and the central three-stranded anti-parallel b-sheet, followed by a c-terminal a-helix. b-sheet is indicated by yellow arrows, and a-helix and 3 10 -helix are indicated by red cylinders. pathway, resulting in prostate cancer cell migration and tumorigenesis (52) . the cxcr5-mediated pathway also utilizes phosphoinositide 3-kinase (pi3k) to activate akt, resulting in the activation of downstream nf-kb and mammalian target of rapamycin (mtor) pathways involved in cell growth and invasion (52, 53) . recent studies have found that cxcr5 regulates downstream glycogen synthase kinase-3b (gsk-3b) and b-catenin through the pi3k-akt pathway, leading to epithelial cell proliferation and tumorigenesis through upregulating cyclin d1 and c-myc expressions (54) . in addition to the erk1/2 pathway, cxcr5 also activates the mapk pathway utilizing c-jun n-terminal kinase (jnk) and p38, to mediate prostate cancer cell proliferation and inflammatory pain, respectively (55, 56) . the signaling pathways for cxcl13/cxcr5 axis described above are shown in figure 4 . | cxcl13/cxcr5-mediated signaling pathways. cxcl13 exerts its biological functions through activating cxcr5, a chemokine receptor coupled to gprotein heterotrimer. upon activation, cxcr5 undergoes conformation change and induces the cycle of g-protein activation, leading to a cascade of downstream signal transduction pathways including: (1) activation of plc leads to conversion of pip2 to ip3 and dag. ip3 can promote the release of ca 2+ from intracellular stores into the cytoplasm. on the other hand, dag consistent with increased ca 2+ activate pkc, which contributes to the activation of transcription factor nf-kb to promote cell migration; (2) activation of pi3k can trigger the activation of akt, thus stimulating downstream nf-kb, mtor, and gsk-3b/b-catenin/tcf/lef signaling, which play key roles in tumor cell growth, proliferation, invasion, and migration; (3) cxcr5 also activates mapk pathways utilizing jnk, erk, and p38 via g-protein, which may further stimulate ap-1 to promote cell proliferation and inflammation. ap-1, activating protein-1; dag, diacylglicerol; erk, extracellular signal-regulated kinase; gsk-3b, glycogen synthase kinase-3b; ip3, inositol triphosphate; jnk, c-jun n-terminal kinase; mtor, mammalian target of rapamycin; nf-kb, nuclear factor-kb; pi3k, phosphoinositide 3-kinase; pkc, protein kinase c, plc, phospholipase c; pten, phosphatase and tensin homologue; tcf/lef, t-cell factor/ lymphoid enhancer-binding factor. autoimmune diseases are characterized by the presence of autoantibodies or autoreactive t cells that attack self-antigens, and thus lead to damage and dysfunction of tissues and organs (57) . autoimmune diseases comprise a large spectrum of diseases such as ra, sle, ms, pss, mg, and ibd etc. (58) . chemokines play important roles in regulating immune responses and mediating inflammation, as well as being involved in the pathogenesis of autoimmune diseases (2) . for instance, cxcl9, cxcl10, and cxcl11 promote th1 polarization and mediate th1 responses in autoimmune diseases (59) . cxcl13 is vital in the lymphoid neogenesis, maintaining the architecture of secondary lymphoid tissues, and immune responses (24) . cxcl13 is also involved in the initiation and organization of ectopic lymphoid-like structures (elss), the organized lymphocyte aggregates developed at sites of inflammation in target tissues of autoimmune diseases (60) . elss are dynamic structures resembling secondary lymphoid organs in both histological structures and gene expression profile (60) . in elss of inflammatory tissues, elevated expression of cxcl13 can regulate b-cell infiltration and positioning, and can also regulate b-cell shuttle inside the ectopic gcs (61). in addition, tfh cells can also be attracted by cxcl13 towards the proximity of b cells in els, and further exert b-cell help (62) . elss can serve as functional gcs that promote autoreactive b-cell response and increase the local autoantibody production (62) . the pivotal role cxcl13/cxcr5 axis in elss development has been detected in several autoimmune diseases such as ra and pss. in ra, mrna and protein levels of cxcl13 are increased in ectopic lymphoid follicles in the synovial tissues, especially in the area with b cells accumulation (63, 64) . in addition, the mrna expression levels of cxcl13 are positively correlated with elss development in inflamed synovial tissues of ra patients (65) . these studies indicate that cxcl13 may attract b cells and contribute to the formation elss in chronic arthritis (65) . cxcl13 is overexpressed in elss in salivary glands of patients with pss (66) . in salivary gland tissues of pss patients, cxcl13 expression is associated with increased size of lymphoid aggregates and the progressive organization lymphoid-like structures (67) . the elss in pss can further promote b-cell expansion, local antibody production, and lymphomagenesis (68) . besides, cxcl13/cxcr5 axis plays similar roles in other autoimmune diseases, such as ms (69), sle (70), mg (71) , and so on. hence, cxcl13 may affect the pathogenesis of autoimmune diseases through regulating elss development (68) . in addition, the cxcl13/cxcr5 axis may also have disease-specific pathogenetic mechanisms in certain autoimmune diseases, which will be discussed further below. (figure 2 and table 1 ). ra is a chronic inflammatory autoimmune disease characterized by inflammation of synovium and progressive destruction of joint, bone, and cartilage (113) . b cells are critical contributors to ra, as they secrete proinflammatory cytokines, present antigens, and interact with other inflammatory cells (114) . cxcl13, a regulator of b-cell homing and activation, is regarded as a novel biomarker of ra (115) . through single-cell rna-seq analyses, tph cells have been identified to be the predominant source of cxcl13 in ra synovial tissues, while cxcl13 expression was less detectable in synovial fibroblasts, vascular cells, macrophages, dcs, or other lymphocytes (116) . through secreting cxcl13, tph cells can attract cxcr5 + cells towards inflamed synovium tissues (13) . in vitro studies showed that tph cells could induce the differentiation of memory b cells into plasma cells in il-21 and signaling lymphocytic activation molecule (slam) family dependent manners (11) . furthermore, the frequency of tph cells is robustly expanded in seropositive ra patients with higher disease activity, and is significantly declined after effective treatment, further supporting the pathogenetic role of these cells in ra (11) . serum cxcl13 levels are found to be increased in ra patients, and are further elevated in rheumatoid factor (rf) positive and anti-citrullinated peptide antibodies (acpa) positive patients (72) . in both early and established ra cohorts, serum cxcl13 exhibits a strong correlation with serum igm and iga rf in seropositive ra patients (117) . in early ra, baseline serum cxcl13 levels are correlated with elevated rates of joint damage during the 7-year follow-up period (118) . moreover, plasma cxcl13 concentrations are found to be higher in active ra patients when compared to quiescent ra or healthy controls. moreover, plasma cxcl13 concentrations are positively correlated with clinical parameters, including creactive protein (crp), erythrocyte sedimentation rate (esr), rf, and the tender joint count in 68 joints (73) . cxcl13 has also been detected in synovial tissues and synovial fluids (sf) in ra patients (63, 74) . furthermore, synovial cxcl13 expression is associated with markers that reflect immune cell activation and bone destruction in early ra (63) . these results suggest that cxcl13 is a predictor of more severe and erosive ra (63) . studies exploring the possible association between cxcl13 levels and drug responsiveness have also been conducted. notably, consistent results have also shown that the levels of cxcl13 in patient serum are markedly declined during therapy of disease modifying anti-rheumatic drugs (dmards) (119) (120) (121) . in addition, higher baseline serum cxcl13 levels enable to predict faster return of circulating b cells following a course of rituximab, a b-cell depletion therapy (119) . collectively, these studies report the role of cxcl13 as a biomarker for treatment response in ra. cxcl13 regulates lymphocyte aggregation and ectopic gcs formation in ra. cxcl13 and ccl20 synergistically drive the recruitment of b-cell towards inflammatory synovium of ra patients (122) . histological analysis indicates that cxcl13 is preferentially expressed in ectopic gcs within the ra synovium (65, 123) . in addition, cxcl13 expression is associated with cluster enlargement and progressive organization of elss in arthritic joints, and has been identified to be an independent predictor for ectopic gcs formation (123, 124) . furthermore, in a rodent model of chronic antigen-induced arthritis, cxcr5 -/mice showed impaired development and organization of elss along with alleviated joint destruction (125) . cxcl13 also drives the preferential migration and expression of il-10 in b10 + cells, a subtype of regulatory b cells (bregs) that plays an essential role in maintaining immune tolerance (75) . however, it was found that cxcr5 expression was decreased in b10 + cells of ra patients, leading to impaired migration of b10 + cells toward cxcl13-rich synovial fluid, consistent with less il-10 secretion (75) . in addition to the regulation of immune cells, cxcl13 also regulates endothelial progenitor cell (epc) homing and angiogenesis during the development of ra (76) . blood-derived leukocytes enter into the synovial tissues through the vessels, which makes angiogenesis an important driver of ra progression (126). tsai et al. found that cxcl13 promoted the expression of vascular endothelial growth factor (vegf) in epc via activation of plc and mek, with concomitant upregulated activator protein-1 (ap-1) signaling pathway, therefore contributing to epc homing and angiogenesis during ra progression (76) . inhibition of cxcl13 using an short hairpin rna (shrna) in collagen-induced arthritis (cia) mice markedly reduced epc homing, angiogenesis, and arthritis severity (76) . interestingly, antibodies against cxcl13 show beneficial effect in animal models of ra. in cia mice, both prophylactic and therapeutic administration of cxcl13 neutralizing antibody curtailed the disease development, and alleviated the joint inflammation and cartilage damage (77) . anti-cxcl13 antibody reduced b cells in the lymphoid infiltration, and significantly attenuated cartilage damage and joint destruction (77, 127) . similarly, cxcr5-deficient mice showed severely impaired cia, characterized by lower serum anti-collagen ii antibody levels (78) . in addition, selective ablation of cxcr5 expression in b cells or t cells also suppressed arthritis in cia mice (78) . taken these studies into consideration, cxcl13/ cxcr5 axis may be a novel treatment target for ra (57) . multiple sclerosis (ms) is an autoimmune-mediated disease of the central nervous system (cns), characterized by inflammation, demyelination, and neurodegeneration, which can lead to muscle weakness, visual dysfunction, sensory loss, ataxia, and cognitive impairment (128) . increased cxcl13 concentrations have been detected in blood, cerebrospinal fluid (csf), and actively demyelinating brain lesions in ms patients (79, 80) . notably, in patients with relapsing-remitting ms (rrms), csf cxcl13 levels are associated with increased relapse rate and disease severity measured by the expanded disability status scale (edss) (129) . in addition, csf cxcl13 levels are correlated with intrathecal immunoglobulin production, oligoclonal bands (ocbs), and the presence of lymphocytes (80, 130) . in addition, csf cxcl13 levels are reduced by treatment of methylprednisolone (131), daclizumab (132) and natalizumab (131, 133) in ms patients. moreover, independent rituximab (134) and fingolimod therapy (135) also decreased csf cxcl13 levels in rrms. collectively, cxcl13 is considered to be a potential marker for ms disease severity, prognosis, and clinical therapeutic responses. the pathogenesis role of cxcl13 in ms is further demonstrated in experimental autoimmune encephalomyelitis (eae) mice, a commonly used experimental animal model for human ms. bagaeva et al. found that cxcl13 -/mice showed a milder and self-limited form of eae, as demonstrated by reduced clinical signs; attenuated inflammation, demyelination, gliosis, and fibrosis of white matter; and more complete recovery (81) . however, the exact mechanisms of cxcl13/cxcr5 axis in ms are still unknown. cxcl13 is involved in elss formation in ms, which may further lead to local humoral responses and inflammation. elss with a network of cxcl13 + cells, have been detected in the meninges of approximately 40% of secondary progressive ms (spms) patients, and are associated with more severe disease course and cortical lesions (69, (136) (137) (138) . in addition, columba-cabezas et al. found that blocking the lt-ltbr signaling pathway in eae mice inhibited cxcl13 production and elss formation in the meninges and suppressed eae symptoms (139) . th17 cell-driven eae mice model (140) . however, the predominant role of cxcl13 in ms is assumed to be attracting tfh cells but not b cells. quinn et al. explored the role of anti-cxcl13 antibody in eae induced by adoptive transfer of myelin-specific th17 cells (82) . they found reduced tfh cells infiltration in cns, and decreased eae severity, while infiltrated th17 cells and b cells were largely unaffected (82) . however, anti-cxcl13 antibody treatment was not effective in b-cell deficient eae mice (82) . similarly, rainey-barger et al. demonstrated no difference in cns b-cell infiltration between cxcl13 -/mice and wild-type mice in actively induced eae (141) . however, the th1 responses and th17 responses were weakened two weeks after the peak of the disease in eae mice with cxcl13 deficiency (141) . these results suggest that cxcl13 may attracts tfh cells toward the cns, which further maintains immune responses mediated by b cells, th1 cells, and th17 cells in eae. however, further studies are required to confirm this hypothesis in human ms. sle is a chronic autoimmune disease that affects multiple organs and tissues, such as the skin, joints, the cns, and kidneys (142) . lupus nephritis (ln) is a glomerulonephritis caused by sle, and can lead to irreversible kidney injury (143) . in the last couple of decades, increasing evidence has linked cxcl13/cxcr5 axis to sle and its major manifestations. serum cxcl13 levels are found to be significantly higher in sle patients, especially in those with renal involvement, as compared with healthy controls (83, 84) . serum cxcl13 is positively correlated with the sle disease activity index (sledai) (84, 144, 145) , anti-double-stranded dna (anti-dsdna) antibodies titers, and prevalence of inflammatory arthritis, while it is inversely correlated with serum levels of complement factors c3 and c4 (146) (147) (148) . cxcl13 is also a potential differentiation marker to identify active sle from inactive sle, and to identify ln from non-ln in sle patients (148) . expressions of cxcl13 and cxcr5 are also detected in the renal cortex from patients with ln (83) . renal cxcl13 mrna levels were higher in ln patients with abundant intrarenal b cells infiltrate than those without (70) . in addition, most infiltrating b cells express cxcr5 and are colocalized with cxcl13-abundant regions, suggesting that the cxcl13/cxcr5 axis plays a significant role in the recruitment of b cells in these inflammatory lesions (70) . in addition to b-cell attraction, cxcl13 also induces double-negative (dn) t cells infiltration within inflammatory kidney tissues (85) . in sle, dn t cells could produce igg anti-dna antibody and inflammatory cytokines, and lead to tissues damages in the kidneys of sle patients (149) . in cxcr5 -/-b6/lpr mice, b-cell count and b-cellmediated immune responses were diminished, and dn t cells accumulation was also reduced (85) . b cells and dn t cells from cxcr5 -/-b6/lpr mice failed to migrate toward cxcl13 in vitro (85) . moreover, cxcl13 also regulates the balance between th17 and regulatory t cells (tregs) in sle (86) . in mrl/lpr lupus-prone mice, blockade of cxcl13 was found to significantly reduce spleen th17/tregs ratio, renal inflammatory cytokines production, and alleviated kidney damage (86) . macrophages and renal dcs are reported to be the main sources of cxcl13 in lupus-prone mouse models (150, 151) . in nzb/w f1 mice, a mouse model that resembles human sle, elevated tlr7 and tlr9 responses induced increased cxcl13 expression in cd11b + cd11 chi dcs, associated with dysregulated nf-kb signaling pathway (152) . in addition, moreth et al.'s study showed that elevated proteoglycan biglycan found in ln could trigger cxcl13 expression in macrophages and dcs in a tlr2-and tlr4-dependent manner (153) . the cxcl13/cxcr5 axis also contributes to the proliferation of mesangial cells and to transforming growth factor-b1 (tgf-b1) production by activating erk signaling pathway in ln. this process seems to be targeted by microrna-155 (mir-155) (84, 87 ). an in vitro study found that cxcl13 induced secretion of proinflammatory cytokines and chemokines in human podocytes, via erk signaling pathway (88) . furthermore, the cytokine and chemokine cocktail produced by activated podocytes leads to a respiratory burst in isolated human neutrophils (88) . in addition, cxcl13 expression is significantly increased in circulating tph cells and tfh cells from sle patients compared with controls (154) . the frequency of tph cells, but not tfh cells, is positively correlated with both disease activity and cd11c + b cells frequency in sle patients (88) . tph cells may further support memory b-cell differentiation in sle via il-21 and maf (88) . however, further studies are needed to explore the regulatory roles of the cxcl13/ cxcr5 axis in sle, and whether this axis could be a novel target in human sle also needs more evidence. the pss is a chronic autoimmune disease characterized by lymphocytic infiltration within lacrimal and salivary glands, dryness of mouth and eyes, and elevated incidence of lymphoma (155) . in pss, dysregulated immune responses to the auto-antigens, such as ro/ssa and la/ssb, result in epithelial destruction of the exocrine glands (155) . cxcl13 is proposed to be a biomarker of pss severity. serum cxcl13 levels are significantly elevated in pss patients, and are found to correlate with clinical parameters, including rheumatoid factor (rf), k-to-l free light chain ratio, b2microglobulin, g-globulins, anti-ro/ssa, anti-la/ssb, and erythrocyte sedimentation rate (esr) (89, 90) . increased cxcl13 protein levels are also detected in pss patients' saliva (91) , salivary glands tissues (92) , and salivary gland secretome (i.e., salivary gland biopsy supernatants) (93) . interestingly, the serum concentrations of cxcl13 are also associated with the risk and occurrence of lymphoma in pss patients (89, 90) . collectively, these observations highlight the potential role of cxcl13 in pss. cxcl13 is also described to be a biomarker of histological involvement in sjögren's syndrome. in pss patients, cxcl13 is also associated with elss, a key mediator in pss pathogenesis (156) . serum cxcl13 is associated with increased levels of lymphocytic infiltration, lymphoid organization, and the presence of ectopic gcs in pss salivary glands (157) . in pss salivary gland tissues, cxcr5 + b cells are organized into gcslike clusters (92) . moreover, blokland et al. found a positive correlation between cxcl13 mrna expression and tfh cell frequencies in salivary gland of pss patients (158) . in addition, administration of abatacept, a recombinant fusion protein that selectively inhibited t-cell activation via binding to cd80 and cd86, significantly downregulated cxcl13 levels in pss patients (159) . in a virus-induced murine model of lymphoid neogenesis in the salivary glands, il-22 induced the expression of cxcl12 and cxcl13 in epithelial and fibroblastic stromal cells respectively, which is pivotal for b-cell recruitment, elss formation, and the aberrant autoimmune response (160) . cxcl13 neutralization results in a modest reduction of glandular inflammation (91) . in addition, concomitant blockade of cxcl13 and b cell activating factor receptor (baffr) led to reduced salivary gland inflammation, total serum antibodies and anti-nuclear autoantibodies (ana)specific igg autoantibody titers, along with preventing xerostomia development in a pss mouse model (161) . recently, paeoniflorin-6'-o-benzene (cp-25) has been found to limit the target organ index and b cell activities in experiment sjögren's syndrome (ess) by inhibiting cxcr5-g protein-coupled receptor kinase 2 (grk2)-erk/p38 signaling pathway (162) . in addition, cp-25 also alleviated the salivary gland indexes; improved tissue integrity and saliva flow; and reduced the igg antibodies, anti-ssa, and anti-ssb antibodies in ess mice, by inhibiting jak1-signal transducer and activator of transcription 1 and 2 (stat1/2)-cxcl13 signaling and interfering with b-cell migration (163) . these results suggest that the cxcl13/cxcr5 axis may serve as a new biomarker and a potential therapeutic target in pss (91) . cxcl13 axis may also be implicated in the lacrimal gland pathology in pss. ltbr blockade in the non-obese diabetic (nod) model of sjögren's syndrome significantly reduced cxcl13 protein levels in lacrimal glands, accompanied by reduced b-cell accumulation, eliminated high endothelial venule (hev) in lacrimal glands, reduced entry rate of lymphocytes into lacrimal glands, and improved tear-fluid secretion and corneal integrity (164) . however, further studies are required to elucidate the role of the cxcl13/cxcr5 axis in keratoconjunctivitis sicca of human pss. myasthenia gravis (mg) is an autoimmune disease caused by autoantibodies against acetylcholine receptors (achr) or other functionally related molecules in the postsynaptic membrane at the neuromuscular junction (165) . muscle weakness is the prominent feature of mg, and typically develops with repetitive muscle use and as the day progresses (166) . thymic lymphoid hyperplasia, characterized by the presence of ectopic gcs, has been found in most of the patients with earlyonset mg (165) . thymic cxcl13 expression levels are significantly elevated, and are correlated with disease severity in mg patients with thymic hyperplasia (71, (94) (95) (96) (97) . furthermore, serum cxcl13 levels are increased in mg patients (95, 96, 98) , and are strongly correlated with disease severity and the frequency of circulating tfh cells (98) . in addition, corticosteroid treatment markedly reduced thymic cxcl13 expression and gcs formation in mg patients (95) . moreover, dna microarray analysis showed that cxcl13 was the gene on which glucocorticoids exert the most significant effect, suggesting that cxcl13 is a major target of glucocorticoids in mg treatment (95) . cxcl13 is a target gene of mir-143, a tumor suppressor microrna (167) . mir-143 can repress proliferation and enhance apoptosis of thymocytes through negatively targeting cxcl13 (167) . cxcl13 is also negatively regulated by mir-548k (168) . downregulated expression of mir-548k may contribute to elevated cxcl13 levels in thymus of mg patients with thymus hyperplasia (168) . cxcl13 is constitutively produced by thymic epithelial cells (tecs) in the normal thymus and is overexpressed in mg tecs, which may contribute to the elevated cxcl13 levels in mg thymus (95) . polyinosinic-polycytidylic acid (poly(i:c)) injection triggers a local increase in type i interferons (ifn-i) in mouse thymus, associated with overexpression of cxcl13, ccl21, baff, and increased recruitment of b cells (169) . in addition, an in vitro study showed that ifn-b induced cxcl13 and ccl21 expressions in tecs and lymphatic endothelial cells respectively (169) . these results suggest that ifn-i can trigger chemokine expressions that favor ectopic gcs development in mg thymus (169) . however, in a transgenic mouse model with overexpressed cxcl13 in the thymus, thymic cxcl13 overexpression by itself is not sufficient to induce b-cell recruitment (66) . interestingly, under the inflammatory conditions induced by poly(i:c), this thymic cxcl13 overexpression model gets a strong recruitment of b cells to the thymus, and is more susceptible to developing mg, as demonstrated by elevated clinical signs, anti-achr antibody levels, and thymic gcs-like structures, when compared with wild-type mice (66) . these observations suggest that cxcl13 can exaggerate the pathogenesis of mg, rather than initiating the onset of the disease. a subtle dual-regulated effect of estrogen has been found on cxcl13 expression in mg (170) . in the resting state, estrogen inhibits cxcl13 expression of tecs (170) . however, under inflammatory conditions, the direct inhibited effect of estrogen is overpassed by inflammatory pathways, such as tlr/ifn-i related pathways (170) . consequently, estrogen contributes to the sustained activation of these pathways and promotes the expression levels of some chemokines including cxcl13 (170) . emerging studies have also found that cxcl13 can be served as a potential biomarker for other autoimmune diseases and autoimmune-related disorders such as type 1 diabetes mellitus (t1dm), inflammatory bowel disease (ibd), primary biliary cholangitis (pbc), graves' disease (gd), bullous pemphigoid (bp), psoriasis, systemic sclerosis (ssc), autoimmune pancreatitis (aip), and common variable immunodeficiency (cvid). t1dm is a chronic autoimmune disease that results from pancreatic islet b-cells destruction, leading to insulin deficiency and hyperglycemia (171) . several studies conducted on animal models aimed to elucidate the role of cxcl13 in t1dm. however, the results seem to be equivocal. the initial study utilized transgenic mice that expressed cxcl13 in islet b-cells, and found b-cell infiltration within the islets of these mice (99) . furthermore, the study also found the presence of wellcharacterized elss in large-sized infiltration within the pancreas of these transgenic mice (99) . however, this model failed to mimic a diabetes phenotype of impaired b-cells and increased blood glucose (99) . in the autoimmune non-obese diabetic (nod) t1dm mouse model, cxcl13 and its cognate receptor, cxcr5, were highly expressed during the development of elss, especially in the early onset of diabetes (100) . in addition, a high expression of cxcr5 was also observed in islet-infiltrating b220 + cells (100) . in nod mice, cxcl13 blockade disrupted b-cell organization in islet elss, however, neither the proportion of b-cell within the islets, nor the t1dm disease progression were affected (172) . these results suggest that cxcl13 plays role in the neogenesis and maintenance of elss in islets of t1dm, but whether cxcl13 is involved in the development of t1dm remains to be further studied. in addition, vecchione et al. evaluated plasma cxcl13 levels of children with new-onset t1dm, autoantibody-positive at-risk children, and autoantibody-negative control children (173) . however, the results showed no significant difference in plasma cxcl13 levels among the three groups (173) . in a recent study of vecchione et al., plasma cxcl13 levels were evaluated in t1dm, autoantibody-positive, and autoantibodynegative adult subjects (174) . the study found that plasma cxcl13 levels in t1dm subjects were significantly decreased compared to non-diabetic autoantibody-negative and autoantibody-positive at-risk subjects (174) . thus, the role of cxcl13 in human t1dm also needs to be further studied to take further conclusions. ibd, mainly comprised of crohn's disease and ulcerative colitis (uc), are chronic inflammatory disorders of the gastrointestinal tract (175) . although the exact etiology of ibd remains unknown, it involves a complex interaction between the genetic, environmental, epithelial, microbial, and immune factors (175) . circulating cxcl13 levels are increased in both pediatric (101) and adult (102) patients with ibd. abundant expressions of cxcl13 and cxcr5 were also detected within aberrant lymphoid aggregates in uc lesions (103) . in addition, cxcr5 gene polymorphisms can influence ibd susceptibility. the cxcr5 rs6421571 allele c is associated with increased risks of crohn's disease in the japanese population (176) . these results support the possible involvement of cxcl13 and cxcr5 in ibd. monocytes/macrophages seem to be the main producers of cxcl13 in inflammatory uc lesions where lymphoid neogenesis occurs (177) . however, in a uc mice model induced by dextran sulfate sodium (dss), cxcl13 is produced by gp38 + colonic stromal cells, mediated by the innervation of the vagus nerve (178) . selective surgical ablation of vagus nerve innervation inhibited local cxcl13 expression and abrogated elss formation but did not affect colitis (178) . in addition, in the dss-induced uc mice model, the ox40/ox40l axis can induce cxcr5 expression on cd4 + t cells, and further promote their migration toward gcs in the lesioned colonic mucosa (179) . anti-cxcl13 antibody treatment reduced the disease severity in the dss-induced uc, suggesting cxcl13 as a potential target for treating uc (101) . pbc is the prototype of an autoimmune disease characterized by destructive lymphocytic cholangitis and the presence of serum anti-mitochondrial antibodies (amas) targeting at the e2 subunit of the pyruvate dehydrogenase complex (pdc-e2) (180) . serum cxcl13 is increased in patients with pbc, and is associated with total bilirubin levels, and is gradually decreased during the longitudinal phase of ursodeoxycholic acid treatment (104) . elevated cxcl13 expression was also found in liver tissues of pbc patients, especially in portal tracts areas (104) . in addition, expanded cxcr5 + cd4 + t cells, cd19 + b cells, and cd19 + cd38 + b cells were detected in portal tracts of pbc, accompanied with increased expression of intrahepatic il-21 (104) . these results suggest that cxcl13 may serve as a key regulator for bcell dysregulation in pbc. in addition, genome-wide association studies have identified cxcr5 (rs6421571) as a risk locus for pbc in both uk and japanese populations (176, 181) . gd is an autoimmune thyroid disease characterized by the presence of autoantibodies against the thyroid-stimulating hormone receptor (tsh-r), thyroglobulin and thyroperoxidase (182) . cxcl13 and cxcr5 expressions is increased in gd thyroid tissues (105) (106) (107) . cxcl13 expression levels are significantly associated with focal lymphocytic infiltrates, the presence of ectopic gcs, and anti-thyroperoxidase autoantibodies (105, 106) . the above-mentioned evidence suggests that dysregulated cxcl13 expression is related to the pathogenesis of gd. bp is the most common type of pemphigoid diseases which is characterized by autoantibodies against structural proteins of the hemidesmosomes, leading to tense blisters and erosions on the skin or mucous membranes (183) . serum cxcl13 levels and the proportion of cxcl13 + cells among leukocytes in lesioned skin are both elevated in patients with bp, and are positively correlated with anti-bp180-nc16a titers in bp patients (108) . psoriasis is a chronic immune-mediated inflammatory skin disease, characterized by clinical features of erythema, thickening, and scale (184) . cxcl13 circulating levels and cxcl13 expression levels in cutaneous lesions are increased in psoriasis, and are positively correlated with disease severity (109, 110) . moreover, cxcl13 expression levels in psoriatic lesions are decreased after anti-il23 treatment (109) . plasma cxcl13 levels are positively correlated with the frequency of peripheral helper t 17 (tph17) cells and tfh cells in peripheral blood of psoriasis patients (110) . additionally, subsets of cd8 + t cells, characterized by expression of il-17 pathway cytokines, cytolytic genes, and cxcl13, were specifically detected in psoriasis lesions (109) . ssc, also known as scleroderma, is characterized by immune dysregulation, fibrosis of the skin and internal organs, and vasculopathy (185) . serum cxcl13 levels are increased in patients with ssc compared with healthy controls (111) . notably, serum cxcl13 levels are associated with tissue fibrosis, vasculopathy and immune activation, especially with interstitial lung disease and digital ulcers in ssc patients (111) . recently, gaydosik et al. identified a cluster of recirculating cxcl13 + t cells, characterized by a tfh-like gene expression profile, which may promote b-cell responses within the lesioned skin in ssc (186) . aip is the pancreatic manifestation of systemic igg4-related disease (igg4-rd), characterized by increased serum igg4 levels and lymphoplasmacytic sclerosing pancreatitis (187) . in patients with aip, mrna levels of cxcl13, consistent with ccl19, ccl21, ccl17, baff, lt-a, and lt-b expression were increased in pancreatic tissues, compared with controls (112) . in addition, serum levels of cxcl13, ccl19, tnf-a, and il-6 were also elevated in aip patients (112) . overexpression of lt-a, and lt-b in pancreatic acinar cells of mice induced cxcl13 expression and other features resembling human aip (112) . furthermore, these features were significant ameliorated through inhibiting ltbr-signaling (112) . therefore, lt-ltbr axis plays a pivotal role in aip by mediating chemokines and pro-inflammatory cytokines such as cxcl13. at present, there is still a lack of study on the role of cxcl13 in other types of igg4-rd such as orbital disease, sclerosing cholangitis, retroperitoneal fibrosis, and interstitial nephritis, and more attention is needed to address this issue. cxcl13 is also associated with immunodeficiency associated autoimmune diseases such as cvid. cvid is a heterogeneous group of primary immunodeficiency diseases characterized by hypogammaglobinemia (188) . patients with cvid are often complicated with respiratory tract infection, inflammatory diseases, and autoimmune diseases (188) . hultberg et al. evaluated plasma protein profiles of patients with cvid through proximity extension assay, and found that cxcl13 was the top upregulated extracellular protein in cvid, as compared with healthy controls (189) . due to the heterogeneity of cvid, the role of cxcl13 in the disease still needs to be further studied. however, there has been only limited research on the function of cxcl13/cxcr5 axis in these autoimmune diseases, and more studies are required to provide further insights. due to its essential role in immune regulation and inflammatory response, cxcl13/cxcr5 axis may serve as a potential therapeutic target for autoimmune diseases ( table 2) . indeed, numerous preclinical studies support the concept of suppressing the cxcl13/cxcr5 axis as a novel therapeutic approach for autoimmune diseases treatment. for example, in cia mice, prophylactic or therapeutic administration of anti-cxcl13 monoclonal antibody curtailed the disease development, consistent with alleviated joint inflammation and cartilage damage (77) . in addition, tsai et al. reported that inhibition of cxcl13 action through an shrna markedly reduced angiogenesis and arthritis severity in cia mice (76) . moreover, several studies have revealed that cxcl13 neutralization by anti-cxcl13 antibody also attenuates disease progression and severity in eae mice (77, 81, 82) . to assess the role of cxcl13 in ln, wu et al. used neutralizing anti-cxcl13 antibodies to block mrl/lpr lupus-prone mice (86) . the results showed that cxcl13 neutralization significantly improved kidney injury, and reduced serum anti-dsdna titers, renal immune complex deposition, renal inflammatory cytokines production, and spleen th17/tregs ratio (86) . similarly, in a mouse model of pss, cxcl13 neutralization by anti-cxcl13 monoclonal antibody results in a modest reduction of glandular inflammation (91) . in addition, in a uc mice model induced by dss, anti-cxcl13 antibody significantly reduced the disease severity (101) . to summarize, these results suggest that the cxcl13/cxcr5 axis is a promising target for autoimmune diseases, which may open avenues for novel strategies for autoimmune diseases treatment. this review highlights the biological functions, protein structures, signaling transduction pathways, and roles of cxcl13/cxcr5 in the pathogenesis of autoimmune diseases. autoimmune diseases are characterized by immune dysfunction, autoantibody production and chronic inflammation. in this context, the role of cxcl13 as a b-cell chemokine and key regulator of humoral immunity has attracted considerable attention in the study of the development of autoimmune diseases. abnormal expression of cxcl13, consistent with that of other chemokines and cytokines, contributes to ectopic lymphoid neogenesis in local lesioned tissues, and promotes the production of autoantibodies in autoimmunity (62) . in addition, recent studies have revealed that cxcl13/cxcr5 may also have a disease-specific pathological mechanism in some autoimmune diseases. for example, in ra, cxcl13 regulates epc, thereby promoting angiogenesis (76) . in ln, cxcl13 regulates dn t cells, mesangial cells and podocytes and leads to renal damage (85, 87, 88) . however, the specific mechanism through which cxcl13 plays a role in autoimmune diseases is still largely unknown. it is necessary to utilize better animal models, human pathological specimens, and in vitro research to further investigate the regulatory role of cxcl13 in ectopic lymphoid neogenesis, immune cell activation and other key pathological processes in autoimmunity. in addition to autoimmune diseases, cxcl13 is also regarded as a prognostic biomarker for ipf (27, 190) . cxcl13 was overexpressed in lung tissues and plasma of patients with ipf (27) . plasma cxcl13 levels were positively correlated with disease manifestations and prognoses of ipf patients (27) . during pneumocystis infection, cxcl13 was required for the formation of inducible bronchus associated lymphoid tissues (ibalt), the elss in the lung (191) . in pneumocystis-infected rag2 −/− il2rg −/− mouse, the expression of cxcl13 was increased (191) . in addition, cxcr5 -/mice lacked draining lns and presented poorly organized ibalt structures in the lung after pneumocystis infection (191) . cxcl13 can be produced by pulmonary macrophages under the stimulation of tnf-a, il-10 (192) , and can also be produced by pulmonary fibroblasts under the stimulation of il-13 and il-17a (191) . in activated alveolar macrophages (am) and monocyte-derived macrophages (modm), the cxcl13 expression was induced by nf-kb and jak/stat pathways (192) . besides, both il-13 and il-17a synergistically upregulated cxcl13 expression in pulmonary fibroblasts in stat3-and gata3-dependent manner (191) . clinical studies have shown that cxcl13 expression is elevated in autoimmune diseases patients, and is correlated with clinical parameters that are related to disease severity, activity, and prognosis (73, 129, (193) (194) (195) . in addition, in the clinical setting, glucocorticoid and dmards can alleviate the symptoms of autoimmune diseases and reduce the expression of cxcl13, suggesting that cxcl13 may reflect treatment response (95, 134) . however, due to the heterogeneity of autoimmune diseases, the clinical significance of cxcl13 may be different for patients with different disease subtypes, different stages of progression, and different genetic backgrounds. further studies are required to explore this issue. moreover, cxcl13 combined with other biomarkers may be helpful for the diagnosis of some autoimmune diseases, but the optimal cut-off value should be determined according to specificity and sensitivity, and their diagnostic efficacy should be carefully evaluated. in animal models of autoimmune diseases, knockout or neutralization of cxcl13/cxcr5 significantly improve clinical symptoms, suggesting that cxcl13/cxcr5 can be used as a therapeutic target for autoimmune diseases (77) . however, due to the gap between animal models and human diseases, further studies are required to assess if this therapeutic effect can be reflected in humans and whether the drugs targeting cxcl13/ cxcr5 are safe. contrastingly, neutralizing antibodies are the most frequently used methods to target cxcl13/cxcr5, although there is still a lack of studies on rna interference (rnai) or small molecule inhibitors targeting cxcl13/cxcr5. the development of highly selective and stable small molecule inhibitors targeting cxcl13/cxcr5 will become a focus of future research. in addition, the protein structure of cxcr5 is still largely unknown, and analysis of the protein structure of cxcr5 will accelerate the development of relevant drugs. in brief, although numerous challenges remain, cxcl13/cxcr5 axis is undoubtedly a promising therapeutic target for human autoimmune diseases. zp, tz, and yl responsible for literature research and writing. nz reviewed the manuscript, made significant revisions on the drafts, and supervised and finalized this work. all authors have read and agreed to the published version of the manuscript. chemokines and chemokine receptors: positioning cells for host defense and immunity exploring the complex role of chemokines and chemoattractants in vivo on leukocyte dynamics the role of chemokines and chemoki ne receptors in multiple sclerosis b cell-attracting chemokine 1, a human cxc chemokine expressed in lymphoid tissues, selectively attracts b lymphocytes via blr1/cxcr5 a b-cell-homing chemokine made in lymphoid follicles activates burkitt's lymphoma receptor-1 blc (cxcl13) is expressed by different dendritic cell subsets in vitro and in vivo follicular stromal cells and lymphocyte homing to follicles follicular helper cd4 t cells (tfh) definition of b cell helper t cells in rheumatoid arthritis and their behavior during treatment t(fh)-derived dopamine accelerates productive synapses in germinal centres pathologically expanded peripheral t helper cell subset drives b cells in rheumatoid arthritis cxcl13-producing pd-1(hi)cxcr5(-) helper t cells in chronic inflammation a distinct human cd4+ t cell subset that secretes cxcl13 in rheumatoid synovium a regulatory effect of il-21 on t follicular helper-like cell and b cell in rheumatoid arthritis human sox4 facilitates the development of cxcl13-producing helper t cells in inflammatory environments two populations of circulating pd-1hicd4 t cells with distinct b cell helping capacity are elevated in early rheumatoid arthritis expression of the g-protein-coupled receptor blr1 defines mature, recirculating b cells and a subset of t-helper memory cells cxc chemokine receptor 5 expression defines follicular homing t cells with b cell helper function human blood t(fr) cells are indicators of ongoing humoral activity not fully licensed with suppressive function a migratory population of skin-derived dendritic cells expresses cxcr5, responds to b lymphocyte chemoattractant in vitro, and co-localizes to b cell zones in lymph nodes in vivo chemokines and chemokine receptors in lymphoid tissue dynamics cxcl13/cxcr5 axis in autoimmune diseases cxcl13 is required for b1 cell homing, natural antibody production, and body cavity immunity cxcl13/cxcr5 signaling enhances bcr-triggered b-cell activation by shaping cell dynamics cxcl13/cxcr5 signaling axis in cancer cxcl13-mediated recruitment of intrahepatic cxcr5(+)cd8(+) t cells favors viral control in chronic hbv infection altered expression of the receptor-ligand pair cxcr5/cxcl13 in b cells during chronic hiv-1 infection c-x-c motif chemokine 13 (cxcl13) is a prognostic biomarker of idiopathic pulmonary fibrosis lyme neuroborreliosis: clinical outcomes, controversy, pathogenesis, and polymicrobial infections chemokine 13 is a noninvasive biomarker of antibody−mediated renal allograft rejection cxcl13 drives spinal astrocyte activation and neuropathic pain via cxcr5 the potential for cxcl13 in csf as a differential diagnostic tool in central nervous system infection a putative chemokine receptor, blr1, directs b cell migration to defined lymphoid organs and specific anatomic compartments of the spleen a chemokine-driven positive feedback loop organizes lymphoid follicles chemokine cxcl13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation multiple roles of lymphatic vessels in peripheral lymph node development receptor-alpha cells that differentially engender lymph nodes and peyer's patches lymph node stromal cells: cartographers of the immune system how follicular dendritic cells shape the b-cell antigenome live imaging of il-4-expressing t follicular helper cells in explanted lymph nodes role of cxcr5 and ccr7 in follicular th cell positioning and appearance of a programmed cell death gene-1high germinal center-associated subpopulation helper cell heterogeneity: time, space, and function the germinal center response is impaired in the absence of t cell-expressed cxcr5 a novel role for c5a in b-1 cell homeostasis chemokine and chemokine receptor structure and interactions: implications for therapeutic strategies the n-terminal length and side-chain composition of cxcl13 affect crystallization, structure and functional activity solution structure of cxcl13 and heparan sulfate binding show that gag binding site and cellular signalling rely on distinct domains sequence variation of a novel heptahelical leucocyte receptor through alternative transcript formation regulation, signaling, and physiological functions of g-proteins signal transduction by the chemokine receptor cxcr5: structural requirements for g protein activation analyzed by chimeric cxcr1/cxcr5 molecules ebi2 regulates cxcl13-mediated responses by heterodimerization with cxcr5 cxcl13/ cxcr5 interaction facilitates vcam-1-dependent migration in human osteosarcoma protein kinase c epsilon cooperates with pten loss for prostate tumorigenesis through the cxcl13-cxcr5 pathway cxcl13/cxcr5 axis predicts poor prognosis and promotes progression through pi3k/ akt/mtor pathway in clear cell renal cell carcinoma cxcl13 promotes intestinal tumorigenesis through the activation of epithelial akt signaling cxcl13 mediates prostate cancer cell proliferation through jnk signalling and invasion through erk activation chemokine cxcl13 activates p38 mapk in the trigeminal ganglion after infraorbital nerve injury clinical implications of shared genetics and pathogenesis in autoimmune diseases human autoimmune diseases: a comprehensive update th1 chemokines in autoimmune endocrine disorders role of chemokines in ectopic lymphoid structures formation in autoimmunity and cancer ectopic lymphoid structures: powerhouse of autoimmunity ectopic lymphoid neogenesis in rheumatic autoimmune diseases high expression levels of the b cell chemoattractant cxcl13 in rheumatoid synovium are a marker of severe disease effects of cxcl13 inhibition on lymphoid follicles in models of autoimmune disease lymphoid chemokine b cell-attracting chemokine-1 (cxcl13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients novel cxcl13 transgenic mouse: inflammation drives pathogenic effect of cxcl13 in experimental myasthenia gravis association of cxcl13 and ccl21 expression with the progressive organization of lymphoid-like structures in sjögren's syndrome activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large b cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and malt lymphoma in sjögren's syndrome detection of ectopic b-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis analysis and classification of b-cell infiltrates in lupus and anca-associated nephritis regulatory and pathogenic mechanisms in human autoimmune myasthenia gravis the predictive value of serum soluble icam-1 and cxcl13 in the therapeutic response to tnf inhibitor in rheumatoid arthritis patients who are refractory to csdmards potential novel biomarkers of disease activity in rheumatoid arthritis patients: cxcl13, ccl23, transforming growth factor alpha, tumor necrosis factor receptor superfamily member 9, and macrophage colony-stimulating factor cell chemoattractant cxcl13 is preferentially expressed by human th17 cell clones #cxcr5/cxcl13 pathway, a key driver for migration of regulatory b10 cells, is defective in patients with rheumatoid arthritis cxcl13/ cxcr5 axis facilitates endothelial progenitor cell homing and angiogenesis during rheumatoid arthritis progression cxcl13 antibody for the treatment of autoimmune disorders cerebrospinal fluid and blood cytokines as biomarkers for multiple sclerosis: a systematic review and meta-analysis of 226 studies with 13,526 multiple sclerosis patients chemokines in multiple sclerosis: cxcl12 and cxcl13 up-regulation is differentially linked to cns immune cell recruitment cxc chemokine ligand 13 plays a role in experimental autoimmune encephalomyelitis role of tfh cells in promoting t helper 17-induced neuroinflammation serum blc/ cxcl13 concentrations and renal expression of cxcl13/cxcr5 in patients with systemic lupus erythematosus and lupus nephritis cxcl13 promotes proliferation of mesangial cells by combination with cxcr5 in sle cxcr5 is critically involved in progression of lupus through regulation of b cell and double-negative t cell trafficking cxcl13 blockade attenuates lupus nephritis of mrl/lpr mice microrna-155 suppresses mesangial cell proliferation and tgf-b1 production via inhibiting cxcr5-erk signaling pathway in lupus nephritis pathogenetic role of glomerular cxcl13 expression in lupus nephritis cxcl13 and ccl11 serum levels and lymphoma and disease activity in primary sjögren's syndrome. arthritis rheumatol serum cxcl13 levels are associated with lymphoma risk and lymphoma occurrence in primary sjögren's syndrome cxcl13 is elevated in sjögren's syndrome in mice and humans and is implicated in disease pathogenesis ectopic expression of the b cell-attracting chemokine bca-1 (cxcl13) on endothelial cells and within lymphoid follicles contributes to the establishment of germinal center-like structures in sjögren's syndrome salivary gland secretome: a novel tool towards molecular stratification of patients with primary sjögren's syndrome and non-autoimmune sicca microarrays reveal distinct gene signatures in the thymus of seropositive and seronegative myasthenia gravis patients and the role of cc chemokine ligand 21 in thymic hyperplasia the chemokine cxcl13 is a key molecule in autoimmune myasthenia gravis ectopic and high cxcl13 chemokine expression in myasthenia gravis with thymic lymphoid hyperplasia expression of immune molecules cd25 and cxcl13 correlated with clinical severity of myasthenia gravis expansion of circulating counterparts of follicular helper t cells in patients with myasthenia gravis blc expression in pancreatic islets causes b cell recruitment and lymphotoxin-dependent lymphoid neogenesis evolution of ectopic lymphoid neogenesis and in situ autoantibody production in autoimmune nonobese diabetic mice: cellular and molecular characterization of tertiary lymphoid structures in pancreatic islets loss of n-6 fatty acid induced pediatric obesity protects against acute murine colitis chemokine and cytokine levels in inflammatory bowel disease patients b cell attracting chemokine 1 (cxcl13) and its receptor cxcr5 are expressed in normal and aberrant gut associated lymphoid tissue chemokine (c-x-c motif) ligand 13 promotes intrahepatic chemokine (c-x-c motif) receptor 5+ lymphocyte homing and aberrant b-cell immune responses in primary biliary cirrhosis chemokines determine local lymphoneogenesis and a reduction of circulating cxcr4+ t and ccr7 b and t lymphocytes in thyroid autoimmune diseases the role of cxcr5 and its ligand cxcl13 in the compartmentalization of lymphocytes in thyroids affected by autoimmune thyroid diseases elevated follicular helper t cells and expression of il-21 in thyroid tissues are involved in the pathogenesis of graves' disease possible roles of cxcl13/cxcr5 axis in the development of bullous pemphigoid single-cell rna sequencing of psoriatic skin identifies pathogenic tc17 cell subsets and reveals distinctions between cd8(+) t cells in autoimmunity and cancer increased peripheral helper t cells type 17 subset correlates with the severity of psoriasis vulgaris cxcl13 produced by macrophages due to fli1 deficiency may contribute to the development of tissue fibrosis, vasculopathy and immune activation in systemic sclerosis lymphotoxin b receptor signaling promotes development of autoimmune pancreatitis a systematic review of cxcl13 as a biomarker of disease and treatment response in rheumatoid arthritis increased circulating cxcl13 levels in systemic lupus erythematosus and rheumatoid arthritis: a meta-analysis single-cell rna-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation serum c-x-c motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels identification of cxcl13 as a marker for rheumatoid arthritis outcome using an in silico model of the rheumatic joint cxcl13: a novel biomarker of b-cell return following rituximab treatment and synovitis in patients with rheumatoid arthritis thu0130 distinct biomarkers enrich for clinical response to tocilizumab (tcz) and adalimumab (ada) in adacta: a head-to-head monotherapy study in patients (pts) with methotrexate (mtx)-ir rheumatoid arthritis (ra) efficacy and safety of pateclizumab (anti-lymphotoxin-a) compared to adalimumab in rheumatoid arthritis: a head-to-head phase 2 randomized controlled study (the altara study) role of cxcl13 and ccl20 in the recruitment of b cells to inflammatory foci in chronic arthritis lymphoid neogenesis in rheumatoid synovitis systematic microanatomical analysis of cxcl13 and ccl21 in situ production and progressive lymphoid organization in rheumatoid synovitis cxcr5-and ccr7-dependent lymphoid neogenesis in a murine model of chronic antigen-induced arthritis implications of angiogenesis involvement in arthritis cxcl13 neutralization reduces the severity of collagen-induced arthritis diagnosis and treatment of multiple sclerosis: a review cerebrospinal fluid cxcl13 in multiple sclerosis: a suggestive prognostic marker for the disease course the influence of disease duration, clinical course, and immunosuppressive therapy on the synthesis of intrathecal oligoclonal igg bands in multiple sclerosis increased cerebrospinal fluid concentrations of the chemokine cxcl13 in active ms inhibition of lti cell development by cd25 blockade is associated with decreased intrathecal inflammation in multiple sclerosis natalizumab in progressive ms: results of an open-label, phase 2a, proof-of-concept trial changes in b-and t-lymphocyte and chemokine levels with rituximab treatment in multiple sclerosis cerebrospinal fluid biomarkers of inflammation and degeneration as measures of fingolimod efficacy in multiple sclerosis meningeal b-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology a gradient of neuronal loss and meningeal inflammation in multiple sclerosis meningeal inflammation is widespread and linked to cortical pathology in multiple sclerosis suppression of established experimental autoimmune encephalomyelitis and formation of meningeal lymphoid follicles by lymphotoxin beta receptor-ig fusion protein integration of th17-and lymphotoxin-derived signals initiates meningeal-resident stromal cell remodeling to propagate neuroinflammation the lymphoid chemokine, cxcl13, is dispensable for the initial recruitment of b cells to the acutely inflamed central nervous system protecting the kidney in systemic lupus erythematosus: from diagnosis to therapy elevation of serum cxcl13 in sle as well as in sepsis the correlational research among serum cxcl13 levels, circulating plasmablasts and memory b cells in patients with systemic lupus erythematosus: a strobe-compliant article cxcl13 is an activity marker for systemic, but not cutaneous lupus erythematosus: a longitudinal cohort study cytokine profiling in active and quiescent sle reveals distinct patient subpopulations the cxcl13 chemokine serves as a potential biomarker to diagnose systemic lupus erythematosus with disease activity expanded double negative t cells in patients with systemic lupus erythematosus produce il-17 and infiltrate the kidneys aberrant high expression of b lymphocyte chemokine (blc/cxcl13) by c11b +cd11c+ dendritic cells in murine lupus and preferential chemotaxis of b1 cells towards blc activated renal macrophages are markers of disease onset and disease remission in lupus nephritis heightened tlr7/9-induced il-10 and cxcl13 production with dysregulated nf-ҝb activation in cd11c(hi)cd11b(+) dendritic cells in nzb/w f1 mice the proteoglycan biglycan regulates expression of the b cell chemoattractant cxcl13 and aggravates murine lupus nephritis pd-1hicxcr5-t peripheral helper cells promote b cell responses in lupus via maf and il-21 b cells in the pathogenesis of primary sjögren syndrome cxcl13 as biomarker for histological involvement in sjögren's syndrome epigenetically quantified immune cells in salivary glands of sjögren's syndrome patients: a novel tool that detects robust correlations of t follicular helper cells with immunopathology attenuation of follicular helper t cell-dependent b cell hyperactivity by abatacept treatment in primary sjögren's syndrome. arthritis rheumatol il-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs early baff receptor blockade mitigates murine sjögren's syndrome: concomitant targeting of cxcl13 and the baff receptor prevents salivary hypofunction alleviating effect of paeoniflorin-6'-o-benzene sulfonate in antigen-induced experimental sjögren's syndrome by modulating b lymphocyte migration via cxcr5-grk2-erk/p38 signaling pathway cp-25 alleviates antigen-induced experimental sjögren's syndrome in mice by inhibiting jak1-stat1/2-cxcl13 signaling and interfering with b-cell migration lymphotoxin-beta receptor blockade reduces cxcl13 in lacrimal glands and improves corneal integrity in the nod model of sjögren's syndrome micro-rna-143 inhibits proliferation and promotes apoptosis of thymocytes by targeting cxcl13 in a myasthenia gravis mouse model mir-548k regulates cxcl13 expression in myasthenia gravis patients with thymic hyperplasia and in jurkat cells central role of interferon-beta in thymic events leading to myasthenia gravis type 1 diabetes mellitus cxcl13 blockade disrupts b lymphocyte organization in tertiary lymphoid structures without altering b cell receptor bias or preventing diabetes in nonobese diabetic mice reduced pd-1 expression on circulating follicular and conventional foxp3(+) treg cells in children with new onset type 1 diabetes and autoantibody-positive at-risk children reduced follicular regulatory t cells in spleen and pancreatic lymph nodes of patients with type 1 diabetes pathophysiology of inflammatory bowel diseases disease susceptibility genes shared by primary biliary cirrhosis and crohn's disease in the japanese population monocyte-like and mature macrophages produce cxcl13 (b cell-attracting chemokine 1) in inflammatory lesions with lymphoid neogenesis vagal innervation is required for the formation of tertiary lymphoid tissue in colitis ox40/ox40l interaction induces the expression of cxcr5 and contributes to chronic colitis induced by dextran sulfate sodium in mice primary biliary cholangitis: pathogenesis and therapeutic opportunities association study identifies 12 new susceptibility loci for primary biliary cirrhosis autoimmune bullous skin diseases, pemphigus and pemphigoid clinical presentation, and treatment of psoriasis: a review systemic sclerosis-associated interstitial lung disease single-cell transcriptome analysis identifies skin-specific t-cell responses in systemic sclerosis mechanistic insights into autoimmune pancreatitis and igg4-related disease autoimmunity and infection in common variable immunodeficiency (cvid) plasma protein profiling reflects t(h)1-driven immune dysregulation in common variable immunodeficiency circulating plasma biomarkers of survival in antifibrotic-treated patients with idiopathic pulmonary fibrosis pneumocystis-driven inducible bronchus-associated lymphoid tissue formation requires th2 and th17 immunity tnf-a and il-10 control cxcl13 expression in human macrophages cxcl13 as a marker for outcome of rheumatoid arthritis: comment on the article by meeuwisse et al serum levels of cxcl13 are elevated in active multiple sclerosis cxcl13 is a biomarker of inflammation in multiple sclerosis, neuromyelitis optica, and other neurological conditions conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest we would like to thank editage (www.editage.cn) for english language editing. figures 2 and 4 were produced using servier medical art (www.servier.com). key: cord-0028379-w4okyd70 authors: paul, alok k.; jahan, rownak; paul, anita; mahboob, tooba; bondhon, tohmina a.; jannat, khoshnur; hasan, anamul; nissapatorn, veeranoot; wilairatana, polrat; de lourdes pereira, maria; wiart, christophe; rahmatullah, mohammed title: the role of medicinal and aromatic plants against obesity and arthritis: a review date: 2022-02-25 journal: nutrients doi: 10.3390/nu14050985 sha: 299c04b1526c19c41f0f6e59465342a958bb98e8 doc_id: 28379 cord_uid: w4okyd70 obesity is a significant health concern, as it causes a massive cascade of chronic inflammations and multiple morbidities. rheumatoid arthritis and osteoarthritis are chronic inflammatory conditions and often manifest as comorbidities of obesity. adipose tissues serve as a reservoir of energy as well as releasing several inflammatory cytokines (including il-6, ifn-γ, and tnf-α) that stimulate low-grade chronic inflammatory conditions such as rheumatoid arthritis, osteoarthritis, diabetes, hypertension, cardiovascular disorders, fatty liver disease, oxidative stress, and chronic kidney diseases. dietary intake, low physical activity, unhealthy lifestyle, smoking, alcohol consumption, and genetic and environmental factors can influence obesity and arthritis. current arthritis management using modern medicines produces various adverse reactions. medicinal plants have been a significant part of traditional medicine, and various plants and phytochemicals have shown effectiveness against arthritis and obesity; however, scientifically, this traditional plant-based treatment option needs validation through proper clinical trials and toxicity tests. in addition, essential oils obtained from aromatic plants are being widely used as for complementary therapy (e.g., aromatherapy, smelling, spicing, and consumption with food) against arthritis and obesity; scientific evidence is necessary to support their effectiveness. this review is an attempt to understand the pathophysiological connections between obesity and arthritis, and describes treatment options derived from medicinal, spice, and aromatic plants. obesity can be characterized as a body mass index (bmi) of 25 or more in adults, who are classified as overweight, or a bmi of 30 or more, classified as obesity [1] . in 2016, around 1.9 billion adults (aged > 18 years) were overweight, but >650 million people were (e.g., transforming growth factor β 2 ), food antigens, and essential microbiota supplements (e.g., non-sterile breast milk contains long-chain polyunsaturated fatty acids (lcpufas), which impede the production of proinflammatory cytokines [24] [25] [26] (figure 1 ). human milk intake in infancy can also protect children against various pathogens, including but not limited to bordetella pertussis, campylobacter, haemophilus, salmonella, streptococcus, shigella, vibrio cholerae, and respiratory viruses [23, 27, 28] . noticeably, formula-fed children show reduced transforming growth factor β 2 compared to breast milk-fed children; instead, they display higher levels of plasma proinflammatory cytokines (e.g., il-2 and tnf-α) than breast-fed children [29] . inadequate intake of lcpufas in the body/diet can influence the development of obesity and arthritis (figure 1 ). resistin-an adipokine-along with other senescence-associated secretory phenotype factors, regulates glucose metabolism, oxidative stress, inflammatory responses, and autoimmune diseases. [30, 31] . high plasma resistin concentrations can increase the possibility of inflammation, insulin resistance, and the aging process [30] . resistin possibly interacts with tlr-4 receptors, influences the transcription of proinflammatory genes, inflammatory cytokines, and chemokines, and causes osteoclastogenesis via the nf-κb pathway [30] . sedentary lifestyle and increased calorie intake are related to the progression of adipocyte hypertrophy and low-grade inflammation via the recruitment of antigen-producing cells in adipose tissues [31, 32] . resistin, adiponectin, tnf-α (released by adipocytes), and proinflammatory cytokines (e.g., il-1β, il-6) derived from adipokines increase muscle and bone metabolism [30, 32] . this biological pathway is responsible for the generation of several chronic diseases, including obesity, diabetes, and arthritis [32]. there are various types of arthritis, and these are multifactorial, with the common features of chronic intense pain and inflammation [33] . osteoarthritis (oa) is a chronic painful disorder that increases with age and is common in adults aged over 55 years [34] . the mechanisms of oa are not completely understood, but its clinical features include irreversible age-related damage to the joint cartilage, pain, and low-grade inflammation over a period of many years [35] . the pathogenesis of oa can also be caused by cellular stress produced by the activation of endogenous cytosolic proteins such as nucleotide-binding domain, leucine-rich repeat/pyrin domain-containing-3 (nalp3) inflammasome [36] [37] [38] [39] [40] , proinflammatory cytokines released by macrophages [41] [42] [43] , or the production of proinflammatory cytokines induced by uric-acid-crystal-induced inflammasome assembly [40] . there is a positive correlation between osteoarthritis (oa) severity, uric acid levels (in synovial fluid), and proinflammatory cytokines (e.g., il-18, il-1β) [38, 44] . monosodium uric acid (msu) can accumulate in joints as crystals when its plasma concentrations exceed its solubility (≥70 mg/l) [45] , stimulating the synthesis of different inflammatory cytokines [46] . the inflammatory processes are also triggered by chemokines, proteases, and oxidative materials that cause osteoporosis, cartilage degradation, and inflammation in the synovial joints [44, 47] . this process is further exaggerated when toll-like receptors recognize msu (monosodium urate crystals), and when lymphocytes and macrophages in synovial fluids uptake msu. these interactions ultimately release various inflammatory cytokines (especially il-1β, il-6, tnf-α, and il-18) via nucleotide-binding domain and the leucine-rich repeat/pyrin domain-containing-3 (nalp3) inflammasome [36] [37] [38] [39] [40] . oa is also influenced by calcium-oxalate-containing crystals that stimulate the production of il-1β, causing cartilage damage [48] . oa is also caused by mutations of genes encoding collagens (e.g., types ii, iv, v, and vi) [33, 49] . the pathogenesis of oa can cause neuronal damage in joint tissues, causing intense pain, limited mobility, depression, and anxiety in elderly people ( figure 2) [50,51]. people experiencing oa also often have multiple comorbidities, such as obesity [52] [53] [54] , diabetes [4] , cardiovascular diseases [3] , cancers [5] , and musculoskeletal disorders [2] . currently, analgesics such as non-steroidal anti-inflammatory drugs (nsaids) and corticosteroids are used to manage oa, but these drugs have no effect on the prevention of oa's pathogenesis, and they are mainly for symptomatic management. in addition, these drugs have adverse effects on the gut, liver, kidneys, and heart [55] [56] [57] [58] . long-term use of nsaids to manage arthritis provides poor pain relief, major discomfort for patients, and can lead to invasive procedures, such as surgeries [59] [60] [61] (figure 3) . a clinical study showed that paracetamol alone provided insufficient analgesia in oa, but an nsaid such as diclofenac (0.15 g daily) showed noticeable efficacy in oa management [62] . it is also important to note the adverse effects of diclofenac, including gastrointestinal toxicity, liver toxicity, and renal impairment ( figure 3 ). another study reported that celecoxib (an nsaid) caused lower cardiovascular, renal, and gastrointestinal adverse reactions than ibuprofen or naproxen (nsaids), which was similar in patients experiencing oa or ra [63] . it is understood that there are significant variations between drugs within a group of drugs (e.g., nsaids), and the efficacy of a particular drug may depend on its molecular structure, formulation, route of administration, dosage, and duration of treatment [64] . the bioactivity and efficacy of a drug also depend on its metabolic capabilities (e.g., hepatic or renal impairment, aging-related) and bioavailability at the target site. importantly, chronic treatment with nsaids for oa can result in adverse outcomes and cause adverse events in older adults [65, 66] . opioids are not recommended to manage oa [51,67], but these drugs are widely used in oa-related chronic pain management for older adults, despite their potential adverse effects-such as addiction, dependence, analgesic tolerance, respiratory depression, and behavioral disorders over long-term usages ( figure 3 ) [66, [68] [69] [70] [71] . routes of administration of commonly used drugs for the treatment of oa, and some adverse effects associated with these drugs. abbreviations-nsaids: non-steroidal anti-inflammatory drugs (nsaids); mab: monoclonal antibody. this figure was made with www.biorender.com (accessed on 25 january 2022). despite differences in the initiation and progression mechanisms between oa and ra-the latter of which is another type of arthritis that is multifactorial, and whose root causes remain to be elucidated-long-term low-grade inflammation is the common ground in the pathogenesis of obesity, oa, and ra [52] [53] [54] 72] . as with the pathogenesis of oa, ra manifests with increased secretion of proinflammatory cytokines (e.g., il-1, il-6, il-12, il-17, il-18, and tnf-α). in parallel, secretion of immunomodulatory cytokines (e.g., il-10, il-11, and il-13) is reduced in the blood, along with stiffness, swollen joints, and impaired movement of the affected person [73] [74] [75] [76] (figure 1 ). defensive cells, such as t helper 1 (th1) and t helper 17 (th17) cells, produce an inflammatory response via il-17a, ifn-γ, and tnf-α, leading to the pathogenesis of ra [77, 78] . toll-like receptors (tlrs) regulate the functions of the nuclear factor kappa b ligand (nf-κb), osteoclastogenesis, and generation of proinflammatory cytokines [79] [80] [81] . as a result, joint pain, inflamed joints, and damage to cartilage can be seen during clinical symptoms of ra [82] . inflammatory cytokines such as il-17 or tnf-α can influence the upregulation of matrix metalloproteinase (mmp) enzymes, which irreversibly damage the extracellular matrix and the cartilage of joints [74, 83] (figure 1 ). apart from inflammatory or genetic mechanisms, fat-rich food intake, smoking, and periodontal infections also affect the generation and progression of ra [84, 85] . women are more prone to ra than men. citrullination of proteins in lung macrophages, along with neuropathic pain and osteoporosis, can potentially influence the pathogenesis of ra [84, [86] [87] [88] . ra also manifests as a result of excess inflammatory cytokines, with the influence of major changes in the microbial population of the gut. for example, faecalibacterium spp. are a part of the healthy gut microbiota that is responsible for butyrate production [89] [90] [91] , and helps in the secretion of mucin-a natural lubricator of gut epithelial cells. if the abundance of faecalibacterium spp. decreases, other opportunistic bacteria such as collinsella, eggerthella, haemophilus, prevotella, and streptococcus can grow and produce inflammatory cytokines and/or cause citrullination of proteins, leading to ra [90, 92] . prevotella copri (p. copri) is a part of our normal gut microbiota and oral cavity, and can grow massively with the influence of change in diet, stress, lack of oral hygiene, and microbial infection [85, [93] [94] [95] . as a result, p. copri can cause increased production of t helper cells (e.g., th1, th17) and inflammatory cytokines (e.g., il-1β, il-6, il-17, and il-23), leading to an inflammatory response in the gut, and can possibly migrate to inflammatory joint tissues [96, 97] (figure 1 ). prevotella spp. can produce increased prostaglandin e2 in joint tissues, and has been observed in ra, causing joint pain, inflammation, and bone degradation [98, 99] (figure 1 ). the simultaneous growth of porphyromonas gingivalis in the mouth and p. copri in the intestine are noticed in ra patients [100] . p. gingivalis possibly translocates to synovial joints via phagocytosis, causes citrullination of proteins in joints, and increases inflammatory cytokine production [101, 102] . proper management and restoration of healthy gut microbiota by using probiotic supplements as food can reduce the population of prevotella spp. and increase the gut population of lactobacillus spp. [95] . oa and ra are both prevalent in older adults (>55 years), and especially in the elderly with frailty syndromes (e.g., falls, immobility, delirium, incontinence, and adverse effects of medications) [72, 103] . obesity is also a common comorbidity of this population cohort for various reasons, including inactivity, diet, diabetes, and aging [104, 105] . tumor necrosis factor α (tnf-α)-a proinflammatory cytokine-from the adipose tissues of obese animals can cause low-grade inflammation in adipocytes [52, 106] . adipose tissues mainly produce inflammatory biomarkers such as tnf-α, and macrophages and other immune cells are partially responsible for oxidative damage and low-grade inflammation in the body [52, 106, 107] . nlrp3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3)-a polyprotein complex inflammasome found in macrophages-is also responsible for releasing proinflammatory cytokines. nlrp3 is stimulated by the activation of nf-κb (nuclear factor kappa b, which tnf-α stimulates), and causes the secretion of the proinflammatory cytokines pro-il-1β and pro-il-18 [108] ( figure 4 ). nlrp3 is matured by pamps (pathogen-associated molecular patterns) and damps (damage-associated molecular patterns) or lipopolysaccharides. nlrp3 maturation stimulates the release of cytokines (e.g., il (interleukin)-1β, il-6, and il-18) and low-grade inflammation in multiple organs, including joints ( figure 4 ) [109] . clinical studies show strong positive connections between obesity, osteoarthritis, and rheumatoid arthritis [52, 53, [110] [111] [112] (figure 4 ). people with a body mass index of >30 kg/m 2 show higher incidence of knee oa than people of normal weight, and it is recommended to reduce weight in order to improve clinical symptoms of oa in obese patients [113, 114] . a clinical study showed that obesity was present (33.4%) in ra patients (n = 11,406) at a significantly higher rate than obesity (31.6%) in the control group (n = 54,701) [112] . obesity causes inflammation and autoimmune conditions in ra patients [112] . obese ra patients experience more tender joints and swelling in joints than non-obese ra patients [115] . obesity is a common comorbidity of ra patients, and it also reduces the efficacy of drugs working against tnf-α, but losing body weight improves the success of treatment with these drugs [111] . importantly, no association with bmi was found in this review with drugs other than anti-tnf-α drugs, such as biologics that act against il-6, cd4, or cd20 [111] . studies have reported that the ra patients experience lower grip strength and fatigue (40-80%), and these decrease their strength and their interest in being involved in various physical activities [116, 117] . similarly, a later study showed that patients who also experienced ra displayed fatigue (40%) and anxiety/depression (52%) as comorbidities [118] . obese ra patients experienced less remission (improvement of symptoms and pain relief) and lower disease activity scores than non-obese (control) ra patients [110] . van beers-tas et al. mentioned that reduced smoking increased arthritis remission, but obesity increased arthritis progression and delayed its remission [119] . another study on a small number (n = 19) of obese ra patients (aged 55 years on average; range: 34-71) observed that reduction in dietary energy intake and moderate physical exercise led to a 9% reduction in fat mass and improved physical fitness of the participants [120]. conversely, another study with a comparatively large number (n = 192) of participants (aged 64.5 years on average, range: 50-78) in a similar weightreduction program did not improve structural joint damage, muscle strength, or knee joint alignment, but achieved some benefits in terms of overall health improvement [121]. noticeably, age was an important factor in the performance of the participants, and there were differences in the measurements of performance, as the previous study measured outcomes such as the capability to ride a bicycle, whereas the later study investigated using mri and radiographs [120, 121] . collectively, management of obesity may improve the clinical symptoms of obese oa and ra patients. a few anti-obesity drugs have now been approved for human use, and most of these show various side effects. according to the national institute of diabetes and digestive and kidney diseases (niddk), the united states food and drug administration (usfda) has so far approved five drugs-namely, orlistat, phentermine/topiramate, lorcaserin, naltrexone/bupropion, and liraglutide-to treat obesity [122] . importantly, the european medicines agency (ema) has approved three drugs to fight symptoms of obesity: orlistat, bupropion/naltrexone, and liraglutide [123] . orlistat reduces intestinal absorption of fat content from food, as it is a pancreatic lipase inhibitor; side effects of this drug include diarrhea, oily stools, abdominal pain and, less frequently, cholelithiasis, cholestatic hepatitis, and subacute hepatitis [124] . people feel less hungry when using the drug combination phentermine/topiramate, as phentermine decreases one's appetite, while topiramate reduces seizures and migraine headaches. noticeably, this drug combination can cause serious side effects, including dysgeusia (taste alteration), paresthesia (burning sensation in hands and feet), hypoesthesia (loss of sensation of a body part), attention deficiency, dizziness, constipation, and dry mouth [125] . there are serious safety concerns with respect to the long-term efficacy of anti-obesity medications; the european medicines agency refused the approval of phentermine/topiramate, while for lorcaserin, authorization was previously withdrawn for a low overall benefit/risk ratio [126] . lorcaserin (for the risk of cancer), rimonabant, and sibutramine have been withdrawn from the us market for safety concerns [126, 127] . mitral regurgitation is a serious side effect of lorcaserin, and may lead to other complications, such as increased risk of cardiovascular complications [127, 128] . the naltrexone/bupropion drug combination has little effect against obesity individually. longterm opioid treatment causes various behavioral adverse effects, addiction, and tolerance, but naltrexone-as an opioid antagonist-shows efficacy against dependency on opioids and alcoholic beverages [64, 69, 122, 129] . patient management using these analgesics should also consider the reduced metabolic capability of people such as the elderly, or people suffering from chronic kidney or liver diseases [70, [130] [131] [132] [133] . bupropion is used for treating depression and for help with giving up smoking. individually, these drugs have no or little effect on obesity; used in combination, they form a safe anti-obesity polypharmacy drug with no serious side effects except for nausea [134, 135] . liraglutide-an anti-diabetic drug-works as an anti-obesity drug as well, and shows side-effects such as nausea, diarrhea, abdominal pain, and constipation. acute pancreatitis and rare thyroid tumors are severe adverse effects that may arise from the use of liraglutide [136] . rheumatoid arthritis (ra) is currently treated with disease-modifying anti-rheumatic drugs (dmards) such as methotrexate, non-steroidal anti-inflammatory drugs (such as paracetamol, ibuprofen, naproxen, diclofenac, indomethacin, ketoprofen, and meloxicam), janus kinase (jak) inhibitors (e.g., baricitinib and upadacitinib), anti-malarial drugs (e.g., hydroxychloroquine and chloroquine), tnf-α inhibitors, and glucocorticoids (e.g., prednisone, hydrocortisone, and dexamethasone). all of these drug types produce severe adverse effects ( figure 5 ), limiting their efficacy, and scientists are looking for safe alternative drugs or food supplements for the prevention or cure of ra [95, [137] [138] [139] . overall, anti-obesity drugs are effective in reducing body weight, but in consideration of their adverse effect profiles, the only possible alternative to these drugs is bariatric surgery, which also increases the risk of developing alcohol use disorders [140] . thus, scientists, naturopaths, and traditional medicinal practitioners are investigating some suitable plants that have the ability to reduce weight. a single plant may contain hundreds of secondary metabolites, a few of which may be effective against obesity. plants are readily available from nature; many plants can be cultivated and extracted to isolate active ingredients for various purposes. to select the information on medicinal or aromatic plants for use against obesity and rheumatoid arthritis (ra) for this review, data from recent literature (no time limit used) were gathered from the pubmed, scopus, and google scholar databases. the keywords used for the literature research included the terms obesity, anti-obesity, rheumatoid arthritis, ra, medicinal plant, essential oils, and preclinical and clinical studies. (table 1) . curcuma longa has shown anti-inflammatory and anti-arthritic effects in various clinical and preclinical studies. the rhizome of curcuma longa is traditionally used as a spice in indian cuisine and for medicines in ayurveda. the rhizome of this plant is known to be effective against asthma, allergies, rheumatism, liver disorders, and inflammation in ayurvedic medicines. a recent clinical trial demonstrated that 0.5 g twice daily consumption of curcuma longa extract (composition: 80% wt/wt aqueous-based extract standardized to turmerosaccharides, and 20% wt/wt curcuminoids) over 12-week period improved symptoms (such as knee pain using both the visual analogue scale (vas) and western ontario and mcmaster universities osteoarthritis index (womac) pain values) of patients experiencing symptomatic knee osteoarthritis and knee effusion synovitis [151] . boswellia serrata is known as an ayurvedic medicine used against rheumatic pain and inflammatory diseases. in recent years, several randomized clinical trials found that the extracts of boswellia serrata provided relief from arthritis-related pain and stiffness from knee osteoarthritis [155], reduced inflammatory cytokines, and improved western ontario and mcmaster universities osteoarthritis index and visual analog scale scores [157] . supplementation of 100 mg of boswellia serrata extract with 300 mg of hyaluronic acid (1 tab/day for 20 days) improved arthritis-pain-related visual analogue scales (e.g., the american knee society score (akss) and visual analogue scale (vas) for pain) [167] . curcuma longa (350 mg extract) and boswellia serrata extract (150 mg) twice daily for 12 weeks also improved oa pain in patients with moderate knee oa using the western ontario and mcmaster universities osteoarthritis (womac) index and visual analogue scale (vas) [168] . [166] ginger (rhizomes of zingiber officinale) improved ra by increasing expression of the forkhead-box-p3 (foxp3) gene, and by reducing the expression of retinoic-acid-receptorrelated orphan nuclear receptor gamma (rorγt) and t-bet genes [165] (figure 6 ). foxp3 is an essential transcription factor of regulatory t (t-reg) cells, and expression of this factor helps the development and function of t-reg cells [169] . activation of t-reg cells produces the immunomodulatory cytokines transforming growth factor (tgf)-β and interleukin-10 (il-10), and reduces inflammation [169] . the transcription factor rorγt is considered to be a major regulator of the differentiation of t helper 17 cells (th17 cells) and the production of il-17 family cytokines, which play an essential role in the development of a number of autoimmune disorders, including arthritis. t-bet is an immune cell transcription factor; in dendritic cells, t-bet reportedly regulates the production of the proinflammatory cytokine il-1α [170] . cumulatively, the decrease in the expression of these two transcription factor genes can be helpful in ameliorating ra ( figure 6 ) [171] . in another study with knee oa patients, 12 weeks of treatment with ginger (750 mg capsule) and ginger supplemented with diclofenac (750 mg capsule with a 50 mg diclofenac tablet) improved oa [163] . noticeably, no severe adverse effects were recorded in these studies. importantly, a tinospora cordifolia-, zingiber officinale-, and semecarpus anacardiumcontaining ayurvedic drug reportedly improved the symptoms of oa [158] . furthermore, another clinical study over a period of 24 weeks with co-treatment with an ayurvedic formulation containing mixed extracts of tinospora cordifolia, boswellia serrata, emblica officinalis, and zingiber officinale, along with glucosamine sulphate (2 g/day) and celecoxib (0.2 g/day), reduced symptoms of knee oa [160]. a three-month treatment with a herbal formulation containing mixtures of powders of withania somnifera (roots), boswellia serrata (stem), curcuma longa (rhizomes), and zinc produced better pain relief and reduced the disability scores of patients with knee oa [172] . commiphora species of plants have been used in traditional medicine as painkillers and anti-inflammatory agents. in a recent clinical trial, a tcm medicinal formulation containing extracts of commiphora myrrha (gum resin) and paeonia lactiflora (root) showed pain relief and no severe adverse effects when given over a period of 12 weeks in people experiencing knee oa [161] . piper nigrum is traditionally used in many ayurvedic formulations [166] . the main alkaloid of fruits of this plant is piperine. daily treatment twice for 4 weeks with herbal capsules containing curcumin (300 mg), gingerols (7.5 mg), and piperine (3.75 mg) reduced the prostaglandin e2 levels in people experiencing chronic knee oa [166] . another plant-typhonium trilobatum (l.) schott (ghatkul, ghetkun)-is known to have antiarthritic and anti-rheumatic effects (leaf and whole plant, respectively). a preclinical study confirmed that the plant showed anti-inflammatory and analgesic effects, but the study needs further evidence from actual clinical trials [173, 174] . it can be concluded that various trials have shown the efficacy of a number of ayurvedic and other traditional medicinal formulations in the treatment of arthritis; however, more clinical trials are necessary, as there are various discrepancies between the settings of clinical trials, such as the population sizes, types of patients, methodologies, and duration of treatment. using modern formulations, such as implementation of nanotechnology-based formulations, would increase the bioavailability of some of these phytochemicals (such as curcumin), as shown in experimental studies [175, 176] . in addition to the anti-arthritic plants mentioned above, mukhopadhyay et al. (2019), in their list of anti-arthritic plants, mentioned cuscuta reflexa roxb., piper longum l., coriandrum sativum l., cinnamomum zeylanicum blume, caesalpinia pulcherrima (l.) sw., asparagus racemosus willd., abutilon hirtum (lam.) sweet, terminalia pallida brandis, lawsonia inermis l., trigonella foenum-graecum l., punica granatum l., ruta graveolens l., terminalia chebula retz., sida rhombifolia l., xanthium strumarium l., vitex negundo l., lantana camara l., and citrullus colocynthis (l.) schrad. for their uses in the alleviation of arthritis [177] [178] [179] ; their phytoconstituents and other details are shown in table 2 . [190, 191] apocynaceae calotropis procera leaves, seeds, roots benzoyllineolone, benzolisolineolone [192] apocynaceae roots terpenoids [193] araliaceae acanthopanax chiisanensis leaves chiisanoside, chiisanogenin [194] araliaceae panax notoginseng ethanol extract, n-butanol extract ginsenoside [195] [196] [197] [198] asparagaceae anemarrhena asphodeloides roots mangiferin, polysaccharides, fructan [199, 200] asparagaceae asparagus racemosus hydroalcoholic extract shatavarin, saponin [201, 202] apart from the intake of traditional medicines, massages and complementary therapies using essential oils are also claimed by traditional medicinal practitioners (tmps) to improve the symptoms of various diseases-especially from arthritis or chronic pain. these beliefs stem from the practices and customs learned in various human societies over hundreds or thousands of years, and oral passage of the knowledge gained from generation to generation before the arrival of writing and record keeping on clay tablets, papyrus, or paper [353] . essential oils are volatile aromatic oils isolated from flowers, barks, leaves, and other parts of specific plants. many of these oils have antimicrobial, emollient, palatable, and lipophilic permeability through the skin. essential oils give people a good feeling at spiritual, physical (via massaging), and olfactory levels. the efficacy of these oils against chronic arthritic pain is yet to be established. some preliminary clinical trials with a few essential oils have shown some benefits against arthritis, but their efficacy over a long period of time is unknown (table 3 ). in our search of the pubmed search engine, we found four randomized clinical trials (table 3 ) of essential oil therapy. out of these four trials, aromatherapy with essential oils was used in two studies, whereas oral or gargling administrations were used in the other two trials [354] [355] [356] [357] . a recent systematic review reported that essential oils have been used to treat ra, mainly with a small number of subjects for a short duration of observation (2-12 weeks), and mostly with women (60-100%) [358] . the typical oil used for aromatherapy to treat ra was lavender, ginger, or rosemary oil, and a single study showed efficacy against ra [358] . table 3 . essential oils used to treat ra in randomized clinical trials (rcts). evening primrose oil patients with ra (n = 40 total) and nsaid-induced gi lesions treated with γ-linolenic acid 540 mg/day (evening primrose oil 6 g/day) for 3 months slightly improved ra-related morning stiffness. [355] lavender oil aromatherapy with lavender oil improved arthritic pain (against placebo) in patients (n = 30 each group) with knee osteoarthritis, but no proof of its long-term efficacy. [356] lavender oil aromatherapy with lavender oil improved daily routine activities of patients (n = 30 each group) with knee osteoarthritis (against placebo), but no proof of its long-term efficacy. [357] mouthwash with essential oils and curcumin gargling with mouthwash containing essential oils and curcumin (mec) over 6 weeks reduced periodontal disease and ra-related parameters (n = 15 each group) [354] the efficacy of essential oils in anti-obesity trials has been mostly based on in vitro experimental and preclinical studies (table 4 ). essential oils from various plants, flowers, leaves, and roots have been experimentally proven to be effective against obesity based on their anti-inflammatory effects in mice or rats via a common mechanism sharing the pathogenesis of obesity, oa, and ra (figures 1 and 2) . oral consumption of ginger or garlic oils, inhalation of certain species of lavender oil, or injections of certain citrus essential oils have been shown to result in reductions in body weight, lipid profile, fatty liver disease, and arthritis (table 4 and references therein). table 4 . essential oils used to treat obesity and arthritis in preclinical trials. garlic essential oil daily consumption of garlic essential oil (25, 50, and 100 mg/kg) or diallyl disulfide (10 and 20 mg/kg) for 12 weeks in c57bl/6j mice prevented the development of non-alcoholic fatty liver disease. the oil and its major compound also significantly prevented the release of proinflammatory cytokines from murine livers. [359] ginger essential oil ginger essential oil (28 mg/kg/day i.p. for 4 weeks) treatment improved joint inflammation caused by streptococcal cell-wall-induced arthritis in female lewis rats. [351] pogostemon cablin benth. or patchouli essential oil inhalation of the oil reduced food intake, systolic blood pressure, and plasma low-density lipoprotein cholesterol levels in sd rats. [360] rhaponticum acaule (l.) dc. treatment inhibited xanthine oxidase and turkey pancreatic lipase, thus reducing oxidative stress and pancreatitis. [361] ginger essential oil (geo) male c57bl/6j mice with a high-fat diet (hfd) mixed with geo (12.5, 62.5, and 125 mg/kg) or citral (2.5 and 25 mg/kg) for 12 weeks showed improved hfd-induced obesity by reducing triglyceride and total cholesterol levels. in addition, the treatment reduced inflammatory response in murine liver. [362] pinus koraiensis siebold and zucc. leaf essential oil treatment inhibited the level of cholesterol acyltransferase-1 and -2, as well as low-density lipoprotein (ldl) oxidation activity; thus, it may act against hyperlipidemia. [363] citrus aurantifolia (christm.) swingle essential oil forty-five days of treatment with this oil (125 mg/kg/day, s.c.) prevented ketotifen (32 mg/kg/day s.c.)-induced body weight gain and food intake in mice. [364] artemisia annua l. essential oil treatment reduced obesity-related ppar-γ, c/ebp-α, srebp-1c, fas, and acc levels in vitro using 3t3-li cells. [365] lavandula pubescens decne. essential oil l. pubescens eo was assessed against pancreatic lipase inhibitory activity with an ic 50 of 1.08 µl/ml (in vitro). [366] various plant materials produced better results in terms of anti-obesity and antiinflammatory properties compared to their respective placebo or control groups, as observed in various clinical studies. all of these plants are indispensable parts of different traditional and complementary medicines, and recently, in various randomized clinical trials, they have shown some promising results against obesity and/or arthritis (table 5 ). nigella sativa oil daily consumption of capsules with oil (2 mg/day over a period of 8 weeks) improved hdl-c and lowered ldl-c and tc/hdl-c ratio compared to placebo in obese and overweight women. [367] nigella sativa oil patients were given a low-calorie diet supplemented with nigella sativa oil (3 g/day for 8 weeks) (n = 45 each group), which reduced tnf-α and hepatic c-reactive protein levels, but no changes were observed in plasma il-6 levels in obese (bmi: 30-35 kg/m 2 ) women aged 25-50 years old. [368] opuntia ficus-indica natural fiber complex (litramine) was 3 g/day with a low-calorie diet for 12 weeks, which reduced body weight compared to placebo in obese women (total n = 133) [369] camellia sinensis green tea (n = 32; 1 g of dry green tea extract in capsule/day) reduced total cholesterol (tc) and ldl-c after 12 weeks of treatment in non-diabetic obese women. [370] saffron reduced hyperglycaemia and hyperlipidaemia and improved liver function in patients with type 2 diabetes in an 8-week randomized clinical trial. [371] laminaria digitata (brown seaweed) treatment with sodium alginate from laminaria digitata over a period of 10 days showed no effects in an anti-obesity related trial. [372] lycium barbarum (fruit juice) a single-day bolus drink increased metabolic rate; 120 ml of fruit juice per day for 2 weeks reduced waist circumference in overweight men and women (n = 15, bmi = 29, age = 34 years). [373] consumption of 1.6 g of garlic powder (4 × 400 mg tablets daily, for 12 weeks) produced significant decreases in waist circumference and body fat percentage in patients with non-alcoholic fatty liver disease (n = 45). [374] allium cepa onion powder (9 g per day for 12 weeks) did not cause any major changes between groups. [375] avocados are a natural source of lutein. daily oral consumption of 300 mg/day of asu-e (avocado-soybean unsaponifiables, expanscience-a formula with a 1:3 ratio of avocado: soybean oil) for 3 years did not cause any changes in joint space width loss compared to the placebo group. [376] oral consumption of momordica charantia (3 × 500 mg per capsule daily for 3 months) taken thrice daily reduced body weight, body mass index, fasting blood glucose levels, and knee injury and osteoarthritis outcome scores. [377] consumption of (n = 35) aqueous extract of cissus quandrangularis (300 mg/day, over 8 weeks) reduced body fat and improved blood parameters related to metabolic syndrome in overweight patients. [378] flavonoids are natural polyphenolic compounds with antioxidant, anti-inflammatory, and antiviral properties, as well as protective effects on the gastrointestinal tract [379] [380] [381] [382] . apigenin, cyanidin, (-)-epigallocatechin-3-o-gallate (egcg), genistein, kaempferol, luteolin, puerarin, and quercetin are all antioxidants (table 6) ; therefore, these compounds demonstrate an inverse relationship between oxidative stress and arthritis, with or without obesity [383] [384] [385] [386] [387] . metabolic syndrome (mets) is a combination of obesity along with high blood pressure and diabetes; obesity can be a driving factor behind the occurrence of mets. it is said that around one-quarter of the world's adult population now suffers from mets. there are conventional drugs for the treatment of obesity, such as orlistat or semaglutide, but these drugs either have adverse effects or are not affordable to the general obese people of low-income countries (lics) and low-middle-income countries (lmics) [398] . to reduce obesity, the common, illiterate people, with less means to afford expensive conventional drugs, mostly rely on tmps, who treat obesity, cardiovascular disorders, and diabetes with medicinal plants. a recent survey lists 16 plants/plant parts used in south africa for weight loss [398] . these plants include leaves of aloe vera mill., rosmarinus officinalis l., and moringa oleifera lam. over 20 plants used to reduce obesity were listed in a review published in 2013; the authors concluded that among the significant anti-obesity plants were cissus quandrangularis l., asparagus officinalis l., and zingiber officinale roscoe [399] . another review listed curcuma longa l. rhizomes (active ingredient curcumin) and leaves of salvia officinalis l. (active ingredient: carnosic acid) as anti-obesity plant parts [400] . obesity as a disorder has been recognized in ayurveda-the ancient medical treatise of india-where it is described as "meda". some ayurvedic plants/plant parts used to treat obesity in india include the fruits of garcinia cambogia l. (active ingredient: (-)-hydroxycitric acid), cyperus rotundus l. rhizomes (active ingredient: cyperine), the roots of embelia ribes burm.f., whole plants of boerhaavia diffusa l., seeds of achyranthes aspera l., and roots of withania somnifera (l.) dunal. [401] . table 6 . role of flavonoids against obesity and arthritis (preclinical and clinical studies). apigenin ra was induced by 0.1 ml freund's complete adjuvant (fca) injections in the palmar surface of paws of sprague-dawley (sd) rats. apigenin suppressed the expressions of p2x7/nf-κb signaling and associated ra-related inflammatory reactions (e.g., reduced il-1β, il-6 and tnf-α) [388] apigenin ra was induced in a murine collagen-induced arthritis (cia) model. apigenin inhibited cia by repressing synovial hyperplasia (by reducing the multiplication of fibroblast-like synoviocytes), causing the growth of new blood vessels and osteoclastogenesis. [389] the effects of cyanidin-3-o-glucoside were investigated in a murine high-fat-diet-induced non-alcoholic fatty liver disease (nafld) model. treatment with this flavone reduced nlrp3 inflammasome activation, oxidative stress, and steatosis in mice. [390] table 6 . cont. over a period of 3 days, 300 mg of egcg drink increased postprandial fat oxidation in obese men similarly to 200 mg of caffeine, but the effect was not observed with 600 mg of egcg drink. limitation: total n = 10, pilot study. [391] (-)-epigallocatechin-3-o-gallate (egcg) consumption of egcg and resveratrol (282 mg and 80 mg/day over a period of 12-week accordingly) increased oxidative capacity in permeabilized muscle fibers, but showed reduced plasma triacylglycerol concentration in a high-fat mixed-meal assay in obese men (n = 18). [392] genistein consumption of 15 g of genistein for 3 months (5 days of daily administration per week plus 2 days without treatment) in adult patients (53% men) reduced blood glucose and malondialdehyde levels, but did not impact on lipid profile. [393] kaempferol treatment with 200 mg/kg of kaempferol (over eight weeks) with a high-fat diet in c57bl/6 mice reduced the increases in body and liver weight, serum cholesterol, and triglyceride levels [394] luteolin luteolin increased the expression of liver x receptor (lxr)-α (in vitro). luteolin (0.05% w/w in high fat diet) reduced plasma cholesterol and lowand very-low-density lipoprotein cholesterols in male c57bl/6 mice. [395] puerarin obese women with polycystic ovary syndrome (pcos) took 150 mg/d of puerarin tablets for 3 months in addition to their standard treatment, and showed decreased total cholesterol and systolic blood pressure compared with their pre-treatment levels. [396] quercetin quercetin (500 mg/day for 8 weeks) reduced ra symptoms (based on an assessment questionnaire) and high-sensitivity tumor necrosis factor α (hs-tnf-α) in women with ra. [397] some natural anti-obesity agents have been described from dietary sources. these include flavonoids from citrus depressa hayata, anthocyanins from vaccinium ashei rehder and morus australis poir., and gingerol, paradol, and shogaol from zingiber officinale roscoe [402] . it is evident from several clinical and preclinical trials that essential oils or extracts from aromatic and medicinal plants demonstrate potential therapeutic value against obesity and arthritis (tables 1-5) . these plants and phytochemicals should be considered as functional foods rather than therapeutics, and warrant further extensive clinical studies for dosage and safety determinations for chronic conditions. importantly, traditional medicines have been used as medicines and foods since prehistoric times. a number of these plant materials (e.g., flavonoids) are used almost every day as a part of our foods, drinks, or spices, and their consumption as medications or therapeutic supplements can help people to avoid the severity of obesity or arthritis ( table 6 ). the famous greek physician hippocrates in 440 bc stated "let food be thy medicine, and let medicine be thy food", which is still applicable today. whether knowingly or unknowingly, human beings do consume at least some bioactive compounds with their daily diet. traditional medicinal doctors and even scientists recommend that certain foods are beneficial during certain dis-eases. although the daily intake of plants containing requisite phytochemicals for a given disorder is also recommended by the authors (tables 1, 3, 5 and 6) , we would like to point out that such intake should have scientific evidence behind it, including determination of dosage, frequency of eating, toxicity, and any adverse reactions when taken alone or with other foods. we need to take a closer look at the dietary factors that influence obesity and other inflammatory diseases, obesity and the development of metabolic syndrome, and obesity itself. from this viewpoint, flavonoids such as quercetin, genistein, apigenin, and cyanidin deserve a closer look [403] . the association between musculoskeletal disorders and obesity. aust. health rev obésité et maladies cardiovasculaires. m/s méd. sci obesity and type 2 diabetes mellitus obesity and cancer risk: recent review and evidence mechanisms, pathophysiology, and management of obesity mechanisms and metabolic implications of regional differences among fat depots the pathophysiology of obesity and its clinical manifestations inflammatory cytokines in general and central obesity and modulating effects of physical activity adipokines: inflammation and the pleiotropic role of white adipose tissue adipokinome signatures in obese mouse models reflect adipose tissue health and are associated with serum lipid composition de novo lipogenesis in human fat and liver is linked to chrebp-β and metabolic health adipokines and non-alcoholic fatty liver disease: multiple interactions the adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ obesity and inflammation: the linking mechanism and the complications is obesity an inflammatory condition? nutrition a new way of looking at obesity circulating interleukin-6 in relation to adiposity, insulin action, and insulin secretion weight loss reduces interleukin-18 levels in obese women pro-inflammatory cytokines: the link between obesity and osteoarthritis osteoarthritis is a serious disease efficacy and safety of turmeric extracts for the treatment of knee osteoarthritis: a systematic review and meta-analysis of randomised controlled trials nonsteroidal antiinflammatory drug-related injury to the gastrointestinal tract: clinical picture, pathogenesis, and prevention piroxicam-β-cyclodextrin: a gi safer piroxicam non-steroidal anti-inflammatory drug use in chronic pain conditions with special emphasis on the elderly and patients with relevant comorbidities: management and mitigation of risks and adverse effects pain treatment in arthritis-related pain: beyond nsaids key questions concerning paracetamol and nsaids for osteoarthritis efficacy and safety of oral nsaids and analgesics in the management of osteoarthritis: evidence from real-life setting trials and surveys effectiveness of non-steroidal anti-inflammatory drugs for the treatment of pain in knee and hip osteoarthritis: a network meta-analysis differences in safety of nonsteroidal antiinflammatory drugs in patients with osteoarthritis and patients with rheumatoid arthritis. arthritis rheumatol morphine dosing strategy plays a key role in the generation and duration of the produced antinociceptive tolerance towards a mechanism-based approach to pain management in osteoarthritis management of osteoarthritis during the covid-19 pandemic guideline for the management of knee and hip osteoarthritis prevalence of opioid and benzodiazepine prescriptions for osteoarthritis opioid analgesia and opioid-induced adverse effects: a review pain management in older adults: facts to consider non-surgical treatment of osteoarthritis-related pain in the elderly rheumatoid arthritis in the elderly: characteristics and treatment considerations il-29 enhances toll-like receptor-mediated il-6 and il-8 production by the synovial fibroblasts from rheumatoid arthritis patients anti cytokine therapy in chronic inflammatory arthritis discovery of il-6 and development of anti-il-6r antibody understanding the role of cytokines in the pathogenesis of rheumatoid arthritis molecular mechanisms underpinning t helper 17 cell heterogeneity and functions in rheumatoid arthritis rheumatoid arthritis: recent advances on its etiology, role of cytokines and pharmacotherapy toll-like receptor pathways in autoimmune diseases toll-like receptor 2 (tlr2) induces migration and invasive mechanisms in rheumatoid arthritis investigating the role of toll-like receptors in models of arthritis the pathogenesis of rheumatoid arthritis matrix metalloproteinases and synovial joint pathology a new model for an etiology of rheumatoid arthritis: smoking may trigger hla-dr (shared epitope)-restricted immune reactions to autoantigens modified by citrullination infectious triggers in periodontitis and the gut in rheumatoid arthritis (ra): a complex story about association and causality. front sex and management of rheumatoid arthritis estrogens in rheumatoid arthritis; the immune system and bone insights into the study and origin of the citrullinome in rheumatoid arthritis action and function of faecalibacterium prausnitzii in health and disease an expansion of rare lineage intestinal microbes characterizes rheumatoid arthritis faecalibacterium prausnitzii: a next-generation probiotic in gut disease improvement. can the oral and gut microbiome in rheumatoid arthritis patients: a systematic review the relationship of prevotella intermedia, prevotella nigrescens and prevotella melaninogenica in the supragingival plaque of children, caries and oral malodor periodontitis and rheumatoid arthritis: the same inflammatory mediators? probiotics and amelioration of rheumatoid arthritis: significant roles of lactobacillus casei and lactobacillus acidophilus. microorganisms dysbiosis contributes to arthritis development via activation of autoreactive t cells in the intestine the immune response to prevotella bacteria in chronic inflammatory disease prevotella intermedia induces prostaglandin e2 via multiple signaling pathways cyclooxygenase in biology and disease oral-gut microbiota and arthritis: is there an evidence-based axis? arthritic role of porphyromonas gingivalis in collagen-induced arthritis mice microbial carriage state of peripheral blood dendritic cells (dcs) in chronic periodontitis influences dc differentiation, atherogenic potential frailty syndrome in rheumatoid arthritis and symptomatic osteoarthritis: an emerging concept in rheumatology obesity and weight management in the elderly obesity and weight management in the elderly: a focus on men does oxidative stress management help alleviation of covid-19 symptoms in patients experiencing diabetes? the nlrp3 inflammasome instigates obesity-induced inflammation and insulin resistance modulatory mechanisms of the nlrp3 inflammasomes in diabetes. biomolecules impact of obesity on remission and disease activity in rheumatoid arthritis: a systematic review and meta-analysis efficacy and tolerability of ginger (zingiber officinale) in patients of osteoarthritis of knee the amazing and mighty ginger the effect of ginger supplementation on some immunity and inflammation intermediate genes expression in patients with active rheumatoid arthritis herbal formulation "turmeric extract, black pepper, and ginger" versus naproxen for chronic knee osteoarthritis: a randomized, double-blind, controlled clinical trial clinical comparison of oral administration and viscosupplementation of hyaluronic acid (ha) in early knee osteoarthritis clinical evaluation of a formulation containing curcuma longa and boswellia serrata extracts in the management of knee osteoarthritis regulatory t cell and forkhead box protein 3 as modulators of immune homeostasis transcription factor t-bet regulates inflammatory arthritis through its function in dendritic cells antagonizing retinoic acid-related-orphan receptor gamma sctivity blocks the t helper 17/interleukin-17 pathway leading to attenuated pro-inflammatory human keratinocyte and skin responses treatment of osteoarthritis with a herbomineral formulation: a double-blind, placebo-controlled, cross-over study a survey of plant items eaten by the low income groups of the rural population of talbunia village in bagerhat district, bangladesh with an account of their folk medicinal applications analgesic, anti-inflammatory and anti-diarrheal activities of ethanolic leaf extract of typhonium trilobatum l. schott. asian pac curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model therapeutic potential of hyaluronic acid/chitosan nanoparticles for the delivery of curcuminoid in knee osteoarthritis and an in vitro evaluation in chondrocytes antiarthritic medicinal plants: a review. res anti-inflammatory and anti-arthritic activity of type-a procyanidine polyphenols from bark of cinnamomum zeylanicum in rats ameliorative effects of a polyphenolic fraction of cinnamomum zeylanicum l. bark in animal models of inflammation and arthritis andrographolide benefits rheumatoid arthritis via inhibiting mapk pathways efficacy of an andrographis paniculata composition for the relief of rheumatoid arthritis symptoms: a prospective randomized placebo-controlled trial allium sativum, rosmarinus officinalis, and salvia officinalis essential oils: a spiced shield against blowflies effect of the milk extract of semecarpus anacardium nut on adjuvant arthritis-a dose-dependent study in wistar albino rats anti-inflammatory effect of semecarpus anacardium linn. nut extract in acute and chronic inflammatory conditions evaluation of antioxidant effect of semecarpus anacardium linn. nut extract on the components of immune system in adjuvant arthritis immunomodulatory activity of semecarpus anacardium extract in mononuclear cells of normal individuals and rheumatoid arthritis patients a standardized extract of centella asiatica (eca 233) prevents temporomandibular joint osteoarthritis by modulating the expression of local inflammatory mediators in mice intra-articular route for the system of molecules 14g1862 from centella asiatica: pain relieving and protective effects in a rat model of osteoarthritis attenuation of collagen induced arthritis by centella asiatica methanol fraction via modulation of cytokines and oxidative stress evaluation of disease modifying activity of coriandrum sativum in experimental models anti-granuloma activity of coriandrum sativum in experimental models a review on plants having anti-arthritic potential anti-arthritis activity of roots of hemidesmus indicus r.br. (anantmul) in rats. asian pac antiinflammatory effects of chiisanoside and chiisanogenin obtained from the leaves of acanthopanax chiisanensis in the carrageenan-and freund's complete adjuvant-induced rats n-butanol extracts of panax notoginseng suppress lps-induced mmp-2 expression in periodontal ligament fibroblasts and inhibit osteoclastogenesis by suppressing mapk in lps-activated raw264.7 cells panax notoginseng saponins alleviate osteoporosis and joint destruction in rabbits with antigen-induced arthritis panax notoginseng saponins prevent senescence and inhibit apoptosis by regulating the pi3k-akt-mtor pathway in osteoarthritic chondrocytes anti-arthritic effect of ginsenoside rb1 on collagen induced arthritis in mice anemarrhenae asphodeloides rhizoma extract enriched in mangiferin protects pc12 cells against a neurotoxic agent-3-nitropropionic acid the analgesic and anti-inflammatory effect of win-34b, a new herbal formula for osteoarthritis composed of lonicera japonica thunb and anemarrhena asphodeloides bunge in vivo in-vivo anti-inflammatory and anti-arthritic activity of asparagus racemosus roots chemical constituents of asparagus yuccalechins a-c from the yucca schidigera roezl ex ortgies bark: elucidation of the relative and absolute configurations of three new spirobiflavonoids and their cholinesterase inhibitory activities anti-inflammatory and anti-arthritic effects of yucca schidigera: a review influence of phenolic constituents from yucca schidigera bark on arachidonate metabolism in vitro immunosuppressive properties of pluchea lanceolata leaves pluchea lanceolata (rasana): chemical and biological potential of rasayana herb used in traditional system of medicine protective effect of pluchea lanceolata against aluminum chloride-induced neurotoxicity in swiss albino mice efficacy of xixiancao (herba siegesbeckiae orientalis) on interactions between nuclear factor kappa-b and inflammatory cytokines in inflammatory reactions of rat synovial cells induced by sodium urate topical anti-inflammatory and analgesic activity of kirenol isolated from siegesbeckia orientalis convergence chromatographic determination of camphor in the essential oil of tanacetum parthenium l ): a systematic review parthenolide inhibits proliferation of fibroblast-like synoviocytes in vitro immunomodulatory activity of acidic polysaccharides isolated from tanacetum vulgare l antioxidant principles of tanacetum vulgare l. aerial parts anti-arthritic activity of xanthium strumarium l. extract on complete freund's adjuvant induced arthritis in rats traditional uses, botany, phytochemistry, pharmacology, pharmacokinetics and toxicology of xanthium strumarium l.: a review pdk1 in nf-κb signaling is a target of xanthium strumarium methanolic extract-mediated anti-inflammatory activities berberine inhibits the gene expression and production of proinflammatory cytokines by mononuclear cells in rheumatoid arthritis and healthy individuals study on the anti-inflammatory action of berberis vulgaris root extract, alkaloid fractions and pure alkaloids effectiveness of a hydroxynaphthoquinone fraction from arnebia euchromain rats with experimental colitis activity study of a hydroxynaphthoquinone fraction from arnebia euchroma in experimental arthritis bromelain, the enzyme complex of pineapple (ananas comosus) and its clinical application. an update proteinase activity and stability of natural bromelain preparations. int. immunopharmacol antiinflammatory activities of the triterpene acids from the resin of boswellia carteri boswellia frereana (frankincense) suppresses cytokine-induced matrix metalloproteinase expression and production of pro-inflammatory molecules in articular cartilage anti-arthritic and anti-inflammatory constituents from medicinal plants boswellia serrata extract attenuates inflammatory mediators and oxidative stress in collagen induced arthritis anti-arthritic activity of glycyrrhiza glabra, boswellia serrata and their synergistic activity in combined formulation studied in freund's adjuvant induced arthritic rats cassane diterpenoids from caesalpinia pulcherrima and their anti-inflammatory and α-glycosidase inhibitory activities evaluation of anti-arthritic activity of caesalpinia pulcherrima in freund's complete adjuvant induced arthritic rat model oral anti-inflammatory activity of cannabidiol, a non-psychoactive constituent of cannabis, in acute carrageenan-induced inflammation in the rat paw. naunyn-schmiedebergs arch cannabis-based medicinal products in arthritis, a painful conundrum patterns of medicinal cannabis use, strain analysis, and substitution effect among patients with migraine, headache, arthritis, and chronic pain in a medicinal cannabis cohort anti-inflammatory activity of some saudi arabian medicinal plants anti-arthritic active fraction of capparis spinosa l. fruits and its chemical constituents acute effect of capparis spinosa root extracts on rat articular pain anti-hyperuricemic and anti-gouty arthritis activities of polysaccharide purified from lonicera japonica in model rats a herbal formula comprising rosae multiflorae fructus and lonicerae japonicae flos, attenuates collagen-induced arthritis and inhibits tlr4 signalling in rats identification of novel compounds against three targets of sars cov-2 coronavirus by combined virtual screening and supervised machine learning gastroprotective and safety effects of win-34b, a novel treatment for osteoarthritis, compared to nsaids tripterygium wilfordii: an inspiring resource for rheumatoid arthritis treatment tripterygium wilfordii hook f. versus sulfasalazine in the treatment of rheumatoid arthritis: a well-designed clinical trial of a botanical demonstrating effectiveness therapeutic effect of tripterine on adjuvant arthritis in rats the chinese anti-inflammatory and immunosuppressive herbal remedy tripterygium wilfordii hook f. rheum tripterygium wilfordii inhibiting angiogenesis for rheumatoid arthritis treatment anti-inflammatory and lysosomal stability actions of cleome gynandra l. studied in adjuvant induced arthritic rats a placebo-controlled double-blind study demonstrates the clinical efficacy of a novel herbal formulation for relieving joint discomfort in human subjects with osteoarthritis of knee hydrolysable tannin-rich fraction from terminalia chebula retz. fruits ameliorates collagen-induced arthritis in balb/c mice anti-arthritic and analgesic effect of ndi10218, a standardized extract of terminalia chebula, on arthritis and pain model anti-arthritic and disease modifying activity of terminalia chebula retz. in experimental models the morphology, extractions, chemical constituents and uses of terminalia chebula: a review anti-inflammatory, anti-lipid peroxidative, antioxidant and membrane stabilizing activities of hydroalcoholic extract of terminalia chebula fruits anti-arthritic effect of scopoletin, a coumarin compound occurring in erycibe obtusifolia benth stems, is associated with decreased angiogenesis in synovium scopolin isolated from erycibe obtusifolia benth stems suppresses adjuvant-induced rat arthritis by inhibiting inflammation and angiogenesis anti-inflammatory and analgesic activities of tunisian citrullus colocynthis schrad. immature fruit and seed organic extracts screening of analgesic and anti-inflammatory activities of citrullus colocynthis from southern tunisia the analgesic and anti-rheumatic effects of thladiantha dubia fruit crude polysaccharide fraction in mice and rats in vitro evaluation of anti-arthritic activity in different solvent extracts from cuscuta reflexa evaluation of anti-rheumatoid activity of cuscuta reflexa in freund's adjuvant induced arthritic rats a parasitic medicinal plant cuscuta reflexa: an overview extracts and constituents of lavandula multifida with topical anti-inflammatory activity anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of sophora flavescens the protective effect of sophocarpine in osteoarthritis: an in vitro and in vivo study anti-inflammatory and antioxidative effects of mucilage of trigonella foenum graecum (fenugreek) on adjuvant induced arthritic rats evaluation of anti-arthritic potential of trigonella foenum graecum l. (fenugreek) mucilage against rheumatoid arthritis effect of ethanol extract of trigonella foenum graecum (fenugreek) seeds on freund's adjuvant-induced arthritis in albino rats essential oil composition of lavandula dentata, l. stoechas and l. multifida cultivated in tunisia leucas aspera inhibits the dalton s ascitic lymphoma in swiss albino mice: a preliminary study exploring possible mechanism of action modulation of inflammatory markers by the ethanolic extract of leucas aspera in adjuvant arthritis water soluble compounds of rosmarinus officinalis l. improve the oxidative and inflammatory states of rats with adjuvant-induced arthritis evaluation of the antinociceptive effect of rosmarinus officinalis l. using three different experimental models in rodents carnosic acid inhibits inflammation response and joint destruction on osteoclasts, fibroblast-like synoviocytes, and collagen-induced arthritis rats carnosic acid (ca) attenuates collagen-induced arthritis in db/db mice via inflammation suppression by regulating ros-dependent p38 pathway. free radic pf2401-sf, standardized fraction of salvia miltiorrhiza shows anti-inflammatory activity in macrophages and acute arthritis in vivo salvia miltiorrhiza injection restores apoptosis of fibroblast-like synoviocytes cultured with serum from patients with rheumatoid arthritis identification of the molecular mechanisms of salvia miltiorrhiza relevant to the treatment of osteoarthritis based on network pharmacology therapeutic effects of standardized vitex negundo seeds extract on complete freund's adjuvant induced arthritis in rats therapeutic effects of the total lignans from vitex negundo seeds on collagen-induced arthritis in rats norisoboldine suppresses osteoclast differentiation through preventing the accumulation of traf6-tak1 complexes and activation of mapks/nf-κb/c-fos/nfatc1 pathways norisoboldine, an anti-arthritis alkaloid isolated from radix linderae, attenuates osteoclast differentiation and inflammatory bone erosion in an aryl hydrocarbon receptor-dependent manner norisoboldine ameliorates collagen-induced arthritis through regulating the balance between th17 and regulatory t cells in gut-associated lymphoid tissues cechinel-filho, v. antiinflammatory and anti-hyperalgesic evaluation of the condiment laurel (litsea guatemalensis mez.) and its chemical composition anti-arthritic activity of bartogenic acid isolated from fruits of barringtonia racemosa roxb. (lecythidaceae). evid.-based complement therapeutic effect of nux vomica total alkali gel on adjuvants arthritis rats a review on medicinal uses, analytical techniques and pharmacological activities of strychnos nux-vomica linn.: a concise report analgesic and anti-inflammatory properties of brucine and brucine n-oxide extracted from seeds of strychnos nux-vomica evaluation of antiarthritic activity of butanol fraction of punica granatum linn. rind extract against freund's complete adjuvant-induced arthritis in rats consumption of hydrolyzable tannins-rich pomegranate extract suppresses inflammation and joint damage in rheumatoid arthritis therapeutic applications of pomegranate (punica granatum l.): a review potential effects of pomegranate (punica granatum) on rheumatoid arthritis: a systematic review anti-arthritic activity of abutilon hirtum phytochemical and pharmacological profile of abutilon indicum l. sweet: a review anti-arthritic activity of various extracts of sida rhombifolia aerial parts anti-inflammatory and anti-oxidant properties of sida rhombifolia stems and roots in adjuvant induced arthritic rats cyclin-dependent kinase inhibition by flavoalkaloids unsaponifiable fraction of unripe fruits of olea europaea: an interesting source of anti-inflammatory constituents tunisian olea europaea l. leaf extract suppresses freund's complete adjuvant-induced rheumatoid arthritis and lipopolysaccharide-induced inflammatory responses the role of omega-3 fatty acids contained in olive oil on chronic inflammation olea europaea leaf (ph.eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase double-blind placebo-controlled trial of hydroxytyrosol of olea europaea on pain in gonarthrosis phytochemistry and pharmacology of biophytum sensitivum dc influence of paeonia lactiflora roots extract on camp-phosphodiesterase activity and related anti-inflammatory action mechanisms involved in the therapeutic effects of paeonia lactiflora pallas in rheumatoid arthritis chondroprotective potential of phyllanthus amarus schum. & thonn. in experimentally induced cartilage degradation in the explants culture model phonophoresis of phyllanthus amarus nanoparticle gel improves functional capacity in individuals with knee osteoarthritis: a randomized controlled trial anti-arthritic activity of standardised extract of phyllanthus amarus in freund's complete adjuvant induced arthritis suppressive effects of the standardized extract of phyllanthus amarus on type ii collagen-induced rheumatoid arthritis in sprague dawley rats antirheumatoid activity of aqueous extract of piper longum on freunds adjuvant-induced arthritis in rats a systematic review on piper longum l.: bridging traditional knowledge and pharmacological evidence for future translational research medicinal plants with potential anti-arthritic activity effects of a combined therapy with d-002 (beeswax alcohols) plus d-003 (sugarcane wax acids) on osteoarthritis symptoms effects of a mixture of fatty acids from sugar cane (saccharum officinarum l.) wax oil in two models of inflammation: zymosan-induced arthritis and mice tail test of psoriasis chemical constituents and pharmacological properties of poria cocos clematis vitalba l. aerial part exhibits potent anti-inflammatory, antinociceptive and antipyretic effects coptidis rhizoma and its main bioactive components: recent advances in chemical investigation, quality evaluation and pharmacological activity anti-inflammatory, anti-arthritic and anti-nociceptive activities of nigella sativa oil in a rat model of arthritis phytochemistry, pharmacology, and therapeutic uses of black seed (nigella sativa). chin nigella sativa fixed oil as alternative treatment in management of pain in arthritis rheumatoid effects of nigella sativa oil extract on inflammatory cytokine response and oxidative stress status in patients with rheumatoid arthritis: a randomized, double-blind, placebo-controlled clinical trial effects and mechanisms of glucosides of chaenomeles speciosa on collagen-induced arthritis in rats glucosides of chaenomeles speciosa remit rat adjuvant arthritis by inhibiting synoviocyte activities anti-inflammatory and analgesic activities of chaenomeles speciosa fractions in laboratory animals sclareol-loaded lipid nanoparticles improved metabolic profile in obese mice rosa canina-rose hip pharmacological ingredients and molecular mechanics counteracting osteoarthritis-a systematic review a systematic review on the rosa canina effect and efficacy profiles the effects of rose hip (rosa canina) on plasma antioxidative activity and c-reactive protein in patients with rheumatoid arthritis and normal controls: a prospective cohort study a new sesquiterpene lactone from the roots of lasianthus acuminatissimus three new and antitumor anthraquinone glycosides from lasianthus acuminatissimus merr 8-methoxycoumarin enhances melanogenesis via the mapkase signaling pathway. die pharm protective effects of isolated polyphenolic and alkaloid fractions of ruta graveolens l. on acute and chronic models of inflammation methanolic extract of ruta graveolens l. inhibits inflammation and oxidative stress in adjuvant induced model of arthritis in rats s. anti-inflammatory and nf-κb inhibitory activity of aerial parts of cestrum diurnum amelioration of adjuvant-induced arthritis by ursolic acid through altered th1/th2 cytokine production inhibition of the nemo/ikkβ association complex formation, a novel mechanism associated with the nf-κb activation suppression by withania somnifera's key metabolite withaferin a evaluation of anti-inflammatory effect of withania somnifera root on collagen-induced arthritis in rats extract from withania somnifera root ameliorates arthritis via regulation of key immune mediators of inflammation in experimental model of arthritis evaluation of nitric oxide scavenging activity, in vitro and ex vivo, of selected medicinal plants traditionally used in inflammatory diseases bioactive triterpenoids from lantana camara showing anti-inflammatory activities in vitro and in vivo antiulcerogenic activity of lantana camara leaves on gastric and duodenal ulcers in experimental rats anti-arthritic activity of hydroalcoholic extract of lawsonia innermis preliminary investigation of the effects of topical mixture of lawsonia inermis l. and ricinus communis l. leaves extract in treatment of osteoarthritis using mia model in rats aloe emodin glycosides stimulates glucose transport and glycogen storage through pi3k dependent mechanism in l6 myotubes and inhibits adipocyte differentiation in 3t3l1 adipocytes aloe vera as a treatment for osteoarthritis: a summary anti-inflammatory, anti-nociceptive, and anti-psychiatric effects by the rhizomes of alpinia officinarum on complete freund's adjuvant-induced arthritis in rats curcumin protects against collagen-induced arthritis via suppression of baff production oral administration of curcumin (curcuma longa) can attenuate the neutrophil inflammatory response in zymosan-induced arthritis in rats curcumin loaded solid lipid nanoparticles ameliorate adjuvant-induced arthritis in rats ginger phenylpropanoids inhibit il-1β and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases a2 zingiber officinale: a potential plant against rheumatoid arthritis anti-inflammatory effects of the essential oils of ginger (zingiber officinale roscoe) in experimental rheumatoid arthritis gingerols and shogaols: important nutraceutical principles from ginger traditional medicine: past, present and future research and development prospects and integration in the national health system of cameroon efficacy of a mouthwash containing essential oils and curcumin as an adjunct to nonsurgical periodontal therapy among rheumatoid arthritis patients with chronic periodontitis: a randomized controlled trial evening primrose oil in patients with rheumatoid arthritis and side-effects of non-steroidal anti-inflammatory drugs effect of aromatherapy massage with lavender essential oil on pain in patients with osteoarthritis of the knee: a randomized controlled clinical trial aromatherapy massage with lavender essential oil and the prevention of disability in adl in patients with osteoarthritis of the knee: a randomized controlled clinical trial essential oil therapy in rheumatic diseases: a systematic review garlic essential oil protects against obesity-triggered nonalcoholic fatty liver disease through modulation of lipid metabolism and oxidative stress inhalation of patchouli (pogostemon cablin benth.) essential oil improved metabolic parameters in obesity-induced sprague dawley rats rhaponticum acaule (l) dc essential oil: chemical composition, in vitro antioxidant and enzyme inhibition properties ginger essential oil ameliorates hepatic injury and lipid accumulation in high fat diet-induced nonalcoholic fatty liver disease essential oil of pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme a: cholesterol acyltransferase essential oil from citrus aurantifolia prevents ketotifen-induced weight-gain in mice anti-adipocyte differentiation activity and chemical composition of essential oil from artemisia annua downy lavender oil: a promising source of antimicrobial, antiobesity, and anti-alzheimer's disease agents. evid.-based complement the effect of nigella sativa supplementation on cardiovascular risk factors in obese and overweight women: a crossover, double-blind, placebo-controlled randomized clinical trial nigella sativa oil with a calorie-restricted diet can improve biomarkers of systemic inflammation in obese women: a randomized double-blind, placebo-controlled clinical trial a natural fiber complex reduces body weight in the overweight and obese: a double-blind, randomized, placebo-controlled study green tea extract outperforms metformin in lipid profile and glycaemic control in overweight women: a double-blind, placebo-controlled, randomized trial saffron improves life and sleep quality, glycaemic status, lipid profile and liver function in diabetic patients: a double-blind, placebo-controlled, randomised clinical trial effect of alginate on satiation, appetite, gastric function, and selected gut satiety hormones in overweight and obesity lycium barbarum increases caloric expenditure and decreases waist circumference in healthy overweight men and women: pilot study effects of garlic powder supplementation on insulin resistance, oxidative stress, and body composition in patients with non-alcoholic fatty liver disease: a randomized controlled clinical trial effect of daily ingestion of quercetin-rich onion powder for 12 weeks on visceral fat: a randomised, double-blind, placebo-controlled, parallel-group study controlled trial of avocado-soybean unsaponifiable (piascledine) effect on structure modification in hip osteoarthritis: the eradias study the effects of momordica charantia (bitter melon) supplementation in patients with primary knee osteoarthritis: a single-blinded, randomized controlled trial the use of a stem and leaf aqueous extract of cissus quadrangularis (cqr-300) to reduce body fat and other components of metabolic syndrome in overweight participants potential role of flavonoids against sars-cov-2 induced diarrhea health effects of quercetin: from antioxidant to nutraceutical precision and advanced nano-phytopharmaceuticals for therapeutic applications nanotechnology applications of flavonoids for viral diseases study of antioxidant effect of apigenin, luteolin and quercetin by dna protective method antitumor, antioxidant and anti-inflammatory activities of kaempferol and its corresponding glycosides and the enzymatic preparation of kaempferol antioxidant properties of puerarin and genistein from white kwao krua induced by elicitors and their antihyperglycemic effect on rats bioavailability and antioxidant effect of epigallocatechin gallate administered in purified form versus as green tea extract in healthy individuals color and antioxidant properties of cyanidin-based anthocyanin pigments protective effect of apigenin on freund's complete adjuvant-induced arthritis in rats via inhibiting p2x7/nf-κb pathway the roles of synovial hyperplasia, angiogenesis and osteoclastogenesis in the protective effect of apigenin on collagen-induced arthritis cyanidin-3-o-glucoside improves non-alcoholic fatty liver disease by promoting pink1-mediated mitophagy in mice epigallocatechin-3-gallate and postprandial fat oxidation in overweight/obese male volunteers: a pilot study combined epigallocatechin-3-gallate and resveratrol supplementation for 12 wk increases mitochondrial capacity and fat oxidation, but not insulin sensitivity, in obese humans: a randomized controlled trial efecto de una intervención dietética con un producto alimenticio a base de leguminosas sobre los niveles de malondialdehído, índice homa y perfil de lípidos preventive ffects of kaempferol on high-fat diet-induced obesity complications in c57bl/6 mice luteolin improves hypercholesterolemia and glucose intolerance through lxrα-dependent pathway in diet-induced obese mice therapeutic effects of puerarin on polycystic ovary syndrome the effect of quercetin on inflammatory factors and clinical symptoms in women with rheumatoid arthritis: a double-blind, randomized controlled trial the ppotential therapeutic value of medicinal plants in the management of metabolic disorders a systematic review of anti-obesity medicinal plants-an update natural anti-obesity agents and their therapeutic role in management of obesity: a future trend perspective herbal medicines used in the traditional indian medicinal system as a therapeutic treatment option for overweight and obesity management: a review natural dietary and herbal products in anti-obesity treatment role of flavonoids in the interactions among obesity, inflammation, and autophagy the authors declare no conflict of interest. key: cord-0075776-p15c4r7q authors: khaleel, anas; alkhawaja, bayan; al-qaisi, talal salem; alshalabi, lubna; tarkhan, amneh h. title: pathway analysis of smoking-induced changes in buccal mucosal gene expression date: 2022-03-17 journal: egypt j med hum genet doi: 10.1186/s43042-022-00268-y sha: 8ddb7af28a3c3dd6ca8014a65d6c40043ba10d5d doc_id: 75776 cord_uid: p15c4r7q background: cigarette smoking is the leading preventable cause of death worldwide, and it is the most common cause of oral cancers. this study aims to provide a deeper understanding of the molecular pathways in the oral cavity that are altered by exposure to cigarette smoke. methods: the gene expression dataset (accession number gse8987, gpl96) of buccal mucosa samples from smokers (n = 5) and never smokers (n = 5) was downloaded from the national center for biotechnology information's (ncbi) gene expression omnibus (geo) repository. differential expression was ascertained via ncbi’s geo2r software, and ingenuity pathway analysis (ipa) software was used to perform a pathway analysis. results: a total of 459 genes were found to be significantly differentially expressed in smoker buccal mucosa (p < 0.05). a total of 261 genes were over-expressed while 198 genes were under-expressed. the top canonical pathways predicted by ipa were nitric oxide and reactive oxygen production at macrophages, macrophages/fibroblasts and endothelial cells in rheumatoid arthritis, and thyroid cancer pathways. the ipa upstream analysis predicted that the tp53, app, smad3, and tnf proteins as well as dexamethasone drug would be top transcriptional regulators. conclusions: ipa highlighted critical pathways of carcinogenesis, mainly nitric oxide and reactive oxygen production at macrophages, and confirmed widespread injury in the buccal mucosa due to exposure to cigarette smoke. our findings suggest that cigarette smoking significantly impacts gene pathways in the buccal mucosa and may highlight potential targets for treating the effects of cigarette smoking. supplementary information: the online version contains supplementary material available at 10.1186/s43042-022-00268-y. tobacco smoking is responsible for one in six of all deaths from non-communicable diseases, leading experts to identify tobacco control as the highest priority public health intervention [1, 2] . the prevalence of smoking has fallen around the world over the past three decades, but the absolute number of people who smoke has increased [3] . despite a coordinated worldwide effort against smoking, there are around 1.1 billion current smokers, and it is expected that this number would reach 1.9 billion by 2025 if current smoking patterns are maintained [4] . cigarette smoke contains over 5000 chemicals, of which 98 have been identified as carcinogenic or probably carcinogenic to humans [5] . the plethora of carcinogens in cigarette smoke perturbs biological pathways related to cellular proliferation, inflammation, and tissue injury, with strong links to various types of cancer [6, 7] . in cancer patients, cigarette smoking has been associated with an increased symptom burden as well as a reduced efficacy of chemotherapy [6, 8] . smoking-induced differential gene expression has been well-documented in previous studies. in fact, smoking has a characteristic impact on the transcriptome, as it activates inflammatory and oxidative responses, changes airway structures, and alters gene expression across tissue types [9] . previous studies have shown that cigarette smoking significantly alters the gene expression profiles of adipose tissue, buccal cells, nasal epithelial cells, lung tissue, and whole blood [10] [11] [12] [13] [14] . the aim of the current study is to broaden the understanding of the molecular pathways that are altered in buccal mucosa after exposure to cigarette smoke. gene expression data from smokers and never smokers were analyzed via ingenuity pathway analysis (ipa), which is a web-based software application that identifies new targets within the context of biological systems. the microarray dataset investigated in the present study was obtained from the national center for biotechnology information's (ncbi) gene expression omnibus (geo) repository (accession number gse8987). this dataset included gene expression data of buccal mucosa samples from smokers (n = 5) and never smokers (n = 5) [15] . smokers were classified as those who had smoked at least 10 cigarettes per day and who had a cumulative smoking history of at least 10 pack years [15] . table 1 shows the gene expression data samples included in the current study. as per the original study by sridhar et al., buccal mucosa samples were collected from the study participants by scraping the inside of their mouths with a concave plastic tool with serrated edges. total rna was extracted from buccal mucosa samples using trizol reagent (invitrogen, carlsbad, ca), and rna integrity was assessed using a denaturing agarose gel. the affymetrix human genome u133a (hg-u133a) array (affymetrix, santa clara, ca) was then used to profile the gene expression of the extracted total rna samples [15] . the demographics of the 10 subjects varied with regard to sex, age, and race. among the 5 smokers, the mean age was 36 years old (± 8 years), with 1 male and 4 females. similarly, the mean age of the 5 never smokers was 31 years old (± 9 years), with 2 males and 3 females. in terms of race, the smoker group comprised 3 caucasians and 2 african americans, while the never-smoker group consisted of 2 caucasians and 3 african americans. demographic data for individual subjects were not provided in the dataset, but statistical comparisons of the smoker and never-smoker groups revealed not significant p values for sex (p = 0.42), age (p = 0.36), and race (p = 0.40) [15] . the geo2r software, which is available on the ncbi website, was used to create a list of 15,000 differentially expressed genes between smoker and never-smoker buccal samples. the 15,000 genes were inputted into a microsoft excel spreadsheet and sorted by significance (additional file 1: table s1 ). after applying strict cut-off criteria (p < 0.05 and absolute fold change between − 0.5 and 1.5), the list of de genes was narrowed down to 459 genes. the bioconductor package enhanced volcano was used to visualize the 459 de genes in the form of a labelled volcano plot [16] . the list of de genes was inputted into ipa software (qiagen, hilden, germany), where the 'core analysis' function of the software was used to interpret the data in terms of canonical pathways and upstream regulators. the bioconductor package clusterprofiler was used to carry out an over-representation analysis of the de genes [17, 18] . similarly, the signaling network open resource 2.0 (signor 2.0) was used to explore the signaling networks that exist between the de genes [19] . figure 1 displays a volcano plot of the full list of de genes. however, only 459 genes exhibited significant differential expression, with 261 genes found to be overexpressed and 198 found to be under-expressed. figure 2 illustrates the chromosomal location, molecular class, and cellular location of the 459 de genes. chromosome 1 had the highest number of significantly de genes (n = 63), followed by chromosome 6 (n = 30), chromosome 2 (n = 29), and chromosome 19 (n = 27). similarly, the most represented molecular classes among the significantly de genes were enzymes (19.6%) and transcription regulators (12%). lastly, the majority of the significantly de genes were located either in the cytoplasm (40.5%) or the nucleus (25.7%). table 2 lists the most significantly de genes between smoker and never smoker buccal mucosa samples, showing that protein-coding genes occupy the top ranks in terms of significance. figure 3a demonstrates the interplay between the de oncological pathways, cytokines, and genes in smoker buccal mucosa, namely the il2, egfr, and esr2 genes. other than timp3, all the proteins in the pathway were predicted to be inhibited in smoker buccal mucosa. figure 3b illustrates the results of an interaction network analysis of the de genes in smoker buccal mucosa. interestingly, the rpa1 gene was shown to have the highest number of interactions with the other de genes in smoker buccal mucosa, but it did not have a significant level of differential expression (p > 0.05). the top 20 regulators predicted by ipa included the tp53, app, smad3, and tnf proteins as well as the drug dexamethasone, among other molecules (table 3 ). figure 4 illustrates the data in table 3 and emphasizes the predicted activation status of the top upstream regulators as revealed by ipa. as can be seen from fig. 4 , the most inhibited upstream regulator in smoker buccal mucosa is predicted to be the tp63 protein. dexamethasone was predicted to be a top upstream regulator and affected a total of 78 genes via indirect interactions (fig. 5a) . likewise, microrna-8 (mir-8) was found by ipa to be among the top upstream regulators to be activated, as mir-8 targeted 7 of the de genes between smokers and never smokers (fig. 5b) . of those genes, 5 (ccnd2, itgav, qki, rps6kb1, and smad2) were under-expressed and 2 (bmp2 and cldn3) were over-expressed. further analysis of the top upstream regulator proteins resulted in the construction of gene-gene (fig. 6 ) and protein-protein (fig. 7) interaction networks. figure 6 shows that the 36.04% of the top upstream regulator proteins were predicted to have interactions with one another, 26.19% have shared protein domains, and 22.85% were co-expressed. similarly, fig. 7 shows that the tp53 and tnf proteins had the highest number of interactions with the other top upstream regulator proteins. the most significant canonical pathway was identified as the nitric oxide and reactive oxygen production at macrophages ( table 4) . the de genes in smoker buccal mucosa are significantly associated with cancer and organismal injury, among other diseases (table 5) . pathway and functional enrichment analysis figure 8 illustrates the most over-represented biological processes in smoker buccal mucosa. interestingly, craniosynostosis and fibroid tumors were revealed to be the topmost significantly over-represented biological processes. figure 9 shows the results of signaling network analysis of the 459 significantly de genes, with the smad2 gene having the most interactions. smad2 is directly downregulated by the ctdspl and skil genes and indirectly upregulated by the bmp2 gene. the most significantly differentially expressed (de) protein-coding genes in smoker buccal mucosa were the chd5, qki, batf3, and il6r genes, which have previously reported associations with smoking and related diseases. the chd5 gene, which is a tumor suppressor gene that is preferentially expressed in the nervous system and testis, was significantly upregulated in smoker buccal mucosa [20, 21] . chd5 is believed to serve as a master regulator in tumor-suppressive networks, and chd5 expression levels are strongly associated with the prognosis of several cancers, including hepatocellular carcinoma and non-small cell lung cancer [20, [22] [23] [24] . one study found that a rare chd5 variant, rs12564469-rs9434711, contributed to the risk of hepatocellular carcinoma, a risk effect which was statistically significant in alcohol drinkers but not smokers [25] . the qki gene contributes to a number of human diseases, including cancers, myelin disorders, and schizophrenia, and it is a critical regulator of alternative splicing in cardiac myofibrillogenesis and contractile function [26] . qki has also been identified as a master regulator of alternative splicing in human lung cancer cell lines, but no significant statistical association was found between qki expression and smoking status in lung tumors [27, 28] . moreover, qki was identified as a significantly altered gene in the ciliated epithelial cells of lungs affected by chronic obstructive pulmonary disease (copd), a disease that is primarily caused by tobacco smoking [29] . the batf3 gene belongs to the ap-1 transcription factor family, whose members respond to a range of pathological and physiological stimuli by mediating gene expression [30] . batf3 controls the differentiation of dendritic cells, inhibits the differentiation of regulatory t cells, and critically regulates the development of memory t cells [31, 32] . batf3 expression in the lungs was necessary in order to induce protection against allergic airway inflammation through tolerization with helicobacter pylori extract [33] . moreover, the acute inhalation of electronic cigarette smoke by healthy never smokers led to the significant upregulation of batf3, among other genes that play a role in promoting tumorigenesis [34] . the il6r gene is a pleiotropic regulator of both acquired and innate immune responses, and it is believed to be expressed in the lungs [35] . there have been conflicting findings regarding the benefits of anti-il-6r therapy for covid-19-induced acute respiratory distress syndrome [36, 37] . in the context of smoking, exposure to cigarette smoke led to increased il6r mrna levels in primary bronchial epithelial cell lines [38] . moreover, a certain il6r haplotype (rs6684439-rs7549250-rs4129267-rs10752641-rs407239) has been associated with a lower copd risk in a mexican mestizo population, while the il6r variant asp358ala did not show any association with copd [39, 40] . pseudogene expression was also altered in smoker buccal mucosa, most notably in the upregulation of fmo6p, znf259p1, and znf702p and the downregulation of aldoap2 and pnliprp2. fmo6p has significant sequence homology with the fmo3 gene, the latter of which functions to metabolize a small amount of nicotine [41] . a single nucleotide variation in the fmo6p pseudogene, rs6608453, was associated with nicotine dependence in african americans [42] . likewise, aldoap2 was over-expressed in both healthy and non-healthy smokers compared to non-smokers, while exposure to cigarette smoke resulted in the upregulation of the pnliprp2 polymorphic pseudogene in a murine model [43, 44] . in contrast, znf259p1 and znf702p did not have previously reported associations with smoking. znf259p1 was significantly correlated with the tumor size of primary lung adenocarcinomas, while znf702p was found to be upregulated after bcl2l10 knockdown in two ovarian cell lines [45, 46] . analysis of upstream regulators revealed that the tumor protein 53 (tp53) gene was the most significantly de regulator in smoker buccal mucosa. tp53 contains cellular proliferation by guarding against genomic mutation, and tp53 mutations are among the most common genetic alterations in human cancers [47] . tobacco smoking is known to influence tp53 mutation patterns and frequencies in lung cancer and urothelial cell carcinoma patients [48, 49] . in fact, a large proportion of tp53 mutations in the lung cancers of smokers were g → t transversions, a primary mutagenic signature that is caused by dna damage from tobacco smoke [50] . the most significant canonical pathway identified by ipa was the "nitric oxide and reactive oxygen production at macrophages". nitric oxide and reactive oxygen species are essential for maintaining redox balance, but they also act in pathological processes [51] . tobacco smoke contains large numbers of free radicals, including nitric oxide and reactive oxygen species (ros), that cause oxidative stress on the cellular and sub-cellular levels [52, 53] . in turn, smoking-induced oxidative stress activates inflammatory response pathways that produce endogenous ros at the site of oxidative stress, potentially causing further oxidative damage to that site [53] . smoking also reduces the production of nitric oxide while also elevating the production of ros in endothelial cells [54, 55] . smoking-induced ros production is especially concerning as it may contribute to the progression of endometrial adenocarcinoma [56] . among the de genes, those associated with craniosynostosis and fibroid tumors were over-represented in smoker buccal mucosa. craniosynostosis, which is caused by the premature fusion of cranial sutures, is the second-most common cranio-facial anomaly [57] . smoking during pregnancy was associated with an increased risk of craniosynostosis, while exposure to secondhand smoke modestly increased the risk of this birth defect [58] . maternal smoking impacts cranio-facial development by acting upon variant alleles of the transforming growth factor alpha (tgf-α) gene, and genetic variation of the tgf-α gene is associated with increased risk of cranio-facial defects [59, 60] . fibroid tumors are non-cancerous growths that develop inside or on the uterus and are the most common type of pelvic tumor detected in women [61] . previous studies that investigated the impact of smoking on fibroid tumors yielded conflicting results. earlier studies suggested that smoking had a protective effect against fibroid tumors, but subsequent studies have shown either a negative effect or no relationship at all [61, 62] . it is worthwhile to note that smoking has been shown to have an anti-estrogenic effect in women, resulting in an earlier natural menopause as well as protective associations with the risk of estrogen-related cancers [63, 64] . pathway network analysis revealed that the smad2 gene had the highest number of interactions with other de genes, and it was also a target of mir-8. smad3 was predicted by ipa to be an inhibited upstream regulator. the smad family member 2 (smad2) gene encodes for a protein that is vital for early development, and smad2 mutations were associated with complex cranio-facial defects in a murine model [65] . smad2, smad3, and smad4 mediate the signal transduction of transforming growth factor-β (tgf-β) superfamily members, the latter of which induce a range of effects that involve cellular differentiation, proliferation, migration, and apoptosis [66] . the present study is affected by a few limitations. the sample size was relatively small, and the patient samples differed in terms of sex and race, which could confound the interpretation of the genetic variation. additionally, several differentially expressed genes in smoker buccal mucosa were uncharacterized or unmapped to pathways, meaning that their effects are not considered in the current analysis. the current findings signify the importance of inflammatory response and oxidative stress as a major component of smoking-induced tissue injury. most significantly, nitric oxide-related inflammation stands as one of the canonical pathways underlying genetic and molecular pathways changes coupled with exposure to cigarette smoke. future lines of research should focus on validating the results of the current study in a larger population to ascertain potential therapeutic targets in the context of smoking-induced damage. effective tobacco control is key to rapid progress in reduction of non-communicable diseases priority actions for the non-communicable disease crisis spatial, temporal, and demographic patterns in prevalence of smoking tobacco use and attributable disease burden in 204 countries and territories, 1990-2019: a systematic analysis from the global burden of disease study lung cancer 2020: epidemiology, etiology, and prevention hazardous compounds in tobacco smoke effects of tobacco smoke on gene expression and cellular pathways in a cellular model of oral leukoplakia cigarette smoking: cancer risks, carcinogens, and mechanisms the effect of cigarette smoking on cancer treatment-related side effects effect of smoking on gene expression profile -overall mechanism, impact on respiratory system function, and reference to electronic cigarettes tobacco smoking-response genes in blood and buccal cells smoking induces coordinated dna methylation and gene expression changes in adipose tissue with consequences for metabolic health tobacco smoking increases the lung gene expression of ace2, the receptor of sars-cov-2 tobacco-related alterations in airway gene expression are rapidly reversed within weeks following smoking-cessation a whole-blood transcriptome meta-analysis identifies gene expression signatures of cigarette smoking smoking-induced gene expression changes in the bronchial airway are reflected in nasal and buccal epithelium enhancedvolcano: publication-ready volcano plots with enhanced colouring and labeling clusterprofiler: an r package for comparing biological themes among gene clusters 0: a universal enrichment tool for interpreting omics data signor 2.0, the signaling network open resource 2.0 2019 update role of chd5 in human cancers: 10 years later chd5, a new member of the chromodomain gene family, is preferentially expressed in the nervous system chd5 a tumour suppressor is epigenetically silenced in hepatocellular carcinoma the single-nucleotide polymorphisms in chd5 affect the prognosis of patients with hepatocellular carcinoma chd5 is a potential tumor suppressor in non small cell lung cancer (nsclc) a rare chd5 haplotype and its interactions with environmental factors predicting hepatocellular carcinoma risk qki is a critical pre-mrna alternative splicing regulator of cardiac myofibrillogenesis and contractile function the rnabinding protein qki suppresses cancer-associated aberrant splicing a large-scale analysis of alternative splicing reveals a key role of qki in lung cancer single cell rna sequencing identifies igfbp5 and qki as ciliated epithelial cell genes associated with severe copd ap-1 subunits: quarrel and harmony among siblings the transcription factor batf3 inhibits the differentiation of regulatory t cells in the periphery cutting edge: batf3 expression by cd8 t cells critically regulates the development of memory populations effective treatment of allergic airway inflammation with helicobacter pylori immunomodulators requires batf3-dependent dendritic cells and il-10 altered lung biology of healthy never smokers following acute inhalation of e-cigarettes framingham heart study genome-wide association: results for pulmonary function measures genetic il-6r variants and therapeutic inhibition of il-6 receptor signalling in covid-19 anti-il6r role in treatment of covid-19-related ards adam17 and egfr regulate il-6 receptor and amphiregulin mrna expression and release in cigarette smoke-exposed primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (copd) genetic variants in il6r and adam19 are associated with copd severity in a mexican mestizo population neutrophil-mediated il-6 receptor trans-signaling and the risk of chronic obstructive pulmonary disease and asthma genetic susceptibility to nicotine and/or alcohol addiction: a systematic review targeted sequencing identifies genetic polymorphisms of flavin-containing monooxygenase genes contributing to susceptibility of nicotine dependence in european american and african american modulation of atherogenic lipidome by cigarette smoke in apolipoprotein e-deficient mice genomic mechanisms of transformation from chronic obstructive pulmonary disease to lung cancer bcl2l10 induces metabolic alterations in ovarian cancer cells by regulating the tca cycle enzymes sdhd and idh1 hidden treasures in "ancient" microarrays: gene-expression portrays biology and potential resistance pathways of major lung cancer subtypes and normal tissue tp53 mutations in human cancers: origins, consequences, and clinical use tp53 mutation spectrum in smokers and never smoking lung cancer patients mutations in tp53, but not fgfr3, in urothelial cell carcinoma of the bladder are influenced by smoking: contribution of exogenous versus endogenous carcinogens tp53 mutation spectrum in lung cancers and mutagenic signature of components of tobacco smoke: lessons from the iarc tp53 mutation database oxidative damage in alveolar macrophages exposed to cigarette smoke extract and participation of nitric oxide in redox balance active smoking causes oxidative stress and decreases blood melatonin levels relationships among smoking, oxidative stress, inflammation, macromolecular damage, and cancer cigarette smoke adversely influences nitric oxide bioavailability by effects on l-arginine transport and oxidative stress in endothelial cells reactive oxygen species are involved in smoking-induced dysfunction of nitric oxide biosynthesis and upregulation of endothelial nitric oxide synthase: an in vitro demonstration in human coronary artery endothelial cells the cigarette smoke components induced the cell proliferation and epithelial to mesenchymal transition via production of reactive oxygen species in endometrial adenocarcinoma cells craniosynostosis as a clinical and diagnostic problem: molecular pathology and genetic counseling craniosynostosis and maternal smoking association of genetic variation of the transforming growth factoralpha gene with cleft lip and palate gene/environment interactions in craniosynostosis: a brief review epidemiology and risk factors of uterine fibroids environmental tobacco smoke and risk of late-diagnosis incident fibroids in the study of women's health across the nation (swan) the antiestrogenic effect of cigarette smoking in women cigarette smoking and estrogen-related cancer smad2 role in mesoderm formation, left-right patterning and craniofacial development tgf-beta receptor-mediated signalling through smad2, smad3 and smad4 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to acknowledge the efforts of the university of petra's faculty of pharmacy and medical sciences, its dean, and its department head of pharmacology and biomedical sciences. the online version contains supplementary material available at https:// doi. org/ 10. 1186/ s43042-022-00268-y.additional file 1. list of de genes downloaded from geo2r. a complete list of gene names and their abbreviations are delivered in the supplementary data files. ak was involved in conceptualization, writing (review & editing), formal analysis, validation, and visualization. bk and ls were involved in formal analysis, methodology, and writing (original draft). at and tq were involved in visualization and writing (review & editing). all authors have read and approved the manuscript. this study was not funded. the current report utilized a previously published dataset for the analysis. the dataset used in this work was acquired from the national center for biotechnology information's (ncbi) gene expression omnibus (geo) depository (accession number gse8987). original article j fac med baghdad vol. 48 , no. 3 , 2006 293 some immunological profile of rheumatoid arthritis nahida r. abbas* ph.d khalida m. mousawy *ph.d sami salman ** ph.d jassim al-kafaji ph.d summary: seventyfour cases of clinically diagnosed rheumatoid arthritis (ra), fifty cases of systemic lupus erythematosus (sle), and thirty healthy normal controls were investigated for detection of rheumatoid factor (rf), total serum immunoglobulins (igs), antinuclear antibody (ana), and ana subtype anti-double stranded dna (anti-ds dna). patients with ra showed 58.1% positive for rf comparable with 14% positivity in sle patients and 6.6% in normal individuals. serum igs (iga,igg) were found to be elevated in ra and sle patients (62.2% , 36.5%) (54% , 38%) respectively. this study revealed that ana is found in 88% of sle patients sera and 78% of these ana is ds dna in comparison with only 6.8% of ra sera were found positive for ana. __________________________________________________________________________________________ introduction:______________________________ ra is a chronic systemic inflammatory autoimmune disorders of mysterious aetiology, which is dominated by joint inflammation accompanied by several peripheral inflammatory manifestations . in this disease patients develop distinct immuno abnormalities espe-cially autoantibodies, among these antibodies is rf which is a major immunological abnormality in ra and regarded as one of the diagn-ostic criteria of ra which is included in the american college of it may occur even in some normal individuals (4). it is well accepted that anti-ds dna antibodies are rather specific to sle with occurance in 70% but detected in less than 5% in ra patients (5). rf, ana and anti-ds dna appear in ra as well as sle patients sera. patients and methods study population : the study cohort comprised 74 sera samples for patients (19male and 55 female), their age range (18-67 years) with ra who met (acr) 1987 reversed criteria (1) attending the rheumatology consultation clinic or admitted to baghdad teaching hospital in the period between november 2001 and february 2002.  control group: were sex and age matched. they were included : _________________________________________ * dep. of microbiology of medicine of baghdad of univ. ** dep. of medicine of baghdad univ. of medicine patient control group of 50 sera samples for clinically diagnosed sle patients according to acr criteria 1997 for classification of sle (6). healthy control group of thirty healthy individuals collected from the blood banking donors. all these sera samples have been collected and stored at-20c° for analysis. laboratory investigations : rf was detected using the latex test rheumatology(acr)1987 reversed criteria (1), though non specific because present in 5% of healthy individuals and in many auto immune rheumatic diseases in addition in some chronic bacterial infections (2), while ana was directed against a variety of nuclear antigens has been identified in the serum of patients with many rheumatic and non rheumatic diseases (3). moreover; several studies denote that these antibodi-es are not specific for the disease, supplied by biokit company, spain and results were expressed in international units (iu/ml) . total igs concentration were estimated by single radial immuno diffusion (srid)test (biomaghreb), and results were expressed in mg/dl. ana and anti-ds dna were performed by enzyme-linked immunesorbent assay (elisa) using purified antigens (extracted from the hep-2 nucleus) are bound to microwells.the results expressed in iu/ml. in addition to that ana was detected by immunofluorescence antibody test (ifat) as well as using substrate (mouse kidney). results : from 74 ra patients the maximum incidence of the disease was observed among age 30-49 years, with mean age 42.1±11.3. there were 55 females and 19 males with a female to male ratio 2.9:1 as shown in table 1&2. j fac med baghdad 2006; vol. 48, no.3 received oct 2005 accepted feb. 2006 some immunological profile of rheumatoid arthritis nahida khalida sami jassim j fac med baghdad vol. 48 no. 3 , 2006 294 among the laboratory tests performed. � rheumatoid factor: 43 patients were rf positive (58.1%), while the rest (41.9%) were negative, while in sle patients only 7 patients were rf positive (14%) as observed in table 3. table 1: age distribution of studied groups. ra sle healthy control age in years n % n % n % <20 1 1.4 4 8.0 2 6.7 20-29 9 12.2 18 36.0 6 20.0 30-39 23 31.0 17 34.0 13 43.3 40-49 19 25.7 10 20.0 7 23.3 50-59 16 21.6 1 2.0 2 6.7 60+ 6 8.1 total 74 100.0 50 100.0 30 100.0 range 18-67 9-59 18-56 mean 42.1 30.6 33.8 sd 11.3 10.1 9.4 p (anova) < 0.001 table 2: distribution of the studied groups by gender. ra sle healthy control n % n % n % gender female 55 74.3 44 88 23 76.7 male 19 25.7 6 12 7 23.3 total 74 100 50 100 30 100 � total concentration of immuno-globulins level (iga, igg, and igm): the serum of iga and igg level in ra patients were significantly higher than those in healthy control group, while there was no difference in comparison to sle patients, where as igm level was normal in all studied groups. auto antibodies such as ana and ds-dna: were not present in ra patients except in few cases in comparison to patients control with highly significant difference p<0.001 as clearly shown in table 3. while in table 5 the data showed that these abs were detected by 2 different methods, either by elisa as showed in 5 (6.8%) ra patients cases as compared to 44 (88%) in sle patients, or by ifat was revealed in 4 (5.5%) ra patients in comparison to 39 (78%) sle patients table 3: the difference in positivity rate of different parameters measurement between ra patients and control groups. ra sle healthy control p (fisher's exact) for the difference between ra and n % n % n % (n=74) (n=50) (n=30) controls sle rf by latex 43 58.1 7 14.0 2 6.7 <0.001 <0.001 ** high serum immunoglobulins level serum iga 46 62.2 27 54.0 5 16.7 <0.001 0.24[ns] serum igg 27 36.5 19 38.0 1 3.3 <0.001 0.51[ns] serum igm 6 8.1 1 2.0 0 .0 0.12[ns] 0.15[ns] positive autoantibodies * ana 5 6.8 44 88.0 0 .0 0.17[ns] <0.001 * anti ds dna antibodies 0 .0 39 78.0 0 .0 *** <0.001 * detected by elisa ** detected by srid some immunological profile of rheumatoid arthritis nahida khalida sami jassim j fac med baghdad vol. 48 no. 3 , 2006 295 table 4 : the difference in mean serum immunoglobulin concentration (mg/dl) between studied groups. serum igs (mg/dl) ra (n=74) sle (n=50) healthy control (n=30) p (anova) * serum iga <0.001 range 62.3-633.3 48-633.3 90-540 mean 350.9 358.3 208.8 sd 129.3 174.7 105.9 * serum igg 0.006 range 643.7-2965.9 295.9-3042.3 700-1614.6 mean 1462.8 1481 1114.5 sd 515.9 728.2 282.9 * serum igm 0.43[ns] range 48.1-277.3 40.8-277.3 93.2-205.2 mean 151.6 140.5 146.1 sd 48.9 49.9 33.1 * normal range of igs iga: 90-540 mg/dl igg: 700-1620 mg/dl igm: 50-250 mg/dl table 5: the difference in positivity rate of anas cases detected using two different parameters. ra sle technique n % n % elisa positive 5 6.8 44 88 nagative 69 93.2 6 12 total 74 100 50 100 ifa positive 4 5.5 39 78 nagative 70 94.5 11 22 total 74 100 50 100 ddiissccuussssiioonn now as ever, autoimmune diseases constitute one of the main problem in human clinical medicine. this is because our knowledge of their aetiology and pathogenesis is still not sufficient enough to provide concepts toward specific therapy, moreover it’s importance resides not only from the fact that it is fairly prevalent but rather because the victims are usually young, and economically active people. it is generally accepted that the incidence of ra is usually at the fourth decade of life,which is rather consistent with iraqi studies (7,8), and abroad studies (2,9). however, in the present study, the maximum incidence of the disease observed among 30-49 with a mean age 42.1±11.3 years and predominantly female. thus our patients are younger than those in most westren and american countries, where their range was 44-67 years (10,11). this might be related to the increased average of middle age in these countries due to advanced health education services, or probably related to environmental influence during the last decade. however female to male ratio in this study was 2.9:1 which is somewhat comparable to 2.7:1 reported by ubaid, and higher than farhat, 2.1:1(12). however abroad studies showed the ratio of 2.1:1, and 3.4:1 had been reported by saraux, and constantin, respectively (11,13) and this is generally accepted to be related to sex hormones e.g. estrogen. it is generally agreed that rfs have been widely used as a immunologic marker. latex agglutination test was showed 58.1% that showed to be less than other iraqi studies where 72.6%, 75.3% had been reported by ubaid, al-rawi, respectively (8,14), while abroad studies 56.6%,and 70% that had been reported by adhya, and tighe and carson, respectively (15,16). regarding immunoglobulins (iga, igg, and igm) concentration levels; our results were similar to abroad study (19), which denote no correlation in between igs themselves and different rf isotype and their concentrations. related to this study, the concentration levels of iga & igg were significantly higher in ra patients compared to some immunological profile of rheumatoid arthritis nahida khalida sami jassim j fac med baghdad 296 vol. 48 no. 3 , 2006 control groups, possible explanation of the above data propose that high level of igg related to denaturation of igg during initiation phase, while iga concentration is proportionally associated with it’s consumption in the synovium due to alternative pathway complement activation. rrr eee fff eee rrr eee nnn ccc eee sss 1arnett, f.c., edworthy, s.m., bloch d.a., mc.shane, d.j., fries, j.f., cooper, n.s. et al . the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis . arthritis rheum 1988;31:315-24. 2goronzy, j.j., weyand, c.m. epidemiology, pathology, and pathogenesis. primer on the rheumatic disease 12th ed. athlanta georgia. arthritis foundation. 2001:209-17. 3craft, j., hardin, j.a: antinuclear antibodies. in kelley w.n., harris, b., ruddy, s., sledge, c.b. (eds) : textbook of rheumatology, 4th ed. philla-dephia, wb saunders, 1993, p164. 4tan, e.m.; feltkamp, t.e.w; smolen, j.s; butcher, b.; dawkins, r. etsal. range of antinuclear antibodies in healthy individual. arthritis and rheum. 1997; 40:1601-11. 5ghiran, i., barbashov, s.f., klickstein, l.b., tas s.w., jensenius, j.c., nicholson-weller, a. complement receptor 1/cd35 is a receptor for mannanbinding lectin. j exp med 2000; 192:1797-807. 6hochberg, m.c. updating the american college of rheumatology revised criteria for the classification of systemic lupus erthymatosus. arthritis and rheum. 1997; 40: (17251734). 7al-shihabi, w.m. evaluation of safty and efficacy of auranofin in 20 iraqi patients with rheumatoid arthritis. iraqi medical journal. 1988;43:17-28. 8ubaid, a.h: formal education level as a marker of clinical status in ra among iraqis. a thesis submitted to the college of medicine, university of baghdad for the partial fulfillment of the master degree in community medicine (1996). 9lipsky, p.e. pathology and pathogenesis in rheumatoid arthritis in: harrisons principles of internal medicine 15th ed. vol.3,2001:1928-37. 10desirre, m., van der heijde, piet l. van riel, ike h. nuver zwart, frank w. gribnan, levinus b. van de peutte, effects of hydroxychloroquine and sulfasalazien on progression of joint damage in rheumatoid arthritis. the lancet 1999;may 13:1036-38. 11saraux, a., berthelot, j.m., charles, g., et al. value of laboratory tests in early prediction of rheumatoid arthritis. j, arthi & rheum. 2002; 47(2):155-65. 12farhat, h.f: antiphospholipid antibodies in rheumatoid arthritis. clinical and laboratory correlations. a thesis submitted to the college of medicine, university of baghdad in partial fulfillment of the requirements for the degree of master of science in pathology. (2000). 13constantin, a., cances, v.l., navaux, f., et al.(). stromelysin 1 (mafrix metalloproteinase 3) and hla-drb1 gene polymorphismsj. arth. & rheum. 2002; 46(7):1754-62. 14al-rawi,z.s., al azzawi, a.j., al ajili, f.m., al wakil, r. “rheumatoid arthritis in population samples in iraq “ann rheum. dis. 1978;37:73-75. 15adhya, s., chakraborty, g., hajra, b. bhattacharya, s., sikdar, p./k., sinha, s., banerjee, p.p., ghosh, e. serology and immunoglobulin profile in rheumatoid arthritis. indian j-pathol-microbid. 1998; 41(1):43-7. 16tighe, h., carson d.a: rheumatoid factors in: kelly, w.n; ruddy, s: harris, e.d and sledge, c.b. textbook of rheumatology (5th ed). sanders, w.b. company. 1997: 241-49. 17jorgensen, c., legouffe, m.c., bolongna, c., brochier, j. sany, j. iga isotypes rheumatoid factor in rheumatoid arthritis: clinical implications. clin. exp. rheumatol. 1996; 14(3):301-4. key: cord-0006875-hfm80xoq authors: banerjee, shubhasree; biehl, ann; gadina, massimo; hasni, sarfaraz; schwartz, daniella m. title: jak–stat signaling as a target for inflammatory and autoimmune diseases: current and future prospects date: 2017-03-03 journal: drugs doi: 10.1007/s40265-017-0701-9 sha: 277575898ce3b2eee6789233fc65d439c15858a2 doc_id: 6875 cord_uid: hfm80xoq the janus kinase/signal transduction and activator of transcription (jak–stat) signaling pathway is implicated in the pathogenesis of inflammatory and autoimmune diseases including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. many cytokines involved in the pathogenesis of autoimmune and inflammatory diseases use jaks and stats to transduce intracellular signals. mutations in jak and stat genes cause a number of immunodeficiency syndromes, and polymorphisms in these genes are associated with autoimmune diseases. the success of small-molecule jak inhibitors (jakinibs) in the treatment of rheumatologic disease demonstrates that intracellular signaling pathways can be targeted therapeutically to treat autoimmunity. tofacitinib, the first rheumatologic jakinib, is us food and drug administration (fda) approved for rheumatoid arthritis and is currently under investigation for other autoimmune diseases. many other jakinibs are in preclinical development or in various phases of clinical trials. this review describes the jak–stat pathway, outlines its role in autoimmunity, and explains the rationale/pre-clinical evidence for targeting jak–stat signaling. the safety and clinical efficacy of the jakinibs are reviewed, starting with the fda-approved jakinib tofacitinib, and continuing on to next-generation jakinibs. recent and ongoing studies are emphasized, with a focus on emerging indications for jak inhibition and novel mechanisms of jak–stat signaling blockade. cytokines are the soluble messengers that immune cells use to communicate, ultimately modulating protective responses against pathogens [1] . yet, cytokines may also drive the dysregulated immune responses that characterize autoimmune diseases such as rheumatoid arthritis (ra). inflammatory cytokines such as tumor necrosis factor (tnf)-a and interleukin (il)-6 maintain and aggravate inflammation in ra: they induce activation of leukocytes, endothelial cells, and fibroblasts like synoviocytes, promote differentiation of pathogenic immune cells, and promote synthesis of metalloproteinases that erode the joint [2] . the development over the past decade and a half of agents targeting cytokines or their receptors, denoted 'biologics', represents a landmark advancement in the treatment of autoimmune and inflammatory diseases. these agents include monoclonal antibodies and recombinant proteins that target cytokines or cytokine receptors, such as tnf-a, il-6, and its receptor, il-1, and others. however, despite the therapeutic success of biologics, it has become evident that targeting a single cytokine does not completely abrogate the pathology of rheumatologic disease for all patients. in addition, many of these agents lose efficacy over time owing to immunogenicity [3] and the intravenous or subcutaneous administration of these agents is an obstacle for some patients. over the past 20 years, our knowledge of the intracellular pathways downstream of cytokine receptors has greatly increased, and the inhibition of intracellular enzymes such as receptor-associated kinases represents a novel way to simultaneously inhibit multiple cytokines. small orally available molecules can be passively transported through the cellular membrane and block the intracellular activity of their targets [3] [4] [5] [6] . the type i/ii cytokine receptor family is used by several cytokines implicated in the pathogenesis of rheumatologic disease [1, 5] . this family of cytokine receptors employs the janus kinase-signal transduction and activation of transcription (jak-stat) pathway to effect signal transduction [7] . upon binding of a type i/ii cytokine to its cognate receptor, receptorassociated jaks are activated and phosphorylate stats, transcription factors critical for the activation of cytokinespecific genetic programs. given the major role played by jaks and stats in the pathogenesis of autoimmunity [8, 9] , it is perhaps no surprise that small molecules targeted against jaks, or jakinibs, represent an emerging treatment for autoimmune and inflammatory disease. in this review, we have described the jak-stat pathway and its role in human diseases, and then we have discussed the efficacy and safety of individual jakinibs in different diseases. electronic databases of embase, pubmed, and scopus were searched to identify all reports published in the english language that described 'janus kinase', 'jak/stat pathway', 'role of jak/stat pathway in human diseases', and 'jak inhibitors'. we also searched relevant conferences for abstracts describing jakinibs or the jak-stat pathway, searched clinicaltrials.gov for individual jakinibs, and reviewed press releases from pfizer, lilly, galapagos, incyte, and abbvie. 2 structure of jaks and stats, and implications for targeted therapy jaks belong to the family of tyrosine kinases (tyks). the basic structure of all jaks consists of four structural domains composed of seven homologous regions [jh1-7] (fig. 1 ). jh1 and jh2 denote the kinase and pseudokinase domains: the name janus is an allusion to the double-faced roman god of gates and doors owing to the presence of these two kinase domains. jh1 is the active catalytic phosphotransferase domain and the target of the jakinibs developed so far, which compete with adenosine triphosphate at the catalytic site. because the jh1 domains of the four jaks exhibit a high degree of homology not only within the jaks but also with other tyks, development of a selective jakinib has been challenging. the pseudokinase domain (jh2) was thought to have a regulatory function rather than a catalytic activity [5] : jh2 suppresses ligand-independent kinase activity through direct interactions with jh1 but is also required for ligandinduced jak activation [10] . recent work, however, has demonstrated that in jak2, the jh2 has low-level catalytic activity [11, 12] . jh3 and jh4 are primarily involved in stabilizing the structural conformation of the enzyme whereas the jh5, jh6, and jh7/four-point-one protein, ezrin, radixin, and moesin domain (ferm) are critical for the association of the jaks with their cognate receptors [13] . notably, recent mapping of the ferm domains of receptor-bound jak1 and tyk2 revealed striking differences in the structure that confers binding specificity, which might be exploited for the generation of selective inhibitors [5, [14] [15] [16] . the stat transcription factors transmit type i/ii cytokine signals downstream of the jaks. stat proteins contain an amino terminal, a coiled coil, a dna-binding domain (dbd), a linker, a src-homology2 (sh2), and a transcriptional activation domain (tad) [17] (fig. 1 ). inactive cytoplasmic stats exist primarily as monomers or preformed dimers [18] ; upon activation, stat dimers form a nutcracker-like structure. the highly conserved sh2 domain forms the hinge of this structure and is the target of most patented stat inhibitors [19] . the linker region and dbd surround the centrally located chromatin like the jaws of the nutcracker, whereas the tad is located at the c terminus and undergoes serine phosphorylation to recruit additional transcriptional activators and enhance transcriptional activity. these areas display the lowest sequence conservation [20] . given the involvement of stats in signaling events downstream of cytokine receptors as well as growth factors, stats have long been considered as potential therapeutic targets for cancer and autoimmune disease [19] . therefore, low-sequence conservation areas could represent a target for inhibitors, such as dna-binding decoy oligonucleotides, with increased selectivity [21] . the jak-stat pathway has been used for over 500 million years as a means of intracellular signal transduction in response to cytokines and growth hormones, evolving before the divergence of protostomes from deuterostomes [22] . the canonical signaling cascade is initiated when type i/ii cytokines bind to their cognate receptors (fig. 2) . type i/ii receptors are composed of distinct chains, which oligomerize upon binding of the cytokine. oligomerization causes separation of the intracellular subunits of the cytokine receptor, which moves the receptor-associated jaks apart from each other, relieving constitutive inhibition and resulting in their activation [1, 7, 23, 24] . the jaks phosphorylate themselves and the intracellular portion of the receptors, which serve as docking sites for stat transcription factors [25] , which, in turn, are also phosphorylated. when phosphorylated by jaks, inactive cytosolic stat monomers undergo a conformational change that allows for the formation of active homodimers, heterodimers, or tetramers. the active stats can then translocate into the nucleus where they act as transcription factors to regulate gene expression [1, 26, 27] . there are four jak namely, jak1, jak2, jak3, and tyk2 [5] . different jak-dependent cytokine receptors signal through different jaks. each receptor is composed of multiple subunits, and each subunit associates with a jak (fig. 3) . some receptor chains associate selectively with a specific jak, whereas some are less selective. thus, the extent to which a particular type i/ii cytokine depends on a specific jak to transduce signals is determined by the subunits of that cytokine's receptor (tables 1, 2) . for example, the common c-chain (cc), used by il-2, il-4, il-7, il-9, il-15, and il-21, associates exclusively with jak3 and is the only receptor subunit that uses jak3 [28] . other functions. an alternative nomenclature for the domains based on their amino acid sequence classifies them as seven janus homology (jh) domains. b simplified three-dimensional image of jak. the crystal structure of the ferm and sh2 domain was recently described and may contribute to receptor recognition. jh1 and jh2 are the kinase and pseudokinase domains respectively. c linear structure of stat molecule showing different domains. stat proteins contain an amino terminal, a coiled coil, a dna-binding domain (dbd), a linker, an sh2, and a transcriptional activation domain. the tad domain is located at the c terminus and undergoes serine phosphorylation to recruit additional transcriptional activators. d simplified three-dimensional image of stat. activated stat dimers form a nutcracker-like structure as shown in this figure. the hinge of the nutcracker is formed by highly conserved sh2 domain and is commonly the target of stat inhibitors. the linker region and dbd surround centrally located chromatin like the jaws of the nutcracker receptor chains are able to associate with more than one jak isoform: for instance, the gp130 subunit can use jak1, jak2, and possibly tyk2 [16] . a further layer of specificity is conferred by the pairing of specific receptor chains with each other: thus, the cc pairs exclusively with jak1-associated receptor subunits. most type i/ii cytokine receptors signal through multiple jaks as a result of this pairing of distinct jak-associated subunits. jak2-associated growth factor and hormone-like cytokine receptors are the exception and their subunits have the unique property of self-pairing [5] . there are seven members of the mammalian stat family: stat1, stat2, stat3, stat4, stat5a, stat5b, and stat6. as mentioned above, upon activation of jak-associated cytokine receptors, cytosolic stats undergo tyrosine phosphorylation and dimerize. however, it is important to recognize a number of additional non-canonical roles for stats. for example, stats act not only as homodimers or heterodimers, but also as tetramers [29] . stats can be phosphorylated by kinases other than jaks, including flt3r and pyruvate kinase [29] . in addition to tyrosine phosphorylation, stats undergo serine phosphorylation in response to various external stimuli, which can augment transcriptional responses [30] . serine phosphorylation also appears to be important for the ability of certain stats to promote oxidative phosphorylation in mitochondria [29] . finally, non-phosphorylated stats are capable of dimerizing and acting as transcriptional regulators [31, 32] . stats do not physically associate with a specific cytokine receptor but can be phosphorylated on specific tyrosine and serine residues. this results in a certain degree of functional overlap between stats. each member of the stat family can be activated by multiple cytokines and their associated jaks [33] , and, in certain situations, one stat protein can transmit signals that would normally be transduced by a different stat. mutations and polymorphisms in jak and stat genes have been linked with several human diseases, which is not surprising as a large number of cytokines and soluble factors signal through the jak-stat pathway [5] . hematopoietic growth factors, including erythropoietin and thrombopoietin, signal through jak2 [25] , thus gain-offunction (gof) mutations in jak2 cause hematologic disorders. the most extensively described jak2 mutation, v617f, causes polycythemia vera, essential thrombocythemia, and myelofibrosis [34, 35] . somatic gof mutations in jak1 and jak3 are also associated with hematologic malignancies such as t-cell acute lymphoblastic leukemia and solid organ malignancies such as breast cancer [36] [37] [38] . jak1 and jak2 deficiency phenotypes have not been described in humans, likely because the phenotype is incompatible with life: loss-of-function (lof) mutations in either jak is embryonically lethal in mice [39] . lof tyk2 mutation causes a milder immunodeficiency characterized by susceptibility to viral infection [40, 41] because cells cannot respond to interferon (ifn)-c or ifna/b [42] . lof mutations in jak3 cause autosomal recessive severe combined immunodeficiency, which recapitulates the phenotype observed in patients with mutations in the cc subunit [41, 43] . t-cell and natural killer (nk) cell maturation are profoundly impaired given the importance of cc cytokines such as il-7 and il-15 in their development and b-cell functions are also affected. patients present with severe recurrent infection, failure to thrive, atopic dermatitis, and chronic diarrhea. at this time, the only definitive treatment for this disease is hematopoietic stem cell transplantation. because jak3 is highly expressed in immune cells, patients with autosomal recessive severe combined immunodeficiency are spared from extra-immune disease manifestations. this observation formed the basis for interest in jak3 blockade as a potential immunosuppressive therapy [5] with limited off-target effects. binding of cytokine to the receptor leads to activation and phosphorylation of jak and phosphorylation of the receptor. this in turn leads to phosphorylation and dimerization of stat. activated stat dimer migrates to the nucleus and binds to specific dna-binding sites regulating gene transcription. this culminates in alteration of cellular function stat1 mutations resulting in either lof or gof have been described. autosomal dominant lof mutations cause mendelian predisposition to mycobacterial diseases [33] because responses to ifnc are impaired [44] : the mutation is dominant negative for type ii ifn responses. however, signaling downstream of ifna/b is unaffected because the mutation is autosomal recessive for type i ifn signaling. therefore, heterozygous patients are not susceptible to viral infection. complete biallelic stat1 deficiency, by contrast, is purely autosomal recessive for type i and ii ifn signaling. affected patients therefore exhibit fatal susceptibility to viral disease in addition to mycobacterial infections [45] . gof stat1 mutations cause chronic mucocutaneous candidiasis because increased signaling downstream of ifnc inhibits il-17 production, ultimately causing defective responses to fungal infection. chronic mucocutaneous candidiasis patients are also predisposed to autoimmunity, and gof stat1 mutations have been reported to cause a number of other autoimmune manifestations [46] . lof mutation in the stat2 gene causes increased susceptibility to viral infection and has also been described as a cause of sepsis-like syndrome following immunization with a live viral vaccine [47, 48] . this is consistent with the role of stat2 in signaling downstream of type i ifns, which are critical for immune responses to virus. stat3 signals downstream of il-6 and is critical for the differentiation of t-helper (th)17 cells, which secrete cytokines such as il-17 and il-22 [49, 50] , among others. il-17 is critical for immune responses to extracellular bacteria and fungi, and il-22 promotes barrier integrity [51, 52] . dominant-negative stat3 mutations cause hyper immunoglobulin e syndrome, also known as job's syndrome, which is characterized by recurrent sinopulmonary infections, mucocutaneous candidiasis, dermatitis, elevated serum immunoglobulin e levels, and connective tissue abnormalities [53] . activating mutations in stat3, by contrast, cause early-onset autoimmune disease [54] with neonatal diabetes and autoimmune lymphoproliferative disease [55] . this phenotype is driven by increased stat3 transcriptional activity, concomitant defective stat1/5 phosphorylation, and reduced differentiation of regulatory t cells, which are important for immune tolerance [55] . however, jak1 has a broader role in cytokine signaling. newer generation jakinibs block specific jak molecules compared with the first-generation jakinibs that are non-selective. thereby, the newgeneration jakinibs should have fewer side effects while maintaining similar efficacy as first-generation jakinibs. however, some degree of off-target side effects such as cytopenias are seen even with selective jakinibs such as decernotinib and abt494. epo erythropoietin, gh growth hormone, gm-csf granulocyte macrophage-colony stimulating factor, ifn interferon, th t-helper, tpo thrombopoietin, tyk tyrosine kinase somatic stat3 mutations are also associated with a broad range of hematologic and solid organ malignancies, which make stat3 blockade an active area of research for therapeutic agents [56, 57] . there are two stat5 genes, stat5a and stat5b. stat5b signals downstream of many cytokines critical for immune cell growth and immune response and is particularly important for t and nk cells. as expected, lof stat5b mutations cause immunodeficiency [58] . however, stat5 is also crucial for the differentiation and function of regulatory t cells, which constrain autoreactive immune responses. thus, patients with stat5b deficiency develop autoimmune disease. moreover, because growth hormone signals through stat5, the clinical spectrum of stat5b deficiency also includes dwarfism [59] . the variety of pathology caused by jak and stat mutations dramatically illustrates the criticality of jak-stat signaling both for the normal and aberrant immune responses. furthermore, a large body of genome-wide association studies also implicates the jak-stat pathway in the pathogenesis of common rheumatologic diseases [33] . for example, jak2 polymorphisms are associated with behçet's disease, while single nucleotide polymorphisms in the tyk2 gene have been implicated in crohn's disease (cd) and lupus [25, 60, 61] . stat3 polymorphisms are linked to cd, psoriasis, and behcet's disease [62, 63] , whereas ra and systemic lupus erythematosus (sle) are associated with stat4 polymorphisms [64] , and stat6 polymorphisms have been linked to ra, atopy, and asthma [65] . g-csf granulocyte-colony stimulating factor, il interleukin, tpo , tyk tyrosine kinase 5 inhibitors of the jak-stat pathway what we have described so far illustrates the importance of jaks and stat in the homeostasis of the immune system and provides the rationale for targeting jak-stat signaling to treat autoimmune and inflammatory diseases. the potential of jak inhibition as a therapeutic strategy was recognized in the 1990s [66] and fewer than 20 years later two small-molecule jakinibs were approved by the us food and drug administration (fda): ruxolitinib for the treatment of myeloproliferative neoplasm and tofacitinib for the treatment of ra. recently tofacitinib received positive opinion from european medicine agency (ema) for its use in ra [67] . current jakinibs act by competitively blocking the adenosine triphosphate-binding site in the jh1 domain through non-covalent interactions [5] . structural similarities of this binding site to the active domains of several other tyrosine kinases presented a challenge for the development of a compound that would specifically block jaks without off-target effects [5] . furthermore, given the high conservation of the jh1 domain among jaks, developing a jakinib that would selectively block one jak was even more challenging [5] . despite these difficulties, multiple jakinibs have been developed with reasonable specificity [5] and it has become apparent that pan-jakinibs, with activity against multiple jaks, are efficacious with an acceptable adverseeffect profile. it is still unclear if selective inhibition of a specific jak translates into therapeutic specificity [16] . tofacitinib was the first jakinib approved for use in autoimmune diseases. it is a jak1/jak3 inhibitor with some activity against jak2 [5, 16, 68] and negligible activity towards tyk2 [69] . metabolism and pharmacokinetics the majority of the metabolism of tofacitinib occurs via cytochrome p450 (cyp) 3a4 and, to a lesser extent, cyp2c19 [70] . in vitro cyp3a4 inhibition with ketoconazole resulted in over 70% inhibition of metabolism [70] , which supports in vivo data estimating a 103% increase in the tofacitinib area under the concentration-time curve (auc) following ketoconazole administration [71] . drug interaction studies with fluconazole also showed significant increases in the maximum plasma concentration (c max ) and auc of tofacitinib, resulting in dosing adjustment recommendations in the package insert for concomitant use with strong cyp3a4 inhibitors or moderate inhibitors of cyp3a4 and strong inhibitors of cyp2c19 [72, 73] . interaction studies with rifampin, a cyp3a4 inducer, yielded lower c max and auc values for tofacitinib; however, the pharmacodynamic and clinical significance of these changes are unknown [74] . tofacitinib is not an inhibitor of cyp3a4 itself, as evidenced in a lack of alteration of midazolam pharmacokinetics, a cyp3a4 substrate, when co-administered with tofacitinib [75] . tofacitinib extended release, the recently fda-approved formulation suitable for once-daily dosing, relies on extrudable core system osmotic-delivery technology, which confers improved upper limits of drug loading vs. bilayer push-pull osmotic tablets [76] . the extended-release formulation provides equivalent total systemic exposure, c max , and minimum plasma concentration when compared with the immediate-release tablet dosed at 5 mg twice daily (bid) [76] . as expected, time to c max and elimination half-life are prolonged for the extended-release formulation [76] . pharmacokinetic considerations and implications for drug interactions of the fda-approved jakinibs are shown in table 3 . topical tofacitinib ointment is currently being studied for plaque psoriasis. pharmacokinetic data from a phase iia trial showed quantifiable systemic concentration of the drug in 60% of the patients at one time point. the two different formulations of 2% tofacitinib ointment, composed of different vehicles, had time to maximum plasma concentration values of 0.5 and 2 h [77] . efficacy of tofacitinib in rheumatoid arthritis various phase ii and phase iii trials showed the safety and effectiveness of tofacitinib as monotherapy and in combination with other disease-modifying anti-rheumatic drugs (dmards) in the treatment of rheumatoid arthritis [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] (table 4 ). the dose of tofacitinib in the reported phase ii studies ranged between 1 and 30 mg bid. a doseranging study by tanaka and colleagues showed incremental response in american college of rheumatology 20% (acr 20) with increasing doses of tofacitinib [79] , but this observation was not confirmed in other clinical trials [80] . such trials, including the phase iii trials under the oral ra trials (oral) series, showed increased attainment of acr 20 response with tofacitinib relative to placebo. other measures of improvement as defined by the american college of rheumatology (acr 50, acr 70), and functional status measured by the health assessment questionnaire-disability index (haq-di) and the 36-item short form health survey were also improved ( table 4) . several studies have examined the effect of tofacitinib on structural joint disease, assessed radiographically, with promising results. a phase ii randomized controlled trial compared the effects of tofacitinib monotherapy, tofacitinib and methotrexate combination therapy, and methotrexate monotherapy on the musculoskeletal system. magnetic resonance imaging (mri) outcomes were reported as outcome measures in rheumatology clinical trials ra mri score (ramris), quantitative ramris, and dynamic contrast-enhanced mri [90] . the study showed significant improvement in ramris bone marrow edema at 6 months and improvement in synovitis scoring at 3 months in both tofacitinib monotherapy and tofacitinib with methotrexate combination therapy compared with methotrexate monotherapy. a significant difference was noted in synovitis scores by quantitative ramris at 3 months. erosive damage was significantly lower at 6 and 12 months in both the tofacitinib monotherapy and combination therapy groups compared with methotrexate monotherapy [91] . the phase iii oral-scan trial also used radiographic outcomes and showed slower rates of radiographic progression of disease in patients treated with tofacitinib at 5 or 10 mg bid with background methotrexate as measured by erosion score and joint space narrowing scores. the change in the joint space narrowing score was statistically significant at 12 months [88] . the long-term efficacy of tofacitinib in moderate to severe ra in 4000 patients was reported in the oral sequel study, which showed continued efficacy of the drug over 48 months as measured by acr 20/50/70, disease activity score 28-4-erythrocyte sedimentation rate (das28-4-esr), and haq-di. [92] . finally, a long-term extension trial collecting data on open-label tofacitinib following blinded treatment with adalimumab or tofacitinib for moderate to severe ra was reported recently, from patients in the oral standard and oral sequel trials. results supported long-term efficacy of tofacitinib, with improved physical function and disease signs and symptoms as measured by acr response criteria and das28-esr in tofacitinib-treated patients [93] . tofacitinib in psoriasis and psoriatic arthritis many inflammatory cytokines critical to the pathogenesis of psoriasis signal through the jak-stat pathway, including type i/ii ifns, il-12, il-22, and il-23 [94] . thus, it is not unexpected that jakinibs are an effective treatment for psoriatic skin and joint disease. a phase i trial conducted on medically stable patients with mild to moderate psoriasis demonstrated the efficacy of tofacitinib at a dose of 10 mg bid or higher, as measured by patient global assessment and histology [95] . a subsequent phase ii trial using a range of 2-15 mg bid doses of tofacitinib to treat more severe plaque disease also demonstrated statistically significant improvement in the psoriasis area and severity index (pasi) 75 and in other outcome measures including physician global assessment, pasi 50, and pasi 90 compared with placebo [96] . phase iii trials (opt pivotal 1 and opt pivotal 2 and long term extension study) using oral tofacitinib at 5-and 10-mg bid dosing in patients with moderate to severe plaque psoriasis demonstrated efficacy of both doses over placebo as measured by standard criteria described above. the efficacy was maintained at 2 years in the long-term extension trial. the higher dose of 10 mg bid was found to be more efficacious [97, 98] . indeed, comparison of both 5and 10-mg bid doses with weekly etanercept established non-inferiority of only the 10-mg dose, whereas the 5-mg dose was inferior to tnf blockade [99, 100] . it was concluded from this result that the effective dose of tofacitinib in psoriasis would be 10 mg bid. owing to potential safety concerns regarding the use of higher doses of tofacitinib, the drug failed to obtain fda approval for the treatment of psoriasis. it remains to be seen whether this decision will be re-evaluated as more extensive safety data from longterm extension studies clarify the long-term risks of such treatment. a 52-week, phase iii, multisite, randomized, doubleblind trial was conducted in 16 centers in japan to study the efficacy, safety, and tolerability of tofacitinib in the treatment of psoriatic arthritis. the results showed that 62.8 and 72.7% of plaque psoriasis patients on 5 and 10 mg of tofacitinib, respectively, achieved a pasi 75. while only 12 patients had joint disease, 100% of these patients achieved acr 20 response. preliminary results from the larger phase iii opal-broaden and opal-beyond studies were similarly encouraging, meeting their primary efficacy endpoints for dmard-refractory and anti-tnf-refractory psoriatic arthritis [101] . these studies demonstrated that tofacitinib may represent a promising therapy for psoriatic arthritis, although more extensive results and future longterm studies may be needed to clarify the effects of tofacitinib on structural joint disease [102] . topical tofacitinib in psoriasis a phase iia, multi-center, double-blind, vehicle-controlled study was conducted to evaluate the efficacy, safety, tolerability, and systemic pharmacokinetics of topical tofacitinib in mild-to-moderate plaque psoriasis. two different tofacitinib ointment formulations were assessed, both were administered bid for 4 weeks to a single fixed 300-cm 2 area with one target plaque with or without one or more non-target plaques and normal skin. the primary endpoint, defined as the percentage change from baseline in the target plaque severity score, showed statistically significant improvement by about 50% only for one of the ointments at week 4 [77, 103] . tofacitinib in inflammatory bowel disease inflammatory bowel disease (ibd) comprises two major distinct entities: ulcerative colitis (uc) and cd. while the two forms of ibd exhibit many common clinical features, they are pathophysiologically distinct and may not respond to tofacitinib in the same way. although a full understanding of ibd immunopathogenesis is lacking, it appears that both forms result from dysregulated immune responses. the role of type i/ii cytokines in ibd is well established: il-12, type ii ifn, and il-6 promote the function of pathogenic innate lymphoid cells and t cells [104] . however, patients are also immunodeficient: they are unable to constrain pathogenic microbiota. moreover, anti-inflammatory cytokines such as il-22 and il-17a may have a protective role [104] . thus, type i/ii cytokine blockade may have unintended consequences. tofacitinib is being considered for the treatment of uc. after promising results from initial phase ii trials [105] [106] [107] , the efficacy of tofacitinib as induction therapy was assessed in two phase iii trials in patients with moderate-to-severe uc (octave induction 1 and 2), which were recently completed. preliminary reports indicate that the trials met their primary and secondary endpoints, although full results are not yet available [108] . the octave sustain trial, which examines the efficacy of tofacitinib as maintenance therapy, and the long-term extension octave open trial will provide more data regarding the efficacy of jak inhibition in the treatment of uc. approval of tofacitinib for uc will likely depend on the final outcomes of these studies [109] . results for patients treated with tofacitinib for cd are less clear. a randomized, multi-center, phase ii clinical trial including 139 patients with moderate-to-severe active cd receiving tofacitinib 1, 5, or 15 mg or placebo bid for 4 weeks did not show clinical efficacy. however, the study showed a statistically significant reduction in serum c-reactive protein (crp) and fecal calprotectin levels in subjects receiving the 15-mg dose. the reasons for the negative results of the trial are unclear but may be related to the short study duration, possibly limiting the ability of tofacitinib to show any significant improvement in clinical response [109, 110] . preliminary data from subsequent studies indicate a small effect for tofacitinib treatment in cd [111] ; however, it is not clear whether such effects are clinically significant. results also indicate that tofacitinib may be effective as maintenance therapy [112] , although further data from an ongoing long-term clinical trial will answer this question more definitively (http://www. clinicaltrials.gov nct01393626). the first fda-approved jakinib, ruxolitinib is a jak1 and jak2 inhibitor [5] with moderate inhibitory activity against tyk2 [113] . as mentioned above, ruxolitinib was developed for the treatment of polycythemia vera and intermediate-and high-risk primary myelofibrosis [33, 114] , where inappropriate activation of jak2 underlies disease pathogenesis, and is fda approved for these diseases. ruxolitinib is also effective in the treatment of essential thrombocythemia [115] and has been granted breakthrough therapy designation for the treatment of graft vs. host disease [116] . metabolism and pharmacokinetics ruxolitinib and tofacitinib have similar pharmacokinetic profiles but ruxolitinib has more active metabolites and lower renal excretion [70] . ruxolitinib is metabolized primarily by cyp3a4 and to a lesser extent, cyp2c19 (table 3) . pharmacokinetic and pharmacodynamic studies using ketoconazole and erythromycin, strong and intermediate inhibitors of cyp3a4 as well as rifampin, a cyp3a4 inducer, were conducted to determine the impact on the metabolism of ruxolitinib [117] . co-administration of ketoconazole with single-dose ruxolitinib resulted in an increase in drug exposure for ruxolitinib of 91% and a prolongation of the elimination half-life of ruxolitinib of approximately 2 h. co-administration of erythromycin, a moderate cyp3a4 inhibitor, with ruxolitinib exhibited much less significant impact on c max and drug exposure of ruxolitinib. pharmacokinetic studies of ruxolitinib with rifampin resulted in a 52% decrease in c max of ruxolitinib with a decrease in terminal half-life of approximately 50% [117] . the pharmacodynamic impact of these interactions were assessed through an assay evaluating the extent of inhibition of stat3 phosphorylation. co-administration of ketoconazole resulted in a doubling of stat3 phosphorylation inhibition, which was clinically and statistically significant. co-administration of erythromycin and rifampin resulted in a 13% increase or a 10% decrease of pharmacodynamic activity respectively. these changes were not considered to be clinically significant [117] . further pharmacokinetic modeling studies evaluated the effect of fluconazole, a moderate inhibitor of both cyp3a4 and cyp2c19, and estimated a two-fold increase in ruxolitinib auc with fluconazole doses of the 100-200 mg total daily dose. this same modeling study also evaluated the impact of ruxolitinib on p-glycoprotein efflux pumps and did not predict a significant impact of ruxolitinib co-administration with p-glycoprotein substrates including digoxin [118] . use of ruxolitinib in autoimmune diseases studies using ruxolitinib to treat various inflammatory and autoimmune diseases have been promising. a phase iia trial of ruxolitinib in ra showed encouraging results with improvement in acr 20, 50, and 70 and haq-di as compared with placebo after 28 days [113, 119] . a case of chilblain lupus erythematosus has been successfully treated with oral ruxolitinib [120] . improvement in muscle strength and skin lesions was also reported in a patient with dermatomyositis and post-polycythemia vera jak2 v617f-positive myelofibrosis [121] . moreover, remarkable improvements in patients with alopecia areata treated with oral ruxolitinib for 3-5 months have been reported. comparison of biopsy samples at baseline and after 12 weeks of treatment demonstrated decreased inflammation post-treatment [122] . ruxolitinib has been reported to have significant cutaneous anti-inflammatory action [123] , and effects on plaque psoriasis were investigated in a small placebo-controlled clinical trial (86) . topical ruxolitinib was well tolerated and superior to placebo in reducing the plaque area [124] . topical ruxolitinib has also been reported to be useful in alopecia [125] . baricitinib is another selective jak1/jak2 inhibitor that inhibits intracellular signaling of multiple proinflammatory cytokines including il-6, il-12, il-23, and ifnc [126] . metabolism and pharmacokinetics renal clearance is the primary route of excretion for baricitinib; therefore, the role of cyp-mediated medication interactions are thought to be minimal for this drug [127] . it is speculated that the half-life would be prolonged in disease states with reduced renal function [127] (table 3) . efficacy of baricitinib in ra baricitinib has progressed to phase iii studies in ra. phase iib studies have demonstrated the efficacy of baricitinib at 4-and 8-mg dosing in ra unresponsive to methotrexate over a 12-to 24-week study period [128, 129] . improvement in musculoskeletal mri findings was demonstrated along with clinical response in a phase iib substudy [130] . table 5 summarizes important phase iii trials on baricitinib in ra [131] [132] [133] [134] . the trials unequivocally established the efficacy of baricitinib in active ra with improvement in all the measures of acr response criteria. there was significant improvement in acr 20/50/70, das28, and haq-di in the subjects treated with baricitinib compared with placebo. the ra-beam (a study in moderate to severe rheumatoid arthriris) study demonstrated superiority of baricitinib over adalimumab, a landmark finding not achieved with any other disease-modifying agent. the long-term extension of this study at 24 and 52 weeks showed prevention of progressive radiographic structural joint damage with baricitinib [134] . similar radiographic improvement was shown in the ra-build (a study in moderate to severe rheumatoid arthritis participants) study as well, where a change in the medial tibia stress syndrome score at week 24 was significantly lower in the baricitinib group compared with placebo [133] . an interesting finding in this study was the rapid improvement in acr criteria within a week whereas, inability to respond to the drug within 4 weeks was predictive of future failure. this information could be used to prevent unnecessary drug exposure in non-responders [135] . following the ra-build study, patient-reported outcome measures including pain, functional disability, and fatigue showed significant improvement with baricitinib therapy [136] . an extension study in participants with moderate to severe rheumatoid arthritis (ra-beyond) is currently recruiting participants. the purpose of this study is to investigate the long-term safety and any side effects of baricitinib in participants who have completed a previous baricitinib ra study. the study will provide for 4 years of additional treatment with baricitinib (http:// www.clinicaltrials.gov nct01885078). baricitinib and other diseases baricitinib has been shown to improve pasi 75 scores in plaque psoriasis in a phase iib trial [137] . baricitinib is also extremely effective in the treatment of autoinflammatory diseases characterized by an ifn gene signature, such as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature syndrome [138] . similar to ruxolitinib, baricitinib was found to be effective in alopecia areata in a patient who received this drug for the treatment of chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature syndrome [139] . oclatinib is a pan-jakinib approved for canine eczema and atopic dermatitis [25, 140] . the efficacy of oclacitinib for canine atopic disease hints that jakinibs may represent a promising therapeutic strategy for the treatment of allergic diseases in humans, and preliminary results indicate that tofacitinib may also be efficacious for atopic dermatitis [141] . clinical trials are ongoing to further evaluate the efficacy of systemic and topical jakinibs for this class of diseases. because jakinibs simultaneously block signaling downstream of cytokines important for a range of physiological functions, their side effects can often be directly linked to their mechanism of action. safety concerns include effects on hematopoiesis, innate and adaptive host defense, as well as growth. because tofacitinib is the most extensively studied jakinib, most available safety data are derived from clinical trials where this drug was used. these studies have demonstrated an acceptable safety profile [33] , and the safety profiles of other jakinibs appear comparable. infection secondary to immunosuppression represents a major concern in jakinib-treated patients. common side effects in ra clinical trials included infections such as nasopharyngitis or upper respiratory infections, bronchitis, and gastroenteritis. a number of opportunistic infections such as herpes zoster, tuberculosis, cellulitis, panniculitis, septic shock, and osteomyelitis were also reported [3, 142] . the observed risk was similar to that with other dmards, and a retrospective meta-analysis of pooled data from all the trials and extension studies indicated a lower risk of infection in tofacitinib-treated patients than for patients treated with biological dmards [142] . the exception to this is varicella zoster virus, for which the risk of reactivation is substantially higher in tofacitinib-treated patients [143, 144] . this increased risk may be in part owing to the importance of jak3-dependent cytokines in driving the development and functions of nk cells, which are important for controlling viral infections such as herpes zoster [142] . however, nk cell counts are not markedly reduced in tofacitinibtreated patients; therefore, the etiology of zoster reactivation may be related to nk cell function or to effects on a different leukocyte population. among other serious opportunistic infections, bk viremia and nephropathy have been reported in kidney transplant recipients treated with highdose tofacitinib (30 and 15 mg bid) in combination with mycophenolate mofetil [145] . a larger multi-center clinical trial also showed a higher incidence (14-18%) of bk nephropathy in renal transplant recipients treated with tofacitinib compared with cyclosporine (6%) [146] , also in combination with mycophenolate mofetil and at relatively high doses. progressive multifocal leukoencephalopathy has been reported in a 75-year-old man treated with ruxolitinib for myelofibrosis [147] . like infectious complications, jakinib-driven cytopenias were expected because many hematopoietic growth factors including erythropoietin and granulocyte macrophage-colony stimulating factor signal through jak2. neutropenia and anemia were indeed observed albeit sporadically. a higher incidence of mild-to-moderate anemia, leukopenia, neutropenia, lymphopenia, and thrombocytopenia was observed in patients receiving tofacitinib 30 mg compared with patients on lower doses of tofacitinb [78] . in patients taking lower doses, cytopenias were typically mild and did not necessitate discontinuation of the drug. one particular concern with long-term suppression of the jak-stat pathway is the possible development of malignancies. both type i and ii ifns play an important role in the process of immunoediting, which is critical for the anti-tumor immune response [148] . in post-transplant patients treated with tofacitinib, the risk of lymphoproliferative malignancy was increased by jak inhibition [149] . however, the phase ii and iii trials for autoimmune diseases have not shown an increased cancer risk associated with tofacitinib treatment [92] . the incidence of malignancy, including lymphoma and non-melanomatous skin cancer, is similar to that seen with other biologics. the increase in low-density lipoprotein (ldl), highdensity lipoprotein (hdl), and a few cardiovascular events such as atrioventricular block, congestive heart failure, and myocardial infarction were observed in tofacitinib-treated patients. however, long-term extension studies have not shown evidence of an increased rate of cardiovascular events [92] . combined data from phase iii trials on tofacitinib demonstrated stabilization of lipid levels after 3 months of treatment and the incidence of cardiovascular adverse events was similar to placebo [150] . the effect of tofacitinib on the lipid profile is similar to tocilizumab and may be secondary to blocking il-6 signaling. the physiological role of il-6 on hepatic lipid metabolism is complex and incompletely understood [151] . il-6 is known to cause insulin resistance and high il-6 levels have been noted in obesity. il-6 also support the redistribution of fatty acids from the blood to peripheral tissues, which results in low serum levels of ldl, hdl, and triglycerides [25] . to date, long-term extension studies have not shown an increase in major cardiovascular events in tofacitinib-treated patients relative to those treated with placebo [150, 152] . tofacitinib was actually shown to reduce vascular stiffness in ra patients in a small study of 18 patients [153] . assessment of cholesterol and lipoprotein kinetics in ra patients before and after tofacitinib treatment in comparison to healthy volunteers revealed increased levels of cholesterol in ra patients after treatment and is secondary to reduced cholesterol ester catabolism and anti-atherogenic hdl level improvement [154] . other changes in laboratory parameters included sporadic elevations in transaminases and creatinine. clinically significant hepatic and renal compromise have not been reported; however, [92] a phase i randomized controlled trial assessed changes in serum creatinine and glomerular filtration rate in ra patients treated with tofacitinib and compared them with patients who received placebo. tofacitnib treatment caused mild increases in creatinine (5%) and decreases in glomerular filtration rate (8%), which reversed rapidly upon drug discontinuation [155] . whereas first-generation jakinibs including tofacitinib have shown efficacy in the treatment of inflammatory conditions like ra, nonselective pan-jak blockade can be associated with unwanted adverse effects such as cytopenias. this raises the potential utility of next-generation jakinibs with selective inhibitory activity for a specific jak (fig. 3) , which, in principle might be used to treat selected autoimmune disorders with fewer adverse effects [156] . however, increased selectivity may also translate into reduced efficacy. decernotinib is a next-generation jakinib with in vitro kinase assays demonstrating five-fold selectivity towards jak3 compared with jak1, jak2, and tyk2 [157] . decernotinib showed promising results in animal models of autoimmune diseases [156] and was therefore moved into clinical trials for the treatment of ra. metabolism and pharmacokinetics decernotinib possesses a unique pharmacokinetic profile with potential implications for medication interactions. the major metabolite of decernotinib, m3, acts as a potent inhibitor of cyp3a4 [158] . cyp3a4 is the most prevalent hepatic cyp enzyme and is implicated in metabolism for over 50% of currently marketed medications [159] . the clinical implications of m3's inhibition of cyp3a4 may be far reaching, as one of the phase iib dercernotinib studies excluded not only subjects taking moderate or strong inhibitors or inducers of cyp3a4 and p-glycoprotein, but also subjects taking any medication metabolized via cyp3a4 with the potential for toxicity at high levels of exposure [160] . in addition, adverse effects of this drug included elevations in lipid parameters [80, 132, 160] . notably, several high-potency, commonly used hydroxymethylglutaryl co-a reductase inhibitors (statins) including simvastatin and atorvastatin are metabolized via cyp3a4 [161] . therefore, concomitant use with decernotinib could potentially increase the risk for statin-associated toxicity. decernotinib in ra clinical data using decernotinib in ra have initially been promising. phase ii trials demonstrated efficacy at doses of 50-150 mg bid, with improvement of acr response criteria and das28 joints using the crp level (das28-crp) compared with placebo. adverse events reported were similar to first-generation jakinibs such as infections, transaminitis, and hyperlipidemia [126, 160, 162] . anemia was not observed, consistent with selectivity for jak3 over jak2. surprisingly, neutropenia was seen in a large number of patients, which may indicate that the drug could have some offtarget effects [157] . a phase iib study recently demonstrated improvement in synovitis and osteitis with decernotinib along with conventional dmards in ra patients [163] . filgotinib inhibits both jak1 and jak2 in whole blood cell-based assays and kinase assays but displays an 30-fold selectivity towards jak1 [126] . in vitro studies also demonstrated a dose-dependent inhibition of th1 and th2 and, to lesser extent, th17 cell differentiation. metabolism and pharmacokinetics in vitro analysis of the impact of filgotinib and its active metabolite on cyp enzymes indicate that neither agent inhibits nor induces cyp activity at clinically relevant concentrations [164] . this conclusion was confirmed for cyp3a4 in a study on the impact of filgotinib on midazolam clearance in healthy volunteers, which showed no changes in midazolam metabolism [164] . the potential of filgotinib to impact cell-based transport systems including p-glycoprotein and breast cancer resistance protein were examined in vitro, with the authors concluding that filgotinib was unlikely to inhibit these transport systems. the effects of filgotinib on organic cation transporters including organic cation transporter 2 were also examined in vitro with observed inhibition, but this is of unknown clinical significance. the potential for filgotinib to affect organic anion transporters implicated in methotrexate (mtx) clearance was explored, with no changes in mtx clearance observed in doses of filgotinib up to 300 mg [164] . it appears that the unique pharmacokinetic profile of filgotinib may provide flexibility in dosing regimens (146) . filgotinib in autoimmune diseases filgotinib is currently is being investigated for the treatment of ra [165] . phase iia studies in patients with active ra and inadequate response to mtx showed efficacy of filgotinib over placebo at doses of 30 mg daily and higher [126, 166] . this was followed by two phase iib trials: darwin1 and darwin 2. darwin1 is a study on 595 mtx-treated ra patients where filgotinib was added at a range of doses from 50 mg daily to 100 mg bid. the darwin2 study assessed filgotinib monotherapy in 280 ra patients with doses ranging from 50 to 200 mg daily [126] . in both studies, filgotinib was found to be superior to placebo in controlling disease activity as measured by acr 20/50, das28-crp, simple disease activity index (sdai), and clinical disease activity index [167, 168] . filgotinib is also being investigated in moderate to severe cd (fitzroy study) [109, 169] . preliminary data from the trial showed significantly improved clinical outcomes and quality of life as measured by the clinical disease activity index and the inflammatory bowel disease questionnaire, respectively, with figlotinib compared with placebo [170] . safety profiles in both darwin trials and the fitzroy trial were all favorable. in the fitzroy study, filgotinib showed a favorable lipid profile with an increase in hdl and no change in ldl, resulting in an improved atherogenic index. an increase in hemoglobin was also observed and no clinically significant changes from baseline in neutrophils or liver function tests were observed, consistent with intact signaling through jak2. notably, a trial in patients with sle was closed for lack of efficacy [171] . abt-494 is a next-generation jakinib with 74-fold selectivity for jak1 over jak2, based on the drug's ability to bind jak1 outside the adenosine triphosphate-binding site of jh1 in addition to the adenosine triphosphate binding site. because the binding occurs with a less conserved domain, it is described as being specific for jak1 [172] . importantly, as jak2 and jak3 signaling remain unaffected, abt-494 does not affect erythropoietin signaling or reduce peripheral nk cell counts at therapeutic doses [126] . two multi-center, randomized, double-blind, placebocontrolled phase iib studies (balance i and ii) were conducted in subjects with moderate to severe ra taking mtx who did not respond to either anti-tnf therapy (balance i) or mtx (balance ii) (http://www. clinicaltrials.gov nct01960855). both studies demonstrated rapid improvement in acr 20/50/70 and das28-crp with abt-494 compared with placebo. improvements were observed as early as week 2 with abt4-94 [173, 174] . patient recruitment is currently ongoing for a phase iii, double-blind, placebo-controlled study in ra with inadequate response to mtx, comparing abt-494 with adalimumab on a stable background dose of mtx (http://www.clinicaltrials.gov nct02629159). peficitinib (asp015k) is a novel, orally bioavailable jak inhibitor that inhibits jak1, jak2, jak3, and tyk2 enzyme activities with moderate selectivity for jak3 inhibition. inhibition of jak2 by peficitinib is relatively mild, which confers an acceptable safety profile [175] and some potential advantages over first-generation inhibitors. metabolism and pharmacokinetics similar to other jakinibs, peficitinib exhibits rapid oral absorption. interestingly, in metabolic studies, peficitinib possessed no single dominant clearance pathway [176] . to date, clinically significant drug interactions with peficitinib have not been identified, and the major phase ii trial with this agent lacked exclusion criteria based on potential drug interactions that may affect clearance of the jakinib [175] . peficitinib in autoimmune/inflammatory diseases peficitinib reduced paw swelling and ankle bone destruction in a preclinical model of rat adjuvant-induced arthritis [177] . early clinical studies have also been promising. a phase ii trial in ra patients showed a statistically significant acr 20 response compared with placebo at a range of doses from 25 to 150 mg [175] . a phase iia trial in plaque psoriasis demonstrated dose-dependent efficacy of peficitinib in pasi, body surface area (bsa), physician static global assessment, and histological measures of severity after 6 weeks [178] . no major adverse events were reported by either of these trials. solcitinib is another selective jak1 inhibitor that has been evaluated for the treatment of moderate-to-severe plaquetype psoriasis. a 12-week, randomized, placebo-controlled clinical trial in moderate-to-severe plaque psoriasis revealed significant improvement in pasi 75 scores with solcitinib compared with placebo [179] . because jak1 transmits signals downstream of type i ifns, and patients with sle have evidence of aberrant type i ifn signaling, solcitinib was therefore assessed in a phase ii, randomized, placebo-controlled study of patients with moderate-to-severe sle. however, two cases of drug reaction with eosinophilia and systemic symptoms syndrome and severe but reversible liver function test abnormalities in six subjects were reported, necessitating early termination of the study [180, 181] . what this means for the use of other jakinibs in sle remains unclear. incb039110 is a next-generation jak inhibitor with selective inhibitory action against jak1. incb-039110 was demonstrated as having a [20-fold selectivity for jak1 over jak2 and a [200-fold selectivity over jak3. preclinical studies supported its efficacy in mouse adjuvant arthritis models, at doses that did not inhibit the biological activity of erythropoietin. incb039110 has also been shown to inhibit inflammatory pathways involved in the pathogenesis of psoriasis [182, 183] . a phase ii, multi-center clinical trial using this drug to treat active ra demonstrated clinically significant improvement in acr 20/50/70 and das28-crp compared with placebo (http://www.clinicaltrials.gov nct01626573) [126, 184] , at doses ranging from 100 mg bid to 600 mg daily. a phase ii clinical trial in plaque psoriasis was also encouraging, with improvement in pasi 50/75, psga, and affected body surface area in patients receiving incb039110 compared with placebo [182] . common adverse effects were similar to those seen with nonselective jakinibs and included infectious nasopharyngitis, elevated transaminases, and hypertriglyceridemia [182] . several biotechnology companies are in the process of developing jak inhibitors with the goal of creating a molecule with maximum efficacy and minimum off target effects [126] . the newer jakinibs are isoform specific, which is postulated to diminish adverse events found with first generation non-selective jakinibs. some of the newgeneration jakinibs are covalently bound to specific sequences of the jaks, resulting in better selectivity [126] . table 6 summarizes additional jak inhibitors in various stages of clinical and preclinical development. as jak substrates and key signaling molecules downstream of type i/ii cytokine receptors, stats have been investigated as an attractive target in the treatment of inflammation and autoimmunity, as well as malignancy. stat3 is essential for signaling downstream of il-6, which regulates the production of il-17 by t cells and other immune cells, implicating stat3 in the pathogenesis of many rheumatologic diseases [185, 186] . moreover, constitutive activation of stat3 and stat5 has been observed in several human cancers and cancer cell lines [50] . blocking the action of transcription factors, however, is much more challenging than inhibiting the activity of enzymes such as kinases. challenges to the development of stat inhibitors include issues with bioavailability and selectivity. for example, considerable homology exists between stat1 and stat3. stat1 facilitates vital functions including cell death, apoptosis, and pathogen defense, thus off-target stat1 blockade by a stat3 inhibitor can lead to a host of undesired adverse effects such as increased survival of tumor cells [33, 187, 188] . another challenge in developing stat inhibitors is functional redundancy in the action of different stats. specifically, although stat3 is critical for signaling downstream of il-6, stat3-deficient cells continue to respond to il-6 stimulation through activation of stat1 [189] . thus, selective blockade of one stat molecule may not be clinically useful, as another stat might compensate for the inhibited protein. despite these limitations, several small-molecule inhibitors targeting the sh2 domain have been developed and tested in phase i and ii clinical trials. one of these, opb-31121, was studied in a phase i/ii trial for hepatocellular carcinoma but had an unacceptable adverse effect profile, particularly peripheral neuropathy, and limited efficacy [190] . another inhibitor, opb-51602, appears safe and effective in the treatment of solid organ malignancies such as lung cancer [57] but is also associated with a high risk of peripheral neuropathy and has poor bioavailability, further limiting its tolerability [57, 191] . stat6 plays an important role in allergic pathways acting downstream to il-4 and il-13. phosphopeptides blocking the sh2 domain of stat6 are being developed to inhibit phosphorylation and further downstream signaling of this pathway, which may be useful in allergic diseases such as bronchial asthma [192] . because of difficulties with small-molecule inhibitors that target the sh2 domain, several other mechanisms of inhibition are being explored. stat3 decoy oligonucleotides target the dna-binding domain of the stat and are much more selective than inhibitors that target the sh2 domain. however, phase 0 studies revealed that the first generation of decoys degraded quickly in vivo, limiting their effectiveness [19] . development of intrabodies against phosphorylated forms of the stat3 molecule have been shown to be effective in in vitro studies [193] and may represent another method of successfully targeting stats therapeutically. nonetheless, however promising stat inhibition may be in the treatment of malignancy and autoimmunity, there are currently no clinical trials of stat inhibitors actively recruiting patients. given the number of cytokines that signal through the jak-stat pathway, it is no surprise that jakinibs have become the first kinase inhibitors used successfully in the treatment of rheumatologic disease. at present, tofacitinib is the only jakinib approved for autoimmune disease, but several more may soon follow as new data emerge and the development of novel agents continues. clinical trials are ongoing with various jakinibs in several autoimmune conditions ranging from rheumatoid arthritis to psoriasis. similarly, diseases characterized by serum elevations of jak-dependent cytokines could respond well to jakinibs. these include diseases for which a type i ifn signature has been defined such as sle, myositis, scleroderma, and primary sjogren's syndrome, as well as diseases driven by il-6 such as relapsing polychondritis and large-vessel vasculitis. several trials are indeed probing the efficacy of jakinibs in the treatment of diseases characterized by an ifn signature, based on preclinical data [194] and on the observation that baricitinib is effective for the treatment of monogenic interferonopathies such as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature syndrome and savi [stimulator of interferon genes (sting) associated vasculopathy with onset in infancy], which are also associated with a type i ifn signature [138] . moreover, the preliminary success of jakinibs in the treatment of alopecia and other skin-related diseases such as vitiligo is very exciting as no existing therapy has been effective in these relatively common conditions so far [195] [196] [197] . optimal dosing strategies of jakinibs also deserve further study, whereas currently approved regimens rely on a single dose for induction of remission and maintenance, it is not clear that this is the optimal strategy. it is possible that loading patients with high doses to induce remission, followed by a lower maintenance dose, would be a more effective strategy, as is the case for corticosteroids. investigations of non-oral formulations of jakinibs are also in the preliminary stages for diseases including psoriasis, allergic dermatitis, and ocular disease. finally, biomarker development is ongoing to identify groups of patients with disease likely to respond to jakinibs, as complex rheumatologic diseases may be driven by jak-dependent cytokines to a different degree in different patients [198] . after the era of biologics, the development of jakinibs and their successful use in autoimmune and inflammatory diseases heralds an exciting new chapter in rheumatology. these drugs are unique both structurally and functionally, as small molecules that can be administered orally rather than injected, and which simultaneously block multiple cytokines downstream of their receptors. in the coming years, jakinibs are poised to change the field of autoimmune and rheumatologic diseases as ongoing basic, preclinical, and clinical research will surely determine the feasibility and benefits of selectivity, optimized dosing, formulation, and patient selection to minimize undesirable off-target effects and maximize clinical efficacy. janus kinase inhibitors in autoimmune diseases systemic lupus erythematosus janus kinase inhibitors for rheumatoid arthritis the mechanism of action of tofacitinib: an oral janus kinase inhibitor for the treatment of rheumatoid arthritis discovery and development of janus kinase (jak) inhibitors for inflammatory diseases tofacitinib for the treatment of psoriasis jaks and stats: biological implications characterisation of a dendritic cell subset in synovial tissue which strongly expresses jak/stat transcription factors from patients with rheumatoid arthritis the activity of jak-stat pathways in rheumatoid arthritis: constitutive activation of stat3 correlates with interleukin 6 levels atp binding to the pseudokinase domain of jak2 is critical for pathogenic activation the pseudokinase domain of jak2 is a dual-specificity protein kinase that negatively regulates cytokine signaling structural and functional characterization of the jh2 pseudokinase domain of jak family tyrosine kinase 2 (tyk2) jak1 takes a ferm hold of type ii cytokine receptors the structural basis for class ii cytokine receptor recognition by jak1. structure structural basis of recognition of interferon-alpha receptor by tyrosine kinase 2 treating inflammation with the janus kinase inhibitor cp-690,550 structure, function, and regulation of stat proteins the molecular regulation of janus kinase (jak) activation a stat inhibitor patent review: progress since signaling via the cytor/jak/ stat/socs pathway: emergence during evolution first-in-human trial of a stat3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy evolution of the jak-stat pathway mechanism of activation of protein kinase jak2 by the growth hormone receptor. science intracellular signal pathways: potential for therapies type i/ii cytokines, jaks, and new strategies for treating autoimmune diseases structural bases of unphosphorylated stat1 association and receptor binding jaks and stats in immunity, immunodeficiency, and cancer cytokines and their role in lymphoid development, differentiation and homeostasis mechanisms of jak/stat signaling in immunity and disease stat4 serine phosphorylation is critical for il-12-induced ifn-gamma production but not for cell proliferation emancipation from transcriptional latency: unphosphorylated stat5 as guardian of hematopoietic differentiation dynamics and noncanonical aspects of jak/stat signalling the jak-stat pathway: impact on human disease and therapeutic intervention clinical significance of v617f mutation of the jak2 gene in patients with chronic myeloproliferative disorders jak-mutant myeloproliferative neoplasms somatic mutations of jak1 and jak3 in acute leukemias and solid cancers jak mutations in high-risk childhood acute lymphoblastic leukemia ferm domain mutations induce gain of function in jak3 in adult t-cell leukemia/lymphoma disruption of the jak1 gene demonstrates obligatory and nonredundant roles of the jaks in cytokine-induced biologic responses mutations of jak-3 gene in patients with autosomal severe combined immune deficiency (scid) mutation of jak3 in a patient with scid: essential role of jak3 in lymphoid development a restricted role for tyk2 catalytic activity in human cytokine responses revealed by novel tyk2-selective inhibitors inborn errors of human jaks and stats impairment of mycobacterial but not viral immunity by a germline human stat1 mutation dominant gain-offunction stat1 mutations in foxp3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-x-linked-like syndrome stat2 deficiency and susceptibility to viral illness in humans gone fission: an asymptomatic stat2 mutation elongates mitochondria and causes human disease following viral infection stat3: a stat family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6 stats: an old story, yet mesmerizing signaling through il-17c/il-17re is dispensable for immunity to systemic, oral and cutaneous candidiasis il-22 increases the innate immunity of tissues the hyper-ige syndromes activating germline mutations in stat3 cause early-onset multi-organ autoimmune disease early-onset lymphoproliferation and autoimmunity caused by germline stat3 gain-offunction mutations somatic stat3 mutations in large granular lymphocytic leukemia phase i and biomarker study of opb-51602, a novel signal transducer and activator of transcription (stat) 3 inhibitor, in patients with refractory solid malignancies cutting edge: decreased accumulation and regulatory function of cd4 ? cd25(high) t cells in human stat5b deficiency growth hormone insensitivity associated with a stat5b mutation polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus genome-wide metaanalysis increases to 71 the number of confirmed crohn's disease susceptibility loci jak2 and stat3 polymorphisms in a han chinese population with behcet's disease combined analysis of genome-wide association studies for crohn disease and psoriasis identifies seven shared susceptibility loci stat4 and the risk of rheumatoid arthritis and systemic lupus erythematosus stat6 as an asthma candidate gene: polymorphism-screening, association and haplotype analysis in a caucasian sib-pair study autosomal scid caused by a point mutation in the n-terminus of jak3: mapping of the jak3-receptor interaction domain pfizer receives positive chmp opinion in europe for xeljanz ò (tofacitinib citrate) for the treatment of moderate to severe active rheumatoid arthritis receives positive chmp opinion in europe for xeljanz ò (tofacitinib citrate) for the treatment of moderate to severe active rheumatoid arthritis pfizer receives positive chmp opinion in europe for xeljanz ò (tofacitinib citrate) for the treatment of moderate to severe active rheumatoid arthritis receives positive chmp opinion in europe for xeljanz (tofacitinib citrate) for the treatment of moderate to severe active rheuamtoid arthritis prevention of organ allograft rejection by a specific janus kinase 3 inhibitor tofacitinib for the treatment of rheumatoid arthritis the pharmacokinetics, metabolism, and clearance mechanisms of tofacitinib, a janus kinase inhibitor, in humans a phase 1 study to estimate the the effect of ketoconazole on the pharmacokinetics of tofacitinib (cp-690,550) in healthy volunteers an open label study to estimate the effect of fluconazole on the pharmacokinetics of cp-690,550 in healthy adult subjects (abstract) the effect of rifampin on the pharmacokinetics of tofacitinib (cp-690,550) in healthy volunteers (abstract no. pi-73) lack of effect of tofacitinib (cp-690,550) on the pharmacokinetics of the cyp3a4 substrate midazolam in healthy volunteers: confirmation of in vitro data extended-release once-daily formulation of tofacitinib: evaluation of pharmacokinetics compared with immediate-release tofacitinib and impact of food a randomized phase 2a efficacy and safety trial of the topical janus kinase inhibitor tofacitinib in the treatment of chronic plaque psoriasis the safety and efficacy of a jak inhibitor in patients with active rheumatoid arthritis: results of a double-blind, placebo-controlled phase iia trial of three dosage levels of cp-690,550 versus placebo tofacitinib study i: phase ii study of tofacitinib (cp-690,550) combined with methotrexate in patients with rheumatoid arthritis and an inadequate response to methotrexate a phase iib dose-ranging study of the oral jak inhibitor tofacitinib (cp-690,550) versus placebo in combination with background methotrexate in patients with active rheumatoid arthritis and an inadequate response to methotrexate alone improved pain, physical functioning and health status in patients with rheumatoid arthritis treated with cp-690,550, an orally active janus kinase (jak) inhibitor: results from a randomised, double-blind, placebo-controlled trial placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis phase iib doseranging study of the oral jak inhibitor tofacitinib (cp-690,550) or adalimumab monotherapy versus placebo in patients with active rheumatoid arthritis with an inadequate response to disease-modifying antirheumatic drugs tofacitinib (cp-690,550) in combination with methotrexate in patients with active rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitors: a randomised phase 3 trial tofacitinib or adalimumab versus placebo in rheumatoid arthritis tofacitinib in combination with nonbiologic disease-modifying antirheumatic drugs in patients with active rheumatoid arthritis: a randomized trial cp-690,550) in patients with rheumatoid arthritis receiving methotrexate: twelve-month data from a twenty-four-month phase iii randomized radiographic study tofacitinib versus methotrexate in rheumatoid arthritis tofacitinib or adalimumab versus placebo: patient-reported outcomes from a phase 3 study of active rheumatoid arthritis an introduction to the eular-omeract rheumatoid arthritis mri reference image atlas comparing the effects of tofacitinib, methotrexate and the combination, on bone marrow oedema, synovitis and bone erosion in methotrexate-naive, early active rheumatoid arthritis: results of an exploratory randomised mri study incorporating semiquantitative and quantitative techniques safety and efficacy of tofacitinib, an oral janus kinase inhibitor, for the treatment of rheumatoid arthritis in open-label, longterm extension studies switching from adalimumab to tofacitinib in the treatment of patients with rheumatoid arthritis psoriasis, psoriatic arthritis, and rheumatoid arthritis: is all inflammation the same? semin arthritis rheum double-blind, placebocontrolled, dose-escalation study to evaluate the pharmacologic effect of cp-690,550 in patients with psoriasis efficacy and safety of tofacitinib, an oral janus kinase inhibitor, in the treatment of psoriasis: a phase 2b randomized placebo-controlled doseranging study an oral janus kinase inhibitor, for the treatment of chronic plaque psoriasis: results from two randomized, placebo-controlled, phase iii trials tofacitinib, an oral janus kinase inhibitor, for the treatment of chronic plaque psoriasis: long-term efficacy and safety results from 2 randomized phase-iii studies and 1 open-label long-term extension study tofacitinib versus etanercept or placebo in moderate-to-severe chronic plaque psoriasis: a phase 3 randomised non-inferiority trial emerging drugs for psoriatic arthritis pfizer announces positive top-line results from the first phase 3 trial of investigational tofacitinib in adults with psoriatic arthritis oral tofacitinib efficacy, safety and tolerability in japanese patients with moderate to severe plaque psoriasis and psoriatic arthritis: a randomized, double-blind, phase 3 study emerging drugs for the treatment of axial and peripheral spondyloarthritis cytokines in inflammatory bowel disease promising biological therapies for ulcerative colitis: a review of the literature tofacitinib, an oral janus kinase inhibitor, in active ulcerative colitis randomized trial of tofacitinib in active ulcerative colitis: analysis of efficacy based on patient-reported outcomes pfizer (pfe)'s oral tofacitinib meets primary and key secondary endpoints in two late-stage trials will novel oral formulations change the management of inflammatory bowel disease? a phase 2 study of tofacitinib, an oral janus kinase inhibitor, in patients with crohn's disease op021 efficacy and safety of oral tofacitinib for induction therapy in patients with moderate-to-severe crohns disease: results of a phase 2b randomised placebo-controlled trial. 11th congress of european crohns and colitis organization op022 efficacy and safety of oral tofacitinib for maintenance therapy in patients with moderate-to-severe crohns disease: results of a phase 2b randomised placebo-controlled trial. 11th congress of european crohn's and colitis organization janus kinase inhibitors for the treatment of myeloproliferative neoplasias and beyond food and drug administration approval: ruxolitinib for the treatment of patients with intermediate and high-risk myelofibrosis long-term results from a phase ii open-label study of ruxolitinib in patients with essential thrombocythemia refractory to or intolerant of hydroxyurea fda grants breakthrough therapy designation for incyte's ruxolitinib (jakafi ò ) in acute graft-versus-host disease (gvhd) the effect of cyp3a4 inhibition or induction on the pharmacokinetics and pharmacodynamics of orally administered ruxolitinib (incb018424 phosphate) in healthy volunteers predicting drug-drug interactions involving multiple mechanisms using physiologically based pharmacokinetic modeling: a case study with ruxolitinib kinase inhibitors: the next generation of therapies in the treatment of rheumatoid arthritis jak-1/2 inhibitor ruxolitinib controls a case of chilblain lupus erythematosus remission of recalcitrant dermatomyositis treated with ruxolitinib alopecia areata is driven by cytotoxic t lymphocytes and is reversed by jak inhibition upcoming therapeutic targets in cutaneous lupus erythematous preliminary clinical activity of a topical jak1/2 inhibitor in the treatment of psoriasis topical ruxolitinib for the treatment of alopecia universalis selective jak inhibitors in development for rheumatoid arthritis the pharmacokinetics, pharmacodynamics, and safety of baricitinib, an oral jak 1/2 inhibitor, in healthy volunteers safety and efficacy of baricitinib at 24 weeks in patients with rheumatoid arthritis who have had an inadequate response to methotrexate efficacy and safety of baricitinib in japanese patients with active rheumatoid arthritis receiving background methotrexate therapy: a 12-week, doubleblind, randomized placebo-controlled study small molecular compounds in development for rheumatoid arthritis baricitinib, methotrexate, or baricitinib plus methotrexate in patients with early rheumatoid arthritis who had received limited or no treatment with disease-modifying anti-rheumatic drugs (dmards): phase 3 trial results baricitinib in patients with refractory rheumatoid arthritis lb0001 baricitinib, an oral janus kinase (jak)1/jak2 inhibitor, in patients with active rheumatoid arthritis (ra) and an inadequate response to cdmard therapy: results of the phase 3 ra-build study baricitinib versus placebo or adalimumab in patients with active rheumatoid arthritis (ra) and an inadequate response to background methotrexate therapy: results of a phase 3 study swift response seen for baricitinib in ra: response by week four predicts subsequent low disease activity, remission patient-reported outcomes from a phase 3 study of baricitinib in patients with rheumatoid arthritis with inadequate response to conventional synthetic disease-modifying antirheumatic drugs a randomized phase 2b trial of baricitinib, an oral jak1/jak2 inhibitor, in patients with moderate-to-severe psoriasis preliminary response to janus kinase inhibition with baricitinib in chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (candle) reversal of alopecia areata following treatment with the jak1/2 inhibitor baricitinib an update on the treatment of canine atopic dermatitis treatment of recalcitrant atopic dermatitis with the oral janus kinase inhibitor tofacitinib citrate systematic review and meta-analysis of serious infections with tofacitinib and biologic disease-modifying antirheumatic drug treatment in rheumatoid arthritis clinical trials herpes zoster and tofacitinib therapy in patients with rheumatoid arthritis real-world comparative risks of herpes virus infections in tofacitinib and biologic-treated patients with rheumatoid arthritis calcineurin-inhibitorfree immunosuppression based on the jak inhibitor cp-690,550: a pilot study in de novo kidney allograft recipients randomized phase 2b trial of tofacitinib (cp-690,550) in de novo kidney transplant patients: efficacy, renal function and safety at 1 year progressive multifocal leukoencephalopathy associated with ruxolitinib new insights into cancer immunoediting and its three component phases: elimination, equilibrium and escape evaluation of the effect of tofacitinib exposure on outcomes in kidney transplant patients cardiovascular safety findings in patients with rheumatoid arthritis treated with tofacitinib, an oral janus kinase inhibitor. semin arthritis rheum interleukin-6: from basic biology to selective blockade of pro-inflammatory activities effects of tofacitinib on cardiovascular risk factors and cardiovascular outcomes based on phase iii and long-term extension data in patients with plaque psoriasis tofacitinib improves arterial stiffness despite up-regulating serum cholesterol with chronic cardiovascular disease in methotrexate-resistant active rheumatoid arthritis patients: a cohort study potential mechanisms leading to the abnormal lipid profile in patients with rheumatoid arthritis versus healthy volunteers and reversal by tofacitinib evaluation of the effect of tofacitinib on measured glomerular filtration rate in patients with active rheumatoid arthritis: results from a randomised controlled trial vx-509 (decernotinib) is a potent and selective janus kinase 3 inhibitor that attenuates inflammation in animal models of autoimmune disease editorial: decernotinib. a next-generation jakinib the effect of deuteration of vx-509 (decernotinib) on drug-drug interactions (ddi) with midazolam human cytochrome p450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors vx-509 (decernotinib), an oral selective jak-3 inhibitor, in combination with methotrexate in patients with rheumatoid arthritis drug-drug interactions that interfere with statin metabolism a randomized, double-blind, placebo-controlled, twelve-week, doseranging study of decernotinib, an oral selective jak-3 inhibitor, as monotherapy in patients with active rheumatoid arthritis efficacy of vx-509 (decernotinib) in combination with a disease-modifying antirheumatic drug in patients with rheumatoid arthritis: clinical and mri findings clinical confirmation that the selective jak1 inhibitor filgotinib (glpg0634) has a low liability for drug-drug interactions pharmacokinetics and pharmacokinetic/pharmacodynamic modeling of filgotinib (glpg0634), a selective jak1 inhibitor, in support of phase iib dose selection the jak1-selective inhibitor glpg0634 is safe and rapidly reduces disease activity in patients with moderate to severe rheumatoid arthritis; results of a 4-week dose ranging study filgotinib (glpg0634), an oral jak1 selective inhibitor is effective as monotherapy in patients with active rheumatoid arthritis: results from a phase 2b dose ranging study filgotinib (glpg0634), an oral jak1 selective inhibitor is effective in combination with methotrexate in patients with active rheumatoid arthritis: results from a phase 2b dose ranging study efficacy and safety of glpg0634 in subjects with active crohn's disease filgotinib (glpg0634), an oral jak1 selective inhibitor, induces clinical remission in patients with moderate-to-severe crohn's disease: results from the phase 2 fitzroy study interim analysis galapagos. glpg0778/555. immuno and inflammatory thu0127 pharmacodynamics of a novel jak1 selective inhibitor in rat arthritis and anemia models and in healthy human subjects a phase 2b study of abt-494, a selective jak1 inhibitor, in patients with rheumatoid arthritis and an inadequate response to anti-tnf therapy a randomized phase 2b study of abt-494, a selective jak1 inhibitor in patients with rheumatoid arthritis and an inadequate response to methotrexate efficacy and safety of the oral janus kinase inhibitor peficitinib (asp015k) monotherapy in patients with moderate to severe rheumatoid arthritis in japan: a 12-week, randomised, double-blind, placebo-controlled phase iib study human mass balance, metabolite profile and identification of metabolic enzymes of asp015k: a novel jak inhibitor demonstrated potent efficacy in adjuvant-induced arthritis model in rats a phase 2a randomized, double-blind, placebo-controlled, sequential dose-escalation study to evaluate the efficacy and safety of asp015k, a novel janus kinase inhibitor, in patients with moderate-to-severe psoriasis investigation of selective jak1 inhibitor gsk2586184 for the treatment of psoriasis in a randomized placebo-controlled phase 2a study dress syndrome and reversible liver function abnormalities in patients with systemic lupus erythematosus treated with the highly selective jak-1 inhibitor gsk2586184 safety, tolerability, efficacy and pharmacodynamics of the selective jak1 inhibitor gsk2586184 in patients with systemic lupus erythematosus a randomized, double-blind, placebo-controlled, dose-escalation study of the safety and efficacy of incb039110, an oral janus kinase 1 inhibitor, in patients with stable, chronic plaque psoriasis novel oral therapies for psoriasis and psoriatic arthritis a randomized, doseranging, placebo-controlled, 84-day study of incb039110, a selective janus kinase-1 inhibitor sta-21, a promising stat-3 inhibitor that reciprocally regulates th17 and treg cells, inhibits osteoclastogenesis in mice and humans and alleviates autoimmune inflammation in an experimental model of rheumatoid arthritis regulation of pre-b cell colony-enhancing factor by stat-3-dependent interleukin-6 trans-signaling: implications in the pathogenesis of rheumatoid arthritis absence of stat1 disturbs the anticancer effect induced by stat3 inhibition in head and neck carcinoma cell lines selective inhibition of stat3 with respect to stat1: insights from molecular dynamics and ensemble docking simulations mutational switch of an il-6 response to an interferon-gamma-like response phase 1 and pharmacological trial of opb-31121, a signal transducer and activator of transcription-3 inhibitor, in patients with advanced hepatocellular carcinoma phase i study of opb-51602, an oral inhibitor of signal transducer and activator of transcription 3, in patients with relapsed/refractory hematological malignancies targeting the src homology 2 (sh2) domain of signal transducer and activator of transcription 6 (stat6) with cell-permeable, phosphatasestable phosphopeptide mimics potently inhibits tyr641 phosphorylation and transcriptional activity selective inhibition of the function of tyrosine-phosphorylated stat3 with a phosphorylation site-specific intrabody tofacitinib ameliorates murinelupus and its associated vascular dysfunction treatment of an alopecia areata patient with tofacitinib results in regrowth of hair and changes in serum and skin biomarkers rapid skin repigmentation on oral ruxolitinib in a patient with coexistent vitiligo and alopecia areata (aa) pharmacologic inhibition of jak-stat signaling promotes hair growth in search of magic bullets: the golden age of immunotherapeutics discovery of a jak3-selective inhibitor: functional differentiation of jak3-selective inhibition over pan-jak or jak1-selective inhibition the selective pan-janus kinase (jak) inhibitor vr588 demonstrates potent anti-inflammatory activity in a murine chronic house dust mite (hdm) model of asthma (abstract) jak1-stat3 blockade by jak inhibitor shr0302 attenuates inflammatory responses of adjuvant-induced arthritis rats and decreases th17 and total b cells shr 0302. jiangsu hengrui medicine co jte 052. originator: japan tobacco acknowledgements we are grateful to dr. john o'shea for his supervision and critical revision in the preparation of the manuscript. funding no sources of funding were used to support the writing of this article. key: cord-0023487-351097vu authors: radu, andrei-flavius; bungau, simona gabriela title: management of rheumatoid arthritis: an overview date: 2021-10-23 journal: cells doi: 10.3390/cells10112857 sha: 3a9ee1a9ab51dc479bbcb13d6a3152bc82c78e37 doc_id: 23487 cord_uid: 351097vu rheumatoid arthritis (ra) is a multifactorial autoimmune disease of unknown etiology, primarily affecting the joints, then extra-articular manifestations can occur. due to its complexity, which is based on an incompletely elucidated pathophysiological mechanism, good ra management requires a multidisciplinary approach. the clinical status of ra patients has improved in recent years due to medical advances in diagnosis and treatment, that have made it possible to reduce disease activity and prevent systemic complications. the most promising results were obtained by developing disease-modifying anti-rheumatic drugs (dmards), the class to which conventional synthetic, biologic, and targeted synthetic drugs belong. furthermore, ongoing drug development has led to obtaining molecules with improved efficacy and safety profiles, but further research is needed until ra turns into a curable pathology. in the present work, we offer a comprehensive perspective on the management of ra, by centralizing the existing data provided by significant literature, emphasizing the importance of an early and accurate diagnosis associated with optimal personalized treatment in order to achieve better outcomes for ra patients. in addition, this study suggests future research perspectives in the treatment of ra that could lead to higher efficacy and safety profiles and lower financial costs. rheumatoid arthritis (ra) is defined as a systemic autoimmune pathology associated with a chronic inflammatory process, which can damage both joints and extra-articular organs, including the heart, kidney, lung, digestive system, eye, skin and nervous system [1, 2] . numerous types of arthritis have been investigated and described in order to classify them into non-inflammatory arthritis (osteoarthritis) and inflammatory arthritis caused by crystal deposition (pseudogout, basic calcium phosphate disease, gout), by bacterial and viral infections (staphylococcus aureus, neisseria gonorrhea, complications of lyme disease, parvovirus, enterovirus) or by autoimmune processes. the heterogeneous group of autoimmune rheumatic diseases also includes systemic lupus erythematosus (sle), sjögren's syndrome, adult-onset scleroderma, spondylarthritis (spa), psoriatic arthritis (psa), polymyositis (pm), etc. due to the fact that they may be similar in signs and symptoms, differential diagnosis is essential [3] . although a number of biomolecular mechanisms have been proposed, the etiology of ra is not yet fully elucidated, a current hypothesis being that dysregulated citrullination leads to the production of anti-citrullinated protein antibodies (acpas) [4, 5] . the evolution of ra is fluctuant with episodic exacerbations and in the absence of optimal treatment symptoms gradually worsen until the joints are irreversibly damaged and physical and psychological functioning is affected [6] . moreover, ra complications and comorbidities reduce the life expectancy of patients by a few years [7] . existing statistical analysis and interpretation of quantitative data show that ra represents not only a medical feature, but also a public health issue. the most common epidemiological studies measuring the prevalence of ra in a few european, asian, north american, and south american countries between 1990 and 2005 reported pertinent and relevant results. low prevalence ratios were reported in serbia (0.18%) [16] , china (0.28%) [17] , france (0.31%) [18] , italy (0.33%) [19] , and the us (0.41%) [17] , while higher prevalence ratios were observed in japan (1.7%) [20] and argentina (1.97%) [21] . it is worth pointing out that older studies can face methodological biases resulting in differences in the prevalence of ra because the types of studies conducted were significantly different: cross-sectional studies, random selection, telephone survey, postal questionnaire, inception cohort, outpatient, and hospitalization medical records. moreover, it was observed that gender differences exist in the prevalence of ra. all the studies reported a three-to fivefold higher prevalence of ra in females than males. the most significant difference was reported by the argentinian study (women 3.2%, men 0.6%), while the closest values were reported in serbia (women 0.29%, men 0.09%) [17] . trends in the prevalence of ra have been assessed over the years, and the results are presented in table 1 . serbia 1991 0.18 0.17 [16] 2013 0.35 [22] italy 1991 0.33 0.07 [19] 2011 0.4 [23] japan 1996 1.7 −0.95 [20] 2016 0.75 [24] china 1997 0.28 0.14 [17] 2013 0.42 [25] argentina 1998 1.97 −1.03 [21] 2010 0.94 [26] france 2001 0.31 0.03 [18] 2013 0.34 [22] spain 2002 0.5 0.32 [27] 2017 0.82 [28] turkey 2004 0.49 0.07 [29] 2017 0.56 [30] the prevalence of ra has been rising almost unanimously since 1990 up to date. the largest increase was observed in the spanish population. however, in japan and argentina the prevalence ratios have decreased over the years. nowadays, the global prevalence ratio of ra is about 1% and it is more common in women, with small continuous fluctuations and an apparent growth from south to north, and from countryside to metropolitan areas [31] . from an epidemiological perspective, the incidence of ra varies by age and population. studies have been conducted over years to measure the incidence in certain geographical areas and for identifying variables that have led to different results. the data collection methods used were types of observational studies, including inception cohort, longitudinal population-based study, review of medical records, and prospective case-control studies, and were conducted between 1985 and 2002 [17] . lower incidence rates have been reported in japan (8 cases per 100,000 inhabitants) [20] , and france (8.8 cases per 100,000 inhabitants) [32] . the highest incidence rate has been observed in the us (44.6 cases per 100,000 inhabitants) [33] . it has also been reported that the incidence in women is significantly higher than in men. however, recent studies have reported a fluctuating incidence over the past three decades. therefore, the incidence ratios in the us ranged from 40 cases per 100,000 inhabitants in 1994 to 43 cases per 100,000 inhabitants in 2004 and nowadays ra has an incidence of 41 cases per 100,000 inhabitants [34] . the influence of age on the incidence of ra has been assessed by studies that have shown an increase with age up to 80 years when it begins to decline. moreover, the incidence rate has decreased progressively in the last 60 years, being much more significant among women [17] . several studies have reported differences in incidence rates at the regional level within countries. one potential explanation for these variations may have been environmental exposure to chemicals, climatic changes, infectious diseases, and food [35, 36] . furthermore, it has been reported that people with a low socio-economic background, living in rural areas during childhood, are at a higher risk of developing ra in adulthood [37] . the latest studies have reported that the united kingdom has the highest standardized incidence rate (27.5 cases per 100,000 inhabitants) and canada has had the biggest rise in the incidence rate in the last 30 years [15, 38] . the reasons for the increase in the incidence rate have no unequivocal explanation, but risk factors may play an important role. ra is a multifactorial disease caused by genetic, environmental and stochastic factors [39] . the genetic risk for ra that has been estimated by scientific studies is about 50% [40, 41] . the presence or absence of rheumatoid factor (rf) and acpas can divide ra into two types (seropositive and seronegative) and there are also differences between the risk factors involved [42, 43] . tyrosine phosphatase non-receptor type 22 (ptpn22) risk alleles [44, 45] , human leukocyte antigen d-related (hla-dr) alleles [42] , and tumors necrosis factor-receptor associated factor 1 and complement component 5 (traf1/c5) related genes are the main genetic factors associated with an acpa-positive subtype [46], while interferon regulatory factor 5 (irf-5) is confined to the acpa-negative subtype [47] . as significant contributors to population health, environmental risk factors play an important role in the management of ra. like other diseases, smoking is linked to the development or exacerbation of ra. the first evidence of the association of smokers with an increased risk of ra was observed by serendipity in a study with a different purpose [48] . since then, it has become the best described risk factor for ra. the harmful chemicals in tobacco products have been comprehensively evaluated and the results suggest that smoking delivers a specific signal. smoking might be related to a genetic context with a specific role in triggering a particular subtype of ra [49] . it has been reported that smoking affects rf-or acpa-positive ra [50], and has no or very little effect on acpa-negative ra [51] . moreover, the risk of developing acpa-positive ra is much higher in smokers who carry hla-dr beta 1 shared epitope alleles [52]. it has not been observed that any association exists between passive smokers and the risk of developing ra [53] . exposure to silica dust is an occupational type of exposure that impacts ra. it has been reported there is an association between silicosis and ra, mainly affecting patients with acpa-positive ra [54] . chronic exposure to silica can lead to rheumatoid pneumoconiosis, also known as caplan's syndrome, a rare disease of ra patients who have developed silicosis [55] . dietary factors and consuming habits have also been evaluated over time. dietary agents influence ra and the evidence has shown that fasting periods and vegetarian diets can decrease the evolution of ra. moreover, avoiding red meat and increasing fruit and oily fish consumption can be associated with a decreased risk for ra [56, 57] . coffee consumption may be a risk factor for ra, a possible explanation being the involvement in the production of rf [58] . it has been reported in a case-control study that alcohol consumption may have a beneficial effect on ra by lowering the risk of developing acpa-positive ra, but this hypothesis requires additional investigation [49] . therefore, a personalized diet for each person should be considered. infections are biological risk factors that might trigger the development of ra. a comparative cohort study reported that the risk of joint, skin and bone infections is much higher in patients with ra compared with non-inflammatory rheumatic diseases [59] . moreover, bacterial triggers have also been identified in the case of lyme arthritis, a pathology with many similarities to ra [60] . porphyromonas gingivalis is a pathogenic bacterium that causes periodontal disease. due to its role in inducing citrullination and promoting osteoclast genesis, an association between ra and periodontal disease has been reported [61] . a comprehensive characterization of the interaction between environment, genes and stochastic factors may be the basis for understanding the complexity of the biomolecular mechanisms that coordinate ra. although the pathophysiological mechanisms for ra are not fully elucidated, several hypotheses have been postulated. it has been reported that immunological processes can occur many years before symptoms of joint inflammation are noticed, the so-called pre-ra phase [62] . the interactions between epigenetic modifications on the genomic structure and environmental factors can lead to modified self-antigens as in the case of immunoglobulin g (igg), type 2 collagen and vimentin. these proteins with arginine residues can be converted to citrulline by peptidyl arginine deiminases in a post-translational modification called citrullination [63, 64] . moreover, joint disorders like synovial hyperplasia or synovial infections can trigger cytokine release that may cause joint inflammation and also modified self-antigens [65] . due to the susceptibility genes hla-dr1 and hla-dr4, the immune system is no longer able to recognize citrullinated proteins (vimentin, type ii collagen, histones, fibrin, fibronectin, epstein-barr nuclear antigen 1, α-enolase) as self-structures [66] . antigens are taken up by antigen-presenting cells (apc), which are dendritic cells that are activated to initiate an immune response. the whole complex migrates to the lymph node, where the activation of cd4 + helper t cells takes place. furthermore, the germinal center of the lymph node contains b cells that get activated by reciprocal and sequential signals with t cells, an immunological process called costimulation. an example of costimulation is the interaction between cd28 and cd80/86 [67, 68] . at this level, b cells undergo somatic hypermutation or class-switch recombination and start to proliferate and differentiate intro plasma cells that produce autoantibodies depending on the receptors of the precursor cells [69] . autoantibodies are proteins produced by an immune system that no longer discriminates self from non-self-structures, so self-tissues and organs are accidentally targeted. rf and acpa are the most studied autoantibodies involved in ra. rf is an igm antibody with a testing specificity of 85% in ra patients, which targets the fc portion of igg, also called the constant region [70] . it also forms an immune complex with igg and complement protein, a complex able to migrate in the synovial fluid. however, acpa is more specific for ra and targets citrullinated proteins and after their binding interactions, immune complexes are formed with an accumulation in the synovial fluid [71] . all the features of an immune response in the pre-ra phase are summarized in figure 1 . in the realm of ra, air pollution, which consists of a mixture of suspended particulate materials (pm) of various sizes and gases (nitrates, ozone, sulfur dioxide and carbon monoxide), has recently received increasing attention. pollutants are released into the air through a variety of man-made and natural sources, including agriculture, fossil fuel combustion, chemical industries, use of solvents, volcanic eruptions, windblown dust, emissions from plants, etc. the clinical impact of air pollution is primarily considered in relation to respiratory diseases. the alveoli, an important part of the respiratory system that filters oxygen and carbon dioxide, have been reported to be damaged by ozone. pollutants can also cause secondary harm to lung tissue by reacting with different enzymes, resulting in pulmonary inflammation or infection. three major epidemiological investigations conducted in the united states, canada and sweden have demonstrated that air pollutants can be linked to the pathogenesis of ra [72] . alsaber et al. (2020) , conducted a study to investigate the correlations between air pollutants and ra activity through regression models. nitrates and sulfur dioxide were discovered to be important risk factors for the development of ra [73] . one of the latest research studies that has been published is a case-crossover study (which assessed a potential association between air pollutants in the verona area and ra evolution) in 888 ra patients, showed that air pollution is linked to high c-reactive protein levels (crp), to the severity of ra illness and its reactivations due to a poor response to biological therapies [74] . the involvement of air pollutants in the pathogenesis of ra may be based on a few mechanistic processes. free reactive oxygen species (ros) generated by pm inhalation can activate nuclear factor kappa b (nf-kb), which activates t helper cell type 1 (th1) to produce tumor necrosis factor alpha (tnf-α), interleukin-1 (il-1) and interleukin-6 (il-6). these cytokines promote the maturation of resting monocytes into mature dendritic cells, which then offer auto-antigens to self-reactive t lymphocytes, causing them to move to target tissues and promote joint inflammation and erosion. moreover, the citrullination of arginine amino acid residues into citrullinated peptides is also aided by ros, which promotes chronic lung disease and systemic inflammation. acpas, which are generated by biochemical reactions, trigger an immunological response by binding to cellular fc receptors and activating complement, resulting in joint inflammation and bone erosion [72] . reduced ultraviolet b (uvb) radiation causes a decrease in 1,25-dihydroxyvitamin d3 production in the skin, which functions as an immunomodulator by activating the vitamin d receptor (vdr). as a result, the immunomodulatory functions are not optimal, and this can trigger ra [75] . another important element with major implications in the pathogenesis of ra is the gut microbiota, the most densely colonized bacterial population within the human body [76] . ra etiology is also related to intestinal dysbiosis, which leads to certain autoimmune pathways and mechanisms, such as stimulation of apc by activating toll-like receptors (tlrs) or nod-like receptors (nlrs), molecular mimicry, alterations in intestinal permeability, promotion of t cell differentiation and amplification of mucosal inflammation via certain pathways [77] . it has been demonstrated that immunological, metabolic, and neurobehavioral features are influenced by gut microorganisms. when compared to healthy controls, ra patients showed significant differences in the gut microbiota composition, being associated with an increase or a decrease in certain bacterial populations [78] . the gastrointestinal microbiota can impact the development of ra through proximal intestinal immunomodulatory cells, which are found in specific locations within the gut. several case-control studies have demonstrated quantitative changes in specific bacteria in ra patients by 16s rrna sequencing and metagenomic shotgun sequencing. according to the results of the studies, prevotella copri, collinsella and lactobacillus salivarius were found to be more abundant in ra patients, while bacteroides, faecalibacterium, veillonella and haemophilus were lower in quantity [76, 79] . the mechanisms underlying the involvement of air pollutants and gut microbiota in the pathogenesis of ra are shown in figure 2 [72, 76, 78] . ra is generally characterized by an insidious onset of symptoms, but over time the disease progresses and gradually worsens. the trigger for ra symptoms is unknown, but the immunological processes that take place in the synovium and in the synovial fluid have been described [80] . synovial macrophages release cytokines like tumor necrosis factor alpha (tnf-α), interleukin-1 (il-1) and interleukin-6 (il-6), which are associated with inflammatory processes, stimulation of fibroblast-like synoviocytes (fls) and stimulation of osteoclast activity [81] . increased osteoclast activity and maturation leads to bone erosion. once activated, fls are specialized cells that can produce matrix metalloproteinase (mmp) [82] . mmp can lead to cartilage degradation and the cartilage also secrets proteases in a feedback mechanism [83, 84] . fls can migrate from joint to joint, creating a pattern of symmetrical ra [12] . moreover, fls stimulates receptor activator of nuclear factor-kb ligand (rankl) expression, allowing t cells to bind proteins on the surface of osteoclasts, which also leads to bone erosion by increasing osteoclast activity [85] . cd4 + t cells promote inflammation, bone erosion and cartilage degradation by stimulating rankl expression and producing interleukin 17 (il-17), with an important role in the stimulation of synovial macrophages and fls [86, 87] . plasma cells also promote inflammation through cytokines and autoantibodies [88] . in the synovial fluid the presence of neutrophils has been reported, which produce proteases and reactive oxygen species (ros) that may cause bone erosion and cartilage degradation [89, 90] . immune complexes have also been identified in the synovial fluid such as antibodies that bind one to another, promote inflammation and over-activate the complement system [91] . angiogenesis is a process of forming new blood vessels from existing ones, which also occurs in ra. in contrast to its beneficial role in many physiological processes, in ra it plays a critical role because the immune cells can migrate into the joints due to the increase in vascular permeability and the expression of adhesion molecules (vascular adhesion molecule 1) [92, 93] . furthermore, vascular endothelial growth factor (vegf) is a proangiogenic factor located in the synovium in ra patients, which has a potent role in bone destruction as a promoter of osteoclast genesis [92] . the pathophysiological processes that lead to the appearance of symptoms in ra are summarized in figure 3 . the complexity of this pathology is also based on numerous signaling molecules with specific roles in inflammatory processes. janus kinases (jaks) are small signaling proteins with pathophysiological relevance because they can represent molecular targets for many therapeutic agents [94] . thus, further research is needed to elucidate all the pathological mechanisms and to optimize future therapies with high safety and efficacy profiles. an essential part of ra management is the evaluation of clinical aspects, including signs and symptoms, prognostic laboratory biomarkers, differential diagnosis, complications, and extra-articular manifestations, etc. early and accurate diagnosis of ra is highly important in order to differentiate between types of arthritis and types of autoimmune disease and to promptly establish the correct treatment and prevent long-term complications [95] . the 2010 american college of rheumatology (acr) and european league against rheumatism (eular) classification criteria for ra evaluates a set of variables, such as risk factors, number and type of joints involved and the duration of symptoms, in order to redefine the focus from late-stage phase management to the early detection of ra [96] . the classification system exposes conditions to which a certain score corresponds and must be re-examined over time: • 2-10 large joints corresponding to 1; • 1-3 small joints (±large joints) corresponding to 2; • 4-10 small joints (±large joints) corresponding to 3; • >10 joints (≥1 small joint + any others) corresponding to 5; • negative rf and negative acpa corresponding to 0; • low-positive rf and/or acpa ≤3× upper limit of normal for local laboratory assay corresponding to 2; • high-positive rf and/or acpa >3× upper limit of normal corresponding to 3; • abnormal erythrocyte sedimentation rate (esr) and/or abnormal c-reactive protein (crp) corresponding to 1; • normal crp and normal esr corresponding to 0; • patient reported pain, swelling and tenderness ≥6 weeks corresponding to 1 [97] . patients with a score of ≥6 are classifiable as having ra. to be eligible for a new series of tests, two mandatory conditions must be met. the first one is the need of evidence of synovitis, with a swelling in at least one joint as evaluated by a specialist, not including the typical joints involved in osteoarthritis: the first metatarsophalangeal joint, the first carpometacarpal joint and distal interphalangeal joint. the second condition for applying the criteria is that the patient does not have another diagnosis for synovitis. moreover, the large joint category includes ankles, hips, elbows, shoulders, and knees, while the small joints category consists of proximal interphalangeal joints, wrists and second through fifth metatarsophalangeal joints [98] . algorithms have been reported for the diagnosis of early ra, with different development, depending on the features of each patient [96] . as a general disease pattern, ra presents an insidious onset with gradual progression, being associated with joint pain, tenderness, swelling and symmetrical joint damage [98] . ra is predominantly observed in the elderly group and, if left untreated, it can lead to loss of function, disability, and an increased burden of disease [15, 99] . using differential diagnosis to confirm ra is a challenge and represents the optimal medical approach. in order to make a differentiation of ra from other similar diseases, certain features must be evaluated. a biopsy is required sometimes to differentiate diseases with similar conditions. the distribution of synovitis is different in ra (symmetric, great, and small joints including wrist and elbow) than in ankylosing spondylitis (limited to small joints) and psoriatic arthropathy (asymmetric, including toes). inflammation is more intense in ra than in osteoarthritis [95] . the presence of rf is associated predominantly with ra, but also with sjögren's syndrome and sle [100] . antinuclear antibodies are more common in sle than in ra. the most intense erosive changes on x-rays are found in ra. cutaneous signs suggest sle, psa [101, 102] or systemic sclerosis. pm usually primarily affects the shoulders and hips. in spa, the most common inflammatory processes affect the eye and the back [95] . patients who cannot be classified according to acr-eular criteria due to a duration of symptoms of less than 6 weeks may be suspected of having a viral infection (parvovirus, enterovirus) or lyme arthritis [103] . when more than four joints are affected by arthritis, the disease is called polyarthritis and it is difficult to differentiate it from osteoarthritis and fibromyalgia, when pain is the only symptom. furthermore, blood and urine tests may help in establishing an accurate diagnosis. other differences concerning extra-articular manifestations have also been reported [95] . the identification and optimization of biomarker panels represents a promising medical tool, due to their diagnostic, prognostic, predictive and therapeutic role. the acr 1987 criteria included only rf as a biomarker. the last established classification includes four biomarkers (rf, acpa, esr, crp), all with certain limitations [104] . more recent studies have identified other diagnostic proteins with roles in early diagnosis of ra: antibodies against mutated citrullinated vimentin (anti-mcv), antibodies against carbamylated proteins (anti-carp) and 14-3-3 eta protein. a systematic review of studies in which biomarkers were tested for their potential diagnostic role has reported no difference between cyclic acpa and anti-mcv. thereby, it can represent a diagnostic tool when ra and acpa are negative [105] . moreover, the diagnostic accuracy of 14-3-3 eta protein has been evaluated in experimental studies and it has been reported that for all ra patients with a negative rf and acpa, 14-3-3 eta protein was positive [106, 107] . anti-carp was detected in ra patient serum and several studies have reported that the presence of anti-carp is associated with pre-symptomatic phases and may also be used as a prognostic tool [108] [109] [110] . more recent studies have demonstrated that gene profiles can represent important diagnostic tools. by comparing fls from healthy individuals to fls-ra it has been demonstrated that statistically significant differences can occur in heat-shock protein family a members, matrix metalloproteinase 1 (mmp1), matrix metalloproteinase 13 (mmp13) and tumor necrosis factor ligand superfamily member 10 (tnfsf10) genes [111] [112] [113] [114] . furthermore, the technical development of proteomics enables the identification of protein panels, with an important role in early diagnosis. an experimental study used label-free quantitative proteomics to characterize proteins with diagnostic potential, especially for seronegative ra patients. it has been demonstrated that serum amyloid a-4 protein (saa4), retinol-binding protein-4 (rbp4), angiotensinogen (agt) and vitamin d-binding protein (vdbp) are accurate enough to be used as diagnostic tools [115] . glycoprotein ykl-40 can be used as a diagnostic biomarker because of its promising results [116] . a few biomarkers (anti-mcv, rf, 14-3-3 eta protein, acpa) can also be used as prognostic tools because they are associated with severe phases of ra [117, 118] . further research is needed in order to identify new potential prognostic biomarkers. predictive biomarkers are essential in the therapeutic management of ra because they are used to establish an effective treatment that all patients will respond to. it has been reported by several studies that anti-ccp, anti-mcv, 14-3-3 eta, cartilage oligomeric matrix protein (comp), survivin and calprotectin are correlated with a good predictability of treatment response [119] [120] [121] [122] . an accurate diagnosis involves the association between the detection and quantification of biomarkers with imaging tools. the acr-eular 2010 classification includes ultrasonography, computed tomography (ct) and magnetic resonance imaging (mri) as imaging tools for establishing an early diagnosis, due to their much higher accuracy than in the case of conventional radiographs [97] . x-ray examinations of joints cannot reveal the early presence of degradations and erosions [123] . even though x-ray is still used as a diagnosis technique of late changes in the joints because of its availability, low cost, and more medical records, it has limitations due to the radiations that are used, low sensitivity in detecting early erosion processes and because 3d anatomical structures are shown only in 2d [124] . moreover, a few radiographic hallmarks of ra have been identified, including symmetrical abnormalities, periarticular osteopenia, narrowing of the joint spaces and marginal degradation, swelling of the soft tissue and synovial cysts and nodules [125, 126] . ultrasonography is a diagnosis technique that characterizes the interaction between tissues and sound waves to produce an image of the tissue. it can detect small bone and cartilage erosions and explore the structures in great detail. doppler ultrasound may differentiate active from inactive inflammatory tissues [127] . the inherent advantage of ultrasonography over x-ray has been demonstrated in a case-control study, where sonography detected more erosions, especially in early ra [128] . ct is a rarely used imaging technique which, due to its ionizing radiation, can damage the deoxyribonucleic acid (dna) of human cells, and it has limited soft tissue contrast [126, 129] . however, it can be successfully used in medical cases where 3d imaging is required. clinical trials conducted over time have demonstrated similarities between ct and mri [130, 131] . however, mri is the most accurate imaging tool for the detection of early ra. contrast enhanced mri can generate a differential diagnosis between joint effusion and synovitis. furthermore, it can detect early erosions and hypertrophies and it is the gold standard for bone marrow edema detection. a recent longitudinal study evalu-ated the role of mri in predicting ra progression in patients with clinical symptoms, but showed no correlation between them, even though the detection accuracy was high [132] . the utility of various imaging tools in ra depends on the ra stage of progression. mri is the most suitable imaging method for the detection of early changes in ra patients, except for detecting joint space widening, where ct is more appropriate. for late changes that occur in the joints, all the imaging tools mentioned above can be used with good results. future challenges and optimization strategies in medical imaging include thermography, near infrared imaging (nir), positron emission tomography (pet) and single-photon emission computerized tomography (spect) [126] . ra is an autoimmune disease that primarily affects the small joints and then the large ones, but as a systemic disorder, extra-articular structures can be involved. affected joints may be in the upper extremity (hand, wrist, elbow, shoulder), lower extremity (foot and ankle, forefoot, midfoot, hindfoot, knees, hips) and spine and axial joints (cspine, atlantoaxial subluxation, basilar invagination, sub axial subluxation, thoracic spine, sternoclavicular spine, manubriosternal joints, lumbar spine, temporomandibular joint, sacral spine, cricoarytenoid joint, ossicles of the ear). extra-articular manifestations (eams) in ra are serious conditions correlated with high morbidity and mortality rates. eams may result from the release of proinflammatory cytokines in the bloodstream [133] . various tissues and organ systems can be affected. the most severe manifestations include vasculitis, felty's syndrome, pericarditis and pleuritis and it has been reported in a retrospective study conducted in a cohort of 424 cases, that 39.85% of patients developed severe eams [134] . moreover, a multicenter research trial evaluated the frequency of eams in 587 ra patients and showed that 40% of patients developed extra-articular features [135] . systemic vasculitis may result in skin manifestations, gastrointestinal complications, cardiac disease, and pulmonary manifestations. the most common skin manifestations are rheumatoid nodules located in different areas, which can occur mainly in seropositive patients with erosive disease. other skin manifestations include periungual inflammation, ulcerations and digital gangrene [2] . ocular manifestations are not as common as those on the skin and include kerato-conjunctivitis sicca as the most frequent manifestation of this subset, episcleritis, scleritis and keratitis. the swelling of the salivary gland and xerostomia are oral manifestations that can occur. however, ocular and oral manifestations can also be found in sjögren's syndrome [133] . pulmonary complications are frequent, but asymptomatic, including pleural effusions, pulmonary fibrosis, interstitial lung disease and arteritis. smokers are at greater risk of developing life-threatening complications of ra [2] . ra patients can be associated with an increased risk of cardiovascular mortality because numerous cardiac structures are involved in the pathological processes, which may lead to atherosclerosis, arterial stiffness, coronary arteritis, congestive heart failure, valvular disease and fibrinous pericarditis [136] . it could potentially contain prognostic markers of diseases like hypertension and dyslipidemia [137] . it has been reported in a meta-analysis of 14 controlled observational studies involving 41,490 patients, that the risk of cvd increased by over 48% in ra patients compared to the general population [138] . moreover, a case-control study assessed potential cardiac abnormalities in 47 ra patients without manifested cardiovascular symptoms using doppler echocardiography technique and showed a high incidence of pulmonary hypertension and left ventricular diastolic dysfunction [139] . renal manifestations are rare, including glomerulonephritis and interstitial renal disease, which are correlated with the presence of vasculitis, while neurological complications may result in peripheral neuropathy and cervical myelopathy [140] . the most common hematologic abnormality in ra patients is anemia, due to hepcidin stimulation that inhibits iron transport. moreover, it has been reported that hepcidin may be a valuable prognostic biomarker in ra [141] . other eams include malignancies, neutropenia, eosinophilia, and thrombocytopenia [2] . felty's syndrome is a severe eam, which can occur mainly in seropositive patients with a low white blood cell count and an enlargement of the spleen. thus, these patients are more susceptible to opportunistic infections [142] . since both ra and ageing are linked to emerging comorbidities, such as cardiovascular disease (cvd), infections, interstitial lung disease and cancer, these elements will have a significant impact on ra global management. moreover, knowing the current status of the management of ra-associated comorbidities and the formation of a multidisciplinary team of medical specialists, are essential parts in an attempt to lower morbidity and mortality rates [143] . the rbsmr study managed to define the profile of comorbidities by following 225 ra patients. the prevalence of cvd was 23.1% and of pulmonary disease was 5.77% [144] . comorbidities and associated risk factors should be screened for and evaluated on a regular basis, as well as the management of these conditions. lifestyle advice about regular physical activity, balanced diets, quitting smoking and vaccine updates should be parts of the management program. controlling the inflammatory process with dmards, especially targeted therapy, is linked to a lower risk of cvd [145, 146] . a recent cross-sectional real-life study reported that the use of less glucocorticoids and an increasing use of bdmards in patients with cardiovascular comorbidities suggested that rheumatologists have become aware of the potential influence that ra drugs may have on comorbidities [147] . current cardiovascular risk management involves the evaluation of conventional cardiovascular risk factors (diabetes, obesity, dyslipidemia, hypertension) using heartscore ® and the control of inflammatory states. according to eular recommendations, the rheumatologist is in the most suitable position to organize risk factor assessment and care in ra patients [148] . the results of the cardiovascular risk assessment guide the ldlcholesterol level, the frequency of testing, and show whether a cardiologist's opinion is necessary. moreover, according to eular, the presence of carotid artery plaque implies a high level of cardiovascular risk. about 60% of ra patients have carotid artery plaque [149] . however, scientific evidence indicates that statins reduce ra-related cardiovascular risk by reducing cholesterol, but also provide angioprotective, anti-inflammatory and antioxidative effects. because of this, as well as their safety profile, statins can be a good option for comprehensive control of ra vascular comorbidities [150] . the measurement of cardiovascular risk is only one aspect of comorbidity screening and management. infections, lung disease and malignancies are among risks associated with ra, which can be exacerbated by ra or its treatments [151] . immunization is a very important aspect to consider because the risk of infection is given both by the pathology per se, but especially by immunosuppressive agents. in ra patients, the influenza vaccine should be given annually, diphtheria-polio-tetanus every 10 years and the pneumococcal vaccine every 5 years, in accordance with the vaccines recommended for the general population [152] . in ra patients, lung disease is one of the most common causes of extra-articular morbidity and mortality. the six-minute walk test and the five-point medical research council breathlessness scale should be used in clinical evaluations to quantify exercise tolerance [153] . due to the limited evidence, there are no international guidelines for the treatment of interstitial lung disease, as a comorbidity of ra. therefore, the management of ra patients with moderate to severe lung disease should include a collaboration with a respiratory physician, especially given that evidence shows serious respiratory adverse events when using drugs for the treatment of ra [154] . screening tests for malignancies applied to the general population are also useful for ra patients. mammograms are the most suitable diagnostic tools for detecting breast cancer, the pap and the hpv test for detecting cervical cancer and low-dose computed tomography for lung cancer screening [155] . the existing medical evidence showed that the management of comorbidities is not efficient enough and further research is needed. incorporating comorbidities into the daily management of ra would lead to a more complete standard of care and screening tests should be included in diagnostic procedures for ra patients. the canadian dermatology-rheumatology comorbidity initiative provided 19 evidence-based recommendations for managing comorbidities in ra patients, emphasizing the importance of differentiating comorbidities due to ra per se from those caused by therapeutic agents [156] . a few cohort studies have reported decreases in the incidence and prevalence of eams of ra in the last decades [134, 157, 158] . the management of eams in ra has improved as technological and medical advances have emerged providing a better understanding of the mechanism underlying the effects. different treatment strategies have been used over time in order to improve patients' quality of life, to reduce the risk of eams and to determine the safety and efficacy profile of new active molecules. the principle established by the acr is "treat to target", which refers to the choice of a good treatment to achieve remission, or a reduced disease activity as an alternative. therapeutic intervention must be aggressive and rapid because already existing erosions are not reversible [97] . the general approach to treatment starts with a highly accurate diagnosis and includes prevention strategies, nonpharmacological and pharmacological therapies, in order have a quick result. the 2021 acr guideline for the treatment of ra updated the pharmacological management of ra, providing seven strong recommendations and 37 conditional ones [159] . the characterization of risk factors provides tools for preventing ra. focusing on prevention may be an important part of the general management of ra. four levels of prevention (primary, secondary, tertiary, clinical) have been shown. primary prevention is focused on not allowing pathological processes to begin, the secondary one manages the risk factors to detect and reduce them, and tertiary prevention deals with damagelimiting mechanisms. clinical prevention includes reducing complications and stopping relapses [160] . screening strategies of people at risk of developing ra may result in lower incidence and prevalence rates. blood relatives, twins of ra patients and seropositive individuals should be closely monitored because they are in the risk category [161] . the goals of nonpharmacological approaches are to decrease anxiety and depression, to reduce pain and to increase mobility. polyunsaturated fatty acids (pufas) have gained wider attention because of their links to a variety of brain disorders, including anxiety and depression. these pufas include docosahexaenoic acid (dha) and eicosapentaenoic acid (epa) in the series of omega-3 fatty acids [162] . to determine the efficacy of pufas, particularly dha and epa, in the treatment of depression, a meta-analysis of 26 doubleblind randomized placebo-controlled trials was conducted. the results showed that omega-3 pufas improved depression significantly. the ratio of epa to dha with the most effective antidepressant properties was 2:1 or 3:1 [163] . in addition, two meta-analyses estimated that the most effective formulations are those containing ≥60% epa [164, 165] . another meta-analysis of 19 clinical trials assessed the potential of pufas to relieve anxiety symptoms. existing medical evidence suggested that pufas may influence several neural processes that underlie anxiety. even though diagnoses were diverse, the major conclusion was that omega-3 pufas were linked to a significant reduction in anxiety symptoms when compared to controls [166] . moreover, a recent prospective study including 36 patients with jaki treatment discovered an inverse association between patients' pain scores and dha serum levels and showed that pain relief can be promoted by supplementation with pufas. due to the small number of patients included in the study, large prospective studies need to be done in order to confirm the hypothesis [167] . in ra patients, anxiety, depression, and pain are associated with disease activity and a poor functional status. according to the medical evidence existing so far, pufas can become useful tools in controlling the symptoms, but additional studies are needed. rest, occupational therapy, physical exercise, and surgery can also be useful. most studies that have evaluated the role of physical activity and psychological interventions for ra-related fatigue patients, have demonstrated their effectiveness and associated with rest, they may relieve stress on inflamed tissues and slow down the progression of the disease [168, 169] . a systematic review of 42 articles about the advantages of occupational therapy for ra patients showed an increase in joint function [170] . joint surgery is used only in severe stages of ra. however, the rates of surgery in ra have low values in patients under 60 years. surgical approaches provide pain relief and restore the function of joints. due to recent advances in the surgical field, numerous procedures are available: tens-synovectomy, radio synovectomy, arthroscopy, osteotomy, joint fusion, metatarsal head excision arthroplasties or total joint replacement [171] . scientific evidence suggests that massage, positioning, hot and cold therapy, acupuncture, transcutaneous electrical nerve stimulation and progressive muscle relaxation are complementary therapies that might be useful in nonpharmacological pain management [172] . nonpharmacological approaches should be associated with pharmacological treatments in order to maximize therapeutic success. continuous improvement in the procedures and techniques in drug design strategies has led to considerable progress in pharmacological approaches towards finding a cure for ra. the new therapeutic options have managed to reduce the symptoms, slow the progression and prevent complications. current treatment options in accordance with acr and eular recommendations manage ra from two perspectives: symptomatic treatment (nsaids and gcs) and disease modifying management (dmards) [159, 173] . the symptomatic management of ra consists primarily of nsaids and gcs, but weak opioid analgesics may also be considered for short-term management of pain after an accurate assessment of the benefit-risk balance [14, 174] . nsaids (naproxen, ibuprofen, coxibs) are used in the acute phase response to reduce pain by decreasing inflammation. nsaids exert their pharmacological effect by inhibiting cyclooxygenase (cox), especially cox-2 which is increased during inflammation. however, the risk of harm should be considered because the inhibition of prostaglandins can lead to serious side effects, such as bleeding, gastrointestinal ulceration, renal failure, heart failure, rashes, dizziness, confusion, seizures, etc. some of the side effects can be avoided by using cox-2-selective nsaids (celecoxib, rofecoxib, valdecoxib) [14, 175] . the effectiveness of nsaids in ra has been demonstrated in placebo-controlled trials in which patients without gc treatment were included [176] . gcs (prednisone, hydrocortisone, prednisolone, dexamethasone) have greater potency and efficacy than nsaids, due to the complex mechanisms of their anti-inflammatory and immunosuppressive effects, but the safety profile of nsaids is slightly better [177] . long-term side effects of gcs include weight gain, water retention, muscle weakness, diabetes, bone thinning, etc. thus, they have a short-term use and can be administered orally, intravenously, intramuscularly, and intra-articularly [178] . gcs have two major roles in the treatment of ra, as bridging therapy for dmards until their effects start and as adjunctive therapy for active ra that persists despite using dmards. it is critical not to abruptly discontinue corticosteroid therapy due to negative feedback in the regulation of hypothalamic-pituitary-adrenal (hpa) axis pulsatility [177] . dmards are pharmacological agents that are used to promote remission by suppressing autoimmune activity and by delaying or preventing joint degeneration. the treatment should be initiated as soon as possible because early implementation leads to better results, especially given that dmards are slow acting drugs with a delayed onset of between 6 weeks and 6 months. dmards have been classified as conventional synthetic dmards (csdmards), biologic dmards (bdmards) and targeted synthetic dmards (tsdmards) [179] . csdmards are typically used as a first-line therapy for newly diagnosed ra patients. bdmards or tsdmards are recommended if first-line therapy is not tolerated or is ineffective. tsdmards, including the class of janus kinase inhibitors (jaki), have the advantage of being orally administered [180] . csdmards are a heterogeneous class of drugs including methotrexate (mtx), leflunomide (lef), hydroxychloroquine (hcq) and sulfasalazine (ssz), which are more frequently used than other agents with a lower efficacy and safety profile, such as gold salts, azathioprine, d-penicillamine, cyclosporine, minocycline, and cyclophosphamide. their mechanisms of action lead to a non-targeted suppression of the overactive immune system [12, 177] . the 2021 acr guideline for the treatment of ra claims mtx as a first-line treatment for ra, both as a monotherapy and associated with other molecules as well, due to its efficacy and safety profile, flexible administration, and low cost. moreover, the guideline strongly recommends the use of mtx monotherapy over hydroxychloroquine, sulfasalazine, bdmards, and tsdmards for ra dmards-naive patients with moderateto-high inflammatory activity. furthermore, its conditional recommendations include the use of mtx over lef for ra patients untreated with dmards, and the use of mtx monotherapy over dual or triple csdmards therapy or over mtx associated with bd-mards or tsdmards. the inhibition of purine biosynthesis and cytokines production, as well as the activation of adenosine receptors lead to the anti-inflammatory properties of mtx. oral administration of mtx is conditionally recommended over other administration routes for dmards-naive patients [159] . a recent systematic review of 73 clinical trials assessing the efficacy and safety profile of mtx showed it had the safest profile of any csdmards used for ra and great efficacy rates [181, 182] . toxic effects identified over time are rare and are mainly gastrointestinal, hepatic, hematologic and pulmonary, consisting of diarrhea, nausea, liver damage with a lower incidence of cirrhosis, thrombocytopenia, leukopenia, pulmonary fibrosis, and pneumonitis [183] . a meta-analysis performed in order to compare the efficacy and safety profiles of lef and mtx demonstrated the similarity of their efficacy profiles and a slightly lower safety profile for lef, due to a higher increase in liver enzymes [182] . thus, lef can be used as an alternative initiating treatment option for patients with poor tolerance to mtx [184] . hcq is a drug used for malaria, but due to its immunomodulatory effects with a decreased secretion of cytokines, can be an alternative option in the treatment of ra. a multicentric, randomized, double-blind, placebo controlled clinical trial assessed the efficacy and safety of hcq in ra and demonstrated that the drug was effective and welltolerated in the patients included in the study [185] . the principal benefit of hcq is that it has no myelosuppressive, renal or hepatic side effects. however, at higher dosages, the eye can be affected, and pre-retinopathy can develop [177, 186] . ssz is a two-metabolite prodrug with anti-inflammatory and immunosuppression effects. its similar efficacy to lef has been demonstrated in a multicentric, randomized, double-blind, placebo controlled clinical trial, but the use of ssz is limited by its side effects, such as rash, serum sickness-like reactions, urticaria, nausea, and diarrhea. serial monitoring of certain laboratory tests and managing the changes that can occur may reduce the side effects [12] . for dmard-naive patients with low disease activity, the 2021 acr guideline for the treatment of ra suggests that hcq is conditionally indicated over other csdmards, ssz is conditionally suggested over mtx, and mtx is conditionally recommended over lef. the chemical structures of the most prescribed csdmards are depicted in figure 4 [187] . further treatment options including bdmards, tsdmards, biosimilars or combination therapy are available when csdmards are ineffective or poorly tolerated. bdmards are a newer option for the treatment of ra and provide a targeted therapy on the structures of the immune system [12] . bdmards are genetically engineered protein molecules divided into several classes, depending on the mechanism of action, as follows: granulocyte-macrophage colony-stimulating factor inhibitor (mavrilimumab, otilimab); • rankl inhibitor (denosumab) [12, 159, 188, 189] . since their discovery, the use of bdmards has been on an upward trend. this statement is supported by a study the aim of which was to analyze prescription patterns for csdmards and bdmards between 2004 and 2011 by using yearly cross-sectional investigations [189, 190] . the use of csdmards is still much higher than bdmards. over the 7 years of the study, there was a permanent increase in the use of csdmards, from 6.53% in 2004 to 8.93% in 2011. the study also showed a significant increase in the annual prevalence of bdmards use from 2004 (0.35%) to 2011 (1.54%). the most prescribed bdmards were adalimumab (between 0.07-0.35%), etanercept (between 0.16-0.46%) and rituximab (between 0.03-0.21%). moreover, the prevalence of prescriptions has increased over the years, except for anakinra for which a constant value has remained (0.01%) [189] . a randomized, double-blind, placebo controlled, phase iii clinical trial evaluated the efficacy and safety profile of adalimumab as a monotherapy in patients with ra who had failed to respond to csdmards [191] . the results showed both statistically significant improvement in the disease activity and a good safety profile. however, due to the suppression of the immune system, bdmards influence the susceptibility to infections and this aspect should be carefully monitored. a meta-analysis of nine clinical trials of adalimumab in the treatment of ra demonstrated its association with a greater risk of serious infection, which increased with dose [192] . one of the shortcomings of bdmards, especially tnf-α inhibitors, is the risk of developing tuberculosis (tb). the use of tnf-α inhibitors was associated with an 18-fold increased tb incidence in a population-based cohort investigation of ra patients from a high-incidence area. when compared to etanercept, adalimumab was linked to a higher and earlier diagnosis of tb [193] . adalimumab-atto, adalimumabadbm, adalimumab-adaz, adalimumab-bwwd, adalimumab-afzb and adalimumab-fkjp are biosimilars approved by the fda for the treatment of ra [194] . etanercept was the first anticytokine medication approved by the fda for ra treatment [195] . it is the only tnf-α inhibitor that is not an antibody, but a dimeric fusion protein. a long-term evaluation of the safety and efficacy profile of etanercept in 549 ra patients was made through an open-label trial, which showed that after 36 months of treatment, etanercept demonstrated long-term efficacy, as well as a favorable safety profile [196] . it is administered twice weekly via subcutaneous injection and has a toxicity profile like infliximab. it has also demonstrated a beneficial role in reducing radiographic progression in ra patients. according to the medical literature, the number of patients who achieved clinical remission with etanercept varied between 50% and 75%. etanercept-szzs and etanercept-ykro are biosimilars approved by the fda for the treatment of ra. even though a meta-analysis of all cochrane reviews on bdmards for ra estimated that adalimumab, etanercept and infliximab had similar efficacy profiles [197] , etanercept had the best drug survival of all tnfi, according to the ssatg and danbio registries [198] . as bdmards become more widely used and for longer periods of time, studies of their long-term safety and efficacy are becoming increasingly important. an example of such a study was conducted in order to assess the safety and efficacy profile of etanercept beyond 10 years of therapy in 1272 north american ra patients treated with 25 mg of etanercept twice a week for 10 years. the study reported 5 opportunistic infections, 29 cases of sepsis, 14 lymphomas and 61 deaths, but the occurrence of serious adverse events was higher in longstanding ra patients that in early ra patients. however, it was demonstrated that etanercept provided a good risk/benefit ratio due to its efficacy and safety profile and can be a long-term therapeutic option [199] . infliximab is a chimeric monoclonal antibody with a human antibody backbone that binds to all forms of tnf-α, neutralizing its biological function. it is administered by intravenous infusion. a decrease in adhesion molecules, il-1, il-6 and il-8 was found after therapy with infliximab in ra patients [12] . medical evidence indicates that patients treated with infliximab have a quick response and it has a good preventive effect on joint degeneration. a cohort study assessed 24 cases of ra patients with medium and high disease activity, despite the use of bdmards like adalimumab, golimumab, tocilizumab, etanercept or abatacept. the medical intervention was the switch to infliximab and the results showed a good efficacy profile due to the 37.5% of patients who achieved a low disease activity and 70.8% of patients who achieved a moderate or good eular response [200] . the safety profile was also good because only one serious adverse event was identified (infection with hospitalization). infliximab-dyyb, infliximab-abda, infliximab-qbtx and infliximab-axxq are biosimilars approved by the fda for the treatment of ra. recent studies demonstrated that there are no statistically significant differences in terms of efficacy and safety between bio-original and infliximab biosimilars [201] . golimumab is a human monoclonal antibody administered once a month by subcutaneous injection. even though it has a similar safety and efficacy profile to other tnfi, golimumab is less effective than other tnfi in individuals who have failed multiple biological treatments. however, due to its high mass it can be a good therapeutic option during lactation. the biosimilar products of golimumab are still in a preclinical phase [202] . certolizumab is a human monoclonal antibody administered every 2 weeks by subcutaneous injection. it is a biological molecule that can be safely used during pregnancy due to its lack of placental transfer and it has been approved for the treatment of ra in pregnant women [203] . the biosimilar products of certolizumab are still in a preclinical phase [202] . abatacept is a fusion protein that inhibits t cell activation by blocking the interaction with cd28. it is administered by intravenous infusion and should be administered 2 and 4 weeks after the first infusion, then every 4 weeks. numerous phase 3 trials have examined the safety and efficacy profiles of abatacept. a double-blind trial including ra patients with a poor response to mtx therapy assessed the safety and efficacy of abatacept or infliximab versus placebo. the results of the study estimated a similar efficacy profile of abatacept and infliximab, but a better safety profile for abatacept, with fewer adverse events [204] . however, an observational post-marketing study analyzed individual case safety reports provided by vigibase, to compare the incidence of cancer reported in ra patients receiving abatacept compared to those receiving other bdmards. abatacept was only substantially related with an elevated risk of reporting melanoma in ra patients when compared to other bdmards [205] . tocilizumab is a monoclonal antibody with an il-6 inhibition mechanism. it is available on the pharmaceutical market as an infusion and can be administered subcutaneously and intravenously. evidence from 14 phase 3 clinical trials suggested that the immunogenicity risk of tocilizumab is low, regardless of the route of administration [206] . according to the adacta study, tocilizumab therapy was found to be more effective than adalimumab monotherapy in terms of reducing signs and symptoms in ra patients with an inadequate response to mtx therapy [207] . the most common side effects reported in clinical trials are upper respiratory tract infections, nasopharyngitis, cellulitis, and high blood pressure [202] . rituximab is a well-tolerated molecule, not associated with an increased risk of infection. to evaluate infection rates between rituximab and placebo, a fixed-effect metaanalysis was conducted. the study's findings showed that the risk of serious infection when rituximab is administered is low, even at higher doses [208] . furthermore, rituximab is a monoclonal antibody with good efficacy in ra as demonstrated by a prospective, noninterventional study and can be an alternative for patients with an inadequate response to treatment with mtx or tnf-α inhibitors [209] . a network meta-analysis of the most suitable cochrane reviews on bdmards for ra compared the efficacy and safety profile of six bdmards (abatacept, adalimumab, anakinra, infliximab, rituximab, etanercept). the results showed that adalimumab and etanercept were more effective than anakinra, and that adalimumab, infliximab and anakinra are less safe than etanercept [197] . bdmards are scientific breakthroughs that have revolutionized the treatment of ra. numerous benefits have been reported in ra patients who had a poor response to csdmards. the focus of therapeutic management is on a rapid and aggressive intervention with the most effective drugs. the high cost of biologics is one the major factors limiting patient access to bdmards. however, patient profiles have changed over time and now include shorter disease progression times and lower disease activity. a trend of prescribing bdmards as the first line of treatment in ra patients with comorbidities has been observed [210] . the newest therapeutic approach to ra approved by the fda and ema involves the use of jaki. these molecules are divided into two groups based on their selectivity, the first one consisting of inhibitors with low selectivity, inhibiting the signaling of a broad range of cytokines, and the second generation that can selectively inhibit signaling processes. jaks are cytoplasmic proteins that connect cytokine signaling from membrane receptors to transcription factors known as signal transducers and activators of transcription (stat), so that an optimal control of the inflammatory response can be achieved, and they can also become a valuable tool for the management of autoimmune diseases [211] . moreover, there are four members in the jaks family (jak1, jak2, jak 3 and tyrosine kinase 2, tyk2) and seven types of stats (stat1, stat2, stat3, stat4, stat5a, stat5b, stat6), that can be targets for jaki [212] . in addition to the good efficacy and safety profiles, other important advantages of jaki are their oral route of administration and that their production costs are lower than those of bdmards [213] [214] [215] . the 2021 acr guideline for the treatment of ra updated the recommendations on using jaki when csdmards are ineffective. furthermore, patients' compliance to jaki monotherapy may be higher than with multiple therapies with csdmards, in this case with the safety profile being lower [159, 216] . due to the suppression of the immune system, the risk of infections, especially pulmonary ones, can be high and therefore vaccination before initiating treatment is recommended [217, 218] . there has also been a reported dosedependent alteration of lipid metabolism, but without a correlation with an increased risk of cardiac disease [212] . table 2 provides an overview of jaki, emphasizing their novelty in ra management and presenting their molecular targets and safety profiles [212, 219] . clinical trials have provided complex information on the safety and efficacy profile of jaki and an increase in their future use is expected as the pathophysiological processes of ra and the pharmacological properties of therapeutic agents are fully elucidated. in this context, results of post-marketing surveillance (pms) are becoming an important tool for ensuring that therapeutic approaches remain safe and effective but can also reveal areas where improvements can be made. to assess the safety of tofacitinib, baricitinib and upadacitinib in a post-marketing setting, researchers analyzed pms reports and then provided valuable information. a meta-analysis systematically assessed tofacitinib pms data obtained in the pfizer safety database from november 2012 (approval date) to november 2015. reporting rates (rr) were computed by dividing the number of serious adverse events by the predicted 100 patient-years of exposure. moreover, patient exposure was determined by using estimated worldwide sales and a twice-daily regimen of tofacitinib 5 mg. worldwide postmarketing exposure to tofacitinib since approval was estimated to be 34,223 patient-years, throughout the 3-year reporting period. in total, 9291 case reports were analyzed (82.9% non-serious) and 102 fatal cases were reported. the calculated rrs were 2.57 for infections, 0.45 for neoplasms and 0.43 for cardiac disorders. the most frequently reported adverse events out of a total of 25,417 reports were drug ineffectiveness (13.2%), headache (9.0%) and pain (6.4%). the types of events analyzed from the pms data for tofacitinib in ra were correlated with the established tofacitinib safety profile, with no novel safety hazards identified [225] . another recent meta-analysis of observational studies evaluated the risk of malignancy in ra patients exposed to non-tnfi or tofacitinib therapy, in comparison with csdmards or tnfi. a total of 10 studies out of 2819 identified articles involving 40,587 patients exposed to non-tnfi and 2221 patients exposed to tofacitinib were included. abatacept exposure was associated with a minor risk of developing cancer, but tofacitinib, rituximab and tocilizumab were not associated with an elevated cancer risk [226] . however, a recent phase 4 study conducted between 2014 and 2020 assessed the safety of tofacitinib versus adalimumab and etanercept, in terms of major adverse cardiovascular events (mace) and malignancies, excluding non-melanoma skin cancers. the goal of this study was to show the non-inferiority of tofacitinib compared to tnfi related to these pathologies. results showed that the non-inferiority criteria that had been set were not met [227] . furthermore, in pms studies, jaki, particularly tofacitinib, have been linked to an elevated risk for venous thromboembolisms [228] . however, this increased risk was only observed when tofacitinib was given at a dose of 10 mg twice a day, which is higher than the level recommended for ra in most countries [229] . at the moment, baricitinib is used less than tofacitinib. an all-case pms study of baricitinib analyzed its safety and efficacy profile in japanese patients with ra from september 2017 to june 2020. the data collected demonstrated no additional safety issues, indicating that baricitinib should be used in accordance with guidelines [230] . because jaki represent new therapeutic options, most phase 4 studies are ongoing. two major phase 4 studies designed to compare the safety of baricitinib versus tnfi with respect to venous thromboembolic events are currently ongoing [231, 232] . moreover, there are also two ongoing studies on the use of upadacitinib. the first one started in october 2020 and assessed the change in disease symptoms in adult canadian participants with moderate to severe ra (closeup). the estimated enrollment in multiple sites within canada is about 390 participants and the estimated study completion date is september 2024 [233] . the second phase 4 study is more recent (january 2021) and larger (3000 participants). it is a pms to evaluate the efficacy and safety profile of upadacitinib in korean adult participants with ra [234] . additionally, pms are very necessary, especially for new therapies, and the data they bring will further guide jaki therapy. due to the current epidemiological context, studies have been conducted to determine whether there are certain correlations between ra and coronavirus disease 2019 (covid-19), starting from the evaluation of the cytokine storm and other data that showed an increase in the serum acpa level after infection with sars-cov-2 [235, 236] . even though ra patients appeared to be more vulnerable due to their autoimmune disorder, the epidemiological parameters and the evolution of sars-cov-2 infection are not different from those reported in the general population, according to data from cross-sectional and cohort studies published so far. furthermore, immunosuppressive agents do not appear to be linked to covid-19 progression, so that the treatment of non-infected patients can carefully continue. however, further research is needed in order to investigate the influence of ra medications for patients infected with sars-cov-2, which is still controversial, so monitoring each therapeutic stage becomes essential [237] . the management of ra has changed significantly over the last decades, resulting in improved quality of life and outcomes for ra patients. this has been made feasible by the successful discovery of several pathways involved in the pathogenesis of ra. however, both the mechanisms underlying the inflammatory processes and the pharmacological effects of therapeutic molecules are still incompletely elucidated, leading to some unmet needs in the management of ra. these include a deeper understanding of how different therapies have such comparable efficacies; elucidating why certain patients become less responsive over time; detecting pre-ra and establishing an early and aggressive treatment; and improving the efficacy and safety profiles of novel compounds, especially jakis [31, 238] . several ways to improve ra treatment are now being tested in different experimental models. numerous new therapeutic targets are being researched and potential therapeutic agents are in various stages of testing in order to obtain a complete remission of ra. the present and future of targeted therapies are summarized in figure 5 [12, 219] . , updated and considered complex information about small molecular metabolite targets (prostaglandins, thromboxane a2, leukotriene b4 receptor, plateletactivating factor, cannabinoid receptors, inducible nitric oxide, etc.) and epigenetic targets (dna methylation, rna methylation, histone modification, etc.) and other protein targets (p38 mitogen-activated protein kinase, complex g protein-coupled receptor kinase 2, granulocyte-macrophage colony-stimulating factor), along with potential therapeutic agents [219] . mesenchymal stem cells (mscs) are also a promising therapeutic approach due to their ability to differentiate into new tissues like bone and cartilage and they have been reported to have immunosuppressive properties in vitro by suppressing t cell activation. moreover, treatment with mscs in both animal model studies and clinical trials in ra patients have shown a decrease in the proinflammatory response and an improvement of ra symptoms by reducing the blood levels of il-1, il-6, il-8 and tnf-α [239] . toll-like receptor 4 has a confirmed role in ra pathogenesis, promoting joint inflammation. thus, therapeutic compounds targeting this receptor or its ligands, such as heat-shock protein crystalline or tenascin c can be optimized [239] . therapeutic options for ra are increasingly diverse, and numerous ongoing studies can contribute to a significant improvement for ra patients by discovering new molecular targets, new therapeutic agents, and new methods to counteract side effects. a personalized approach based on genetic studies doubled by evidence-based medicine can transform the future of medicine and succeed in curing the incurable [240] . in this review, an overview was presented of the management of ra, centralizing updated information. in recent years, interest in controlling autoimmune disease has grown, with numerous studies testing various approaches to patients with ra. even though it is still incurable, patients' quality of life has improved considerably. disease prevention strategies, screening programs of people at risk of developing ra and comprehensive information on the disease monograph provided to the population can significantly improve epidemiological parameters [12] . the first step to effective disease management is an early and correct diagnosis, especially as several signs and symptoms are also associated with other diseases. the proper use and interpretation of acr-eular criteria, the identification and quantification of diagnostic biomarkers and the association of the obtained results with imaging techniques contribute to the establishment of an accurate diagnosis. according to existing data, the benefits of nonpharmacological interventions are greater the faster the diagnosis is established [239] . the ultimate goal of ra management is to start an aggressive drug treatment in order to obtain full remission or at least a significant reduction in symptoms and clinical signs. the results of the conducted studies facilitated the understanding of the pathophysiological mechanisms and developed new therapeutic approaches, which made ra become a manageable pathology. however, many ra patients continue to be unresponsive to current medications. there are still insufficient data to achieve complete control of the disease, highlighting the need for new drugs to be developed and a greater focus on personalized medicine [240] . beyond the joints, the extra-articular manifestations in rheumatoid arthritis extra-articular manifestations in rheumatoid arthritis immunologic rheumatic disorders the role of anti-citrullinated protein antibodies (acpa) in the pathogenesis of rheumatoid arthritis autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis cognitive dysfunction in patients of rheumatoid arthritis how many life years are lost in patients with rheumatoid arthritis? secular cause-specific and all-cause mortality in rheumatoid arthritis, and their predictors in a long-term australian cohort study a public health approach to addressing arthritis in older adults: the most common cause of disability which one? what kind? how many? types, causes, and prevalence of disability among u.s. adults. disabil resource utilization and cost of care for rheumatoid arthritis and osteoarthritis in a managed care setting. the importance of drug and surgery costs economic burden of rheumatoid arthritis: a systematic review rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies american college of rheumatology guideline for the treatment of rheumatoid arthritis use of steroid and nonsteroidal anti-inflammatories in the treatment of rheumatoid arthritis: systematic review protocol global, regional and national burden of rheumatoid arthritis 1990-2017: a systematic analysis of the global burden of disease study prevalence of rheumatoid arthritis in belgrade, yugoslavia descriptive epidemiology of rheumatoid arthritis prevalence of rheumatoid arthritis in italy: the chiavari study changes in the incidence and prevalence of rheumatoid arthritis in kamitonda prevalence of rheumatoid arthritis in tucumán prevalence of rheumatoid arthritis in serbia prevalence and incidence of rheumatoid arthritis in italy epidemiological characteristics of rheumatoid arthritis in japan: prevalence estimates using a nationwide population-based questionnaire survey chinese guidelines for the diagnosis and treatment of rheumatoid arthritis the prevalence of rheumatoid arthritis in argentina: a capture-recapture study in a city of buenos aires province the prevalence of rheumatoid arthritis in the general population of spain the prevalence of rheumatoid arthritis in spain the prevalence of rheumatoid arthritis in an urban population of izmir-turkey prevalence of rheumatoid arthritis and spondyloarthritis in turkey: a nationwide study rheumatoid arthritis low incidence of rheumatoid arthritis in france trends in incidence and mortality in rheumatoid arthritis in rochester, minnesota, over a forty-year period is the epidemiology of rheumatoid arthritis changing? results from a population-based incidence study geographic variation in incidence and prevalence rates for rheumatoid arthritis in saskatchewan geographic variation in rheumatoid arthritis incidence among women in the united states porphyromonas gingivalis and rheumatoid arthritis immunopathogenesis of rheumatoid arthritis pad enzymes in rheumatoid arthritis: pathogenic effectors and autoimmune targets the etiology of rheumatoid arthritis modeling rheumatoid arthritis in vitro: from experimental feasibility to physiological proximity hla-disease associations in rheumatoid arthritis activation and inhibition of lymphocytes by costimulation therapeutic t-cell manipulation in rheumatoid arthritis: past, present and future mechanism and regulation of class switch recombination rheumatoid factors: clinical applications the roles of anti-citrullinated protein antibodies in the immunopathogenesis of rheumatoid arthritis is air pollution a risk factor for rheumatoid arthritis? influence of ambient air pollution on rheumatoid arthritis disease activity score index association between environmental air pollution and rheumatoid arthritis flares association between vitamin d intake and the risk of rheumatoid arthritis: a meta-analysis host-microbiota interactions in rheumatoid arthritis ra and the microbiome: do host genetic factors provide the link? role of intestinal microbiota on gut homeostasis and rheumatoid arthritis quantitative microbiome profiling links gut community variation to microbial load puxeddu, i. one year in review 2019: pathogenesis of rheumatoid arthritis synovial macrophages: from ordinary eaters to extraordinary multitaskers fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis metalloproteinases in rheumatoid arthritis: potential therapeutic targets to improve current therapies cartilage destruction by matrix degradation products the role of rank ligand/osteoprotegerin in rheumatoid arthritis cells of the synovium in rheumatoid arthritis. t lymphocytes expression, modulation and signalling of il-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis pathogenic role of immune cells in rheumatoid arthritis: implications in clinical treatment and biomarker development oxidative stress, consequences and ros mediated cellular signaling in rheumatoid arthritis the role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis complement in the initiation and evolution of rheumatoid arthritis angiogenesis in rheumatoid arthritis the pathogenic role of angiogenesis in rheumatoid arthritis molecular structure and function of janus kinases: implications for the development of inhibitors diagnosis and management of rheumatoid arthritis performance of the 2010 acr/eular classification criteria for rheumatoid arthritis: a systematic literature review rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative clinical features of rheumatoid arthritis diagnosis and management of rheumatoid arthritis: a review previous diagnosis of sjögren's syndrome as rheumatoid arthritis or systemic lupus erythematosus differential diagnosis of rheumatoid and psoriatic arthritis at an early stage in the small hand and foot joints using magnetic resonance imaging differential diagnosis between rheumatoid arthritis and psoriatic arthritis: the value of ultrasound findings at metacarpophalangeal joints level diagnosis and treatment of lyme arthritis sensitivity and specificity of the american college of rheumatology 1987 criteria for the diagnosis of rheumatoid arthritis according to disease duration: a systematic literature review and meta-analysis does anti-mutated citrullinated vimentin have additional value as a serological marker in the diagnostic and prognostic investigation of patients with rheumatoid arthritis? a systematic review serum 14-3-3η is a novel marker that complements current serological measurements to enhance detection of patients with rheumatoid arthritis 14-3-3η protein: a promising biomarker for rheumatoid arthritis anti-carbamylated protein antibodies in the pre-symptomatic phase of rheumatoid arthritis, their relationship with multiple anti-citrulline peptide antibodies and association with radiological damage diagnostic value and clinical significance of anti-carbamylated protein (anti-carp) antibodies in egyptian patients with rheumatoid arthritis anti-carbamylated protein (anti-carp) antibodies precede the onset of rheumatoid arthritis serum mmp-3 and mmp-1 and progression of joint damage in early rheumatoid arthritis effect of the oral application of a highly selective mmp-13 inhibitor in three different animal models of rheumatoid arthritis identification of novel markers in rheumatoid arthritis through integrated analysis of dna methylation and microrna expression heat shock proteins and their immunomodulatory role in inflammatory arthritis serum biomarker panel for the diagnosis of rheumatoid arthritis glycoprotein ykl-40: a potential biomarker of disease activity in rheumatoid arthritis during intensive treatment with csdmards and infliximab. evidence from the randomised controlled neo-raco trial diagnostic tests for rheumatoid arthritis: comparison of anti-cyclic citrullinated peptide antibodies, anti-keratin antibodies and igm rheumatoid factors poor prognostic factors guiding treatment decisions in rheumatoid arthritis patients: a review of data from randomized clinical trials and cohort studies sat0070 levels of 14-3-3eta predict good eular response to anti-tnf treatment in patients with rheumatoid arthritis low serum level of comp, a cartilage turnover marker, predicts rapid and high acr70 response to adalimumab therapy in rheumatoid arthritis serum levels of anti-ccp antibodies, anti-mcv antibodies and rf iga in the follow-up of patients with rheumatoid arthritis treated with rituximab high survivin levels predict poor clinical response to infliximab treatment in patients with rheumatoid arthritis the role of imaging in rheumatoid arthritis detection of bone erosions in rheumatoid arthritis wrist joints with magnetic resonance imaging, computed tomography and radiography ultrasound versus conventional radiography in the assessment of bone erosions in rheumatoid arthritis rheumatoid arthritis revisited-advanced imaging review laboratory biomarkers or imaging in the diagnostics of rheumatoid arthritis the value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: a comparison with conventional radiography rheumatoid arthritis bone erosion volumes on ct and mri: reliability and correlations with erosion scores. on ct, mri and radiography a comparative analysis of magnetic resonance imaging and high-resolution peripheral quantitative computed tomography of the hand for the detection of erosion repair in rheumatoid arthritis are bone erosions detected by magnetic resonance imaging and ultrasonography true erosions? a comparison with computed tomography in rheumatoid arthritis metacarpophalangeal joints do magnetic resonance imaging-detected erosions predict progression to rheumatoid arthritis in patients presenting with clinically suspect arthralgia? a longitudinal study extra-articular manifestations and comorbidity in rheumatoid arthritis: potential impact of pre-rheumatoid arthritis prevention occurrence of extraarticular disease manifestations is associated with excess mortality in a community based cohort of patients with rheumatoid arthritis extra-articular manifestations in 587 italian patients with rheumatoid arthritis cardiovascular manifestations of rheumatologic diseases the impact of traditional cardiovascular risk factors on cardiovascular outcomes in patients with rheumatoid arthritis: a systematic review and meta-analysis risk of incident cardiovascular events in patients with rheumatoid arthritis: a meta-analysis of observational studies echocardiographic and doppler findings in long-term treated rheumatoid arthritis patients without clinically evident cardiovascular disease neuropathy in rheumatoid arthritis serum levels of hepcidin in rheumatoid arthritis and its correlation with disease activity and anemia: a meta-analysis felty's syndrome as an initial presentation of rheumatoid arthritis: a case report the key comorbidities in patients with rheumatoid arthritis: a narrative review comorbidities in rheumatoid arthritis: the rbsmr study the effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis the lipid paradox as a metabolic checkpoint and its therapeutic significance in ameliorating the associated cardiovascular risks in rheumatoid arthritis patients cardiovascular risk comorbidities in rheumatoid arthritis patients and the use of anti-rheumatic drugs: a cross-sectional real-life study eular recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders application of the 2015/2016 eular recommendations for cardiovascular risk in daily practice: data from an observational study the role of statins in disease modification and cardiovascular risk in rheumatoid arthritis points to consider for reporting, screening for and preventing selected comorbidities in chronic inflammatory rheumatic diseases in daily practice: a eular initiative eular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases an official ats/ers/jrs/alat statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management practical management of respiratory comorbidities in patients with rheumatoid arthritis cancer screening and early detection in the 21st century evidence-based recommendations for the management of comorbidities in rheumatoid arthritis, psoriasis, and psoriatic arthritis: expert opinion of the canadian dermatology-rheumatology comorbidity initiative rheumatoid arthritis in spain: occurrence of extra-articular manifestations and estimates of disease severity extra-articular manifestations and complications of rheumatoid arthritis american college of rheumatology guideline for the treatment of rheumatoid arthritis can rheumatoid arthritis be prevented? prevention of autoimmune rheumatic disease: state of the art and future perspectives food for mood: relevance of nutritional omega-3 fatty acids for depression and anxiety efficacy of omega-3 pufas in depression: a meta-analysis efficacy of omega-3 highly unsaturated fatty acids in the treatment of depression meta-analysis of the effects of eicosapentaenoic acid (epa) in clinical trials in depression association of use of omega-3 polyunsaturated fatty acids with changes in severity of anxiety symptoms: a systematic review and meta-analysis increased levels of omega-3 fatty acids and dha are linked to pain reduction in rheumatoid arthritis patients treated with janus kinase inhibitors the role of non-pharmacological interventions in the management of rheumatoid-arthritis-related fatigue fatigue in rheumatoid arthritis: time for a conceptual model occupational therapy for rheumatoid arthritis: a systematic review treatment strategies in surgery for rheumatoid arthritis nonpharmacologic pain management in inflammatory arthritis eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update multinational evidence-based recommendations for pain management by pharmacotherapy in inflammatory arthritis: integrating systematic literature research and expert opinion of a broad panel of rheumatologists in the 3e initiative use of nsaids in treating patients with arthritis new directions in symptomatic therapy for patients with osteoarthritis and rheumatoid arthritis rheumatoid arthritis: a brief overview of the treatment glucocorticoids in rheumatoid arthritis: current status and future studies factors influencing the choice of first-and second-line biologic therapy for the treatment of rheumatoid arthritis: real-life data from the italian lorhen registry patient preferences on rheumatoid arthritis second-line treatment: a discrete choice experiment of swedish patients long-term safety of methotrexate in the treatment of rheumatoid arthritis systematic review and meta-analysis of the efficacy and safety of leflunomide and methotrexate in the treatment of rheumatoid arthritis side effects of methotrexate therapy for rheumatoid arthritis: a systematic review the efficacy and safety of leflunomide in patients with active rheumatoid arthritis: a five-year followup study efficacy and safety of hydroxychloroquine sulphate in rheumatoid arthritis: a randomized, double-blind, placebo controlled clinical trial-an indian experience hydroxychloroquine decreases th17-related cytokines in systemic lupus erythematosus and rheumatoid arthritis patients liquid chromatography based methods for analysis of disease-modifying antirheumatic drugs (dmards) in biological matrices new therapeutic approaches in rheumatoid arthritis frequency and trends of disease-modifying antirheumatic drug (dmard) use in germany the use of biologics in rheumatoid arthritis: current and emerging paradigms of care efficacy and safety of adalimumab as monotherapy in patients with rheumatoid arthritis for whom previous disease modifying antirheumatic drug treatment has failed anti-tnf antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials a population-based study of tuberculosis incidence among rheumatic disease patients under anti-tnf treatment adalimumab biosimilars in the treatment of rheumatoid arthritis: a systematic review of the evidence for biosimilarity etanercept for the treatment of rheumatoid arthritis a long-term, open-label trial of the safety and efficacy of etanercept (enbrel) in patients with rheumatoid arthritis not treated with other disease-modifying antirheumatic drugs a network meta-analysis of randomized controlled trials of biologics for rheumatoid arthritis: a cochrane overview eq-5d utility, response and drug survival in rheumatoid arthritis patients on biologic monotherapy: a prospective observational study of patients registered in the south swedish ssatg registry safety and efficacy of etanercept beyond 10 years of therapy in north american patients with early and longstanding rheumatoid arthritis efficacy of infliximab as a switched biologic in rheumatoid arthritis patients in daily clinical practice a comparative study of pf-06438179/gp1111 (an infliximab biosimilar) and reference infliximab in patients with moderate to severe active rheumatoid arthritis: a subgroup analysis biologic drugs for rheumatoid arthritis in the context of biosimilars, genetics, epigenetics and covid-19 treatment lack of placental transfer of certolizumab pegol during pregnancy: results from crib, a prospective, postmarketing, pharmacokinetic study efficacy and safety of abatacept or infliximab vs placebo in attest: a phase iii, multi-centre, randomised, double-blind, placebo-controlled study in patients with rheumatoid arthritis and an inadequate response to methotrexate abatacept in rheumatoid arthritis and the risk of cancer: a world observational post-marketing study low immunogenicity of tocilizumab in patients with rheumatoid arthritis tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind risk of serious infections during rituximab, abatacept and anakinra treatments for rheumatoid arthritis: meta-analyses of randomised placebo-controlled trials rituximab in patients with rheumatoid arthritis in routine practice (gerinis): six-year results from a prospective, multicentre, non-interventional study in 2,484 patients janus kinase inhibitors for rheumatoid arthritis jak-inhibitors for the treatment of rheumatoid arthritis: a focus on the present and an outlook on the future a phase iib study of abt-494, a selective jak-1 inhibitor, in patients with rheumatoid arthritis and an inadequate response to anti-tumor necrosis factor therapy efficacy and safety of tofacitinib, baricitinib, and upadacitinib for rheumatoid arthritis: a systematic review and meta-analysis comparison of janus kinase inhibitors in the treatment of rheumatoid arthritis: a systemic literature review role of janus kinase inhibitors in rheumatoid arthritis treatment growing evidence of the safety of jak inhibitors in patients with rheumatoid arthritis safety and efficacy of tofacitinib for up to 9.5 years in the treatment of rheumatoid arthritis: final results of a global, open-label, long-term extension study promising therapeutic targets for treatment of rheumatoid arthritis adverse drug events associated with 5mg versus 10mg tofacitinib (janus kinase inhibitor) twice daily for the treatment of autoimmune diseases: a systematic review and meta-analysis of randomized controlled trials risk of adverse drug events observed with baricitinib 2 mg versus baricitinib 4 mg once daily for the treatment of rheumatoid arthritis: a systematic review and meta-analysis of randomized controlled trials efficacy and safety of peficitinib (asp015k) in patients with rheumatoid arthritis and an inadequate response to methotrexate: results of a phase iii randomised, double-blind, placebo-controlled trial (raj4) in japan effect of filgotinib vs placebo on clinical response in patients with moderate to severe rheumatoid arthritis refractory to disease-modifying antirheumatic drug therapy: the finch 2 randomized clinical trial decernotinib: a next-generation jakinib worldwide, 3-year, post-marketing surveillance experience with tofacitinib in rheumatoid arthritis risk of malignancy with non-tnfi biologic or tofacitinib therapy in rheumatoid arthritis: a meta-analysis of observational studies safety study of tofacitinib versus tumor necrosis factor (tnf) inhibitor in subjects with rheumatoid arthritis incidence of venous and arterial thromboembolic events reported in the tofacitinib rheumatoid arthritis, psoriasis and psoriatic arthritis development programmes and from real-world data 5 mg film-coated tablets-summary of product characteristics ab0249 safety of baricitinib in japanese patients with rheumatoid arthritis (ra): the 2020 interim report from all-case post marketing surveillance in clinical practice a study of baricitinib in participants with rheumatoid arthritis (ra-branch) a study of baricitinib (ly3009104) in participants with rheumatoid arthritis (ra-bridge) a study of oral upadacitinib tablets to assess the change in disease symptoms in adult canadian participants with moderate to severe rheumatoid arthritis (closeup) a study of oral upadacitinib (rinvoq) tablets to assess adverse events and change in disease symptoms in korean adult participants with moderate to severe active rheumatoid arthritis first flare of acpa-positive rheumatoid arthritis after sars-cov-2 infection covid-19 infection and rheumatoid arthritis: mutual outburst cytokines and remedies clinical management of patients with rheumatoid arthritis during the covid-19 pandemic update on the pathomechanism, diagnosis, and treatment options for rheumatoid arthritis current therapeutic options in the treatment of rheumatoid arthritis the authors thank the university of oradea, considering the logistic facilities they used and for supporting the publication of this manuscript. the authors declare no conflict of interest. althea medical journal. 2015;2(3) 423 identification of risk factors for osteoporotic fracture using fracture risk assessment tool in dr. hasan sadikin general hospital, bandung, indonesia from june to december 2013 nik fatin farhana binti mohd rahhim,1 bambang tiksnadi,2 eppy buchori3 1faculty of medicine, universitas padjadjaran, 2department of orthopaedic & traumatology, faculty of medicine universitas padjadjaran/dr. hasan sadikin general hospital bandung, 3department of radiology faculty of medicine universitas padjadjaran/dr. hasan sadikin general hospital bandung abstract background: osteoporosis has become a growing public health problem in indonesia. a definite estimation of osteoporosis prevalence in indonesia is not available due to the limited access of dual energy x ray absorptiometry (dxa) facilities. in 2008, the world health organization has developed a tool called fracture risk assessment tool to identify fracture risk based on the clinical risk factors. the study aimed to identify the risk factors of osteoporotic fracture using fracture risk assessment tool in dr. hasan sadikin general hospital, bandung, indonesia. methods: this descriptive study was conducted from june–december 2013 in orthopedic & traumatology, internal medicine, geriatric and surgery polyclinics dr. hasan sadikin general hospital, bandung to 77 respondents, aged 40–90 years, using the random sampling method. fracture risks were calculated online, and the data obtained were analyzed and presented using frequency distribution in tables. results: most of the respondents had low risk for osteoporotic fracture, and only 5.19% of them had moderate risk. the main risk factors were rheumatoid arthritis (57.14%), followed by current smoking (27.27%) and prolonged glucocorticoids consumption (25.98%). the moderate risk group was females, above 60 years old and with normal bmi or underweight with risks of previous fracture, parent’s previous hip fracture, rheumatoid arthritis and prolonged glucocorticoids exposure. conclusions: majority of the respondents have low risk for osteoporotic fracture. it must be taken into consideration that increasing age, rheumatoid arthritis, current smoking, prolonged glucocorticoids consumption, previous fracture and parent’s previous hip fracture can cause increased risk. [amj.2015;2(3):423–28] keywords: fracture, fracture probability, fracture risk assessment, osteoporosis correspondence: nik fatin farhana binti mohd rahhim, faculty of medicine, universitas padjadjaran, jalan raya bandung-sumedang km.21, jatinangor, sumedang, indonesia, phone: +62 85624820691 email: kin_frhan@yahoo.com introduction osteoporosis is a systemic skeletal disease characterized by low bone density and micro architectural deterioration of bone tissue with a consequent increase in bone fragility.1 osteoporosis has become a growing public health concern in asia. more than 50% of all osteoporosis-related fractures are estimated to occur in asia by 2050.2 in 2005, 2 out of 5 indonesians were reported to be at risk of osteoporosis. however, a definite estimation of osteoporosis prevalence in indonesia is not available due to the limited access of dual energy x ray absorptiometry (dxa) facilities; it makes this disease be considered as an under diagnosed disease.2 measurement of bone mineral density (bmd) using the dxa technology can identify individuals with low bone mass, which is a major characteristic of osteoporosis, yet, this technology is relatively expensive and widely unavailable in most developing asian countries. 2,3 osteoporosis is a silent disease. the occurrence of low trauma or fragility fracture has been identified as the outcome of osteoporosis. an estimation of one half of all clinical fractures occurs in persons without althea medical journal. 2015;2(3) 424 amj september, 2015 osteoporosis based on bmd. this indicates that the identification of other risk factors is required in order to identify individuals that are at highest risk for fracture.4 the who5 has developed a computer-based algorithm program called the fracture risk assessment (frax) tool which integrates the weight of clinical risk factors with or without the bmd results to predict the absolute percentage of risk for major osteoporotic fracture and hip fracture over the next 10 years.6-8 major osteoporotic fracture is defined as any osteoporosis related fractures such as clinical spine, wrist, proximal humerus and hip fracture.9 the frax tool is validated globally, applicable to both men and women from 40 to 90 years of age, cost-effective and countryspecific.3 this study was conducted to identify the distribution of risks for major osteoporotic fracture using frax tool among patients from 40 to 90 years of age in the polyclinics of dr. hasan sadikin general hospital, and could provide an overview of a rough estimation of the risk for osteoporotic fracture in bandung. methods a descriptive study was conducted from june–december 2013 in several polyclinics of dr. hasan sadikin general hospital. among selected polyclinics were the polyclinic of orthopedic & traumatology, polyclinic of internal medicine, polyclinic of geriatric and polyclinic of surgery. this study was conducted under a permission letter (no.: lb.02.01/c02/12526/xl/2013) approved by the health research ethics committee of dr. hasan sadikin general hospital. a total of 77 respondents from 40 to 90 years of age in the selected polyclinics were included using the random sampling method and all of them were informed of the objectives and benefits of the study before the interview. written informed consent was obtained prior to the interview. those refusing to be interviewed were excluded from this study. the interview was conducted based on the questions provided in the frax tool.5 questions included age, gender, weight, height, smoking status, three or more alcoholic units per day, parent with a hip fracture, previous fracture, rheumatoid arthritis, prolonged glucocorticoid consumption, secondary osteoporosis and bone mass density score (bmd). next, online calculation was conducted to calculate the respondent's percentage risk for major osteoporotic fracture over the next 10 years based on the integration of the clinical risk factors with or without the bone mass density (bmd) score. furthermore, fracture risks were classified into three groups: low risk (frax score of less than 10% of osteoporotic fracture), moderate risk (frax score between 1019% of osteoporotic fracture) and high risk (frax score of 20% or more for osteoporotic fracture).10 percentage of fracture risks obtained from the online calculation was then informed to the respondents. additionally, education on prevention of osteoporosis and fracture associated was also given after the interview to raise the awareness of the respondents on osteoporosis itself. the respondents were given education on lifestyle and dietary modifications as well as preventive measures such as fall preventions. apart from that, the respondents with moderate fracture risk were given recommendation to perform bone mass density (bmd) testing for further evaluation of their risk of osteopenia and osteoporosis. data obtained from all the respondents were recorded and analyzed descriptively using microsoft excel. results were then classified into risk groups, age specific, gender specific and body mass index related and presented using frequency distribution in the form of tables and chart. results most of the respondents had never performed any bmd testing before and only a few had done table 1 distribution of risk groups for major osteoporotic fracture* risk groups frequency (n) percentage (%) low 73 94.81 moderate 4 5.19 high 0 0.00 total 77 100 *examples are clinical spine, wrist, proximal humerus and hip fracture althea medical journal. 2015;2(3) 425 it, but failed to remember the score. therefore, the answers for the question on bmd were recorded as a 'no' for all respondents. the percentage of fracture risks calculated online using the frax tool was classified into 3 groups: low risk, moderate risk and high risk according to the standardized fracture risk categorization. the study showed that from all of the respondents, only 2 classifications were identified: low risk and moderate risk. low risk covered 94.81% of the two classifications. (table 1) in table 2, it is shown that all of the respondents had one or more risk factors of osteoporotic fracture although most of them were included in low risk group. the main risk factors occurred were rheumatoid arthritis (57.14%), followed by current smoking and prolonged glucocorticoids consumption (27.27% and 25.98%, respectively). four risk factors in the moderate risk group were identified: previous fracture, parent’s previous hip fracture, rheumatoid arthritis and prolonged glucocorticoids consumption. neither gender has high risk for major osteoporotic fracture based on frax tool. in addition, none of the male respondents have moderate risk for major osteoporotic fracture as all four of the respondents with moderate risk are females. (table 3). table 2 distribution of osteoporotic fracture risk factors risk factors alternative answers risk groups low moderate total frequency (n) percentage (%) frequency (n) percentage (%) frequency (n) percentage (%) previous fracture yes 8 10.39 1 1.30 9 11.69 no 65 84.42 3 3.90 68 88.32 previous yes 2 2.60 2 2.60 4 5.19 parent's hip fracture no 71 92.21 2 2.60 73 94.81 current smoking yes 21 27.27 0 0.00 21 27.27 no 52 67.53 4 5.19 56 72.73 glucocorticoids yes 19 24.68 1 1.30 20 25.98 no 54 70.13 3 3.90 57 74.02 rheumatoid arthritis yes 41 53.25 3 3.90 44 57.14 no 32 41.56 1 1.30 33 42.86 secondary yes 2 2.60 0 0.00 2 2.60 osteoporosis no 71 92.21 4 5.19 75 97.40 alcohol yes 0 0.00 0 0.00 0 0.00 no 73 94.81 4 5.19 77 100.0 table 3 distribution of risk groups based on gender gender risk groups total low moderate frequency (n) percentage (%) frequency (n) percentage (%) frequency (n) percentage (%) male 34 44.16 0 0.00 34 44.16 female 39 50.65 4 5.19 43 55.84 total 73 94.81 4 5.19 77 100 nik fatin farhana binti mohd rahhim, bambang tiksnadi, eppy buchori: identification of risk factors for osteoporotic fracture using fracture risk assessment tool in dr. hasan sadikin general hospital, bandung, indonesia from june to december 2013 althea medical journal. 2015;2(3) 426 amj september, 2015 the study focused on population with age range from 40 to 90 years old in which frax tool is only applicable. age was further subdivided into 5 groups. the age group of 60–69 years has the most number of respondents with moderate risk whereas for age group of 70–79 and 80–89 years, each has one respondent with moderate risk for major osteoporotic fracture. distribution of risk groups related to the body mass index (bmi) of respondents is shown in table 5. respondents with moderate risk for major osteoporotic fracture were found to have underweight and normal weight status. discussion in the present study, the frax tool provided the assessment of fracture probability in both genders, and has been calibrated to the epidemiology of indonesia which is available online.5 interaction of each of the clinical risk factors with or without the presence of bone mass density (bmd) score improved the accuracy for the fracture risk to be computed. some of the respondents with presence of two risk factors such as previous fracture, parent’s previous hip fracture, prolonged glucocorticoid exposure and rheumatoid arthritis had moderate risk for 10-year fracture probability. these four risk factors were considered as strong risk factors in which each of them had their significance for major osteoporotic fracture probability. a previous study also showed that individuals especially women with presence of one of these four risk factors without bmd score had moderate risk of 10-year major osteoporotic fracture probability.7 the distribution of risks for major osteoporotic fracture has been identified for all the respondents using the online calculation of frax tool. all the clinical risk factors on the frax tool such as age, bmi, previous fracture, parent’s previous hip fracture and prolonged glucocorticoid exposure have been integrated in order to calculate the fracture risk percentage. in the present study, it is identified that presence of more than one risk factor will subsequently cause an increment in the tenyear major osteoporotic fracture probability. for example, 3 out of 4 respondents with moderate risk for major osteoporotic fracture table 4 distribution of risk groups based on age age groups risk groups total low moderate frequency (n) percentage (%) frequency (n) percentage (%) frequency (n) percentage (%) 40–49 20 25.97 0 0.00 20 25.97 50–59 26 33.77 0 0.00 26 33.77 60–69 21 27.27 2 2.60 23 29.87 70–79 4 5.19 1 1.30 5 6.49 80–89 2 2.60 1 1.30 3 3.90 total 73 94.81 4 5.19 77 100 table 5 distribution of risk groups based on body mass index (bmi) body mass index (bmi) risk groups total low moderate frequency (n) percentage (%) frequency (n) percentage (%) frequency (n) percentage (%) underweight 7 9.09 1 1.30 8 10.39 normal 37 48.05 3 3.90 40 51.95 overweight 21 27.27 0 0.00 21 27.27 obese 8 10.39 0 0.00 8 10.39 total 73 94.81 4 5.19 77 100 althea medical journal. 2015;2(3) 427 had two risk factors present in which the tenyear fracture probability ranged from 11–12%. in a previous study, it showed the presence of more than one risk factor increased fracture probability in an incremental manner.7 all of the respondents with moderate risk for major osteoporotic fracture were female suggesting that women had higher risk of 10year fracture probability compared to men. a study reported that the probabilities for a major osteoporotic fracture ranged from 3.5% to 31% in women, and from 2.8% to 15% in men.7 another study showed, the 10-year probability of major osteoporotic fracture risk for all women was 12.0% and 10.7% for men.11 two of the respondents with moderate risk for major osteoporotic fracture within age range of 60 to 69 years old had fracture risk of 11%. the present study observed that as age increases, the 10-year fracture risk probability will also increase. age is indeed a strong risk factor that brings significance to the computed fracture risk. the result from a previous study conducted in united kingdom has reported that respondents with moderate risk for major osteoporotic fracture within age range of 6069 years old have fracture risk of 11%.7 the bmi of respondents were classified according to the center for disease control and prevention (cdc) into 4 categories: underweight (bmi below 18.5), normal weight (bmi 18.5–24.9), overweight (bmi 25.0–29.9) and obese (bmi of 30.0 and above). findings from the present study showed that respondents with lower bmi had higher fracture risk compared to those with higher bmi. fracture results from increased skeletal fragility that can be due to loss of bone mass. individuals with higher bmi tended to have higher bmd. hence, low bone mass and low body weight increased the risk for osteoporosis and fracture of an individual.2 despite the fact that majority of the respondents had low risk for major osteoporotic fracture based on the frax tool, education on lifestyle and preventive measures for osteoporosis was given to each one of them. these individuals did not require pharmacologic therapy but rather the knowledge on how to prevent fracture that is due to osteoporosis such as increasing the amount of daily calcium intake, doing physical activities and smoking cessation. as for respondents with moderate risk, it is recommended to have their bmd and additional clinical risk factors evaluated to prevent fracture and to decide whether or not pharmacologic therapy is required.10,12 several limitations were identified during the course of this study. although frax tool is well validated, there are several other risk factors that may be important in assessing individuals for 10-year fracture probability such as exercise and nutritional status. apart from that, time constraint had also been recognized as one of the study limitations as there was lack of time for data collection and analysis. the sample population of the study was imbalance in terms of the distribution between age groups and genders. since this study was conducted in the polyclinics of dr. hasan sadikin general hospital, the results of this study could not be generalized to the population of indonesia. in brief, this study identified that 95% of the respondents had low risk for major osteoporotic fracture and the remaining 5% had moderate risk of major osteoporotic fracture for the next ten years to come. the respondents identified with moderate risk were all females. apart from that, fracture risk probability increased as the age increased. this could be seen as all of the respondents with moderate risk for major osteoporotic fracture were above 60 years old. majority of the respondents with moderate risk were either underweight or with normal bmi. four strong risk factors in the moderate risk group were identified whereby each one of them brought significance in calculating the fracture risk probability. the risk factors were previous fracture, parent’s previous hip fracture, rheumatoid arthritis and prolonged glucocorticoids exposure. references 1. who. prevention and management of osteoporosis. geneva: who technical report series. 2003 2. mithal a, dhingra v, lau e. the asian audit epidemiology, costs and burden of osteoporosis in asia 2009. switzerland: arch osteoporos. 2009 3. watts nb, bilezikian jp, camacho rm, greenspan sl, harris st, hodgson sf, et al. american association of clinical endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of p o s t m e n o p a u s a l osteoporosis. endocr pract. 2010;16(3):1– 37. 4. donaldson mg, palermo l, schousboe jt, ensrud ke, hochberg mc, cummings sr. frax and risk of vertebral fractures: the nik fatin farhana binti mohd rahhim, bambang tiksnadi, eppy buchori: identification of risk factors for osteoporotic fracture using fracture risk assessment tool in dr. hasan sadikin general hospital, bandung, indonesia from june to december 2013 althea medical journal. 2015;2(3) 428 amj september, 2015 fracture intervention trial. j bone miner res. 2009;24(11):1793–9. 5. kanis ja. frax ® who fracture risk assessment tool. united kingdom: w o r l d health organization collaborating centre for metabolic bone diseases. 2008. 6. noor z, sumitro sb, hidayat m, rahim ah, taufiq a. assessment of microarchitecture and c r y s t a l structure of hydroxyapatite in osteoporosis. univ med. 2011;30(1):29–35. 7. kanis ja, johnell o, oden a, johansson h, mccloskey e. frax and the assessment of fracture probability in men and women from the uk. o s t e o p o r o s i s int. 2008;19(4):385–97. 8. horikawa a, miyakoshi n, shimada y, kodama h. frax and exercise : s h o u l d exercise be categorized as a risk factor in osteoporotic patients. open journal of orthopedics. 2013;3(2):133–36. 9. mccloskey e. frax ® identifying people at high risk of fracture. who fracture risk assessment tool, a new clinical tool for informed treatment d e c i s i o n s . switzerland: arch osteoporos. 2009 10. papaioannou a, morin s, cheung am, atkinson s, brown jp, feldman s, et al. 2010 clinical practice guidelines for the diagnosis and management o f osteoporosis in canada: summary. cmaj. 2010;182(17):1864–73. 11. leslie wd, lix lm, johansson h, oden a, mccloskey e, kanis ja. i n d e p e n d e n t clinical validation of a canadian frax tool: fracture prediction and model calibration. j bone miner res. 2010;25(11):2350–8. 12. kanis ja, oden a, johansson h, borgström f, ström o, mccloskey e. frax and its applications to clinical practice. bone. 2009;44(5):734–43. key: cord-0030275-bpdng4wm authors: pulito-cueto, verónica; remuzgo-martínez, sara; genre, fernanda; atienza-mateo, belén; mora-cuesta, víctor m.; iturbe-fernández, david; lera-gómez, leticia; rodriguez-carrio, javier; prieto-peña, diana; portilla, virginia; blanco, ricardo; corrales, alfonso; gualillo, oreste; cifrián, josé m.; lópez-mejías, raquel; gonzález-gay, miguel a. title: angiogenic t cells: potential biomarkers for the early diagnosis of interstitial lung disease in autoimmune diseases? date: 2022-04-05 journal: biomedicines doi: 10.3390/biomedicines10040851 sha: fa2fc1a2a504d450e44d64cc43b82f5bd51234dc doc_id: 30275 cord_uid: bpdng4wm (1) background: we explored, for the first time, the contribution of angiogenic t cells (tang) in interstitial lung disease associated to autoimmune disease (ad-ild(+)) as potential biomarkers of the disease, evaluating their role in the underlying vasculopathy and lung fibrosis. additionally, the relationship of tang with clinical manifestations and cellular and molecular endothelial dysfunction-related biomarkers was assessed. (2) methods: we included 57 ad-ild(+) patients (21 with rheumatoid arthritis (ra)-ild(+), 21 with systemic sclerosis (ssc)-ild(+) and 15 with other ad-ild(+)) and three comparative groups: 45 ad-ild(−) patients (25 ra-ild(−) and 20 ssc-ild(−)); 21 idiopathic pulmonary fibrosis (ipf) patients; 21 healthy controls (hc). tang were considered as cd3(+)cd184(+)cd31(+) by flow cytometry. (3) results: a similar tang frequency was found between ad-ild(+) and ipf, being in both cases lower than that observed in ad-ild(−) and hc. a lower tang frequency was associated with negative scl-70 status and lower fev1/fvc ratio in ssc-ild(+), as well as with men in ra-ild(+) and non-specific interstitial pneumonia radiological pattern in other ad-ild(+). no relationship between tang and endothelial progenitor cells, endothelial cells and vascular endothelial growth factor gene expression and protein levels was disclosed. (4) conclusions: our findings suggest tang as potential biomarkers for the early diagnosis of ild in ad. interstitial lung disease (ild) is a common and potentially life-threatening complication in patients with autoimmune diseases (ad), mainly in those with systemic sclerosis (ssc) and rheumatoid arthritis (ra) [1] [2] [3] [4] [5] . early diagnosis of ad-ild + is sometimes challenging due to the potential absence of symptoms in early or mild disease and the similarity of radiological features with other entities involving the lung [1] [2] [3] [4] [5] [6] . currently, there is no established protocol to evaluate these patients, although several studies highlight the need for careful follow-up of these patients with both pulmonary function tests (pfts) and high-resolution computed tomography (hrct) [1] [2] [3] [4] [5] [6] [7] . in this sense, the early detection of pulmonary involvement is crucial to start an appropriate therapy and to avoid an irreversible damage to the lung in these patients [1] [2] [3] [4] [5] 7, 8] . a large body of evidence suggests that an impairment of vascular endothelium is a characteristic hallmark of the initial phase in these inflammatory diseases, ultimately resulting in a constitutive activation of fibroblasts in various organs, predominantly in the lung, leading to pulmonary fibrosis. in fact, the damage of the pulmonary endothelium has been described as one of the early key stages for the development of pulmonary lesions and the subsequent onset and progression of ild in ad. however, the mechanisms underlying endothelial cell damage and defective repair remain incompletely understood in ad-ild + [1, 3, [9] [10] [11] [12] [13] . endothelial progenitor cells (epc) and endothelial cells (ec) are key cellular effectors in the homeostasis of the physiologic vascular network, and they have been described as an essential element of the endogenous vascular repair machinery in ad [14] [15] [16] . in this regard, we recently proposed epc as biomarkers to identify the presence of ild in patients with ra and ssc [14, 15] . moreover, it has been reported that a circulating cell population showing both m1 and m2 monocyte/macrophage surface markers characterizes ssc patients with lung involvement [17] . it has been described that a specific t cell population termed angiogenic t cells (tang) cooperate with epc in the endothelial repair function [18] . since then, several studies have supported the notion that tang promote the formation of new blood vessels and enhance the repair of damaged endothelium [9, [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . furthermore, tang exhibit a vasculogenic phenotype characterized by enhanced endothelial proliferation and may function by cell contact-dependent and paracrine mechanisms [9, 18, 28, 29] . specifically, tang secrete a wide array of proangiogenic factors that have been implicated in ad-related angiogenic disturbances such as vascular endothelial growth factor (vegf) [9, 18, 28, 29] . moreover, it has been demonstrated that tang have migratory capacity towards the angiogenic chemoattractant vegf secreted by injured endothelium [28, 30] . interestingly, altered tang frequencies have been linked to ra [20, 23, 24] , ssc [9, 19] , or to other ad [22, 23, [25] [26] [27] . nevertheless, information on their role in the development of ild in ad patients is scarce. it has become apparent that the scarcity of useful markers for the early diagnosis of ad-ild + remains a problem that needs to be solved [1, 2, 4, 8] . with respect to this, tang may have an important role as biomarkers of endothelial damage in ad-ild + . accordingly, the main objective of this study was to determine, for the first time, the contribution of tang in the pathogenesis of ad-ild + as potential biomarkers of the disease. for this purpose, we evaluated the role of tang in the underlying vasculopathy of patients with ad-ild + and in the presence of lung fibrosis in these patients. additionally, we also aimed to assess the relationship of tang with ad-ild + clinical manifestations and endothelial dysfunction-related biomarkers at the cellular (epc, ce) and molecular (vegf mrna expression and vegf protein) level. peripheral venous blood was collected from a total of 144 individuals. specifically, 57 patients with ad-ild + were recruited: 21 with ra-ild + , 21 with ssc-ild + and 15 with other ad-ild + . moreover, to assess the role of tang in ad-ild + , we recruited different comparative groups. a group of ad-ild − patients (n = 45) composed of 25 ra-ild − and 20 ssc-ild − , another group of idiopathic pulmonary fibrosis (ipf) patients (n = 21), and 21 healthy controls (hc). both patients and hc were recruited from the departments of pneumology and rheumatology of hospital universitario marqués de valdecilla (santander, spain). patients with ad had an underlying vasculopathy (clinically evident or not) and met the criteria established by the acr/eular for the classification and diagnosis of each ad [31, 32] . pulmonary involvement was assessed in all the patients by hrct images of the chest and pfts. ad-ild − patients lacked lung involvement, whereas those with ad-ild + fulfilled the ats/ers criteria for ild [33] . ipf patients fulfilled the ats/ers criteria [33] . hrct patterns of ild patients were stratified according to the criteria for usual interstitial pneumonia (uip) pattern of the fleischner society [34] . additionally, in ssc and ipf patients, pulmonary hypertension (ph) was diagnosed by transthoracic echocardiogram. demographic and clinical features of patients including sex, age, smoking history, duration of disease, pfts, pulmonary involvement on hrct and hrct pattern, among others, were collected. the main characteristics of all the patients of the study group (ra-ild + , ssc-ild + and other ad-ild + ) and the comparative groups (ra-ild − , ssc-ild − , ipf patients) are shown in table 1 . furthermore, ph and other clinical manifestations of ssc patients were described in table s1 . hc did not present any history of autoimmune or lung diseases. additionally, their mean age ± standard deviation (sd) was 41.2 ± 12.5 years, 33.3% of them were women, and 31.3 % were smokers. all patients and hc gave their written informed consent to be included in the study. the procedures followed were in accordance with the ethical standards of the approved guidelines and regulations, according to the declaration of helsinki. the ethics committee of clinical research of cantabria, spain (2016.092) approved all experimental protocols. tang quantification was analyzed by direct flow cytometry following a method previously described [24] . briefly, cells obtained from 200 µl of peripheral blood were labelled with vioblue-conjugated anti-cd3 (miltenyi biotec, madrid, spain), apc-conjugated anti-cd184 (miltenyi biotec, madrid, spain) and pe-conjugated anti-cd31 (miltenyi biotec, madrid, spain) monoclonal antibodies. in a further step, incubation with facs lysing solution (bd bioscience, san jose, ca, usa) was performed to lyse red blood cells. after obtaining the white cell pellets, two washes with pbs were carried out. finally, a cytoflex flow cytometer (beckman coulter, brea, ca, usa) and the cytexpert 2.3 analyzer (beckman coulter, brea, ca, usa) were used to assess the labeled cells, acquiring approximately 3 × 10 4 events per sample. cd3 + cells were gated and then assayed for the expression of cd184 and cd31 in the lymphocyte gate. tang were considered as triple-positive for cd3, cd184 and cd31 ( figure s1 ) and expressed as percentage of cells in the lymphocyte gate. epc and ec frequencies were measured by flow cytometry following the method previously described [14, 15] . epc were considered as cd34 + , cd45 low , cd133 + and cd309 + cells and ec were defined as triple-negative for cd34, cd45 and cd133 and positive for cd309, following the nomenclature previously defined [14, 15] . total rna was isolated from peripheral blood by a commercial rna extraction kit (nucleospin rna blood kit, macherey-nagel, neumann-neander-str., düren, germany). the complementary dna (cdna) was obtained using iscript tm advanced cdna synthesis kit for reverse transcription-quantitative real-time polymerase chain reaction (qpcr) (bio-rad, madrid, spain). qpcr was performed in the thermocycler quantstu-dio™ 7 flex real-time pcr system (applied biosystems, foster city, ca, usa) using ssoadvanced tm universal sybr ® green supermix (bio-rad, madrid, spain). all samples were assayed in duplicate and experimental control assays were included. the relative vegf mrna expression was analyzed by the comparative ct method using gapdh as housekeeping gene. vegf levels were measured in serum samples by a commercial quantitative colorimetric sandwich enzyme-linked immunosorbent assay (reddot biotech inc., kelowna, bc, canada) as previously described [35] . data were reported as the number of individuals (n) and percentage (%) or mean ± sd depending on the type of data. differences in tang frequencies between two study groups were calculated and compared by student's t-test. to evaluate the implication of tang in the underlying vasculopathy, we compared all patients with hc, while their role in fibrosis was analyzed by comparing patients with ad-ild + , patients with ad-ild − and patients with ipf. estimation of the pearson's correlation coefficient (r) was used to assess the relationship of tang frequency with continuous variables. to evaluate the association of tang frequency with categorical variables, we employed one-way anova. statistical significance was defined as p-values < 0.05. stata statistical software 12/se (stata corp., college station, tx, usa) was used to perform all statistical analysis. first, we studied the role of tang in the vasculopathy in ad-ild + . patients with ad-ild + showed a significantly lower frequency of tang than hc (11.560 ± 5.242 vs. 16 .500 ± 4.830, p < 0.001, figure 1a and table s2 ). it was also the case when ipf patients were compared with hc (11.340 ± 3.732 vs. 16 .500 ± 4.830, p < 0.001, figure 1a and table s2 ). however, similar frequencies of tang in patients with ad-ild − and hc were found (figure 1a and table s2 ). the same findings were seen when patients were stratified according to the underlying ad. in particular, frequencies of tang were significantly decreased in patients with ra-ild + and ssc-ild + in relation to hc (11.950 ± 5.234 vs. 16 .500 ± 4.830, p = 0.007 and 12.570 ± 5.052 vs. 16 .500 ± 4.830, p = 0.016, respectively), unlike patients with ra-ild − and ssc-ild − who showed no differences with hc (figure 1b,c and table s2) . furthermore, patients with other ad-ild + displayed a lower frequency of tang than hc (10.560 ± 6.684 vs. 16 .500 ± 4.830, p = 0.005, figure 1d and table s2 ). in a second step, we evaluated the implication of tang in the presence of fibrosis in ad-ild + . tang frequencies were similar between patients with ad-ild + and those with ipf, while these frequencies were significantly lower in relation to those with ad-ild − (11.560 ± 5.242 vs. 15.920 ± 4.612, p < 0.001 and 11.340 ± 3.732 vs. 15.920 ± 4.612, p < 0.001, respectively, figure 1a and table s2 ). specifically, patients with ra-ild + exhibited significantly lower tang frequencies than those with ra-ild − (11.950 ± 5.234 vs. 16 .400 ± 4.926, p = 0.006), but no differences were observed when they were compared to patients with ipf ( figure 1b and table s2 ). moreover, a significant increase in the frequency of tang was seen in patients with ra-ild − when compared to those with ipf (16.400 ± 4.926 vs. 11.340 ± 3.732, p < 0.001, figure 1c and table s2 ). patients with ssc-ild + and ipf had the same frequencies, which were significantly lower than those observed in patients with ssc-ild − (12.570 ± 5.052 vs. 16 .070 ± 5.420, p = 0.044 and 11.340 ± 3.732 vs. 16 .070 ± 5.420, p = 0.003, respectively, figure 1c and table s2 ). likewise, tang frequencies of patients with other ad-ild + were similar to the frequency of those with ipf ( figure 1d and table s2 ). regarding ra-ild + patients, men had significantly lower tang frequencies than women (9.75 ± 4.12 vs. 16 .44 ± 5.97, p < 0.01, table 2 ), though no relationship was disclosed between these cells and the duration of ra, c-reactive protein (crp), erytrocyte sedimentation rate (esr) or pfts. no differences were found in the frequency of tang when patients with ra-ild + were stratified according to smoking history, rheumatoid factor/anti-cyclic citrullinated peptide antibodies status or hrct pattern ( table 2 ). with respect to ssc-ild + patients, a positive correlation between the frequency of tang and the forced expiratory volume in one second (fev1)/forced vital capacity (fvc) ratio was observed in these patients (r = 0.48; p = 0.03, table 3 ). anti-scl70 negative ssc-ild + patients presented lower tang frequencies compared to anti-scl70 positive patients (10.30 ± 5.09 vs. 15.73, p = 0.03, table 3 ). no significant relationship was found between the frequency of tang and ssc duration, crp or esr ( table 3 ). the same results were obtained when ssc-ild + patients were stratified according to sex, smoking history, anti-nuclear antibodies/anti-centromere antibodies status, presence of pulmonary hypertension or hrct pattern (table 3) . in relation to patients with other ad-ild + , differences in the frequency of tang were found when these patients were stratified according to the hrct pattern (table 4) . specifically, patients who presented a nsip pattern had lower tang frequencies than those with an uip pattern (6.43 ± 3.99 vs. 15.11 ± 7.69, p = 0.03, table 4 ). nonetheless, no associations of pfts with tang were noted in these patients (table 4) . similarly, we did not disclose an association with tang frequency when these patients with other ad-ild + were analyzed according to sex or smoking history (table 4) . tang: angiogenic t cells; ad: autoimmune disease; ild: interstitial lung disease; fvc: forced vital capacity; fev1: forced expiratory volume in one second; dlco: diffusing capacity of the lung for carbon monoxide; sd: standard deviation; nsip: non-specific interstitial pneumonia; hrct: high resolution computed tomography; uip: usual interstitial pneumonia. significant results are highlighted in bold. tang frequency did not show correlation with epc or ec frequency in ad-ild + patients (table s3) . likewise, no association between tang frequency and vegf, either at mrna expression or at protein level, was observed (table s3) . growing evidence indicates that vascular abnormalities constitute the early phase in the pathogenesis of ad-ild + [1, 3, [9] [10] [11] [12] . to the best of our knowledge, this is the first study exploring the implication of tang, a crucial player in endothelial repair [18] , in the pathogenic processes of lung fibrosis and vasculopathy in patients with ad-ild + . the present findings provide the first evidence that tang may be a relevant factor involved in the processes of lung fibrosis. this idea is supported by the decrease in tang in patients with ad-ild + compared to those with ad-ild − . in line with this notion, patients with ra-ild + and ssc-ild + showed a decrease in tang compared to ra-ild − and ssc-ild − patients, respectively, demonstrating the same behavior of tang regardless of the underlying ad. in keeping with our results, a previous study showed different frequencies of tang in systemic lupus erythematous (sle) depending on the presence or absence of a renal involvement, one of the most severe comorbidities of sle [27] . interestingly, our work disclosed that patients with ipf presented tang frequencies similar to those with ad-ild + and lower than ad-ild − patients. accordingly, we disclosed that tang were decreased in all the individuals with a lung involvement, including both ad-ild + and ipf patients, compared to those unaffected by this condition, highlighting the contribution of tang in the pulmonary complications. therefore, a reduction in tang may indicate the presence of lung fibrosis. based on our results and given that the development of ild is one of the main causes of mortality in ad patients [1, 2, 4] , tang could be used as novel biomarkers for the early diagnosis of ad-ild + . following the same line of evidence, both patients with ad-ild + and ipf showed a remarkable decrease in tang frequency when compared to hc. in accordance with our results, it has been previously reported that tang diminish in response to vascular disease in other disorders [23] [24] [25] 29, 30, 36, 37] . furthermore, our data showed that tang frequency in ad-ild − patients, in particular in ra-ild − and ssc-ild − patients, was not different from hc, as disclosed in other rheumatic diseases [9, 23, 26, 27] . consequently, we could speculate that the decrease in circulating tang in ad-ild + and ipf patients occurs because they are migrating to the site of lung injury to repair the endothelium, constituting a marker of lung vasculopathy. in the present study, we also disclosed a relationship of tang with some characteristics of our patients with ad-ild + . notably, we found a lower frequency of tang in men with ra-ild + , which seems to be expected considering that the male sex is a known ra-ild + risk factor [1] . paradoxically, a higher tang frequency was observed in scl-70-positive when compared with scl-70-negative ssc-ild + patients. additionally, a higher tang frequency was associated with a higher fev1/fvc ratio in ssc-ild + patients. since the scl-70 antibody is a risk factor for the development of ild in patients with ssc and a decrease in fvc is used as a routine measure to assess disease progression in fibrotic ild [5] , it is possible that the relative tang increases in these two situations in ssc-ild + patients may be due to a compensatory mechanism in response to vascular damage. it is worth mentioning that patients with other ad-ild + who presented nsip pattern had the lowest tang frequencies. this is in line with the fact that nsip is the predominant pattern in ad-ild + [1, 2, 8, 33, 34, 38] . finally, a relationship of tang with epc or ec was not found in peripheral blood of our ad-ild + patients. these results are in keeping with other studies in which a lack of association was described in patients with ra and diabetes mellitus [24, 37] . it is possible that the cooperation of tang and epc may take place when they are already in the damaged tissues and not at the blood level. additionally, we did not find an association of tang with vegf. this finding may be explained by the fact that vegf secretion is regulated by many different factors in ad-ild + or even at different molecular levels. in conclusion, our findings suggest, for the first time, that tang play a relevant role in the underlying lung vasculopathy and fibrosis, being potential biomarkers of ild in patients with ad. therefore, the assessment of tang could help to establish an earlier diagnosis of ad-ild + . this may favor the use of appropriate therapy in earlier stages of the disease, preventing progression to an irreversible pulmonary process and, ultimately, contributing to improving the survival of patients with ad. the results of this work were partially presented at the american college of rheumatology (acr) 2021 congress (abstract no. 1508) (view abstract and citation information online https://acrabstracts.org/abstract/decrease-of-angiogenic-t-cells-associated-tothe-presence-of-interstitial-lung-disease-in-patients-with-connective-tissue-diseases/ (accessed on 4 april 2022)), european alliance of associations for rheumatology (eular) 2021 congress (abstract no. ab0026) (view abstract and citation information online https://ard.bmj.com/content/80/suppl_1/1046.3 (accessed on 4 april 2022)) and european respiratory society (ers) virtual congress (abstract no. 27636) (view abstract and citation information online: https://erj.ersjournals.com/content/58/suppl_65/pa3620 (accessed on 4 april 2022)). the following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10040851/s1, figure s1 : representative dot-blots of the strategy used to quantify of tang by flow cytometry; table s1 : clinical manifestations of patients with ssc-ild + and ssc-ild − ; table s2 : frequency of tang (%) in all the individuals included in the study; table s3 : detailed information of cellular and molecular endothelial dysfunction-related biomarkers in the whole cohort of patients with ad-ild + . institutional review board statement: all subjects gave their informed consent to be included in the study. the procedures followed were in accordance with the ethical standards of the approved guidelines and regulations, in accordance with the declaration of helsinki. all experimental protocols were approved by the ethics committee of clinical research of cantabria, spain (2016.092). informed consent was obtained from all subjects involved in the study. data availability statement: all data generated or analyzed during this study are included in this published article. interstitial lung disease in systemic autoimmune rheumatic diseases: a comprehensive review pivotal clinical dilemmas in collagen vascular diseases associated with interstitial lung involvement interstitial lung disease in connective tissue disorders management of interstitial lung disease associated with connective tissue disease interstitial lung disease associated with systemic sclerosis (ssc-ild) the role of chest ct in deciphering interstitial lung involvement: systemic sclerosis versus covid-19 high-resolution computed tomography: lights and shadows in improving care for ssc-ild patients idiopathic interstitial pneumonias with connective tissue diseases features: a review angiogenic t cell expansion correlates with severity of peripheral vascular damage in systemic sclerosis endothelial dysfunction in rheumatic autoimmune diseases endothelial dysfunction and inflammation: immunity in rheumatoid arthritis vasculopathy in scleroderma systemic sclerosis in northwestern spain: a 19-year epidemiologic study endothelial progenitor cells: relevant players in the vasculopathy and lung fibrosis associated with the presence of interstitial lung disease in systemic sclerosis patients endothelial progenitor cells as a potential biomarker in interstitial lung disease associated with rheumatoid arthritis early endothelial progenitor cells (eepcs) in systemic sclerosis (ssc)-dynamics of cellular regeneration and mesenchymal transdifferentiation a circulating cell population showing both m1 and m2 monocyte/macrophage surface markers characterizes systemic sclerosis patients with lung involvement identification of a novel role of t cells in postnatal vasculogenesis. characterization of endothelial progenitor cell colonies international immunopharmacology the risk of circulating angiogenic t cells and subsets in patients with systemic sclerosis cd147 participates in the activation function of circulating angiogenic t cells in patients with rheumatoid arthritis increased circulating cd3+/cd31+ t cells in patients with acute coronary syndrome angiogenic t cells in primary sjögren 's syndrome: a double-edged sword? senescent profile of angiogenic t cells from systemic lupus erythematosus patients angiogenic t cells are decreased in rheumatoid arthritis patients the immense study: the interplay between immune and endothelial cells in mediating cardiovascular risk in systemic lupus erythematosus circulating angiogenic t cells and their subpopulations in patients with systemic lupus erythematosus circulating angiogenic t cells are increased in lupus nephritis patients endothelial function and cardiovascular risk editorial: senescent angiogenic t cells: the use of cd28 makes the difference in endothelial homeostasis cd31+ t cells represent a functionally distinct vascular t cell phenotype rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative classification criteria for systemic sclerosis: an american college of rheumatology/european league against rheumatism collaborative initiative statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias diagnostic criteria for idiopathic pulmonary fibrosis: a fleischner society white paper role of vegf polymorphisms in the susceptibility and severity of interstitial lung disease. biomedicines 2021 angiogenic t-cells and putative endothelial progenitor cells in hypertension-related cerebral small vessel disease angiogenic t cells are decreased in people with type 2 diabetes mellitus and recruited by the dipeptidyl peptidase-4 inhibitor linagliptin: a subanalysis from a randomized, placebo-controlled trial (release study) the spectrum of interstitial lung disease associated with autoimmune diseases: data of a 3.6-year prospective study from a referral center of interstitial lung disease and lung transplantation we thank all subjects that participated in this study. the authors declare no competing interests related to this study. key: cord-0048130-8f463wz0 authors: viedma-poyatos, álvaro; pajares, maría a.; pérez-sala, dolores title: type iii intermediate filaments as targets and effectors of electrophiles and oxidants date: 2020-07-17 journal: redox biol doi: 10.1016/j.redox.2020.101582 sha: 65fae7c9ff2a945347bba77c48e2561fca8b9f0b doc_id: 48130 cord_uid: 8f463wz0 intermediate filaments (ifs) play key roles in cell mechanics, signaling and homeostasis. their assembly and dynamics are finely regulated by posttranslational modifications. the type iii ifs, vimentin, desmin, peripherin and glial fibrillary acidic protein (gfap), are targets for diverse modifications by oxidants and electrophiles, for which their conserved cysteine residue emerges as a hot spot. pathophysiological examples of these modifications include lipoxidation in cell senescence and rheumatoid arthritis, disulfide formation in cataracts and nitrosation in endothelial shear stress, although some oxidative modifications can also be detected under basal conditions. we previously proposed that cysteine residues of vimentin and gfap act as sensors for oxidative and electrophilic stress, and as hinges influencing filament assembly. accumulating evidence indicates that the structurally diverse cysteine modifications, either per se or in combination with other posttranslational modifications, elicit specific functional outcomes inducing distinct assemblies or network rearrangements, including filament stabilization, bundling or fragmentation. cysteine-deficient mutants are protected from these alterations but show compromised cellular performance in network assembly and expansion, organelle positioning and aggresome formation, revealing the importance of this residue. therefore, the high susceptibility to modification of the conserved cysteine of type iii ifs and its cornerstone position in filament architecture sustains their role in redox sensing and integration of cellular responses. this has deep pathophysiological implications and supports the potential of this residue as a drug target. intermediate filaments (ifs) are cytoskeletal structures critical for cell mechanics, which maintain a close interplay with the other cytoskeletal systems, namely, actin microfilaments and microtubules [1, 2] . ifs act as integrators of cytoskeletal responses and cell behavior. they are constituted by homo-or heterooligomers of proteins, which are classified into six different families depending on their structure. keratins (types i and ii), vimentin (type iii) and nuclear lamins (type v) are examples of the different protein families and illustrate the critical roles of ifs in epithelial biology, cytoplasmic and nuclear mechanics and stress sensing, and nuclear support and function, respectively [3] . mutations in many if proteins lead to devastating diseases, ranging from epidermolysis bullosa (keratin) [4] to premature aging syndromes (lamins) [5] , which highlight their key function in cell and organism homeostasis. the type iii if protein family comprises vimentin, glial fibrillary acidic protein (gfap), desmin and peripherin, which are important components of the cytoplasmic cytoskeleton and are expressed in specific cell types. vimentin is present mainly in cells of mesenchymal origin and is the most widely expressed type iii if protein [6, 7] , whereas the expression of desmin, gfap and peripherin is mainly restricted to the muscle and glial cells and neurons of the peripheral system, respectively [6] . vimentin is also the most thoroughly studied member and is frequently considered as a prototype for the assembly, regulation and implications of the constituents of this family [7] . therefore, many of the features outlined below derive from research on vimentin. type iii if proteins form a dynamic network that typically extends from the periphery of the nucleus to the cell membrane, interacting with and seemingly modulating the behavior of the actin and tubulin cytoskeletons [7] [8] [9] [10] . vimentin influences many critical cellular processes including cell plasticity and mechanics, deformability, cell cortex properties in interphase and mitosis, migration and adhesion, cell division, organelle positioning, and nucleus and dna integrity under stress [6] [7] [8] [11] [12] [13] . in turn, gfap has been involved in astrocyte https://doi.org/10.1016/j.redox.2020.101582 received 11 february 2020; received in revised form 5 may 2020; accepted 13 may 2020 only cysteine residue. moreover, this residue is conserved in all species, from zebra fish to humans. the conserved cysteine residue is located in the last coil segment of the rod, facing outwards from the vimentin dimer [25] . notably, whereas in the a11 tetramer cysteine residues from different dimers will be far apart, in the a22 tetramer conformation, the cysteine residues would be closer. nevertheless, information on the three-dimensional organization of tetramers in the body of the filament would be necessary in order to ascertain the relative positions of cysteine residues from different tetramers. from the existing models of vimentin [19, 24] , it could be hypothesized that the cysteine residue would occupy a position close to the space needed for the interdigitation of adjacent ulfs during elongation (fig. 1 ). therefore, bulky modifications of this residue could lead to alterations in assembly. in the context of oxidative modifications, other nucleophilic residues prone to oxidation or adduction of electrophiles, including several histidine, lysine and arginine residues, as well as the single tryptophan (w290 in vimentin), are also conserved among members of the type iii if family, as well as between species [26, 27] . in cells, type iii ifs are under constant remodeling through the exchange between the assembled polymers and the pool of soluble subunits, and their dynamics is tightly controlled by posttranslational modifications (ptms), mainly phosphorylation [28] . regulation of vimentin by phosphorylation is critical in mitosis, where the spatiotemporally concerted action of several kinases allows its reorganization, which is important for completion of cytokinesis [29] . in addition, the extent of phosphorylation together with protein-protein interactions influences whether vimentin is disassembled in mitosis or remains in filaments [30] that intertwine with and modulate the actin cortex, allowing proper mitosis progression [8] . type iii ifs can suffer diverse enzymatic ptms, including ubiquitination and sumoylation [31] , glycosylation [32] , proteolysis [33] , and acetylation [34] . one important modification with significant pathophysiological consequences is citrullination [35] . citrullinated vimentin is an autoantigen involved in the pathogenesis of rheumatoid arthritis and in antitumor immunity [36, 37] . in turn, citrullinated gfap has been detected in several diseases, including alzheimer's disease (ad) and multiple sclerosis, and it has been reported to constitute an early response to retinal injury [38, 39] . enzymatic ptms of type iii if proteins have been considered in several important reviews [31, 40] . nevertheless, type iii ifs are also subjected to numerous non-enzymatic modifications, including those induced by oxidants and electrophiles, which are arising as important determinants in filament structure and function and will be considered in more detail below. all cells are exposed to reactive oxygen and/or nitrogen species (ros/rns) and possess defense mechanisms to avoid excessive oxidation. oxidative stress arises when oxidant levels, either from endogenous or exogenous sources, surpass the cellular antioxidant defenses. protein oxidation can occur upon exposure to diverse oxidant species and induce multiple structural alterations. several excellent alzheimer's disease axd alexander's disease 15d-pgj 2 [41, 42] , and of cysteine residues in particular [43, 44] , and provided detailed methodological information for their assessment. in addition, oxidative stress can increase the production of electrophilic species eliciting additional ptms. depending on their type and/or extent, oxidative and electrophileinduced ptms can contribute to cell homeostasis, signaling or stimulation of antioxidant defenses, or result in protein dysfunction, (caption on next page) á. viedma-poyatos, et al. redox biology 36 (2020) 101582 misfolding or aggregation, and to the impairment of protein interactions and functions, in the cell or in the extracellular medium [45] [46] [47] . importantly, these ptms can lead to structure-specific outcomes. thus, depending on the protein, certain modifications can have virtually no consequences on protein function, whereas others can lead to activation or inhibition, or to the modulation of protein-protein interactions or subcellular localization [45, 48, 49] . cysteines are the most nucleophilic residues and readily attack and form adducts with electrophiles of very diverse structure. therefore, the moieties bound to a protein through cysteine residues can be very varied, and range from the addition of one oxygen atom or one oxygen plus one nitrogen atom, in the case of sulfenylation (sometimes called sulfenation) and nitrosation (frequently referred to as nitrosylation) [50] , respectively, to bulky substitutions by endogenous reactive species or drugs (fig. 2) . formation of disulfide bonds can result in covalent binding of the protein to small molecules, like cysteine or glutathione (gsh) (cysteinylation and glutathionylation, respectively), or to other proteins. importantly, cysteine modifications can sometimes interconvert. disulfide exchanges between proteins and small molecules can occur. moreover, the nitroso moiety can be transferred between proteins or lead to no release. interestingly, nitrosoglutathione (gsno) can induce transnitrosation or glutathionylation of target cysteine residues depending on their nucleophilicity. thus, highly nucleophilic thiols break the s-no bond leading to glutathionylation, whereas moderately nucleophilic thiols are more likely to become transnitrosated [51] . in turn, oxidized and electrophile adducted moieties can suffer further transformations leading to derived species and/ or protein crosslinks [41] . an important aspect of cysteine oxidative modifications is stability. while some modifications are readily reversible (e.g. sulfenylation, disulfide formation and nitrosation), others are considered irreversible due to their high stability under biological conditions (e.g. sulfonylation). some reversible modifications of cysteine residues, including glutathionylation or nitrosation have been considered as protective mechanisms preventing the occurrence of "irreversible" modifications, thus preserving protein function [52] . cysteine residues can also undergo a plethora of enzymatic ptms, including some catalyzed oxidative modifications, acylations, prenylation and methylation (for cterminal isoprenylated cysteine residues), etc. [53] [54] [55] . therefore, in certain cases there can be interplay between these ptms leading to structurally and functionally diverse proteoforms [49] . the term lipoxidation refers to the modification of proteins by reactive products of lipid peroxidation [56] . these electrophilic lipids are generated under physiological conditions at moderate levels and they participate in signaling pathways and adaptive and cytoprotective responses that have been recently reviewed [57] . however, under pathological situations commonly associated with oxidative stress, the levels of these agents increase, favoring their reaction with other biomolecules including other lipids, dna and proteins. the structures and concentrations of electrophilic lipids can be very varied (fig. 3) , and can range from small aldehydes, like acrolein or malondialdehyde (mda) (56 and 72 da, respectively), to cyclopentenone prostaglandins (cypg) or isoprostanes (300-400 da), or even oxidized or nitrated phospholipids (700-900 da). indeed, the number of reactive lipid species expands to several hundred [56] . adducts with protein residues occur mainly through schiff base formation with amino groups or , rod and tail domains is shown for the four members of the type iii class of if (upper panel) . dots indicate the position of the conserved cysteine residue (black) and that of the additional cysteine of peripherin (red). amino acid numbering for the various domains corresponds to the following entries from uniprot: vimentin, p08670; gfapα, p14136; desmin, p17661; peripherin, p41219. the lower panel depicts a cartoon view interpretation of the assembly process of type iii ifs, as inferred from numerous in vitro works (see the text for details). please note that in unit length filaments (ulfs), individual tetramers are deliberately not perfectly aligned. the 2-ulf "filament" shown at the bottom illustrates the potentially critical position of the cysteine residue, which could lie close to the region required for the "plugging" of two ulfs. modifications of this residue with bulky moieties may have important consequences for filament assembly or elongation. although the a11 tetramer assembly is considered the starting unit, once assembled in filaments, vimentin dimers can fall in different relative positions, giving rise to several tetrameric configurations, including those outlined in the figure by color shadowing: a11 tetramer (blue), acn tetramer (yellow) and a22 tetramer (green). these tetrameric configurations have been schematically extracted at the right for easier visualization. depictions of if monomer and ulf are modified from ref. [13] . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) fig. 2 . schematic representation of several modifications of vimentin c328. the figure shows a cartoon of a stretch of the alpha helical structure of a segment of vimentin and the c328 lateral chain prepared using pymol and data from the pdb entry 3klt [202] . structures of nitrosocysteine, acrolein and 15d-pgj 2 have been obtained from pubmed compound. this schematic representation illustrates the diverse size of the moieties that can bind to c328, potentially imposing steric hindrances or altering interactions required for filament assembly. through michael addition with various residues bearing nucleophilic moieties, like the thiol group of cysteine, imidazole of histidine and amino of lysine residues [48] . the stability of adducts formed will be influenced by several factors including the nature of the adducted residue and the occurrence of additional intramolecular reactions [58] . moreover, both, non-enzymatic and enzymatic reversal of certain electrophilic lipid-protein adducts has been reported in vitro as well as in cellular models [59] [60] [61] . therefore, lipoxidation can constitute a dynamic modification contributing to signaling mechanisms [60, 61] . given the structural variety of adducts their functional consequences (structure-activity relationships) can be diverse. there are several examples of differential effects of lipoxidation of cysteine residues, depending on the adducted moiety. adduction of distinct electrophilic lipids to ras proteins can differentially influence activation and subcellular localization [49, 62] . similarly, the aldo-keto reductase enzyme akr1b1 is activated by addition of small moieties but inhibited by larger ones [63] . cypg are electrophilic eicosanoids derived from prostaglandins by non-enzymatic dehydration [64] . they are characterized by the presence of an α,β-unsaturated carbonyl group in the cyclopentane ring that allows michael adduct formation, preferentially with cysteine residues. one example of these eicosanoids is 15-deoxy-δ 12,14 [13] ; network fragmentation alone or accompanied by solubilization, characterized by the diffuse background, correspond to treatment with diamide or diamide plus calyculin a, respectively, as in ref. [91] ; filament bundling is exemplified by treatment with 15d-pgj 2 as in ref. [91] , and peripheral condensation, by treatment with no 2 -popc as in ref. [115] . scale bar, 20 μm. please note that the effects observed in cells can be the consequence of direct and/or indirect modifications of if proteins, as well as of ptm interplay. prostaglandin j 2 (15d-pgj 2 ) (fig. 2) , which has been broadly studied both in cell culture systems and in vivo. the effects of cypg, as those of many lipoxidation agents and oxidants, can have a dual or hormetic nature, depending on time or concentration. low concentrations can activate the cellular antioxidant defenses and potentiate the initial inflammatory response, whereas high levels can elicit anti-inflammatory and antiproliferative effects and induce cytotoxicity [65, 66] . intracellular gsh is an important determinant of cypg distribution and effects through various mechanisms, including the formation of cypg-gsh adducts that can be involved in cypg transport or sequestering, leading to a differential modification of proteins depending on the subcellular compartment [49] . numerous studies on protein oxidative damage use the term carbonylation to refer to the formation of carbonyl groups on proteins. chemically, carbonylation refers to the addition of carbon monoxide to a compound leading to the appearance of a carbonyl group. appearance of carbonyl groups on proteins can occur by several mechanisms, including oxidation of amino acid lateral chains, oxidative deamination or formation of certain adducts with compounds that retain a free carbonyl group, as in some types of lipoxidation, like michael addition of cypg or of hne, or after glycation or glycoxidation. as none of these processes involves carbon monoxide addition the term "formation of protein carbonyls", should be preferred instead of protein carbonylation, which, as stated above is not selective for a particular type of modification, but can arise by multiple mechanisms, as detailed in ref. [67] . early functional studies in cells lacking vimentin showed altered nuclear morphology and impaired distribution of certain organelles [68] . however, the finding that vimentin knockout mice were fertile and apparently developed normally [69] dimmed the interest in this protein. nevertheless, subsequent works have established the role of vimentin as a key player in cellular organization and as a modulator and effector of numerous biological and pathophysiological processes. indeed, vimentin knockout animals do not respond normally to many kinds of stress and show alterations in immune function [70] , autophagy, cell migration and division, wound healing, function of the central nervous system, and arterial remodeling [71] , among others. in addition, vimentin is an important marker and player of epithelial mesenchymal transition and its expression in certain types of cancer can be considered a sign of malignancy and invasiveness [16] . it is also necessary for cellular entry and/or toxic cellular actions of diverse bacterial and viral pathogens [72] [73] [74] . in contrast to other if proteins, only a few mutations in the vimentin sequence have been identified. the e151k mutation [75] , the heterozygous frameshift from v6c [76] and the missense (q208r) [77] mutation have been associated with a hypothetical model to conciliate the differential effects of certain oxidants on the assembly of soluble if proteins and preformed filaments is shown. a very simplified representation of if dimers (rectangles) and if tetramers is depicted, showing the approximate positions of the cysteine residues (black dots). on the left, disulfide formation (red ellipses) or modification of cysteine residues by bulky moieties (yellow shapes) in soluble if tetramers may interfere with their subsequent assembly in ulfs or with ulf elongation upon induction of polymerization, or may impede conformational rearrangements needed for proper assembly. conversely, on the right, preformed filaments could undergo a certain degree of modification on accessible residues, or of disulfide formation between close cysteine residues, without suffering substantial disruption of their assembly. nevertheless, modification above a certain threshold could lead to filament disruption. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) cataract development. recently, a dominant vimentin variant (l387p) causing a rare syndrome with premature aging has been reported [78] . vimentin is the target of numerous enzymatic and non-enzymatic ptms, some of which have been outlined above. accumulating evidence indicates that vimentin behaves as a sensor for several kinds of stress, including oxidative stress and generation of lipid-or sugar-derived electrophiles that target several residues leading to versatile network remodeling [56, 79] (fig. 3) . the single cysteine residue of vimentin (c328) appears to be highly susceptible to oxidation or addition of diverse species, both in vitro and in cells [25, [80] [81] [82] [83] [84] , suggesting that it is a preferred site of modification. moreover, modifications of c328 play a key role in redox-related reorganizations of vimentin filaments, with diverse consequences on cell behavior. disulfide formation and other cysteine oxidations. early studies using cytoskeleton preparations from several cell types subjected to oxidative in vitro crosslinking with cu 2+ -phenanthroline showed the formation of homo-and heterodimers of either vimentin and desmin or vimentin and gfap, which led to propose that both proteins were present in hybrid filaments [85, 86] . these studies indicated that cysteine residues in the "crosslinkable" units were exposed to the protein surface at a distance sufficiently close to allow interaction. oxidized oligomeric species of vimentin were also detected in fibers isolated from eye lenses of several species, when extracted in the absence of beta-mercaptoethanol [87] . later studies putatively identified vimentin as one of the proteins undergoing disulfide bonding in fibroblast-like synoviocytes subjected to oxidative stress by treatment with h 2 o 2 [88] , and soon after, traub et al., showed that oxidized vimentin and desmin species, including homo-and heterooligomers of both proteins, formed abnormal filaments, which could be restored by reducing agents [89] . the involvement of the cysteine residue in disulfide formation and its impact on assembly was confirmed by using a c328a vimentin mutant that showed resistance to crosslinking by oxidation with h 2 o 2 and cucl 2 and formed apparently normal filaments in vitro [25] . it was then postulated that crosslinking of vimentin wild type could occur between neighboring dimers arranged in the a22 conformation, in which the cysteine residues fall in a closer position (fig. 1 ). however, it was noted that this arrangement was still not ideal for disulfide bond formation unless additional flexibility or molecular motion within the tetramer occurred to allow a closer contact between the cysteine lateral chains [25] . dimeric species of vimentin have also been observed in sds-page gels upon treatment with bifunctional cysteine crosslinkers, like dibromobimane (spacer arm 4.66 å), both in vitro and in cells [13] , indicating that in intact cells cysteine residues within filaments can also be present at a close distance. moreover, the use of this reagent allowed obtaining vimentin-gfap heterodimers from intact astrocytoma cells [90] , thus suggesting the presence of both proteins in the same filaments as proposed in earlier in vitro works [85, 86] . recently, the electrophilic reagent diamide has been used to induce vimentin disulfide bonding [91] . this reagent reacts sequentially with two thiol groups facilitating disulfide bond formation. diamide-treated purified vimentin appears as monomer and dimer species in non-reducing sds-page gels in approximately a 1:1 proportion. notably, disulfide formation profoundly impaired subsequent polymerization into filaments of vimentin wild type, but not of a cysteine-deficient mutant. however, preformed filaments underwent disulfide formation to a similar extent than the soluble protein but were not disrupted by short diamide treatments. therefore, these results suggest that promoting disulfide bond formation in soluble tetramers may lead to their association in conformations that are not productive for subsequent assembly, whereas once filaments are formed and the subunits are in place, a certain number of disulfide bonds can form between cysteines that fall within close distance without disrupting the whole filament (fig. 4) . remarkably, although resistant to oxidation by diamide, filaments formed in vitro by a c328s mutant showed subtle, but significant, differences with vimentin wild type, calling attention on the importance of this residue for filament assembly [91, 92] . interestingly, diamide induces a major disruption of vimentin filaments in cells in such a way that filaments rapidly reorganize into discrete dots [13, 91] . in cells, both soluble and polymerized vimentin coexist and exchange. therefore, oxidation of soluble vimentin could hamper its incorporation into filaments leading to network disruption. moreover occurrence of additional ptms as a consequence of oxidative stress, could also contribute to diamide-induced filament fragmentation. nevertheless, the fact that the c328s vimentin mutant is completely resistant to this effect, confirms the critical role of the modification of the cysteine residue in vimentin reorganization [91] . formation of mixed disulfides can also occur between vimentin and small thiol-containing molecules, like gsh (glutathionylation) or cysteine (cysteinylation). vimentin glutathionylation has been observed in several experimental models. stimulation of isolated aortic rings with acetylcholine led to vimentin glutathionylation through an enos-dependent mechanism [93] . notably, in this model system, glutathionylation can occur directly or after denitrosation. as enos possesses basal activity in endothelial cells, these observations suggest that glutathionylation could occur under basal conditions and constitute a physiological regulatory mechanism. indeed, vimentin glutathionylation has been detected in control mouse embryonic fibroblasts [94] . nevertheless, this modification increases under conditions of oxidative stress. in particular, loss of function of the krit1 gene, which is associated with the pathogenesis of cerebral cavernous malformations, induces a decrease in the gsh/gssg ratio and increased glutathionylation of several proteins, including vimentin, in cellular models of the disease [94] . glutathionylation of vimentin has also been observed in oxidatively stressed t-lymphocytes or cos7 cells exposed to no donors [93, 95] , and in human senescent fibroblasts, where it has been connected with cell growth arrest and cell death [96] . moreover, data mining of the oxidized thiol proteome, has found a high proportion of vimentin c328 involved in disulfide bonding in aging and cataractous lenses [82] . from the functional perspective, the vimentin cysteine residue has been compared to cryptic cysteines in other proteins, in which glutathionylation could contribute to protein elasticity [97] . in addition, recent studies indicate that quantitative vimentin glutathionylation impairs vimentin elongation and provokes pre-formed filament severing [98] , supporting the view that modification of c328 with bulky moieties could have important consequences for filament assembly. nevertheless, the functional implications of vimentin glutathionylation in cells have not been fully elucidated and could depend on the proportion of the protein modified, and/or represent a protective mechanism against irreversible modifications. the oxidation of vimentin in the extracellular space deserves special attention. vimentin can be exposed at the cell surface or secreted by several cell types [99, 100] . cell surface vimentin can be labeled by cellimpermeable cysteine reagents. the observation that labeling can be increased by pretreatment with the reducing agent n-acetyl-cysteine suggests that part of the protein exposed is reversibly oxidized [101] . interestingly, oxidative stress increases extracellular exposure of vimentin, frequently in an oxidized form [100] , and it has been proposed that this phenomenon could constitute an early response of cells to oxidants from the extracellular space, and at the same time, generate oxidized and secreted species of vimentin that could be involved in pathogenesis of autoimmune diseases or act as a vehicle amplifying oxidative stress and lipid oxidation [74, 102] . nitrosation. the above observations show that vimentin cysteine can be modified under (patho)physiological conditions. shear flow is a physiological stimulus that activates enos in endothelial cells leading to increased no levels that can induce protein s-nitrosation. indeed, vimentin was identified among the s-nitrosated proteins detected in endothelial cells after treatment with an exogenous no donor or upon stimulation of endogenous no production [103] . moreover, c328 has been identified as the nitrosated residue in endothelial cells after shear stress, depending on enos activity [104] , and after no-donor exposure [105] . vimentin nitrosation can also occur under basal conditions and increase as a consequence of drug treatment. statins, drugs widely used to control cholesterol levels, modulate enos expression in endothelial cells [106] , and lead to increased enos activity and nitrosation of endothelial proteins, including vimentin [107] . nitrosation has been proposed to occur in hydrophobic environments, and indeed, hydrophobicity plots predict c328 to be located in a hydrophobic motif, qsltcevda [104] . interestingly, the sequence -[i/l]-x-c-x 2 -[d/e]-, has been reported to constitute a consensus motif for inos-s100 catalyzed transnitrosation [108] , that can take place for instance in activated peripheral blood monocytes. vimentin c328 is located in one such motif and has been validated as a target for nitrosation through this mechanism. the functional significance of vimentin nitrosation is not fully understood, although in endothelial cells it has been proposed to contribute to endothelial remodeling under flow. a recent study indicates that s-nitrosation of vimentin at c328 does not hinder, but slows down, vimentin elongation in vitro. these results shed light on the different functional outcomes of structurally different c328 modifications [98] . in addition, being a readily reversible modification, snitrosation could also act as a defense mechanism protecting proteins from deleterious oxidative modifications. lipoxidation. the stability of some adducts formed between vimentin and electrophilic lipids has allowed obtaining structural and functional information on the role of the lipoxidation of vimentin cysteine, both in vitro and in cells. modification of vimentin by cypg in vitro occurs selectively at c328 [80, 91] , and disrupts subsequent naclelicited polymerization, inducing filament widening and bundling and formation of aggregates, in wild type but not c328s vimentin. notably, these differential effects occurred in conditions under which the extent of vimentin lipoxidation was estimated to be approximately 10% [80] , which is in the range of that estimated after treatment of cells with biotinylated cypg analogs [109] . nevertheless, at high 15d-pgj 2 concentrations this selectivity is lost, since these compounds can form adducts with other nucleophilic residues, namely, histidine. interestingly, as observed with oxidants, preformed filaments appear to be protected from disruption in vitro, even when modified to the same extent, potentially due to a different topology of the modification (fig. 4) . the amenability of cypg to labeling with biotin or other moieties has allowed their use as probes for lipoxidation. using biotinylated 15d-pgj 2 or pga 1 , vimentin was first identified as one of the major targets for these eicosanoids in mesangial cells, in which they induce a marked cytoskeletal rearrangement leading to perinuclear condensation of vimentin filaments into thick bundles [109] . besides filament condensation, cypg provoke the dislodgement of vimentin from the actin cell cortex in mitosis, which could have deleterious consequences for cell division [8] . the use of a vimentin c238s mutant has allowed identifying the cysteine residue as the key site for adduct formation and/or for the induction of vimentin filament disruption by cypg in several cell types [13, 80] . as other electrophilic lipids, cypg covalently bind to multiple cellular targets and induce oxidative stress on their own, thus the possibility exists that some of their effects on the vimentin network may be indirect. indeed, in addition to lipoxidation, 15d-pgj 2 has been shown to induce reversible cysteine oxidation (cysteinylation) of certain proteins [110] . nevertheless, the fact that the effects of cypg are attenuated in the c328s mutant, both in vitro and in cells, indicate that this residue is a critical site for the action of these eicosanoids. hne is one of the most studied electrophilic lipids. the levels of hne, and of hne-protein adducts, increase under many pathophysiological conditions, including inflammatory, neurodegenerative diseases and aging [56, 111] , in some of which hne can reach micromolar concentrations [112] . lipoxidation of vimentin by hne has been detected in human monocytic thp1 cells treated with this lipid by means of immunological and derivatization methods, and c328 was identified as the modification site using lc-ms/ms [81] . hne-vimentin adducts have also been identified in senescent fibroblasts, mainly in soluble vimentin and vimentin fragments [113] . notably, hne can form several types of adducts with proteins, both through michael addition and schiff base formation [114] . in addition, hne adducts can give rise to protein crosslinks. consistent with this broad reactivity, hne can modify both vimentin wild type and c328s in vitro [91] . however, the functional consequences of the modification are different; whereas hne-pretreated vimentin wild type formed thicker and shorter filaments with a tendency to laterally associate, c328s vimentin was protected from these effects [91] . these results suggest that even though hne can bind to other residues, the modification of c328 is functionally the most important. this is also supported by observations in cells showing that hne treatment induces a juxtanuclear accumulation of vimentin wild type in thick bundles or aggregates, whereas in cells expressing vimentin c328s, the network is preserved and filament retraction is attenuated [13, 56] . among other lipoxidation agents, vimentin can be modified by mda. interestingly, immunization of mice with senescent cells led to the isolation of a polyreactive antibody that recognized vimentin at the surface of senescent primary human fibroblasts. in addition, an increase in vimentin modified by mda at c328 was observed at the cell surface of senescent cells and in plasma of aged senescence-accelerated mice [83] . based on these findings it was proposed that mda-modified vimentin could serve as a senescence marker and potentially be recognized by the innate immune system to eliminate senescent cells. other electrophilic lipids potentially interacting with vimentin include nitrated phospholipids (fig. 3) , which alter the cellular vimentin network in a manner dependent on the presence of c328, and shield this residue from alkylation in vitro [115] . importantly, although the c328s mutant is protected from oxidative insults in cells, it shows a lower efficiency than the wild type at forming extended networks, supporting aggresome formation and promoting organelle position [13] . therefore, even conservative substitutions of the cysteine residue have a measurable impact on vimentin function. in spite of the importance of c328 in vimentin lipoxidation, modification of k235, but not of c328, by nonealdehyde has been detected in hepatic tissue from patients with end stage alcoholic liver disease or non-alcoholic steatohepatitis [116] . this modification eliminates the positive charge of k235, disrupting its electrostatic interaction with e230 [117] . this brings about the potential of lipoxidation to alter protein-protein interactions, with consequences for cell activation and/ or cytotoxicity. indeed, lipoxidized proteins are ligands for the receptor for advanced glycated end products (rage), which has been involved in the pathogenic consequences of hyperglycemia or inflammation. by abolishing the positive charges of lysine residues, lipoxidation disrupts the ion pairs with negative residues in their vicinity, enabling the latter to interact with positive residues in rage, favoring the activation of this receptor [118] . through this mechanism, lipoxidized proteins could contribute to propagate the damage. protein carbonyl formation (pcf). formation of protein carbonyls on vimentin has been reported in numerous studies using derivatization approaches, although, in many instances, the sites of modification have not been identified. among evidence from cellular models, pcf on vimentin has been detected in rat aortic smooth muscle cells treated with homocysteine or bone morphogenic protein-4 (bmp-4) [119, 120] , huvec treated with sodium arsenite [121] , and lung epithelial cancer cells exposed to acrolein or smoke extracts [122, 123] , these latter cells showing also vimentin crosslinked products [122] . vimentin bearing protein carbonyls has been identified in non-tumor lung specimens of patients with lung cancer with or without chronic obstructive pulmonary disease [124] . increased vimentin pcf has also been detected in cultured fibroblasts from ad patients with respect to those of control subjects, after exposure to aβ (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) . these observations suggest a dysfunction of antioxidant responses in ad [125] , and raise the importance of vimentin as a target for oxidation in cns diseases [126] , together with gfap, as it will be detailed below. pcf can occur also as a consequence of glycation. in primary cultured human dermal fibroblasts vimentin was glycated predominantly at lysine residues located in linker regions likely exposed to the cytosol. this caused vimentin redistribution into perinuclear aggregates, which was accompanied by loss of fibroblast contractile capacity. glycated aggregated vimentin was also detected in facial skin biopsies. therefore, the accumulation of life-long vimentin glycation was proposed to contribute to loss of skin contractile properties during aging [79] . other modifications. vimentin tyrosine nitration has been detected by immunoprecipitation with an anti-3-nitrotyrosine antibody in mitochondrial diseases [127] . a similar approach was used to identify tyrosine nitrated vimentin in lung cancer [128] . however, in these studies, identification of the nitration sites was not achieved. tryptamine-4,5-dione is a product of the oxidation of serotonin that is highly reactive with thiols. vimentin has been identified as a target for this putative neurotoxin in sh-sy5y neuroblastoma cells, suggesting the modification of the thiol group, although this was not confirmed [129] . in summary, a plethora of vimentin modifications has been identified to date in many pathophysiological situations. oxidative and nonoxidative modifications can coexist in many instances allowing their interplay, but an understanding of their structural and functional consequences requires further investigation. gfap is the main if expressed in mature astrocytes, which also express vimentin [130, 131] . gfap can be expressed in other cell types outside the nervous system, including fibroblasts and hepatic stellate cells [80, 132] . a particular feature of gfap among type iii if proteins is the existence of multiple isoforms that arise by alternative splicing and differ mainly in the tail domain, some of which are not able to form filaments on their own [14] . here, we will focus on the most abundant isoform that is gfapα (fig. 1) . this protein plays complex functions in astrocyte homeostasis which, to date, have not been totally unraveled. studies in knockout animals have yielded diverse results pointing to the complexity of gfap functions and their dependence on the experimental system. roles in astrocyte migration, organelle distribution and mitosis have been defined. the presence of gfap also influences neuronal functions, axonal and neuronal regeneration and several aspects of neurotransmission [14] . gfap plays an important role in the response to damage, including inflammation, physical or ischemic trauma. in these processes, astrocytes become activated, overexpress gfap and resume the expression of other ifs, like nestin and synemin [130] . gfap appears to play a dual role in trauma, favoring the formation of a glial scar that limits the damage at the early stages, but slowing down regeneration at later stages [133] . the importance of gfap in brain homeostasis is highlighted by the devastating consequences of its mutations, which lead to alexander disease (axd), a rare neurodegenerative disease that causes leukodystrophy, epilepsy, loss of brain white matter and ultimately death [17] . in axd, gfap forms aggregates known as rosenthal fibers that accumulate other proteins as well, and show oxidative damage [134] . interestingly, axd associated mutations often involve substitutions of the wild type residues by cysteines [135] , which raises the possibility that they undergo additional oxidative modifications. gfap is also a hub for ptms and it has been reported to undergo phosphorylation at multiple residues. phosphorylation plays an important role in the regulation of filament assembly and in the progression of mitosis and cytokinesis [136, 137] . moreover, increased phosphorylation of certain residues, in particular s13, has been associated with pathological conditions including frontotemporal lobar degeneration, axd and hypoxia [138] [139] [140] . other ptms have also been associated with disease, including glycosylation and citrullination, the latter, rendering gfap an autoantigen for autoimmune disease or appearing in conditions such as ad or ischemia reperfusion injury [38, 39] . moreover, gfap degradation products have been observed in neurodegenerative diseases [141] as well as in models of viral infection [33] . gfap can be targeted by numerous oxidative modifications and appears frequently in proteomic studies of pathophysiological conditions. nevertheless, most studies focus on the variations of its levels as a marker of nervous system injury and less attention has been paid to its role as a target of damage. as specified above, disulfide formation between gfap monomers, likely belonging to different tetramers, has been observed in vitro upon oxidative crosslinking [86] . moreover, bifunctional cysteine reagents also lead to homo-and heterodimers of gfap and vimentin in vitro and in cells [90] . these studies have substantiated the complex nature of these filaments in astrocytes and in astrocyte models. moreover, results from cellular models have shown the requirement for the presence of the cysteine residue in gfap for proper filament assembly, which highlights the functional importance of this residue. interestingly, in primary astrocytes from mice deficient in both gfap and vimentin, the requirement for the cysteine residue was more marked for gfap than for vimentin, and this latter protein seemed to rescue the defect in c294s gfap assembly to some extent [90] . gfap has been found both in reduced and in reversibly oxidized forms in brain tissue from a subject with mild ad, by employing a differential thiol labeling protocol followed by 2d electrophoresis and ms identification [142] . finally, the appearance of high molecular weight oligomers containing gfap has been noted in several models of disease, including axd [143] , although the nature of these oligomers has not been characterized. gfap appears s-nitrosated in several conditions. a marked increase in gfap nitrosation has been observed in synaptosomes of transgenic mice overexpressing mutated human amyloid precursor protein compared to wild type, suggesting its involvement in pathology [144] . however, s-nitrosation of gfap has also been detected under control conditions and found not to increase in experimental models of murine autoimmune encephalomyelitis [145] . therefore, the role of this modification in pathogenesis or as a defense mechanism requires further investigation. lipoxidation. evidence on gfap lipoxidation is gathering in recent years. pick's disease is a type of fronto-temporal dementia characterized by severe atrophy of frontal and temporal lobes, neuronal loss and astrogliosis [146] . gfap has been shown to be a major target of lipoxidation by hne and glycoxidative damage in brain samples from pick's disease patients compared to control subjects [146] . down's syndrome patients develop symptoms of premature aging, including neuropathological features of ad. therefore, observations in this syndrome have been considered clues for the understanding of ad. a 3.3-fold increase in hne-modified gfap has been observed in brain tissue from down's syndrome patients compared to controls by means of immunological detection with an anti-hne antibody [147] . hne also targets elements of the protein degradation machinery and quality control, the impairment of which could contribute to accumulation of damaged or misfolded proteins. gfap has also been recognized as a major target for hne-protein adduct formation in frontotemporal lobar degeneration [148] , although the site(s) of modification have not been identified. notably, an 8-fold increase in mda-modified gfap has also been reported in brain samples from ad patients compared to agedmatched controls [149] , whereas non-significant differences were found for vimentin modification in this study. nevertheless, it should be taken into account that many of these reports use whole tissue samples for the proteomic studies. although the evidence for lipoxidation is solid, novel approaches able to assess oxidative damage in specific regions and/or cell types or subpopulations in the brain may shed valuable information on pathogenic mechanisms [150] . using cellular models such as fibroblasts and astrocytes, we have recently identified gfap as a target for modification by cypg [80, 90] . in astrocytoma cells, cypg produced an intense disruption of the gfap network with retraction of filaments from the cell periphery and formation of curly bundles or aggregates in the proximity of the nucleus, whereas in mouse primary astrocytes the most obvious effect was filament retraction and perinuclear condensation [90] . biotinylated analogs of cypg bind to gfap in a manner dependent on the presence of its single cysteine, c294. moreover, a cysteine deficient gfap mutant, although showing altered filament assembly per se, is protected against network disruption induced by these electrophilic lipids. similarly, treatment of cells in the presence of dtt attenuated network disruption, pointing to the importance of thiol modification in cypg effect [90] . protein carbonyl formation. most of the evidence on protein carbonyl formation on gfap has been obtained through the use of derivatization techniques combined with immunological detection after immunoprecipitation or 2d analysis, or by using adduct-specific antibodies (reviewed in ref. [151] ). these approaches have demonstrated pcf in ad, tautopathies including progressive supranuclear palsy and pick's disease, and in huntington disease [146, [151] [152] [153] ]. an 8-fold increase in gfap protein carbonyl levels has been found in samples from the frontal cortex of down's syndrome patients compared to controls [154] . moreover gfap has been identified as the major carbonyl containing protein in the cerebral cortex of a patient with aceruloplasminemia, the hallmark of which is intracellular iron overload [155] . from all this evidence, it seems clear that gfap constitutes a highly susceptible target for oxidative modifications in various neurodegenerative diseases. nevertheless, whether the modifications lead to significant network rearrangements and whether they represent a toxic or a defense mechanism requires further study. nitration. nitrated gfap has been identified in normal rat cortex [156, 157] , and marked increases in nitration have been observed in a model of hypobaric hypoxia and reoxygenation [157] . in this model, intense nitration of other cytoskeletal components, including tubulin and γ-actin has been observed, for which a cooperation of these proteins in cytoskeletal disruption has been proposed. although in this review we have not considered the modifications of gfap isoforms, the study of their specific modifications could yield valuable pathogenic information. indeed, in proteomic studies, up to 46 forms of soluble gfap could be separated by 2d immunoblotting from control and ad brain samples, which differed in size, phosphorylation, glycosylation and oxidation state [158] . desmin is the main if protein of mature striated muscle, where it is located in the sarcolemma, z-lines, neuromuscular and myotendinous junctions. this protein provides strength to the muscle fiber during contraction and facilitates mechanochemical signaling [15, 159] . desmin and its protein binding targets form a scaffold that links the contractile system to the nucleus, mitochondria and other organelles through the z-discs, allowing their crosstalk. this interconnecting role also enables transport between the extracellular and nuclear matrix [15, 159] . more than 70 mutations in the desmin gene (des) have been identified, which lead to different types of myopathy (e.g. myotilinopathies and desminopathies), including cardiomyopathy or isolated myopathies. mutations have been identified in all the protein domains. skeletal and cardiac muscle phenotypes involve mainly mutations affecting rod 2b, abnormal phosphorylation levels, and some oxidative modifications that correlate with desmin related myopathies (drms) and cardiac disease [15, 159] . desmin is a target for a variety of ptms including phosphorylation, adp-ribosylation, ubiquitination, glycation, oxidation and nitration. most of them cause disassembly of the desmin network [160] . also in this case, phosphorylation is by far the most studied ptm. although alterations in desmin levels or distribution have been frequently encountered in pathophysiological situations associated with oxidative stress or in cellular models of electrophilic stress [161] , the precise characterization of oxidative modifications and their impact on the function of the protein is not always available. indeed, muscle activity induces thermal, mechanical and redox stress, which in cellular models of desminopathies elicit aggregation of desmin mutants [162] that can be attenuated by treatment with antioxidants, like, n-acetyl-cysteine or tocopherol [163] . as described above, oxidative crosslinking-induced dimers of desmin or of desmin and vimentin have been generated in vitro and showed impaired assembly [85] . moreover, purified desmin subjected to in vitro oxidation with h 2 o 2 and fe 2+ increases its content of random coil and β-sheet, in turn, decreasing its α-helix content according to far-uv cd spectra [164] . also, oxidation can alter its susceptibility to proteolytic cleavage [164] . in myocytes, several redox modifications of desmin have been reported including disulfide linked and sulfenylated forms of the protein, as has been identified in studies using diamide, dimedone analogs or peroxide [165, 166] . desmin oxidation has also been confirmed in muscle biopsies from patients with desminopathies or myotilinopathies [159] . desmin is also a target for lipoxidation. functional derangement by hne treatment has been reported [161] , and acrolein-modified desmin has been identified in myocytes exposed to this toxic agent [167] . among other modifications, desmin has also been identified as a target for nitration [127, 159] , and increased formation of protein carbonyls in skeletal muscle has been detected in sepsis, ischemia-reperfusion, diabetes and chronic obstructive pulmonary disease (reviewed in ref. [168] ). however, the functional consequences of these modifications are not yet clear. peripherin expression is mainly restricted to the peripheral nervous system, but also occurs in neurons of the cns. there are several forms of peripherin generated by alternative splicing, some of which increase in response to traumatic neuronal injury [169] . the role of this protein is unclear, but it seems to contribute to if cytoskeleton organization and axonal elongation. the protein is a major autoantigen in type 1 diabetes [170] and appears in compact inclusions in several diseases, including amyotrophic lateral sclerosis (als). screening for sequence variants in the peripherin gene (prph) has been performed in patients with als and several snps have been identified [171] . these include an insertion in intron 8 (ivs8-36insa), a 1 bp deletion in exon 1 (228delc) leading to a truncated form of the protein, and a homozygous mutation in the first linker generating the d141y mutation. peripherin overexpression induces motor neuron degeneration in transgenic mice, whereas the knockout mice seem to compensate lack of prph expression by inducing that of vimentin and α-internexin, apparently leading to decreased myelination of only a subset of sensory fibers [172] . peripherin is by far the less studied of the type iii if class, and is the only one that possesses two cysteine residues. nevertheless, several oxidative modifications have been reported. a disulfide-linked peripherin dimer of 130 kda has been detected in rat sciatic nerve and dorsal root ganglia [173] . non-differentiated n2a neuroblastoma cells treated with hydrogen peroxide showed concentration-dependent changes in the levels of peripherin, together with alterations in the proportion of the peripherin forms, per-58 and per-45. moreover, peroxide (100-500 μm) induced loss of filament structure and inclusions in nondifferentiated cells, whereas retraction of neurites was detected at 10 μm. however, peripherin filaments and aggregates did not colocalize with carbonyl markers, despite increased total protein carbonyl formation [174] . the best characterized oxidative modification of peripherin is nitration, which has been described upon ngf-induced differentiation in pc12 cells, and occurs at the head (y17) and tail (y376) domains [175] . apparently, the ratio between nitration levels and peripherin expression before and after ngf addition increases during differentiation. remarkably, nitrated peripherin is only present in the insoluble cytoskeletal fraction. heavy tyrosine nitration and phosphorylation of peripherin have been observed in als, in association with disruption of filament association and appearance of inclusions or aggregates [169] . in any given situation, proteins are subjected to multiple ptms that can occur on the same or on different protein molecules in different combinations, giving rise to structurally and functionally different proteoforms. type iii ifs and, in particular, their conserved cysteine residues, are emerging as hot spots for modification by oxidants and electrophiles. indeed, the studies summarized above highlight the susceptibility of these cysteine residues to a plethora of modifications including nitrosation, disulfide formation, sulfenylation and addition of various electrophilic lipids including hne, mda and cypg. interestingly, a complex interplay between modifications can take place both by direct and indirect mechanisms (fig. 5) . reversible cysteine modifications, for instance, glutathionylation, could prevent irreversible oxidations or adduct formation; however, some oxidative modifications can increase or decrease the reactivity of the affected residues [176] . phosphorylation is probably the most studied modification regulating protein function. many oxidant species act on signaling proteins, for instance, kinases and phosphatases, triggering additional modifications of ifs. vimentin is a target for phosphorylation by numerous kinases, including pka and akt, which are directly and/or indirectly regulated by redox mechanisms [177] . the impact of oxidative modifications on kinase activity can lead to activation or inhibition. in turn, tyrosine phosphatases are generally inhibited by oxidative stress, whereas oxidation of serine/threonine protein phosphatases can result in activation or inhibition. interplay of nitric oxide with vimentin phosphorylation has been reported through modulation of pp2a activity [178] . phosphatase inhibition would result in vimentin hyperphosphorylation, which could contribute to network disruption by oxidative modifications. in this context, we have recently observed that the phosphatase inhibitor calyculin a, an inhibitor of pp2a and pp1 phosphatases, potentiated the fragmentation of vimentin network induced by diamide, whereas a phosphorylation-deficient vimentin mutant "sa mutant", in which 11 phoshorylatable residues of the n-terminal domain had been substituted by alanines, was partially protected from the diamide-disruptive effect [91] . a complex interplay can also occur with the protein degradation machinery. certain oxidative insults or other types of stress can lead to the degradation of vimentin and gfap by activating proteases [179] , although some ubiquitin ligases and subunits of the proteasome can be inhibited by lipoxidation [180] , which, as stated above, would result in under oxidative stress, several oxidants are generated that can react with unsaturated lipids in membranes giving rise to the formation of electrophilic lipid species. these can directly form adducts with proteins in a process called lipoxidation. oxidative stress also diminishes the ratio gsh/gssg and favors the formation of disulfide bonds between glutathione and thiol groups in proteins (glutathionylation). this modification can also be mediated by nitrosoglutathione, which can also induce s-nitrosation. oxidative modifications of cysteine residues can also lead to the formation of sulfenic, sulfinic and sulfonic acids. other oxidative modifications include protein carbonyl formation, nitration or formation of protein crosslinks (see ref. [41] for review). other ptms that are under redox control, such as phosphorylation and protein degradation will be modulated in situations of oxidative stress, thus contributing to the diversity and complexity of proteoforms arising under these conditions. the function of type iii ifs will be also affected by oxidative modifications of interacting proteins, such as those described for plectin, actin, and several chaperones. importantly, interplay can also take place with other ptms, including those listed in the lower panel, which can occur on the same or nearby residues, affecting their availability or accessibility to modification. accumulation of damaged proteins. interestingly, due to their polyelectrolyte properties, ifs interact with cations such as ca 2+ , mg 2+ or zn 2+ which, depending on their size and concentration, influence if assembly [13, 92, 181] . importantly, zn 2+ availability can influence the occurrence of certain ptms on vimentin [13] , which could occur through direct interaction or through its capacity to elicit antioxidant or pro-oxidant effects [182, 183] . although zn 2+ is a redox-inert ion, it can influence cellular redox status because it is necessary for the activity of enzymes involved in the antioxidant defense, whereas its deficiency or excess cause oxidative stress [183] . in addition, modification of interacting proteins can have an important impact on the regulation of ifs localization and function. the linker protein plectin has been reported to be nitrosated, affecting vimentin distribution [184] , whereas actin and tubulin are targets for various oxidative and lipoxidative modifications [56] . moreover, certain modifications can compete for the same site, for instance, carbamoylation, resulting from modification by isocyanic acid [185] , can occur at citrullination sites, and tyrosine nitration could preclude phosphorylation. modification of a given residue can also affect that of nearby amino acids, either by altering their reactivity or their accessibility. therefore, bulky moieties can shield nearby residues, but certain modifications can cause unfolding and exposure of additional amino acids for modification. additionally, many conditions associated with oxidative stress and oxidative modifications trigger important changes in gene expression, including the antioxidant stress response [57] , contributing to the complexity of the effects. given their implication in pathological processes, ifs in general, and those belonging to the type iii family in particular, are becoming important drug targets. indeed, hypothesis-driven, as well as virtual and pharmacological screening approaches are being used to find small molecule modulators of these proteins. high expression levels of type iii if proteins have been considered as pathogenic factors in several diseases. therefore, efforts have been devoted to find strategies to modulate their levels or organization in cells [186, 187] . nevertheless, this section will focus on approaches related to ptms and more precisely on drugs that directly bind to these proteins and preferentially interact with their conserved cysteine residue. as vimentin c328 and the equivalent cysteine residues in the other members of the family seem to be highly susceptible to modification by oxidant and electrophilic species, their modification by cysteine-reactive drugs is worth exploring. among the first compounds reported to target vimentin c328 is the withanolide natural product withaferin a (wfa). this molecule was studied based on its antitumor and antiangiogenic properties and was reported to covalently bind to c328 and induce vimentin aggregation in vitro and filament disruption in cells [188] . in addition, wfa caused vimentin downregulation in cells and showed in vivo antiangiogenic effects, reportedly mediated by vimentin [188] . it was later described that this molecule could bind to the single gfap cysteine residue, c294 [189] . moreover, it was shown that wfa reduced the levels of both vimentin and gfap in primary astrocytes as well as in mouse retina in vivo [189] . therefore, these wfa actions could be beneficial in situations associated with increased expression of these proteins, such as reactive gliosis. thus, wfa has become a popular tool employed to target and disrupt the vimentin network in several experimental models [190] . however, being a cysteine reactive molecule, wfa could interact with other cellular targets. in fact, several works have called attention to its lack of specificity [191] . indeed, wfa has recently been reported to covalently bind the heterogeneous nuclear ribonucleoprotein k (hnrnp-k) [192] , and tubulin, and induce the degradation of the latter [193] . moreover a vimentin mutant lacking c328 appears to respond to wfa as well [191] . recent molecular dynamics simulations indicate that wfa binds in the vicinity of c328, but not directly to this residue [192] . therefore, although wfa may be useful to disrupt vimentin and other type iii if proteins, its use as a selective target should be performed cautiously. interestingly, the compound arylquin 1 also binds to vimentin at a region close to the cysteine residue. in this case, interaction of arylquins with vimentin displaced the binding of par4, an apoptosis-inducing protein, with consequences for cell viability [194] . additional evidence of the importance of c328 as a drug target has risen from the garlic compound ajoene [195] , which has been shown to form a disulfide bond with this residue. this modification is associated with condensation of vimentin filaments and with an impairment of several vimentin-related processes, including cell migration and invasion of cancer cells. these effects were attenuated in cells in which vimentin expression had been transiently knocked down, thus pointing to vimentin importance as mediator of ajoene actions. notably, another garlic defense substance, allicin, has been shown to induce s-thioallylation of several cytoskeletal proteins, including vimentin, in jurkat cells [196] . in addition, phenyl vinyl sulfones, mechanism-based inhibitors of protein tyrosine phosphatase, covalently bind vimentin c328 in cells, although the significance of this finding is still not known [197, 198] . a number of proteomic studies searching for protein targets of drugs or natural products have found vimentin [199] , although the precise site of interaction or the functional consequences have not always been elucidated. additional compounds found to bind vimentin include two quinone methide metabolites of the phenolic antioxidant butylated hydroxytoluene. these metabolites are believed to be responsible for promoting lung tumor formation in mice [200] . in contrast, the green tea polyphenol epigallocatechin gallate binds to vimentin and inhibits its phosphorylation in cells, which is associated with an antiproliferative effect [201] . type iii if proteins are arising as integrators of basic cellular processes and as sensors of stress. these proteins, and in particular their conserved cysteine residue, have been proposed as hot spots for ptms. modification of this cysteine residue leads to marked reorganization of type iii ifs in cells, which is supported by the lack of response of cysteine-deficient mutants. remarkably, the nature of the reorganization appears to depend on the extent and the structure of the modification, illustrating an exceptional versatility of type iii ifs remodeling. moreover, this suggests that the conserved cysteine residue of type iii ifs functions as a hub that can accept different types of modifying moieties and transduces them into distinct cytoskeletal responses, either per se or in cooperation with other ptms, thus acting as a sensor for various signals. the position of the conserved cysteine residue in the structure of the protein could be critical for the functional outcome of the modifications, affecting its organization in filaments. nevertheless, many aspects of structure and function of type iii ifs still need investigation. elucidation of the structure of filaments and of their various arrangements in cells will be key to understand the basis of their response to stress. moreover, the downstream consequences of the diverse reorganizations are still poorly understood. the modification of type iii ifs by oxidant and electrophilic species could impact on many cellular processes, but it also could represent a defensive mechanism by which these proteins would act as decoys or scavengers of reactive species. therefore, further identification and characterization of the ptms occurring in vivo, both structurally and quantitatively, will be necessary to elucidate their role in physiological if regulation and their potentially protective or pathogenic effects under pathophysiological conditions. finally, certain oxidative ptms of type iii ifs can be considered as disease biomarkers, whereas their modulation could become a therapeutic strategy in a variety of illnesses. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. intermediate filaments mediate cytoskeletal crosstalk cytoskeletal crosstalk: when three different personalities team up intermediate filaments: structure and assembly, cold spring harbor persp genetic disorders of keratins and their associated proteins a-type lamins and hutchinson-gilford progeria syndrome: pathogenesis and therapy type iii intermediate filaments desmin, glial fibrillary acidic protein (gfap), vimentin, and peripherin, cold spring harbor persp intermediate filaments play a pivotal role in regulating cell architecture and function vimentin filaments interact with the actin cortex in mitosis allowing normal cell division f-actin interactome reveals vimentin as a key regulator of actin organization and cell mechanics in mitosis vimentin intermediate filaments template microtubule networks to enhance persistence in cell polarity and directed migration vimentin supports mitochondrial morphology and organization vimentin protects cells against nuclear rupture and dna damage during migration vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding glial fibrillary acidic protein (gfap) and the astrocyte intermediate filament system in diseases of the central nervous system desmin related disease: a matter of cell survival failure the role of vimentin intermediate filaments in the progression of lung cancer mutations in gfap, encoding glial fibrillary acidic protein, are associated with alexander disease intermediate filament structure: the bottom-up approach a proposed atomic model of the head-to-tail interaction in the filament structure of vimentin completion of the vimentin rod domain structure using experimental restraints: a new tool for exploring intermediate filament assembly and mutations diversity of intermediate filament structure. evidence that the alignment of coiled-coil molecules in vimentin is different from that in keratin intermediate filaments chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations site-directed spin labeling and electron paramagnetic resonance determination of vimentin head domain structure structural dynamics of the vimentin coiled-coil contact regions involved in filament assembly as revealed by hydrogen-deuterium exchange characterization of disulfide crosslink formation of human vimentin at the dimer, tetramer, and intermediate filament levels atomic structure of the vimentin central alpha-helical domain and its implications for intermediate filament assembly expression of vimentin in hair follicle growth cycle of inner mongolian cashmere goats specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments defect of mitotic vimentin phosphorylation causes microophthalmia and cataract via aneuploidy and senescence in lens epithelial cells nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis post-translational modifications of intermediate filament proteins: mechanisms and functions site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton human immunodeficiency virus type 1 protease cleaves the intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein vimentin acetylation is involved in sirt5-mediated hepatocellular carcinoma migration citrullinated autoantigens, from diagnostic markers to pathogenetic mechanisms mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis citrullinated vimentin presented on mhc-ii in tumor cells is a target for cd4+ t cell-mediated antitumor immunity abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with alzheimer's disease citrullination of glial intermediate filaments is an early response in retinal injury providing cellular signpostspost-translational modifications of intermediate filaments detection, identification, and quantification of oxidative protein modifications proteome oxidative modifications and impairment of specific metabolic pathways during cellular senescence and aging quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines activity-based sensing for site-specific proteomic analysis of cysteine oxidation protein oxidation and peroxidation lipoxidation targets: from basic mechanisms to pathophysiology oxidized protein aggregates: formation and biological effects, free radic lipoxidation adducts with peptides and proteins: deleterious modifications or signalling mechanisms? modification of cysteine residues by cyclopentenone prostaglandins: interplay with redox regulation of protein function biological nitric oxide signalling: chemistry and terminology nitric oxide-induced sglutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase regulation of protein function by s-glutathiolation in response to oxidative and nitrosative stress glutathione s-transferase p-mediated protein s-glutathionylation of resident endoplasmic reticulum proteins influences sensitivity to drug-induced unfolded protein response fatty acylation and prenylation of proteins: what's hot in fat mass spectrometry in studies of protein thiol chemistry and signaling: opportunities and caveats, free radic protein lipoxidation: detection strategies and challenges regulation of stress signaling pathways by protein lipoxidation advanced glycoxidation and lipoxidation end products (ages and ales): an overview of their mechanisms of formation molecular interactions and implications of aldose reductase inhibition by pga 1 and clinically used prostaglandins protein alkylation by the alpha,betaunsaturated aldehyde acrolein. a reversible mechanism of electrophile signaling? chemoproteomics reveals chemical diversity and dynamics of 4-oxo-2-nonenal modifications in cells modification and activation of ras proteins by electrophilic prostanoids with different structure are site-selective regulation of aldose reductase and the polyol pathway activity by nitric oxide electrophilic eicosanoids: signaling and targets biphasic effects of 15-deoxy-delta(12,14)-prostaglandin j(2) on glutathione induction and apoptosis in human endothelial cells 15-deoxy-δ 12,14 -prostaglandin j 2 exerts pro-and anti-inflammatory effects in mesangial cells in a concentration-dependent manner modification of proteins by reactive lipid oxidation products and biochemical effects of lipoxidation the presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human sw-13 cells mice lacking vimentin develop and reproduce without an obvious phenotype vimentin regulates activation of the nlrp3 inflammasome vimentin regulates notch signaling strength and arterial remodeling in response to hemodynamic stress vimentin in bacterial infections surface vimentin is critical for the cell entry of sars-cov vimentin as a multifaceted player and potential therapeutic target in viral infections dominant cataract formation in association with a vimentin assembly disrupting mutation sporadic and familial congenital cataracts: mutational spectrum and new diagnoses using next-generation sequencing targeted exome sequencing of congenital cataracts related genes: broadening the mutation spectrum and genotype-phenotype correlations in 27 chinese han families a dominant vimentin variant causes a rare syndrome with premature aging vimentin is the specific target in skin glycation. structural prerequisites, functional consequences, and role in skin aging study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin pga 1 : focus on vimentin sitespecific protein adducts of 4-hydroxy-2(e)-nonenal in human thp-1 monocytic cells: protein carbonylation is diminished by ascorbic acid the oxidized thiol proteome in aging and cataractous mouse and human lens revealed by icat labeling senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody the conserved cysteine residue of type iii intermediate filaments serves as a structural element and redox sensor, free rad heteropolymer filaments of vimentin and desmin in vascular smooth muscle tissue and cultured baby hamster kidney cells demonstrated by chemical crosslinking molecular interactions in intermediate-sized filaments revealed by chemical cross-linking. heteropolymers of vimentin and glial filament protein in cultured human glioma cells studies on lens vimentin oxidation of thiol in the vimentin cytoskeleton separation and characterization of homo and heterooligomers of the intermediate filament proteins desmin and vimentin the cysteine residue of glial fibrillary acidic protein is a critical target for lipoxidation and required for efficient network organization, free rad vimentin disruption by lipoxidation and electrophiles: role of the cysteine residue and filament dynamics zinc differentially modulates the assembly of soluble and polymerized vimentin protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification krit1 loss-of-function associated with cerebral cavernous malformation disease leads to enhanced s-glutathionylation of distinct structural and regulatory proteins identification by redox proteomics of glutathionylated proteins in oxidatively stressed human t lymphocytes cellular redox imbalance and changes of protein s-glutathionylation patterns are associated with senescence induced by oncogenic h-ras s-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding vimentin sglutathionylation at cys328 inhibits filament elongation and induces severing of mature filaments in vitro vimentin is secreted by activated macrophages alpha1-antitrypsin binds hemin and prevents oxidative activation of human neutrophils: putative pathophysiological significance regulation of redox-sensitive exofacial protein thiols in cho cells autoreactivity to malondialdehyde-modifications in rheumatoid arthritis is linked to disease activity and synovial pathogenesis s-nitrosoprotein formation and localization in endothelial cells shear flow increases s-nitrosylation of proteins in endothelial cells s-nitrosoproteome in endothelial cells revealed by a modified biotin switch approach coupled with western blot-based two-dimensional gel electrophoresis effects of the 3-hydroxy-3-methylglutaryl-coa reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells the role of nitric oxide on rosuvastatinmediated s-nitrosylation and translational proteomes in human umbilical vein endothelial cells target-selective protein s-nitrosylation by sequence motif recognition identification of novel protein targets for modification by 15-deoxy-δ 12,14 -prostaglandin j 2 in mesangial cells reveals multiple interactions with the cytoskeleton induction of reversible cysteine-targeted protein oxidation by an endogenous electrophile 15-deoxy-delta(12,14)-prostaglandin j2 siems, 4-hydroxynonenal (hne) modified proteins in metabolic diseases, free radic chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes, free radic protein modification and replicative senescence of wi-38 human embryonic fibroblasts basic aspects of the biochemical reactivity of 4-hydroxynonenal insight into the cellular effects of nitrated phospholipids: evidence for pleiotropic mechanisms of action, free rad increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis, free radic differential carbonylation of proteins in end-stage human fatty and nonfatty nash, free radic advanced lipoxidation end products (ales) as rage binders: mass spectrometric and computational studies to explain the reasons why proteomic screening of antioxidant effects exhibited by radix salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment bone morphogenic protein-4-induced oxidant signaling via protein carbonylation for endothelial dysfunction, free radic oxidative modifications of proteins by sodium arsenite in human umbilical vein endothelial cells intermediate filament carbonylation during acute acrolein toxicity in a549 lung cells: functional consequences, chaperone redistribution, and protection by bisulfite toxicity of smoke extracts towards a549 lung cells: role of acrolein and suppression by carbonyl scavengers redox imbalance in lung cancer of patients with underlying chronic respiratory conditions anti-apoptotic proteins are oxidized by abeta25-35 in alzheimer's fibroblasts vitamin e prevents oxidation of antiapoptotic proteins in neuronal cells increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement abnormalities in nitric oxide and its derivatives in lung cancer covalent modification of cytoskeletal proteins in neuronal cells by tryptamine-4,5-dione intermediate filament protein partnership in astrocytes gfap in health and disease glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis c, liver transplantation absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration advanced lipid peroxidation end-products in alexander's disease alexander disease glial fibrillary acidic protein: dynamic property and regulation by phosphorylation phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and rho-associated kinase phosphoproteomic analysis reveals site-specific changes in gfap and ndrg2 phosphorylation in frontotemporal lobar degeneration site-specific phosphorylation and caspase cleavage of gfap are new markers of alexander disease severity phosphorylation of gfap is associated with injury in the neonatal pig hypoxic-ischemic brain analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in alzheimer's disease serial protein labeling with infrared maleimide dyes to identify cysteine modifications oligomers of mutant glial fibrillary acidic protein (gfap) inhibit the proteasome system in alexander disease astrocytes, and the small heat shock protein alphab-crystallin reverses the inhibition global analysis of s-nitrosylation sites in the wild type (app) transgenic mouse brain-clues for synaptic pathology identification of major s-nitrosylated proteins in murine experimental autoimmune encephalomyelitis glial fibrillary acidic protein is a major target of glycoxidative and lipoxidative damage in pick's disease redox proteomics analysis of hne-modified proteins in down syndrome brain: clues for understanding the development of alzheimer disease, free radic typedependent oxidative damage in frontotemporal lobar degeneration: cortical astrocytes are targets of oxidative damage proteins in human brain cortex are modified by oxidation, glycoxidation, and regional vulnerability to lipoxidative damage and inflammation in normal human brain aging protein targets of oxidative damage in human neurodegenerative diseases with abnormal protein aggregates redox proteomic analysis of carbonylated brain proteins in mild cognitive impairment and early alzheimer's disease proteomic and oxidative stress analysis in human brain samples of huntington disease, free radic impairment of proteostasis network in down syndrome prior to the development of alzheimer's disease neuropathology: redox proteomics analysis of human brain glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain identification of nitrated proteins in the normal rat brain using a proteomics approach proteomic characterization of nitrated cell targets after hypobaric hypoxia and reoxygenation in rat brain proteomic analysis of glial fibrillary acidic protein in alzheimer's disease and aging brain desmin is oxidized and nitrated in affected muscles in myotilinopathies and desminopathies posttranslational modifications of desmin and their implication in biological processes and pathologies cytoskeletal alterations in cultured cardiomyocytes following exposure to the lipid peroxidation product, 4-hydroxynonenal n-acetyl-l-cysteine prevents stress-induced desmin aggregation in cellular models of desminopathy antioxidant treatment and induction of autophagy cooperate to reduce desmin aggregation in a cellular model of desminopathy influence of oxidation on the susceptibility of purified desmin to degradation by mu-calpain, caspase-3 and -6 detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis protein sulfenation as a redox sensor: proteomics studies using a novel biotinylated dimedone analogue mechanisms of acrolein-induced myocardial dysfunction: implications for environmental and endogenous aldehyde exposure protein carbonylation in skeletal muscles: impact on function neuronal intermediate filaments and als: a new look at an old question discovery of phosphorylated peripherin as a major humoral autoantigen in type 1 diabetes mellitus a frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis reduced number of unmyelinated sensory axons in peripherin null mice identification of a peripherin dimer: changes during axonal development and regeneration of the rat sciatic nerve identification and characterization of peripherin isoforms in amyotrophic lateral sclerosis tyrosine nitration is a novel post-translational modification occurring on the neural intermediate filament protein peripherin sulfenic acid chemistry, detection and cellular lifetime redox regulation of protein kinases nitric oxide leads to cytoskeletal reorganization in the retinal pigment epithelium under oxidative stress a proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells 15-deoxy-delta12,14-prostaglandin j2 modifies components of the proteasome and inhibits inflammatory responses in human endothelial cells divalent cations crosslink vimentin intermediate filament tail domains to regulate network mechanics vimentin gets a new glow from zinc the redox biology of redox-inert zinc ions, free radic oxidation and nitrosylation of cysteines proximal to the intermediate filament (if)-binding site of plectin: effects on structure and vimentin binding and involvement in if collapse protein carbamylation: chemistry, pathophysiological involvement, and biomarkers antisense suppression of glial fibrillary acidic protein as a treatment for alexander disease an image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells the tumor inhibitor and antiangiogenic agent withaferin a targets the intermediate filament protein vimentin withaferin a targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis the use of withaferin a to study intermediate filaments withaferin a alters intermediate filament organization, cell shape and behavior 3-dihydro-3beta-methoxy withaferin-a lacks anti-metastasis potency: bioinformatics and experimental evidences the natural compound withaferin a covalently binds to cys239 of beta-tubulin to promote tubulin degradation arylquins target vimentin to trigger par-4 secretion for tumor cell apoptosis the garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells the human allicin-proteome: s-thioallylation of proteins by the garlic defence substance allicin and its biological effects, free radic aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives a vimentin binding small molecule leads to mitotic disruption in mesenchymal cancers immunochemical and proteomic analysis of covalent adducts formed by quinone methide tumor promoters in mouse lung epithelial cell lines the intermediate filament protein vimentin is a new target for epigallocatechin gallate atomic structure of vimentin coil 2 this work was supported by grants from agencia estatal de investigación, ministerio de ciencia e innovación (mci, spain) and european regional development fund, rti2018-097624-b-i00, instituto de salud carlos iii (spain) retic aradyal rd16/0006/0021, and csic pti global health (pie202020e223/csic-cov19-100). av-p is supported by the fpi program from mci, reference bes-2016-076965. feedback from cost action eurocellnet ca15214 is gratefully acknowledged. supplementary data to this article can be found online at https:// doi.org/10.1016/j.redox.2020.101582. key: cord-0012950-p3mb7r0v authors: luo, yan; chalkou, konstantina; yamada, ryo; funada, satoshi; salanti, georgia; furukawa, toshi a. title: predicting the treatment response of certolizumab for individual adult patients with rheumatoid arthritis: protocol for an individual participant data meta-analysis date: 2020-06-12 journal: syst rev doi: 10.1186/s13643-020-01401-x sha: 3597f1835f9887c8eb25073c63b70d14934e2a7d doc_id: 12950 cord_uid: p3mb7r0v background: a model that can predict treatment response for a patient with specific baseline characteristics would help decision-making in personalized medicine. the aim of the study is to develop such a model in the treatment of rheumatoid arthritis (ra) patients who receive certolizumab (ctz) plus methotrexate (mtx) therapy, using individual participant data meta-analysis (ipd-ma). methods: we will search cochrane central, pubmed, and scopus as well as clinical trial registries, drug regulatory agency reports, and the pharmaceutical company websites from their inception onwards to obtain randomized controlled trials (rcts) investigating ctz plus mtx compared with mtx alone in treating ra. we will request the individual-level data of these trials from an independent platform (http://vivli.org). the primary outcome is efficacy defined as achieving either remission (based on acr-eular boolean or index-based remission definition) or low disease activity (based on either of the validated composite disease activity measures). the secondary outcomes include acr50 (50% improvement based on acr core set variables) and adverse events. we will use a two-stage approach to develop the prediction model. first, we will construct a risk model for the outcomes via logistic regression to estimate the baseline risk scores. we will include baseline demographic, clinical, and biochemical features as covariates for this model. next, we will develop a meta-regression model for treatment effects, in which the stage 1 risk score will be used both as a prognostic factor and as an effect modifier. we will calculate the probability of having the outcome for a new patient based on the model, which will allow estimation of the absolute and relative treatment effect. we will use r for our analyses, except for the second stage which will be performed in a bayesian setting using r2jags. discussion: this is a study protocol for developing a model to predict treatment response for ra patients receiving ctz plus mtx in comparison with mtx alone, using a two-stage approach based on ipd-ma. the study will use a new modeling approach, which aims at retaining the statistical power. the model may help clinicians individualize treatment for particular patients. systematic review registration: prospero registration number pending (id#157595). (continued from previous page) systematic review registration: prospero registration number pending (id#157595). keywords: rheumatoid arthritis, certolizumab, individual participant data meta-analysis, prediction model, treatment response background rheumatoid arthritis (ra) is a chronic inflammatory disease, for which we cannot currently expect complete cure. the drugs that can delay disease progression are known as disease-modifying anti-rheumatic drugs (dmards). there are 3 categories: conventional synthetic dmards (csdmards), biologic dmards (bdmards), and targeted synthetic dmards (tsdmards). bdmards can be further divided into several subtypes according to the target, among which the tumor necrosis factor (tnf) α inhibitors are the most classic and widely used. most ra patients undergo long-term treatment. according to the treat-to-target strategy proposed by the eular (european league against rheumatism) practice guideline [1] , repeated assessment of disease activity should be performed every 3 to 6 months after a treatment is given, to evaluate the response and decide the next-step strategy: switching drugs, maintenance, tapering, or discontinuation. hence, the disease course of ra is composed of many short-term (3 to 6 months) intervention-response loops. for the purpose of improving long-term prognosis, such as delaying the progression of bone fusion or functional deficiency, short-term intervention-response loops need to have beneficial outcomes [2] . to find the optimal treatment for a particular patient, it is necessary to personalize the treatment. it would be helpful if we could predict the probability of treatment response based on the patient's genetic, biologic, and clinical features. however, common evidence in the form of randomized controlled trials (rcts) or their meta-analyses (mas) at the aggregate level only reports average results. the drug that works for the average patients might not work or even be harmful for a particular patient. consequently, it is desirable to identify subgroups of patients associated with different treatment effects. individual participant data meta-analysis (ipd-ma) has been previously employed to develop prediction models for treatment effects [3] [4] [5] [6] . previous treatment response prediction models for ra were mainly based on observational studies [7] [8] [9] [10] [11] . observational studies seem suited for predicting the absolute risk of an outcome, but it may be less satisfactory in estimating the relative risk between different drugs, because unknown confounders may persist even when we try to adjust for known confounders. on the other hand, though the population in rcts is highly restricted hence may be less representative, data from rcts are more rigorously collected and more likely to provide an unbiased estimate of the relative treatment effects [12] . the synthesis of rct data via ipd-ma can increase the statistical power [13] and have been used to predict treatment response [6, [14] [15] [16] [17] . to the best of the authors' knowledge, such an approach has not been taken to predict treatment response in ra to date. our aim is to develop a prediction model of treatment effects based on individual characteristics of ra patients through ipd-ma. since tnfα inhibitors are the most classic and widely used bdmards for ra, we will build a model for certolizumab (ctz), a tnfα inhibitor with sufficient ipd data, in this study. we will first estimate the pooled average effect sizes for the primary and secondary outcomes using one-stage bayesian hierarchical ipd-ma. the main objective of the study is to use a two-stage risk modeling approach to predict the individualized treatment effects interest [12] . the first stage is to build a multivariable model aiming to predict the baseline risk for a particular patient blinded to treatment. in the second stage, this baseline risk score will be used as a prognostic factor and an effect modifier in an ipd meta-regression model to estimate the individualized treatment effects of ctz. we consider to validate and optimize the modeling approach in the present study, and plan eventually to expand it to an ipd network meta-analysis to compare several drug types (e.g., interleukin-6 inhibitors, anti-cd20 antibodies) as our future research perspective. the present protocol has been registered within the prospero database (provisional registration number id#157595) and is being reported in accordance with the reporting guidance provided in the preferred reporting items for systematic reviews and meta-analyses protocols (prisma-p) statement [18] (see the checklist in additional file 1). the proposed ipd-ma will be reported in accordance with the reporting guidance provided in the preferred reporting items for systematic reviews and meta-analyses of individual participant data (prisma-ipd) statement [19] . any amendments made to this protocol when conducting the study will be outlined and reported in the final manuscript. studies will be selected according to the following criteria: patients, interventions, outcomes, and study design. we will include adults (18 years or older) who are diagnosed with either early ra (2010 american college of rheumatology (acr)/european league against rheumatism (eular) classification criteria) [20, 21] or established ra (1987 classification criteria) [22] . patients with inner organ involvement (such as interstitial lung diseases), vasculitis, or concomitant other systemic autoimmune diseases will be excluded. we will include both treatment-naïve patients and patients who have insufficient response to previous treatments. we will include patients with moderate to severe disease activity based on any validated composite disease activity measures. patients who have already achieved remission or at low disease activity at baseline will be excluded. patients who have used certolizumab (ctz) within 6 months before randomization will be excluded. we will include rcts which compare certolizumab (ctz) plus methotrexate (mtx) with mtx monotherapy, regardless of doses. if a study compares ctz + any csdmards with any csdmards, we will only include patients on ctz + mtx or mtx from that study. trials investigating the tapering or discontinuation strategy of ctz will be excluded. our primary outcome is efficacy defined by disease states, which is achieving either remission (based on acr-eular boolean or index-based remission definition [23] ) or low disease activity (based on either of the validated composite disease activity measures [24] : das28 (disease activity score based on the evaluation of 28 joints) ≤3.2 [25] , cdai (clinical disease activity index) ≤ 10 [26] , sdai (simplified disease activity index) ≤ 11 [27] ) at 3 months (allowance 2-4 months) after treatment, as a binary outcome. we choose it as our primary outcome because it is suggested as the indicator of the treatment target in both the practice guideline [1] and the guideline for conducting clinical trials in ra [2] , and it has been shown to provide more information for future joint damage and functional outcomes compared to relative response (change from baseline) [28] . we have two secondary outcomes. one is efficacy defined by response (improvement from baseline), for which we will use the acr response criteria acr50 (50% improvement based on acr core set variables) [29] . another is adverse events (aes). we will perform an ipd-ma separately for patients with all kinds of infectious aes within 3 months since it is one of the most important aes for biologic agents. we will also describe other noticeable aes within 3 months reported in the trials. we will not make predictions models for the secondary outcomes. we will include double-blind rcts only. if there are crossover rcts, only the data of the first phase will be used for analysis. cluster rcts, quasi-randomized trials, and observational studies will be excluded. we will conduct an electronic search of cochrane cen-tral, pubmed, and scopus from inception onwards, with the keywords: "rheumatoid arthritis," "certolizumab" or "cdp870" "cimzia", "methotrexate" or "mtx," without language restrictions. a draft search strategy is provided in additional file 2. we will search who international clinical trials registry platform to find the registered studies. we will search the us food and drug administration (fda) reports to see if there are any unpublished reports from the pharmaceutical companies. for ipd, we will contact the company which markets certolizumab and request ipd through http://vivli.org. we will assess the representativeness of the ipd among all the eligible studies by investigating the potential differences between trials with ipd and those without ipd. two independent reviewers will screen the titles and abstracts retrieved from the electronic searches to assess for inclusion. if both reviewers agree that a trial does not meet eligibility criteria, it will be excluded. the full text of all the remaining articles will be obtained for further reading, and the same eligibility criteria will be applied to determine which to exclude. any disagreements will be resolved through discussion with a third member of the review team. two reviewers will independently extract the information for all the included studies at aggregate level. a detailed data extraction template will be developed and piloted on 3 articles; after finalizing the items on the data extraction form, the 3 articles will be re-extracted. the main information includes intervention/control details, trial implementation features (e.g., completion year, randomized numbers, dropouts, follow-up length), baseline demographic and disease-specific characteristics, and outcomes of interested. the above information will be used for: (1) exploring the representativeness of the trials with ipd among all the eligible trials and (2) confirming if the ipd is consistent with the reported results. when the ipd is ready to be used, we will identify the variables of interest before the analysis. the variables regarding intervention, control, and outcomes are defined as the above in the "eligibility criteria" section. with regard to patient or trial characteristics to be used as potential covariates in the prognostic model, based on the literature [30] [31] [32] and our clinical practice, we propose the following factors as candidates of potential prognostic factors (pfs, baseline factors that may affect the response regardless of the treatment) (table 1) , which will be used for baseline risk model development (see the "predicting treatment effect for patients with particular characteristics: a two-stage model" section below). we will try to collect all the information listed in table 1 from the data, but only available factors that have been recorded in the trials will be added into the model. we will decide in which type (e.g., continuous, categorical, binary, etc.) a covariate will be put into the model according to the distribution of that covariate after we obtain the data. two independent reviewers will assess the risk of bias (rob) for each included rct according to "rob 2 tool" proposed by the cochrane group [34] . for the efficacy primary outcome, rcts will be graded as "low risk of bias," "high risk of bias," or "some concerns" in the following five domains: risk of bias arising from the randomization process, risk of bias due to deviations from the intended interventions, missing outcome data, risk of bias in measurement of the outcome, and risk of bias in selection of the reported result. the assessment will be adapted for ipd-ma, i.e., as per the obtained data and not the conducted and reported analyses in the original publications. finally, they will be summarized as an overall risk of bias according to the rob 2 algorithm. since our primary aim is to develop a prediction model and not to get a precise estimation of the treatment effects, all the analyses will be based on ipd only. therefore, we will neither analyze aggregate data together nor investigate the robustness of the ipd-ma including aggregate data, for they are beyond the perspectives of the present study. we first synthesize the data using one-stage bayesian hierarchical ipd-ma [35] . we will estimate the average relative treatment effect in terms of odds ratio (or) for efficacy. let y ij denote the dichotomous outcome of interest (y ij = 1 for remission or low disease activity), for patient i where i = 1, 2, …, n j in trial j out of n trials, t ij be 0/1 for patient in control/intervention group, and p ij is the probability of having the outcome. where α j is the log odds of the outcome for the control group, in trial j, which is independent across trials; δ j is table 1 potential candidates to be involved as prognostic factors in the prognostic model *factors that have been proved to be a prognostic factor for any treatments in previous studies # since genetic tests for ra are not routinely implemented in clinical practice, we anticipate that most studies will not report them. although genetics are often considered critical in precision medicine, we will consider it justifiable if no genetic information is included in our model, because there is no single one that has been proven to be strongly associated with the prognosis or treatment responses, and two studies have indicated that genetic information barely contribute in predicting treatment effects [33] the treatment effect (log or), which we assume to be exchangeable across trials; δ is the summary estimate of the log-odds ratios for the intervention versus the control arm; and τ 2 is the heterogeneity of δ across trials and normally distributed across trials. predicting treatment effect for patients with particular characteristics: a two-stage model data pre-processing within each study, the outcomes and the covariates will be evaluated for missing data, and we will further look at their distributional characteristics and correlations between the covariates (listed in the "at ipd level: for studies with ipd available" section). we will use multiple imputation methods for handling missing data [36] . we will consider data transformation for continuous variables to resolve skewness and re-categorization for categorical variables if necessary. if two or more variables are highly correlated, we will only retain the variable that is most commonly reported across studies and in the literature or the variable that has the least missing values. stage 1: developing a baseline risk model in this step, we will build a multivariable model to predict the probability that a patient, given her or his baseline characteristics, is likely to achieve remission or low disease activity irrespective of treatment; we will refer to this model as the baseline risk model. the risk model can be built using the patients from the control group only, or from both intervention and control group. the former is more intuitive; however, a simulation study indicated that models based on the whole participants produced estimates with narrower distribution of bias and were less prone to overfitting [37] . we will fit a multivariable logistic regression model: r ij is the probability of the outcome for patient i from trial j at the baseline. b 0j is the intercept, which is exchangeable across studies. pf ijk denotes the k prognostic factor (in total, there are p prognostic factors) in study j for patient i, and b kj is the regression coefficient for k prognostic factor in study j and is exchangeable across studies. in order to select the most appropriate model, we propose two approaches: (1) use previously identified prognostic factors and through discussions with rheumatologists to decide the subset of the most clinically relevant factors and estimate the coefficients using penalized maximum likelihood estimation shrinkage method and (2) use lasso penalization methods for variable selection and coefficient shrinkage [38] . for each possible model, we will examine the sample size first, in order to assess the reliability of the model. we will calculate the events per variable (epv) for our model, using all the categories of categorical variables and the degrees of freedom of continuous outcomes [39] . we will calculate efficient sample size for developing a logistic regression model [40] . validation is essential in prediction model development. since no external data is available, we can only use internal validation. via resampling methods like bootstrap or cross-validation, we can estimate the calibration slope and c-statistic for each model, to indicate the ability of calibration and discrimination. stage 2: developing a meta-regression model for treatment effects we use the same notation system as that in the "average relative treatment effect: ipd-ma" section. the logit(r ij ) from stage 1 will be used as a covariate in the meta-regression model, both as a prognostic factor and as an effect modifier. let logitðr ij þ j denote the average of logit-risk for all the individuals in study j. the regression equation will be: γ a j is the log odds in the control group when a patient has a risk equal to the mean risk, which is assumed to be independent across trials. g 0j is the coefficient of the risk score, while g j is the treatment effect modification of the risk score for the intervention group versus the control group; both are assumed to be exchangeable cross trials and normally distributed about a summary estimate γ 0 and γ respectively. predicting the probability of having the outcome for a new patient assume a new patient i who is not from any trial j has a baseline risk score g logitðr i þ calculated from stage-one. in order to predict the absolute logitprobability to achieve the outcome, we use: we would have estimated δ, γ 0 , and γ in the metaregression stage. we will estimate logitðrþ as the mean of logit(r ij ) across all the individuals and studies. for a, we will estimate it by synthesizing all the control arms. then, we can calculate the individual probability of the outcome both for the control and the intervention and estimate the predicted absolute and relative treatment effect. to evaluate the performance of the two-stage prediction model, we will use internally validation methods via both the traditional measures, like c-statistic, and measures relevant to clinical usefulness. publication bias considering that we will probably not be able to include all the relevant research works, as some studies or their results were likely not published owing to non-significant results (study publication bias and outcome reporting bias) [41, 42] , we will evaluate this issue by comparing the search and screening results (as we will try to retrieve possibly unpublished reports) with the ipd we can get. if necessary, we will address it by adding the study's variance as an extra covariate in the final ipd meta-regression model (see the section "predicting treatment effect for patients with particular characteristics: a two-stage model"-"stage 2: developing a meta-regression model for treatment effects"). statistical software we will use r for our analyses. stage 2 will be performed in a bayesian setting using r2jags. for the development of the baseline risk model, we will use the pmsampsize command to estimate if the available sample size is enough. we will examine the linear relationship between each one of the prognostic factors and the outcome via rcs and anova commands. the lasso model will be developed using the cv.glmnet command. we will use the lrm command for the predefined model based on prior knowledge, and then for the penalized maximum likelihood estimation, we will use the pentrace command. for the bootstrap internal validation (both for the baseline risk score and for the two-stage prediction model), we will use self-programmed r-routines. we have presented the study protocol for a prediction model of treatment effects for ra patients receiving ctz plus mtx, using a two-stage approach based on ipd-ma. though there are many optional drugs in treating ra, as treatment failure is relatively high, individualizing the treatment is imperative. many prognostic models for ra have been proposed, but no one is sufficiently satisfactory [31] . we have discovered several problems. most previous models focused on long-term radiographic or functional prognosis. although they are certainly the critical outcomes that both clinicians and patients care about, the complex therapeutic changes during the long treatment process are extremely difficult to handle in developing prediction models. thus, it usually ends up with a simplified strategy, such as taking only the initial treatment into account, which compromises the clinical interpretation and relevancy of the model. on the other hand, a good short-term treatment response is always positively associated with good longterm prognosis [43, 44] . predicting short-term treatment effect is instructive in clinical practice; however, research is lacking. a few established "short-term" diseaseactivity-oriented prediction models used an outcome measured at 6 months or 12 months. the problem is, unless in active-controlled studies, there would be considerable dropouts after 3-4 months; furthermore, due to ethnical issues, many trials would offer the nonresponders other active treatments after 3-4 months. under the itt principle, patients were commonly analyzed as originally allocated; when dropouts were not negligible, imputation methods were usually used, but mostly single imputation such as non-responder imputation or last observation carried forward (locf) [45] . one may argue that these estimates were conservative to the intervention group though not precise. but in fact it is not always conservative for a relative effect estimate, while unbiased relative estimates are of critical interest in building personalized prediction models. as a result, in order to be methodologically rigorous, we choose the outcome measured at 3 months, when the randomization is likely kept, and which is consistent with the assessment time recommended by the guideline [1] . additionally, thanks to the ipd, we will be able to use multiple imputation to handle missing data, rather than the single imputations used in primary rcts. we will use a two-stage approach to construct the prediction model using ipd-ma. unlike the usual approach, which includes baseline features as prognostic factors and effect modifiers (through interaction terms) simultaneously, we first build a risk model for baseline factors, then treating the risk score as both a prognostic factor and an effect modifier. by doing so, overfitting problem caused by too many covariates and interaction terms can be alleviated. moreover, since penalization will only be used in common regression during risk modeling stage but not in meta-regression, the compromised penalization in meta-regression can be avoided. for stage 1, generally there are two types of risk models. one is an externally developed model, which is derived based on data independent from the data used at stage 2, such as established models from previous studies, or using some other studies. the other is an internally developed risk model, for which the same data will be used to build both the risk model and the treatment effects model [37] . because there is no well-established risk model to predict the short-term disease activity for ra patients and also because we will very probably not have sufficient sample size to divide the entire data into two parts, we will use the internal risk model for our study. we acknowledge several limitations in our study. first, we handle effect modification at the level of risk scores, instead of particular covariates. that is, we will not try to identify specific effect modifiers. this may cause some problems in interpretation, as the concept of distinguishing prognostic factors and effect modifiers is well recognized. however, our approach assures the statistical power. second, due to the restricted sample size, only internal validation is planned while external validation is lacking. it needs to be validated on an external dataset in the future. third, we only focus on short-term treatment response for ra patients receiving two kinds of treatment, ctz and mtx. future studies may extend the scope to compare several kinds of therapies and treatment strategies and finally model for the long-term prognosis taking into consideration all the treatment processes. supplementary information accompanies this paper at https://doi.org/10. 1186/s13643-020-01401-x. additional file 1. prisma-p checklist. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update committee for medicinal products for human use (chmp): guideline on clinical investigation of medicinal products for the treatment of rheumatoid arthritis a framework for developing, implementing, and evaluating clinical prediction models in an individual participant data meta-analysis developing and validating risk prediction models in an individual participant data meta-analysis cochrane ipdm-amg: individual participant data (ipd) meta-analyses of diagnostic and prognostic modeling studies: guidance on their use statistical approaches to identify subgroups in meta-analysis of individual participant data: a simulation study arthritis: which subgroup of patients with rheumatoid arthritis benefits from switching to rituximab versus alternative anti-tumour necrosis factor (tnf) agents after previous failure of an anti-tnf agent? prediction of response to methotrexate in rheumatoid arthritis prediction of response to targeted treatment in rheumatoid arthritis association of response to tnf inhibitors in rheumatoid arthritis with quantitative trait loci for cd40 and cd39 assessment of a deep learning model based on electronic health record data to forecast clinical outcomes in patients with rheumatoid arthritis personalized evidence based medicine: predictive approaches to heterogeneous treatment effects meta-analysis of individual participant data: rationale, conduct, and reporting getreal methods review g: get real in individual participant data (ipd) meta-analysis: a review of the methodology quantifying heterogeneity in individual participant data meta-analysis with binary outcomes a critical review of methods for the assessment of patient-level interactions in individual participant data meta-analysis of randomized trials, and guidance for practitioners cognitive-behavioral analysis system of psychotherapy, drug, or their combination for persistent depressive disorder: personalizing the treatment choice using individual participant data network metaregression preferred reporting items for systematic review and meta-analysis protocols (prisma-p) preferred reporting items for systematic review and meta-analyses of individual participant data: the prisma-ipd statement rheumatoid arthritis classification criteria: an american college of rheumatology/ european league against rheumatism collaborative initiative rheumatoid arthritis classification criteria: an american college of rheumatology/ european league against rheumatism collaborative initiative the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis american college of rheumatology/european league against rheumatism provisional definition of remission in rheumatoid arthritis for clinical trials the definition and measurement of disease modification in inflammatory rheumatic diseases the disease activity score and the eular response criteria acute phase reactants add little to composite disease activity indices for rheumatoid arthritis: validation of a clinical activity score remission and active disease in rheumatoid arthritis: defining criteria for disease activity states the importance of reporting disease activity states in rheumatoid arthritis clinical trials american college of rheumatology. preliminary definition of improvement in rheumatoid arthritis remission-induction therapies for early rheumatoid arthritis: evidence to date and clinical implications assessing prognosis and prediction of treatment response in early rheumatoid arthritis: systematic reviews poor prognostic factors guiding treatment decisions in rheumatoid arthritis patients: a review of data from randomized clinical trials and cohort studies crowdsourced assessment of common genetic contribution to predicting anti-tnf treatment response in rheumatoid arthritis rob 2: a revised tool for assessing risk of bias in randomised trials meta-analysis using individual participant data: one-stage and two-stage approaches, and why they may differ missing data in randomised controlled trials: a practical guide using internally developed risk models to assess heterogeneity in treatment effects in clinical trials regression shrinkage and selection via the lasso risk prediction models: i. development, internal validation, and assessing the incremental value of a new (bio)marker minimum sample size for developing a multivariable prediction model: part ii -binary and time-to-event outcomes randomized controlled trials of rheumatoid arthritis registered at clinicaltrials.gov: what gets published and when dissemination and publication of research findings: an updated review of related biases rheumatoid arthritis treatment: the earlier the better to prevent joint damage evaluation of different methods used to assess disease activity in rheumatoid arthritis: analyses of abatacept clinical trial data a systematic review of randomised controlled trials in rheumatoid arthritis: the reporting and handling of missing data in composite outcomes publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions yl and taf conceived the study. kc and gs designed the modeling strategy. ry and sf provided substantial contribution to the design of the study during its development. yl drafted the manuscript, and all the authors critically revised it. all authors gave final approval of the version to be published. this study was supported by the intramural support to the department of health promotion and human behavior, kyoto university graduate school of medicine/school of public health. the funder has no role in the study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit for publication. the data that support the findings of this study are available from http://vivli. org but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. data are however available from http://vivli.org upon reasonable request and application, after their permission. this study does not require institutional review board approval and participant consent.competing interests taf reports personal fees from mitsubishi-tanabe, msd, and shionogi and a grant from mitsubishi-tanabe, outside the submitted work; taf has a patent 2018-177688. gs was invited to participate in two methodological meetings about the use of real-world data, organized by biogen and by merck. all the other authors report no competing interests to declare. key: cord-0072355-wxvkobsi authors: tsiogkas, sotirios g.; grammatikopoulou, maria g.; gkiouras, konstantinos; zafiriou, efterpi; papadopoulos, iordanis; liaskos, christos; dardiotis, efthimios; sakkas, lazaros i.; bogdanos, dimitrios p. title: effect of crocus sativus (saffron) intake on top of standard treatment, on disease outcomes and comorbidities in patients with rheumatic diseases: synthesis without meta-analysis (swim) and level of adherence to the consort statement for randomized controlled trials delivering herbal medicine interventions date: 2021-11-27 journal: nutrients doi: 10.3390/nu13124274 sha: 5230cc2829a1915984d8a10273b82376fac4920a doc_id: 72355 cord_uid: wxvkobsi rheumatic diseases (rds) are often complicated by chronic symptoms and frequent side-effects associated with their treatment. saffron, a spice derived from the crocus sativus l. flower, is a popular complementary and alternative medicine among patients with rds. the present systematic review aimed to summarize the available evidence regarding the efficacy of supplementation with saffron on disease outcomes and comorbidities in patients with rd diagnoses. pubmed, central, clinicaltrials.gov and the grey literature were searched until october 2021, and relevant randomized controlled trials (rcts) were screened for eligibility using rayyan. risk of bias was assessed using the cochrane’s risk of bias-2.0 (rob) tool. a synthesis without meta-analysis (swim) was performed by vote counting and an effect direction plot was created. out of 125 reports, seven fulfilled the eligibility criteria belonging to five rcts and were included in the swim. the rcts involved patients with rheumatoid arthritis, osteoarthritis and fibromyalgia, and evaluated outcomes related to pain, disease activity, depression, immune response, inflammation, oxidative stress, health, fatigue and functional ability. the majority of trials demonstrated some concerns regarding overall bias. moreover, the majority of trialists failed to adhere to the formula elaborations suggested by the consort statement for rcts incorporating herbal medicine interventions. standardization of herbal medicine confirms its identity, purity and quality; however, the majority of trials failed to adhere to these guidelines. due to the great heterogeneity and the lack of important information regarding the standardization and content of herbal interventions, it appears that the evidence is not enough to secure a direction of effect for any of the examined outcomes. rheumatic and musculoskeletal diseases have the highest population impact across all adverse health outcomes, including greater disability-adjusted life years (daly) [1, 2] . due to the chronic nature of these conditions and the frequent side-effects associated with their treatment, patients often resort to complementary and alternative medicines (cams), in search of "less toxic" therapies [3, 4] . garlic, ginger, curcumin, cinnamon, or saffron are a few of the most popular cams used in rheumatic diseases (rds) [5] [6] [7] . saffron, in particular, is the dried stigma of the flowers of crocus sativus l. (family iridaceae), cultivated mainly in southern europe, india and iran, and is considered as one of the most expensive culinary spices globally [8] . the medicinal properties of saffron and its constituents (safranal, crocin, and crocetin) include anti-inflammatory, antioxidant, analgesic, antihypertensive, hypolipidemic, antitussive, anticonvulsant, antidepressant, anxiolytic, anticancer, and antinociceptive characteristics [9] [10] [11] [12] [13] [14] [15] . nevertheless, although saffron supplementation has been tested in patients with various rds employing a randomized controlled trial (rct) design, we have insufficient evidence regarding its efficacy, as no systematic reviews have attempted to synthetize these data in order to aid in the formulation of recommendations. a common issue in cam research, however, is the lack of standardization of the administered products, often resulting in an inability to reproduce the findings and understand which active ingredients may in fact propel the observed outcomes. the standardization of herbal medicine confirms its identity, purity and quality, and for this, relevant trials ought to disclose information regarding formula elaborations [16] . this information is required to judge the internal validity, external validity, and reproducibility of the administered interventions [17, 18] . the aim of the present systematic review was to evaluate the efficacy of saffron oral nutrient supplementation (ons) on top of standard treatment, on disease outcomes and comorbidities in patients with rds and evaluate the quality of these trials. the preferred reporting items for systematic reviews and meta-analyses (prisma) [19] and the synthesis without meta-analysis (swim) extension [20] were used for the presentation of the present review. the study's protocol was published at the center for open science framework (osf) (https://bit.ly/3phesa7, accessed on 26 november 2021). the pico of the study's research question is detailed in table 1 . studies related to the research question were identified through pubmed, the cochrane central register of controlled trials (central), clinicaltrials.gov and grey literature searches from inception until october 2021 by three independent reviewers (s.g.t., m.g.g. and k.g.). any disagreement between reviewers was resolved by a senior researcher (d.p.b.). the search syntax used in each database is presented in figure 1 . rayyan [21] , a web and mobile application for conducting systematic reviews, was used to scan and identify all studies fulfilling the study's criteria. all identified references were imported into rayyan using reference manager software, and duplicate entries were excluded. combinations of relevant keywords were used to identify relevant rcts in the literature. the keywords used included (crocus sativus), (saffron), (crocin), (crocetin), (safranal), (rheumatoid arthritis), (scleroderma), (fibromyalgia), (behçet's syndrome), (osteoarthritis), (hyperuricemia), (gout), (ankylosing spondylitis), (psoriatic arthritis), (psoriasis), (psoriatic plaque), (spondylarthritis), (systemic lupus erythematosus), (lupus), (sle), (sjogren's syndrome), (systemic sclerosis), and (rheumatic disease*). were imported into rayyan using reference manager software, and duplicate entries were excluded. combinations of relevant keywords were used to identify relevant rcts in the literature. the keywords used included (crocus sativus), (saffron), (crocin), (crocetin), (safranal), (rheumatoid arthritis), (scleroderma), (fibromyalgia), (behçet's syndrome), (osteoarthritis), (hyperuricemia), (gout), (ankylosing spondylitis), (psoriatic arthritis), (psoriasis), (psoriatic plaque), (spondylarthritis), (systemic lupus erythematosus), (lupus), (sle), (sjogren's syndrome), (systemic sclerosis), and (rheumatic disease*). although not belonging to the rds, osteoarthritis (oa) was also included in the search strategy, since many patients with ra are often misdiagnosed with oa and vice versa [22] . studies were included in the synthesis when they (1) had an rct design, (2) were parallel or cross-over, (3) used an active per os intervention with saffron in any form (tabs, caps, powder, syrup, sachets, tea), (4) were conducted in patients with a rd diagnosis (or osteoarthritis), (5) examined any age group, and (6) used a placebo or any other intervention as a comparator (comparative effectiveness studies). although not belonging to the rds, osteoarthritis (oa) was also included in the search strategy, since many patients with ra are often misdiagnosed with oa and vice versa [22] . studies were included in the synthesis when they (1) had an rct design, (2) were parallel or cross-over, (3) used an active per os intervention with saffron in any form (tabs, caps, powder, syrup, sachets, tea), (4) were conducted in patients with a rd diagnosis (or osteoarthritis), (5) examined any age group, and (6) used a placebo or any other intervention as a comparator (comparative effectiveness studies). exclusion criteria involved (1) all other study designs (non-interventional) including those lacking a comparator arm, (2) studies not including patients with rds, (3) or using interventions lacking saffron, (4) interventions with curcumin, and (5) published protocols without published results, as well as (6) studies on animals or preclinical studies. special caution was taken not to include rcts investigating the effects of curcumin, which is also known as "indian saffron" [23] . outcomes of interest involved any specific index/score for rds, including disease activity scores, pain, inflammation markers, antioxidant and oxidative stress status, anxiety, depression, quality of life (qol), health assessment, immune response indicators, etc. eligible studies were independently assessed for bias using the cochrane's revised risk of bias (rob) tool 2.0 [24] by two authors (k.g. and m.g.g.). judgments were made if there was a low risk, some concerns or high risk of bias in terms of the randomization process, deviations from intended interventions, missing outcome data, measurement of the outcomes, selection of the reported results and the final assessment regarding the overall bias. two independent researchers (m.g.g. and k.g.) extracted data in excel spreadsheets. information regarding the sample (size, rd diagnosis, age, % female), recruitment, country of origin, funding, design and methodology (randomization particularities, masking, etc.), intervention (standardization particularities and dosage) and comparator arms, outcomes of interest, drop-outs, adverse events, presented analysis, and general results was extracted for all studies. at least three rcts investigating the same outcome for each rd were required for an effective data synthesis. since a meta-analysis was not feasible, vote counting was applied, based on the direction of effect (mean differences) for each outcome [25] in order to accompany the narrative synthesis [26] . the methodological characteristics of each study (rd diagnosis, overall risk of bias, etc.) were used to assess heterogeneity, according to the cochrane handbook [26] and the swim guidelines [20] . out of 139 studies screened in total, five rcts and seven publications (two studies with duplicate publications) [27] [28] [29] [30] [31] [32] [33] fulfilled the protocol's criteria and were included in the systematic review. figure 2 details the prisma 2020 flow diagram of the study selection process [19] . details of the rcts fulfilling the study's criteria, evaluating saffron interventions in patients with rheumatoid arthritis (ra), osteoarthritis (oa), or fibromyalgia (fm), the respective trials are detailed in figure 3 . the effect of saffron supplementation was evaluated in two trials using participants with ra [27, 31, 33] , an additional two rcts with patients with a knee oa diagnosis [28, 29, 32] and finally, on one rct performed in patients with fm [30] (figure 3 ). in ra, two different diagnostic criteria were employed, including the american college of rheumatology/european league against rheumatism (acr/eu-lar) 2010 [34] and the revised acr 2017 [35] . for patients with oa and fb, the acr [36] and acr [37] criteria were employed, respectively. no relevant completed trials were retrieved for spondylarthritis, ankylosing spondylitis, sjogren's syndrome, hyperuricemia, systemic lupus erythematosus (sle), scleroderma, psoriatic arthritis, psoriasis, or behçet's syndrome (bs). all trials were conducted in iran and were published between the years 2018 and 2021. the rcts employed a parallel intervention design. no cross-over trials were retrieved, fulfilling the pico question of the study. all included rcts were double blinded [27] [28] [29] [30] 32 ]. the administered doses of crocus sativus l. ranged between 15 mg/day [28, 30] and 100 mg daily [27, 29, [31] [32] [33] . all studies used pills, tablets or capsules for the delivery of saffron supplements. sahebari and associates [27] used pure saffron powder made of saffron flowers (saharkhiz saffron factory, mashhad, iran), hamidi et al. [31, 33] administered saffron sargol (saharkhiz saffron factory, mashhad, iran), and poursamimi and as details of the rcts fulfilling the study's criteria, evaluating saffron interventions in patients with rheumatoid arthritis (ra), osteoarthritis (oa), or fibromyalgia (fm), the respective trials are detailed in figure 3 . the effect of saffron supplementation was evaluated in two trials using participants with ra [27, 31, 33] , an additional two rcts with patients with a knee oa diagnosis [28, 29, 32] and finally, on one rct performed in patients with fm [30] (figure 3 ). in ra, two different diagnostic criteria were employed, including the american college of rheumatology/european league against rheumatism (acr/eular) 2010 [34] and the revised acr 2017 [35] . for patients with oa and fb, the acr [36] and acr [37] criteria were employed, respectively. no relevant completed trials were retrieved for spondylarthritis, ankylosing spondylitis, sjogren's syndrome, hyperuricemia, systemic lupus erythematosus (sle), scleroderma, psoriatic arthritis, psoriasis, or behçet's syndrome (bs). all trials were conducted in iran and were published between the years 2018 and 2021. the rcts employed a parallel intervention design. no cross-over trials were retrieved, fulfilling the pico question of the study. all included rcts were double blinded [27] [28] [29] [30] 32 ]. the administered doses of crocus sativus l. ranged between 15 mg/day [28, 30] and 100 mg daily [27, 29, [31] [32] [33] . all studies used pills, tablets or capsules for the delivery of saffron supplements. sahebari and associates [27] used pure saffron powder made of saffron flowers (saharkhiz saffron factory, mashhad, iran), hamidi et al. [31, 33] administered saffron sargol (saharkhiz saffron factory, mashhad, iran), and poursamimi and associates [28] applied interventions with krocina tm (samisaz pharmaceutical company, mashhad, iran). in the shakiba trial [30] , dried and milled crocus sativus l. stigma (impi-ran, tehran, iran) was used for the preparation of tablets, and firoozabadi et al. [29, 32] administered saffron pills (not-other defined). extraction information and methods were only provided in two trials [27, 30] . additional compounds in the administered tabs were reported in two trials [27, 30] , but the exact composition of the final products was not declared in any rct. standardization of the final product was only reported by shakiba and associates [30] , based on the crocin and safranal content of the capsules via spectrometry. although poursamimi et al. [28] administered ready-to-buy supplements, no information is currently provided on the manufacturer's website [38] . intervention duration spanned between 8 weeks [30] and 4 months [28] . four rcts used placebos as comparators [27] [28] [29] [31] [32] [33] and one used duloxetine [30] , but the aim in the latter was to assess the comparative effectiveness of saffron versus duloxetine for depression in patients with fm. the sample size was rather small in all rcts, spanning from 40 [28] to a maximum of 82 [27] patients per trial, prior to randomization. the included rcts involved a total of 148 patients with ra, 106 patients with oa, and 54 patients with fm. in the pooled sample, 104 patients received a saffron intervention and 104 were allocated to the control arms. one trial which was only published in abstract format [29, 32] did not report the number of patients allocated in the intervention/comparator arms. one important outcome of interest among the included trials involved pain, which was evaluated using the visual analogue scale (vas) [39] , the pain scale (not defined), the brief pain inventory (bpi) [40] , or the western ontario and mcmaster universities (womac) oa index pain subscale [41] . immune response post-saffron supplementation was evaluated in one rct [28] assessing cd8+ and cd4+ t helper (th) cells, th17 cell percentage (%), t-regulatory (treg) cells percentage (%), and the geometric mean fluorescence intensity (gmfi) of forkhead box protein p3 (foxp3) of treg cells, as well as the treg/th17 ratio. assessed inflammation markers included the erythrocyte sedimentation rate (esr), creactive protein (crp) and hs-crp (high sensitivity crp), tumor necrosis factor-α (tnf-α), interferon-γ (ifn-γ), interleukine-17 (il-17), and interleukine-1β (il-1β) levels. health was self-assessed by the patients themselves using the health assessment questionnaire-disability index (haq-di) [42] , or by their physicians using the physician global assessment (pga) [43] . fatigue was evaluated using the global fatigue index (gfi) [44] in one trial. depression was assessed using beck's depression inventory (bdi), the hamilton depression rating scale [45] , or the hospital anxiety and depression scale (hads) [46] . [40] ; das-28, disease activity score -28 [47] ; elisa, enzyme-linked immunosorbent assay; esr, erythrocyte sedimentation rate; eular, european league against rheumatism; fiq, fibromyalgia impact questionnaire [48, 49] ; fm, fibromyalgia; foxp3, forkhead box [40] ; das-28, disease activity score -28 [47] ; elisa, enzyme-linked immunosorbent assay; esr, erythrocyte sedimentation rate; eular, european league against rheumatism; fiq, fibromyalgia impact questionnaire [48, 49] ; fm, fibromyalgia; foxp3, forkhead box protein p3; gfi, global fatigue index [44] ; gi, gastrointestinal; gmfi, geometric mean fluorescence intensity; hads, hospital anxiety and depression scale [46] ; haq-di, health assessment questionnaire-disability index [42] ; hpmc, hydroxy-propyl methyl-cellulose; hs-crp, high sensitivity c-reactive protein; hrsd, hamilton rating scale for depression [45] ; ifnγ, interferon-γ; ipaq, international physical activity questionnaire [50] ; locf, last-observation carry forward; mda, malondialdehyde; n/a, not applicable; nr, not reported; nsaids, non-steroid anti-inflammatory drugs; itt, intention-totreat; oa, osteoarthritis; ons, oral nutrient supplementation; pe, physical exercise; pga, physician global assessment [43] ; pp, per protocol; ps, pain score; ra, rheumatoid arthritis; sjc, swollen joint count; th, t helper; tjc, tender joint count; tnf-a, tumor necrosis factor α; treg-cells, regulatory t cells; vas, visual analogue scale [39] ; womac, western ontario and mcmaster universities osteoarthritis index [41] . * within the manuscript text, 31 participants were reported to have completed the active arm intervention and 30 controls, in the consort flow chart it appears that 32 women from the intervention and 31 from the control arm were analyzed, but in the tables, the respective number of reported participants in active and comparator arms was 33 and 32. ‡ reported data refer to the pp analysis; r range; m mean ± standard deviation. antioxidant activity and oxidative stress were assessed according to the malondialdehyde (mda) levels, and total antioxidant capacity (tac) via the ferric reducing ability of plasma (frap) method. disease-specific scores were also evaluated, depending on the diagnosis of the participants in each trial. for ra, the disease-specific scores involved the disease activity score-28 (das-28) including the esr assay [47] (das-28-esr) and the swollen and tender joint count (sjc, tjc). for the rct performed in patients with fm [30] , the fibromyalgia impact questionnaire (fiq) [48, 49] was applied. in the case of oa, one trial [29, 32] reported using the womac [41] . the summary of risk of bias for the included rcts is presented in figure 4 . the majority of rcts (60%) exhibited some concerns for overall risk of bias, with the remaining 40% having a high risk for overall bias. the greatest proportion of trials with unclear bias involved the randomization process and the deviations from intended interventions domains. joint count; tnf-a, tumor necrosis factor α; treg-cells, regulatory t cells; vas, visual analogue scale [39] ; womac, western ontario and mcmaster universities osteoarthritis index [41] . * within the manuscript text, 31 participants were reported to have completed the active arm intervention and 30 controls, in the consort flow chart it appears that 32 women from the intervention and 31 from the control arm were analyzed, but in the tables, the respective number of reported participants in active and comparator arms was 33 and 32. ‡ reported data refer to the pp analysis; r range; m mean ± standard deviation. antioxidant activity and oxidative stress were assessed according to the malondialdehyde (mda) levels, and total antioxidant capacity (tac) via the ferric reducing ability of plasma (frap) method. disease-specific scores were also evaluated, depending on the diagnosis of the participants in each trial. for ra, the disease-specific scores involved the disease activity score-28 (das-28) including the esr assay [47] (das-28-esr) and the swollen and tender joint count (sjc, tjc). for the rct performed in patients with fm [30] , the fibromyalgia impact questionnaire (fiq) [48, 49] was applied. in the case of oa, one trial [29, 32] reported using the womac [41] . the summary of risk of bias for the included rcts is presented in figure 4 . the majority of rcts (60%) exhibited some concerns for overall risk of bias, with the remaining 40% having a high risk for overall bias. the greatest proportion of trials with unclear bias involved the randomization process and the deviations from intended interventions domains. treatment adherence was assessed only in two trials [30, 31, 33] , with the remaining studies failing to control for this issue. furthermore, the ban of antioxidant supplements at the beginning of the trials was not reported by any trialist, whereas in shakiba's trial [30] , only a history of treatment with saffron was an exclusion criterion, without controlling for other antioxidants. diet was only recorded and assessed by hamidi et al. [31, 33] , despite the fact that it can alter antioxidant intake. similarly, physical activity a known mediator of disease activity and stress was only assessed by hamidi [31, 33] . among the included trials, the majority failed to adhere to the formula elaborations suggested by the consolidated standards of reporting trials (consort) statement for rcts including herbal medicine interventions [17] (figure 5 ). thus, it appears that the exact composition and dosage of active saffron ingredients, including crocin, crocetin or safranal, cannot be calculated, with the exception of one trial [30] . shakiba's rct [30] adhered to the majority of consort components involving the standardization and procedures required for rcts with herbal medicine interventions. on the other hand, sahebari [27] also reported all added constituents, but failed to define the exact dosage per administered unit. firoozabadi and associates [29, 32] demonstrated the least adherence; however, their results were only published in abstract format, and thus limited space was available. ling for other antioxidants. diet was only recorded and assessed by hamidi et al. [31, 33] , despite the fact that it can alter antioxidant intake. similarly, physical activity a known mediator of disease activity and stress was only assessed by hamidi [31, 33] . among the included trials, the majority failed to adhere to the formula elaborations suggested by the consolidated standards of reporting trials (consort) statement for rcts including herbal medicine interventions [17] (figure 5 ). thus, it appears that the exact composition and dosage of active saffron ingredients, including crocin, crocetin or safranal, cannot be calculated, with the exception of one trial [30] . shakiba's rct [30] adhered to the majority of consort components involving the standardization and procedures required for rcts with herbal medicine interventions. on the other hand, sahebari [27] also reported all added constituents, but failed to define the exact dosage per administered unit. firoozabadi and associates [29, 32] demonstrated the least adherence; however, their results were only published in abstract format, and thus limited space was available. regarding the sensation of pain, ons with crocus sativus l. either reduced [27, 28, 33] , or did not appear to have an effect [27, 29, 30, 32] when administered to patients with ra, oa or fm. the use of nsaids was reduced in one trial using a sample of patients with oa [29, 32] . on the other hand, no change was recorded regarding the sensation of fatigue in fm (one trial) [30] . markers of inflammation were examined in ra and oa and were either reduced or remained unchanged post-intervention, with trials indicating conflicting results. indicators of antioxidant activity and oxidative damage remained unchanged (mda [31, 33] and tac [31, 33] in one rct each), raising concerns regarding the efficacy of saffron. immune response was evaluated in one oa rct [28] , which reported an increase in the percentage of treg-cells, the treg/th17 ratio and a decrease in the th17 cell percentage. in ra, disease-specific indexes such as the das-28-esr, morning stiffness, tjc and sjc were reduced in one rct [31, 33] and were not affected in another [27] . in oa, results concerning the total womac score were not reported by the trialists [29, 32] . in fm, saf regarding the sensation of pain, ons with crocus sativus l. either reduced [27, 28, 33] , or did not appear to have an effect [27, 29, 30, 32] when administered to patients with ra, oa or fm. the use of nsaids was reduced in one trial using a sample of patients with oa [29, 32] . on the other hand, no change was recorded regarding the sensation of fatigue in fm (one trial) [30] . markers of inflammation were examined in ra and oa and were either reduced or remained unchanged post-intervention, with trials indicating conflicting results. indicators of antioxidant activity and oxidative damage remained unchanged (mda [31, 33] and tac [31, 33] in one rct each), raising concerns regarding the efficacy of saffron. immune response was evaluated in one oa rct [28] , which reported an increase in the percentage of treg-cells, the treg/th17 ratio and a decrease in the th17 cell percentage. in ra, disease-specific indexes such as the das-28-esr, morning stiffness, tjc and sjc were reduced in one rct [31, 33] and were not affected in another [27] . in oa, results concerning the total womac score were not reported by the trialists [29, 32] . in fm, saf-reported. regarding the sensation of pain, ons with crocus sativus l. either reduced [27, 28, 33] , or did not appear to have an effect [27, 29, 30, 32] when administered to patients with ra, oa or fm. the use of nsaids was reduced in one trial using a sample of patients with oa [29, 32] . on the other hand, no change was recorded regarding the sensation of fatigue in fm (one trial) [30] . markers of inflammation were examined in ra and oa and were either reduced or remained unchanged post-intervention, with trials indicating conflicting results. indicators of antioxidant activity and oxidative damage remained unchanged (mda [31, 33] and tac [31, 33] in one rct each), raising concerns regarding the efficacy of saffron. immune response was evaluated in one oa rct [28] , which reported an increase in the percentage of treg-cells, the treg/th17 ratio and a decrease in the th17 cell percentage. in ra, disease-specific indexes such as the das-28-esr, morning stiffness, tjc and sjc were reduced in one rct [31, 33] and were not affected in another [27] . in oa, results concerning the total womac score were not reported by the trialists [29, 32] . in fm, saffron ons did not affect the fiq among participants [30] . depression scores remained unchanged in individual trials in patients with ra [27] and fm [30] . ons with saffron failed to induce improvement in physical function among patients with ra (one rct) [27] . last, self-rated health assessment remained unchanged in ra post-intervention [27] , but was improved when assessed by the physicians using the pga in patients with ra [31, 33] . in the present systematic review, two [28, 29, 32] out of five rcts failed to report adverse events. the most frequently reported issues following saffron supplementation involved xerostomia, abdominal pain, vomiting, anxiety, palpitations, etc. figure 6 details the effect direction plot of the outcomes assessed in the included rcts. for the majority of outcomes, conflicting results are apparent. moreover, for most outcomes, less than three rcts have provided results regarding similar outcomes. nutrients 2021, 13, x for peer review 11 of 21 figure 6 . qualitative synthesis without meta-analysis of the outcomes in each rct, favoring the saffron arms post-intervention. all rcts had less than 50 participants in each arm. row background colors denote study quality according to the rob assessment. bdi, beck depression inventory [51] ; bpi, brief pain inventory [40] ; das-28, disease activity score 28 [47] ; esr, erythrocyte sedimentation rate; fiq, fibromyalgia impact questionnaire [48, 49] ; fm, fibromyalgia; foxp3, forkhead box protein p3; gfi, global fatigue index [44] ; gmfi, geometric mean fluorescence intensity; hads, hospital anxiety and depression scale [46] ; haq-di, health assessment questionnaire-disability index [42] ; hdrs, hamilton depression rating scale [45] ; hs-crp, high sensitivity c-reactive protein; ifn-γ, interferon-γ; mda, malondialdehyde; nr, not reported; nsaids, non-steroid anti-inflammatory drugs; oa, osteoarthritis; pga, physician global assessment [43] ; ra, rheumatoid arthritis; rct, randomized controlled trial; rob, risk of bias [24] ; sjc, swollen joint count; tac, total antioxidant capacity; th, t helper; tjc, tender joint count; treg, t regulatory; ‖ intervention duration <3 months; § intervention duration between 3-4 months; ¶ intervention duration 12 months;  increased;  reduced; no change. for the outcome of pain (vas), four rcts provided results, with two indicating a reduction in pain and two failing to reveal an effect. regarding the levels of esr, and tnfα, three and two of the included rcts, respectively, provided results. however, for both esr and tnf-α, the direction plot was similar in all, indicating a lack of effect following saffron supplementation in patients with ra and oa. due to the heterogeneity of the rcts and the lack of data regarding the standardization of the herbal medicine interventions, a meta-analysis was not deemed as a safe option. the present swim assessed the effects of supplementary crocus sativus l. intake on disease-related outcomes among patients with a rd diagnosis. it appears that limited rcts have been performed on this issue, thus demonstrating1 that the evidence is not enough to secure a positive direction of effect for any of the examined outcomes. moreofigure 6 . qualitative synthesis without meta-analysis of the outcomes in each rct, favoring the saffron arms postintervention. all rcts had less than 50 participants in each arm. row background colors denote study quality according to the rob assessment. bdi, beck depression inventory [51] ; bpi, brief pain inventory [40] ; das-28, disease activity score 28 [47] ; esr, erythrocyte sedimentation rate; fiq, fibromyalgia impact questionnaire [48, 49] ; fm, fibromyalgia; foxp3, forkhead box protein p3; gfi, global fatigue index [44] ; gmfi, geometric mean fluorescence intensity; hads, hospital anxiety and depression scale [46] ; haq-di, health assessment questionnaire-disability index [42] ; hdrs, hamilton depression rating scale [45] ; hs-crp, high sensitivity c-reactive protein; ifn-γ, interferon-γ; mda, malondialdehyde; nr, not reported; nsaids, non-steroid anti-inflammatory drugs; oa, osteoarthritis; pga, physician global assessment [43] ; ra, rheumatoid arthritis; rct, randomized controlled trial; rob, risk of bias [24] ; sjc, swollen joint count; tac, total antioxidant capacity; th, t helper; tjc, tender joint count; treg, t regulatory; intervention duration <3 months; § intervention duration between 3-4 months; increased; reduced; thesis without meta-analysis of the outcomes in each rct, favoring the saffron arms post-inters than 50 participants in each arm. row background colors denote study quality according to the k depression inventory [51] ; bpi, brief pain inventory [40] ; das-28, disease activity score 28 [47] ; tation rate; fiq, fibromyalgia impact questionnaire [48, 49] ; fm, fibromyalgia; foxp3, forkhead al fatigue index [44] ; gmfi, geometric mean fluorescence intensity; hads, hospital anxiety and q-di, health assessment questionnaire-disability index [42] ; hdrs, hamilton depression rating sensitivity c-reactive protein; ifn-γ, interferon-γ; mda, malondialdehyde; nr, not reported; i-inflammatory drugs; oa, osteoarthritis; pga, physician global assessment [43] ; ra, rheumaomized controlled trial; rob, risk of bias [24] ; sjc, swollen joint count; tac, total antioxidant c, tender joint count; treg, t regulatory; ‖ intervention duration <3 months; § intervention dura-; ¶ intervention duration 12 months;  increased;  reduced; no change. for the outcome of pain (vas), four rcts provided results, with two indicating a reduction in pain and two failing to reveal an effect. regarding the levels of esr, and tnfα, three and two of the included rcts, respectively, provided results. however, for both esr and tnf-α, the direction plot was similar in all, indicating a lack of effect following saffron supplementation in patients with ra and oa. due to the heterogeneity of the rcts and the lack of data regarding the standardization of the herbal medicine interventions, a meta-analysis was not deemed as a safe option. the present swim assessed the effects of supplementary crocus sativus l. intake on no change. for the outcome of pain (vas), four rcts provided results, with two indicating a reduction in pain and two failing to reveal an effect. regarding the levels of esr, and tnf-α, three and two of the included rcts, respectively, provided results. however, for both esr and tnf-α, the direction plot was similar in all, indicating a lack of effect following saffron supplementation in patients with ra and oa. due to the heterogeneity of the rcts and the lack of data regarding the standardization of the herbal medicine interventions, a meta-analysis was not deemed as a safe option. the present swim assessed the effects of supplementary crocus sativus l. intake on disease-related outcomes among patients with a rd diagnosis. it appears that limited rcts have been performed on this issue, thus demonstrating1 that the evidence is not enough to secure a positive direction of effect for any of the examined outcomes. moreover, serious pitfalls regarding the reporting of the intervention formulas are apparent, further reducing the quality of the trials. consumption of saffron can reduce inflammation through inhibition of the cyclooxygenase enzyme activity [52] . according to a recent meta-analysis [9] , saffron is effective in improving the levels of inflammatory markers such as tnf-α, il-6 and crp when administered at specific doses (≤30 mg/day) in young adults (<50 years old) lacking a diabetes diagnosis. in the present review, only four trials administered a dose not exceeding 30 mg/day [28, 30] , with only one [28] evaluating inflammatory markers among participants. interestingly, crp and il-17 were improved in this trial post-intervention. thus, it is possible that the higher doses administered in the rest of the trials [29, [31] [32] [33] might have produced a negative or null effect. nevertheless, another meta-analysis [9] failed to detect any differences regarding crp, tnf-α, and il-6 between the saffron and placebo arms. these discrepancies, however, may lay in the underline pathologies of the participants, the duration of interventions, or differences in the standardization of the administered supplements. research indicates that saffron can reduce the concentrations of endogenously generated reactive oxygen species, inhibiting oxidative damage, while reducing the production of pro-inflammatory biomarkers [9, 53] . according to a recent meta-analysis [54] , supplementation can induce improvements in the mda and tac levels. however, no improvements were revealed in the present swim, due to the small number of studies evaluating these outcomes, most of which indicated a null effect. depression and anxiety are common problems in patients with chronic disease and rheumatic disease in particular. moreover, recent meta-analyses indicate that ons with crocus sativus l. may improve depressive symptoms and anxiety [12, 55] . this effect is persistent even when used as an adjunct to antidepressants, as in the present rcts. moreover, specific depression batteries such as the bdi appear to be more sensitive to saffron ons, whereas the hdrs has been reported to be less flexible [56] . saffron has been suggested to entail relaxant, inhibitory effects on both histamine (h1) and the muscarinic receptors [57] . by inducing relaxation and reducing anxiety, supplementation with crocus sativus can also improve sleep quality [58] . on the other hand, improved sleep is associated with less fatigue. overall, previous evidence synthesis indicates that saffron is more efficient compared to placebo and additionally equally effective with synthetic antidepressants [59, 60] . these findings, however, were not akin to the present swim due to the probable methodological pitfalls of the included trials, heterogeneity and lack of information regarding the standardization of the intervention formulations. regarding pain, no meta-analyses have evaluated the effect of saffron ons, although individual rcts performed on patients with distinct diagnoses indicate possible improvement in the sensation of pain [61] . according to research, the dried stigmas and tops of the plant styles have the majority of medicinal properties, including immunomodulating responses. saffron contains a variety of mineral agents, glycosides, anthocyanins, alkaloids, carotenoids and flavonoids including quercetin and kaempferol, which further increase its immunoregulatory properties [62, 63] . studies using animal models have revealed that saffron acts on selective th2 upregulation, naming it a "nutraceutical" spice [64] . other preclinical and animal studies showed that saffron can increase the expression levels of foxp3, a transcriptional factor, in treg cells, and suppress il-10 and ifn-γ secretion [65] [66] [67] . in the present swim, only one trial [28] evaluated immune response post-saffron supplementation, indicating improved immunomodulation. however, further studies are required, assessing similar outcomes. according to research, treatment adherence in clinical trials is suboptimal, affecting the economic costs, while impacting the methodological quality of the trials [68] . nearly half of the rcts involving oral pharmacological interventions failed to report adherence rates [69] , indicating that proper adherence consideration is the exception instead of the rule [68] . in the present review 40% (n = 2) of the included rcts reported assessing treatment adherence, although the exact rates were not presented. moreover, none of the trials adhered to the espacomp medication adherence reporting guideline (emerge) reporting guidelines regarding treatment adherence assessment [68] . a high non-adherence rate can reduce a trial's ability to detect a true treatment effect [70] . if adherence was considered and reported, the results regarding crocus sativus l. supplementation in rds might have been different. the standard treatment of participants was not reported in all trials. in the sahebari et al. rct [27] , vitamin d ons was among the standard therapy received by the participants and changes the sensation of pain was one of the outcomes of interest. although pain was improved post-saffron administration [27] , the scientific literature indicates that vitamin d might influence immune cells and pain sensitization through a variety of hormonal and neurological pathways [71, 72] . thus, the improved pain sensation noted in the trial might well be the synergistic result of vitamin d and crocus sativus l. similarly, in the trial conducted by poursamimi and associates [28] , as the authors promptly noted, the improved pain relief observed may be the result of sodium diclofenac, which was administered to all participants during the trial. for this, significant improvements regarding pain were noted in both arms [28] . in the present swim, rcts performed in patients with an oa diagnosis were also included, as often, patients with ra are misdiagnosed with oa, and vice versa [22] . thus, it is possible that some of the patients included in the trials might have belonged in the opposite diagnosis, despite recruitment intentions. among the included rcts, the one conducted by sahebari and associates [27] was the only one where the anti-cyclic citrullinated peptide (anti-ccp)-positive patients were assessed within the sample, reporting that 89.2% of those allocated in the intervention and 81.6% of the controls were positive. the remaining ra/oa trials [28, 29, [31] [32] [33] failed to address this issue. since this is a common problem in arthritis research, including both diagnosis without merging them was deemed as the safest option for the swim. lifestyle has an impact on disease activity and outcomes in patients with rds. in further detail, exercise can reduce disease activity and diet can either improve or amplify symptoms related to the diseases [73] [74] [75] [76] [77] . for this, the diet of participants in each rct with ons interventions must be recorded, and in parallel, physical activity should also be monitored. among the included rcts, however, only one [31, 33] evaluated the diet of participants and their physical activity levels. the remaining failed to control for this important factor, introducing bias to their results. as ali [78] noted, herbal medicines tend to suffer from lack of standardization parameters. in more detail, there appears to be a lack of standardization regarding the raw materials used, the harvesting, drying, storage and processing methods, as well as the final products and dosage formulation [16, 79] . moreover, quality control procedures are inexistent in most of the trials [79] . according to the world health organization (who), all medicines, whether they are of plant origin or synthetic, must fulfill the basic requirements of safety and effectiveness [16, 80] . nevertheless, it appears that trials implementing herbal medicine interventions often fail to report information required to judge internal validity, external validity, and reproducibility [17, 18] . from the bush to the content of a pill, herbal substances undergo a variety of procedures that define the final product's active ingredients and may greatly affect efficacy. as a result, most frequently, batch-to-batch uniformity of the active constituents and quality control using various analytical techniques are inexistent [81] , leading to substantial variations in the formulation and bioactivity of herbal medicine supplements from lot to lot [82] , and it is unclear if single and consistent batches are used for the formulations applied in the trials. moreover, the need to quantify the test substance using high-performance liquid chromatography, gas chromatography, or other techniques is required to understand the exact dose of active ingredient that produces a significant effect [81] . according to guo [83] , the often non-standardized nature of the prepared interventions increases the probability for adverse events, indicating that in all cases of rcts with herbal medicine, standards of safety and efficacy must be implemented. today, poor reporting of adverse events consists of a frequent criticism regarding cam research [84, 85] and in the present systematic review 2/3 of the rcts failed to report any adverse reactions. moreover, serious adverse events have been reported by the fda; however, as they are rare, they often fail to be manifested in small or underpowered rcts [82] . apart from the consort for herbal medicine interventions [17] , a variety of additional guidelines have been published with regard to quality standards and good clinical practice in herbal medicine trials, including who recommendations and international union of pure and applied chemistry (iupac) protocols [86] [87] [88] [89] . furthermore, information regarding fingerprinting analyses for the quality assessment of herbal medicine have also been proposed for interested stakeholders [90] . in the present systematic review, it was shown that regarding rcts with saffron interventions in patients with rds, the majority failed to adhere to the consort-specific requirements for herbal medicine interventions. similar issues have also been reported to exist in cochrane systematic reviews evaluating herbal medicine [91] . for this important limitation, despite the plethora of meta-research evaluating herbal medicine interventions that have been published in high-end academic journals without considering this limitation [84, 92] , we considered that any quantitative synthesis would be misleading for the authors and clinical practice, and was avoided. the duration of the intervention varied greatly in the included rcts, spanning from as low as 8 weeks [30] to 4 months [28] . it is possible that a longer intervention duration might have changed the results in several trials, as other trials administering saffron for other conditions have, in their majority, applied the interventions for 3-4 months [9, 15, 93] , with a respective follow-up session. moreover, according to a recent meta-analysis, longer saffron supplementation durations have been shown to improve outcomes with regard to blood pressure [15] . suffice to say, the exact intervention duration required to produce beneficial effects for each outcome has not yet been delineated. all trials included in the present swim were conducted in iran. today, 80% of the global saffron production is harvested from iran, and this is why iranian researchers are keen on investigating the plant's properties [57] . nevertheless, according to an umbrella review [94] , when studying the available literature, the need to conduct higher-quality trials outside of iran becomes apparent, in order to reduce bias. figure 7 details the ongoing trials investigating the effect of saffron in patients with rds. a total of four rcts were identified in the iranian registry of clinical trials (irct) and none in the clinicaltrials.gov database. these trials are recruiting patients with bs, ra or fm, investigating similar outcomes as in the present review. their results are expected to aid in understanding the possible results of saffron supplementation among patients with rheumatic diseases. beneficial effects for each outcome has not yet been delineated. all trials included in the present swim were conducted in iran. today, 80% of the global saffron production is harvested from iran, and this is why iranian researchers are keen on investigating the plant's properties [57] . nevertheless, according to an umbrella review [94] , when studying the available literature, the need to conduct higher-quality trials outside of iran becomes apparent, in order to reduce bias. the limitations of the present qualitative synthesis primarily involve the lack of an adequate number of trials investigating similar outcomes in distinct rds. furthermore, a gap in the literature is apparent, with null saffron rcts conducted for specific rds (psoriasis, sle, ankylosing spondylitis, sjogren's syndrome, etc.) as in every meta-research, the present review also carries the limitations of the included trials, indicating that there is room for the methodological improvement of rcts investigating saffron in rds. interestingly, most of the included trials failed to assess and report changes in disease-activity specific scores (e.g., womac), an issue that should be accounted for when designing future trials. moreover, the high clinical and methodological heterogeneity among the included trials did not allow for a meta-analysis to be performed. according to a recent umbrella systematic review [94] , rcts evaluating saffron interventions entail a variety of biases, and their methodology should be improved. the need for evaluating herbal medicine interventions is indisputable. today, it is estimated that 2/3 of the global population uses herbal medicines, with some countries having incorporated them into the public health system [88] . nevertheless, serious doubts regarding their safety and effectiveness remain [95] . according to ernst and pittler [96] , the majority of studies published in cam journals report positive findings and the concerns regarding the variation in formulation and bioactivity of some supplements remain a challenge [82] . as suggested by the european research network for cam [97] , cam constitutes a neglected research area requiring more activities; however, specific standards of reporting must be met in advance. although the assessment of the adherence to the consort guidelines for the conduction and reporting of herbal medicine rcts was not included in the initial aims of the present systematic review or the protocol, during the peer review process, it became clear that this issue constitutes an important factor affecting trial quality and intervention efficacy. this additional analysis added value to the present review, highlighting an area in need of improvement regarding the reporting of these trials. pedanio dioscorides, an ancient greek medical practitioner, was the first to report the medicinal properties of saffron [52, 57] . in an extensive review of the history and the literature, christodoulou [57] underlined the value of saffron over the centuries, with the "saffron war" taking place in the middle ages and the execution of those who dared to tamper with saffron's composition due to its medicinal properties. today, in the era of evidence-based medicine, whether this value can also be evidence-based greatly depends on the appraisal of the existing primary studies. research has suggested that crocus sativus can form an effective adjuvant therapy for many conditions, and a promising one for rds. rcts performed in patients with rds indicate that saffron may target many different outcomes, including inflammation, antioxidant status, depression and anxiety, pain, immune response and many others. if its efficacy is demonstrated, then it will undoubtedly be the "golden spice" for rds. nevertheless, at the moment, more primary studies are required to help us find the appropriate dose and conclude with certainty on the efficacy of saffron ons in rheumatic diseases-related outcomes. furthermore, the present systematic review raised concerns regarding the importance of reporting standards in herbal medicine research, with chemical fingerprinting being a required prerequisite for the standardization, safety and efficacy evaluation of the active ingredients. institutional review board statement: ethical review and approval were waived for this study, as this is a meta-research. deaths, and disability-adjusted life years due to musculoskeletal disorders for 195 countries and territories the population impact of rheumatic and musculoskeletal diseases in relation to other non-communicable disorders: comparing two estimation approaches herbal medicine in the treatment of rheumatic diseases herbal medicine for rheumatic diseases: promises kept? systematic review nutritional interventions and supplementation for rheumatoid arthritis patients: a systematic review for clinical application, part 3: fruits and herbs efficacy of spice supplementation in rheumatoid arthritis: a systematic literature review oral adjuvant curcumin therapy for attaining clinical remission in ulcerative colitis: a systematic review and meta-analysis of randomized controlled trials saffron: potential health benefits effects of saffron (crocus sativus l.) supplementation on inflammatory biomarkers: a systematic review and meta-analysis analgesic and anti-inflammatory effects of crocus sativus l. (saffron). in bioactive nutraceuticals and dietary supplements in neurological and brain disease: prevention and therapy saffron supplementation effects on glycemic indices: a systematic review and meta-analysis of randomized controlled clinical trials effect of saffron supplementation on symptoms of depression and anxiety: a systematic review and meta-analysis saffron for mild cognitive impairment and dementia: a systematic review and meta-analysis of randomised clinical trials saffron (adjunct) for people with schizophrenia who have antipsychoticinduced metabolic syndrome the effect of saffron supplementation on blood pressure in adults: a systematic review and dose-response meta-analysis of randomized controlled trials standardization of herbal medicines-a review reporting randomized, controlled trials of herbal interventions: an elaborated consort statement herbal medicine for low back pain: a cochrane review the prisma 2020 statement: an updated guideline for reporting systematic reviews synthesis without meta-analysis (swim) in systematic reviews: reporting guideline rayyan-a web and mobile app for systematic reviews ab0378 osteoarthritis is the most frequent cause of rheumathoid arthritis misdiagnosis in a colombian specialized center curcumin, the golden spice from indian saffron, is a chemosensitizer and radiosensitizer for tumors and chemoprotector and radioprotector for normal organs rob 2: a revised tool for assessing risk of bias in randomised trials the effect direction plot: visual display of non-standardised effects across multiple outcome domains synthesizing and presenting findings using other methods a double-blind placebo-controlled randomized trial of oral saffron in the treatment of rheumatoid arthritis immunoregulatory effects of krocina tm , a herbal medicine made of crocin, on osteoarthritis patients: a successful clinical trial in iran anti-inflammatory effect of saffron in knee osteoarthritis: a double blind clinical trial crocus sativus) versus duloxetine for treatment of patients with fibromyalgia: a randomized double-blind clinical trial the effect of saffron supplement on clinical outcomes, inflammatory and oxidative markers in patients with active rheumatoid arthritis pt3:026 the effectiveness of saffron pill in treatment of osteoarthritis: a randomized, double-blind controlled trial the effect of saffron supplement on clinical outcomes and metabolic profiles in patients with active rheumatoid arthritis: a randomized, double-blind, placebo-controlled clinical trial rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative what is rheumatoid arthritis? considering consequences of changed classification criteria development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee the american college of rheumatology preliminary diagnostic criteria for fibromyalgia and measurement of symptom severity rheumatoid arthritis disease activity measures: american college of rheumatology recommendations for use in clinical practice pain assessment: global use of the brief pain inventory-pubmed clinical evaluation of the womac 3.0 oa index in numeric rating scale format using a computerized touch screen version development and validation of the health assessment questionnaire ii: a revised version of the health assessment questionnaire using the physician global assessment in a clinical setting to measure and track patient outcomes comparison of self-reported fatigue in rheumatoid arthritis and controls-pubmed a rating scale for depression the hospital anxiety and depression scale validation of the 28-joint disease activity score (das28) and european league against rheumatism response criteria based on c-reactive protein against disease progression in patients with rheumatoid arthritis, and comparison with the das28 based on erythrocyte sedimentation rate the fibromyalgia impact questionnaire: development and validation-pubmed the validity and reliability of the persian version of the revised fibromyalgia impact questionnaire international physical activity questionnaire: 12-country reliability and validity beck depression inventory saffron: a potential drug-supplement for severe acute respiratory syndrome coronavirus (covid) management crocin prevents sesamol-induced oxidative stress and apoptosis in human platelets effect of saffron supplementation on oxidative stress parameters: a systematic review and meta-analysis of randomized placebo-controlled trials the efficacy of saffron in the treatment of mild to moderate depression: a meta-analysis the effects of saffron (crocus sativus l.) on mental health parameters and c-reactive protein: a meta-analysis of randomized clinical trials saffron: a natural product with potential pharmaceutical applications effects of saffron extract on sleep quality: a randomized double-blind controlled clinical trial safety and efficacy of saffron (crocus sativus l.) for treating mild to moderate depression: a systematic review and meta-analysis the efficacy of crocus sativus (saffron) versus placebo and fluoxetine in treating depression: a systematic review and meta-analysis comparison of the effects of crocus sativus and mefenamic acid on primary dysmenorrhea flavonols from saffron flower: tyrosinase inhibitory activity and inhibition mechanism saffron (crocus sativus) petal as a new pharmacological target: a review selective th2 upregulation by crocus sativus: a neutraceutical spice crocetin downregulates the proinflammatory cytokines in methylcholanthrene-induced rodent tumor model and inhibits cox-2 expression in cervical cancer cells crocetin activates foxp3 through tipe2 in asthma-associated treg cells a qualitative and quantitative comparison of crocus sativus and nigella sativa immunomodulatory effects how the emerge guideline on medication adherence can improve the quality of clinical trials adherence reporting in randomized controlled trials medication adherence in randomized controlled trials evaluating cardiovascular or mortality outcomes in dialysis patients: a systematic review vitamin d and pain: vitamin d and its role in the aetiology and maintenance of chronic pain states and associated comorbidities vitamin d and its potential interplay with pain signaling pathways. front let food be thy medicine: the case of the mediterranean diet in rheumatoid arthritis is tea consumption associated with reduction of risk of rheumatoid arthritis? a swedish case-control study improvement of inflammation and pain after three months' exclusion diet in rheumatoid arthritis patients curcumin mediates attenuation of pro-inflammatory interferon γ and interleukin 17 cytokine responses in psoriatic disease, strengthening its role as a dietary immunosuppressant do interventions with diet or dietary supplements reduce the disease activity score in rheumatoid arthritis? a systematic review of randomized controlled trials need of standardization of herbal medicines in modern era who guidelines on safety monitoring of herbal medicines in pharmacovigilance systems; world health organization challenges and guidelines for clinical trial of herbal drugs challenges in systematic reviews of complementary and alternative medicine topics a systematic review of randomised clinical trials of individualised herbal medicine in any indication an overview of systematic reviews of complementary and alternative therapies for fibromyalgia using both amstar and robis as quality assessment tools first, do no harm" with complementary and alternative medicine operational guidance: information needed to support clinical trials of herbal products extension for chinese herbal medicine formulas 2017: recommendations, explanation, and elaboration (traditional chinese version) general guidelines for methodologies on research and evaluation of traditional medicine; world health organization annex 1 who guidelines for selecting marker substances of herbal origin for quality control of herbal medicines; world health organization quality assessment of herbal medicines based on chemical fingerprints combined with chemometrics approach: a review reporting quality of cochrane systematic reviews with chinese herbal medicines complementary and alternative medicine therapies for psoriasis: a systematic review kyrou, i. saffron (crocus sativus l.) in ocular diseases: a narrative review of the existing evidence from clinical studies saffron (crocus sativus l.) and health outcomes: a meta-research review of meta-analyses and an evidence mapping study herbal medicine in healthcare-an overview alternative therapy bias research network for complementary and alternative medicine (cam) final report summary data availability statement: all data are presented within the manuscript text. the authors declare no conflict of interest. iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 45 quantitative and qualitative analysis of plumbagin in the leaf and root of plumbago europaea growing naturally in kurdistan by hplc hazhar m. muhammad * , kawkab y. saour ** , and alaadin m. naqishbandi *, 1 * department of pharmacognosy, college of pharmacy, hawler medical university, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad ,iraq. abstract plumbago (plumbaginaceae) is a genus of 10-20 species of flowering plants used in traditional indian medicine, native to warm temperature to tropical regions of the world. the roots of plumbago europaea, the iraqi species of plumbago, have been used for the treatment of cancer, rheumatoid arthritis, and dysmenorrhea. the main active constituents from dried powdered leaves and roots of plumbago europaea were extracted by soxhlet apparatus using ethyl acetate, the main active constituent was characterized by spectroscopic analysis (ir, 1 h nmr, and 13 c nmr) as plumbagin. quantitative and qualitative study of plumbagin in the roots and leaves extracts was carried out by hplc technique using analytical column (eurospher 100, c18, 5 µm, 250 x 4.6 mm) with 10% solvent (b) isocratic elution of methanol (solvent a) and water (solvent b) at flow rate 0.75 ml/min and detection wave length of 270 nm. the percentage of plumbagin in the root and leaf extracts was recorded to be (1.9 %) and (1.5 %) respectively. keywords: plumbago europaea, plumbagin, hplc الخالصة انهُذ, يًُى بصىسة طبيؼيت في يٍ اَىاع انُباحاث انضهشيت انًسخخذيت في انطب انشؼبى فى plumbago 01-01يضى جُس انسشطاٌ ػالج في plumbago europaeaيسخؼًم جزوس انُىع انؼشالي .انًُاطك انحاسة انى انًُاطك االسخيىائيت يٍ انؼانى نك ور plumbago europaea ت لجزوس انًجففانألوساق ونانكيًيائيت انشئيسيت ى اسخخالص انًكىَاثح .فث،انشوياحيضو و طًث انش ) ححهيم األطيافسخخذاو اانكيًيائيت انشئيسيت انًؼضونت ب انًادةحى حشخيض .وانًزيب أثيم اسيخيج soxhletباسخخذاو جهاص 13 c nmr , 1 h nmr ,ir بًادة ) plumbagin . نًادة َىػيت و كًيت دساستاء اجشحى plumbagin االوساق و نًسخخهصاث في ا eurospher 100س )ـــــــــىد انًؼاكــــذاو انؼًـــ( باسخخhplcباسخخذاو حمُيت كشوياحىغشافيا ححج انضغظ انؼاني ) انجزوس ,c18 column ,(4.6 mm i.d. x 250 mm, 5 µm :انً ييخاَىلو ححج انظشوف انخانيت( زيبaو انًاء ) انًمطش (انًزيب b ) ت انفىق انبُفسجيت . حى انكشف باألشؼ .ml/min 1..4 و سشػت جشياٌ b% يٍ انًزيب 01كًحهىل َالم ورنك بانغسم انثابج يغ ػهى % 0.4% و 0.1انجزس وانىسق نًسخخهصيٍ في كم يٍ ا plumbagin كاَج انُسب انًؤيت نًادة .nm 0.1بانطىل انًىجي نخىاني .ا introduction plumbago (plumbaginaceae) is a genus of 10-20 species of flowering plants, native to warm temperature to tropical regions of the world (1) . roots of plumbago species are used in traditional indian medicine, immunosuppressive and antitumour activities have been demonstrated (2) . plumbago europaea have been used extensively in china and other asian countries for treatment of cancer, rheumatoid arthritis, dysmenorrheal (3) . the chemical profile of the plumbago genus is marked by the presence of naphthoquinones, flavonoids and terpenoids (4) . plumbago europaea is naturally occurring plant in kurdistan region (kurdish name: rashky kalak) traditionally used for wart skin infection, hence the aim of this research works directed towards qualitative and quantitative analysis of the main active constituents in the leaf and root of the plumbago europaea by hplc. 1 corresponding author e-mail : alaadinmn@hotmail.com received : 20/1/2009 accepted : 16/6/2009 iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 44 materials and methods plant materials plumbago europaea leaves and roots were collected from susae village kurdistan region in iraq during june 2007, authenticated by the department of biology, college of education, university of salahaddin. extraction of the active constituents plumbago europaea leaves and roots were collected, dried in air for seven days and powdered separately with mechanical grinder. 25 gm of dried powdered leaves and roots were extracted separately in the soxhlet with 400 ml ethylacetate for 5 hr. the extracts were evaporated in vacu by rotary evaporator to yield 2.134 gm of dark green color residue of total leaf extract (p1) and 1.1gm of orange yellow color residue of total root extract (p2). quantitative separation of the major active constituents by tlc the major constituent in p1 and p2 extracts was isolated quantitatively using preparative tlc. fifteen gm of silica gel gf254 was mixed with 30 ml distilled water to prepare the slurry which spread on one glass plate of (20 x 20) cm by desaga spreader to obtain 0.75 mm thickness layer of silica gel sorbent (5) . the plates were activated for 1 hr in oven at 110 ºc before use. the mobile phase used was (toluene: ethyl acetate (93:7)). development of tlc plates four hundred mg of p1 and p2 extracts were dissolved separately in 10 ml of ethyl acetate and applied as a line by a capillary tube on silica gel plate (25 plates) fore each extract. one major band which was detected visually and under uv 366 in the plates of p1 and p2 extracts, scraped off from the plates and silica gel containing the isolated major band was dissolved separately in chloroform: ethanol (3:1). the mixtures were filtered through buckner funnel and the filtrates obtained from p1 and p2 extracts were evaporated to dryness in vacue to get constituent (a). the main active constituent (a) was characterized by spectroscopic analysis (ir, 1 h nmr, and 13 c nmr). hplc analysis hplc technique was used for qualitative and quantitative study of plumbagin in p1 and p2 extracts obtained from plumbago europaea using analytical column (eurospher 100, c18, 5 µm, 250 x 4.6 mm), mobile phase used was methanol: water (90:10), flow rate of 0.75 ml/min, injection volume of 20 µl, detection wave length was set to 270 nm, and temperature adjusted to 33 ºc. sample preparation used in hplc analysis the sample extracts from plumbago europaea were prepared by extraction of 1 g of dried powdered leaf and root separately with 35 ml ethylacetate for two hours by refluxing two times, the extracts were filtered, and evaporated to dryness by rotary evaporator, and dissolved separately in 5 ml methanol, filtered through 0.45 µm membranes before injection to the hplc. the knauer hplc instrument equipped with chromgate software provided by knauer was used for this analysis. the calibration curve was plotted using single level calibration, made by preparation of solution (1mg/ml) of standard plumbagin (sigma aldrich, usa) in methanol. the calibration graph was obtained from chromo gate software, figure (1). figure (1): calibration curve of authentic plumbagin. iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 45 results the major band on the chromatogram corresponding to authentic standard compound was isolated in both chromatograms of p1 and p2 extracts, figure (2). the purity of the isolated constituents was confirmed by tlc.the ir spectrum for the isolated constituent (a) figure (3), showed stretching vibration bands at (3419, 3000, 1725-1710, 1648 and 1611) cm -1 . 1 h-nmr spectrum figure(4), as shown in cdcl3 showed a singlet peak at (3.323-3.332) δ, two doublet strong bands at (6.68 and 7.054) δ, multiplate peaks at (7.314) δ , and a singlet peak at (7.613). while 13 c nmr figure (5), the data were 46.744, 47.028, 47.312, 47.596, 47.880, 48.163, 48.447, 116.104, 118.157, 120.151, 121.891, 125.726, 125.799, 126.332, 128.365, 129.370, 132.088, 146.423, and 150.581. hplc chromatogram of p1 and p2 extracts indicated the presence of plumbagin by comparing the retention time with that of the standard plumbagin (rt 6.567), figure (6), and the concentration of plumbagin was calculated by using chromgate software depending on the calibration curve of the standard plumbagin. the results showed higher concentration of plumbagin in the root part of the plant than in the leaf, table (1). (a) (b) figure (2) tlc chromatogram of preparative separation of the major constituent from a-p1 extract, bp2 extract. figure (3): ir spectrum of isolated constituent (a). iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 4. figure (4): 1 h nmr spectrum of isolated constituent (a). figure (5): 13 c nmr spectrum of isolated constituent (a). iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 45 (a) (b) (c) figure (6): hplc chromatogram, aleaves extracts (p1), broots extracts (p2), cstandard plumbagin minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ua u -200000 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 ua u -200000 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 p lu m b a g in 6 .5 6 7 1 .0 0 0 s 2500 plumbagin authentic name retention time estd concentration minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 u a u 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 u a u 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 p lu m b a g in 0 .0 1 5 7 1 8 8 0 7 s 2500 leave sample3 name estd concentration area minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ua u -5000 0 5000 10000 15000 20000 25000 30000 ua u -5000 0 5000 10000 15000 20000 25000 30000 p lu m b a g in 0 .0 1 9 8 6 9 6 0 1 s 2500 root sample3 name estd concentration area iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 41 table (1): quantitative study of plumbagin in p1 and p2 extracts by hplc discussion preparative tlc technique was used for separation and isolation of the major constituent in p1 and p2 extracts. the spectroscopic data (ir, 1 h nmr, and 13 c nmr) obtained confirmed the chemical structure of the isolated constituent (a) to contain the important functional groups of plumbagin which agrees with the data obtained for the same compound in other research works (6, 7, 8) . hplc technique was used successfully for this study; different conditions of hplc were used for qualitative and quantitative analysis of plumbagin in other species of the genus plumbago (9, 10) . plumbagin was identified qualitatively and quantitatively in p1 and p2 extracts by comparing the retention time with that of the standard sample. the result indicates that the hplc method was efficient for qualitative identification and quantitative determination of plumbagin. references 1. huxley a., ed. (1992), new horticultural society dictionary of gardening, vols.4, macmillan. 2. evans wc (2005): treace and evans pharmacognosy, 15 th ed. london, uk, w.b. saunders company. 3. binita b. chaplot., ashok m., dave and yogesh t., jasrai (2006), a valued medicinal plant-chitrak (plumbago zeylanica l.): successful plant regeneration through various explants and field performance, plant tissue culture and biotechnology, 16(2), 77-84. 4. selma r. de paiva., lucilene a. lima., maria raquel figueiredo and maria axiliadora c. kaplan (2004),plumbagin quantifications in roots of plumbago scandens l.obtained by different extraction techniques, annals of the brazilian academy of sciences, 76(3) 499-504. 5. joseph c. touchstone and murrell f.dobbins (1977), practical of thin layer chromatography, awilly-interscience publication, pages 25, 42,311. 6. nisha mathew., paily k.p., abidha p., kalyanasundaram m., balaraman k. (2002). macrofilaricidal activity of the plant plumbago indica/rosea, drug development research, 56(1), 33-39. 7. nayana s. kapadia., shalini a. isarani., mamta b. shah (2005),a simple method for isolation of plumbagin from roots of plumbago rosea, pharmaceutical biology,43 (6), 551-553. 8. kwang-soo s., samkeun l. and byeongjin c. (2007), antifungal activity of plumbagin from leaves of nepenthes ventricosa x maxima against phytopathogenic fungi, plant pathology journal, 23(2), 113-115. 9. yuan l., fanq d., chao l., qing-yan m., ze-wen g. (2006), determination of plumbagin in different parts of plumbago zeylanica by rp-hplc, zhongguo zhong yaoza zhi. 31(20), 1684-1686. 10. mei z.-n., zhao y.-h., li x.-k. (2007), hplc determination of plumbagin in different sections of plumbago zeylanica linn. from previous sources of yunnan, chinese journal f pharmaceutical analysis, 27(6), 901-903. no extract area under the curve of samples area under the curve of standard plumbagin concentration (%) 1 p1 718807 46608959 1.5 2 p2 896601 1.9 iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis doi: https://doi.org/10.31351/vol28iss2pp134-141 134 belief about medicines among a sample of iraqi patients with rheumatoid arthritis mirna k. faiq *,1, dheyaa j. kadhim** and faiq i. gorial *** * department of clinical pharmacy, college of pharmacy, university of tikrit, salah-din, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. *** rheumatology unit, department of medicine, college of medicine, university of baghdad, baghdad, iraq. abstract rheumatoid arthritis is a chronic, progressive, inflammatory autoimmune disease of unidentified etiology, associated with articular, extra-articular and systemic manifestation that requires long-standing treatment. taking patient’s beliefs about the prescribed medication in consideration had been shown to be an essential factor that affects adherence of the patient in whom having positive beliefs is an essential for better adherence. the purpose of the current study was to measure beliefs about medicines among a sample of iraqi patients with rheumatoid arthritis and to determine possible association between this belief and some patient-certain factors. this study is a cross-sectional study carried out on 250 already diagnosed rheumatoid arthritis patients who attended baghdad teaching hospital/medical city/ rheumatology department. the mean age of the patients was (50.8 ± 13.1 years). belief about medicines was measured via the arabic version of the beliefs about medicines questionnaire. the majority of the patients (88%) had strong beliefs in the necessity of treatment (specific-necessity score greater than specific-concern). there was a significant direct correlation between age, male gender, number of other chronic diseases, disease activity score 28 and clinical disease activity index with specific necessity, and direct correlation between clinical disease activity index with specific concern. future studies should investigate how interventional approaches addressing these predictors may lead to improve beliefs about medicines among rheumatoid arthritis patients and their impression on disease control. keywords: rheumatoid arthritis, beliefs about medicines, specific necessity, specific concern. عن االدوية لدى عينة من مرضى التهاب المفاصل الرثوي العراقيلمعتقدات ا ميرنا كفاح فائق * ،1 ، ضياء جبار كاظم** و فائق ايشو كوريال*** تكريت ، صالح الدين ، العراق. فرع الصيدلة السريرية ،كلية الصيدلة ، جامعة* ** فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق. ***وحدة أمراض المفاصل ،فرع الطب ، كلية الطب ، جامعة بغداد، بغداد، العراق. الخالصة أن التهاب المفاصل الرثوي هو مرض مزمن ومتفاقم يصيب جهاز المناعة غير معروف السبب يصاحب بتأثيرات مفصلية وخارج مفصلية لى ع وجهازية والتي تتطلب عالج طويل المدى. أن أخذ معتقدات المريض حول الدواء الموصوف بعين االعتبار يعد واحد من أهم العوامل التي تؤثر عالج .حيث أن المعتقدات اإليجابية حول االدوية تعد عامال أساسيا اللتزام المريض بالعالج . ان الهدف من الدراسة الحالية هو تقييم االلتزام بال .المعتقدات حول االدوية لدى مرضى التهاب المفاصل الرثوي وتحديد االرتباط المحتمل بين هذا االعتقاد وبعض العوامل الخاصة بالمريض مريض تم تشخيصهم سابقا بمرض التهاب المفاصل الرثوي الذين حضرو الى مستشفى 052اسة الحالية هي دراسة مستعرضة أجريت على الدر سنة(. تم تقييم المعتقدات عن االدوية باستخدام النسخة 1.01± 5205بغداد التعليمي /مدينة الطب /قسم امراض المفاصل . متوسط عمر المرضى ) ( معتقدات قوية في ضرورة العالج )معيار ضرورة العالج كان أكبر 55استبيان المعتقدات عن االدوية. كان لدى غالبية المرضى )%العربية من من معيار القلق من األدوية(. كانت هناك عالقة طردية بين العمر وجنس الذكور و عدد االمراض المزمنة االخرى ودرجة نشاط المرض ومؤشر جث ان تب ري مع معيار ضرورة العالج . وارتباط طردي بين مؤشر نشاط المرض السريري مع معيار القلق من األدوية. يجبنشاط المرض السري يمكن للتداخالت المهتمة بهذه العوامل أن تحسن من المعتقدات حول االدوية لدى مرضى التهاب المفاصل الرثوي وتأثير الدراسات المستقبلية كيف ى المرضذلك على السيطرة عل القلق من العالج. ،ضرورة العالج ،المعتقدات حول االدوية،الكلمات المفتاحية: التهاب المفاصل الرثوي introduction rheumatoid arthritis (ra) is a chronic, progressive, inflammatory autoimmune disease(1) of unknown etiology(2) associated with articular, extra articular and systemic manifestation(1). it occurs worldwide in virtually all ethnic groups, with a prevalence estimated between 0.5% and 1%(3). 1corresponding author: e-mail:mirnapharma@gmail.com received: 29/5 /2019 accepted: 18/ 8 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp134-141 iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 135 the prevalence rate in iraq is 1%(4). a global burden study estimated ra prevalence in middle east north africa (mena) region as among the lowest at 0.16 %(5).like many autoimmune diseases, the etiology of ra is multifactorial(6). inflammation and following destruction of synovial joints is the hallmark of ra(3). morning stiffness in and around the joints, lasting at least one hour is a characteristic sign of ra. rheumatoid arthritis can be associated with variable manifestations of extra-articular involvement such as rheumatoid nodules, vasculitis, hematologic abnormalities, and visceral involvement(7). rheumatoid arthritis is one such illness where patients must take daily medicine to manage their ache and reduce probabilities of physical disability(8). the goals of pharmacologic therapy are to induce remission and prevent further loss of joint tissues or function in daily activities. the main drug classes that are currently used for treatment of ra include non-steroidal antiinflammatory drugs (nsaids), glucocorticoids, disease-modifying anti-rheumatic drugs (dmards), and biological agents(9). the main factor that affects patient adherence is his/ her general view about medicines, since it can overrun most of the other factors(10). accordingly, taking patient’s beliefs about their medication in consideration had been shown to be an important factor that affects adherence of the patient in which holding positive beliefs is essential for better adherence(11-13). the purpose of the current study was to measure beliefs about medicines among a sample of iraqi patients with rheumatoid arthritis and to determine possible association between this belief and some patient-certain factors. patients and method patients the current cross-sectional study was carried out on a convenient sample of 250 already diagnosed ra patients (mean age was 50.8±13.1 years) who attended baghdad teaching hospital/medical city/rheumatology department during october 2018 to january 2019. the number of female patients was 221(88.4%), while the number of male patents was 29 (11.6%). inclusion criteria the inclusion criteria for the current study were: 1-patients with ra as defined by the 1987 revised american rheumatism association (ara) criteria(14). 2-ra patients who were aged 20-80 years of either sex who were accepted to participate in the study. 3-disease duration >1yr. 4-current treatment with steroids, nsaid and/or dmards (including methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, and azathioprine), with or without concomitant administration of biologic agents. 5-patients had not changed their medication in the last three months. exclusion criteria the exclusion criteria for the current study were: 1-patient who had a hearing, speech or cognitive deficits that would impair understanding of the questions. 2-patient who take antidepressant drugs, or being on treatment for any neurological or psychological diseases. 3-if they were receiving no medication. 4-pregnant women. 5-patient who had serious co-morbidity such as, malignancies and end stage organ failure. 6-patients providing incomplete or conflicting information during completion of the questionnaire. method the questionnaires belief about medicine was measured by using an arabic version of the belief about medicines questionnaire (bmq) established by horne et al (figure 1)(15). it has two parts: the bmqspecific evaluating beliefs about medication used for a specific condition and the bmq-general evaluating beliefs about medicines in general(16). the bmq-specific part covers two subjects; specific necessity subject which assesses patients’ view about the necessity and importance of their medicine, while specific concern subject covers patients’ beliefs about potential harm and adverse effects of their own medications and every one of which has a score extend from 5 to 25. a high score in necessity subject means that patients think their medications are essential to them; on the other hand high score in the concern subject implies that patients are worried and stressed over their very own medicines. likewise, bmq-general part has two subjects; general overuse subject which assesses how patients perceive the extent of medication usage, and general harm theme represents patients’ beliefs about unsafe nature of medication in general. the scores of the last two subjects extend from 4 to 20, and high score in each subject means negative perception about medications in general(17). respondents indicate their degree of concurrence with each individual statement about medicines on a 5-point likert scale, extend from 1 (strongly disagree) to 5 (strongly agree)(18). study design administration of questionnaires the information identified with the investigation were gathered by the analyst herself. when the patients arrived to the hospital/rheumatology department, they were asked if they accept to participate in the study, an explanation of the questionnaire was given to each patient who spent about 5 minutes to fill the research questionnaire completely. iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 136 statistical analysis anderson darling test was done to evaluate if continuous variables follow normal distribution, if follow normal distribution than mean and standard deviation used, if did not follow normal distribution than median and interquartile range (25% to 75% percentile range) will be used to present the data. linear regression analysis performed to evaluate the relationship between different variables, r (correlation coefficient or standardized beta is a representative of magnitude and direction of the relationship), 0.00 0.29 = little or no correlation; 0.30-0.49 = weak; 0.50-0.69 = moderate; 0.70-0.89 = strong; and 0.90-1.00 = very strong. negative sign indicate inverse relationship, but positive sign represent direct relationship. spss 22.0.0 (chicago, il), graph pad prism version 8.0.0 for windows, graph pad software, san diego, california usa, software package used to make the statistical analysis, p value considered when appropriate to be significant if less than 0.05 . strongly disagree disagree uncertain agree strongly agree specific necessity 1-my life would be impossible without medicine 2-without medicine i'll be very ill 3-my health , at present depend on my medicine 4-my medicine protected me from becoming worse 5-my health in the future depends on my medicine specific concern 6-i sometimes worry about the long term effect of my medicine 7-having to take medicine scares me 8-i sometimes worry about becoming too dependent on my medicine 9-my medicine disrupt my life 10-my medicines are mystery to me general-harm 11-people who take medicines should stop their treatment for a while every now and again 12-most medicines are addictive 13-medicines do more harm than good 14-all medicines are poison general-overuse 15-natural remedies are safer than medicines 16-doctors use too many medicines 17-doctors place too much trust on medicines 18-if doctors had more time with their patients they would prescribe fewer medicines figure 1. the beliefs about medicines questionnaire (bmq)(15). iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 137 results mean age of patients was 50.8 ± 13.1 years. 89.6% were married, and 35.6% were illiterate. 88.4% were female, the majority lived in urban area of residence, and most of the patients were nonsmokers, as illustrated in table 1. table 1. socio-demographical characteristics of patients. variables value age (years), mean ± sd 50.8 ± 13.1 patient’s age no. (%) <30 years 16 (6.4%) 30 – 39 years 33 (13.2%) 40 – 49 years 58 (23.2%) 50 – 59 years 69 (27.6%) ≥60 years 74 (29.6%) gender no. (%) male 29 (11.6%) female 221 (88.4%) marital status no. (%) single 26 (10.4%) married 224 (89.6%) education level no. (%) illiterate 89 (35.6%) primary 64 (25.6%) secondary 59 (23.6%) college 38 (15.2%) location no. (%) urban 233 (93.2%) rural 17 (6.8%) smoking no. (%) smoker 17 (6.8%) non-smoker 233 (93.2%) the mean of disease duration was (9.6±7.8 years), most patients with disease duration less than ten years (69.6%), and (63.2%) had no other chronic disease. in addition, (52%) of patients had high disease activity (das28-esr) score and (44.8%) had high clinical disease activity index (cdai) score, as illustrated in table 2. table 2. disease characteristics of the patients variables value disease duration (years), mean ± sd 9.6 ± 7.8 patient’s age no. (%) < 10 years 174 (69.6%) 10 – 19 years 43 (17.2%) ≥ 20 years 33 (13.2%) medication use duration (years), mean ± sd 7.0 ± 6.4 number of other chronic diseases, no (%) none 158 (63.2%) 1 disease 63 (25.2%) 2 diseases 29 (11.6%) das 28-esr 3.5 ± 0.6 remission 1 (0.4%) low 11 (4.4%) moderate 108 (43.2%) high 130 (52.0%) cdai low 15 (6.0%) moderate 123 (49.2%) high 112 (44.8%) cdai: clinical disease activity index; das28-esr: disease activity score-erythrocyte sedimentation rate. the total score with the sub-scores of patients believes for all patients are shown in table 3 as well as figure 2. table 3. beliefs about medicines questionnaire scores of patients. scores value necessity score 21.3 ± 2.5 concern score 14.2 ± 3.9 overuse score 10.2 ± 2.7 harm score 13.6 ± 2.1 total score 59.3 ± 6.9 figure 2. beliefs about medicines questionnaire scores of patients iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 138 the majority of the patients (88%) had strong beliefs in the necessity of treatment (scores bmq specificnecessity greater than score bmq specific-concern. however, (4.4%) of the patients reported strong concerns about the treatment (scores bmq specificconcern greater than score bmq specificnecessity).the residual of the patients (7.6%), have equal scores for bmq specific-necessity and specific-concern scores suggests that they have an equal agreement on both concept of the subpart where they share similar score. as clarified in table 4. in univariate analysis; the bmq-specific necessity (bmq-sn) showed significant direct correlation with age, gender (male vs. female), number of other chronic diseases, das28-esr and cdai. the bmq-specific concern (bmq-sc) showed significant direct correlation with cdai. the bmqgeneral overuse (bmq-go) showed a significant direct correlation with gender (male vs. female), and inverse correlation with education levels. while the bmq-general harm (bmq-gh) showed significant inverse correlation with medication use duration, and direct correlation with das28-esr as illustrated in table 5. table 4. beliefs about medicines questionnaire necessity – concern differential table 5.univariate correlation between bmq components with various variable. cdai: clinical disease activity index; das28-esr: disease activity score-erythrocyte sedimentation rate necessity – concern differential no. percentage necessity > concern 220 88% necessity < concern 11 4.4% necessity = concern 19 7.6% bmq-specific necessity bmq-specific concern bmq-general overuse bmq-general harm regression coefficient p-value regression coefficient pvalue regression coefficient pvalue regression coefficient pvalue age 0.161 0.005 [s] 0.045 0.239 0.053 0.201 0.067 0.145 gender(male vs. female) 0.222 <0.001 [s] 0.037 0.558 0.146 0.021 [s] -0.061 0.337 marital status 0.009 0.441 -0.047 0.228 -0.064 0.158 0.073 0.126 education level -0.051 0.210 -0.032 0.306 -0.108 0.044 [s] 0.057 0.186 residence -0.022 0.364 -0.042 0.254 -0.042 0.252 0.008 0.451 smoking 0.022 0.364 0.042 0.254 0.042 0.252 -0.008 0.451 disease duration 0.080 0.104 -0.003 0.483 -0.056 0.189 -0.094 0.070 medication use duration -0.039 0.267 -0.032 0.308 0.037 0.281 -0.109 0.043 [s] number of other chronic disease 0.138 0.015 [s] -0.073 0.125 -0.042 0.253 -0.008 0.450 das28-esr 0.127 0.023 [s] 0.104 0.051 0.101 0.055 0.131 0.019 [s] cdai 0.152 0.008 [s] 0.149 0.009 [s] 0.094 0.070 0.014 0.411 linear regression analysis [s], significant relationship when p value<0.05. iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 139 in multivariate analysis, only age and gender were independently correlate ( direct relationship ) with bmq sn score . while das28 esr was independently correlate (direct relationship) with bmq-gh. as illustrate in table 6. table 6. multivariate linear regression analysis between bmq components and other variables of patients cdai: clinical disease activity index; das28-esr: disease activity score-erythrocyte sedimentation rate. the number of other chronic diseases was excluded from multivariate regression table because the tolerance=0 this means that this variables was already contained in or redundant with other independent variables (predictors) i.e. can be perfectly predicted from one or more of the other independent variables. discussion rheumatoid arthritis is a chronic inflammatory joint disease, with a higher prevalence observed in both older age groups and women(19). patients with rheumatoid arthritis need to take daily medication to relieve their pain and reduce chances of physical disability(8). as shown in sociodemographical date of the patients, about 88.4% of patients were female and 57.2% were above 50 year. study in rochester, minnesota, 1955-1985 found that the incidence of ra was nearly twofold in women compared with that in men and increased steadily with age, until age 85 years, after which the rate of ra diminished(20). findings of the current study showed that positive beliefs about the necessity of medication (specific necessity score) had recorded the highest mean among the rest of the scores followed by specific concern score about potential adverse effects of medication and the majority of the patients (88%) had bmq specificnecessity score greater than bmq specific-concern score. as most of ra medications are life sustaining and it is not surprising that most of the recipients rated their beliefs on the necessity of taking medications higher than concerns about the medication (a positive necessity-concerns differential). study done by (r. neame and a. hammond) demonstrated that 75% of individuals with ra have positive belief about the need of their medicines(21). study done by zwikker he, shown increasing need belief about prescription in clinical practice may be advantageous in improving medicine adherence in ra patients(22). similarly, a study done by mardby, et al, concluded that increase awareness of the patient’s beliefs about medicines is needed and that healthcare providers ought to urge patients to express their perspectives about medicines so as to invigorate concordance and adherence to prescription(23). in this study, there are direct correlations between age, male gender, number of other coexisting chronic disease, and disease activity estimated by both das28-esr and cdai with bmq specific-necessity scores, which could be clarified that when patients became older they tend to be more wiser and had sufficient cognitive function to manage medications(24), and once disease became more violent, or when coexisting with other chronic disorder, patients tend to be more careful and realize the necessity for their medications(25). also, this study showed that male patients had more specific necessity about ra medications than female patients while another study showed no association between them(21). the current study show a significant positive correlation between cdai and specific-concerns. a possible explanation of this result is that patients with high concerns about therapy tend to be nonadherent to their medication which might lead to increase disease activity(26). a study done by (r. bmq-specific necessity bmq-specific concern bmq-general overuse bmq-general harm regressio n coefficient p-value regression coefficient pvalue regression coefficient pvalue regressio n coefficient pvalue age 0.184 0.012[s] gender(male vs. female) 0.238 <0.001[s] 0.121 0.081 education level -0.041 0.584 medication use duration -0.088 0.363 das28-esr 0.010 0.908 0.273 0.004 [s] cdai 0.119 0.187 0.160 0.093 r2 0.172 0.072 0.093 0.080 [s], significant relationship when p value<0.05. iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 140 neame and a. hammond) showed that high levels of concern are associated with helplessness and nonadherence, likewise they detailed a similar finding of the present examination that worries about unfavorable outcomes of prescriptions is independent of patients' age and education level(21). in this way, worries about ra drugs should be tended to paying little mind to the age or education level of the patient. in the current study, patients who lack adequate education had opinion that medicines are overused .a possible reason is the absence of information to assist a greater understanding of medicines and their effects. as medication use duration increase, general harm belief about medicines decrease, a possible is that when patients become more familiar with their treatment, their influence might diminish about general harm belief about medicines(27). also, this study demonstrates a positive association between both general harm belief about medicines and das28-esr score. a conceivable clarification is patients with a view that their medicines are harmful tend to be afraid to use their therapy appropriately as a consequence start to be poor adherent and so the activity of the disease will increase. univariate analysis showed that disease activity estimated by both das28-esr and cdai significant correlate directly with specific necessity belief. however this relationship is absent in multiple regression model. this means that disease activity score is an important predictor for belief about medication necessity when considered as a single factor but such factors become insignificant when considered in the presence of other stronger factors like age and gender. in addition only das28esr is independently correlated (significant direct relationship) with general harm belief in multiple regression regardless of the presence of other factors. the belief about medicine questioner may distinguish individuals in danger of poor prescription adherence and give a concentration to patients to talk about their convictions, giving chances to improve medicine adherence(21 ). limitations of the study this study had some limitations. patients were incorporated from only one department of internal medicine and the main diagnosis was rheumatoid arthritis. subsequently the outcomes can't be summed up to other patient gatherings with other sicknesses. in addition, the sample of ra patients had a disease duration above one year, making the results not generalizable to patients with recently diagnosed ra. lastly, the present investigation was a cross-sectional study, which makes it impossible to draw causal conclusions. more researches are needed to explore other factors (e.g., disease burden) that could affect the degree of belief about medication. conclusions the majority (88%) of iraqi ra patients sample had strong beliefs in the necessity of their ra treatment where the medication-necessity score was greater than medication-concern score, were older people, male gender, presence of other chronic illnesses and those who had high disease activity score tend to had more specific necessity score about ra medications. references 1. choy e. understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. rheumatology. 2012; 51(suppl_5):v311. 2. veehof m. measuring treatment response in rheumatoid arthritis. the use of patient‐reported outcome measures [thesis]. enschede: university of twente. 2008:1-147. 3. kahlenberg jm, fox da. advances in the medical treatment of rheumatoid arthritis. hand clinics. 2011; 27(1):11-20. 4. al-rawi, z. s., al-azawi, a. j., al-ajili, f. m., et al. rheumatoid arthritis in population samples in iraq. ann rheum dis.1978; 37(1):73-5. 5. cross m, smith e, hoy d, et al. the global burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study. ann rheum dis. 2014; 73(7):1316–22. 6. amy m. wasserman, md. diagnosis and management of rheumatoid arthritis. boston university school of medicine. 2011; 84(11):1245-1252. 7. grassi w, de angelis r, lamanna g, et al. the clinical features of rheumatoid arthritis. european journal of radiology. 1998; 27:s1824. 8. majed alsubaie1, waleed alqahtani, waleed alshardi., et al. methotrexate in rheumatoid arthritis patients: common side effects and leading cause of discontinuation. international journal of medical research & health sciences. 2018; 7(1): 116-121. 9. reddy d, trost lw, lee t, et al. rheumatoid arthritis: current pharmacological treatment and anesthetic considerations. middle east journal of anesthesiology. 2007; 19(2):311. 10. tsianou k, giannakeas n, tsipouras mg, et al. accessing patient views about medication in chronic conditions using the beliefs about medicine questionnaire (bmq): a review study. j drug res dev.2017; 3(2):1-9. 11. rob h, sarah c, rhian p, et al. understanding patients’ adherence-related beliefs about medicines prescribed for long-term conditions: a meta-analytic review of the iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 141 necessity-concerns framework. plos one. 2013; 8 (12): 1-24. 12. mohamed m, salmiah m, mohd d. beliefs and adherence to medicines among malaysian malay type 2 diabetes. international journal of current research. 2014; 6(2):5026-33. 13. james e, john d. diabetic patients’ medication underuse, illness outcomes, and beliefs about antihyperglycemic and antihypertensive treatments. diabetes care. 2009; 32(1): 19-24. 14. arnett fc, edworthy sm, bloch da, et al. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum 1988; 31:315–24. 15. horne r, weinman j. patients' beliefs about prescribed medicines and their role in adherence to treatment in chronic physical illness. journal of psychosomatic research. 1999; 47(6):555-67. 16. wei l, champman s, li x, et al. beliefs about medicines and non-adherence in patients with stroke, diabetes mellitus and rheumatoid arthritis: a cross-sectional study in china. bmj open. 2017; 7(10):e017293. 17. alhewiti a. adherence to long-term therapies and beliefs about medications. international journal of family medicine. 2014; 2014(479596):1-8. 18. horne r, graupner l, frost s, et al. medicine in a multi-cultural society: the effect of cultural background on beliefs about medications. social science and medicine. 2004; 59(6):1307-13. 19. agarwal sk. biologic agents in rheumatoid arthritis: an update for managed care professionals. journal of managed care pharmacy. 2011; 17(9 supp b):s14-18. 20. gabriel se, crowson cs, o'fallon m. the epidemiology of rheumatoid arthritis in rochester, minnesota, 1955–1985. arthritis & rheumatism: official journal of the american college of rheumatology. 1999; 42(3):415-20. 21. neame r, hammond a. beliefs about medications: a questionnaire survey of people with rheumatoid arthritis. rheumatology. 2005; 44(6):762-7. 22. zwikker he, van dulmen s, den broeder aa, et al. perceived need to take medication is associated with medication non-adherence in patients with rheumatoid arthritis. patient preference and adherence. 2014; 8:1635. 23. mårdby ac, åkerlind i, jörgensen t. beliefs about medicines and self-reported adherence among pharmacy clients. patient education and counseling. 2007; 69(1-3):158-64. 24. park dc, hertzog c, leventhal h, et al. medication adherence in rheumatoid arthritis patients: older is wiser. journal of the american geriatrics society. 1999; 47(2):172-83. 25. komninis id, micheli k, roumeliotaki t, et al. adaptation and validation of the beliefs about medicines questionnaire (bmq) in primary care patients in greece. european journal for person centered healthcare. 2013; 1(1):22431. 26. pound p, britten n, morgan m, et al. resisting medicines: a synthesis of qualitative studies of medicine taking. social science & medicine. 2005; 61(1):133-55. 27. treharne gj, lyons ac, kitas gd. medication adherence in rheumatoid arthritis: effects of psychosocial factors. psychology, health & medicine. 2004; 9(3):337-49 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ satoh et al.indd review correspondence: dr. hiroyasu satoh, department of pharmacology, nara medical university, kashihara, nara 634-8521, japan. tel: +81-744-29-8831; fax: +81-744-29-0510; email: hysat@naramed-u.ac.jp please note that this article may not be used for commercial purposes. for further information please refer to the copyright statement at http://www.la-press.com/copyright.htm cardiovascular pharmacology of sinomenine: the mechanical and electropharmacological actions seiichiro nishida and hiroyasu satoh department of pharmacology, division of traditional medicine, nara medical university, kashihara, nara 634-8521, japan abstract: sinomenine is one of the alkaloids extracted from chinese medical plant, sinomenium acutum rehder et wilson. sinomenine has been used for rheumatoid arthritis as an anti-infl ammatory and immunomodulative drugs. we have so far been investigated the cardiovascular pharmacological actions of sinomenine. sinomenine dilated ne (5 µm)-, kcl (60 mm)and pdb (300 nm)-induced vasoconstrictions. the pretreatment with nicardipine (0.1 µm), staurosporine (30 nm), l-nmma (100 µm), indomethacin (10 µm) or propranolol signifi cantly attenuated the sinomenine-induced vasorelaxation. therefore, these results indicate that sinomenine causes the vasorelaxation by the involvement with the inhibitions of ca2+ current (ica) and pk-c, β-adrenoceptor stimulation, and the activation of no and pgi2 syntheses in endothelium. on the other hand, in the ventricular cardiomyocytes of guinea pig, sinomenine inhibits ica and simultaneously decreases the delayed rectifi er k+ current (ik), resulting in the prolongation of action potential duration. sinomenine also suppresses the dysrhysmias induced by triggered activities under the ca2+ overload condition. therefore, sinomenine may be expected as one of effective therapeutic drugs for heart failure and dysrhythmias, and may maintain the cardiovascular functions due to modulation of cardiac ionic channels and blood vessels. keywords: sinomenine, vasodilation, cardioprotective action, ca2+ channel, aorta, cardiomyocytes introduction sinomenine (7, 8-didehydro-4-hydroxy-3, 7-dimethoxy-17-methyl-9α,13α,14α-morphinan-6-one) is one of the alkaloids extracted from chinese herbs, sinomenium acutum rehder et wilson (li et al. 2004) or sinomenium acutum var. cinereum (zhao et al. 2005). in chinese traditional medicine, sinomenine acutum, a vine plant, has been used for rheumatic diseases for thousands years (yamasaki, 1976; liu et al. 1996). its main constituent of sinomenine acutum is sinomenine, which has also used for clinical treatment of rheumatoid arthritis (ra), due to the anti-infl ammatory and immunomodulative actions (yamasaki, 1976; liu et al. 1996). we have already investigated the cardiovascular pharmacological actions of sinomenine. mokuboito (mu-fang-yi-tang), a kind of kampo formulation containing sinomenium acutum, has been used for heart failure (inaki et al. 2005), which improves heart failure symptom and reduces new york heart association (nyha) class and plasma brain natriuretic peptide (bnp) concentration (yakubo et al. 2002). mokuboito consists of sinomeni caulis et rhizoma (rhizome of sinomenium acutum rehder et wilson), cinnamomi cortex (bark of cinnamomum cassia blume), ginseng radix (roots of panax ginseng c. a. meyer) and gypsum fibrosum. in our recent reports (satoh, 2005; nishida and satoh, 2006; 2007), mokuboito, sinomenium acutum and sinomenine might improve chronic heart failure, resulting from the modulation of cardiac and vascular systems. mokuboito can exert the vasodilating and cardioprotective actions (satoh, 2005), as discussed in the following parts. in general, the basic treatments of heart failure consist of (1) reducing workload of heart, (2) protection of cardiomyocytes and (3) restriction and control of waters and sodium. in order to reduce both preand after-loads, the dilations of arterioles and veins are strongly required in the case of elevated fi lling pressures and reduced cardiac output. as a cardiovascular protective drug, sinomenine might contribute to clinical treatments via modulation of the ionic currents of cardiac cells, and control of the tension of blood vessels. satoh et al.indd 1 5/14/2007 8:58:41 pm 97drug target insights 2007: 2 97–104 nishida and satoh drug target insights 2007: 2 in this review, the cardiovascular pharmacological actions of sinomenine are mainly shown and discussed. source of sinomenine sinomenine is derived from natural plant such as sinomenium acutum (li et al. 2004) and sinomenium acutum var. cinereum (zhao et al. 2005). sinomenium acutum and sinomenium acutum var. cinereum are widely in a lot of regions of china. caulis sinomeni is the dried plant stems of sinomenium acutum and sinomenium acutum var. cinereum. there is less or no difference in sinomenine content of caulis sinomeni between the species and the varieties of growing regions. the variation is found among the samples collected from different parts of plant. the content of sinomenine is dependent on the size (diameter) of stem. the sinomenine content in sinomenine acutum is 1.63 ± 0.64 (%, w/w) in large (>3 cm) stem, 0.96 ± 0.45 (%, w/w) in 1–3 cm stem, and 0.49 ± 0.16 (%, w/w) in <1 cm stem (zhao et al. 2005). pharmacokinetics of sinomenine the pharmacokinetics and tissue distribution of sinomenine have been studied in rats (liu et al. 2005). sinomenine achieves high bioavilability (about 80%) by oral administration of 90 mg/kg. at 45min later, sinomenine is found widely in internal organs such as kidney, liver, lung, spleen, heart, brain and testis. sinomenine is metabolized and eliminated by kidney and liver. tmax is 39.5 ± 8.49 min, cmax is 13.89 ± 4.29 µg/ml, t1/2a phase is 61.28 ± 53.62 min, auc0-t is 2331.53 ± 1172.77 µg-min/ml, and cl is 42.95 ± 14.4 ml/min per kg. in clinical studies, oral administration of 80 mg sinomenine is performed for healthy volunteers (yan et al. 1997). in the pharmacokinetic parameters, t1/2α is 1.04 ± 0.491h, t1/2β is 9.397 ± 2.425h, tmax is 1.04 ± 0.274h, cmax is 246.6 ± 71.165 ng/ml. furthermore, auc is 2651.158 ± 1039.050 ng.h/ml, and cl is 0.033 ± 0.010 ng/ml. vascular pharmacology male wistar rats (5–10 week-old) were anesthetized with ether, and euthanized by exsanguination. the thoracic aorta was quickly removed, and then the isolated aorta was cut into rings of 3-mm in length. all rings were stretched to generate a resting tension of 1.2 g in krebs solution. after 40 min of resting, norepinephrine (ne) (5 µm) was added to the tissue bath. after the contractile response became steady, the drugs were cumulatively administrated into the bath solution. the similar methods are described in our previous reports (nishida and satoh, 2003; 2006). the aorta ring strip of rat had a strong contraction after an initial application of 5 µm ne. sinomenine (0.1 to 100 µm) applications potently relaxed the contraction induced by ne in a concentration-dependent manner (nishida and satoh, 2006). the relaxation was produced at the concentrations of over 0.3 µm sinomenine, and at 100 µm decreased the contractions by 68.8 ± 5.1% (n = 6, p < 0.001). pdb (300 nm) and kcl (60 mm) also caused strong contractions. sinomenine dilated both pdband kcl-induced contractions in a concentration-dependent manner; at 100 µm by 49.9 ± 9.8% (n = 6, p < 0.001) and 86.9 ± 8.5% (n = 6, p < 0.001), respectively (fig. 1). furthermore, the relaxation of sinomenine (0.1 to 100 µm) was attenuated signifi cantly by the pretreatment with nicardipine (fig. 2). at 100 µm the relaxation decreased from 68.8 ± 5.1% (n = 6) to 35.5 ± 6.9% (n = 5, p < 0.001). since pk-c inhibition may be related to sinomenine-induced vasorelaxation, the pretreatment with staurosporine (30 nm) for 30 min was carried out (satoh, 1996; nishida and satoh, 2004). staurosporine attenuated the sinomenine-induced vasorelaxation; at 100 µm from 68.8 ± 5.1% (n = 6) to 49.5 ± 7.7% (n = 5, p < 0.001) (fig. 2). in addition, propranolol (0.3 µm) also signifi cantly attenuated the sinomenineinduced relaxation. the vasorelaxation at 100 µm sinomenine was attenuated to 45.2 ± 4.2% (n = 5, p < 0.01). therefore, the modulation of both ca2+ channel and pk-c is largely contributed to sinomenineinduced actions, and sinomenine also vasodilates mediated through β-adrenoceptor stimulation of aortic smooth muscle cells. for involvement with endothelium-dependent relaxation via no activation, a pretreatment with 100 µm l-nmma (a non-selective no synthesis inhibitor) was carried out (nishida and satoh, 2003; 2007). under the conditions, the vasorelaxation induced by sinomenine was attenuated from 68.8 ± 5.1% (n = 6) to 25.3 ± 2.3% (n = 5, p < 0.01) (fig. 2). the attenuation was supported by the results using the aorta with removal of endothelium; at 100 µm sinomenine by 53.7 ± 1.8% (n = 5, p < 0.01). indomethacin (10 µm), as an satoh et al.indd 2 5/14/2007 8:59:09 pm 98 cardiovascular pharmacology of sinomenine drug target insights 2007: 2 inhibitor of prostanoid production, also strongly reduced the vasorelaxation induced by 100 µm sinomenine to 37.1 ± 9.3% (n = 5, p < 0.001). thus, both l-nmma and indomethacin affected the sinomenine-induced relaxation significantly. therefore, sinomenine possesses the pharmacological characteristics for modulation of no synthesis and pg production, as well as the inhibitions of ca2+ channel and pk-c and the stimulation of β-adrenoceptor. the possible mechanisms for the vasodilating actions of sinomenine are summarized in fig. 3. cardiac electropharmacology cardiac cells were taken from the ventricle muscles of guinea pig hearts, using methods similar to those described previously (satoh, 2003, 2005). currentclamp and whole-cell voltage-clamp were performed using an axopatch patch-clamp amplifi er (axon instruments, burlingame, c.a, u.s.a.) and standard techniques. current-clamp experiments were carried out to examine the modulation of the action potential confi guration in guinea pig ventricular muscles (fig. 4a) (satoh, 2005). sinomenine at 300 µm and 1 mm increased the 75% repolarization of action potential duration (apd75) by 24.9 ± 3.5% (n = 6, p < 0.05) and by 43.7 ± 3.3% (n = 6, p < 0.001), respectively. sinomenine at 1 mm decreased the amplitude (apa), but not signifi cantly (0.9 ± 2.1%, n = 5). the resting potential was not affected. the modulation of l-type ca2+ current (ica) was investigated. test pulses were applied to 0 mv from a holding potential of –30 mv. the average capacitance was 84.1 ± 2.4 pf (n = 23). at 1 mm sinomenine inhibited the ica at 0 mv by 18.2 ± 2.1% (n = 6, p < 0.05). at 1 mm, the delayed rectifi er k+ currents (ik) at 60 mv was inhibited by 16.2 ± 2.6% (n = 6, p < 0.05) (fig. 4b), and the inwardly rectifying k+ current (ik1) at -120 mv by 47.2 ± 3.8% (n = 6, p < 0.01). s inome nine 0 10 20 30 40 50 60 70 80 90 100 0.1 0.3 1 3 10 30 100 r el ax at io nn (% ) ne p db kc μ m figure 1. concentration-dependent relaxation of sinomenine on ne-, pdband kcl-induced vasorelaxations. symbols used are sinomenine in pretreatments with ne (open circles, n = 6), pdb (triangles, n = 6) and kcl (squares, n = 6). values (%) represent mean ± s.e.m. *: p < 0.05, **: p < 0.01, ***: p < 0.001, with respect to control value. satoh et al.indd 3 5/14/2007 8:59:10 pm 99 nishida and satoh drug target insights 2007: 2 in multicellular preparations, the modulation of action potential confi guration by sinomenine was also examined (satoh, 2005). the preparations were stimulated at 1 hz. sinomenine (100 µm to 1 mm) had inhibitory effects on the action potentials, and tended to increase the action potential durations (apd); at 1 mm, by 4.1± 1.6% (n = 6) in apd50 and by 4.5 ± 1.4% (n = 6) in apd90 (but not signifi cantly). the maximum rate of depolarization (vmax) was also inhibited by 20.0 ± 2.4% (n = 6, p < 0.05) at 300 µm and by 32.1 ± 3.3% (n = 6, p < 0.05) at 1 mm of sinomenine. in high extracellular ca2+ ([ca2+]o) solution (5.4 mm) to cause cellular ca2+ overload, abnormal action potentials occurred irregularly, in spite of constant stimulation (1 hz) (satoh, 2005). application of 300 µm sinomenine suppressed and abolished the abnormal action potentials (dysrhythmias) (fig. 5). the changes in the action potential confi gurations and the modulation of the ionic channel currents on the membrane of cardiomyocyte by sinomenine are summarized in fig. 6. immunomodulative action sinomenium acutum has been used for treatment for various rheumatic diseases as a chinese traditional medicine (yamasaki, 1976; liu et al. 1996). in basic pharmacological studies of sinomenium acutum, the inhibitory actions on some enzymes relating to infl ammation have been shown (li et al. 2003). one of most famous pharmacological actions of sinomenine is an immunomodulative effect. sinomenine is also clinically used for ra treatment (yamasaki, 1976; liu et al. 1996). a lot of reports concerning about not only an anti-rheumatic effect, but also anti-inflammatory and immunomodulative effects have already been shown. as anti-rheumatic direct effects, it has been reported that sinomenine reduces infl ammatory parameters and attenuates proliferation of figure 2. modulation by several inhibitors of the relaxation induced by sinomenine. symbols used are in the absence (open circles, n = 6), in the presence of 100 µm l-nmma (open triangles, n = 5), 10 µm indomethacin (open squares, n = 5), 0.1 µm nicardipine (closed circles, n = 5), 0.3 µm propranolol (closed triangles, n = 5), and 30 nm staurosporine (closed squares, n = 5). values (%) represent mean ± s.e.m. *: p < 0.05, **: p < 0.01, ***: p < 0.001, with respect to control value. satoh et al.indd 4 5/14/2007 8:59:10 pm 100 cardiovascular pharmacology of sinomenine drug target insights 2007: 2 synovial fibroblasts in rat adjuvant arthritis models (liu et al. 1996). the anti-infl ammatory and immunomodulative actions of sinomenine are responsible for various mechanisms via complex modulation of leukocytes and cytokine. sinomenine reduces the production of prostagrandin (pg) e2 and no from macrophage (liu et al. 1994a). also, sinomenine possesses anti-proliferative effects on lymphocytes (liu et al. 1994b), contributing to anti-infl ammatory and anti-rheumatic effects. in addition, sinomenine depressed mrna expression of tumor necrosis factor (tnf)-α and interleukin (il)-β of peritoneal macrophages (wang et al. 2005). therefore, sinomenine may act as an anti-rheumatic drug through the anti-infl ammatory effects on lymphocytes and cytokine. sinomenine inhibited bfgf-induced angiogenesis in vitro and in vivo (kok et al. 2005). sinomenine also attenuates transmigration of granulocyte. the inhibition of leukocytes migration across the vessel wall and anti-angiogeneic effect of sinomenine may also contribute to therapeutic effects for ra. immunomodulative actions have been studied as the other aspect of sinomenine concerning about the cardiac transplantation model. it has been reported that acute and chronic cardiac allograft ejections are blocked by the immunomodulatory effects of sinomenine (mark et al. 2003). conclusion endothelium-dependent and -independent relaxations we have been demonstrated that sinomenine possesses strong vasodilating actions by multiple mechanisms (nishida and satoh, 2006). the summarized mechanisms of sinomenine-induced vasorelaxation are shown in fig. 3. sinomenine possesses endothelium-dependent vasorelaxation via no and pgi2 releasing from endothelium. nos activation and pgi2 release are elicited by an increase in the intracellular ca2+ concentration ([ca2+]i) in endothelium cells (busse et al. 1998; quignard et al. 1999). the mechanisms for the endothelium-dependent relaxations are not yet unclear. however, sinomenine might increase [ca2+]i in endothelium cells and then, activates figure 3. summary of the multiple mechanisms induced by sinomenine. sinomenine produces the vasorelaxation via no and pgi2 releasings from endothelium. also, sinomenine causes the vasorelaxation via modulation of ca2+ channels and pk-c activity in smooth muscle cells. in addition, β-adrenoceptor stimulation is caused. nos: nitric oxide synthetase, pk-c: protein kinase c, pgi2 : prostaglandin i2. satoh et al.indd 5 5/14/2007 8:59:10 pm 101 nishida and satoh drug target insights 2007: 2 nos activity and pgi2 releasing, as reported previously (nishida and satoh, 2003). sinomenine causes the vasorelaxation via modulation of ca2+ channels and pk-c activity in vascular smooth muscle cells. in vascular muscle cells, the contraction systems and the ion channels are regulated through the intracellular signal conditions (satoh and sperelakis, 1991, 1995; satoh, 1996). therefore, sinomenine might modulate the contraction systems, ca2+ and na+ channels, delayed rectifi er k+ channels, and ca2+-activated k+ (kca) channel, accompanied with the activation of pk-c (nishida and satoh, 2007). also, sinomenine possesses β-adrenoceptor stimulating action to inhibit the aortic constriction. clinically possibility of cardiovascular pharmacological effects sinomenine is included in sinomenine acutum of mokuboito. mokuboito is traditionally used for dyspnea and edema (shuji et al. 2002). therefore, sinomenine may be expected as one of the therapeutic agents for heart failure. most recently, satoh (2005) has demonstrated that sinomenine effectively modulates cardiac ionic channels. sinomenine inhibits ica , and simultaneously produces the ik decerease in cardiomyocytes which results in the apd prolongation. modulation of ca2+ channel induced by sinomenine is similarly exerted in vascular smooth muscle cells. in addition, sinomenine possesses the regulatory actions for dysrhythmias under ca2+ overload conditions. it has been well known that under the ischemia and heart failure, the cellular ca2+ overload of heart muscles elicits some arrhythmias and dysfunctions (satoh, 2001; 2003). the regulation of ca2+ infl ux may modulate ca2+ overload in cardiomyocytes and produces protective actions for ca2+-overloaded myocardial cells (satoh and spererakis, 1998). therefore, sinomenine might restrain the cell damages of heart muscles via modulation of [ca2+]i, and as a result, exert a cardioprotective action. cardiopotective action of sinomenine on rat acute myocardial ischemia has also been demonstrated. reperfusion injury is induced by ligating the rat left coronary artery for 15 min and reopening. sinomenine can inhibit the incidence of b a figure 4. modulation by sinomenine on the action potentials in guinea pig ventricular cardiomyocytes. a: concentration-dependent changes in the action potential confi guration by sinomenine. short lines at the left of action potential recordings represent zero mv level. b: the percentage inhibitions by sinomenine of cardiac ionic currents. vertical bars represent mean ± sem. figure 5. antiarrhythmic actions of sinomenine in guinea pig papillary muscles. sinomenine abolishes the abnormal action potentials in high ca2+ concentration (5.4 mm). dots above the action potential recordings are represented the regular rhythms induced by 1 hz stimulation. the horizontal line indicates zero mv. satoh et al.indd 6 5/14/2007 8:59:10 pm 102 cardiovascular pharmacology of sinomenine drug target insights 2007: 2 arrhythmias and reduce intracellular ca2+ concentration (xie et al. 1993), well consistent with our results. sinomenine has multiple vasodilating mechanisms. the vasodilating agent is one of the great useful tools for heart failure and regulates preand afterloads of cardiovascular systems. therefore, sinomenine-induced vasodilating actions may improve cardiac functions via the regulation of both preand after-loads under heart failure. in summary, sinomenine caused a concentrationdependent vasorelaxation on ne-, kcland pdb-induced contractions, and sinomenineinduced vasorelaxation is attenuated by the pretreatments with l-nmma, indomethacin, staurosporine, nicardipine and propranolol. in electropharmacological mechanisms, sinomenine inhibits the ica and the ik in cardiomyocytes which results in the apd prolongation. in addition, sinomenine depressed the dysrhysmias induced by triggered activities under the ca2+ overload. finally, sinomenine also possesses the antiinfl ammatory and immunomodulative actions. in future, therefore, sinomenine as a cardioprotective drug may be expected to the respectable effectiveness for heart failure, mediated through the modulation of cardiac ion channels (including the regulation for dysrhythmias) and blood vessels. further experiments need to elucidate more in detail mechanisms of sinomenine. acknowledgements the authors wish to express thanks for the supply of mokuboito and sinomenium acutum extracts (tsumura co.). this work was in part supported by the research fund of japan kampo medicine manufactures association. references busse, r., fichter, h., luckhoff, a. et al. 1998. hyperpolarizaion and increased free calcium in acethylcholine-stimulated endothelium cells. am. j. physiol., 255:h965–9. inaki, k. 2005. diseases and kampo. in sato, y., hanawa, t., arai, m., cyong, j., fukuzawa, m., mitani, k., ogihara, y., sakiyama, t., shimada, y., toriizuka, k., ymamada, t. ed. introduction to kampo. elsevier japan. p125. kok, t.w., yue, p.y., mak, n.k. et al. 2005. the anti-angiogenic effect of sinomenine. angiogenesis, 8(1):3–12. kubota, y., tanaka, t. and umegaki, k. 2001. ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level. life sci., 69:2327–36. li, r.w., david, lin, g., myers, s.p. et al. 2003. anti-inflammatory activity of chinese medical vine plants. j. ethnopharmacol. 85(1):61–7. li, y., cui, s., cheng, y. et al. 2004. application of nonaqueous capillary electrophoresis for quantitative analysis of quinolizidine alkaloids in chinese herbs. anal. chim. acta., 508:17–22. liu, l., riese, j., resch, k. et al. 1994 (a). impairment of macropharge eicosanoids and nitric oxide production by alkaloid from sinomenium acutum. arzneimittelforschung, 44:1223–26. liu, l., resch, k., and kaever, v. 1994 (b). inhibition of lymphocyte proliferation by the anti-arthritc drug of sinomenine. int. j. immunopharmacol., 16(8):685–91. liu, l., buchner, e., beitze, d. et al. 1996. amelioration of rat experimental arthritides by treatment with the alkaloid sinomenine. int. j. immunopharmacol., 18(10):529–43. liu, z.q., chan, k., zhou, h. et al. 2005. the pharmacokinetics and tissues distribution of sinomenine in rats and its protein binding ability in vitro. life sci., 77(25):3197–209. lopez-jaramillo, p., gonzalez, m.c., palmer, r.m. et al. 1990. the crucial role of physiological ca2+ concentrations in the production of endothelial nitric oxide and the control of vascular tone. br. j. pharmacol., 101(2):489–93. mark, w., schneeberger, s., seiler, r. et al. 2003. sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. transplantation, 15:940–5. nishida, s., and satoh, h. 2003. mechanisms for the vasodilations induced by ginkgo biloba extract and its main constituent, bilobalide, in rat aorta. life sci., 72:2659–67. nishida, s., and satoh, h. 2004. comparative vasodilating actions among terpenoids and fl avonoids contained in ginkgo biloba extract. clinica chimica acta., 339:129–33. nishida, s., and satoh, h. 2006. in vitro pharmacological actions of sinomenine on the smooth muscle and endothelial cell activity in rat aorta. life sci., 79:1203–06. nishida, s., and satoh, h. 2007. vascular pharmacology of mokuboito (mu-fang-yi-tang) and its constituents on the smooth muscle and the endothelium in rat aorta. ecam (in press). quignard, j.f., feleton, m., thollon, c. et al. 1999. pottasium ions and endothelium-derived hyperpolarizing factor in guinea-pig carotid and porcine coronary arteries. br. j. pharmacol., 127:27–37. figure 6. shame for the changes in cardiac ionic channels induced by sinomenine. superimposed action potentials are in control (solid line) and in sinomenine (dash line). ina: the na + channel, ica: the ca 2+ channel, ik: the delayed rectifi er k +channel, ik1: the inwardly rectifying k+channel. satoh et al.indd 7 5/14/2007 8:59:10 pm 103 nishida and satoh drug target insights 2007: 2 satoh, h. 1996. modulation of ca2+-activated k+ current by isoprenaline, carbachol, and phorbol ester in cultured (and fresh) rat aortic vascular smooth muscle cells. gen. pharmacol., 27:319–24. satoh, h. 2001. [ca2+]idependent actions of taurine in spontaneously beating rabbit sino-atrial nodal cells. eur. j. pharmacol., 424:19–25. satoh, h. 2003. effects of ginkgo biloba extract and bilobalide, a main constituent, on the ionic currents in guinea pig ventricular cardiomyocytes. arzneimittelforschung, 53:407–13. satoh, h. 2005. electropharmacology of sinomeni caulis et rhizome and its constituents in cardiomyocytes. am. j. chin. med., 33:967–79. satoh, h., and sperelakis, n. 1991. calcium and potassium currents in cultured rat aortic vascular smooth muscle cell lines. in sperelakis n ed. ion channels of vascular smooth muscle cells and endothelial cells. academic press, new york: p55–63. satoh, h. and sperelakis, n. 1995. modulation of l-type ca2+ current by isoprenaline, carbachol and phorbol ester in cultured rat aortic vascular smooth muscle (a7r5) cells. gen. pharmacol., 26: 369–79. satoh, h. and sperelakis, n. 1998. review of some actions of taurine on ion channels of cardiac muscle cells and others. gen. pharmacol., 30:451–63. shuji, y., kinoshita, y., arakawa, y. et al. 2002. clinical evaluation of moku-boi-to(mu-fang-yi-tang): a japanese and chinese traditional medicine for heart failure. j. trad. med., 19:159–63. wang, y., fang, y., huang, w. et al. 2005. effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. j. ethopharmacol., 98:37–43. yamasaki, h. 1976. pharmacology of sinomenine, an anti-rheumatic alkaloids from sinomenine acutum. acta med okayama, 30:1–20. xie, s.x., and jin, q.q. 1993. prevention of sinomenine on isolated rat myocardial reperfusion injury. zhongguo yao li xue bao, suppl: s12–5. yan, x.h., li, h.d., peng, w.x. et al. 1997. determination of sinomenine hcl in serum and urine by hplc and its pharmacokinetics in normal volunteers. yao xue xue bao, 32(8):620–4. zhao, z.z., liang, z.t., zhou, h. et al. 2005. quantifi cation of sinomenine in caulis sinomenii collected from different growing regions and wholesale herbal markets by a modifi ed hplc method. biol. pharm. bull., 28(1):105–9. satoh et al.indd 8 5/14/2007 8:59:11 pm 104 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preserveepsinfo true /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /apply /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 300 /colorimagedepth -1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasgrayimages false /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 300 /grayimagedepth -1 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasmonoimages false /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 1200 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects false /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /unknown /description << /enu (use these settings to create pdf documents with higher image resolution for high quality pre-press printing. the pdf documents can be opened with acrobat and reader 5.0 and later. these settings require font embedding.) /jpn <feff3053306e8a2d5b9a306f30019ad889e350cf5ea6753b50cf3092542b308030d730ea30d730ec30b9537052377528306e00200050004400460020658766f830924f5c62103059308b3068304d306b4f7f75283057307e305930023053306e8a2d5b9a30674f5c62103057305f00200050004400460020658766f8306f0020004100630072006f0062006100740020304a30883073002000520065006100640065007200200035002e003000204ee5964d30678868793a3067304d307e305930023053306e8a2d5b9a306b306f30d530a930f330c8306e57cb30818fbc307f304c5fc59808306730593002> /fra <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> /deu <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> /ptb <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> /dan <feff004200720075006700200064006900730073006500200069006e0064007300740069006c006c0069006e006700650072002000740069006c0020006100740020006f0070007200650074007400650020005000440046002d0064006f006b0075006d0065006e0074006500720020006d006500640020006800f8006a006500720065002000620069006c006c00650064006f0070006c00f80073006e0069006e0067002000740069006c0020007000720065002d00700072006500730073002d007500640073006b007200690076006e0069006e0067002000690020006800f8006a0020006b00760061006c0069007400650074002e0020005000440046002d0064006f006b0075006d0065006e007400650072006e00650020006b0061006e002000e50062006e006500730020006d006500640020004100630072006f0062006100740020006f0067002000520065006100640065007200200035002e00300020006f00670020006e0079006500720065002e00200044006900730073006500200069006e0064007300740069006c006c0069006e0067006500720020006b007200e600760065007200200069006e0074006500670072006500720069006e006700200061006600200073006b007200690066007400740079007000650072002e> /nld <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> /esp <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> /suo <feff004e00e4006900640065006e002000610073006500740075007300740065006e0020006100760075006c006c006100200076006f0069006400610061006e0020006c0075006f006400610020005000440046002d0061007300690061006b00690072006a006f006a0061002c0020006a006f006900640065006e002000740075006c006f0073007400750073006c00610061007400750020006f006e0020006b006f0072006b006500610020006a00610020006b007500760061006e0020007400610072006b006b007500750073002000730075007500720069002e0020005000440046002d0061007300690061006b00690072006a0061007400200076006f0069006400610061006e0020006100760061007400610020004100630072006f006200610074002d0020006a0061002000520065006100640065007200200035002e00300020002d006f0068006a0065006c006d0061006c006c0061002000740061006900200075007500640065006d006d0061006c006c0061002000760065007200730069006f006c006c0061002e0020004e00e4006d00e4002000610073006500740075006b0073006500740020006500640065006c006c00790074007400e4007600e4007400200066006f006e0074007400690065006e002000750070006f00740075007300740061002e> /ita <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> /nor <feff004200720075006b00200064006900730073006500200069006e006e007300740069006c006c0069006e00670065006e0065002000740069006c002000e50020006f00700070007200650074007400650020005000440046002d0064006f006b0075006d0065006e0074006500720020006d006500640020006800f80079006500720065002000620069006c00640065006f00700070006c00f80073006e0069006e006700200066006f00720020006800f800790020007500740073006b00720069006600740073006b00760061006c00690074006500740020006600f800720020007400720079006b006b002e0020005000440046002d0064006f006b0075006d0065006e0074006500720020006b0061006e002000e50070006e006500730020006d006500640020004100630072006f0062006100740020006f0067002000520065006100640065007200200035002e00300020006f0067002000730065006e006500720065002e00200044006900730073006500200069006e006e007300740069006c006c0069006e00670065006e00650020006b0072006500760065007200200073006b00720069006600740069006e006e00620079006700670069006e0067002e> /sve <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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice althea medical journal. 2016;3(3) 446 amj september 2016 treatment of rheumatoid arthritis with methotrexate only or a combination of methotrexate and hydroxychloroquine natharubini nadarajah,1 riardi pramudyo,2 kuswinarti3 1faculty of medicine universitas padjadjaran, 2department of internal medicine faculty of medicine universitas padjadjaran/dr. hasan sadikin general hospital bandung, 3department of pharmacology and therapy faculty of medicine universitas padjadjaran abstract background: rheumatoid arthritis (ra) is an autoimmune disease that causes significant morbidity in most patients and also premature mortality in some. to prevent this, therapeutic approaches call for the early treatment of ra by using disease-modifying antirheumatic drugs (dmardss), either as single therapy or combination therapy. although in the previous decade dmards were rarely used as combination, now they are used widely to treat ra. the objective of this study was to compare the effectiveness of methotrexate (mtx) on its own and the combination of mtx & hydroxychloroquine (hcq) in ra patients. methods: an analytic retrospective cohort study was conducted from may 2014 to october 2014, on 46 patients with ra at the rheumatology clinic of dr. hasan sadikin general hospital bandung in the period from january 2009 to october 2014, who were taking mtx or mtx & hcq for at least 1 year. the secondary data obtained from these patients’ medical record were then analyzed using the independent t-test and mann-whitney test. results: the study showed that female patients dominated than male patients which were 93.48%. the mean change in disease activity measures was not significant for any of the parameter (p-value for sjc = 0.337; tjc = 0.676; esr = 0.780). in addition, the comparisons of the disease activity score (das 28) before and after therapy were not significant (p-value = 0.584). conclusions: there is no difference in the effectiveness of dmard monotherapy with mtx and combination therapy with mtx & hcq in ra patients. [amj.2016;3(3):446–51] keywords: das 28, dmards, hydroxychloroquine, methotrexate, rheumatoid arthritis correspondence: natharubini nadarajah, faculty of medicine, universitas padjadjaran, jalan raya bandung-sumedang km.21, jatinangor, sumedang, indonesia, phone: +62 81214588020 email: rubini.nadarajah@gmail.com introduction rheumatoid arthritis (ra) is an autoimmune disease1 and also a chronic systemic inflammatory disorder1,2 that causes joint swelling, joint tenderness, and destruction of synovial joints.1 even though the etiology seems to be unknown, however, there are certain risk factors said to be associated with ra, such as genetic and environmental factors.3 although ra affects people of all ages, its onset occurs between 30−55 years of age, with women more likely to suffer from ra compared to men.4 until today, there is no specific cure for ra. however, there are available certain drugs or medications to help decrease symptoms, reduce inflammation, and slow the progression of the disease.5 the treatment approach for ra has two main goals: first, the relief of symptoms (symptomatic treatment) and maintenance of function and second, the slowing of the tissuedamaging process (modifying treatment).6 disease-modifying antirheumatic drugs (dmards) have the capability to arrest or at least to control the disease process in ra by modifying the disease itself.7 diseasemodifying antirheumatic drugs have the ability to reduce signs and symptoms, disability, impairment of quality of life, and progression of joint damage and hence; they interfere with the entire disease process. disease-modifying antirheumatic drugs can be classified into two major groups: synthetic dmards (sdmards) althea medical journal. 2016;3(3) 447 and biologic dmards (bdmards).8 the disease activity score (das, das28) that is used to record disease activity and determine the therapeutic efficacy consists of four items; the number of swollen and tender joints (sjc, tjc), the visual analogue scale of patients’ assessment of their general health (vas-gh), and the erythrocyte sedimentation rate (esr) in the first hour.9 early treatment with dmards has shown improved outcomes of signs and symptoms and also slower progression of damage to joints10 by changing the course of ra itself.11 currently, mtx (methotrexate) is the most preferred synthetic dmard (dmard of first choice)12 because many patients on mtx have good response and serious toxicities are rare.13 however, not all patients have good treatment response with mtx monotherapy.12 this study was conducted to compare the effectiveness of mtx on its own and the combination of mtx & hcq in ra. methods an analytic retrospective cohort study was conducted from august 2014 to october 2014 at the rheumatology clinic of dr. hasan sadikin general hospital bandung. this study was conducted after it was approved by ethical clearance committee of dr. hasan sadikin general hospital bandung. the research population in this study was ra patients who came to the rheumatology clinic at dr. hasan sadikin general hospital bandung. however, the samples of this study were new ra patients in the rheumatology clinic from january 2009 to october 2013 who had a follow-up minimum one year. the inclusion criteria for the study were ra patients aged 19–80 years old whose cases were being followed up at the rheumatology clinic. all ra patients with complete data in their medical records and patients who had not previously received a combination therapy with any of the medications were studied in this study. the exclusion criteria for the study were patients who had stage iv disease. a concurrent therapy with systemic corticosteroids was allowed if the dosage remained stable throughout the study period and the patient took ≤ 10 mg of prednisone (or its equivalent) per day. based on these criteria, 46 samples were obtained from a calculated sample size of 92 whereby 23 of them took mtx monotherapy and the other 23 took the combination therapy of mtx & hcq. the starting dose for mtx was 7.5-10mg per week and was increased gradually depending on the patients’ respsonse to therapy meanwhile the dosage of hcq was 200mg per day. the type of study chosen to compare the effectiveness of the monotherapy and combination therapy was analytic retrospective cohort study. the response to treatment was studied in the 6th and 12th month. variables of this study were type of dmard therapy (monotherapy or combination therapy) and the disease activity table 1 characteristics of respondents according to study group* characteristics mtx mtx & hcq all patients no. of patients 23 23 46 gender male 2 (8.70%) 1 (4.35%) 3 (6.52%) female 21 (91.30%) 22 (95.65%) 43 (93.48%) age, years, mean (range) 44.1 (19−78) 43.8 (24−65) 44 (19−78) 19−28 years (n) 3 3 6 29−38 years (n) 5 6 11 39−48 years (n) 8 7 15 49−58 years (n) 3 5 8 59−68 years (n) 3 2 5 69−78 years (n) 1 0 1 mean dosage of corticosteroids, mg/ day ± sd 5.4 ± 2.1 5.6 ± 1.9 5.5 ± 2.0 note: * mtx = methotrexate; hcq = hydroxychloroquine althea medical journal. 2016;3(3) 448 amj september 2016 score, das28. the effectiveness of the therapy was seen based on the comparison of the das 28 before and after the dmard therapy (after 12 months of therapy) whereby the das 28 of each patient was calculated using the online das 28 calculator for three variables (sjc, tjc, and esr) at http://www.das-score. nl/das28/dascalculators/dasculators.html and then categorized as follows: low (das28 ≤ 3.2), moderate (3.2 < das28 ≤ 5.1), or high (das28 > 5.1). the data from the medical records were statistically analyzed using independent t-test for data that were normally distributed and mann whitney non-parametric test if there were anomalies in the data distribution. statistical significance was considered when p ≤ 0.05. analysis was performed by comparing the both treatment groups. results of the 46 patients in this study, 23 took mtx while the other 23 took mtx & hcq. the distribution of sex, age, and daily corticosteroid usage were roughly balanced across the treatment groups (table 1). the individual parameters of the disease activity measures were shown in table 2, representing the findings at study entry. both groups were roughly balanced in terms of these parameters in study entry. table 3 showed the mean change in the individual parameters of the disease activity measures at the end of the study, by treatment group. the comparison on the effectiveness of monotherapy with mtx and combination therapy with mtx & hcq based on das28 was shown in table 4 whereby a p-value ≥ 0.05 was obtained indicating the insignificant results discussion from the overall data obtained for the outpatients’ medical record installation, there were a total of 213 patients who visited the rheumatology clinic from january 2009 to october 2014 whereby the total of female patients were 165 (77.5%) and male patients were 48 (22.5%). the incidence was a lot higher in females compared to males with a ratio of 3:1. in fact, this is true whereby other autoimmune diseases, ra has a higher occurrence in females compared to males with a ratio of 2−3:1. meanwhile, studies from certain latin american and african countries showed a considerably greater predominance of ra in females compared to males with a ratio of 6−8:1. many theories have proposed the role of estrogens in this disease said that estrogens enhance the immune response by stimulating the production of tumor necrosis factor α (tnf−α) which is an important cytokine in the pathological process of ra.2 the average age of patients in this study was 44 years old and mostly (32.61%) were in the age range of 39−48 years old. most of the studies conducted on ra in europe showed that the average age of the patients is between 48.9−52.1 years.14,15 besides, a study by alam et al.16 in bangladesh to compare the effectiveness of mtx against hcq showed that table 2 initial values for disease activity measures by treatment group* parameters mtx mtx & hcq sjc (maximum 28) 2.52 ± 5.66 3.48 ± 4.22 tjc (maximum 28) 8.83 ± 7.42 9.57 ± 5.62 esr (mm/h) 44.83 ± 34.49 53.57 ± 31.20 note: * values are the mean ± sd. mtx = methotrexate; hcq = hydroxychloroquine; sjc = swollen joint count; tjc = tender joint count; esr = erythrocyte sedimentation rate table 3 changes in values for disease activity measures by treatment group parameters mtx mtx & hcq p-value sjc (maximum 28) 1.52 ± 4.73 2.22 ± 4.07 0.337** tjc (maximum 28) 5.74 ± 7.37 6.57 ± 5.85 0.676* esr (mm/h) 15.61 ± 33.68 13.09 ± 26.74 0.780** note: * mann whitney test; ** independent t-test . mtx = methotrexate; hcq = hydroxychloroquine; sjc = swollen joint count; tjc = tender joint count; esr = erythrocyte sedimentation rate althea medical journal. 2016;3(3) 449natharubini nadarajah, riardi pramudyo, kuswinarti: treatment of rheumatoid arthritis with methotrexate only or a combination of methotrexate and hydroxychloroquine the average age of patients in their two study groups is 41.7 and 42.9 years old respectively. it was said that ra mostly occurs between 40−70 years old and it als occurs later in life for men.2 this may be due to the fact that women begin to experience menopause around the fifth decade of life causing a decrease in their estrogen levels. hence, it can be said that women are likely to suffer from ra when their estrogen level is still high, which is before 50 years old (average). the changes in value for sjc and tjc at the end of this study were -1.5 and -5.7 for the mtx group and -2.2 and -6.6 for the combination therapy group, respectively. in a study conducted by o’dell et al,17 the mean changes in value for sjc and tjc after taking mtx & hcq are -14.0 and -10.0, respectively, which are higher compared to the results in this study. however, it should be noted that the study conducted by o’dell jr et al.15 used the 38 joint count compared to the 28 joint count used in this study. this difference in joints count could be one of the reasons for the low changes in sjc and tjc values in this study. many research and studies have been conductedto determine the effectiveness of dmards. o’ dell jr et al.17 conducted two different studies to determine the effectiveness of triple therapy against dual and monotherapy. in their first study, they demonstrated the superior efficacy of the triple combination therapy (mtx, hcq, & sulfasalazine, ssz) over both mtx on its own and the double combination of mtx & ssz.17 in their second study, they concluded that the efficacy of the triple combination of mtx, ssz, & hcq is superior to the double combination of mtx & ssz and marginally superior to the double cobination of mtx & hcq.15 this is supported by the treach trial conducted by de jong et al.18 that concluded that therapy with a combination of dmards is better than mtx monotherapy. in this study, it was showed that double therapy is similar in effectiveness to monotherapy. the results of this study contradict the results of previous studies. one of the main reasons is the small sample obtained in this study; larger samples increase the chance of finding a significant difference because they more reliably reflect the population mean. in this study, although the calculated sample size was 92, however, only 46 samples were obtained. comparing to other studies, the sample size used was large (about 150−200 patients).15 it is not known why certain patients with ra respond to treatment better compared to others. a study by anderson et al.19, showed certain factors to answer this question and one of the factors is the disease duration. their study showed that patients with longer disease duration have a poorer response to treatment compared to patients with shorter disease duration. in their study, they stated that there are indications that the biologic process of ra change early in the disease, so that patients may be less responsive to treatment over time. other factors that were said to decrease response to treatment were female sex, prior dmard use, and worse functional class. it was also stated that rheumatoid factor affects response to treatment.19 in another study table 4 comparison on the effectiveness of monotherapy with mtx and combination therapy with mtx & hcq based on das28 * category of das 28 score treatment group total p-valuemtx mtx & hcq f % f % f % before treatment low 0 0.00 1 100.00 1 100 moderate 17 58.62 12 41.38 29 100 high 6 37.50 10 62.50 16 100 after treatment low 9 60.00 6 40.00 15 100 0.584 moderate 13 46.43 15 53.57 28 100 high 1 33.33 2 66.67 3 100 note: * das 28 = disease activity score 28; mtx = methotexate; hcq = hydroxychloroquine; f = frequency althea medical journal. 2016;3(3) 450 amj september 2016 by radovits et al.20, to find out the influence of age and gender on das28 in rheumatoid arthritis, they concluded that age and gender do not affect the das28 of ra patients. due to the unavailable complete data in the medical record, this study could not conclude if these factors influence the response to treatment in ra. it was concluded that there is no difference in the effectiveness of dmard monotherapy with mtx and combination therapy with mtx & hcq in ra patients. the limitations of this study are some of the medical records were not available in the storage room where there are possibilities that the medical records are misplaced by the staffs of medical record. besides, some of the medical records also do not have the complete or required data and, hence, cannot be used in this study.the number of the study sample can be one of the limitations of the study which a larger number of study sample can be used reflecting the population clearly. lastly, time constraint is also one of the limitations with the tight academic schedules and short duration for data collection and analysis for this study. certain recommendations can be considered to improve this study. first and foremost is improving the management of medical record at rheumatology clinic of dr. hasan sadikin general hospital bandung so that the data of the patients that are obtained from the medical record can be used and analyzed properly. next a standardized medical form can be used so that physicians attending to patients at the rheumatology clinic will not forget or over look to fill up the relevant data in the medical record. increasing the period of data collection is also suggested to enable the researcher to get more patients to meet the targeted sample size. lastly, further studies about another dmard therapy for ra should be conducted to get more information about the effectiveness of other dmards. references 1. aletaha d, neogi t, silman aj, funovits j, felson dt, iii cob, et al. 2010 rheumatoid arthritis classification criteria: an american college of rheumatology/ european league against rheumatism collaborative initiative. ann rheum dis. 2010;69(10):1580−8. 2. shah a, clair ews. rheumatoid arthritis. in: longo dl, kasper dl, jameson jl, fauci as, hauser sl, loscalzo j, editors. harrison’s principles of internal medicine. 18th ed. new york: mcgraw-hill companies, inc.; 2010. p. 2738−52. 3. mcinnes ib, schett g. the pathogenesis of rheumatoid arthritis. n engl j med. 2011;365(23):2205−19. 4. donahue ke, gartlehner g, jonas de, lux lj, thieda p, jonas bl, et al. systematic review: comparative effectiveness and harms of disease-modifying medications for rheumatoid arthritis. ann intern med. 2008;148(2):124−34. 5. o’dell jr. therapeutic strategies for rheumatoid arthritis. n engl j med. 2004;350(25):2591−602. 6. emery p. treatment of rheumatoid arthritis. bmj. 2006;332(7534):152−5. 7. swierkot j, szechinski j. methotrexate in rheumatoid arthritis. pharmacol rep. 2006;58(4):473−92 8. smolen js, landewé r, breedveld fc, buch m, burmester g, dougados m, et al. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update. ann rheum dis. 2014;73(3):492-509. 9. leeb bf, andel i, sautner j, nothnagl t, rintelen b. the das28 in rheumatoid arthritis and fibromyalgia patients. rheumatology. 2004;43(12):1504−7. 10. siegel j. comparative effectiveness of treatments for rheumatoid arthritis.ann intern med. 2008;148(2):162−3. 11. breedveld fc, kalden jr. appropriate and effective management of rheumatoid arthritis. ann rheum dis. 2004;63(6):627−33 12. smolen j, keystone ec. future of ra: building on what we know and tailoring treatment; biologic therapies beyond conventional dmards. rheumatology. 2012;51(suppl 5):v55−6 13. katchamart w, trudeau j, phumethum v, bombardier c. efficacy and toxicity of methotrexate (mtx) monotherapy versus mtx combination therapy with nonbiological disease-modifying antirheumatic drugs in rheumatoid arthritis: a systematic review and meta-analysis. ann rheum dis. 2009;68(7):1105−12. 14. breedveld fc, weisman mh, kavanaugh af, cohen sb, pavelka k, vollenhoven rv, et al. the premier study; a multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients althea medical journal. 2016;3(3) 451 with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. arthritis rheum. 2006;54(1):26−37. 15. o’dell jr, leff r, paulsen g, haire c, mallek j, eckhoff pj, et al. treatment of rheumatoid arthritis with methotrexate and hydroxychloroquine, methotrexate and sulfasalazine, or a combination of the three medications: results of a two year, randomized, double blind, placebo controlled trial. arthritis rheum. 2002;46(5):1164−70. 16. alam mk, sutradhar sr, pandit h, ahmed s, bhattacharjee m, miah a, et al. comparative study on methotrexate and hydroxychloroquine in the treatment of rheumatoid arthritis. mymensingh med j. 2012;21(3):391−8. 17. o’dell jr, haire ce, erikson n, drymalski w, palmer w, eckhoff pj, et al. treatment of rheumatoid arthritis with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three medications. n engl j med. 1996;334(20):1287−91. 18. de jong ph, hazes jm, barendregt pj, huisman m, van zeben d, van der lubbe pa, et al. induction therapy with a combination of dmards is better than methotrexate monotherapy: first results of the treach trial. ann rheum dis. 2013;72(1):72−8 19. anderson jj, wells g, verhoeven ac, felson dt. factors predicting response to treatment in rheumatoid arthritis: the importance of disease duration. arthritis rheum. 2000;43(1):22−9. 20. radovits bj, fransen j, van riel plcm, laan rfjm. influence of age and gender on the 28-joint disease activity score (das28) in rheumatoid arthritis. ann rheum dis. 2008;67(8):1127−31 natharubini nadarajah, riardi pramudyo, kuswinarti: treatment of rheumatoid arthritis with methotrexate only or a combination of methotrexate and hydroxychloroquine iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 77 evaluation of trace elements in iraqi patients with rheumatoid arthritis by using atomic absorption spectrophotometer (aas) # huda m. ali* and mohammed a. al-zubaidi** ,1 * department of pharmaceutical chemistry and pharmacognosy , college of pharmacy , al-mustansiriya university, baghdad, iraq ** department of clinical laboratory sciences, college of pharmacy, almustansiriya university, baghdad, iraq abstract zinc, copper, selenium, magnesium, manganese, chromium, iron, nickel, cobalt, vanadium and germanium were determined by atomic absorption spectrophotometer (aas) in blood serum of patients with rheumatoid arthritis, (30) patients (14male and 16female) with age range (37-60) years compared with normal tensive control. the analysis of results showed that the mean value of concentration (magnesium, manganese and nickel) were significantly higher in patients with rheumatoid arthritis compared to that of healthy, while the mean levels of serum (zinc, copper, selenium, chromium, iron, cobalt and germanium) were significantly lower than controls. there were no significant changes in overall mean concentration of serum vanadium in patients with rheumatoid arthritis and control group. there were no significant variations in trace elements levels in relation to sex.our results suggest that low level of trace elements of zinc, copper, selenium, chromium, iron, cobalt and germanium , and high levels of magnesium, manganese and nickel may provide good clues to the physician about the high probability of the individual for developing of rheumatoid arthritis disease unless the imbalance of these elements in blood serum to be corrected. key words: trace elements, rheumatoid arthritis,atomic absorption spectrophotometer (aas). باستخذام ثىٌالنادرة فٍ المزضً العزاقُُن المصابُن بالتهاب المفاصل الز تقذَز العناصز مطُاف االمتصاص الذرٌ الشبُذٌ** عبذ الزضاهذي محمذ علٍ* و محمذ ،1 .، ميُح اىصُذىح ، اىداٍعح اىَسرْصشَح ، تغذاد ، اىعشاقفشع اىعقاقُش واىْثاذاخ اىطثُح * .شَح ، ميُح اىصُذىح ، اىداٍعح اىَسرْصشَح ، تغذاد ، اىعشاقفشع اىعيىً اىَخرثشَح اىسشَ ** الخالصت ح اىثاىغح فٍ وقاَح وعالج أٍشاض اىَفاصو واىرٍ ذٌ اسرخذاٍها عاىَُا عيً شنو ُذٌ قُاط اىعْاصش اىْادسج راخ االهَ ,zinc, copper, selenium, magnesium, manganese, chromium, iron, nickelعقاقُش ٍع اىفُراٍُْاخ وهٍ cobalt, vanadium and germanium تىاسطح خهاص االٍرصاص اىزسٌ اىيهثٍ وغُش اىيهثٍ, ذٌ قُاط هزٓ اىعْاصش فٍ ٍصو ( سْح, وذٌ 13-03ذرشاوذ تُِ ) اّثً( وفٍ اعَاس41 و رمش 41 ) (ٍشَط 03 دً اىَشظً اىَصاتُِ تاىرهاب اىَفاصو اىشثىٌ, ) magnesium, manganese andْادسج ٍع اىَسرىي اىطثُعٍ.أوظحد ّرائح اىرحيُو أُ ٍسرىي اىعْاصش ٍقاسّح ّرائح اىعْاصش اى nickel راخ ٍعذه ٍشذفع تشنو ٍعْىٌ ىذي اىَشظً ٍقاسّح تاألصحاء تَُْا ماُ ٍعذهzinc, copper, selenium, chromium, iron, cobalt and germanium ألصحاء. وىٌ َرٌ اىرىصو اىً فشق ظاهش ٍْخفط تشنو ٍعْىٌ عْذ ٍقاسّرها تا قذ ذعطٍ هزٓ اىعْاصش . الَىخذ هْاك اخرالف فٍ ذشمُض اىعْاصش تُِ اىزمش واالّثً.vanadiumتُِ اىَشظً واألصحاء فٍ ٍعذه ىً اىىظع دىُال واظحا ىيطثُة عِ وخىد احرَاىُح مثُشج إلصاتح األفشاد تَشض اىرهاب اىَفاصو اىشثىٌ ٍا ىٌ ذعذه هزٓ اىْسثح إ اىطثُعٍ ورىل تىاسطح اخز هزٓ اىعْاصش حسة اىحاخح. العناصز النادرة ، التهاب المفاصل الزثىٌ ،جهاس االمتصاص الذرٌ.: الكلماث المفتاحُت introduction rheumatoid arthritis had a worldwide distribution and affects all ethnic groups. rheumatoid arthritis is a chronic inflammatory, systemic disease that produces its most prominent manifestations in the diarthrodial (1,2) . typical form of the disease is symmetrical, destructive and deforming polyarthritis affecting small and large synovial joints with associated systemic disturbances, a variety of extra-particular features and the presence of circulating anti globulins antibodies (rheumatoid factor) (3) .meanwhile, trace elements are widely distributed in a variable proportion in human body and they play a vital role in growth. zinc is a part of every cell in the body and forms a part of over 300 enzymes that have functions ranging from proper action of the body hormones to cell growth. zinc deficiency can cause growth retardation (4,5) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : mohammedizubaidy@yahoo.com received : 21/5/2011 accepted : 30/6/2012 iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 78 zinc is important in the maintenance of proper immune response (5) . copper is an essential part of key metalloenzymes as ceruluplasmine, cytochrome, oxidase, tyrosinase and monamine oxidase (6) . copper enters in a large number of enzymes in addition ceruloplasmin, as it will be necessary for the work of an enzyme super oxide dismutase (sod) as well as an oxidation enzyme lysyl oxidase, which is one of the necessary enzymes in the synthesis of connective tissue, it is believe that lack of this enzyme leads to decrease of copper, which leads to adverse effects in bone and connective tissue (7) . excess copper as with excess iron can cause free radical production and damage, also deficiency of copper results in poor collagen integrity with resultant blood vessel rupture (7,8) .selenium is an essential element of one of the main antioxidant enzymes in the human body, glutathione peroxidase (gsh-px). this explains its benefit in a number of “oxidative” conditions including cancer, cardiovascular disease and rheumatoid arthritis (1) , selenium is incorporated at four active site in the antioxidant enzyme glutathione peroxidase (gsh-px). this enzyme protects against free radical and oxidative damage by catalyzing the destruction of hydrogen peroxides (h2o2) and lipid peroxides, this in turn protects membrane lipids and hemoglobin against oxidation by peroxides (9,10) . magnesium is involved in at least 300 enzymatic processes; magnesium participates in a number of biochemical reactions that take place in bone. alkaline phosphatase, enzyme involved in forming a new calcium crystals, is activated by magnesium. the conversion of vitamin d to its biologically active form 1,25 dihydroxy vitamin d3, also appears to require magnesium. deficiency of magnesium can produce a syndrome of vitamin d resistance. the concentration of intracellular magnesium is very high as compared with concentration of extracellular magnesium for that reason can be attributed the increase in the output cell concentration to the damage done to the cells and then increase its concentration in the blood serum (1,11) . magnesium an important in bone structure. deficiency results in tetany and can lead to calcium deficiency. magnesium is essential to maintain both acidbase balance in the body, and healthy functioning of nerves and muscles (12) . diets that provide recommended levels of magnesium are beneficial for bone health, but further investigation on the role of magnesium in bone metabolism and osteoporosis is needed (8,13) . manganese is biochemically essential as a constituent of metalloenzymes and as an enzyme activator (14) . a deficiency of manganese concentration may affect brain health and skeletal and cartilage formation, antioxidant enzyme superoxide dismutase (sod) which prevents damage by superoxide free radicals (15,16) .chromium is one of the newer essential trace elements. have a great role in maintaining good health; chromium may have a function in the control of glucose and lipid metabolism (17) .iron carries oxygen to the cells and is necessary for the production of energy (18-19) . iron is available in both a ferrous and ferric. iron in the ferrous form is better absorbed than ferric iron. many people with iron deficiency anemia die from infection because of weak end immune systems. iron's role in maintaining immunity covers every aspect of how the systems work (20) . iron is also needed to help produce antibodies and to maintain your white blood cell count (18) .a significant amount of iron is stored as ferritine and hemosiderin. iron played a potential role in oxidative stress mediated injuries and pathologies e.g. rheumatoid arthritis (21,22) . nickel has been shown to be essential to man, is present in all tissues of the body. it is firmly attached to dna and a protein that binds to it in the blood (23) . nickel is used for increasing iron absorption, preventing iron-poor blood (anemia) and treating weak bone and bone structure, it may also be involved in iron metabolism, as it influences iron absorption from foods and may also play a role in production of red blood cell. nickel may then bind with one of the body neutral proteins, this nickel-protein complex may not be recognized by the immune system, and this may tiger signals to the body's defense mechanism to respond to the complex as if were an intruding antigen. nickel may deplete glutathione and protein-bound sulf hydryl group, resulting in the production of reactive oxygen species, such as superoxide anion, hydrogen peroxide and hydroxyl radical. other changer observed in a nickel deficient state include change in skin color, hormone imbalance and abnormal bone growth. liver function is impaired and iron metabolism is affected, resulting in poor absorption of iron. several publication review the evidence for the essentiality and proposed functions of this elements (24,25) .cobalt is essential for humans only as an integral part of vitamin b12 (cobalamin), no other function for cobalt in the body is known. deficiency of the vitamin b12 causes a megaloblastic anemia and in several cases, sub acute combined degeneration of the spinal cord (18,12) . cobalt essential for the growth and development of iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 79 healthy nervous system. deficiency of vitamin b12 and hyperplesia of the bone narrow. cobalt is one of the essential trace elements, human body need essential elements to grow. it is also necessary co-factor for making the thyroid hormone and deficiency lead to slow growth and development conditions (26) . cobalt as a co-factor in antioxidant therapy for the enzyme catalase. deficiencies are reflected in abnormal bone development (26) .vanadium biochemical function as peroxide of vanadate activated insulin receptor in animal studies (27) . vanadium is another trace mineral that we do not understand the role of very well yet. it clearly has an effect on the health of our bones. in research with goats scientists have noted skeletal deformations in the legs of vanadium a compound containing vanadium has been shown to stimulate bone cell proliferation. it was also shown to stimulate collagen (the organic part of bone that provides strength and flexibility) synthesis. vanadium compounds were shown to increase bone formation without any adverse health effect (27,28) . vanadium is necessary for bone and tooth development. high doses of vanadium improve the strength of bone and teeth in experimental (27) . it is needed for cellular metabolism and for the formation of bones. vanadium may be required for glucose, cholesterol and bone metabolism (28) . high doses of vanadium may deplete vitamin c (29) . the chemical properties of germanium are similar to those of silicon (30) . according to kidd (31) , germanium normalizes many physiological functions such as lowering high blood pressure in human. seaborn and nielsen (30) investigated whether germanium could substitute for silicon in bone formation. a most interesting characteristic of germanium is it ability to relieve a great deal of pain it does this by inhibiting the natural body enzyme that in turn inhibits production of the body endorphins (28) . germanium has the ability of stimulate immune function, supplement tissue oxygen and help inhibit rheumatoid arthritis. it has been suggested that germanium deficiency could be a contributory factor in kashin-beck disease (arthritis disease) (31) . arthritis is a disease of the immune system, commonly referred to as an (autoimmune) disorder, the membrane surrounding a joint becomes inflamed, resulting in a build up of lymphoid cells, resulting in the degeneration of bone, cartilage, ligaments and tendons. some agent, perhaps the epstein-barr virus, may tigger the initial joint inflammation, resulting in the production away results in the painful swelling and of antibodies. this immune response gone inflammation characterized by arthritis. materials and methods patients and method a shimadzu model aa6200 flame atomic absorption spectrophotometer faas and graphic furnace shimadzu flameless atomic absorption gfa6200 were used for analysis of blood serum samples, which were centrifuged through the preparation of samples then dilution the serum with deionized water for analysis. samples were taken from patients who had been admitted to baghdad teaching hospital with positive diagnosis of rheumatoid arthritis disease from (february december) 2010, the analysis of trace elements was done in ministry of science and technology labs.zinc, copper, magnesium and iron were analyzed by flame atomic absorption spectrophotometer, but selenium, manganese, chromium, nickel, cobalt, vanadium and germanium were analyzed by flameless atomic absorption spectrophotometer at their specific wavelength.standard of sample zn, cu, se mg, mn, cr, fe, ni, co, v and ge are obtained from analytical reagent using solution (aldrich, 1000µg / l) for each, and subsequent dilution is then carried out to obtain a calibration curve. all other reagent used was of analytical grade, distilled deionized water was used to ensure no leaching of any trace elements to the measured standard and samples polyethylene containers are used to maintain clean samples.serum samples were obtained from 30 patients (14 males and 16 females) their ages ranged between (37-60) years, with means ± sd (45.667±6.875). the mean duration of the disease was one year.the control group consisted of 30 healthy (14 male and 16 female) individuals with no general complications and who were receiving no medication, their ages ranged between (35-55) years, with means ± sd, ( 44.667 ± 66.874) . all statistical work and reporting of obtained data were carried out by using spss program version 10.0. difference of the means considered of significance according to the ttest at level of p<0.05 and p<0.01. result and discussion this study was performed for to compare between in apparently healthy subject with patients with rheumatoid arthritis. the role of trace metallic elements in chronic inflammatory states is of great interest because many of them are co-factors in metabolic processes involving articular tissues and immune system function (18) . however, many studies showed that there was a relationship between trace elements levels and period of iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 80 rheumatoid arthritis diseases.the role of zinc and copper in chronic inflammatory disease is of interest because they are co-factor of important enzymes involved in collagen and bone metabolism, immune functions and antioxidant protection. an excess of zinc may cause anemia or reduce bone formation. zinc also plays an important role in the catabolism of rna by regulation rnaase activity. the zinc and copper metals prevent the formation of free radicals capable of inducing mutation and have antioxidant effects (7,8,20) .results of these studies have demonstrated that both copper and zinc alteration can be explained by the active inflammatory process and that serum trace elements are measures of disease activity, and immune system function (7) .one hypothesis mentioned that a decreased zinc and increase of copper in sera of acute or chronic inflammatory processes cause an accumulation of copper and zinc in many body compartments and in the inflamed areas (7) . supporting the hypothesis that the development of inflammation induces an increase body requirement of copper and zinc. the results obtained for zinc concentrations in healthy and patients group which are shown in table (1), show high significant difference in zinc levels decreased (p<0.001). concentration of zinc is so low in patients group as compared with those in control group. the result obtained in this study are similar to those published in the literature (7,8,26) . zinc deficiency is common that effect obviously apparent in figure (2) which represented a histogram for data of table (2) means of zinc colak et al. (7) reported two effect of copper deficiency on iron metabolism, the first occurring early was an adverse effect of copper deficiency on iron absorption (or mobilization), the second was inadequate erythropoiesis, even in the presence of a abundant iron stores. serum copper level for patients and controls are shown in table (1), and figure (2) a histogram indicates decrease serum copper levels in the patients groups in comparison with controls groups in both sex.the decrease of copper level was statistically significant (p<0.001). the results of this study indicate that copper serum level decrease significantly during rheumatoid arthritis these results are in agreement with the reported (6,8) .protects cells from oxidative damage (7) . selenium has been shown to have anti (proliferative, inflammatory, viral) and immune altering effects. dietary selenium is essential for an optimum immune response, although the mechanisms of this requirement are not always fully understood (18) . figure (1) represents a histogram for data of table (1) shows the result of selenium determination in human sera. the mean±sd for control group is (102±15) serum selenium, high significant differences were found (p<0.001), decreased in all patients group compared with control group, these results proved the possible relationship between low levels of serum selenium and rheumatoid arthritis (7) .table (1) comprise the results obtained for the magnesium content in the sera of all groups involved in the present study. therefore, the difference in the mean serum magnesium concentration between the two groups is highly significant increased (p<0.001). these results are in agreement with the previous studied (8) . figure (3) shows a histogram which contains the mean values of serum magnesium concentration in control and patients groups.manganese is one of several trace elements that are necessary for bone health. one study (15) found that taking a combination of calcium, zinc, copper and manganese helped lessen spinal bone loss in a group of post menopausal women. people with arthritis tend to have low level super oxide dismutase (sod) (an oxidant that helps protect the joints from damage during inflammation). manganese is a required mineral in the metabolism of protein, fat, healthy immune also required in normal bone growth, energy production. the estimated results of serum manganese are listed in table (1), these results clearly show that high significant differences, elevated (p<0.001) in patients group with rheumatoid arthritis as compared with control group, very little information was found in the literature about this point (8) . manganese is required for the utilization of vitamin b1 and vitamin e, it is used in the formation of cartilage and synovial fluid of the joints. manganese deficiency can lead to improper bone formation and production disorder. an excess of manganese can lead to poor iron metabolism (14) .chromium is widely distributed throughout the body, infants have a higher chromium concentration than adults (1) . the basis for the suggestion that chromium may effective in preventing rheumatoid arthritis is that post menopausal women taking a chromium supplement exhibited increased plasma dehydro epiandrosterone, a precursor of estrogen which inhibits bone loss, and decreased urinary calcium and hydroxy proline excretion, which are indirect rather variable indicators of bone loss (26) . these provocative findings need to be confirmed, and the prevention of bone loss needs to be validated by the use of methods that can directly detect iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 81 changes or no changes in bone composition with chromium supplementation; chromium supplementation should view as only one of a number of speculative method that may help in maintaining healthy bones (17,28) . figure (1) shows a histogram which contains the mean values of serum chromium concentration in control and patients groups, chromium deficiency in patients with rheumatoid arthritis disease. table (1) shows the results for mean values of serum chromium concentrations of these elements, so low in patients group as compared with those in control group. the results are in agreement with the data (20) . there were no significant difference in serum trace elements level between male and female patient group according table (2) and table (3). studies show people who have both rheumatoid arthritis and anemia tend to have more severe arthritis than people without anemia. they are more likely to have serious joint symptoms; anemia is the most common problem for people with rheumatoid arthritis. studies show as many as 60% of people with rheumatoid arthritis are anemic. table 1: serum trace elements concentration in patients with rheumatoid arthritis and healthy control group in serum (ng/ml) *. trace elements studied groups no. mean(ng/ml) ± sd comparison of significance t-test p-value zn control 30 975.3 ± 170.1 30.45 0.001 patients 30 710.2 ± 200.76 cu control 30 1110 ± 250.3 20.17 0.001 patients 30 872.13 ± 146.72 se control 30 102 ± 15 6.82 0.01 patients 30 92.8 ± 12.13 mg control 30 16526.67 ± 1056 20.72 0.001 patients 30 20984.92 ± 5260 mn control 30 26.5 ± 6.25 6.45 0.01 patients 30 31.26 ± 8.14 cr control 30 46.5 ± 3 85.79 0.001 patients 30 40.2 ± 2.21 fe control 30 1150 ± 300.4 50.69 0.001 patients 30 712.33 ± 152.06 ni control 30 24.5 ± 4.7 20.12 0.001 patients 30 33.46 ± 9.88 co control 30 38.5 ± 4.3 7.87 0.001 patients 30 35.73 ± 3.28 v control 30 33 ± 1.2 0.42 0.51 patients 30 32.66 ± 2.61 ge control 30 42.1 ± 1.6 159.71 0.001 patients 30 31.73 ± 4.2 *concentration are expressed as mean± sd table 2 : mean serum levels (ng/ml) between different genders of patients * zn cu se mg mn cr male 712±220.56 878.15±151.62 93.65±12.5 22113.7±5773 32.4±8.4 40.6±2.92 female 708±180.83 866.11±143.21 91.95±10.2 19856.14±4939 30..12±7.8 39.8±3.05 *concentration are expressed as mean±sd p value >5% for all elements between male and female table 3: mean serum levels (ng/ml) between different genders of patients* fe ni co v ge male 725.82±158.4 34.77±9.23 36.65±3.57 33.48±3.13 32.81±4.5 female 698.84±146.3 32.15±8.71 34.81±2.98 31.84±2.32 30.65±4.1 *concentration are expressed as mean±sd p value >5% for all elements between male and female iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 82 0 20 40 60 80 100 120 mean serum trace elements level (ng/ml) studied groups control patients 0 200 400 600 800 1000 1200 meanserum trace elements level (ng/ml) studied groups control patients table (1) and figure (2) show a histogram which contained the mean values of serum iron concentration in control and patients groups, iron deficiency in patients with rheumatoid arthritis disease (18,28) . iron plays potential role in oxidative stress, mediated injuries and pathologies e.g. rheumatoid arthritis, decades ago it was suggested that iron may have a crucial role in progression of inflammation in rheumatoid arthritis. indeed free radical generated by iron can cause damage to lipids, proteins, carbohydrates and dna. it is this destruction process that it believe to occur in rheumatoid joint (1,18) .research showed that nickel was to be found in blood and tissue at quite consistent physiological significance. nickel is required for normal growth and reproduction in animals and presumably in human being as well it appears to have role in the modulation of the immune system. nickel plays some important role in biological system such as in enzyme activity, hormonal control also in structure or function of rna, dna and protein (24,25) .metabolism of other nutrients like calcium and vitamin b12 is also altered due to nickel deficiency. bone development, resistance to infection and immune function are some of the problems associated with nickel. table (1) figure (1) represented a histogram for mean values of serum nickel concentration in blood sera of control and patients groups.table (1) gives serum cobalt concentrations in patients with rheumatoid arthritis and control subject. attempt to measure serum cobalt levels in patients with rheumatoid arthritis disease were reported only by few investigators (18,11) .table (1) contains the results of serum vanadium concentrations in controls and patients with rheumatoid arthritis disease. as shown above these results are also included in figure (1) which represented a histogram for serum vanadium levels of control and patients groups. this study involved measurement of serum vanadium in patients described above, the results nosignificant difference decrease (p>0.05) compared with control.figure (1) represents a histogram for data of table (1) which contains all results of germanium determination in blood sera of control and patients. figure1: mean serum trace elements level (ng/ml) among the studied groups figure 2: mean serum trace elements level (ng/ml) among the studied groups se cr mn ni v co ge zn cu fe iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 83 figure 3: mean of serum trace elements level (ng/ml) among the studied groups conclusion the conclusions obtained from this study can be summarized: accurate, sensitive and reliable methods had been adopted for measurement of eleven essential and trace elements using both flame and flameless atomic absorption spectrophotometer in blood sera of normal and patients with rheumatoid arthritis.the results observed that the levels of serum zinc, copper, selenium, chromium, iron, cobalt and germanium were significantly decreased in patients with rheumatoid arthritis as compared to a control group,the results show that the level of serum magnesium, manganese and nickel were highly increased in the patients. the results of this study show no significant change in the concentrations of serum vanadium in all patients compared with normal subjects. there were no significant differences in sex related to trace elements. the alteration of trace elements in sera of patients with rheumatoid arthritis can enable us to shed more height on the role of trace elements in both physiological and pathological states. since there is a significant decrease in zinc, copper, selenium, chromium, iron, cobalt and germanium, so supplementation of these trace elements could be necessary to get a beneficial from trace elements rebalance in blood serum. we suggest that the deficiency of serum zinc, copper, selenium, chromium, iron, cobalt and germanium might be used in early diagnosis and treatments of rheumatoid arthritis. references 1. michael lb, edward pf and larry gs, "clinical chemistry” lippincott william and wilkins 2010, 403-668. 2. smith sm, donald a and webb d "complete book of vitamins and minerals" publications international, ltd 1998, 210264. 3. cerhan jr, saag kg, merlino la, mikuls tr and criswell la. "antioxidant micronutrients and risk of rheumatoid arthritis in a cohort of older women" am. j. epidiol 2003; 157(4): 345-354. 4. florianczyk b. "trace elements as constituents of antioxidative proteins", journal of pre-clinical and clinical research 2008; 2( 1): 25-27, 5. rink l and haase h. "zinc homeostasis and immunity", trends in immunology 2006; 28(1):1-4 6. ala s, shokrzadeh m, pur shoja am and saravi sss "zinc and copper plasma concentrations in rheumatoid arthritis patients from a selected population in iran" pakistan journal of biological sciences 2009; 12(14): 1041-1044. 7. colak m, bingol nk, ayhan o and avci s, "serum copper, zinc and selenium levels in rheumatoid arthritis" rhomatizma 2001; 16( 2):66-71. 8. satish kt and reshu m, “assessment of mineral status (zn, cu, mg and mn) in rheumatoid arthritis patients in chandigarh, india”, rheumatology report 2009,1(5):16-20. 9. kaplan l a, and pesce aj “clinical chemistry” 5 th edition, mosby elsevier 2008, 804-821 10. joshi yk, joshi m, singh n, benjamin j and sharma t “basics of clinical nutrition” jaypee brothers medical publishers ltd, 2 nd edition 2008, 190-202. 11. thomas md, "textbook of biochemistry", 7 th ed. john and wiley 2011, 1078-1097. iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 84 12. lengman m. "safe upper levels for vitamins and minerals expert group on vitamins and minerals" 2003, 164-287. 13. nielsen fh. "trace minerals dificiencies”, by crc press llc 2002,1463-1487 14. santamaria ab. “manganese exposure, essentiality and toxicity” indian j.medzres 2003; 128: 484-500. 15. de carvalho pr, gonçalves pita mc, loureiro je, tanaka hr and ribeiro js, “manganese deficiency in bovines: connection between manganese metalloenzyme dependent in gestation and congenital defects in new born calves”, pakistan journal of nutrition 2010, 9(5): 488-503. 16. tuormaa te. “ the adverse effect of manganese deficiency on reproduction and health: a literature review”, journal of orthomolecular medicine 1996; 11( 2): 69-79. 17. jonathan cl, ” trace elements in the fetus and young infant, copper, manganese, selenium and chromium" am. j. dis.child 1981; 134: 74-81. 18. brig mn, chatterje a and shinde r, "text book medical biochemistry", 6 th ed. published by jitendar prij 2005, 178-557. 19. khanna s. “immunological and biochemical markers in oral carcinogenesis; the public health perspective”, int. j. environ. res. public health 2008; 5(5): 418-422. 20. cerhan jr, saag kg and criswell la. “antioxidant micronutrients and risk of rheumatoid arthritis in cohort of older women” am. j. epidemiol 2003; 157(4): 345-354. 21. john lb “institute of medicine, dietary reference intakes for vitamins a, k, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium and zinc”. washington dc, nutritional academy press 2001, 101-122. 22. majhi t and srivastava ak. “ iron deficiency in rheumatoid arthritic patients especially with in the middle age”, international journal of systems biology 2010; 2(1): 1-5. 23. das kk, das sn and dhundasi sa. “ nickel, its adverse health effects and oxidative stress”, indian j.med res. 2008; 128: 421-425. 24. spiewak r, pietowska j and curzytek k. “ nickel: a unique allergic from molecular structure to european legislation, epert rev. immunol. 2007; 3(6): 851-859. 25. cempel m and nikel g. ” nickel: review of its sources and environmental toxicology”, polish j. of environ. stud. 2006; 15(3): 375-382. 26. carl ab and edward ra “ tietz fundamentals of clinical chemistry”, 6 th ed., saunders an imprint of elsevier 2008, 496-507. 27. ferestein gs, larry a, cruz tf, cheng tp, banqueriso ml and boyle dl "vanadium an inhibitor of stromelysin and collagenase expression, suppresses collagen induced arthritis" j. rheumatology 2007; 34(9): 1802-1809. 28. hathcock jn, "vitamins and minerals safety", 2 nd ed. 2004, 35-49. 29. poucheter p, verma s, grynpas md and neill jh, “vanadium and diabetes”, mol. cell biochem. 2000; 208 (1-2): 167-168. 30. seaborn cd and neilson fh. "effect germanium and silicon on bone mineralization", biological trace elements research 1994; 42: 151-164. 31. kidd p m., germanium 132 (ge 132): homeolatic normalizer and immunostimulant: a review of its prevention efficacy. international clinical nutrition review 1987; 7: 11. key: cord-0065950-wxmnx7or authors: wang, zunlian; huang, min; yu, bin; huang, yilan; zheng, silin; yang, xuping; ning, hong title: comparison of the efficacy and safety indicators of dmards for rheumatoid arthritis: a network meta-analysis date: 2021-07-23 journal: medicine (baltimore) doi: 10.1097/md.0000000000026524 sha: 1c6e28bf8e7180338bfd57839601d610b4411122 doc_id: 65950 cord_uid: wxmnx7or objective: to compare efficacy and safety indicators of disease-modifying antirheumatic drugs, sarilumab, sirukumab, baricitinib, tocilizumab and adalimumab in rheumatoid arthritis treatment by a network meta-analysis. methods: medline, embase, web of science, the food and drug administration web site, and cochrane library were searched from build to june 1, 2020. clinical randomized controlled trails of these 5 drugs for rheumatoid arthritis were collected for network meta-analysis. results: a total of 4 randomized controlled trails with 2070 patients were obtained. the results of the network meta-analysis showed that: (1).. there was no significant difference between the 4 drugs (sarilumab, sirukumab, adalimumab, and tocilizumab) (p > .05) in terms of american college of rheumatology 20. (2).. there was no significant difference between the 5 drugs in the aspect of the america college of rheumatology 50% and 70% (american college of rheumatology 50, american college of rheumatology 70) (p > .05). (3).. there was no significant difference between the 3 drugs (sarilumab, sirukumab, adalimumab) in terms of reducing disease activity score 28-erythrocyte sedimentation rate in patients (p > .05). (4).. no significant difference was observed among the 5 drugs in terms of incidence of adverse reactions, serious adverse reactions and withdrawal adverse reactions (p > .05). the results of the ranked probability plot indicated that tocilizumab and sarilumab outperform other drugs in terms of efficacy and safety. conclusion: the results of the ranking of the 5 drugs showed that tocilizumab and sarilumab had the best efficacy and safety. rheumatoid arthritis (ra) is the most common autoimmune inflammatory arthritis. [1] ra can lead to progressive joint disability, systemic inflammation, anemia and cardiovascular disease. the prevalence of ra ranges from 0.4% to 1.3% and is associated with gender (2-3 times higher in women than men) and region of population residence (higher in the north than in the south and higher in urban people than in rural areas). [2] [3] [4] [5] [6] currently, there are more biological disease-modifying antirheumatic drugs (dmards), but these drugs can only relieve the symptoms, not stop the progression of the disease, and have a high incidence of side effects. [7, 8] in the recent year, it has been have shown that interleukin 6 (il-6) and tumour necrosis nactor (tnf) have an important role in the ra pathogenesis. tnf inhibitor (adalimumab), il-6 inhibitor (sarilumab, sirukumab, tocilizumab) and the janus kinase (jak)1/2 inhibitor (baricitinib) has been developed and used clinic. [9, 10] tocilizumab can be used in anti-ra resistant to methotrexate and tnf inhibitors with no difference in safety and efficiency compared to rituximab and abatacept. [11] for refractory ra, rituximab and tocilizumab have better clinical outcomes than abatacept. [12] sarilumab is the first humanized monoclonal antibody that directly binds to the alpha subunit of il-6 receptor complex and blocks the cytokine-mediated inflammatory signaling cascade for use against ra. adalimumab has been widely used worldwide as a tnf inhibitor against -ra. [11] adalimumab and the biosimilar sb5 are safer and tolerated in terms of clinical efficiency, safety and immunogenicity of against ra. [13, 14] baricitinib is an oral reversible inhibitor of jak1 and jak2. it is more efficiency than adalimumab for against ra. [15, 16] sirukumab is a human anti-il-6 monoclonal immunoglobulin g 1 kappa antibody that inhibits il-6-mediated effects. [17] some research has already shown the efficiency of sirukumab against ra. [18] however, these drugs have different clinical profiles. tocilizumab and adalimumab have been in clinical use for many years. [19] the efficacy and safety of the other 3 drugs are unclear. [20] no studies on network meta-analysis of these 5 drugs for the treatment of ra have been reported. in this study, their effectiveness and safety of dmards for the treatment of ra were evaluated using a network meta-analysis of the clinical data ( fig. 1) . the comparative study of dmards will not only help to increase the understanding of the efficacy and safety of new drugs, but also provide clinical evidence for clinicians to treat ra patients who are not responding to remitting antirheumatic drugs. this manuscripts' data is based on the studies of the published/ publicly reported literature. ethical approval (review) was not required by ethics committee of affiliated hospital of southwest medical university, china. the data used in this manuscript are all data involving secondary use, without any personal identifiers, and without access to signed informed consent literatures published before june 1, 2020 were searched in medline (via pubmed), embase (via ovid), web of science, the food and drug administration web site and cochrane library. the search results were restricted to studies conducted in humans, regardless of language or ethnicity. the search terms used were "sarilumab," "sirukumab," "baricitinib," "tocilizumab," "adalimumab," "ra" and "rheumatic arthritis," and were adjusted to the relevant regulations of each database. the inclusion criteria were as follows: (1) randomized controlled trails (rcts); (2) patients over 18 years of age diagnosed with ra; (3) complete experimental data; (4) cases met the american rheumatology association 1987 criteria for ra diagnosis [21] ; (5) the study's medication was in accordance with the medication guidelines. the exclusion criteria were (1) studies with unreliable literature based on the jadad scoring [22] ; (2) studies with incomplete processes; (3) retrospective studies; (4) combination of medication; (5) observation clinical trials; (6) systematic reviews; (7) literature duplication; (8) animal experiment. data were abstracted independently by 2 reviewers, according to the inclusion and exclusion criteria, and checked by a third reviewer. for each study, the data extracted included study design, baseline characteristics, interventions, efficacy outcomes and safety. primary outcome indicators included american college of rheumatology (acr) 20, american college of rheumatology 50 (acr50), american college of rheumatology 70 (acr70); acr criteria are commonly used to assess the improvement in tender or swollen joint counts, acute phase reactant, patient and physician global assessments, pain scale, and disability/functionality questionnaire. american college of rheumatology 20 (acr20), 50 and 70 indicated 20%, 50% and 70% improvement in acr criteria. the secondary outcome indicators included disease activity score 28-erythrocyte sedimentation rate (das28-esr), the incidence of adverse reactions, incidence of serious adverse reactions, and incidence of patients who withdraw from treatment due to adverse reactions. two investigators assessed the quality of eligible studies using the scores, [23] which ranges from 0-7 and which assesses random sequence generation, double-blinding, allocation concealment, patient dropout, and dropout rates. for studies with a jadad scores of 4-7 the quality was classified as high, while studies with a score of 0-3 were classified as low. [22] 2.6. statistical analysis direct and indirect results were compared using r language and r studio 1.1.464 software, and the associated 95% confidence intervals were calculated for data analysis. the network drawing was carried out using stata16.0 software, with each node representing the intervention, the size of the node representing the size of the sample size, and the thickness of the connecting line representing the number of studies included in the study. for continuous variables, the weighted mean difference was used as the effect size. for dichotomous variables, the odds ratio was used as the effect size, expressed as a 95% confidence intervals. the sorted probability plot was used to rank the efficacy of interventions, and a = 0.05 was set as the test level of significance. according to the searching strategy, 2159 relevant literatures, including 4 rcts [24] [25] [26] [27] were initially obtained for a total of 2070 patients. the literatures were published from 2013 to 2020 for 5 interventions, with sirukumab in 2 dose groups and the remaining drugs 1 dose group. the jadad score included in the literature ranged from 5 to 7, and the overall literature quality was high. the flow chart of literature search was shown in figure 1 . the basic characteristics of the included literature and the literature quality evaluation were shown in table 1 . a total of 4 rcts [24] [25] [26] [27] and 5 interventions were included in this study including 3 two-arm studies and 1 three-arm study. the network was plotted with acr20, acr50, and acr70 indicators, respectively. one study [26] did not describe the changes in acr20 in patients after treatment. adalimumab had a large number of studies (fig. 2 ). three [24, 25, 27] reported acr20 before and after treatment, and 4 [24] [25] [26] [27] reported acr50 and acr70 before and after treatment. four of these drugs (sarilumab, sirukumab 50 mg and 100 mg, adalimumab, tocilizumab) were not significantly different in reducing acr20 (p > .05); there was no significant difference among the 5 drugs in acr50 and acr70 (p > .05, tables 2-4). only two literatures [24, 27] described the das28-esr level in patients after treatment, and their network meta-analysis showed no significant difference between sarilumab, sirukumab, and adalimumab in reducing das28-esr (p > .05). all included literature [24] [25] [26] [27] reported adverse reactions, serious adverse events, and patient's withdrawal due to adverse reactions after drug administration. the side effects are mainly in the central nervous system, digestive system, cardiovascular system and urinary system, etc. the network meta-analysis results showed that the incidence of adverse reactions, serious adverse events, and patient withdrawal due to adverse reactions were not significantly different among the 5 drugs (p > .05). the results of the incidence of adverse reactions were shown in table 5 . tocilizumab and sarilumab outperformed the other drugs in terms of efficacy and safety. among them, the probability ranking plots for acr20, acr50 and acr70 were shown in , and the safety levels were shown in table 6 . ra is a highly disabling disease driven by multiple inflammatory cells and a complex network of cytokines. [28] the basic pathological changes are synovial swelling, granulocyte infiltration in the acute phase and synovial hypertrophy in the chronic phase. [29] if the symptoms are severe, it can lead to the inability to take care of oneself, increasing the psychological burden of patients and their families, as well as increasing the financial burden of the family. the development of ra is mainly related to genetic factors, environmental factors and various cytokines in the body. among the cytokines of ra, tnf-a, mainly produced by monocytes/ macrophages, plays an extremely critical role. therefore, blockadalimumab 40 mg q2w 35/150 53.6 ± 11.9 27.3 ± 6.5 6.0 ± 0.9 6.8 ± 0.8 6.6 ± 7.8 cem 2013 [25] tocilizumab8mg ing tnf-a production can inhibit the inflammatory response of ra and achieve the purpose of treating ra. [30] [31] [32] [33] many cytokines involved in the pathogenesis of autoimmune and inflammatory diseases use jak to transmit intracellular signals. [34] mutations in jak cause many immune defects that are associated with ra. [35] il-6 can promote synovial fibroblast proliferation and pannus formation and induce osteoclast formation. [36] with the induction of various inflammatory factors, the il-6 in the synovial fluid of the joints increases, and its levels correlated with the degree of disease activity and joint destruction, [37] so the development of biological agents that block these factors would be an avenue for clinical treatment of ra. meanwhile, given that the therapeutic effects of conventional drugs such as non-steroidal anti-inflammatory drugs, slow-acting antirheumatic drugs, glucocorticoids, and botanical drugs are still not very satisfactory. dmards targeting these cytokines continue to be introduced. the tnf inhibitor adalimumab has been widely used in clinic to replace conventional drugs in the treatment of ra. tocilizumab was the first il-6 antibody to be marketed. with the successful development of tocilizumab, biological agents such as sarilumab, sirukumab and baricitinib, which act on jak1/2 have also been developed for ra treatment with good application prospects. there have been studies on the efficacy and safety of macromolecular biological agents, but most of them have focused on the efficacy and safety of a particular biological agent for the treatment of ra, or a combination of biological agents and biological agents. compared to the efficacy and safety of conventional drugs for the treatment of ra and long-term safety research/systematic evaluation of macromolecular biological agents, there are few trials based on the efficacy evaluation of multiple biological agents for treatment of ra. therefore, the aim table 3 acr50 network meta-analysis results: acr response rates (or, 95% ci). achieving ≥ 20% acr response. acr20 = american college of rheumatology 20, ci = confidence intervals, or = odds ratio. table 5 the incidence of adverse reactions network meta-analysis results (or, 95% ci). of this study was to compare the efficacy and safety of these 5 anti-rheumatic biologics for treatment of ra based on the existing trials with available metrics. in terms of effectiveness, the efficacy of 5 anti-ra drugs was analyzed by online meta-analysis. four of them (sarilumab, sirukumab, adalimumab, tocilizumab) did not differ significantly in reducing acr20 (p > .05). there was no significant difference in reducing acr50 and acr70 (p > .05). these 5 biological agents have good effect in the treatment of ra, which is generally consistent with the findings of bae. [38] and lee [39] there was no significant difference in das28-esr levels of 3 drugs, sarilumab, sirukumab and adalimumab in this study. in terms of safety, there were no significant differences among the 5 anti-ra drugs, indicating that they have similar safety profiles. in the probability ranking, tocilizumab ranked first in acr20 and acr50 with 56.41% and 48.92%, sarilumab ranked second with 43.44% and 32.57%. however, in acr70, tocilizumab was ranked second (40.03%) and sarilumab was ranked first (54.29%). in the incidence of adverse reactions, sirukumab ranked first with 40.43%, suggesting a higher risk of adverse events with this drug; tocilizumab and sarilumab and ranked 5th and 6th, suggesting tocilizumab and sarilumab are superior to the other 3 drugs (sirukumab, adalimumab, and baricitinib) in terms of efficacy and safety. this is in agreement with tocilizumab, sarilumab and sirukumab by bae [38] et al. in this study, the evaluation results of adverse events were similar. tocilizumab, a humanized monoclonal antibody, [40] fights against ra by binding to il-6 receptor and inhibiting downstream il-6 signaling. [41] lee et al [42] conducted a metaanalysis of the tocilizumab, rituximab, abatacept and tofacitinib networks and found that tocilizumab to be relatively safe, similar to this study. sarilumab is a human immunoglobulin g 1 monoclonal antibody. [43] the results of this drug for treatment of ra are similar to those of choy, [44, 45] who analysed sarilumab's treatment of ra. in addition, the safety of sirukumab for ra is similar to the results of sirround-d [46] and others. when using this drug, special attention should be paid to its adverse effects. further rigorous genetic and molecular studies will improve our understanding of the problem in order to update therapeutic approaches. [47] this study also has certain limitations: (1) among the included studies, only taylor et al [26] had 2 courses of treatment and the rest had 1 course of treatment; (2) the literature was incomplete, for example, no post-treatment acr20 was reported in taylor et al [26] and no post-treatment disease activity score 28-c-reactive protein (das28-crp) was reported in all included studies [23] [24] [25] [26] ; (3) the small number of included there may be a risk of publication bias. our study found that tocilizumab ranked first and sarilumab ranked second in response to acr20 and acr50. while for acr70 treatment response, tocilizumab ranked second and sarilumab ranked first. the incidence of adverse reactions was most prominent with sirukumab and lowest with tocilizumab and sarilumab, suggesting that tocilizumab and sarilumab were superior to sirukumab, adalimumab and baricitinib in terms of efficacy and safety. overall, of the dmards studied, tocilizumab and sarilumab are the most prominent. however, due to the small amount of literature included in the network meta-analysis, the conclusions of this study need to be further validated by highquality, long-term follow-up randomized controlled study. the pathogenesis of rheumatoid arthritis monrad seetha u. early diagnosis and treatment of rheumatoid arthritis prevalence of specific types of arthritis and other rheumatic conditions in the ambulatory health care system in the united states epidemiology of inflammatory rheumatic diseases is the incidence of rheumatoid arthritis rising? : results from olmsted county the pathogenesis of rheumatoid arthritis: new insights from old clinical data? innovated formulation of tcm pangolin scales to develop a nova therapy of rheumatoid arthritis profile of sarilumab and its potential in the treatment of rheumatoid arthritis clinical outcomes in patients switched from adalimumab to baricitinib due to non-response and/or study design: phase iii data in patients with rheumatoid arthritis tocilizumab in the treatment of rheumatoid arthritis: an evidence-based review and patient selection comparative effectiveness of rituximab, abatacept, and tocilizumab in adults with rheumatoid arthritis and inadequate response to tnf inhibitors: prospective cohort study adalimumab long-term safety: infections, vaccination response and pregnancy outcomes in patients with rheumatoid arthritis phase iii randomized study of sb5, an adalimumab biosimilar, versus reference adalimumab in patients with moderate-to-severe rheumatoid arthritis patient-reported outcomes from a phase 3 study of baricitinib versus placebo or adalimumab in rheumatoid arthritis: secondary analyses from the ra-beam study baricitinib for rheumatoid arthritis pharmacokinetics and safety of sirukumab following a single subcutaneous administration to healthy japanese and caucasian subjects sirukumab: a novel therapy for lupus nephritis a review of recent advances using tocilizumab in the treatment of rheumatic diseases evaluation of the efficacy and safety of sarilumab combination therapy in patients with rheumatoid arthritis with inadequate response to conventional disease-modifying antirheumatic drugs or tumour necrosis factor a inhibitors: systematic literature review and network meta-analyses the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis assessing the quality of reports of randomized clinical trials: is blinding necessary? preferred reporting items for systematic reviews and meta-analyses: the prisma statement efficacy and safety of sarilumab monotherapy versus adalimumab monotherapy for the treatment of patients with active rheumatoid arthritis (monarch): a randomised, double-blind, parallel-group phase iii trial adacta study investigators. tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind, controlled phase 4 trial baricitinib versus placebo or adalimumab in rheumatoid arthritis efficacy and safety of monotherapy with sirukumab compared with adalimumab monotherapy in biologic-naïve patients with active rheumatoid arthritis (sirround-h): a randomised, double-blind, parallel-group, multinational, 52-week, phase 3 study cytokines, inflammation, and pain rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies tumor necrosis factor-a signaling in macrophages cytokine pathways and joint inflammation in rheumatoid arthritis mechanism of action of certolizumab pegol (cdp870): in vitro comparison with other antitumor necrosis factor alpha agents tumor necrosis factor-alpha (tnfalpha) converting enzyme contributes to production of tnf-alpha in synovial tissues from patients with rheumatoid arthritis jak-stat signaling as a target for inflammatory and autoimmune diseases: current and future prospects jak-stat signaling as a target for inflammatory and autoimmune diseases: current and future prospects interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation serum interleukin 6 levels in rheumatoid arthritis: correlations with clinical and laboratory indices of disease activity comparison of the efficacy and tolerability of tocilizumab, sarilumab, and sirukumab in patients with active rheumatoid arthritis: a bayesian network meta-analysis of randomized controlled trials comparison of the efficacy and safety of tofacitinib and filgotinib in patients with active rheumatoid arthritis: a bayesian network meta-analysis of randomized controlled trials improvements in health-related quality of life after treatment with tocilizumab inpatients with rheumatoid arthritis refractory to tumor necrosis factor inhibitors: results from the 24-week randomized controlled radiate study novel therapeutic agents in clinical development for systemic lupus erythematosus comparative efficacy and safety of tocilizumab, rituximab, abatacept and tofacitinib in patients with active rheumatoid arthritis that inadequately responds to tumor necrosis factor inhibitors: a bayesian network meta-analysis of randomized controlled trials room for more il-6 blockade? sarilumab for the treatment of rheumatoid arthritis evaluation of the efficacy and safety of sarilumab combination therapy in patients with rheumatoid arthritis with inadequate response to conventional disease-modifying antirheumatic drugs or tumour necrosis factor (inhibitors: systematic literature review and network meta-analyses clinical evaluation of the safety, efficacy and tolerability of sarilumab in the treatment of moderate to severe rheumatoid arthritis sirukumab for rheumatoid arthritis: the phase iii sirround-d study the genomic and structural organization of sars-cov-2: a mutational perspective medicine (2021) 100:29 www.md-journal key: cord-0069949-cqbij1vk authors: lara-barba, eliana; araya, maría jesús; hill, charlotte nicole; bustamante-barrientos, felipe a.; ortloff, alexander; garcía, cynthia; galvez-jiron, felipe; pradenas, carolina; luque-campos, noymar; maita, gabriela; elizondo-vega, roberto; djouad, farida; vega-letter, ana maría; luz-crawford, patricia title: role of microrna shuttled in small extracellular vesicles derived from mesenchymal stem/stromal cells for osteoarticular disease treatment date: 2021-11-01 journal: front immunol doi: 10.3389/fimmu.2021.768771 sha: 0838f2bb835301a4f183bee0f44e508481fd526c doc_id: 69949 cord_uid: cqbij1vk osteoarticular diseases (od), such as rheumatoid arthritis (ra) and osteoarthritis (oa) are chronic autoimmune/inflammatory and age-related diseases that affect the joints and other organs for which the current therapies are not effective. cell therapy using mesenchymal stem/stromal cells (mscs) is an alternative treatment due to their immunomodulatory and tissue differentiation capacity. several experimental studies in numerous diseases have demonstrated the mscs’ therapeutic effects. however, mscs have shown heterogeneity, instability of stemness and differentiation capacities, limited homing ability, and various adverse responses such as abnormal differentiation and tumor formation. recently, acellular therapy based on msc secreted factors has raised the attention of several studies. it has been shown that molecules embedded in extracellular vesicles (evs) derived from mscs, particularly those from the small fraction enriched in exosomes (sevs), effectively mimic their impact in target cells. the biological effects of sevs critically depend on their cargo, where sevs-embedded micrornas (mirnas) are particularly relevant due to their crucial role in gene expression regulation. therefore, in this review, we will focus on the effect of sevs derived from mscs and their mirna cargo on target cells associated with the pathology of ra and oa and their potential therapeutic impact. an excessively prolonged imbalance of the immune system response can lead to a vast array of inflammatory and autoimmune disorders. moreover, genetic predisposition and epigenetic regulations, including environmental factors and age, promote autoimmune, inflammatory, and degenerative diseases development (1) . these illnesses imply a high economic burden for the healthcare system and those who suffer from them (2, 3) . osteoarticular diseases (od), such as osteoarthritis (oa), and rheumatoid arthritis (ra), have raised particular concern in the last decades due to the increase of medical consults. they affect roughly 23% of the population over 40 worldwide for knee oa (the most common articulation affected by oa) (4, 5) , and around 0.5% of the worldwide population for ra (6) . moreover, both oa and ra cause a great deal of pain and discomfort to the patients, impacting their quality of life (7) . without a cure for od, patients rely mainly on non-steroidal anti-inflammatory drugs (nsaids), analgesics, and glucocorticoids as the primary options to manage the symptoms (8, 9) . unfortunately, these treatments lack diseaseand structural-modifying capabilities and even worse, their prolonged use is associated with severe side effects (9, 10) . thus, alternative therapies are still needed to treat autoimmune/inflammatory and degenerative diseases like oa and ra. both diseases are mainly defined by the loss of articular cartilage and are known to affect people of all races, genders, and ages (11, 12) . numerous therapeutic efforts have been made to restore the affected joints, including tissue engineering to promote tissue regeneration. recently, cell-based therapies have had a considerable rise, such as the regulatory t cell therapy. however, their high cost and the technical difficulties in producing off-the-counter cell therapies remain significant hurdles for their clinical application (13) . three types of cell treatment are used in clinical trials for oa or degenerative environments; articular chondrocytes, meniscal fibrochondrocytes, and mesenchymal stem/stromal cells (mscs), where the latter has shown encouraging results (11, (14) (15) (16) (17) . mscs are multipotent stem cells of mesodermal origin that can be defined as a cell population with the hallmark self-renewal properties and differentiation into chondrogenic, osteogenic, and adipogenic lineages (18) . although therapy using mscs has achieved significant progress, stem cell-based therapies have not fulfilled the initial promise. some remaining drawbacks include the inconveniences associated with high costs and potential side effects, leading to inconsistency among preclinical and clinical trials (19) . in recent years, the therapeutic benefit of mscs has been attributed to their functions through cell-to-cell contact and, more prominently, paracrine communication. the main mediators of paracrine communication are small extracellular vesicles (sevs), which play an essential role as an alternative mechanism by which mscs regulate different biological processes (20, 21) . sevs are heterogeneous particles that are delimited by a lipid bilayer membrane, whose primary function is to act as vehicles of cellular communication, transporting and transferring several bioactive molecules, such as proteins, peptides, lipids, messenger rna (mrna), and microrna (mirna) (22) . mirnas are small 20-22-nucleotide-long noncoding rnas, which mediate post-transcriptional gene silencing by binding to the 3'-untranslated region (utr) or open reading frame (orf) region of target mrnas (23) unpairing protein translation and causing a rapid tuning of cell fate decisions in response to environmental cues (24) . although sevs can carry different types of cargo, increasing evidence points at mirnas as significant mediators for the effects of these vesicles over the target cells (25, 26) . noteworthy, mirnas regulate the immune system and signaling pathways related to extracellular matrix synthesis, chondrocyte survival, and proliferation (27) (28) (29) . in addition, the auspicious use of sevs as "cell-free cellular therapies'' provides substantial advantages in contrast to whole-cell therapy, such as their easy handling and minimizing the risks of rejection (30) . this review summarizes the current knowledge of msc derived sevs (msc-sevs) and their mirna cargo as a potential and attractive substitute for treating autoimmune/inflammatory and degenerative disorders. mscs are multipotent fibroblast-like cells of mesodermal origin that have been described in several mammals, including humans and mice (31) . according to the international society of cell therapy (isct), three major criteria define mscs: their capacity to adhere to plastic surfaces under culture conditions (32) , their ability to self-renew and differentiate toward mesodermal lineages, such as adipogenic, chondrogenic and osteogenic (33) lineages, as well as the expression of surface markers cd105, cd73, and cd90 in the absence of hematopoietic markers including cd45, cd34, cd14 or cd11b, cd19, and hla-dr (18, 34) . these cells are found in various tissues, including bone marrow, adipose tissue, dental pulp, endometrium, amniotic fluid, placenta, and umbilical cord, among others (35) . however, bone marrow and adipose tissues represent the most common sources for mscs isolation because of their availability (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47) . mscs display a wide variety of biological functions, such as secretory (48) , immunomodulatory (49) and homing (50) properties, representing a stem cell population with demonstrable progenitor cell functionality (33, 51) and a promising candidate for cell-based therapies. illustrating this, clinicaltrial.gov (https://clinicaltrials.gov/) lists 10406 phase i or ii trials using mscs in skin, bone, cartilage, heart, kidney, lung, liver, diabetes, immune/autoimmune diseases and even for covid-19. among these trials, 222 registered studies are using mscs for oa and 55 for ra. od are well-documented candidates for msc treatment. recent studies have shown that oa patients treated with an intra-articular injection of mscs display a substantial enhancement in cartilage coverage and quality, relieving pain, ameliorating disability, and significantly improving their quality of life (11, 12, 52, 53) . similarly, a phase ia clinical trial in ra demonstrated the reduction of proinflammatory cytokines in patients injected with mscs and revealed no short-term safety concerns (54) . this data supports the potential of mscs as an effective treatment for oa and ra patients. several studies have shown that mscs can replace several damaged tissues in vivo. mirza and collaborators showed that undifferentiated mscs seeded on a graft were able to grow and restore a thick multicellular layer mimicking mature vascular tissue (55) , whereas sheng and collaborators were able to successfully transplant mscs and regenerating sweat glands in patients in vivo (55, 56) . previous studies have demonstrated that mscs can regulate the inflammatory response by suppressing mononuclear cells and promoting anti-inflammatory subsets from innate and adaptive immunity, including t-cells (57, 58) . it has been well described that mscs regulate t-cells activation and proliferation without the need for the cell to cell contact, suggesting the involvement of secreted soluble factors as the mechanism of action (59, 60) . additionally, mscs negatively regulate natural killer cells (nk) activity, dendritic cells (dc) maturation, and b-cells proliferation while promoting treg induction [reviewed in (61, 62) ]. it has also been shown that one of the hallmarks of msc therapeutic potential is the regulation of cytokine production, including ifn-g, tnf-a, and il-10 (62). by modulating different immune cells involved in autoimmune diseases' pathogenesis, mscs have a promising therapeutic potential. although some mechanisms require the cell to cell contact, mscs secretome seems to mediate most of their therapeutic effects in several pathologies, including od (63, 64) . in the last few years, several studies suggest that msc therapies in clinical applications do not show severe adverse effects showing promising therapeutic benefits (65) . nonetheless, the clinical application of mscs and the fast development of commercial products show contradicting outcomes in clinical application and unsatisfactory therapeutic effects, primarily due to their low survival and homing capacity in vivo (19) . sitespecific injection seems to be better to obtain more efficiency results [reviewed in (66, 67) ]. therefore, to use mscs as a successful treatment, these difficulties must be overcome. the most critical challenges are donor heterogeneity, stemness stability and differentiation capacities, limited expansion capacities, homing capacity, and rejection risks (68) . in this regard, their derivatives including extracellular vesicles come as a promising solution as a cell-free based therapy due to their role as molecule delivery vehicles that mimic the effects of the parent on the target cell (66) . extracellular vesicles (evs) are membrane-bound nanostructures released that act as essential mediators of cell-to-cell communication under physiological and pathological conditions (69) . according to their size, evs can be classified as apoptotic bodies (more than 1000nm), microvesicles (between 40-1000nm), and exosomes (50-200nm) (70) . evs can be generated directly by budding from the plasma membrane (microvesicles) or after fusion of multivesicular bodies (related to the endocytic pathway) with the plasma membrane to release intraluminal vesicles (exosomes). evs are normally obtained by differential centrifugation protocols and the exosome enriched fraction also contains small microvesicles (smvs) commonly referred to as small extracellular vesicles (sevs) (71, 72) . sevs can be further characterized by the expression of exosomeassociated markers such as tsg101, alix, and tetraspanin proteins such as cd9, cd63 or cd81 (70) . released sevs can either be readily taken up by neighboring or by distant cells due to their ability to travel through body fluids and mimic the parent cell's effect on the target cell (70) . due to the natural role of sevs in cell-to-cell communication, they are readily taken up through phagocytosis, micropinocytosis, and endocytosis mediated by lipid raft, caveolin or clathrin (73, 74) . although sevs can be delivered to any cell type, they are internalized in a highly cell type-specific manner that depends on recognizing typical sev surface molecules by the cell or tissue, making them ideal therapeutic delivery systems [reviewed in (74) ]. a substantial advantage of using sevs as therapeutic carriers is that they are nearly non-immunogenic and are capable of homing to distant tissues where the inflammation is located (75, 76) indeed, mice injected with both wild-type and engineered sevs showed no toxicity nor a significant immune response, further adding to the safety of sev based therapies (77) . however, the delivery and the frequency of sevs injection on patients still needs to be addressed, in order to determine the most efficient strategy to obtain positive clinical outcomes. in preclinical models, it has been described that mscs-sevs inhibit tnf-a induced collagenase activity and promote cartilage regeneration in chondrocytes derived from oa patients in vitro (75, 76) . moreover, mscs-sevs significantly improve oa progression by inhibiting cartilage degeneration in the collagenase-induced oa murine model (78) . mscs-sevs were also shown to enhance the production of immature dcs that secrete il-10, which are involved in suppressing inflammatory t-cell responses (76, 79, 80) . on the other hand, zhu and colleagues demonstrated that sevs could reduce arthritis index, leukocyte infiltration, and, most importantly, destruction of the joint in a cia mice model. these sevs lowered th1 and th17 cells' frequencies through mirna targeting of stat3 and t-bet, having a potential role in treating arthritis (81) . munir and colleagues also proved that treating cia in mice with mscs decreased the severity of the disease by dampening the pathogenic immune response. mice that received this treatment had reduced il-6 and tnf-a, increased il-10 in their joints and increased the frequency of tregs in their spleen and lymph nodes, and a lower th1:th17 ratio (66) . other studies have demonstrated that sevs can decrease the clinical signs of inflammation present in the cia model by polarizing b lymphocytes into breg-like cells (82) . therefore, evidence supports the repairing properties of mscs-sevs in joint tissue, especially after intra-articular administration (83) . these and other preclinical studies of mscs-sevs show that these potential treatments are safe and scalable for clinical application (20) . since phase iii clinical trials have shown inconsistent results in ra and oa without cartilage regeneration despite the promising preclinical studies (52, 84) , their derived sevs could also display conflicting results for ra and oa treatment. several techniques to improve mscs therapy have been recommended to overcome these issues [reviewed in (85) ]. for example, hypoxia preconditioning and 3d culture can increase the production of pro-chondrogenic factors (86) . additionally, sevs action can be strengthened by modifying their specific cargo (87, 88) , or by treatment with immunosuppressive cytokines, such as il-10 (89), enhancing their antiinflammatory and chondroprotective properties. moreover, it has been shown that the genetic engineering of mscs affects their derived sevs, improving their immunosuppressive and chondroprotective abilities (87), where sevs demonstrated to enhance chondrogenesis and suppress cartilage degradation (88) . the therapeutic effect of sevs in the target cell is directly dependent on their cargo, which can be composed of a wide variety of molecules, including proteins, peptides, lipids, and several nucleic acids such as dna, messenger rna and micrornas [reviewed in (21) ]. although the effects of other sev cargos cannot be excluded, proteins and mirnas are considered the main mediators of the effect of sevs in target cells. proteomic analysis in sevs has identified thousand proteins implicated in key biological processes such as sev biogenesis, cellular structure, tissue repair and regeneration, and inflammatory response [reviewed in (90) ] indeed, chaubey and collaborators, validated tsg-6 as one of the protein mediators of msc-sev for immunomodulation by inducing a decrease in neutrophil infiltration in a murine model of hyperoxia-induced lung injury (91) . however, to determine the role of proteins and mirna in mediating the therapeutic efficacy of sevs, a relation between the concentration of mirna and proteins in their cargo is needed (92) . moreover, it is not well defined whether proteins and mirnas work independently or synergistically in target cells, indicating that further studies are needed in this field. on the other hand, mirnas encompass an important fraction of the exosome content and arise as the main regulators of msc-sevs function (26, 93) . mirnas are small non-coding rna highly conserved among species, which control gene expression through its binding capacity to the three prime untranslated region (3'-utr) of the targeted mrnas, for repressing the expression of the corresponding gene at a posttranscriptional level (94) . compared with transcriptional and epigenetic regulation, post-transcriptional processes are fast and therefore can instantly tune cell fate decisions in response to environmental cues (94) . moreover, mirnas contained in sev are protected from rnase degradation and through their integrins and opsonins the delivery of their internal content is efficient (24) . indeed, neviani and collaborators demonstrated that sevs derived from inactivated natural killer (nk) cells showed an equal cytotoxic activity when compared to sevs derived from activated nk cells. indeed, inactivated nk derived sevs showed low levels of killer proteins in their cargo (perforin 1, granzyme a, granzyme b) while still retaining their cytotoxic activity, showing that the protein cargo is not the main bioactive mediator (95) . in line with these results, rna-depleted sevs lose their immunosuppressive activity on t-cells, demonstrating their pivotal role on msc-sevs immunoregulation. mirnas are critical regulators in maintaining a healthy joint as they participate in chondrocyte homeostasis and in the regulation of inflammatory mediators (96, 97) . proof of this is the phenotype observed in dicer (a key enzyme in the mirna biosynthesis pathways) knock-out mice, whose growth plates exhibited a reduction in proliferating chondrocytes and accelerated differentiation into a hypertrophic type, resulting in severe skeletal growth defects and premature death (98) . accordingly, an imbalance of some mirnas has been associated with od in both human and murine models. illustrating this, a study using the serum transfer mouse model of ra in c57bl/6 mice identified a total of 536 upregulated genes and 417 downregulated genes that are predicted targets of mirnas with reciprocal expression in arthritic mice (99) . twenty-two mirnas whose expression was most significantly changed between nonarthritic and arthritic mice regulated the expression of proteins involved in bone formation, specifically wnt and bmp signaling pathway components. while activation of canonical wnt signaling promotes bone formation (100), wnt signaling antagonists such as dkk inhibit this pathway and have been shown to regulate the erosive process in ra (101, 102) . among the most upregulated mirnas found by maeda and colleagues was mir-221-3p, which is induced in the tnf-driven model of arthritis and fibroblast-like synoviocytes (fls) from ra patients (103) . in bone, synovium-derived mirnas, including mir-221-3p, may control skeletal pathways that inhibit osteoblast differentiation from augmenting bone erosion in ra by regulating dkk2. similar studies in oa patients have revealed significant mirna imbalance in cartilage, synovial fluid, and plasma (104) . several studies have shown that there is differential expression of several mirnas in oa versus a healthy joint. by evaluating the expression of 365 mirna in oa patients versus healthy donors, iliopoulos and colleagues found 16 altered mirna, providing one of the earliest insights on the osteoarthritic chondrocytes mirna signature (105) . a subsequent study showed that a set of 17 mirna that contribute to cartilage remodeling presented an altered expression and suggested that these changes were due to epigenetic regulation (106) . murata and colleagues investigated whether, in plasma and synovial fluid, mirna could be used as possible biomarkers for ra and oa, finding that some mirnas can effectively differentiate between both diseases (107). interestingly, 12 mirna were overexpressed under the oa condition, all targeting important genes in chondrocyte maintenance and differentiation such as smad1, il-1b, col3a, vegfa, and fgfr1 (104) . other reports point out imbalances in mirnas associated with the regulation of ecm degradation enzymes. for example, the increase of mir-146a/ mir-145/mir-22 and the decrease in mir-149/mir-125b/mir-558 causes ecm degradation. some mirnas such as mir-27b, mir-140, and mir-320 have been reported to target mmp13, a regulator of tissue repair and remodeling (108) (109) (110) , while mir-92a-3p and mir-27b regulate adamts expression, an enzyme that plays an important function in the degeneration of cartilage in ra and oa (111) . furthermore, it has been shown that the down-regulation of mir-140 inhibits il-1b by inducing adamts expression and that mir-27b regulates mmp-13 expression in human chondrocytes. importantly, mir-27b, mir-140, and mir-146a are dysregulated in oa patients, suggesting a role for them in oa pathogenesis (108, 112, 113) . it has been widely reported that tgf-bs and bmps regulate postnatal joint cartilage homeostasis and that dysregulated tgfb and bmp signaling are often associated with od [reviewed in (114) ]. these tgf-b superfamily members bind to the heteromeric receptor complex, comprised type i and ii receptors at the cell surface, that transduce intracellular signals by activating smad complex or mitogen-activated protein kinase (mapk) cascade. bmps have a chondroprotective role in different animal models of ra (115) ; specifically, it has been suggested that endogenous expression of bmps is required to maintain chondrocytes phenotype in vitro (116, 117) . however, its dynamic regulation has been observed in the cia murine model, supporting a role for this pathway in ra (118) . during cia, bmp-2 and bmp-7 are upregulated in a tnf-dependent manner, a phenomenon accompanied by an increase in smad-5 phosphorylation: thus, there is an increase in bmp signaling activity. similarly, in an oa rat model, it was shown that il1b upregulated bmp-2 through the mek/erk/sp1 signaling pathways and that the administration of the bmp antagonist noggin prevented cartilage degeneration and oa development (119) . an observational study in oa patients showed that the levels mir-22, which targets bmp2, are increased in the progression of the disease (120). furthermore, the inhibition of mir-22 has been shown to prevent inflammatory activity (105, 121) . on the contrary to mir-22, mir-140 also targets bmp2 but in a different position of the 3′-utr region and is associated with increased bmp2 expression (120) . notably, the levels of synovial mir-140 were significantly reduced in the patients with oa and were negatively correlated with oa severity compared to controls (120, 122) . furthermore, after arthroscopic debridement, the levels of these mirnas and bmp2 were restored (120), suggesting mir-22 and mir-140 play a role in the development of oa by regulating bmp-2. it has also been shown that bmp targeting mirnas' dysregulation is associated with the pathogenesis of ra. it has been demonstrated that sevs derived from fibroblast-like synoviocytes with elevated levels of mir-486-5p promoted osteoblast differentiation and proliferation by repressing tob1, thus activating the bmp/smad signaling pathway, alleviating the severity of ra in the cia model (123) . on the other hand, tgf-b has been implicated in cartilage ecm production and maintenance, specifically by increasing col2a1, perlecan, fibronectin, and hyaluronan (124, 125) . furthermore, tgf-b also has anti-inflammatory functions, counteracting il1b and il-6 mediated inflammation in the joint (124, 125) . importantly, several mirnas target different proteins of these pathways, which has been reviewed elsewhere (126) . it has been shown that mir-455-3p promotes tgf-b/ smad signaling in chondrocytes and inhibits cartilage degeneration by directly suppressing pak2, a kinase that inhibits tgf-b signaling. accordingly, the mir-455-3p levels were decreased, and both pak2 and phospho-pak2 were increased in oa cartilage compared with control cartilage. moreover, mir-455-3p ko mice displayed significant degeneration of the knee cartilage (127) . in oa cartilage, mir-150-5p is overexpressed. it has been shown that mir-140-5p directly targets tgf-b3 signaling by altering the expression of tgf-b3 and smad-3 in mandibular condylar chondrocytes, thus having a role in the regulation of mandibular cartilage homeostasis and development (128) . furthermore, this mirna is increased in the cartilage of oa patients compared to control cartilage from femoral neck fracture patients, where it suppresses the smad2/3 pathway, a process that promotes cartilage destruction and the progression of the disease (129). using mir-140-null mice, which showed different changes related to oa such as fibrillation of articular cartilage, miyaki and collaborators demonstrated that mir-140 regulates cartilage development and homeostasis (113) . interestingly, mir-140 knockout mice presented proteoglycan loss and fibrillation of articular cartilage emulating age-related oa. on the contrary, transgenic mice overexpressing mir-140 in cartilage were resistant to antigen-induced arthritis. another mirna involved in tgf-b signaling modulation is mir-125-5p, which downregulates the smad2 expression and leads to the dysfunction of tgf-b signaling. noteworthy, the circular ribonucleic acids (circrnas), circcdk14, which is down-regulated in the joint wearing position, regulates metabolism, inhibits apoptosis, and promotes chondrocyte proliferation by mir-125a-5p sponging (130) . taking together, studying mirna dysregulation in od and the underlying mechanisms could provide new insights towards more effective treatments. at the same time, tgf-b exerts an anabolic repairing response on articular cartilage. on the other hand, proinflammatory cytokines such as il-1b and tnf-a which exert a strong catabolic effect (131) . as follows, the balance between tgf-b and the il-1b or tnf-a signaling pathways is a critical regulator of articular cartilage homeostasis (131) , thereby its disruption contributes to the pathogenesis of oa. in oa, nf-kb signaling orchestrates chondrocyte catabolism, survival, and synovial inflammation. growing evidence suggests that mirnas targeting either matrix-degrading enzymes or components of the nf-kb pathway can suppress chondrocyte catalytic activity. while some mirnas such as mir-138 and mir-9 directly suppress the nf-kb subunits p65 or p105/50 (132, 133) , others like mir-210, mir-26a/b, mir-93, mir149, and mir-146a act indirectly by targeting upstream regulators of nf-kb (134) such as death receptor 6 (dr6), kpna3, toll-like receptor 4 (tlr4), tak1, and tnf-receptor associated factor 6 (traf6)/interleukin-1 receptor-associated kinase 1 (irak1). additionally, synovial inflammation in the context of oa or osteoblastogenesis is associated with mir-146/mir-155/mir-218/mir-135, among others (135) (136) (137) . in ra, mirna dysregulation is implicated in the activation of multiple cytokine-signaling pathways that leads to synovial tissue lesions and dysregulation of immune cells, thereby contributing to pathogenesis (139) . many studies have demonstrated that mir-16, mir-146a, mir-155, and mir-223 present an increased expression level in synovial fluid of ra patients. moreover, inflamed joints of ra patients show an increased expression of mir-133a, mir-142-3p, mir-142-5p, mir-146a, mir-155, mir-203, mir-221, mir-222, mir223 (103, 107, 140, 141) . on the other hand, the expression of mir-124a and mir-34a is decreased in the context of ra (142, 143) . furthermore, mir-181a, mir-17-92 overexpression enhances the inflammation, while upregulation of mir-146a and mir-573 suppresses the autoimmunity (144) . although several mirnas related to inflammation are dysregulated in ra, mir-146a appears to be essential in controlling the inflammation. mir-146a targets tnfa/tnf receptor-associated factor 6 (traf6) and il-1 receptorassociated kinase 1 (irak1), elevating tnf-a production through traf6/irak1 mediated pathway [reviewed in (126, 145) ]. mir-146a is also able to regulate genes such as faf1, irak2, fadd, irf-5, stat-1, and ptc-1 (146), making it a possible therapeutic target for the treatment of ra. besides mir-146, mir-155 can also stimulate the proinflammatory mediators tnf-a, tlrs, lps, and il-1 [reviewed in (145) ]. upregulation of mir-155 has been observed in synovial tissue, fls, peripheral and blood mononuclear cells. supporting a role for targeting mir-155 in ra, mir-155 knockout mice do not develop collagen-induced arthritis (146) . therefore, mir-155 may be a promising therapeutic target for ra. mirnas and their levels in plasma and synovial fluids are associated with the occurrence of od. therefore they could serve as predictive biomarkers and even as therapeutics targets. owing to the fact that mirnas play a crucial role in the maintenance of healthy joints, restoring their balance could be an effective way to treat oa and ra. to accomplish an effective therapeutic strategy, the delivery system is the main barrier that has to be overcome (147) . given that mirnas are naturally carried by sevs, they are protected from rnase degradation and the delivery to target cells is efficient thanks to the integrins and opsonins (147) (148) (149) (150) . since msc-sevs are natural carriers of therapeutic mirna, they have arisen as an attractive therapeutic tool to treat several diseases including od. there are copious amounts of studies reporting the different effects of mirna transfer via sevs, and their relevance in cell to cell communication. indeed, mirnas have gained more attention than proteins or other variety molecules contained in sevs, due to their regulatory roles in gene expression. goldie and collaborators demonstrated that the proportion of mirna is higher in sevs than in their parent cells (151) . moreover, a profiling study of mirnas has demonstrated that mirnas are not randomly packaged into sevs. guduric-fuchs and collaborators have shown that a subset of mirnas (mir-150, mir-142-3p, and mir-451) are preferentially incorporated in sevs (152) . although the effects of other sev cargos cannot be excluded, mirnas are considered the key functional elements on recipient cells. several thousand mirnas have been identified in humans, and their studies have increased in the last decade, moreover mirnas are frequently deregulated in multiple human diseases which offers many opportunities for diagnosis and treatment for various pathological conditions. the use of sevs as a therapeutic treatment for different immune diseases is still challenging, since safety evaluations are still pending. multiple experiments must be done in large and proper animal models in order to prove their therapeutic efficacy and safety in this area before applying this approach in the clinic. given that it primarily affects the joints, we suggest that the optimal form of delivery should be intraarticular injection. chen and collaborators, have shown that, both in vitro and in vivo, bm-msc-sev enriched in mir-150-5p suppress the expression of mmp14 and vegf, and decrease the expression levels of il-b, tnf-a, and tgf-b, resulting in the inhibition of the proliferation and migration of fibroblast-like synoviocytes (fls) and alleviation of inflammation (153) . similarly, bm-msc sev derived mir-320a targets cxcl9 and thereby suppresses fls activation, migration and invasion in ra (154) . additionally, the overexpression of mir-124a in msc-sev significantly increased the expression of apoptosis-related proteins inducing an inhibition on the proliferation, invasion and migration of ra-fls cells (155) . it has been well documented that mirnas in msc-sevs have a chondroprotective role in oa (156) . illustrating this, msc-sevs shuttled mir-92a-3p increases chondrocyte proliferation and the levels of col9a2 and aggrecan, and effect mediated by targeting noggin3 and wnt5a while activating the pi3k/akt/ mtor pathway, thus increasing the levels of [reviewed in (21) ], (88) . on the other hand, msc-sevs-derived mir-135b stimulates cartilage regeneration by binding to the transcription factor sp1 (sp1), which regulates apoptosis and proliferation (157) . moreover, mir-140-5p upregulates sox9 and promotes mscs chondrogenesis ( figure 1) . additionally, recent studies show that sev-mediated transfer of mir-140 from dendritic cells improves oa in vitro by inhibiting proteases associated with cartilage degradative processes in the joint and alleviates the progression of oa in a rat model in vivo (158) . in contrast, another study reported that mir155 on the other hand, the involvement of msc-sevs-derived mirnas in the context of immune modulation has been reported (149) . msc-sevs are immunologically active, meaning that they can attenuate the immune system through increasing antiinflammatory cytokines, such as il-10 and tgf-b and the induction of tregs, modulating immune activity. indeed, rnadepleted sevs lose their immunosuppressive activity on t-cells (161) , demonstrating their pivotal role on msc-sevs immunoregulation and therefore their potential use on autoimmune diseases such as ra (75, 80, 83) . indeed, the downregulation of mir-192-5p has been reported in ra patients, and its transfer via sevs derived from bm-mscs reduced the inflammatory response by downregulating the rasrelated c3 botulinum toxin substrate 2 protein (rac2) (figure 1) , attenuating the severity of the disease in rats (162) . it has been reported that sevs derived from tnfa and ifng pretreated-mscs improve their suppressive activity over t cells (75) . this pretreatment was associated with a higher expression of mir-155 and mir-146, two mirnas involved in activating and inhibiting t cells inflammatory reactions (163) . similarly, mir-155-5p loaded in sevs derived from lps-stimulated periodontal ligament stem cells (pdlscs) inhibited pro-inflammatory th17 cells favoring their conversion into treg through inhibition of sirtuin-1 (sirt1) (164) . moreover, the therapeutic role of mir-146a-5p contained in msc-sevs has been shown in vivo in a model of allergic airway inflammation (161) . in this study, the authors demonstrated that the mirna signature of msc-sevs was enriched in mir-146a-5p compared to sevs derived from less immunosuppressive cells such as fibroblasts (161) . in addition, mir-146a-5p mimic improves the immunosuppressive capacities of fibroblast sevs, while mir-146a-5p inhibition impairs the immunosuppressive activity of msc-sevs on t-cell proliferation (161) . in ra, mir-146a is downregulated, but its upregulation associated with the administration of msc-sevs increased the frequency of treg cell population by increasing the expression of some key autoimmune response genes and their protein products, such as tgfb, il-10 and foxp3 (figure 1) , resulting in a beneficial anti-inflammatory response (165, 166) . rong and collaborators showed that the hypoxic pre-treatment of rat bm-msc (a known method for the improvement of the therapeutic properties of mscs [reviewed in (167) ]) promotes the release of mir-216a-5p enriched sevs that target jak2 in chondrocytes, resulting in an increase in chondrocyte proliferation and migration, while inhibiting their apoptosis. the mir-216a-5p enriched sevs also reduced ecm degradation through the inhibition of mmp expression and increasing col-ii expression levels (168) . in summary, several mirnas are known to be associated with different processes relevant to od (169) , such as inflammation (mir-22, mir-320) (105, 110) , extracellular matrix synthesis (mir-148a, mir-27, mir-218) (170, 171) and chondrocyte proliferation. additionally, several mirnas have been shown to be involved in processes associated with mscs differentiation into chondrocytes (mir-19a, mir-410) (172, 173) , and processes such as chondrocyte hypertrophy (mir-381, mir-140) (174, 175) , apoptosis and autophagy (mir-30b) (176) ( table 1) . the therapeutic potential of mirnas both in degenerative diseases such as oa and autoimmune diseases such as ra is very promising, and their delivery through sevs greatly facilitates escalation to later-stage clinical trials. still, more work needs to be done concerning the full effect of mirnas both in target cells and other types of cells to assess the safety of the therapeutic application of mirnas. as mentioned in the previous sections, msc-sevs arise as a potential cell-free based therapy that can reduce the risks associated with msc. strikingly, several reports show that msc-sevs mimic the biological effects of mscs. therefore, msc-sevs represent a hopeful alternative to msc therapy. the main functional components of msc-sevs are mirnas, which can regulate the expression of multiple target genes and participate in various cell signaling processes. the mirna profile of msc-sevs is associated with their effect. although there are tools to identify mirnas in sevs, the principal target genes of sevs derived mirnas remain unspecified. however, this work summarizes some of the mirnas involved in od pathogenesis and some of the mirnas that mediate the therapeutic effects of sevs in od. these mirna could be considered as promising candidates to use for effective treatment of these diseases. further studies in this field are required to develop msc-sevs therapeutics based on mirna delivery for autoimmune/inflammatory and degenerative diseases. furthermore, delving into the role of mirnas in the pathogenesis of disease, would also improve therapeutic strategies that can restore their normal levels, because not all mirnas have beneficial effects. in this context it is also important to study the regulation of mirnas and their biological functions, and also increase the knowledge of other non-coding rnas that can be involved in od. on the other hand, studies on the enrichment of sevs in beneficial mirnas and/or other non-coding rnas that regulate disease-promoting mirnas and evaluating strategies for the targeted delivery of sevs to particular cell types to increase efficiency remain one of the following challenges. el-b and ma wrote the main part of the manuscript with inputs from ch, fb-b, ao, cg, fg, cp, nl-c, gm, re-v, and fd. pl-c and av-l design the original idea of the review and critical review the manuscript. all authors contributed to the article and approved the submitted version. this review was supported by grants from the agencia nacional de investigation y desarrollo (anid) from chile through the fondecyt regular program grant number 1211353 pi pl-c. we thank the "agence nationale de recherche" for the anr metab-oa (anr-20-ce18-0014) and pri-mitomir. adipose derived mesenchymal stem cell exosomes loaded with mir-10a promote the differentiation of th17 and treg from naive cd4+ t cell economic burden of rheumatoid arthritis: a systematic review of literature in biologic era economic costs of osteoarthritis: a systematic review of cost-of-illness studies global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the global burden of disease study regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies the prevalence of rheumatoid arthritis: a systematic review of population-based studies oarsi guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis early diagnosis and treatment of rheumatoid arthritis concomitant use of low-dose methotrexate and nsaids and the risk of serious adverse events among patients with rheumatoid arthritis clinical efficacy and safety of mesenchymal stem cell transplantation for osteoarthritis treatment: a meta-analysis cartilage repair by mesenchymal stem cells: clinical trial update and perspectives mesenchymal stem cell therapy for the treatment of inflammatory diseases: challenges, opportunities, and future perspectives extracellular vesicles isolated from mesenchymal stromal cells modulate cd4+ t lymphocytes toward a regulatory profile extracellular vesicles from mesenchymal stem cells prevent contact hypersensitivity through the suppression of tc1 and th1 cells and expansion of regulatory t cells mesenchymal stem cell subpopulations: phenotype, property and therapeutic potential immunomodulatory effects of mesenchymal stem cell-derived exosomes on experimental type-1 autoimmune diabetes mesenchymal stem cells for regenerative medicine analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation mesenchymal stem cell-derived exosomes for clinical use msc exosome as a cell-free msc therapy for cartilage regeneration: implications for osteoarthritis treatment mesenchymal stem cell exosomes: the future msc-based therapy? in: mesenchymal stem cell therapy exosome and exosomal microrna: trafficking, sorting, and function microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microrna-dependent reprogramming of resident renal cells mesenchymal stem cell-derived extracellular vesicles affect disease outcomes via transfer of micrornas epigenetics in osteoarthritis: novel spotlight human bone mesenchymal stem cells-derived exosomes overexpressing microrna-26a-5p alleviate osteoarthritis via down-regulation of ptgs2 macro view of microrna function in osteoarthritis mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine mammalian msc from selected species: features and applications dynamic surfaces for the study of mesenchymal stem cell growth through adhesion regulation advances in mesenchymal stromal cell therapy in the management of crohn's disease concise review: multifaceted characterization of human mesenchymal stem cells for use in regenerative medicine eminent sources of adult mesenchymal stem cells and their therapeutic imminence functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers isolation and hepatocyte differentiation of mesenchymal stem cells from porcine bone marrow-"surgical waste" as a novel msc source comparison of proliferative and multilineage differentiation potential of sheep mesenchymal stem cells derived from bone marrow, liver, and adipose tissue biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue immunosuppressive activity of adipose tissue-derived mesenchymal stem cells in a rat model of hind limb allotransplantation differentiation of canine adipose tissue-derived mesenchymal stem cells towards endothelial progenitor cells morphologic and transcriptomic comparison of adipose-and bone-marrow-derived porcine stem cells cultured in alginate hydrogels isolation and characterization of adipose-derived mesenchymal stem cells (adscs) from cattle comparative immunophenotyping of equine multipotent mesenchymal stromal cells: an approach toward a standardized definition immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells in vitro comparison of feline bone marrow-derived and adipose tissue-derived mesenchymal stem cells proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential immunomodulatory properties of mesenchymal stromal cells: still unresolved "yin and yang mesenchymal stromal cell homing: mechanisms and strategies for improvement self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment umbilical cord-derived mesenchymal stromal cells (mscs) for knee osteoarthritis: repeated msc dosing is superior to a single msc dose and to hyaluronic acid in a controlled randomized phase i/ii trial adipose mesenchymal stromal cell-based therapy for severe osteoarthritis of the knee: a phase i dose-escalation trial intravenous infusion of umbilical cord blood-derived mesenchymal stem cells in rheumatoid arthritis: a phase ia clinical trial undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall regeneration of functional sweat gland-like structures by transplanted differentiated bone marrow mesenchymal stem cells mesenchymal stromal cells induce peculiar alternatively activated macrophages capable of dampening both innate and adaptive immune responses the immunomodulatory potential of mesenchymal stromal cells human bone marrow stromal cells suppress t-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals interplay between mesenchymal stem cells and lymphocytes immunomodulatory properties of mesenchymal stem cells and their therapeutic applications human mesenchymal stem cells: the present alternative for high-incidence diseases, even sars-cov-2. ballini a, editor safety of cell therapy with mesenchymal stromal cells (safecell): a systematic review and meta-analysis of clinical trials challenges and controversies in human mesenchymal stem cell therapy mesenchymal stem cell therapy for autoimmune disease: risks and rewards mesenchymal stem cell-derived exosomes: a new therapeutic approach to osteoarthritis? challenges and advances in clinical applications of mesenchymal stromal cells exosomes as mediators of intercellular communication: clinical implications minimal information for studies of extracellular vesicles 2018 (misev2018): a position statement of the international society for extracellular vesicles and update of the misev2014 guidelines rna delivery by extracellular vesicles in mammalian cells and its applications defining mesenchymal stromal cell (msc)-derived small extracellular vesicles for therapeutic applications exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating mir-21 delivery specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on t, b and nk cell functions human mesenchymal stem cell microvesicles for treatment of escherichia coli endotoxin-induced acute lung injury in mice comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from hek293t cells mesenchymal stem cells derived exosomes and microparticles protect cartilage and bone from degradation in osteoarthritis interactions between mesenchymal stem cells and the immune system mesenchymal stem cells secrete immunologically active exosomes g-mdsc-derived exosomes attenuate collagen-induced arthritis by impairing th1 and th17 cell responses mesenchymal stem cells-derived exosomes are more immunosuppressive than microparticles in inflammatory arthritis exosomes: effectual players in rheumatoid arthritis the mesenchymal stromal cells dilemma-does a negative phase iii trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? mesenchymal stem cell perspective: cell biology to clinical progress increased mesenchymal stem cell functionalization in three-dimensional manufacturing settings for enhanced therapeutic applications exosomes derived from genetically modified dc expressing fasl are anti-inflammatory and immunosuppressive exosomes derived from mir-92a-3p-overexpressing human mesenchymal stem cells enhance chondrogenesis and suppress cartilage degradation via targeting wnt5a exosomes derived from il-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis msc exosome works through a protein-based mechanism of action early gestational mesenchymal stem cell secretome attenuates experimental bronchopulmonary dysplasia in part via exosome-associated factor tsg-6 quantitative and stoichiometric analysis of the microrna content of exosomes the long and short of it: the emerging roles of non-coding rna in small extracellular vesicles microrna: key gene expression regulators natural killer-derived exosomal mir-186 inhibits neuroblastoma growth and immune escape mechanisms micrornas in cartilage development, homeostasis, and disease the role of mirnas in cartilage homeostasis dicer-dependent pathways regulate chondrocyte proliferation and differentiation synovium-derived micrornas regulate bone pathways in rheumatoid arthritis wnt signaling in bone homeostasis and disease: from human mutations to treatments resolution of inflammation induces osteoblast function and regulates the wnt signaling pathway dickkopf-1 is a master regulator of joint remodeling identification of microrna-221/222 and microrna-323-3p association with rheumatoid arthritis via predictions using the human tumour necrosis factor transgenic mouse model altered expression of circulating microrna in plasma of patients with primary osteoarthritis and in silico analysis of their pathways integrative microrna and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks the identification of differentially expressed microrna in osteoarthritic tissue that modulate the production of tnf-alpha and mmp13 plasma and synovial fluid micrornas as potential biomarkers of rheumatoid arthritis and osteoarthritis microrna-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes mirna-140 is a negative feedback regulator of mmp-13 in il-1b-stimulated human articular chondrocyte c28/i2 cells microrna-320 regulates matrix metalloproteinase-13 expression in chondrogenesis and interleukin-1b-induced chondrocyte responses microrna-92a-3p regulates aggrecanase-1 and aggrecanase-2 expression in chondrogenesis and il-1b-induced catabolism in human articular chondrocytes expression of microrna-146a in osteoarthritis cartilage microrna-140 plays dual roles in both cartilage development and homeostasis skeletal development, and bone formation, homeostasis and disease noggin haploinsufficiency differentially affects tissue responses in destructive and remodeling arthritis bone morphogenetic protein (bmp)-2 enhances the expression of type ii collagen and aggrecan in chondrocytes embedded in alginate beads phenotypic maintenance of articular chondrocytes in vitro requires bmp activity dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis and disease noggin inhibits il-1b and bmp-2 expression, and attenuates cartilage degeneration and subchondral bone destruction in experimental osteoarthritis detection of mir-22, mir-140 and bone morphogenetic proteins (bmp)-2 expression levels in synovial fluid of osteoarthritis patients before and after arthroscopic debridement inflammatory cytokines induce specific time-and concentration-dependent microrna release by chondrocytes, synoviocytes, and meniscus cells expression of mirna-140 in chondrocytes and synovial fluid of knee joints in patients with osteoarthritis exosomal mirna-486-5p derived from rheumatoid arthritis fibroblast-like synoviocytes induces osteoblast differentiation through the tob1/bmp/smad pathway tgfb/bmp signaling pathway in cartilage homeostasis tgf-b dampens il-6 signaling in articular chondrocytes by decreasing il-6 receptor expression micrornas regulating tgfb and bmp signaling in the osteoblast lineage microrna-455-3p promotes tgf-b signaling and inhibits osteoarthritis development by directly targeting pak2 potential novel prediction of tmj-oa: mir-140-5p regulates inflammation through smad/tgf-b signaling circcdk14 protects against osteoarthritis by sponging mir-125a-5p and promoting the expression of smad2 modulation of tgf-beta signaling by proinflammatory cytokines in articular chondrocytes microrna-9 regulates the development of knee osteoarthritis through the nf-kappab1 pathway in chondrocytes mir-138 suppressed the progression of osteoarthritis mainly through targeting p65 microrna-153 suppresses the osteogenic differentiation of human mesenchymal stem cells by targeting bone morphogenetic protein receptor type ii mir-149 suppresses the inflammatory response of chondrocytes in osteoarthritis by down-regulating the activation of tak1/nf-kb microrna−93 inhibits chondrocyte apoptosis and inflammation in osteoarthritis by targeting the tlr4/nf−kb signaling pathway nf-b-dependent induction of microrna mir-146, an inhibitor targeted to signaling proteins of innate immune responses reduced mir-26a and mir-26b expression contributes to the pathogenesis of osteoarthritis via the promotion of p65 translocation identification of pbmc-expressed mirnas for rheumatoid arthritis altered expression of microrna in synovial fibroblasts and synovial tissue in rheumatoid arthritis altered expression of microrna-203 in rheumatoid arthritis synovial fibroblasts and its role in fibroblast activation microrna-124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis down-regulation of microrna-34a* in rheumatoid arthritis synovial fibroblasts promotes apoptosis resistance altered expression of micrornas in rheumatoid arthritis mirna-regulated key components of cytokine signaling pathways and inflammation in rheumatoid arthritis essential role of microrna-155 in the pathogenesis of autoimmune arthritis in mice microrna delivery through nanoparticles microrna shuttle from cell-to-cell by exosomes and its impact in cancer extracellular vesicles for drug delivery secretory mechanisms and intercellular transfer of micrornas in living cells activity-associated mirna are packaged in map1b-enriched exosomes released from depolarized neurons selective extracellular vesicle-mediated export of an overlapping set of micrornas from multiple cell types therapeutic potential of mesenchymal cell-derived mirna-150-5p-expressing exosomes in rheumatoid arthritis mediated by the modulation of mmp14 and vegf exosomal microrna-320a derived from mesenchymal stem cells regulates rheumatoid arthritis fibroblast-like synoviocyte activation by suppressing cxcl9 expression the inhibition by human mscs-derived mirna-124a overexpression exosomes in the proliferation and migration of rheumatoid arthritis-related fibroblast-like synoviocyte cell mir-100-5p-abundant exosomes derived from infrapatellar fat pad mscs protect articular cartilage and ameliorate gait abnormalities via inhibition of mtor in osteoarthritis tgf-b1 promoted chondrocyte proliferation by regulating sp1 through msc-exosomes derived mir-135b chondrocyte-targeted microrna delivery by engineered exosomes toward a cell-free osteoarthritis therapy a comprehensive analysis of micrornas in human osteoporosis mesenchymal stem cell-derived exosomal microrna-136-5p inhibits chondrocyte degeneration in traumatic osteoarthritis by targeting elf3 small extracellular vesicles derived from human mesenchymal stromal cells prevent group 2 innate lymphoid cell-dominant allergic airway inflammation through delivery of mir-146a-5p bone marrow-derived mesenchymal stem cells-secreted exosomal microrna-192-5p delays inflammatory response in rheumatoid arthritis control of immunoregulatory molecules by mirnas in t cell activation exosomal microrna-155-5p from pdlscs regulated th17/treg balance by targeting sirtuin-1 in chronic periodontitis mirna-146a improves immunomodulatory effects of msc-derived exosomes in rheumatoid arthritis tregs: where we are and what comes next? front immunol preconditioning influences mesenchymal stem cell properties in vitro and in vivo hypoxic pretreatment of small extracellular vesicles mediates cartilage repair in osteoarthritis by delivering mir-216a-5p engineered-extracellular vesicles as an optimistic tool for microrna delivery for osteoarthritis treatment mir-27 inhibits the nf-kb signaling pathway by targeting leptin in osteoarthritic chondrocytes microrna-218 promotes early chondrogenesis of mesenchymal stem cells and inhibits later chondrocyte maturation microrna-410 promotes chondrogenic differentiation of human bone marrow mesenchymal stem cells through down-regulating wnt3a microrna-19a promotes cell viability and migration of chondrocytes via up-regulating sox9 through nf-kb pathway microrna-381 regulates chondrocyte hypertrophy by inhibiting histone deacetylase 4 expression microrna-140 suppresses human chondrocytes hypertrophy by targeting smad1 and controlling the bone morphogenetic protein pathway in osteoarthritis antimir-30b inhibits tnf-a mediated apoptosis and attenuated cartilage degradation through enhancing autophagy exosomes may be the potential new direction of research in osteoarthritis management micrornas' involvement in osteoarthritis and the prospects for treatments microrna-194 promotes osteoblast differentiation via downregulating stat1 mir-216a rescues dexamethasone suppression of osteogenesis, promotes osteoblast differentiation and enhances bone formation, by regulating c-cbl-mediated pi3k/akt pathway the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ..... ..... ......pdf 57 proinflammatory cytokines profile in patients with rheumatoid arthritis shahlaa m.saleh* phd jabbar r. zangor** phd summary: background: rheumatoid arthritis (ra) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. it is a disabling and painful inflammatory condition, which can lead to substantial loss of mobility due to pain and joint destruction. ra is a systemic disease, often affecting extra-articular tissues throughout the body including the skin, blood vessels, heart, lungs, and muscles. patients and methods: enzyme immunoassay for determination of human tnf, il-1 and gm-csf in serum samples from 50 patients with a diagnosis of rheumatoid arthritis results: of cytokines showed a significant increase in tnf-alpha, il-1 beta and gm-csf in patients with rheumatoid arthritis (70.98 12.08) pg/ml,(238.6 116.4)pg/ml and (96.1 12.08)pg/ml respectively. when compared with the control group (7.0 3.09)pg/ml, (15.4 3.8)pg/ml and (6.8 3.03)pg/ml respectively. conclusion: increased serum levels of proinflammatory cytokine such as tnf, il-1 and gm-csf probably play important role in driving inflammatory process and promoting joint destruction in rheumatoid arthritis. regulation of these cytokines is a crucial importance in the ra disease showing pleiotropic actions and many different targets. keywords: rheumatoid arthritis, tnf, il-1 and gm-csf. introduction: rheumatoid arthritis (ra) is a chronic systemic disorder of unknown etiology (1). this disease effects about 1% of the population worldwide most commonly middle-aged women (2). it is characterized by chronic inflammation of the synovium, particularly of small joints, which often leads to destruction of articular cartilage and juxtaarticular bone (3). cytokines regulate a broad range of inflammatory processes that are implicated in the pathogenesis of rheumatoid arthritis (4). in rheumatoid joints, it is well known that an imbalance between pro-and anti inflammatory cytokine activities favors the induction of autoimmunity (5). chronic inflammation and thereby joint damage (6).the cytokine network in rheumatoid arthritis is a complex field, with a lot of cytokines. pro-inflammatory cytokines in ra are il-1 and tnf(6). to keep it simple, the network can be divided in two groups, the proinflammatory and antiinflammatory cytokines (7) (8). controlling the balance between these two groups is considered as an important therapeutic goal (9). tnfis a substance made by cells of the body that has an important role in promoting inflammation (10).tnf promotes the inflammation and its associates fever and signs * (pain,tenderness, and swelling) in several inflammatory conditions (11). il-1 is a polypeptide with pro-inflammatory and immunopotentiating effects and (12). with relevance to rheumatoid arthritis il-1 augments release of prostanoids , proteinases and oxygen metabolites and is a potent inducer of bone and cartilage resorption(13). the colony stimulating factors (csfs) are glycoproteins believed to essential for the survival, proliferation, and differentiation of haematopoietic progenitor cells into monocyte/macrophage and granulocytes (14). they include granulocyte macrophage colony stimulating factor. gm-csf can prime monocyte/ macrophages for the production of these and other proinflammatory mediators, led to the proposal of a csf network loop in ra where gmcsf has a central role in mainting joints inflammation and is a potent inducer of bone and cartilage resorption (15). material and methods: subjects: patient study group: serum samples from 50 patients with a diagnosis of rheumatoid arthritis (they had positive rf test) were collected during the period between january and may 2008. the range of age (35-56) years. in addition to patient group 20 individuals (blood donors) were further investigated, and were considered as a control group. kits used in this study (tnf, il-1 , and gm-csf) by immunotech a beckman coulter company,france. methods: enzyme immunoassay for quantitative determination of human tnf, il-1 , and gm-csf in serum. original article 58 principle: the immunoenzyme assay of tnf, il-1 , and gm-csf is a sandwich type assay with two immunological steps.the first step leads to capture tnf, il-1 , and gm-csf by monoclonal anti tnf, il-1 , and gm-csf, antibody bound to the wells of microtiter plate. in the second step, a second monoclonal antitnf, il-1 , and gm-csf antibody, which is biotinylated is added together with streptavidinperoxidase conjugate. the biotinylated antibody is bound to the solid phase antibody antigen complex and in turn, binds the conjugate. after incubation period, the wells are washed and the binding of the streptavidin peroxiase via biotin is followed by the addition of chromogenic substrate of the peroxidase. bimmunoassay procedure: procedure, according to the information supplied immunotech a beckman coulter company. ccalculation of results: the standards curve was drawn by plotting on horizontal axis the tnf, il-1 , and gm-csf concentrations of the standards and on the vertical axis the corresponding average absorbance. to locate the concentration of tnf, il1 , and gm-csf in the sample, the average absorbance for each sample on the vertical axis was located and the corresponding tnf, il-1 , and gmcsf concentrations were located on the horizontal axis. statistical analysis: data have been analyzed statistically using upss program version 10. results were expressed using simple statistical parameters such as mean and standard deviation. analysis of quntitation data was done using t-test and anova. acceptable level of significance was considered to be less than o.o5 results: serum levels of tnf, il-1 , and gm-csf were significantly higher in patients with ra (70.98 12.08) pg/ml, (238.6 116.4) pg/ml and (96.1 12.08) pg/ml respectively when compared with control group which were significantly lower (7.0 3.09) pg/ml,(15.4 3. pg/ml 8) and (6.8 3.03) pg/ml respectively(p< 0.001). see figures (1, 2, and 3). figure1: serum levels of tnfmeasured by elisa in patients with ra and control group. figure2: serum levels of il-1 measured by elisa in patients with ra and control group. figure3: serum levels of gm-csf measured by elisa in patients with ra and control group. discussion: the current concept is that inflammation and tissue destruction results from complex cell-cell interactions between antigen presenting cells (apc) and cd4+ t cells; apc display complexes of class ii major histocompatibility complex (mhc) molecules and peptide antigens that bind to specific receptors on the t cells. macrophage activation occurs, with abundant secretion of proinflammatory cytokines such as il-1 and tnf . these cytokines stimulate synovial fibroblasts and chondrocytes in the nearby articular cartilage to secrete enzymes that degrade proteoglycans and collagen, leading to tissue destruction (16). in this study the level of serum proinflammatory cytokines (tnf-alpha, il-1 beta and gm-csf) was significantly higher compared with healthy control. this result agrees with previous study which concluded that the development of arthritis in tnf transgenic mice could be prevented with pg/m l pg/ml pg/ml 59 antibodies to tnf , more interestingly, pathology could also be fully blocked with antibodies against the il-1 receptor (17) (18) this strongly indicates that il1 is the secondary mediator responsible for the arthritic changes, and tnf alone is neither arthritogenic nor destructive towards joints (19). another study showed that the concept that tnf alpha is pathogenic in inflammatory arthritis has been validated by showing that neutralizing monoclonal anti-tnf antibodies significantly attenuate collageninduced arthritis in mice (20) in preliminary trials in rheumatoid patients anti-tnf appears to have an impressive effect on indices of disease activity including c-reactive production and serum amyloid-a production. tnf alpha appears to be a relevant therapeutic target in rheumatoid disease (21). il-1 and tnfplay a pivotal role in the ra. tnfis responsible for the inflammatory and proliferative aspects, and il-1 is responsible for the destructive aspects of ra (22). other proinflammatory cytokines also play very important role in ra, because they are responsible for activation of enzymes in synovial fluid, which induce degradation of bone (23). conclusion: increased serum levels of proinflammatory cytokine such as tnf, il-1 and gm-csf probably play important role in driving inflammatory process and promoting joint destruction in rheumatoid arthritis. regulation of these cytokines is a crucial importance in the ra disease showing pleiotropic actions and many different targets. references: 1. key: cord-0032906-7zlx183p authors: robak, ewa; robak, tadeusz title: bruton’s kinase inhibitors for the treatment of immunological diseases: current status and perspectives date: 2022-05-16 journal: j clin med doi: 10.3390/jcm11102807 sha: 0afef4025dff5add39243ea48f89ed318aed1b2e doc_id: 32906 cord_uid: 7zlx183p the use of bruton’s tyrosine kinase (btk) inhibitors has changed the management of patients with b-cell lymphoid malignancies. btk is an important molecule that interconnects b-cell antigen receptor (bcr) signaling. btk inhibitors (btkis) are classified into three categories, namely covalent irreversible inhibitors, covalent reversible inhibitors, and non-covalent reversible inhibitors. ibrutinib is the first covalent, irreversible btk inhibitor approved in 2013 as a breakthrough therapy for chronic lymphocytic leukemia patients. subsequently, two other covalent, irreversible, second-generation btkis, acalabrutinib and zanubrutinib, have been developed for lymphoid malignancies to reduce the ibrutinib-mediated adverse effects. more recently, irreversible and reversible btkis have been under development for immune-mediated diseases, including autoimmune hemolytic anemia, immune thrombocytopenia, multiple sclerosis, pemphigus vulgaris, atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus, sjögren’s disease, and chronic spontaneous urticaria, among others. this review article summarizes the preclinical and clinical evidence supporting the role of btkis in various autoimmune, allergic, and inflammatory conditions. in 1952, dr. ogden bruton first reported a case of an 8-year-old boy who presented with recurrent bacterial sepsis, osteomyelitis, and otitis, with the lack of a gamma globulin fraction and failure to produce antibodies [1] . the disease was then described as x-linked agammaglobulinemia (xla), manifested by decreased serum immunoglobulin and a markedly decreased b-cell number in the peripheral blood. subsequently, mutations of the gene coding tyrosine kinase, named bruton's tyrosine kinase (btk), were identified [2, 3] . bruton's tyrosine kinase is a member of the tec kinase family of nonreceptor tyrosine kinases and is expressed in all hematopoietic cells except t cells, terminally differentiated plasma cells, and natural killer cells [4] . btk contributes significantly to the proliferation and differentiation of b cells and plays a major role in both the innate and the adaptive immune responses and cytokine production. btk inhibitors (btkis) suppress b-cell receptor and myeloid fragment crystallizable receptor mediated signaling, thus inhibiting b-cell activation, antibody class-switching, expansion, and cytokine production. btkis can affect autoimmune diseases involving b cells and non-b cells through b-cell receptor, fc receptor, and rank receptor signaling. therefore, btk is currently being investigated as a promising target for the treatment of immunological disorders in addition to b-cell hematological malignancies [5, 6] . btk inhibitors have been investigated in various autoimmune disorders, including autoimmune hemolytic anemia (aiha), immune thrombocytopenia (itp), multiple sclerosis (ms), atopic dermatitis (ad), rheumatoid arthritis (ra), systemic lupus erythematosus (sle), sjögren's disease (sd), multiple sclerosis (ms), and pemphigus vulgaris (pv). in addition, btkis are under investigation for use in allergic and inflammatory diseases including chronic spontaneous urticaria (csu), asthma, and graft versus host disease (gvhd) [7] . this article summarizes the recent data obtained from preclinical studies and clinical trials of btkis in various immune-mediated diseases, and this introduction should briefly place the study in a broad context and highlight why it is important. bruton's tyrosine kinase plays a critical role in the b-cell receptor (bcr) signaling pathway. btk influences the production of messenger molecules, which can abnormally activate the bcr signaling pathway and transform b cells into self-reactive b cells responsible for autoimmune diseases [8] [9] [10] . the increased activity of btk has been observed in several autoimmune disorders, including sle and ra [11, 12] . the signaling potential of btk, and its targetable nature, have been found to be immensely valuable for a wide range of clinical applications. the development of novel, more specific, and less toxic btkis could potentially revolutionize the treatment of a significant number of diseases with yet unmet treatment needs [10] . the majority of btkis target the atp-binding site. these agents are classified as reversible or irreversible btkis according to their binding mode. in addition, reversible btkis are divided into two categories, namely covalent reversible inhibitors and noncovalent reversible inhibitors [13, 14] . btkis have shown remarkable efficacy in the treatment of b-cell malignancies, including chronic lymphocytic leukemia (cll), mantle cell lymphoma (mcl), waldenstrom macroglobulinemia (mw), and various other b-cell lymphomas. in addition, they have also been investigated in patients with immune disorders, including autoimmune conditions, allergic diseases, and inflammatory disorders. however, long term data are not yet available. the recent discoveries and significance of btk inhibitors in the treatment of various immune diseases are summarized in this review. covalent, irreversible btkis bind cysteine 481 (c481) in the atp binding pocket of btk, thus inhibiting the phosphorylation of downstream kinases in the bcr signaling pathway and blocking b-cell activation [13] . the c481 in btk forms a covalent bond with the inhibitor by acting as a nucleophile [15] . three irreversible covalent btkis, viz. ibrutinib, acalabrutinib, and zanubrutinib, have been approved for the treatment of lymphoid malignancies [6] . in addition, ibrutinib has been approved for the treatment of chronic gvhd. of the three, ibrutinib is the most potent btk inhibitor, followed by zanubrutinib and acalabrutinib, while acalabrutinib is the most selective, followed by zanubrutinib and ibrutinib [16, 17] . recently, several irreversible btkis have been developed and are under clinical investigation (table 1) [6, 18] . these drugs inhibit btk activity by irreversible covalent binding with high affinity to the same cysteine 481 in btk as ibrutinib. some of the newer btkis are more selective than currently approved drugs. in addition, the difference in pharmacodynamics and pharmacokinetics between btk inhibitors may influence their efficacy and safety in clinical use. table 1 . irreversible btk inhibitors studied in immune disorders. ibrutinib (pcyc-1102, imbruvica, pharmacyclics/janssen) [19] covalent btki with off-target activity ( ibrutinib (pcyc-1102, imbruvica ® , pharmacyclics/janssen, sunnyvale, ca, usa) was discovered in 2007 as an irreversible btki with an ic50 for cys481 binding of 0.5 nm [51] . however, ibrutinib has off-target activity and can block other kinases, including egfr, erbb2, itk, and tec [15] . this off-target activity is responsible for an increased risk of bleeding, with an inhibition of collagen-induced platelet aggregation and platelet adhesion via the inhibition of intracellular molecules involved in platelet signaling [52] . in addition, ibrutinib affects pi3k-akt signaling, which is responsible for atrial fibrillation complications in ibrutinib-treated patients [19] . ibrutinib was approved in 2013 for the treatment of mantle cell lymphoma (mcl) and in 2014 for chronic lymphocytic leukemia (cll)/small lymphocytic lymphoma (sll). subsequently, ibrutinib was approved for waldenström's macroglobulinemia (wm), gvhd, and marginal zone lymphoma (mzl). phase 2 trials in aiha are ongoing (table 1) . acalabrutinib (acp-196, calquence ® , acerta pharma, astrazeneca, cambridge, uk) is a second-generation oral, highly selective, covalent btki approved for the treatment of cll/sll and mcl [17] . acalabrutinib covalently binds to c481 in btk with an ic50 of 3 nm [20] . the drug demonstrates less off-target binding and a much higher specificity for btk than ibrutinib. in contrast to ibrutinib, acalabrutinib does not inhibit the tec-family kinases (itk and txk), erbb2, and src-kinases (src, lyn, fyn, yes and lck) [21] ; however, it inhibits the epidermal growth factor receptor (egfr), which can be associated with a rash and severe diarrhea [22] . in addition, acalabrutinib has a shorter half-life than ibrutinib and is given twice daily [23] . in a phase 3 trial (elevate rr), acalabrutinib was better tolerated and had similar efficacy to ibrutinib in patients with previously-treated cll when compared with ibrutinib [24] . in particular, cardiovascular events were less common in the acalabrutinib group compared to ibrutinib. acalabrutinib was approved in 2017 for the treatment of patients with mcl who have received at least one prior therapy, and in 2019 for the treatment of cll/sll. it is also under investigation in phase 2 trials in ra, aiha, and gvhd (table 1) . zanubrutinib (bgb-3111, brukinsa ® , beigene beijing, china) is a second-generation irreversible btki developed by the baekje shenzhou company of china for the treatment of b-cell lymphoid malignancies [25] . zanubrutinib demonstrated potent activity and more selectivity against btk over other tec, egfr, and src families. in addition, compared to ibrutinib, zanubrutinib induced more selective btk inhibition, with more complete and sustained btk occupancy, as well as improved oral absorption and better target occupancy. recent clinical trials have found it to demonstrate excellent efficacy and good tolerability in patients with cll and wm. in previously treated cll patients, zanubrutinib demonstrated better efficacy and safety than ibrutinib (alpine study) [26] . currently, zanubrutinib is under investigation in phase 2 trials in patients with immune disorders including itp, as (antiphospholipid syndrome), igg4-rd, and active proliferative lupus nephritis (table 1) . spebrutinib (cc-292, avl-292, avila therapeutics/celgene, summit, ny, usa) is an oral, irreversible, covalent btki (cys481 ic50 < 0.5 nm) that binds covalently with high affinity to the btk atp binding site (cys481 ic50 < 0.5 nm) [27, 28] . spebrutinib has been found to inhibit b-cell proliferation and reduced both lymphoid and myeloid cytokine production and degranulation in vitro, and to influence osteoclastogenesis [28] . spebrutinib also reduces disease symptoms in experimental ra in mice and in humans [27] . in a first-in-human study performed in healthy volunteers, spebrutinib led to near-complete btk occupancy for eight to 24 h. spebrutinib is currently under investigation in a phase 2a, randomized, placebo-controlled study in patients with active ra (table 1 ). in a clinical trial with female ra patients on stable methotrexate (mtx), the numbers of naive b cells in circulation and transitional b cells were found to decrease, as was the collagen-degradation product ctx-1, indicating reduced osteoclastogenesis [28] . evobrutinib (a18, m2951, merck, readington township, nj, usa) is a highly selective, irreversible, covalent btki inhibitor (cys481 ic50 = 9 nm) with high selectivity in inhibiting both bcr and fc receptor signaling [29, 53] . evobrutinib inhibits b-cell activation and differentiation, as well as m1 macrophage polarization. it also has greater btk selectivity than ibrutinib. in mouse models of ra and sle, evobrutinib reduced the disease severity and histological damage [29, 54] . in the sle model, evobrutinib inhibited b-cell activation, reduced autoantibody production, and normalized b-and t-cell subsets. in a phase 1, double-blind, dose-escalation study in healthy participants, evobrutinib showed linear and time-independent pk which induced long-lasting btk inhibition. the drug was welltolerated and showed no prolongation of the qt/qtc interval [55] . in this study, full btk occupancy was achieved with 25 mg of evobrutinib after multiple daily dosing. treatmentemergent adverse events (aes) were observed in 25% of volunteers after single dosing and in 48.1% after multiple dosing; however, these tended to be mild in most participants without any dose dependency. evobrutinib is one of the first btk inhibitors to be studied in ms. it has recently completed its phase 2 trial in ms patients [30] . clinical trials of evobrutinibin in relapsing ms (rms) and sle are ongoing (table 1) . remibrutinib (lou064, novartis, cambridge, ma, usa) is a highly selective, irreversible covalent btki with cys481 ic50 1.3 nm [31] . remibrutinib very potently inhibits btk and tec in vitro and inhibits btk-dependent platelet activation [32] . in healthy volunteers, single doses of 30 mg remibrutinib or higher resulted in a greater than 95% blood btk occupancy for at least 24 h. with multiple ascending doses greater than or equal to 10 mg q.d., remibrutinib reached near complete blood btk occupancy at day 12 [33] . the drug was developed for the treatment of autoimmune disorders, including csu. in a phase 1 study, remibrutinib showed a favorable safety profile in adult healthy subjects with or without asymptomatic atopic diathesis (nct03918980). a phase 3 clinical trial in csu, inadequately controlled by h1 antihistamine (remix-1), and a phase 2 study in patients with moderate to severe sd (louisse) are ongoing (table 1) . tirabrutinib (velexbru ® , ono/gs-4059, ono pharmaceutical, osaka, japan) is another very potent and specific covalent, irreversible btki that demonstrates greater selectivity than ibrutinib (cys481 ic50 2 nm) [34] . this agent was developed for the treatment of autoimmune disorders and hematological malignancies. tirabrutinib has demonstrated significant cytotoxic activity in several types of b-cell neoplasms in vitro and in vivo in mouse models [35] . in mice treated with tirabrutinib, the suppression of osteoclastic bone resorption was observed together with the inhibition of serum trapcp5b and urinary ctx-1 [36] . it is currently approved for the treatment of central nervous system (cns) lymphoma, wm, and cll [37] . a phase 2 clinical trial in sd is ongoing (nct03100942). elsubrutinib (abbv-105, abbvie, chicago, il, usa) is an irreversible, non-covalent, highly selective, and potent btki with the ic50 of cys481 binding being 180 nm [38] . the drug inhibits a histamine release from ige-stimulated basophils and an il-6 release from igg-stimulated monocytes. elsubrutinib also inhibits tnf-release from cpg-dna stimulated pbmcs [38] . in a preclinical study, the btk occupancy correlated with the in vivo efficacy. phase 2 trials to investigate the safety and efficacy of elsubrutinib in ra (nct03682705) and sle (nct03978520) have been initiated. tolebrutinib (sar 442168, prn 2246, sanofi/principia biopharma, san francisco, ca, usa) is an investigational irreversible btk inhibitor. it covalently binds to a specific conserved cysteine present in only 11 kinases, with potential immunomodulatory and anti-inflammatory activities [39, 40] . tolebrutinib can cross the blood-brain barrier and inhibit the activity of btk in the central nervous system and has potential efficacy in ms. in the first-in-human randomized, double-blind, placebo-controlled study, tolebrutinib was well-tolerated with only mild treatment-related adverse events. phase 2 and 3 studies against ms and myasthenia gravis (mg) are ongoing (table 1) . branebrutinib (bms-986195, bristol myers squib, new york, ny, usa) is a potent, highly selective, oral btki that covalently modifies a cysteine residue in the active site of btk [42] . branebrutinib has demonstrated 5000-fold higher selectivity for btk over 240 other kinases. in murine models of collagen-induced arthritis (cia), branebrutinib reduced clinically evident joint damage and inhibited loss of bone mineral density. branebrutinib is better tolerated than currently approved, less-selective btk inhibitors. a randomized phase 1, placebo-controlled trial in healthy participants found btk to demonstrate a rapid and high occupancy without notable safety findings [43] . currently, branebrutinib is under evaluation as monotherapy in clinical trials in patients with moderate to severe psoriasis, active sle and sd, ad, and ra (table 1) . orelabrutinib (icp-022, biogen/innocare pharma, beijing, china) is another highly selective irreversible, covalent btki (cys481 ic50 = 1.6 nm) under investigation for the treatment of b-cell malignancies and autoimmune diseases [41] . in a kinome scan assay conducted in parallel against numerous kinases at a drug concentration of 1 µm, orelabrutinib was found to be more selective than ibrutinib. in this study, btk was the only kinase targeted by orelabrutinib (with >90% inhibition). in 2020, orelabrutinib was approved in china for the treatment of patients with mcl and cll who had previously received at least one line of anticancer therapy [56] . it is currently investigated in clinical trials for lymphoid malignancies and autoimmune disorders, including itp (nct05020288, nct05124028), sle and sd (nct04305197, nct04186871), and relapsing-remitting ms (nct04711148). poseltinib (hm71224, ly3337641, hanmi pharmaceutical, eli lilly, indianapolis, in, usa) is an experimental, selective irreversible, non-covalent btki with potential anti-inflammatory activity which was developed for the treatment of ra and sle [44, 45] . the drug is relatively selective for a conversed cysteine shared by only ten kinases within the kinome. in a preclinical study, poseltinib effectively inhibited the production of tumor necrosis factor (tnf)-α, interleukin (il)-6, and il-1β by human monocytes, as well as osteoclast formation by human monocytes. it also reduced the signs and symptoms of arthritis and prevented joint destruction in a dose-dependent manner in a murine and rat cia model [44] . a clinical phase 1 trial for healthy volunteers, showed a dose-dependent and persistent btk occupancy in the peripheral blood mononuclear cells of all patients receiving poseltinib [46] . a first-in-human healthy volunteer study confirmed poseltinib as a potential btk inhibitor for the treatment of autoimmune diseases [46] . shr1459 (tg 1701, ebi-1459; reistone biopharma, jiangsu hengrui medicine co, lianyungang, china) is a covalently bound and irreversible btki currently under clinical development. this agent exhibits superior btk selectivity compared to ibrutinib in in vitro kinase screening [47, 48] . shr1459 is currently under clinical development in phase 2 trials in membranous glomerulonephritis and neuromyelitis optica (table 1) . tas5315 (sat0056, taiho pharmaceutical co, tokyo, japan) is a novel covalent, irreversible, highly selective btki with significant efficacy in a mouse cia model for ra. tas5315 treatment resulted in a dose-dependent reduction of the clinical score in a mouse cia model compared with vehicle-treated mice. when applied at 0.4 mg/kg, tas5315 completely ameliorated arthritic symptoms on day 14 [49] . in a histopathological analysis, the mice treated with tas5315 demonstrated a marked reduction in the severity of inflammation, pannus, cartilage destruction, and bone destruction in a dose-dependent manner. tas5315 inhibited rankl-dependent osteoclast activation and osteoclast activation induced by inflammatory cytokines [50] . a phase 2 study comparing tas5315 with placebo in participants with ra is ongoing (table 1) . ac0058 (acea biosciences, san diego, ca, usa) is a covalent, irreversible btki. in preclinical studies, ac0058 inhibited b-cell activation and inflammatory cytokine production in monocytes [5] . the drug is in development for the treatment of b cell-related autoimmune diseases, including ra and sle. in a double-blind, placebo-controlled phase 1 trial, ac0058 was found to be safe and well-tolerated (nct02847325). a phase 1b double-blind, randomized, placebo-controlled study of the safety, pharmacokinetics, and pharmacodynamics of ac0058 in patients with sle is ongoing (table 1) . reversible btkis can be divided into covalent reversible inhibitors and non-covalent reversible inhibitors (table 2) , with the two acting through different mechanisms: the noncovalent inhibitors do not bind to the c481 site on btk. they hence provide an effective alternative to patients with b-cell malignancies who have developed resistance following prior therapy with covalent btkis [57, 58] . some reversible btkis (fenebrutinib, nemtabrutinib, rilzabrutinib, pirtobrutinib, and prn473) have entered early clinical trials [13, 57] . they were found to required more frequent and sustained dosing but have less off-target effects on other kinases. reversible inhibitors seem to be more effective in the treatment of autoimmune diseases such as ra, different types of ms, gvhd, and sle [18] . however, while clinical trials with these drugs are promising, they are less advanced than those with irreversible btk inhibitors. these agents have shown encouraging results in preclinical studies and some in early clinical trials. fenebrutinib (gdc-0853, roche/chugai pharmaceutical, tokyo, japan) is a selective, reversible, non-covalent btki that has a distinct kinase selectivity profile, with strong inhibitory activity against several key oncogenic drivers from the tec, trk, and src family kinases [59] . fenebrutinib forms hydrogen bonds with k430, m477, and d539. the agent blocks the ige-mediated histamine release from the mast cells in vitro, and it was found to inhibit ige-mediated basophil activation in healthy individuals in a phase 1 study [60] . in ra patients with an inadequate response to methotrexate, fenebrutinib caused dose-dependent reductions in the rheumatoid factor with comparable efficacy to adalimumab [61] . a similar response was observed in a phase 2 study performed in sle patients, where fenebrutinib caused dose-dependent reductions in anti-double-stranded dna autoantibodies [62] . however, in moderate to severe sle patients, no improvement was observed in the symptoms (table 2 ) [63] . in a placebo-controlled, randomized phase 2 trial, fenebrutinib decreased the disease activity in patients with antihistamine-refractory csu [64] , and the drug is currently undergoing clinical development for autoimmune disorders such as ra and sle (table 2 ). nemtabrutinib (mk1026, arq 531, arqule, inc., woburn, ma, usa) is a potent, reversible inhibitor of both a wild-type and ibrutinib-resistant c481s-mutant btk [69] , which binds to btk by forming hydrogen bonds with e475 and y476. it has a distinct kinase selectivity profile, with strong inhibitory activity against several key oncogenic drivers from the tec, trk, and src family kinases. in addition to inhibiting btk, nemtabrutinib also inhibits other kinases involved in bcr signaling including the src family kinase lyn and the immediate upstream kinase of erk and mek1 [70] . studies evaluating the safety, tolerability, and pharmacokinetics (pk) of nemtabrutinib have been initiated in patients with hematologic malignancies and mild to moderate persistent asthma ( table 2 ). pirtobrutinib (loxo-305, loxo oncology, stamford, ct, usa) is a reversible, noncovalent, next-generation btki which blocks the atp binding site of btk by noncovalent, non-c481-dependent binding [74] . pirtobrutinib is under evaluation in patients with bcell malignancies. however, clinical trials in immune-mediated diseases have not been initiated yet. rilzabrutinib (prn1008, principia biopharma/sanofi, paris, france) is another reversible, covalent btki with a cys481 ic50 of 1.3 nm [65] . the drug has shown high affinity and selectivity for the btk, together with a long duration of action due to prolonged, but reversible, target occupancy [66, 67] . in contrast to ibrutinib, rilzabrutinib does not interfere with normal platelet aggregation. the agent has demonstrated anti-inflammatory effects, the neutralization of pathogenic autoantibodies, and the blockade of new autoantibody production. in a preclinical study, rilzabrutinib inhibited the activation and inflammatory activities of b cells, macrophages, basophils, mast cells, and neutrophils in human and rodent assay systems [68] . in addition, administration improved joint pathology in a rat cia model and reduced autoantibody mediated fcγr signaling in vitro and in vivo. in contrast to ibrutinib, preclinical studies showed simultaneous rapid anti-inflammatory effects, as well as the neutralization and prevention of autoantibody signaling without any effect on collagen-induced platelet aggregation [65] . the preclinical and clinical data suggest that rilzabrutinib has the potential to treat a wide range of immune-mediated diseases. a phase 1 study in healthy volunteers and patients with immune-mediated diseases found rilzabrutinib to have a promising safety profile [66, 67] . in earlier studies, rilzabrutinib did not impact platelet aggregation in blood samples from either normal healthy volunteers or itp patients [68] . it also inhibits platelet destruction, mainly via the inhibition of autoantibody/fcγr signaling in splenic macrophages [68] . this observation is important in itp patients because of the associated reduction of the risk of bleeding and bruising typically associated with most btk inhibitors. rilzabrutinib is currently under investigation for itp in phase 2 and phase 3 studies (table 2 ). it is also the subject of phase 2 studies for aiha, igg4-related disease (igg4rd), pv, asthma, csu, and ad (table 2 ). prn473 (sar 444727, principia/sanofi, paris, france) is a topical, reversible covalent btki designed for immune-mediated diseases that could benefit from a localized application to the skin with low to no systemic exposure [71] . prn473 inhibits the ige (fcεr)-mediated activation of mast cells and basophils, igg (fcγr)-mediated activation of monocytes, and neutrophil migration. when administered orally, prn473 was found to be effective and well tolerated in the treatment of canine pemphigus foliaceus [72] . a phase 2a study of the safety, tolerability, and pharmacokinetics of topically administered prn473 in patients with mild to moderate atopic dermatitis is ongoing (table 2) . bms-986142 (bristol-myers squibb, new york, ny, usa) is a reversible covalent btki found to reduce fcr-mediated cytokine production and bcr-induced cytokine production by normal b cells in vitro [73] . in animal models of ra, bms-986142 reduced joint inflammation and destruction. however, in a clinical trial with active ra patients, treatment with bms-986142 and mtx had no advantage over placebo and mtx ( [76] . tas5315 was found to selectively inhibit btk with less off-target activity than other kinases. in addition, it was more active against osteoclastogenesis and bone resorbing activity in osteoclasts compared with cc-292. in a cia model, tas5315 significantly reduced paw swelling, and treatment was associated with the repair of bone erosion and improved bone mineral density from joint destruction. these results indicate that tas5315 is a very potent agent for use against joint bone damage and inflammation. it also improved bone erosion in a murine model for ra, suggesting that it may merit clinical studies. gdc-0834 (genentech, san francisco, ca, usa) is a highly potent, selective, reversible adenosine triphosphate (atp)-competitive btk inhibitor developed as a potential drug for treating ra and other immune and inflammatory diseases [77] . this agent inhibited btk with an in vitro ic 50 of 5.9 nm in a biochemical assay and 6.4 nm in a cellular assay. it was found to have an ic 50 of 1.1 in mice and 5.6 microm in rats in vivo. gdc-0834 inhibits toll-like receptor 4 and tumor necrosis factor-α (tnf-α) in monocytes, which may also contribute to pathogenesis in ra [78] . in cia rat models, treatment with oral gdc-0834 demonstrated a robust anti-arthritis effect with a dose-dependent decrease of ankle swelling and a reduction of morphologic changes. gdc-0834 has recently been in phase 1 clinical trials for the treatment of ra but the study has been discontinued. cgi-1746 (cgi/genetech, san francisco, ca, usa) is a reversible, non-covalent atp-competitive btki with anti-inflammatory and anti-arthritic activity in experimental mouse models. this agent reduced cytokine and autoantibody levels in the joints in a mouse arthritis model induced by anti-collagen ii antibodies [79] . cgi-1746 inhibited bcr-mediated b-cell proliferation and suppressed fcγriii-induced tnfα, il-1β, and il-6 production in macrophages in cellular assays [80] . however, clinical trials have not yet been initiated. biib068 (biib068, biogen, cambridge, ma, usa) is a reversible btki showing over 400-fold selective inhibition for btk compared to other kinases [81] . it has the potential for autoimmune diseases. a phase 1, single-ascending-dose, safety, tolerability, pharmacokinetic (pk), and pharmacodynamic (pd) study of biib068 in healthy participants is ongoing ( table 2 ). rn-486 (roche, basel, switzerland), is a selective and reversible btki that potently and competitively binds to the kinase. preclinical studies indicate that the drug has therapeutic activity in ra in rodents and ra synovial tissue explants [82, 83] . in addition, in mouse models of sle, rn486 decreased sle symptoms by inhibiting b-cell activation and reducing the secretion of igg anti-double-stranded dna (anti-dsdna) [84] . however, clinical trials with rn486 have not yet been registered. autoimmune hemolytic anemia is an autoimmune disease characterized by antibodies directed against red blood cells (rbcs). aiha is idiopathic (primary) in 50% of patients, and secondary to an underlying condition, including lymphoproliferative disease, infections, immunodeficiency, and other diseases, in the other 50% [85] . aiha is also divided into the more common (70-80%) warm aiha and less common (20-30%) cold aiha, according to the thermal characteristics of the autoantibodies [86] . humoral, cellular, and innate immunity, including warm polyclonal high-affinity igg autoantibodies, are believed to play a role in the pathogenesis of warm aiha, while in cold aiha, monoclonal igm is also able to fix complement at low temperatures [87] . in patients with coexisting aiha and progressive cll, no progression of cll was observed following ibrutinib treatment [88, 89] . in addition, several recent studies have demonstrated that ibrutinib is an effective drug in patients with aiha complicated lymphoid malignancies [90, 91] . it was also found that aiha was suppressed during ibrutinib therapy, but worsened when ibrutinib was withdrawn [90, 92] . aiha has been found to develop in patients treated with ibrutinib [93, 94] ; however, only 1.28% of aiha were attributed to the use of ibrutinib in one study [89] . ibrutinib has also been successfully used to treat patients with both aiha and cll; after treatment, they discontinued therapy specifically for aiha [89, 95] . ibrutinib may be also an effective drug in the treatment of patients with cold aiha, with all 13 patients with cold aiha in a recent study showing an improvement in hemoglobin levels, including 12 complete responses (cr) and one partial response (pr), after the initiation of ibrutinib [96] . acalabrutinib, a second-generation selective btk inhibitor, also reduced concomitant aiha in patients with relapsed/refractory cll [97] . this drug is currently under evaluation in patients with aiha and cll in a phase 2 study (table 1) . rilzabrutinib, a reversible btk inhibitor, is also under investigation in patients with idiopathic warm aiha in a multicenter, phase 2b study (table 2) ; however, the results are not yet available. immune thrombocytopenia is an acquired disease characterized by the autoimmune destruction of platelets and megakaryocytes by igg autoantibodies targeting surface antigens such as glycoprotein (gp) αiibβ3 (gpiibiiia) and gpib-ix-v [98] . patients with b-cell malignancies treated with irreversible btkis such as ibrutinib, acalabrutinib, zanubrutinib, and tirabrutinib often demonstrate mild bleeding [21] ; however, no bleeding has been reported in the clinical trials of tolebrutinib, evobrutinib, branebrutinib, bms-986142, fenebrutinib, and rilzabrutinib. ibrutinib administration inhibits platelet aggregation, and this precluded its use during itp. however, it has shown beneficial effects in autoimmune cytopemias in patients with cll [95] . hampel et al. reported that among 193 patients treated with ibrutinib for cll, 29 demonstrated autoimmune cytopenias prior to the initiation of treatment, eight had itp, and five had evans' syndrome [95] . no progression of autoimmune cytopenias was observed. in addition, 42% of the patients underwent the reduction of treatments specific for autoimmune disorders and 25% discontinued therapy dedicated to autoimmune cytopenia. another irreversible btki under investigation for primary itp in a phase 2 clinical trial is zanubrutinib (table 1) . zanubrutinib is less potent than ibrutinib in inducing the shedding of human and mouse gpibα and gpix in vitro and in vivo from the platelet surface. in contrast to ibrutinib, zanubrutinib does not induce the shedding of human and mouse integrin α iib β 3 in vitro or in vivo [99] . these results can explain a lower risk of bleeding complications in zanubtutinib-treated cll patients. rilzabrutinib is a reversible btk inhibitor that influences mechanisms driving itp [65] . in contrast to ibrutinib, rilzabrutinib has no effect on collagen-induced platelet aggregation, nor bruising, nor any fluctuation of the platelet count in healthy people [32, 100] . recently, the preliminary results of a phase 1/2 study of rilzabrutinib in 32 chronic itp patients were reported [101] . rilzabrutinib was well-tolerated at all studied doses, and an optimal dose of 400 mg bid was established. treatment resulted in a rapid and durable platelet response across several subgroups of patients and with extended treatment. half of the patients achieved a platelet count of ≥30 g/l in the first week of treatment, with 42% achieving ≥50 g/l. in addition, 71% of the patients demonstrated a persistent response lasting several weeks. rilzabrutinib is currently being compared with placebo in adult and adolescent patients with persistent or chronic itp in the ongoing phase 3 luna3 study, the first randomized, multicentered study evaluating its efficacy and safety, initiated in 2020 (nct04562766; eudract 2020-002063-60). eligible patients are randomized into receiving oral rilzabrutinib 400 mg bid or placebo for up to 24 weeks, followed by openlabel treatment for 28 weeks, and then a four-week safety follow-up. their findings should deliver data on the role of btkis in the treatment of itp [67, 102] . rilzabrutinib is being investigated in a phase 2 and phase 3 trial in patients with itp (table 2) . orelabrutinib is another selective btk inhibitor of potential value in the treatment of itp. in a preclinical study, orelabrutinib effectively suppressed the activation and differentiation of b cells in vitro and in vivo, and reduced thrombocytopenia in active itp murine models [103] . phase 2 clinical trials in patients with primary, refractory itp are ongoing (table 1 ). rheumatoid arthritis is a systemic autoimmune disease that causes chronic inflammation of the joints and surrounding tissues, as well as pathological bone resorption due to osteoclast activity, resulting in joint destruction and disability. the disease is characterized by b-cell dysregulation via bcr signaling, and dysregulation of t cells, leading to the production of autoantibodies and inflammatory cytokines involved in the development and progression of ra. this may indicate that btkis can be useful drugs in the treatment of ra [104] . several preclinical studies indicate that btkis may have therapeutic activity in ra. it is believed to act by decreasing the formation of myeloid immune complexes, inhibiting b-cell activation and inflammatory cytokines, and suppressesing osteoclastic bone erosion [28, 73] . most studies investigating btk inhibitors in ra, based on a collagen induced arthritis (cia) rodent model, indicate that btkis inhibit b-cell activation, myeloid immune complexes' formation, decrease inflammatory cytokine activity, and stimulate osteoclastogenesis [28, 73, 78, 79, 105] . evobrutinib, spebrutinib, and gdc-0834 significantly reduced the development and disease activity in cia in rodents [27, 29] ; however, gdc-0834 yielded similar effects to the controls treated with methotrexate [78] . the irreversible tricyclic btki somcl-17-016 was found to ameliorate arthritis and bone damage in cia mice more effectively than corresponding doses of ibrutinib and acalabrutinib; it was also found that somcl-17-016 demonstrated therapeutic effects in the cia model, inhibiting the b-cell function and osteoclastogenesis [105] . several btkis, such as spebrutinib (cc-292), abbv-105, evobrutinib, bms-986142, rilzabrutinib, and acalabrutinib, have been studied in phase 1 trials in recent years. all these trials reported positive results without serious safety concerns. they also achieved sufficient receptor occupancy with satisfactory pharmacokinetic (pk) and pharmacodynamic (pd) endpoints and were found to be effective in animal models [20, 28, 29, 38, 106, 107] . more recently, irreversible and reversible btk inhibitors, including acalabrutinib, spebrutinib, evobrutinib, tirabrutinib subrutinib, branebrutinib, poseltinib, tas5315, and fenebrutinib, are undergoing phase 2 clinical trials (tables 1 and 2) [36, 104, [107] [108] [109] . shafer et al. evaluated the pharmacology and mechanism of the action of spebrutinib in patients with active ra based on the american college of rheumatology acr20 endpoint [28] . it was found that 41.7% (10/24) of ra patients treated with spebrutinib and 21.7% (5/23) of placebo patients demonstrated ≥ 20% improvement at week four; however, this difference was not statistically significant (p = 0.25). even so, a subgroup analysis indicated potential efficacy in female patients with ra. fenebrutinib has shown efficacy in reducing ra activity in patients with an inadequate response to either methotrexate or tnf inhibitors. in a multicenter, randomized, doubleblind phase 2 study (andes), fenebrutinib was compared to placebo and adalimumab in patients with an inadequate response to either methotrexate or tnf inhibitors [61] . patients with ra and an inadequate response to methotrexate received fenebrutinib (50 mg once daily, 150 mg once daily, 200 mg twice daily), 40 mg adalimumab every other week, or placebo. in the second cohort, patients with ra and an inadequate response to tumor necrosis factor inhibitors were treated with fenebrutinib (200 mg twice daily) or placebo. both groups continued treatment with methotrexate. in cohort 1, those receiving fenebrutinib 50 mg once daily demonstrated a similar acr50 at week 12 to placebo (15%); however, higher acr50 scores were observed for those receiving fenebrutinib 150 mg once daily (28%) and 200 mg twice daily (35%) (p = 0.017; p = 0.0003). in cohort 2, more patients treated with fenebrutinib 200 mg twice daily achieved acr50 (25%) than those with placebo (12%) (p = 0.072). the most common adverse events for fenebrutinib were nausea, headache, anemia, and upper respiratory tract infections. bms-986142, another reversible, covalent btki, was evaluated in a phase 2 study; the participants included patients with moderate to severe ra with an inadequate response to methotrexate or methotrexate and who had received up to two tnf inhibitors ( table 2 ). in the phase 2 study, the patients were randomized to either one of the three doses of bms-986142 or placebo as 1:1:1 randomization for 12 weeks. although the study has been completed, the results are not available yet. the results of the phase 2 ra-juvenate trial investigating poseltinib in patients with active ra were recently published [109] . the trial included patients with active ra, and with either an inadequate response or loss of response to at least one disease-modifying antirheumatic drug, or who had an intolerance to these agents. no statistically significant difference in the acr20 response was observed between any dose of poseltinib and placebo at week 12 (p > 0.05 for all comparisons) in the interim analysis, and part b of the study was discontinued. gdc-0834 is a highly selective, reversible btki that was developed as a potential drug for ra [77] . it inhibited btk phosphorylation in a preclinical study in vitro and suppressed arthritis in a rat cia model in vivo [78] . moreover, gdc-0834 significantly inhibited joint inflammation, cartilage damage, and bone resorption. systemic lupus erythematosus (sle) is an autoimmune disease characterized by autoantibody formation, immune complex deposition, multisystem involvement, and tissue damage [110] . several components of btk signaling pathways are altered in b cells from patients with sle, and btkis are promising for the treatment of sle. btk inhibitors (pf-06250112, m7583, bi-btk-1, rn-486, ibrutinib, poseltinib, and evobrutinib) have demonstrated the prevention or amelioration of lupus nephritis and other sle symptoms in mouse models of sle [45, [111] [112] [113] . m7583 was investigated in the bxsb-yaa and pristane-dba/1 mouse lupus models [114] . in bxsb-yaa lupus mice, m7583 reduced autoantibody levels, nephritis, and mortality. in the pristane-induced dba/1 lupus model, this agent suppressed arthritis but did not affect autoantibodies or the ifn gene signature. another irreversible btk inhibitor, bi-btk-1, ameliorated multiple pathological endpoints associated with kidney disease in two distinct murine models of spontaneous lupus nephritis in mouse models of sle: nzb × nzw f1 (nzb/w) and mrl/lpr [111] . treatment with bi-btk-1 significantly protected both strains from the development of proteinuria, decreased anti-dna titers, and increased survival. however, lupus nephritis or kidney damage is often reduced, despite the persistence of autoantibodies in the serum, especially anti-rna antibodies [114] . fenebrutinib was investigated in a phase 2, multicenter, randomized, placebo-controlled study in patients with moderately-to-severely active sle. the study demonstrated significant reductions in cd19+ b cells and anti-dsdna autoantibodies, but no significant clinical response was observed [62] . treatment was well tolerated but the primary end point of the study, sle responder index 4 (sri-4) response at week 48, was not met. a phase 1 clinical trial with ac0058 in sle patients is ongoing (table 1) . sjogren's disease (sd) was first described by swedish physician henrik sjögren, who reported the coincidence of profound dry eyes and dry mouth with chronic arthritis in a group of women. primary sd is a chronic inflammatory rheumatic disease resulting in immune-mediated injury to the lacrimal and salivary glands. clinical trials have been initiated in sd, with these acting on the autoimmune processes believed to play a role in its pathogenesis. autoreactive cd19-hbtk mice develop spontaneous sd/sle-like autoimmune symptoms, including lymphocytic infiltrates in the salivary glands [115] . in addition, patients with active sd had increased btk levels in circulating b cells, correlating with the numbers of infiltrating t cells in the parotid gland [116] . bms-986142, branebrutinib, remibrutinib, and tirabrutinib have been tested in sd in several clinical trials. the efficacy of treatment with either lulizumab or bms-986142 versus placebo was evaluated in a phase 2 study in patients with moderate to severe primary sd. the efficacy was measured by the change from the baseline in the essdai score at week 12 between active treatment arms (lulizumab or bms-986142) and the placebo arm. however, the study was terminated prematurely due to a lack of therapeutic activity. in addition, treatment with branebrutinib and tirabrutinib did not yield any clinical improvement [9] . a phase 2 randomized double-blind, placebo-controlled multi-center study evaluating the safety and efficacy of multiple remibrutinib doses in patients with moderate to severe sd (louisse) is ongoing (table 1 ) and should be completed in 2024. systemic sclerosis (ss) is a connective tissue disease affecting the skin, blood vessels, and internal organs, with key roles in its pathogenesis played by immune dysregulation and autoantibody production [117] , vasculopathy, and chronic fibroblast activation. an in vitro study found ibrutinib to reduce the production of the profibrotic hallmark cytokines il-6 and tnf-α from the effector b-cell population in primary samples from patients with ss. in addition, small doses of ibrutinib (0.1µm) preserved the production of immunoregulatory il-10 and inhibited hyperactivated, profibrotic effector b cells. it appears that ibrutinib improves the b-cell pathology contributing to ss pathogenesis and progression, and the findings provide support for the initiation of clinical trials of btk inhibitors in humans. multiple sclerosis is an autoimmune disorder caused by the action of environmental factors in genetically predisposed hosts. the b cells in the brain play a central role in antigen presentation, cytokine secretion and antibody production, and in the pathogenesis of the disease [118] . in response, btkis can penetrate to the brain and spine, reducing inflammation and neurodegeneration within the central nervous system, and target the microglia of brain-resident b cells; they can also attenuate b-cell:t-cell interactions [119] . preclinical studies in animal models of ms indicate that btkis influence meningeal inflammation and cortical demyelination [120, 121] . hence, several irreversible (evobrutinib, tolebrutininb, and orelabrutinib) and reversible btkis (fenebrutinib and biib091) might be effective in the treatment of patients with ms [122] . in a preclinical study, evobrutinib inhibited antigen-presenting b-cells for the development of encephalitogenic t cells in mice and reduced disease severity [123] . in a double-blind, randomized, phase 2 trial, performed in relapsing ms, patients received evobrutinib at a dose of 25 mg once daily, 75 mg once daily, 75 mg twice daily, or open-label dimethyl fumarate (dmf). another group received a placebo [124] . patients who received 75 mg of evobrutinib once daily had significantly fewer enhancing lesions during weeks 12 through 24 in comparison with the placebo group. however, no significant improvement was observed among patients treated with evobrutinib at doses of 25 mg oncedaily or 75 mg twice-daily, nor was there any improvement in the annualized relapse rate or the disability progression observed at any dose. subsequently, the phase 3 evolution trial evaluating the efficacy and safety of evobrutinib, given orally twice daily, versus teriflunomide (aubagio ® ), given orally once daily, has been initiated in patients with relapsing ms (rms) ( table 1) . tolebrutinib (prn2246, sar442168) is a covalent btki that crosses the blood-brain barrier and potently inhibits btk in microglial cells isolated from the central nervous system (cns) [40] . tolebrutinib was investigated in a phase 2b, randomized, doubleblind, placebo-controlled trial in relapsing-remitting or relapsing secondary progressive ms [125] . treatment with tolebrutinib for 12 weeks led to a dose-dependent reduction in new gadolinium-enhancing lesions and in new or enlarging t2 lesions. the most common ae during tolebrutinib treatment was a headache. no safety-or treatment-related discontinuations were observed. phase 3 trials in rms, primary progressive (ppms), and secondary progressive (spms) forms of ms are ongoing. evobrutinib was found to be effective against experimental autoimmune encephalomyelitis, an animal model of brain inflammation [123] . the drug was also evaluated in a phase 2 clinical trial in relapsing-remitting ms and active spms (table 1 ) [124] . in this trial, the patients received placebo or evobrutinib at a dose of 25 mg once daily, 75 mg once daily, 75 mg twice daily, or open-label dimethyl fumarate (dmf) as a reference. the primary end point was the total (cumulative) number of gadolinium-enhancing lesions identified on t 1 -weighted magnetic resonance imaging at weeks 12, 16, 20, and 24. treatment with evobrutinib significantly reduced relapse rates and brain lesions at 48 weeks in a dosedependent manner in relapsing ms compared with the placebo. the mean total number of gadolinium-enhancing lesions during weeks 12 through 24 was 4.06 in the evobrutinib 25-mg group, 1.69 in the evobrutinib 75-mg once-daily group, 1.15 in the evobrutinib 75-mg twice-daily group, 3.85 in the placebo group, and 4.78 in the dmf group. no significant change in the edss score from the baseline was observed in any trial group. this reduction was sustained for about two years. the improvement was especially apparent in patients with a more advanced disease [126] . evobrutinib is currently under investigation in patients with relapsing ms in two identically-designed phase 3 trials: evolution rms 1 and evolution rms 2 (table 1) . pemphigus vulgaris is a potentially life-threatening autoimmune disease characterized by acantholysis, which results in erosions and blisters of the skin and mucous membrane. in this disease, pathogenic autoantibodies act against intercellular adhesion proteins in the epidermis of the skin and mucous membrane. it is found in two major clinical forms: pemphigus vulgaris and pemphigus foliaceus (pf). pemphigus vulgaris affects both the skin and mucous membrane when pf affects only the skin. currently, the standard of care for the treatment of this disease is systemic corticosteroid treatment [127] . btk inhibitors are attractive drugs for the treatment of pemphigus as they influence the innate immune system. early studies performed in canine pemphigus showed that rilzabrutinib treatment was safe and effective clinically. it is also being investigated in pv (table 2) [65, 67, 128] . in addition, the efficacy and safety of oral rilzabrutinib in pv was confirmed in a recent multicenter, phase 2 trial, with 52% and 70% of patients showing control of disease activity at four weeks and eight weeks, respectively [67] . however, the phase 3 placebo-controlled trial (pegasus) evaluating rilzabrutinib in patients with pemphigus did not show any difference between rilzabrutinib and the placebo with regard to its primary endpoint, i.e., complete remission from week 29 to week 37 ( table 2 , sanofi press release (https://www. sanofi.com/en/media-room/press-releases/2021/2021-09-09-07-00-00-2293920, accessed on 9 september 2021. prn-473 was active in the treatment of canine pf [128] . the treated dogs showed a reduction in lesions and a modified version of a validated human pemphigus disease activity index (cpdai) score during the first two weeks of treatment in four of nine good responses at the end of treatment after a maximum of 20 weeks. in rats, the topical administration of prn-473 inhibited an igg-mediated passive arthus reaction. similarly, topical prn473 administration caused a significant dose-dependent inhibition of the passive cutaneous anaphylaxis ige-mediated reaction in mice. a low systemic accumulation of the drug was observed, if any. these experiments deliver strong support for local activity in innate immune cell responses with minimal systemic exposure. btk inhibitors offer several advantages over other drugs commonly used for treating allergic diseases, including the inhibition of ige-fc ri-mediated activation of both mast cells and basophils. the initial results of a phase 2 trial showed that treatment with ibrutinib therapy suppresses skin test responses and eliminates ige-mediated basophil activation in adults with peanut or tree nut allergy [129, 130] . btkis may be effective in the treatment of allergic and atopic skin reactions, as well as in ige-mediated anaphylaxis. short treatment with ibrutinib transiently reduced skin prick test responses to foods and aeroallergens in allergic patients. these observations indicate that short courses of ibrutinib could be effective in preventing anaphylaxis to foods or drugs. however, btkis are not likely to be effective in ige-independent allergic diseases. preclinical studies and clinical trials suggest that btk inhibitors can be useful for the treatment of ige-dependent anaphylaxis, food and drug allergy, asthma, csu, and other allergic diseases. atopic dermatitis is a chronic, pruritic inflammatory skin disease typically affecting the face, neck, arms, and legs [131] . in a study of adults with a peanut or tree nut allergy, testing allergic patients before and after the initiation of treatment with ibrutinib showed the elimination of both the aeroallergen skin test and the basophil activation test reactivity, along with ige-mediated basophil activation [129, 132] . tolebrutinib has also been evaluated against ad. it is a reversible covalent btki in a topical formulation designed for the treatment of immune-mediated skin diseases that could benefit from a localized application to the skin. a phase 2a, randomized, double-blind, placebo-controlled trial was designed to evaluate the safety, tolerability, and pharmacokinetics of topical prn473 in healthy adult participants (table 1) . asthma is a complex airways disease with a wide spectrum which ranges from eosinophilic (th2 driven) to mixed granulocytic (th2/th17 driven) phenotypes. a particular cause for concern is mixed granulocytic asthma, as it is often unresponsive to corticosteroids. however, bruton's tyrosine kinase plays an important role in shaping allergic airway inflammation [133] . ibrutinib was able to suppress both th17/th2 and neutrophilic/eosinophilic inflammation in a mouse model of mixed granulocytic asthma, suggesting that this drug may be an alternative therapeutic option in corticosteroid treatmentresistant asthma. chronic spontaneous urticaria is characterized by itching and hives lasting for more than six weeks, with or without angioedema [134] . btk inhibitors can be effective for treating both autoallergic and autoimmune csu, as btk mediates degranulation in mast cells and participates in mcs and autoantibody production in b cells. the efficacy and safety of remibrutinib was tested in a randomized, double-blind, placebo-controlled phase 2b trial over 12 weeks in patients with at least moderately active csu and an inadequately controlled disease. the study showed a statistically significant dose response measured as a change in the urticaria activity score from baseline (uas7) compared to placebo at the fourth week [135] . fenibrutinib was also tested in csu and negative results were reported [9] . an igg4-related disease is a newly recognized fibroinflammatory disorder characterized by elevated igg4 serum levels, increased infiltration of igg4-positive plasma cells, and fibrosis in involved organs and swelling of organs and tissues [136] . treatment is typically based around systemic glucocorticoids, immunosuppressive drugs, biological agents, and surgery [137] . however, despite improvements being seen in most patients, relapse remains common after igg4-rd. btk inhibitors can be potentially useful in treatment and phase 2 clinical trials with rilzabrutinib and zanubrutinib were recently initiated ( table 2 ) [138] . however, the results are not available yet. acute and chronic graft versus host disease is a life-threatening complication after allogenic hematopoietic cell transplantation (allo-hct). currently, approved strategies for gvhd treatment are immunosuppressive therapies, mainly glucocorticoids. however, although therapeutic options for glucocorticoid-refractory patients are limited, btk inhibitors have shown promising results in preclinical animal models and clinical trials [139] . treatment with ibrutinib improved acute gvhd clinical scores and led to a longer survival in mice models of acute gvhd [140] . a phase 1b/2 study was conducted to determine the safety and efficacy of ibrutinib in chronic gvhd patients who had failed at least one line of treatment (table 1 ) [141] . the overall response (or) rate was 76%, and 71% of patients showed a response for more than 20 weeks. at a median follow-up of 13.9 months, 29% of patients were still on ibrutinib treatment. the integrate phase 3 clinical trial evaluated the combination with prednisone in patients with newly diagnosed, moderate to severe chronic gvhd after allo-hct (table 1 ). the ibrutinib arm yielded a higher response rate; in this group, corticosteroids could be withdrawn at 21 and 24 months and the patients demonstrated improved lee symptom scores [142] . in 2017, ibrutinib was approved by the fda for chronic gvhd after the failure of one or more lines of systemic therapy. acalabrutinib is also under evaluation for gvhd prophylaxis following allogeneic sct in a phase 2 study (table 1) . btk inhibitors have a specific safety profile that requires monitoring and management. indeed, the most common reason for treatment discontinuation is toxicity, with btkispecific adverse events (aes) including atrial fibrillation (af), bleeding events, arthralgias, rash, diarrhea, and cytopenias [6] . acalabrutinib and zanubrutinib have less off-target activity than ibrutinib and are better tolerated. the most common aes are headaches, observed in patients treated with acalabrutinib, and neutropenia and infections, in patients treated with zanubrutinib [6, 143] . patients treated with btkis are immunocompromised and have a high risk of infections, which develop in more than 50% of patients [143] . treatment with btkis is also associated with multiple immune defects, including decreased immunoglobulin levels and neutrophil and macrophage dysfunction; however, the iga level has been observed to increase in some patients. in a recent cross-trial comparison of btk inhibitors, similar immunologic changes were observed with both ibrutinib and acalabrutinib; both were associated with a sustained increase in serum immunoglobulin a [144] . however, in a recent preclinical study, ibrutinib reduced the primary antibody responses against an adeno-associated virus (aav) capsid [145] . it is not clear whether btkis affect the risk or severity of covid-19 or reduce vaccine efficacy [146] . btk regulates the activity of macrophages, and it has been suggested that btkis reduce inflammatory symptoms in covid-19 patients with a hyperinflammatory immune response [147] . moreover, acalabrutinib induced a clinical improvement, and reduced inflammation in patients with severe covid-19. however, it is important to note that btk inhibitors impair the innate immunity and increase susceptibility to infections. despite this, long-term btki therapy may improve the recovery of humoral immune function, decrease infection rates, and protect patients from lung injury in the event of covid-19 infection [148] . btkis are likely to decrease direct b-cell-mediated responses to covid-19 vaccines, as they inhibit the proliferation, differentiation, and class switching essential for antibody production [149] . in a recent study performed in cll patients, treatment with btk inhibitors or iga deficiency were independently associated with failure to generate an antibody response after the second vaccine [150] . ibrutinib and other btkis are not approved for the treatment of ra, sle, ms, or other autoimmune diseases, mainly due to toxicities related to their off-target effects. however, the development of new, more specific drugs can change this situation. btk is a signaling kinase that plays a crucial role in the activation of pathogenic b cells and autoantibody production in human autoimmune disorders. it also plays an important role in the production of pathogenic cytokines. in the past few years, btk has become a new target for the treatment of b-cell lymphoid malignancies. ibrutinib, a first-generation btki, revolutionized the treatment of such diseases. the second-generation btkis, such as acalabrutinib and zanubrutinib, offer greater btk selectivity. third-generation agents, including the non-covalent btk inhibitors pirtobrutinib and nemtabrutinib, have entered later-stage clinical development in lymphoid malignancies. more recently, btk inhibitors have shown promise for treating autoimmune diseases and other immune disorders in preclinical studies and early clinical trials. the results of preclinical studies from autoimmune animal models are promising, and several phase 1 and phase 2 clinical trials with btk inhibitors were initiated in patients with immune disorders, such as ra, sle, sm, pv, aiha, itp, and gvhd. among these, positive results have been obtained for ms, pv, itp, aiha, ra, and gvhd, while sle and sd have not been so promising. it is not currently clear why these agents demonstrate lower efficacy against some immunologic diseases. it is possible that btki treatment may be associated with a low btk occupancy in these disorders, or that the suppressive activity of btk inhibition in autoreactive b lymphocytes can partly be reduced by the inhibitor-induced activation of autoreactive t cells. the new generation of irreversible and reversible btkis are currently under investigation in immunological disorders; some of them, including acalabrutinib and zanubrutinib, have been found to be more specific than ibrutinib and induce less adverse events, including atrial fibrillation and hemorrhage. novel btkis with a higher specificity are currently being developed and are under evaluation in clinical trials. in addition, the combination of btkis with other agents may be more effective than monotherapy. future research should examine the pathogenic role of btk signaling and its inhibition in autoimmune and inflammatory disorders. in addition, combination strategies based on btk inhibitors and more specific novel drugs may yield positive results in immunological diseases. author contributions: e.r. and t.r. contributed equally to the conception/design, writing, and approval of the final version of the paper. all authors have read and agreed to the published version of the manuscript. expression of the gene defect in x-linked agammaglobulinemia the gene involved in x-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases the role of bruton's tyrosine kinase in b-cell development and function: a genetic perspective btk inhibitors: a medicinal chemistry and drug delivery perspective the role of bruton's kinase inhibitors in chronic lymphocytic leukemia: current status and future directions btk signaling in b cell differentiation and autoimmunity kinase inhibition in autoimmunity and inflammation bruton's tyrosine kinase (btk) inhibitors and autoimmune diseases: making sense of btk inhibitor specificity profiles and recent clinical trial successes and failures the role of bruton's tyrosine kinase in the immune system and disease rheumatoid arthritis is associated with signaling alterations in naturally occurring autoreactive b-lymphocytes b-cell subsets, signaling and their roles in secretion of autoantibodies structure-function relationships of covalent and non-covalent btk inhibitors novel synthetic drugs currently in clinical development for chronic lymphocytic leukemia the bruton tyrosine kinase inhibitor pci-32765 blocks b-cell activation and is efficacious in models of autoimmune disease and b-cell malignancy potency and selectivity of btk inhibitors in clinical development for b-cell malignancies acalabrutinib: a bruton tyrosine kinase inhibitor for the treatment of chronic lymphocytic leukemia the development of btk inhibitors: a five-year update ibrutinib increases the risk of atrial fibrillation, potentially through inhibition of cardiac pi3k-akt signaling acp-196): a covalent bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile bleeding by bruton tyrosine kinase-inhibitors: dependency on drug type and disease epidermal growth factor receptor inhibitor-associated cutaneous toxicities: an evolving paradigm in clinical management pharmacodynamic analysis of btk inhibition in patients with chronic lymphocytic leukemia treated with acalabrutinib acalabrutinib versus ibrutinib in previously treated chronic lymphocytic leukemia: results of the first randomized phase iii trial discovery of zanubrutinib (bgb-3111), a novel, potent, and selective covalent inhibitor of bruton's tyrosine kinase first interim analysis of alpine study: results of a phase 3 randomized study of zanubrutinibvs ibrutinib in patients with relapsed/refractory chroniclymphocytic leukemia/small lymphocytic lymphoma inhibition of btk with cc-292 provides early pharmacodynamic assessment of activity in mice and humans cc-292) affects markers of b cell activation, chemotaxis, and osteoclasts in patients with rheumatoid arthritis: results from a mechanistic study efficacy and pharmacodynamic modeling of the btk inhibitor evobrutinib in autoimmune disease models ectrims/ean guideline on the pharmacological treatment of people with multiple sclerosis discovery of lou064 (remibrutinib), a potent and highly selective covalent inhibitor of bruton's ty-rosine kinase effects of the btk-inhibitors remibrutinib (lou064) and rilzabrutinib (prn1008) with varying btk selectivity over tec on platelet aggregation and in vitro bleeding time. front. cardiovasc remibrutinib (lou064): a selective potent oral btk inhibitor with promising clinical safety and pharmacodynamics in a randomized phase i trial bio-chemical characterization of tirabrutinib and other irreversible inhibitors of bruton's tyrosine kinase reveals differences in on-and off-target inhibition tirabrutinib hydrochloride for b-cell lymphomas bruton's tyrosine kinase (btk) inhibitor tirabrutinib suppresses osteoclastic bone resorption tirabrutinib: first approval abbv-105, a selective and irreversible inhibitor of bruton's tyrosine kinase, is efficacious in multiple preclinical models of inflammation cysteine mapping in conformationally distinct kinase nu-cleotide binding sites: application to the design of selective covalent inhibitors phase 1 clinical trial evaluating safety, exposure and pharmacodynamics of btk orelabrutinib, a potent and selective bruton's tyrosine kinase inhibitor with superior safety profile and excellent pk/pd properties discovery of branebrutinib (bms-986195): a strategy for identifying a highly potent and selective covalent inhibitor providing rapid in vivo inactivation of bruton's tyrosine kinase (btk) safety, pharmacokinetics and pharmacodynamics of branebrutinib (bms-986195), a covalent, irreversible inhibitor of bruton's tyrosine kinase: randomised phase i, placebo-controlled trial in healthy participants hm71224, a novel bruton's tyrosine kinase inhibitor, suppresses b cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis hm71224, a selective bruton's tyrosine kinase inhibitor, attenuates the development of murine lupus target modulation and pharmacokinetics/pharmacodynamics translation of the btk inhibitor poseltinib for model-informed phase ii dose selection tg-1701 a novel, orally available, and covalently-bound btk inhibitor antitumor activity of the novel btk inhibitor tg-1701 is associated with disruption of ikaros signaling in patients with b-cell non-hodgkin lymphoma utsugi, t. tas5315, a novel bruton's tyrosine kinase (btk) inhibitor, demonstrates potent efficacy in mouse collagen-induced arthritis model sat0056 tas5315, a novel bruton's tyrosine kinase inhibitor, demonstrates potent efficacy against bone destruction in an animal model for rheumatoid arthritis discovery of selective irreversible inhibitors for bruton's tyrosine kinase ibrutinib-related bleeding: pathogenesis, clinical implications and management evobrutinib, a covalent bruton's tyrosine kinase inhibitor: mass balance, elimination route, and metabolism in healthy participants recent advances in btk inhibitors for the treatment of inflammatory and autoimmune diseases safety, tolerability, pharmacokinetics, target occupancy, and concentration-qt analysis of the novel btk inhibitor evobrutinib in healthy volunteers non-covalent btk inhibitors-the new btkids on the block for b-bell malignancies review of the development of btk inhibitors in overcoming the clinical limitations of ibrutinib discovery of gdc-0853: a potent, selective, and noncovalent bruton's tyrosine kinase inhibitor in early clinical development safety, pharmacokinetics, and pharmacodynamics in healthy volunteers treated with gdc-0853, a selective reversible bruton's tyrosine kinase inhibitor fenebrutinib versus placebo or adalimumab in rheumatoid arthritis: a randomized, double-blind, phase ii trial (andes study). arthritis rheumatol efficacy, safety, and pharmacodynamic effects of the bruton's tyrosine kinase inhibitor fenebrutinib (gdc-0853) in systemic lupus erythematosus: results of a phase ii, randomized, double-blind, placebo-controlled trial bruton's tyrosine kinase inhibition as an emerging therapy in systemic autoimmune disease fenebrutinib in h1 antihistamine-refractory chronic spontaneous urticaria: a randomized phase 2 trial preclinical efficacy and anti-inflammatory mechanisms of action of the bruton tyrosine kinase inhibitor rilzabrutinib for immune-mediated disease a phase i trial of prn1008, a novel reversible covalent inhibitor of bruton's tyrosine kinase, in healthy volunteers proof of concept for the clinical effects of oral rilzabrutinib, the first bruton tyrosine kinase inhibitor for pemphigus vulgaris: the phase ii believe study phase i/ii, open-label, adaptive study of oral bruton tyrosine kinase inhibitor prn1008 in patients with relapsed/refractory primary or secondary immune thrombocytopenia bruton tyrosine kinase inhibitors in b-cell malignancies: their use and differential features the btk inhibitor arq 531 targets ibrutinib-resistant cll and richter transformation preclinical mechanisms of topical prn473, a bruton tyrosine kinase inhibitor, in immune-mediated skin disease models efficacy of a bruton's tyrosine kinase inhibitor (prn-473) in the treatment of canine pemphigus foliaceus bruton's tyrosine kinase inhibitor bms986142 in experimental models of rheumatoid arthritis enhances efficacy of agents representing clinical standard-of-care pirtobrutinib shows evidence to inaugurate a third generation of btk inhibitors a phase 1, single and multiple ascending dose study of tas5315-a novel highly selective inhibitor of bruton's tyrosine kinase in healthy male volunteers tas5315, a novel bruton's tyrosine kinase inhibitor, ameliorates inflammation and bone erosion in murine model for rheumatoid arthritis potent and selective bruton's tyrosine kinase inhibitors: discovery of gdc-0834 antiarthritis effect of a novel bruton's tyrosine kinase (btk) inhibitor in rat collagen-induced arthritis and mechanism-based pharmacokinetic/pharmacodynamic modeling: relationships between inhibition of btk phosphorylation and efficacy specific btk inhibition suppresses b cell-and myeloid cell-mediated arthritis ibrutinib and novel btk inhibitors in clinical development discovery of biib068: a selective, potent, reversible bruton's tyrosine kinase inhibitor as an orally efficacious agent for autoimmune diseases rn486, a selective bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in ra synovial tissue explants suppression of glomerulonephritis in lupus-prone nzb × nzw mice by rn486, a selective inhibitor of bruton's tyrosine kinase autoimmune hemolytic anemia in chronic lymphocytic leukemia: a comprehensive review the diagnosis and management of primary autoimmune haemolytic anaemia the role of novel agents in treating cll-associated autoimmune hemolytic anemia ibrutinib in previously treated chronic lymphocytic leukemia patients with autoimmune cytopenias in the resonate study incidence and description of autoimmune cytopenias during treatment with ibrutinib for chronic lymphocytic leukemia ibrutinib is an effective treatment of autoimmune haemolytic anaemia in chronic lymphocytic leukaemia ibrutinib is effective in the treatment of autoimmune haemolytic anaemia in mantle cell lymphoma autoimmune hemolytic anemia (aiha) associated with chronic lymphocytic leukemia in the current era of targeted therapy autoimmune haemolytic anaemia occurring during ibrutinib therapy for chronic lymphocytic leukaemia a case of chronic lymphocytic leukemia complicated by autoimmune hemolytic anemia due to ibrutinib treatment autoimmune cytopenias in patients with chronic lymphocytic leukaemia treated with ibrutinib in routine clinical practice at an academic medical centre effect of ibrutinib treatment on hemolytic anemia and acrocyanosis in cold agglutinin disease/cold agglutinin syndrome acalabrutinib monotherapy in patients with relapsed/refractory chronic lymphocytic leukemia: updated phase 2 results autoimmune thrombocytopenia: current treatment options in adults with a focus on novel drugs ibrutinib, but not zanubrutinib, induces platelet receptor shedding of gpib-ix-v complex and integrin α iib β 3 in mice and humans prn1008, a reversible covalent btk inhibitor in clinical development for immune thrombocytopenic purpura oral rilzabrutinib, a bruton tyrosine kinase inhibitor, showed clinically active and durable platelet responses and was well-tolerated in patients with heavily pretreated immune thrombocytopenia luna3 phase iii multicenter, double-blind, randomized, placebo-controlled trial of the oral btk inhibitor rilzabrutinib in adults and adolescents with persistent or chronic immune thrombocytopenia orelabrutinib, a selective bruton's tyrosine kinase (btk) inhibitor in the treatment of primary immune thrombocytopenia (itp) bruton's tyrosine kinase inhibition for the treatment of rheumatoid arthritis a novel tricyclic btk inhibitor suppresses b cell responses and osteoclastic bone erosion in rheumatoid arthritis discovery of prn1008, a novel, reversible covalent btk inhibitor in clinical development for rheumatoid arthritis immunosuppressive agents for rheumatoid arthritis: a systematic review of clinical trials and their current development stage a highly sensitive lc-ms/ms method to determine novel bruton's tyrosine kinase inhibitor spebrutinib: application to metabolic stability evaluation safety and efficacy of poseltinib, bruton's tyrosine kinase inhibitor, in patients with rheumatoid arthritis: a randomized, double-blind, placebo-controlled, 2-part phase ii study an insight on the pathogenesis and treatment of systemic lupus erythematosus btk inhibition ameliorates kidney disease in spontaneous lupus nephritis btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in ifnα-driven lupus nephritis highly selective inhibition of bruton's tyrosine kinase attenuates skin and brain disease in murine lupus btk inhibition treats tlr7/ifn driven murine lupus btk levels set the threshold for b-cell activation and negative selection of autoreactive b cells in mice enhanced bruton's tyrosine kinase activity in peripheral blood b lymphocytes from patients with autoimmune disease inhibition of effector b cells by ibrutinib in systemic sclerosis bruton's tyrosine kinase inhibitors: a new generation of promising agents for multiple sclerosis therapy btk inhibition limits b-cell-t-cell interaction through modulation of b-cell metabolism: implications for multiple sclerosis therapy bruton's tyrosine kinase inhibition in the treatment of preclinical models and multiple sclerosis btk inhibitors as potential therapies for multiple sclerosis btk blockers make headway in multiple sclerosis inhibition of bruton's tyrosine kinase interferes with pathogenic b-cell development in inflammatory cns demyelinating disease placebo-controlled trial of an oral btk inhibitor in multiple sclerosis safety and efficacy of tolebrutinib, an oral brain-penetrant btk inhibitor, in relapsing multiple sclerosis: a phase 2b, randomised, double-blind, placebo-controlled trial effects of evobrutinib, a bruton's tyrosine kinase inhibitor, on slowly expanding lesions: an emerging imaging marker of chronic tissue loss in multiple sclerosis tyrosine kinase inhibition and its role as an emerging treatment in pemphigus open trial of bruton's tyrosine kinase inhibitor (prn1008) in the treatment of canine pemphigus foliaceus short-term ibrutinib therapy suppresses skin test responses and eliminates ige-mediated basophil activation in adults with peanut or tree nut allergy the use of bruton's tyrosine kinase inhibitors to treat allergic disorders the heterogeneity of atopic dermatitis inhibition of bruton's tyrosine kinase and il-2 inducible t-cell kinase suppresses both neutrophilic and eosinophilic airway inflammation in a cockroach allergen extract-induced mixed granulocytic mouse model of asthma using preventative and therapeutic strategy bruton's tyrosine kinase inhibitor used for treatment of lymphoproliferative disorders, eliminates both aeroallergen skin test and basophil activation test reactivity chronic spontaneous urticaria: latest developments in aetiology, diagnosis and therapy the bruton's tyrosine kinase inhibitor remibrutinib (lou064) in chronic spontaneous urticaria: top-line results of a phase 2b dose-finding study igg4-related disease glucocorticoids and steroid sparing medications monotherapies or in combination for igg4-rd: a systematic review and network meta-analysis b cell targeted therapy for immunoglobulin g4-related disease kinase inhibition as treatment for acute and chronic graft-versus-host disease inhibition of btk and itk with ibrutinib is effective in the prevention of chronic graft-versus-host disease in mice ibrutinib for chronic graft-versus-host disease after failure of prior therapy how ibrutinib, a b-cell malignancy drug, became an fda-approved second-line therapy for steroid-resistant chronic gvhd managing toxicities of bruton tyrosine kinase inhibitors reconstitution of humoral immunity and decreased risk of infections in patients with chronic lymphocytic leukemia treated with bruton tyrosine kinase inhibitors the effect of rapamycin and ibrutinib on antibody responses to adeno-associated virus vector-mediated gene transfer. hum. gene ther. 2022. ahead of print balancing potential benefits and risks of bruton tyrosine kinase inhibitor therapies in multiple sclerosis during the covid-19 pandemic the btk inhibitor ibrutinib may protect against pulmonary injury in covid-19 infected patients btk inhibitors in cancer patients with covid-19: "the winner will be the one who controls that chaos bruton's tyrosine kinase is involved in innate and adaptive immunity antibody responses after first and second covid-19 vaccination in patients with chronic lymphocytic leukaemia we thank edward lowczowski and from the medical university of lodz, poland, for editorial assistance. the authors declare no conflict of interest. writing assistance was not utilized in the production of this manuscript. key: cord-0030918-jxxzx886 authors: han, peng; hou, chao; zheng, xi; cao, lulu; shi, xiaomeng; zhang, xiaohui; ye, hua; pan, hudan; liu, liang; li, tingting; hu, fanlei; li, zhanguo title: serum antigenome profiling reveals diagnostic models for rheumatoid arthritis date: 2022-04-20 journal: front immunol doi: 10.3389/fimmu.2022.884462 sha: 6807226f0f93fd90f93a72edb6c6aa8c47cb269c doc_id: 30918 cord_uid: jxxzx886 objective: the study aimed to investigate the serum antigenomic profiling in rheumatoid arthritis (ra) and determine potential diagnostic biomarkers using label-free proteomic technology implemented with machine-learning algorithm. method: serum antigens were captured from a cohort consisting of 60 ra patients (45 acpa-positive ra patients and 15 acpa-negative ra patients), together with sexand age-matched 30 osteoarthritis (oa) patients and 30 healthy controls. liquid chromatography-tandem mass spectrometry (lc-ms/ms) was then performed. the significantly upregulated and downregulated proteins with fold change > 1.5 (p < 0.05) were selected. based on these differentially expressed proteins (deps), a machine learning model was trained and validated to classify ra, acpa-positive ra, and acpa-negative ra. results: we identified 62, 71, and 49 deps in ra, acpa-positive ra, and acpa-negative ra, respectively, as compared to oa and healthy controls. typical pathway enrichment and protein–protein interaction networks were shown among these deps. three panels were constructed to classify ra, acpa-positive ra, and acpa-negative ra using random forest models algorithm based on the molecular signature of deps, whose area under curve (auc) were calculated as 0.9949 (95% ci = 0.9792–1), 0.9913 (95% ci = 0.9653–1), and 1.0 (95% ci = 1–1). conclusion: this study illustrated the serum auto-antigen profiling of ra. among them, three panels of antigens were identified as diagnostic biomarkers to classify ra, acpa-positive, and acpa-negative ra patients. rheumatoid arthritis (ra) is a chronic autoimmune disease that leads to joint damage, systemic inflammation, and early mortality (1) . the prevalence of ra was approximately 0.5%-1% worldwide and 0.28% in china (2, 3) . the joint inflammation, combined with extra-articular complications, causes disability and reduces quality of life (4) . early diagnosis and subsequent treatment can substantially slow the progression of joint damage, thereby preventing irreversible disability (5) . though the precise molecular mechanism in the triggering and progression of systemic immune response is not fully understood, the emergence of antibodies against self-antigens marks the loss of self-tolerance and can serve as a diagnostic biomarker (6) . among these are rheumatoid factor (rf) and anti-citrullinated protein antibodies (acpas), which are currently used as biomarkers for diagnostics, and other antimodified protein antibodies (ampas) (7) (8) (9) . the combination of autoantibody and self-antigen could form immune complexes that significantly augment the immune response and contribute to the inflammatory process of ra (10) . multiple antigens have been confirmed such as a-enolase, fibrinogen, filaggrin, vimentin, and type ii collagen (11, 12) . however, the profiling of serum antigen, antigenome, remains poorly known. for decades, research has focused on single antigen identified as biomarkers (13). however, none of those achieves better specificity and sensitivity than acpa alone. in this study, we broadened the focus by addressing the entire repertoire, aiming to capture the enormous biodiversity of antigens, with the goal to find a panel of diagnostic biomarkers instead of a single candidate. moreover, the approach allows for finding differences of immune response by clustering the antigen repertoire that share certain function and pathway, providing further evidence in understanding of ra pathophysiology. the robust growth of quantitative proteomic methods enables researchers to discover indicator proteins for diagnosis and treatment of diseases. there has been a recent expansion in proteomics research on a number of different rheumatic diseases (14) (15) (16) . due to the large datasets generated by proteomics, it requires informatic approaches such as machine learning techniques to analyze and interpret data, which have been exploited to predict biomarkers to accurate classify different diseases (17) (18) (19) . we employed a robust mass spectrometry (ms)based proteomics strategy to delineate the serum antigenomic profiling. by applying a widely used machine-learning algorithm, random forest, we described 3 panels of biomarkers to distinguish ra, acpa-positive ra, and acpa-negative ra. these biomarkers were further validated in a cohort using proteomic data. these findings provided knowledge about serum antigen in ra and might reveal potential therapeutic targets. serum from 60 ra patients, as well as sex-and age-matched 30 osteoarthritis (oa) patients and 30 healthy controls were collected at the department of rheumatology and immunology, peking university people's hospital, beijing, china. the study was approved by the research ethics committee of peking university people's hospital. informed consent was obtained from all patients and healthy donors. the study population was randomly split into a test cohort (36 ra, 18 oa, and 18 hc) and a validation cohort (24 ra, 12 oa, and 12 hc). detailed clinical and demographic characteristics are summarized in table 1 . all ra patients met the 2010 american college of rheumatology (acr)/european league against rheumatism (eular) classification criteria (20) . the exclusion criteria include active infection, malignancy, and other known autoimmune or immune-mediated diseases, such as systemic lupus erythematosus, sjogren's syndrome, and type i diabetes. the individuals (n = 10 in each group) selected for igg purification were required to be free from monoclonal antibody treatment in at least 6 months. igg from human serum was purified using protein g spin kit (catalog no.22852, thermo fisher scientific). igg was purified from 500 ml of pooled serum of ten patients according to the manufacturer's instrument, representing the repertoire of antibodies of each group. the eluted igg was then washed and concentrated using 30-kda mwco filters (catalog no. ufc803096, amicon, millipore). to capture serum antigen, 5 mg of igg was coupled to 1 ml of cnbr-activated sepharose 4b column (catalog no.17043001, ge). by pretreating the igg column with acidic elution buffer (10 mmol/l gly-hcl, ph = 2.8), the antigens bound to igg were eluted. then, the diluted serum of one patient was incubated at room temperature with end-over-end mixing for 1 h. bound antigens were eluted with acidic elution buffer (10 mmol/l gly-hcl, ph = 2.8) and immediately neutralized by tris-hcl (1 mmol/l, ph = 9.1). the concentration of the protein was determined by bradford protein assay (catalog no. dq101-01, transgen biotech) and then stored at −80°c . protein (10 mg) was hydrolyzed with trypsin. digested products were separated by a 120-min gradient elution at a flow rate of 0.300 µl/min with the thermo ultimate 3000 nano-uplc system, which was directly interfaced with the thermo fusion lumos mass spectrometer. the analytical column was an acclaim pepmap rslc column (75 µm id, 250 mm length, c18). mobile phase a consisted of 0.1% formic acid, and mobile phase b consisted of 100% acetonitrile and 0.1% formic acid. the single full-scan mass spectra were acquired in a data-dependent manner in the orbitrap at a mass resolution of 60,000 at 375-1500 m/z. xcalibur 4.1.50 software was used for data acquisition. protein identification was carried out using mascot and sequest search algorithms through the proteome discovery software (version 2.4). searches were carried against human refseq protein database. ms tolerance was set to 10 ppm while ms/ ms tolerance was set to 0.02 da. the peptide-spectrum match allowed 1% target false discovery rate (strict). we used label-free quantification (lfq) algorithm to quantify protein expression and peptide-spectrum matching. normalization was performed against the total peptide amount. immunoglobulins and posttranslational modifications are not analyzed in the study, but could be potentially analyzed in the future. to obtain the intersection of antigen among ra, oa, and healthy controls, we used the venn diagram software (http:// bioinformatics.psb.ugent.be/webtools/venn/). pathway enrichment analysis was performed to classify proteins based on molecular function and biological processes by metascape web-based platform (21) . protein-protein interaction of differentially expressed proteins was performed using search tool for the retrieval of interaction gene/protein (string) database (ppi enrichment p-value < 1.0e-16) and visualized by cytoscape plug-in cytohubba (22, 23) . missing values were imputed with the minimal values for each feature. to get differentially expressed proteins, the fold change and t-test p-value were calculated between ra, acpa-positive ra, acpa-negative ra, and control (oa and healthy controls). the protein whose p-value < 0.05 and fold change > 1.5 was defined as differentially expressed protein. the heatmaps were drawn using the r package "pheatmap" (version 1.0.12), the sum of z-scores of log-transformed values were displayed, and the rows were sorted by fold changes. the pca was performed using the function "decomposition.pca" in scikit-learn (version 0.23.1) with default parameters. the log-transformed values were used as input for pca. the random forest classifiers were build using the function "ensemble.randomforestclassifier" in scikit-learn (version 0.23.1), with 101 trees, and the max depth for the trees was set to 4 to avoid overfitting. the log-transformed values of differentially expressed proteins were used as input features, and the number of features to consider in each tree was sqrt (number of features). we deleted saa (d3dqx7) as the sequence was very similar to saa1 and saa2. the importance of proteins was calculated using the build-in function "feature_importances_", which provides the impurity-based feature importance. the train-test split and classification process were repeated 500 times to calculate the auc and feature importance. we procured a cohort of patients containing 60 ra, 30 oa, and 30 healthy controls. the detailed clinical and demographic characteristics are shown in table 1 . the median age was 61.77 years and 81.7% of the patients were female. thirty oa patients and 30 healthy controls were all age-and sex-matched. the disease duration ranged from 1 to 42 years, with a mean duration of 12.37 years. seventy-five percent (45 of 60) of ra patients were acpa-positive. the mean esr (erythrocyte sedimentation rate) and crp (c-reactive protein) were 39.28 mm/h and 23.85 mg/dl, respectively. the mean das28 score (disease activity score-28) was 4.258. the workflow employed for this study is shown in figure 1 . briefly, iggs were purified from 500 ml of pooling mixture serum of 10 individuals in each group, respectively. these iggs were bound to the protein g column and then were treated to remove the antigens potentially bound to the antibodies first. after that, serum antigens from 120 samples were purified and collected individually. the antigen peptide mixture of each sample was then analyzed and quantified by high-resolution liquid chromatography with tandem mass spectrometry (lc-ms/ ms) (24) . applying this workflow, we quantified 4,475 proteins and 12,217 peptides from 120 samples. with immunoglobulins excluded, 461 proteins in acpa-positive ra, 409 proteins in acpanegative ra, 427 proteins in oa patients, and 422 proteins in healthy control were identified. a total of 360 proteins were in common among the 3 groups, while 35 proteins were specific for acpa-positive ra and 15 for acpa-negative ra. eight proteins were found only in the acpa-negative group and 28 in the acpa-positive group (figure 2a) . proteins with high confidence and could be detected in more than 20% in a particular patient group were chosen for further analyses. the principal component analysis (pca) showed that the clustering of samples is clearly classified into different groups as ra, oa, and healthy controls ( figure 2b ). however, pca analysis could not distinguish acpa-negative ra from acpapositive ra patients, demonstrating their similar antigenome pattern. taken together, these data presented a deep antigenome coverage, a promising basis for discovery of biomarkers. we next assessed significant quantitative differences between ra, oa, and healthy controls. we selected significantly upregulated and downregulated proteins by >1.5-fold (p < 0.05). a total of 62 differentially expressed proteins (deps) such as fibrinogen alpha chain, lipopolysaccharide-binding protein, and serum amyloid protein in ra were identified and shown in the volcano plot ( figure 3a) . heatmap analysis was performed to visualize those proteins ( figure 3a) . we next found 71 proteins differentially expressed in acpa-positive and 49 proteins differentially expressed in acpanegative patients, using the same filter criteria (figures 3b, c) . the deps were then subjected to enrichment analysis ( figure 4) . the analysis revealed that deps of these 3 groups were significantly enriched in pathways associated with immunology and inflammatory response, "acute inflammatory response", "activation of complement system", and "humoral immune response". deps in ra were enriched in pathways including "cell-cell adhesion" and "il-4 and il-13 signaling". the pathways of deps in acpa-positive ra were enriched in processes involved in "binding and uptake of ligand of scavenger receptors" and "il-6 pathway". some pathways associated with metabolic process were enriched in deps of acpa-negative ra, such as "folate metabolism", which might be interesting in future studies. as shown by protein-protein interaction (ppi) analysis of deps by the string database, the antigenome possessed abundant interactions. to recognize the key antigens lying in an essential position, we exploited cytoscape plugin cytohubba, which identified hub proteins in the networks. as shown in figure 5 , deps such as haptoglobin and itih4 were screened out as top hub proteins based on the connectivity degree. both haptoglobin and itih4 could function as acute-phase reactants (25, 26) . it was previously reported that the levels of haptoglobin were elevated in ra serum (27) . itih4 was found to be a serum biomarker for a variety of malignancies including gastric cancer and hepatocellular carcinoma (28, 29) . however, there is limited research investigating their detailed role in ra. these proteins might be essential in the pathogenesis of ra and utilized as biomarkers after rigorous validation. next, we attempted to discriminate ra from oa and healthy controls based on the deps. a widely used machine learning algorithm, random forest, was used to classify the patients. the (figure 6a) , and the top 15 dominant antigens in the model were saa2, c-reactive protein (crp), leucine-rich alpha-2glycoprotein, fibrinogen alpha chain, annexin a1, complement component c9, complement c4-a, saa1, carbonic anhydrase, testicular tissue protein li 70, ficolin-3, acx136, hemoglobin subunit alpha, paired like homeobox 2b, and beta-actin-like protein 2 ( figure 6a) . next, we investigated the possibility of discriminating acpapositive and acpa-negative ra patients from oa patients and healthy controls based on the deps. random forest algorithm was employed as well; 60% of the samples were used to train and 40% of the samples were used to evaluate. for acpa-positive ra patients, the model reached an auc of 0.9913 (95% ci = 0.9653-1) (figure 6b) , and the top 15 best-performing proteins were leucine-rich alpha-2-glycoprotein, saa2, crp, complement component c9, fibrinogen alpha chain, annexin a1, c4a anaphylatoxin, arfaptin-1, testicular tissue protein li 70, saa1, itih4 protein, ficolin-3, carbonic anhydrase, hemoglobin subunit alpha, and complement c4-a ( figure 6b ). moreover, for the classification of acpa-negative ra patients, the auc was calculated as 1.0 (95% ci = 1-1) ( figure 6c ). the top 15 proteins were saa2, fibrinogen alpha chain, saa2-saa4 readthrough, alpha-1-antitrypsin mbrescia variant, platelet glycoprotein v, saa1, fc-gamma receptor iiib, beta-actin-like protein 2, testicular tissue protein li 70, complement c1r subcomponent-like protein, aep4d11, crp, procollagen cendopeptidase enhancer, keratin type ii cytoskeletal 6b, and caspase 14 ( figure 6c ). our study presented a serum antigenomic investigation of ra using label-free global proteome strategy, which offered a landscape view of antigens. using random forest, an ensemble, supervised machine learning algorithm, 3 diagnostic signatures were built to classify ra, acpa-positive ra, and acpanegative ra patients. our findings might help to understand the pathogenesis of ra and provide novel and specific diagnostic targets for the disease. early diagnosis and immediate, effective therapy are crucial to gain control of inflammation and prevent deterioration, functional disability, and unfavorable progression in ra patients. to carry out personalized medicine for ra, clinical practice requires the use of biomarkers to ensure diagnosis, accurate stratification, and the high efficacy of treatment. current clinically used biomarkers including anti-ccp and rf only shows a modest discriminating power due to the lack of sensitivity and specificity (30) . searching biomarkers for diagnosis is a continuous effort, but none of those translate into routine clinical use (31) . therefore, searching reliable biomarkers for ra in a large population is highly desirable. to address the problem, we have combined cutting-edge mass spectrometry hardware, ms data processing, and bioinformatic analysis to build a high-performance serum antigenomic workflow. other groups have also performed proteomic studies for ra. mun et al. performed a quantitative proteomic study and identified 5 biomarkers using ra serum, which were quantitively verified by multiple reaction monitoring (mrm) (32). colasanti et al. discovered that anti-hcy-a1at (homocysteinylated alpha 1 antitrysin) autoantibody could be considered as a potential biomarker for ra by using matrixassisted laser desorption/ionization-time of flight (maldi-tof/ tof) (33) . however, we used a novel approach to capture the set of serum antigens, which was advantageous as it focused on a more targeted set of proteins, compared to entire serum proteome. we identified confirmed and putative antigens as candidates of novel potential biomarkers. in total, 4,475 proteins were identified by label-free comparative proteomic analysis of antigen profiling of ra, oa, and healthy control serum. compared to oa and healthy controls, we found 62 deps (fc > 1.5, p < 0.05) in ra, 71 deps that were specific in acpa-positive patients, and 49 deps specific in acpa-negative patients. we tried to gain insight into the functional roles of these deps associated with ra via pathway enrichment analysis. the interaction of deps was shown based on ppi networks. moreover, we identified hub proteins in the interaction networks. these avenues of enquiry may provide insight into the underlying mechanisms of ra. as a single biomarker may hardly achieve satisfactory discriminating power, seeking multiple biomarkers and developing a combinatorial model is a compromising strategy. by virtue of comprehensive antigenome profiling and random forest algorithm, we revealed 3 predictive models for ra, acpa-positive ra, and acpa-negative ra. the models have achieved low classification errors and resulted in very high auc levels. compared to acpa-positive ra, acpa-negative ra is poorly understood in etiology and pathogenesis. lack of effective biomarkers impedes early diagnosis and treatment, highlighting the importance of identifying specific antigens in this subset (34) . it is worth noting that several antigens are unique in the acpa-negative model, such as saa2-saa4 readthrough, platelet glycoprotein v, fc-gamma receptor iiib, complement c1r subcomponent-like protein, procollagen cendopeptidase enhancer, and caspase 14 ( figure 6c) . these proteins participate in various biological processes including acute phase response, cornification, complement activation, innate immune response, platelet activation, and collagen binding (35, 36) . there is limited research investigating their function and association with autoimmune or inflammatory diseases (37) . validation by elisa in larger acpa-negative ra cohorts and exploration of their detailed functions in the disease are needed. hopefully, these newly identified antigens may help early diagnosis and hint underlying mechanism of acpa-negative ra. though assay results were promising, this study does have limitations. first, we utilized igg purified from acpa-positive patients to capture antigens rather than anti-ccp2 antibody, which resulted in an incomplete reactivity pattern of acpa-igg due to its lower affinity toward citrullinated antigens. besides, by using an igg column to capture serum antigens from the corresponding group of patients, the deps identified between groups might be partially due to the differences of igg binding ability from various patients rather than antigens that were purely from the capture. second, if the model is to be applied in clinic, more rigorous quantification and extensive validation by targeted protein quantification or elisa are still needed. the detailed roles of antigens in the pathogenesis of ra should be further elucidated or experimentally validated. third, this was a single-center study, and the results merit validation in a larger, multicenter study that involve oa, systemic lupus erythematosus (sle), ankylosing spondylitis (as), psoriatic arthritis (psa), gout patients, etc. lastly, this work establishes the foundation for longitudinal studies geared toward the development of models predictive of disease onset or progression, and efficacy after treatment. the sera samples were collected at a single time point in both ra patients and control, and future studies of sera from more time points along the disease course are required, which could be potentially utilized to explore molecular dynamics during disease progression. in summary, we employed label-free global proteomics technology to analyze serum antigenome profiling of ra. the study increased our understanding of ra antigens and identified potential biomarkers to provide novel and specific diagnostic targets for the disease. we suggest that these panels identified here could be utilized as multiplex protein microarray platforms that have potential for scalability and contribute toward improved decision-making. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found at: http://www.proteomexchange. org/, pxd031498. the studies involving human participants were reviewed and approved by the research ethics committee at peking university of people's hospital. the patients/participants provided their written informed consent to participate in this study. fh and zl designed the research. ph, xz, and lc collected blood sample and ph summarized clinical data. ph, xs, and xhz performed lc-ms/ms. ph, ch, xs, xhz, xz, lc, tl, and fh analyzed and interpreted the data. ch and tl performed machine learning. ph and ch wrote the manuscript. hy, fh, hp, ll and zl edited the manuscript. all authors contributed to the article and approved the submitted version. rheumatoid arthritis the environment, geo-epidemiology, and autoimmune disease: rheumatoid arthritis epidemiology of eight common rheumatic diseases in china: a large-scale cross-sectional survey in beijing the course of established rheumatoid arthritis diagnosis and management of rheumatoid arthritis: a review the immunology of rheumatoid arthritis rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations anti-ccp antibodies: the past, the present and the future different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modification the pathogenesis of rheumatoid arthritis novel multiplex technology for diagnostic characterization of rheumatoid arthritis antibodies to a new viral citrullinated peptide, vcp2: fine specificity and correlation with anti-cyclic citrullinated peptide (ccp) and anti-vcp1 antibodies proteomic analysis of synovial fluid in osteoarthritis using swath−mass spectrometry proteomic analysis of synovial fluid: current and potential uses to improve clinical outcomes quantitative proteomic analysis of peripheral blood mononuclear cells in ankylosing spondylitis by itraq application of machine learning to proteomics data: classification and biomarker identification in postgenomics biology high-throughput proteomics and ai for cancer biomarker discovery proteomic and metabolomic characterization of covid-19 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative metascape provides a biologist-oriented resource for the analysis of systems-level datasets string v9.1: protein-protein interaction networks, with increased coverage and integration cytoscape: a software environment for integrated models of biomolecular interaction networks accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed maxlfq application of acute phase protein measurements in veterinary clinical chemistry inter-alpha-trypsin inhibitor heavy chain 4 is a novel marker of acute ischemic stroke abnormal function of high-density lipoprotein is associated with poor disease control and an altered protein cargo in rheumatoid arthritis itih4 is a novel serum biomarker for early gastric cancer diagnosis effective serum marker, early warning and diagnosis, hepatocellular carcinoma meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis precision medicine in the care of rheumatoid arthritis: focus on prediction and prevention of future clinically-apparent disease proteomics approach for the discovery of rheumatoid arthritis biomarkers using mass spectrometry homocysteinylated alpha 1 antitrypsin as an antigenic target of autoantibodies in seronegative rheumatoid arthritis patients novel autoantibodies identified in acpa-negative rheumatoid arthritis caspase-14-from biomolecular basics to clinical approach. a review of available data a novel human complement-related protein, c1r-like protease (c1r-lp), specifically cleaves pro-c1s psoriasis is characterized by altered epidermal expression of caspase 14, a novel regulator of keratinocyte terminal differentiation and barrier formation conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest key: cord-0030481-sjadkhd6 authors: pistorius, kimberly; ly, lucy; souza, patricia r.; gomez, esteban a.; koenis, duco s.; rodriguez, ana r.; foster, julie; sosabowski, jane; hopkinson, mark; rajeeve, vinothini; spur, bernd w.; pitsillides, andrew; pitzalis, costantino; dalli, jesmond title: mctr3 reprograms arthritic monocytes to upregulate arginase-1 and exert pro-resolving and tissue-protective functions in experimental arthritis date: 2022-04-14 journal: ebiomedicine doi: 10.1016/j.ebiom.2022.103974 sha: b58272d1c757e504df4975cbb4aef7c94a560d8e doc_id: 30481 cord_uid: sjadkhd6 background: rheumatoid arthritis (ra) is a progressive degenerative disorder that leads to joint destruction. available treatments only target the inflammatory component with minimal impact on joint repair. we recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (mctr), that display both immunoregulatory and tissue-protective activities. thus, we queried whether the production of these autacoids is disrupted in ra patients and whether they can be useful in treating joint inflammation and promoting joint repair. methods: using a highly phenotyped ra cohort we evaluated plasma mctr concentrations and correlated these to clinical markers of disease activity. to evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models. findings: herein, we observed that plasma mctr3 concentrations were negatively correlated with joint disease activity and severity in ra patients. evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that mctr3 reprograms monocytes to confer enduring joint protective properties. single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with mctr3-reprogrammed monocytes revealed a role for arginase-1 (arg-1) in mediating their joint reparative and pro-resolving activities. arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of mctr3-reprogrammed monocytes. interpretation: our findings demonstrate that circulating mctr3 levels are negatively correlated with disease in ra. when administered to mice in vivo, mctr3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. these activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses. funding: this work was supported by funding from the european research council (erc) under the european union's horizon 2020 research and innovation programme (grant no: 677542) and the barts charity (grant no: mgu0343) to j.d. j.d. is also supported by a sir henry dale fellowship jointly funded by the wellcome trust and the royal society (grant 107613/z/15/z). background rheumatoid arthritis (ra) is a progressive degenerative disorder that leads to joint destruction. available treatments only target the inflammatory component with minimal impact on joint repair. we recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (mctr), that display both immunoregulatory and tissue-protective activities. thus, we queried whether the production of these autacoids is disrupted in ra patients and whether they can be useful in treating joint inflammation and promoting joint repair. methods using a highly phenotyped ra cohort we evaluated plasma mctr concentrations and correlated these to clinical markers of disease activity. to evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models. findings herein, we observed that plasma mctr3 concentrations were negatively correlated with joint disease activity and severity in ra patients. evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that mctr3 reprograms monocytes to confer enduring joint protective properties. single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with mctr3-reprogrammed monocytes revealed a role for arginase-1 (arg-1) in mediating their joint reparative and pro-resolving activities. arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of mctr3-reprogrammed monocytes. interpretation our findings demonstrate that circulating mctr3 levels are negatively correlated with disease in ra. when administered to mice in vivo, mctr3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. these activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses. rheumatoid arthritis (ra) is a chronic inflammatory disorder characterized by dysregulated immune activation and unremitting inflammation. [1] [2] [3] [4] [5] this persistent inflammatory response is linked with a progressive destruction of joints, leading to substantial morbidity and a reduction in quality of life. significant progress has been made in the last few decades in both the early diagnosis and treatment of patients with ra. [1] [2] [3] [4] [5] in particular the development of biological drugs that target molecules linked with the propagation of arthritic inflammation, including tumour necrosis factor (tnf) and interleukin (il)-6, has transformed the management of patients with this otherwise debilitating condition. despite these major leaps forward in the treatment of patients with ra, available therapeutics only target the inflammatory component of the disease without rectifying the extensive damage that occurs within the joints. 6 furthermore, a significant portion of patients build resistance to many of these biological drugs with time, limiting their effectiveness. thus, there is a need for the development of therapeutics that not only limit arthritic inflammation but also promote joint repair. the immune system plays a central role in both the propagation of joint inflammation as well as the observed tissue destruction. [7] [8] [9] [10] [11] among the immune cells known to participate in both the onset and termination of rheumatoid arthritis are monocytes [10] [11] [12] findings made in experimental systems suggest that nonclassical monocytes contribute to the onset of inflammatory arthritis, whereas classical monocytes are linked with the resolution of joint disease. 10, 12 elegant investigations with human synovial tissues also suggest a role for monocyte-derived macrophages (mdm) in both the propagation and termination of human disease. single cell rna-sequencing (scrna-seq) analysis of synovial cells from patients with active ra identified a subset of macrophages that displays a pro-inflammatory phenotype and promoted fibroblast invasiveness. 13 on the other hand, scrna-seq analysis of synovial macrophages from ra patients in remission identified two macrophage subsets that were enriched in genes linked with the negative regulation of inflammation and produce higher amounts of the specialized pro-resolving mediator (spm) resolvin d1. 14 these observations are in line with recent findings detailing a link between disruptions in spm pathways, including spm formation and activity, and the onset and progression of inflammatory arthritis. [15] [16] [17] [18] spm are produced via the enzymatic conversion of essential fatty acids, primarily the omega-3 fatty acids eicosapentaenoic acid, n-3 docosapentaenoic acid and docosahexaenoic acid. 19, 20 these autacoids are classified into four main families termed as lipoxins, resolvins, protectins and maresins. studies investigating endogenous mechanisms activated during self-limited inflammation that expedite the resolution of inflammation and promote the repair of damaged tissues uncovered previously unappreciated peptide-lipid conjugated mediators. 21, 22 this family of mediators, named as maresin conjugates in tissue regeneration (mctr), is produced via the stereoselective conversion of the omega-3 fatty acid docosahexaenoic acid. 21, 23 mctrs share key defining biological activities with other spm, such as their ability to counter-regulate the production of inflammatory mediators, including eicosanoids and cytokines. 21, 22 they also limit activity of inflammatory molecules, where for example, in the lung they reduce ltd 4 -induced airway contraction and methacholineinduced hyperreactivity. 24 mctrs also exert potent tissue regenerative activities promoting tissue regeneration in surgically injured planaria. 21, 22 given that mctrs exert both inflammation-modulating and tissue-reparative activities in the present studies we evaluated whether mctr levels are linked with disease severity in patients with ra, finding a significant negative correlation between plasma mctr3 and joint disease activity. administration of mctr3 during peak of arthritic disease led to significant reduction in joint inflammation, as well as cartilage and bone protection. ex vivo treatment of monocytes from arthritic mice with mctr3 was sufficient to reprogram these cells to recapitulate the joint protective activities observed following the systemic administration of mctr3. these results suggest that mononuclear phagocytes play a key role in the observed anti-arthritic and reparative activities exerted by this autacoid. plasma samples were taken from ra patients who were dmards and steroid-naive, had symptoms duration <12 months, and fulfilled the acr/eular 2010 classification criteria for ra and recruited into the pathobiology of early arthritis cohort (peac http://www.peacmrc.mds.qmul.ac.uk). the peac cohort study was approved by the king's college hospital research ethics committee (rec 05/q0703/198). patients provided informed consent. peripheral blood samples were obtained from patients recruited at barts health nhs trust undergoing ultrasound (us)-guided synovial biopsy. 25 targeted lipid mediator profiling all samples were extracted using solid-phase extraction columns as in. 26 in brief, samples were placed in icecold methanol containing deuterated internal standards (d 8 -5s-hete, d 4 -ltb 4 , d 5 -lxa 4 , d 4 -pge 2 , d 5 -rvd2, d 5 -mar1, d 5 -mar2, d 5 -rvd3, d 4 -rve1, d 5 -17r-rvd1, d 5 -ltc 4 , d 5 -ltd 4 and d 5 -lte 4 ) representing each chromatographic region of identified lms. following protein precipitation (-20°c for a minimum of 45 min), samples were centrifuged and supernatants extracted using an extrahera system (biotage) using solid-phase extraction with isolute c18 500 mg columns (biotage). methyl formate and methanol fractions were collected, brought to dryness and resuspended in phase (methanol/water, 1:1, vol/vol) for injection on a shimadzu lc-20ad hplc and a shimadzu sil-20ac autoinjector, paired with a qtrap 5500 or qtrap 6500+ (sciex). in the analysis of mediators eluted in the methyl formate fraction, an agilent poroshell 120 ec-c18 column (100 mm £ 4.6 mm £ 2.7 mm) was kept at 50°c and mediators eluted using a mobile phase consisting of methanol/water/acetic acid of 20:80:0.01 (vol/vol/vol) that was ramped to 50:50:0.01 (vol/vol/vol) over 0.5 min and then to 80:20:0.01 (vol/vol/vol) from 2 min to 11 min, maintained till 14.5 min and then rapidly ramped to 98:2:0.01 (vol/vol/vol) for the next 0.1 min. this was subsequently maintained at 98:2:0.01 (vol/vol/vol) for 5.4 min, and the flow rate was maintained at 0.5 ml/min. qtrap 5500 was operated in negative ionization mode using a multiple reaction monitoring method. in the analysis of mediators eluted in the methanol fraction, an agilent poroshell 120 ec-c18 column (100 mm £ 4.6 mm £ 2.7 mm) was kept at 50°c and mediators eluted using a mobile phase consisting of methanol/water/acetic acid 55:45:0.5 (vol/vol/vol) over 5 min, that was ramped to 80:20:0.5 (vol/vol/vol) for 2 min, maintained at 80:20:0.5 (vol/vol/vol) for the successive 3 min and ramped to 98:2:0.5 (vol/vol/vol) over 3 min. this condition was kept for 3 min. qtrap 6500+ was operated in positive ionization mode using a multiple reaction monitoring method. each lipid mediator was identified using established criteria, including: 1 matching retention time to synthetic or authentic standards, 2 5 data points, and 3 signal to noise ratio 5. calibration curves were obtained for each mediator using synthetic compound mixtures at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg that gave linear calibration curves with an r 2 values of 0.98à0.99. to extract putrescine 50 ml of 200 ng/ml 1,4-diaminobutane-2,2,3,3-d 4 dihydrochloride (d 4 -putrescine) and 250 ml 6% tca were added to pulverized paws tissues, which were further homogenized using a percellys 24 homogenizer. the resultant suspension was incubated on ice for 1 hour, then centrifuged at 10 000 x g for 10 minutes at 4°c. the supernatant was then collected and levels of putrescine and d 4 -putrescine were determined using a shimadzu lc-20ad hplc and a shimadzu sil-20ac autoinjector, paired with a qtrap 5500 (sciex) operated in positive ionization mode using a multiple reaction monitoring method with the following transitions being used for the quantitation of putrescine à m/z 89>72 and d 4 -putrescine à m/z 97>76. an agilent poroshell 120 ec-c18 column (100 mm £ 4.6 mm £ 2.7 mm) kept at 30°c was used in the analysis of these molecules which were eluted using a mobile phase consisting of methanol/water/acetic acid 100:0:0.5 (vol/vol/vol) that was ramped to 0:100:0.5 (vol/vol/vol) over a 5 min period. 10-week-old c57bl/6 mice (charles river, uk), dba/1 mice (charles river, uk) and c57bl/6-ly5.1 mice (charles river, italy) were used in the reported studies. all animal experiments were performed strictly in accordance with united kingdom home office regulations (guidance on the operation of animals, scientific procedures act) and laboratory animal science association guidelines (guiding principles on good practice for animal welfare and ethical review bodies) and according to protocols detailed in a uk home office approved protocol (p998ab295). mice were kept in specific pathogen free housing, food and water were provided ad libitum and kept with a 12h light-dark cycle, with lights on between 7:00 h and 19:00h. all animals were randomized to treatment of vehicle group prior. glucose-6-phosphate isomerase (g6pi) peptide induced arthritis: antigen dba/1 mice were immunised with a g6pi emulsion (100 µl/mouse), prepared by sonication of 10 µg g6pi peptide (sequence: iwyincfgce-thaml; cambridge peptides ltd.) in 50 µl complete freund's adjuvant (cfa) and 50 µl dpbs à/à per mouse (schubert et al., 2004) , via intradermal injection at the base of the tail to initiate inflammatory arthritis. arthritic dba/1 mice were treated with 1 µg/mouse mctr3 or vehicle (dpbs à/à + 0.1 % etoh) on day 24, 26 and 28 intravenously (i.v.). paws were collected for microct analysis and flow cytometry on day 36. k/bxn serum induced arthritis: arthritogenic k/bxn serum (100 µl/mouse) was administered via intraperitoneal (i.p.) injection to c57bl/6 mice on day 0 and 2 to induce self-resolving inflammatory arthritis. 27 disease severity was evaluated using a 26-point arthritic scoring system and ankle and pad oedema was measured daily using callipers. 16 for femur head collection and bm cell isolations for in vitro cell cultures, mice were culled on day 5. otherwise, mice were administered a third k/ bxn serum injection on either day 8 or 9 to prolong inflammatory arthritis. mice were then treated i.v. with vehicle (dpbs à/à + 0.1 % etoh) or 1 µg/mouse mctr3 on day 10, 12 and 14 and on day 25, paws were collected for histology and flow cytometry and blood was collected for elisas. mctr3 was obtained via total organic synthesis as previously reported 28 and its concentration was determined using its uv absorbance characteristics (see figure s2 for compound validation). in separate experiments, arthritis was initiated and prolonged as detailed above. on day 12, mice were treated via i.v injection with 2£10 6 bm derived monocytes, obtained from arthritic c57bl/6 mice that were isolated and trained as detailed below. paws were collected for flow cytometry, single cell rna sequencing and histology on day 22. in other experiments, arthritis was initiated and prolonged as above, on day 12 they were treated with vehicle or 200 µg n v -hydroxy-nor-l-arginine (nor-noha), an arginase 1 inhibitor, administered via i.p. injections daily. on day 22 paws were collected for flow cytometry. bone marrow cells were collected from naive dba/1 mice, arthritic c57bl/6 mice on day 5 after the initial k/bxn injection or arthritic c57bl/6 mice on day 12 after the initiation of arthritis (see above). briefly, femurs, tibiae and humeri were placed in 70 % etoh and rinsed in dpbs à/à . the epiphysis was removed, and a 25g needle was used to flush the bone marrow with 2 ml dpbs à/à per bone. cells were dispersed gently with a 19g needle, filtered through a 70 µm strainer, centrifuged at 400 x g for 5 minutes at 4°c and suspended in dpbs +/+ . for monocyte adoptive transfer experiments, bone marrow-derived monocytes were isolated using easysep tm mouse monocyte isolation kit (stemcell) according to manufacturer's instructions. isolated monocytes from arthritic c57bl/6 mice were labelled with pkh67 red fluorescent cell linker kit (sigma), following manufacturer's instructions. monocytes were then incubated with either vehicle (dpbs +/+ + 0.01 % etoh) or 1 nm mctr3 for 90 min at 37°c. in separate experiments monocytes were first incubated with vehicle (dpbs +/+ + 0.1 % dmso) or 10 µm rg108 (sigma) for 15 min, prior to incubation with mctr3 (1nm) or vehicle (dpbs +/+ + 0.01 % etoh; 37°c). in other experiments bone marrow cells were isolated from bone long bones collected from arthritic mice 5 days after the initiation of arthritis and seeded into 10 cm dishes. these were then incubated at 37°c for 45 minutes in dpbs +/+ , the supernatant was removed and cells were washed with dpbs à/à to remove non-adherent cells. adherent cells were incubated with either 10 µm rg108 or a vehicle (dpbs +/+ + 0.1 % dmso) for 45 minutes in 5 ml dmem containing 1% penicillin and streptomycin (p/s), following which, 1 nm mctr3 or vehicle (dpbs +/+ + 0.1 % etoh) was added to the media. after 2 hours, an additional 5 ml dmem containing 1 % p/s and 0.2 % fbs (for a final concentration of 0.1 % fbs) was added and the cells were incubated at 37°c at 5 % co 2 for a further 22 hours. media was replaced with dmem containing 1 % p/s, 10 % fbs and 20 ng/ml murine gm-csf, and incubated for a further 4 days to allow for macrophage differentiation. subsequently, macrophages were detached using 5 mm edta in dpbs à/à and seeded, at 1.5£10 5 cells/well, into 24-well transwell plates in dmem containing 1 % p/s and 10 % fbs for co-incubations with femoral heads. in separate experiments, bm derived monocytes were treated as described above, and following the replacement of media with dmem articles containing 1 % p/s, 10 % fbs and 20 ng/ml murine gm-csf, monocytes were allowed to differentiate for a further 6 days. media was refreshed after 3 days. to evaluate the role of arg-1 in mediating the joint protective actions of monocyte derived macrophages, bone marrow monocytes were incubated with vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 in 5 ml dmem containing 1% p/s for 2 hours at 37°c at 5 % co 2 , after which an additional 5 ml dmem containing 1 % p/s and 0.2 % fbs (for a final concentration of 0.1 % fbs) was added and the cells were incubated for a further 22 hours. media was then replaced with dmem containing 1 % p/s, 10 % fbs and 20 ng/ml murine gm-csf and incubated for a further 2 days. adherent cells were detached with 5 mm edta in dpbs à/à and seeded into 24-well transwell plates, at 2£10 5 cells/ well. cells were then incubated in serum-free accell sirna delivery medium containing either 1 µm accell anti-mouse arg1 sirna smartpool or mouse control sirna (dharmacon) at 37°c at 5 % co 2 for 48 hours. cells were washed with dpbs à/à and dmem containing 1 % p/s and 10 % fbs was added to the cells for coincubations with femur heads. femur heads were collected as described in. 29 femur heads were removed from arthritic c57bl/6 mice, washed in 70 % etoh, then in dpbs à/à , and incubated in 200 µl pre-warmed serum-free dmem containing high glucose and 1% insulin-transferrin-selenium for 48 hours at 37°c and 5 % co 2 . the medium was replaced with dmem containing 10 % fbs and 10 ng/ml il-1b and femur heads were incubated for a further 72 hours. these were then co-incubated with mdm that were prepared as detailed above for 48 hours at 37°c and 5 % co 2 . tissues were then collected and fixed in 10 % neutral buffered formalin (nbf) for histology. mouse cross linked c-telopeptide of type-i collagen (ctxi; abbexa; 4 x dilution) was evaluated in plasma collected from arthritic c57bl/6 mice treated with vehicle or mctr3, as per manufacturer's instruction. the femur heads and joints were fixed in 10 % nbf, for 72 hours and decalcified in 10 % edta (w/v) in dpbs -/for 2 weeks with shaking. the decalcified tissue was then processed and embedded in paraffin and 4-micron sections were cut. slides were heated at 50°c for 30 minutes, incubated in histoclear twice, then twice in 100 % etoh for 5 minutes each. sections were washed in dh 2 o for 1 minute, air dried at room temperature, fixed in 4 % pfa for 5 minutes and washed in dpbs with on an orbital shaker twice for 5 minute intervals. for digestion of pepsin, slides were incubated in 0.02 % hcl for 7 minutes at 37°c, then for 20 minutes at 37°c in 3 mg/ml pepsin solution in 0.02 % hcl equilibrated to 37°c. this was washed twice in dpbs for 5 minutes on an orbital shaker, quenched by incubating in 50 nm ammonium chloride twice, each for 5 minutes on an orbital shaker. sections were washed as above and blocked in 20 % fbs in dpbs for 1 hour at room temperature. sections were incubated with primary mouse polyclonal anti-collagen type ii (merck millipore; 1:500 in 20% fbs in dpbs) for 1 hour at room temperature in the dark, washed 3 times in dpbs for 10 minutes and incubated with secondary af488 goat anti-mouse igg (1:400 in 20% fbs in dpbs) for 1 hour at room temperature. this was washed in pbs 3 times in the dark for 10 minutes each on an orbital shaker and incubated in the dark with efluoro570 anti-collagen x (1:200 in 20 % fbs in dpbs) at 4°c overnight. dpbs was used to wash the sections 3 times for 10 minutes each on an orbital shaker in the dark and slides were mounted with mowiol with dapi overnight. the siemens inveon ò pet/ct scanner (siemens preclinical solutions, knoxville, tn) with the inveon acquisition workplace software was used perform micro-ct scans of the arthritic dba mice knees at peak of disease on day 24 and following resolution of inflammatory arthritis, on day 35. all procedures were done in accordance with uk home office regulations. before scanning, the center offset and light/dark calibration was performed and a new workflow was created on the scanner. mice were anesthetised with 3-5% inhalation anaesthesia, which was reduced to and maintained at 1.5% during scanning, at a rate of 1.5l/min . mice were laid in prone position on a heating pad at 37°c to maintain body temperature during scanning. scanning was performed at a voltage of 70kv, using an x-ray current of 500 ma and at an exposure time of 2000ms/ projection for 360 projections. hounsfield correction was used for image reconstruction. to evaluate bone callus arthritic joints were collected 25 days after initiation of arthritis using k/bxn serum. samples were wrapped in plastic film prior to scanning to prevent drying and scanned using a skyscan 1172f (bruker, kontich, belgium). the x-ray source was operated at 50kv and 200µa, using an aluminium 0.5mm filter and an exposure time 960ms using a voxel size of 5µm. projection images were reconstructed into tomograms using nrecon 1.7.3.1 (bruker, kontich, belgium) and repositioned using dataviewer 1.5.4 (bruker, kontich, belgium) with bone analysis performed in ctan 1.18.4 (bruker, kontich, belgium). volume rendered 3d visualisations were created using ctvox 3.3 (bruker kontich, belgium). hind paw tissue digestion to isolate leukocytes from arthritic joints was performed as described in. 27 briefly, following the removal of skin and muscle, the hind paw was incubated in 15 ml digestion buffer (rpmi containing 0.5 mg/ml collagenase d and 40 mg/ml dnase) at 37°c for 30 minutes with vigorous agitation. liberated cells within the digestion buffer were passed through a 70 mm strainer into 10 ml 10 % fbs in rmpi on ice. the digestion incubation was repeated and the cell suspension volume was made up to 50 ml with 10 % fbs in rmpi. cells were centrifuged at 400 x g for 10 minutes at 4°c and suspended in dpbs for flow cytometry. tissue was homogenised using a a percellys 24 homogenizer and an rneasy mini kit (qiagen) was used to extract rna, as per manufacturers instruction. cdna synthesis was achieved using superscript iii reverse transcriptase (invitrogen), as per manufacturers instruction. quantitect primer assays (qiagen) for mouse kc, il-6, tnf-a, mmp7, fra-1, dkk1, lef1 and sfrp-1 were used with sybr green i fluorescent dye for realtime pcr (qrt-pcr) evaluation with the stepone tm real-time pcr system (thermofisher). target gene expression was expressed as a value relative to actb expression. isolated cells from arthritic paws were incubated with the following fluorescently conjugated antibodies: pe-cy7 mouse anti-mouse cd64 (biolegend), percp-cy5.5 mouse anti-mouse cd64 (biolegend), pe-cy5 rat antimouse cd11b (biolegend), pe-texas red rat anti-mouse cd11b (biolegend), bv421 rat anti-mouse f4/80 (biolegend), apc/cy-7 rat anti-mouse f4/80 (biolegend), bv711 rat anti-mouse mertk (biolegend) and/or pe-cy7 armenian hamster anti-mouse cd36 (biolegend) at a dilution of 1:100 in dpbs à/à with 0.02 % bsa for 30 minutes at 4°c. cells were incubated with bd fixation/permeabilisation buffer solution and then with bd permeabilisation buffer, each for 20 minutes at room temperature. intracellular staining was performed by incubating cells with bv 421 anti-mouse tgf-b1 (1:50 dilution, biolegend), apc rat anti-mouse inos (1:100 dilution; biolegend), pe sheep anti-mouse arginase 1 (1:50 dilution, r&d) and polyclonal rabbit anti-dbl (1:100 dilution, cell signaling technologies) for 30 minutes at 4°c. rabbit anti-dbl was conjugated with percp/cy5.5 using abcam's percp/cy5.5 conjugation kit, as per manufacturer's instructions. apc-cy7 rat anti-mouse ly6g (1:50 dilution, biolegend) was also incubated with the cells following permeabilisation for intracellular staining. cells were incubated with trustain x to quench non-specific binding. multiparameter analysis was performed with lsr fortessa cell analyser (bd biosciences) and analysed using flowjo (tree star inc., v10). following paw tissue digestion, as described above, live cells were obtained using easysep tm dead cell removal (annexin v) kit (stem cell) according to manufacturer's instruction. cells were incubated with af700 cd45 (biolegend) at a dilution of 1:100 in dpbs à/à with 0.02 % bsa for 30 minutes at 4°c. nonspecific staining was blocked with trustain x. cells were suspended in dpbs à/à with 0.02 % bsa and the bd facs aria ii was used sort for cd45 positive cells, which were collected in dpbs containing 0.1 % bsa for single cell sequencing. 8 single cell suspensions were prepared (one per mouse) and were assessed for cell number using the luna fl automated cell counter (logos biosystems, south korea). cells appeared intact and well distributed with an average count of 148 cells/µl. an equivalent volume of 4000 cells was loaded to the 10x chromium tm single cell a chip (pn-1000009) using the chromium tm 3' library & gel bead kit v2 (pn-120267) as described in the manufacturers user guide (10x genomics, california, usa). gems were recovered from the chip and appeared opaque and uniform in colour. 14 cycles of cdna amplification were performed on the purified gem-rt product, and cdna was examined for quality using the agilent 2200 tapestation with the high-sensitivity d5000 screentape and reagents (agilent technologies, waldbronn, germany), and the qubit ò 2.0 fluorometer and qubit dsdna hs assay kit (life technologies, california, usa). 35 ml of cdna were used to prepare the 10£3'rna libraries and 12 cycles were used for sample index pcr. final cleaned libraries were quantified using the qubit ò 2.0 fluorometer and qubit dsdna hs assay kit and average fragment size checked using the agilent d1000 screentape and reagents. the final pooled library was run on a nextseq500 high-output v2.5 150-cycle kit with a 26 [8] 98 cycle configuration to generate 400 million read pairs in total. the 10x genomics cell ranger pipeline was used to analyse the raw sequence data generated by the single cell rna-seq (10x genomics; https://support.10xgenom ics.com/single-cell-gene-expression/software/). shortly, the pipeline demultiplexes raw base call files generated by illumina sequencers in fastq files and then aligns, filters, and counts (barcode and umi) the reads. the alignment was done using star (https://github.com/ alexdobin/star) and the mus musculus genome (grcm38) 30 as the reference genome. the samples for each group were aggregated (to have a normalized set of cells per group) using "cell ranger aggr" function. using the seurat r package 31 quality control (qc) steps were performance to filter out low quality cells. briefly, cells that contained fewer than 250 expressed genes (low-quality cells or empty droplets), more than 3000 expressed genes (cell doublets), more than 5% mitochondrial transcripts (indication of mitochondrial damage), and less than 500 umi counts were removed. we considered genes as detectable if they were expresses in at least the number of cells that represent 20 % of smallest cell type population. after qc step, expression of each gene was normalized by total expression, then multiplied by a scale factor (1000) and log transformed. for dimensional reduction and clustering, highly variable genes were identified, and principal component analysis (pca) was performance to identify relevant principal components (pc) based on how much the standard deviation of the data was explained by them. pcs were used for data clustering (unsupervised graph-based clustering with resolution 0.5) and dimensionality reduction using the uniform manifold approximation and projection (umap) analysis. data visualization was performance using the "dimplot" function from seurat r package. before differential gene expression analysis, distinct leukocyte subsets were identified based on the expression of specific markers (table s1 -3) and comparing the most expressed genes from each cluster with single cell expression atlas database (https://www.ebi.ac.uk/ gxa/sc/home). differential gene expression analysis was performed using the likelihood ratio test from edge r package (https://bioconductor.org/packages/release/bioc/html/ edger.html). statistical significance was considered with an adjusted (benjamini-hochberg procedure correction) p-value < 0.05. pathway analysis was performed uploading the differentially expressed genes in networkanalyst 3.0 (ora enrichment visualization tab) 32 and searching for the enriched pathways from kegg 33 and string 34 (p value < 0.05, fisher exact test followed by multiple comparison correction using benjamini-hochberg procedure) databases. for evaluation of signalling pathways activated by mctr3, monocytes were isolated from peripheral blood of healthy volunteers, incubated with gm-csf (20ng/ ml, in rpmi containing 10% human serum) for 7 days and then incubated with mctr3 (1nm, in dpbs +/+ ). phosphoproteomics experiments were performed using mass spectrometry as reported. 35, 36 in brief, cells were lysed in 8m urea buffer and supplemented with phosphatase inhibitors (10 mm na 3 vo 4 , 100 mm b-glycerol phosphate and 25 mm na 2 h 2 p 2 o 7 (sigma)). proteins were digested into peptides using trypsin as previously described. 37, 38 phosphopeptides were enriched from total peptides by tio2 chromatography essentially as reported previously 39 ). dried phosphopeptides were dissolved in 0.1% tfa and analysed by nanoflow ultimate 3000 rsl nano instrument which was coupled on-line to a q exactive plus mass spectrometer (thermo fisher scientific). gradient elution was from 3% to 35% buffer b in 120 min at a flow rate 300 nl/min with buffer a being used to balance the mobile phase (buffer a was 0.1% formic acid in water and b was 0.1% formic acid in acetonitrile). the spray voltage was 1.95 kv and the capillary temperature was set to 255 c. the q-exactive plus was operated in data dependent mode with one survey ms scan followed by 15 ms/ms scans. the full scans were acquired in the mass analyser at 375-1500m/z with the resolution of 70 000, and the ms/ms scans were obtained with a resolution of 17 500. ms raw files were converted into mascot generic format using mascot distiller (version 2.5.1) and searched against the swissprot database (release december 2015) restricted to human entries using the mascot search daemon (version 2.5.0). allowed mass windows were 10 ppm and 25 mmu for parent and fragment mass to charge values, respectively. variable modifications included in searches were oxidation of methionine, pyro-glu (n-term) and phosphorylation of serine, threonine and tyrosine. graphpad prism 8 (graphpad software, la jolla, ca, usa) was used to assess differences between the groups. spearman test was used to evaluate the correlation between the lipid mediators and disease parameters. to evaluate difference between experimental groups for group sizes less then 8 we used nonparametric tests. these include one-sample wilcoxon signed rank test for normalized data between 2 groups, mann-whitney u test between 2 groups, a one-way anova between 3 groups or two-way anova for time course analysis. no statistical analysis was performed to determine the sample size for in vivo experiments. sample size was based on effect size observed in published studies evaluating the biological activities of other spm 27, 40 and pilot results whereby the ability of mctr3 to reduce both clinical scores and oedema was evaluated and found to be comparable to that in published studies. therefore, we elected to employ similar sample sizes as those used in published studies. the experimenter was not blinded to treatment allocation. in select experiments mice that did not develop disease were excluded from further analysis. the funders played no part in the design, data collection, data analyses, interpretation, writing of report or in the decision to publish the results. circulating lipid mediator concentrations are linked with peripheral organ disease activity, since these autacoids influence leukocyte recruitment and activation status. [41] [42] [43] [44] [45] to establish whether there was a link between disease activity and mctr concentrations in ra patients we investigated plasma levels of these molecules in relation to both systemic and joint disease activity markers. plasma was obtained from the pathobiology of early arthritis cohort (peac), which is a highly phenotyped patient cohort of disease modifying anti-rheumatic drugs-naÿve patients 25 (see table 1 for patient characteristics). using lipid mediator profiling, we identified all three mctrs in plasma from these patients. notably, while concentrations of all three mediators were observed to display a negative correlation with joint disease activity (i.e. das28 scores), plasma c-reactive protein and erythrocyte sedimentation rate, only correlations between mctr3 and these parameters were statically significant ( table 2) . we also evaluated whether plasma concentrations of these mediators were linked with other clinical features of ra. whilst plasma concentrations were not linked with joint disease pathotype or responsiveness to dmard therapy, we observe a significant reduction in plasma mctr3 concentrations in patients with erosive disease when compared to those that did not display signs of joint erosion ( figure s1 ). having observed a significant relationship between mctr3 concentrations and disease activity we next questioned whether pharmacological administration of mctr3 would modulate joint disease progression and severity in experimental arthritis. for this purpose, we employed a serum transfer model of inflammatory arthritis, which relies on the activation of the innate immune system replicating the effector phase of rheumatoid arthritis. 46 administration of mctr3, obtained via stereoselective total organic synthesis 28 (see figure s2 for physical characterization), immediately after disease onset conferred protection against joint inflammation as observed by a significant reduction in clinical scores and improvements in histological markers of disease. this included a decrease in leukocyte infiltration, and increased safranin staining, a measure of glycosaminoglycan content in the cartilage ( figure s3a-c) . given the roles that lipid mediators have in both propagation (e.g. prostaglandins and leukotrienes) and resolution of joint inflammation (e.g. spm) we next evaluated whether mctr3 administration also regulated the joint lipid mediator profile. using partial least square discriminant analysis, which produces a regression model built using concentrations of lipid mediators differently expressed between the two groups, we found a shift in joint lipid mediator concentrations in mice treated with mctr3. this shift was linked with a downregulation of pro-inflammatory and nociceptive eicosanoids including pge 2 and pgf 2a and an upregulation of pro-resolving and anti-nociceptive mediators such as mar1 and pdx ( figure s3d ,e and table s4 ). we next tested whether mctr3 also displayed joint protective activities when administrated later in the disease course. for this purpose, we used a model of sustained joint inflammation. 47 mctr3 was administered 10 days after disease onset and joint inflammation was evaluated throughout the disease course. here we found that treatment of mice with mctr3 accelerated the resolution of joint inflammation as demonstrated by a shortening of the resolution interval from »9 days to »5 days, a significant reduction in clinical scores and a marked reduction in joint oedema (figure 1a,b) . histological evaluation of joints collected from these mice lymphoid (28), fibroid (28) demonstrated that mctr3 reduced all the parameters evaluated, significantly reducing both leukocyte infiltration and cartilage damage ( figure s4 ). mononuclear phagocytes, in particular mdm, play a central role in the propagation and termination of inflammation, 10,12 as well as tissue repair and regeneration. 48, 49 therefore, we next evaluated whether mctr3 governed mdm phenotype in arthritic joints. flow-cytometric evaluation of phenotypic markers in cells isolated from mice treated with mctr3 demonstrated a marked shift in phenotype as highlighted by a shift in the cluster representing cells obtained from these mice when compared with cells isolated from mice treated with vehicle alone (figure 1c, figure s5 ). to evaluate which of the phenotypic markers were responsible for this shift in macrophage markers we evaluated the variable in importance (vip) scores, whereby a vip score >1 identifies those variables that contribute to the observed separation between the two groups. this demonstrated that the shift in phenotype was primarily linked with the upregulation of two markers in cells from mctr3 treated mice, namely arginase (arg)-1 and interleukin (il)-10 ( figure 1d) . we next assessed whether the protective activities of mctr3 were retained in a model of adaptive immune system-driven arthritis using the glucose-6-phosphate isomerase peptide driven model of inflammatory arthritis. 50 here, administration of mctr3 during the course of arthritic inflammation also led to a reduction in joint inflammation as measured by a decrease in both clinical scores and joint oedema (figure 1e,f) . notably, the mdm-directed activities of mctr3 were retained in this model, as demonstrated by the marked shift in the macrophage phenotype observed in cells isolated from joints of mctr3 treated mice when compared with those isolated from joints of vehicle treated mice (figure 1g ). this shift in phenotype was linked with an upregulation of three phenotypic markers, including arg-1 and cd11b, in cells obtained from joints of mctr3 treated mice (figure 1h ). taken together these findings suggest that mctr3 treatment alters joint mdm phenotype and reduces arthritic inflammation. chronic inflammation in ra is associated with both cartilage and bone degradation which is the main cause of debilitation in patients with ra. 5 thus, we next questioned whether mctr3, in addition to reducing joint inflammation and cartilage damage also promoted joint repair. to address this question, we investigated whether mctr3 regulated cartilage repair in arthritic mice. for this purpose, we used safranin o-staining to evaluate glycosaminoglycan content in joints from arthritic mice. here we observed higher safranin ostaining in joints from mctr3 treated mice when compared with vehicle treated mice in both mice challenged with k/bxn serum and g6pi peptide (figure 2a and figure s6a, b) . furthermore, immunohistochemical staining of joints from mctr3 treated mice demonstrated an increase in the expression of both collagen 2, the principal molecular component in mammalian cartilage, 51 and that of collagen x, which is expressed in the calcified zone of cartilage that interfaces with bone 51 in plasma was collected from a patient cohort of dmard naive patients (n= 99 patients) and concentrations for mctr1, mctr2 and mctr3 were established using lipid mediator profiling (see methods for details). (concentrations for each of these meditators were then correlated with das28 scores as well as plasma c-reactive protein (crp) and erythrocyte sedimentation rate (esr) using spearman correlation, where bold was used to represent significant p values). mice challenged with k/bxn serum and treated with mctr3 (figure 2b-d) . thus, these results demonstrate that mctr3 displays cartilage-protective activities in inflammatory arthritis. since one of the main debilitating features of arthritis is rapid bone remodelling, we next determined whether the joint protective actions of mctr3 extended to the bone. we first evaluated whether mctr3 treatment in k/bxn induced arthritis limited pathological bone formation by evaluating callus cover in long bones from arthritic mice. analysis using microct demonstrated smaller bone calluses on mctr3 treated mice, as measured by the assessment of the total volume and surface area occupied by the callus (figure 2e-h) . notably, these changes were linked with a significant increase in overall surface to volume ratio in the callus, a marker of callus mineralization and therefore bone integrity (figure 2i ). this decrease in callus size was linked with a reduction in plasma concentrations of c-terminal telopeptide of type 1 collagen (ctx-1), a marker of bone resorption, in mctr3 treated mice (figure 2j ). thereby these findings suggest that mctr3 limits bone damage in inflammatory arthritis. given that mctr3 displayed joint protective activities in the g6pi model of chronic arthritic inflammation which is linked with marked bone resorption 52 we next queried whether mctr3 was also able to reverse this process. here, we used microct analysis to investigate bone volume in arthritic joints, comparing bone volumes on day 24, prior to mctr3 treatment, to those at day 35. this analysis demonstrated that bone volume in vehicle treated arthritic mice was reduced, in line with the sustained disease activity (figure 1e,f) . whereas, bone volume in mctr3 treated mice was increased ( figure s6c, d) . these findings indicate that mctr3 reduces the increased bone and cartilage turnover characteristic of arthritic inflammation, thereby suggesting that this autacoid activates protective mechanisms to improve both cartilage and bone integrity in inflammatory arthritis. having observed that mctr3 treatment led to a shift in mdm phenotype, a reduction in disease severity and increased joint repair, we next queried whether the joint protective actions of mctr3 were linked with the reprogramming of circulating monocytes to yield mdm with pro-resolving and tissue reparative properties. to test this hypothesis, we evaluated whether ex vivo treated monocytes from arthritic mice recapitulated the joint protective activities of mctr3. here, we incubated bone-marrow derived monocytes from donor arthritic mice with 1 nm of mctr3 (mctr3-reprogrammed monocytes) or vehicle and after 90 minutes cells were washed and administered to arthritic mice. this concentration was selected since we previously observed that it optimally regulated both tissue regeneration in planaria and macrophage efferocytosis. 22 we then treated mice with 1£10 6 monocytes, a dose that was selected based on published literature demonstrating the ability of mononuclear phagocytes to influence disease activity. 53 in mice administered the mctr3-reprogrammed monocytes we observed a reduction in disease severity, as demonstrated by a significant reduction in clinical scores and oedema, when compared with mice treated with monocytes incubated with vehicle alone (figure 3a , b). the protective activities exerted by mctr3-reprogrammed monocytes were observed at a histological level, where h&e staining revealed a significant reduction in leukocyte infiltration into the inflamed paws in mice treated with mctr3-reprogrammed monocytes (figure 3c ). we also observed that mctr3-reprogrammed monocytes regulated joint lipid mediator concentrations as observed by a shift in the cluster representing lipid mediator profiles obtained from joints of mice treated with these cells compared with mice treated with monocytes incubated with vehicle only (figure 3d) . notably, assessment of the top 15 mediators differentially regulated between the two groups demonstrated a marked upregulation of several joint protective spm, including rvd1, in paws from mice receiving mctr3-reprogrammed monocytes suggesting that these cells exert potent pro-resolving activities (figure 3e, table s5 ). we next evaluated whether mctr3-reprogrammned monocytes regulated tissue repair in arthritic mice. for this purpose, we assessed glycosaminoglycan content using safranin-o staining in articular cartilage. this analysis demonstrated a significant increase in safranin-o staining in mice treated with mctr3-reprogrammed monocytes when compared with mice that were treated with monocytes alone (figure 4a ). this increase in cartilage cover was linked with a significant increase in the expression of collagen 2 and collagen x in mice treated with mctr3-reprogrammed monocytes (figure 4b,c) . to evaluate the mechanisms that lead to both the reduction in inflammatory arthritis and the upregulation of reparative mechanisms, we next evaluated the expression of molecules known to be involved in the propagation of inflammation and in the regulation of joint repair. we first evaluated the expression of kc, the murine homologue of il-8, il-6, tumour necrosis factor (tnf)-a, matrix metalloproteinase (mmp) 7 and fos-related antigen (fra)-1. 54 while mmp7 and il-6 expression in arthritic paws from both groups was essentially similar, the expression of kc, tnf-a and fra-1 was decreased in arthritic paws from mice treated with mctr3-reprogrammed monocytes, reaching statistical significance for tnf-a (figure 4d-h) . having observed a significant regulation of tnf-a in mice receiving the reprogrammed monocytes, we next evaluated the expression of downstream targets of tnf-a which are known to regulate the wnt signalling pathway, a key pathway in both bone and cartilage maintenance. 55, 56 for this purpose, we assessed the expression of dickkopf (dkk)-1, lymphoid enhancer factor (lef)-1, 57 and secreted frizzled-related protein (sfrp)-1. 58 this analysis demonstrated that while lef-1 and sfrp-1 expression was essentially similar between the two groups, dkk-1 expression was significantly downregulated in mice treated with mctr3-reprogrammed monocytes when compared with mice receiving monocytes alone (figure 4i-k) . together these findings indicate that mctr3-reprogrammed monocytes activate reparative mechanisms linked with both bone and cartilage repair. we next sought to determine the mechanism(s) by which mctr3 elicited its protective actions. we first evaluated whether mctr3 reprogramming modulated the ability of these cells to migrate and/or be retained in the arthritic joints. for this purpose, we labelled monocytes with the cell membrane-binding fluorescent dye pkh67 prior to reinjection into arthritic mice and then evaluated their recruitment into the hind leg joints of arthritic mice using flow cytometry. this analysis demonstrated that »2% of the total number of monocytes administered were recoverable from these tissues after 10 days. notably, there was no significant difference in the relative abundance of pkh67+ macrophages found in these tissues between the two groups. these findings suggest that despite the transferred monocytes representing a relatively small subset of total cells in the arthritic joints they display potent antiinflammatory and reparative activities. they also suggest that mctr3-reprogramming does not influence the ability of monocytes to migrate and/or differentiate into macrophages within the arthritic joints and therefore the observed differences are likely to arise from a change in the phenotype of these cells. recent studies demonstrate that changes in the epigenetic landscape of innate immune cells, including monocytes and macrophages, leads to their long term reprograming. 59 having observed that short term incubation of monocytes with mctr3 led to long term protective actions in arthritis we next queried whether this was at least in part linked with the regulation of the epigenetic landscape of the cells. given the central role that dna methyltransferases 59, 60 play in this process we next tested whether inhibition of these enzymes would reverse the protective activities of mctr3-reprogrammed monocytes. indeed, while disease severity was significantly reduced in mice administered mctr3reprogrammed monocytes, incubation of these cells with a dna methyltransferase inhibitor (rg108), abolished the protective actions of mctr3 as observed by a decrease in the ability of these cells to regulate joint inflammation (figure 5a,b) . thus, these findings indicate that mctr3 regulates circulating monocyte responses to limit joint inflammation and promote joint repair in a dna methyltransferase-dependent manner. to further explore the mechanism activated in mctr3-reprogrammed monocytes that contribute to the observed protective actions, we next incubated monocytes with mctr3 as detailed above and administered them to arthritic mice. after 10 days we collected joints, sorted the leukocytes and subjected these cells to single cell rna sequencing. assessment of transcript expression differences between cells isolated from mice receiving mctr3-reprogrammed monocytes and those from mice receiving monocytes incubated with vehicle demonstrated that of the different cell subsets identified, the biggest changes in transcript levels were observed in mdm with 41 differentially regulated genes (figure 5c ,d, figure s7 and table s6 ). notably, out of these differentially expressed genes, arginase-1 (arg-1) was the gene that was upregulated to the greatest extent in mdm isolated from mice receiving mctr3-reprogrammed monocytes (figure 5d ). network analysis of genes that were found to be differentially regulated in mdm from mctr3-reprogrammed monocytes demonstrated a differential regulation of genes linked with several processes involved in joint repair, including k/bxn serum (100 µl, i.p.) was administered to c57bl/6 mice on day 0, 2 and 9 to induce and prolong inflammatory arthritis and, on day 12, mice were treated i.v. with 2£10 6 monocytes isolated from arthritic mice and incubated with either vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 90 min. (a-c) on day 22 hind paws were harvested, fixed, and stained to evaluate a) proteoglycan content using safranin-o staining; (b) col 2 and (c) col x expression was evaluated using immunofluorescence. left panels present representative images from each experimental group, right panels provide a quantitative evaluation of the staining. results are mean § sem osteoclast differentiation and arginine and proline metabolism (figure 5e ). we next evaluated the signaling pathways activated by mctr3 using a phosphoproteomic approach. gene ontology analysis of proteins found to be differentially phosphorylated in mononuclear phagocytes incubated with mctr3 versus those incubated with vehicle alone demonstrated a marked regulation of proteins involved in post-transcriptional regulation and protein translation by mctr3 (figure 5f and table s7 ). this regulation was also observed when using the kyoto encyclopaedia of genes and genomes pathway database that highlighted an enrichment of spliceosome linked proteins as well as proteins involved in mrna surveillance by mctr3 (figure 5g and table s7 ). in these studies, we also found that mctr3 regulated the phosphorylation status of several proteins involved in both epigenetic and chromatin modification, including that of methylases histone-lysine n-methyltransferase setd2 as well as the deacetylases histone deacetylase 1 and histone deacetylase 2 (table s7) . recent studies identified several macrophage subsets in arthritic joints that are linked with different aspects of disease onset/propagation and resolution of joint disease. 14,61 therefore, we evaluated whether the transcriptional changes observed in macrophages from mice receiving mctr3-reprogrammed monocyte were linked with the reprogramming of a specific macrophage population. using the taxonomy published by culemann and colleagues 61 we identified three macrophage populations in our single cell rnaseq dataset. these were mhcii + interstitial macrophages, relm-a + interstitial macrophages, and cx 3 cr1 + lining macrophages, with the latter cells being the most abundant macrophage population in this dataset ( figure s8a) . assessment of the overall number of genes found to be differentially regulated in each of these subsets demonstrated that the largest changes in gene expression were in cx 3 cr1 + lining macrophages. these included the upregulation of arg-1 in cells isolated from arthritic joints of mice that received mctr3-reprogrammed monocytes ( figure s8b-e) . taken together these findings indicate that mctr3 promotes the reprograming of mononuclear phagocytes via the regulation of epigenetic programs and protein translation to facilitate the termination of inflammation and joint protection. they also support the hypothesis that mctr3-reprogrammed monocytes recruited into arthritic joints exert their activities via the upregulation of tissue protective pathways. to evaluate this hypothesis further, we employed an organ culture system whereby monocytes were obtained from the bone marrows of arthritic mice and incubated with or without mctr3 in the presence or absence of a dna methyltransferrase inhibitor. these cells where then differentiated to macrophages for 5 days using gm-csf as the agonist given that this was recently linked with macrophage differentiation in joints from ra patients. 62 we then incubated arthritic femur heads with these cells for 2 days and assessed their glycosaminoglycan expression. assessment of safranin-o staining demonstrated higher glycosaminoglycan content in femur heads incubated with mdm obtained from mctr3-reprogrammed monocytes (figure 6a ). of note, inhibition of methyltransferase activity reversed these protective actions of mctr3-reprogrammed monocytes (figure 6a) . thereby, these findings lend support to the hypothesis that mctr3 reprograms monocyte responses resulting in mdm that display joint protective activities. recent studies demonstrate that arg-1 exerts potent joint protective activities in inflammatory arthritis. 54, 63 having observed an upregulation of arg-1 in joint macrophages from mice treated with mctr3 ( figure 1 ) and those treated with mctr3-reprogrammed monocytes ( figure 5 ) we next questioned whether arg-1 was responsible for the observed anti-arthritic properties of these cells. for this purpose, we repeated the in vivo experiments detailed above, this time labelling the monocytes isolated from arthritic mice with a fluorescent membrane dye, pkh67, to differentiate them from endogenous monocytes. after 10 days we harvested the paws, liberated cells and assessed the expression of arg-1 in pkh67 + mdm. here we observed a significant increase in arg-1 expression in pkh67 + mdm from mice that received mctr3-reprogrammed monocytes when compared with those that received monocytes incubated with vehicle alone (figure 6b ). this observation was also replicated in mdm differentiated in vitro from mctr3-reprogrammed monocytes and previously found to exert cartilage protective activities in our organ culture system (figure 6c) . notably, incubation of monocytes with a methyltransferase inhibitor reversed the ability of mctr3 to upregulate this joint protective enzyme both in vitro and in vivo (figure 6c,d) . intriguingly, the regulation of arg-1 was found to be a specific response elicited by mctr3 in these monocytes, given that inos expression in these cells, an m1-lineage associated marker, was unaffected by mctr3 (figure 6e ). efferocytosis is a key step in the resolution of inflammation and is linked with initiation of reparative responses. 21,64 therefore, we next evaluated whether mctr3 reprogramming enhanced the ability of joint and expressed as percent change vs monocyte group. n = 7-8 mice per group. (statistical differences were evaluated using mann whitney-u test). (d-k) hind paws were also collected on day 22 to evaluate the expression of the indicated genes using quantitative realtime pcr. results are from n = 3-5 mice per group. (statistical differences were evaluated using mann whitney-u test). (c-e) k/bxn serum (100 µl, i.p.) was administered to mice on days 0, 2 and 9 to induce and prolong inflammatory arthritis. on day 12, mice were treated i.v. with 2£10 6 monocytes isolated from arthritic mice that were previously incubated with either vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 90 min. cells were isolated from paw joints on day 22, sorted for cd45 + cells and single cell rna sequencing was performed. c) umap plots of clusters obtained from isolated leukocyte populations, (d) volcano plot highlighted differentially regulated genes in the mdm population (inset) relative gene regulation for arg-1. (statistical differences macrophage to upregulate the clearance of apoptotic cells. flow cytometric analysis demonstrated a significant increase in the levels of macrophages carrying apoptotic neutrophils, as shown by an increase in the number of macrophages expressing the neutrophil marker ly6g (figure 6f ). these results were coupled with a significant increase in the expression of the efferocytosis receptor mertk (figure 6g ). since we observed an increase in both arg-1 and mertk we next queried whether the downstream product of arg-1, putrescine, which is linked with the regulation of mertk expression and efferocytosis in macrophages, 65 was also increased in mice receiving mctr3-reprogrammed monocytes. mass spectrometric analysis demonstrated an upregulation of putrescine in joints from mice receiving mctr3-reprogrammed monocytes when compared with those that received monocytes incubated with vehicle alone, although this increase did not reach statistical significance (figure 6h ). to further investigate the role of arg-1 in mediating the antiarthritic and reparative activities of mctr3reprogrammed monocytes we used an sirna approach to knockdown the expression of this enzyme in mdm and then evaluated the ability of these cells to promote cartilage repair using the organ culture system described above. here we found that while mdm obtained from mctr3-reprogrammed monocytes and transfected with a control sirna markedly increased glycosaminoglycan content in arthritic femur heads, transfection of cells with sirna to arg-1, significantly abrogated the cartilage protective actions of these cells (figure 7a,b) . we next tested whether inhibition of arg-1 activity in vivo would also reverse the protective actions of mctr3reprogrammed monocytes. for this purpose, we treated mice that received mctr3-reprogrammed monocytes with the arg-1 inhibitor n v -hydroxy-nor-ʟ-arginine (nor-noha) and assessed joint disease activity. here we found that as observed in previous experiments, administration of mctr3-reprogrammed monocytes led to a reduction in both clinical scores and joint oedema, when compared with those mice that received monocytes incubated with vehicle alone. of note treatment of mice with the arg-1 inhibitor reversed these joint protective actions of mctr3-reprogrammed monocytes as observed by an increase in clinical scores and oedema (figure 7c-d) . furthermore, inhibition of arg-1 activity also reversed the upregulation of the rac1 guanosine triphosphate (gtp)-exchange factor dbl, an enzyme implicated in mediating the pro-resolving actions of arg-1 in mdm 65 and tgf-b1, a morphogen involved in bone and cartilage maintenance and repair (figure 7e , f). 66 together these findings support a role for arg-1 in mediating the joint protective activities of mctr3reprogrammed monocytes. despite the notion that joint damage in patients with ra leads to significant morbidity, current therapeutic approaches in ra are ineffective at activating joint repair programs. in the present studies we found that mctr3 concentrations were negatively correlated with markers of both systemic and joint inflammation in patients with ra. systemic administration of mctr3 to arthritic mice not only accelerated the resolution of joint inflammation but also activated joint reparative programs. assessment of the cellular mechanisms involved in the observed protective activities demonstrated that mctr3 reprogrammed monocytes upregulated a number of tissue protective mechanisms, including arg-1 expression. intravenous administration of mctr3reprogrammed monocytes recapitulated the protective activities of systemic mctr3 administration. inhibition of dna methyltransferases or arg-1 abrogated both the anti-inflammatory and joint protective actions of mctr3 during inflammatory arthritis. together these findings suggest, that mctr3 reprograms circulating monocytes that when recruited into inflamed joints, differentiate into macrophages with potent anti-inflammatory and tissue reparative activities. unremitting inflammation is a key component in the destruction of joint tissues. in many patients this leads to severe deformation of the articular bones. whilst such deformations in large articular bones, such as hips and knees can be rectified via arthroscopic surgery, smaller joints such as those in the fingers cannot be rectified using these approaches resulting is severe disability in many patients with ra. [1] [2] [3] [4] [5] 48, 67 to date, the only therapeutics that impinge on this process of joint destruction are anti-tnf therapies which have been found to limit the activation of synovial cells and the progression of tissue destruction. 3, 4 nonetheless, these therapeutics do not activate reparative process, and therefore any damage that occurs, especially in those patients with advanced bone and joint destruction are likely to be permanent. furthermore, not all patients treated with anti-tnf therapies go into remission, were evaluated using mann whitney test). (e) the gene network analysis for genes that were found to be differentially regulated in mdm from mice receiving mctr3-reprogrammed monocytes when compared with mdm receiving monocytes incubated with vehicle. results are from n = 4 mice per group. (f-g) monocytes were isolated from human healthy volunteers, these cells were then incubated with gm-csf (7 days, 37°c) then with either vehicle or mctr3 (1nm, 37°c). cells were lysed and the phosphoproteome determined using mass spectrometry. (f) go biological pathway analysis and (g) kegg pathway analysis for proteins found to be differentially phosphorylated in cells incubated with mctr3 when compared to those incubated with vehicle. results are representative of cells from n = 3 healthy volunteers per group. (a) inflammatory arthritis was induced in c57bl/6 mice by administering 100 µl k/bxn serum i.p. on days 0 and 2 and femur heads and bone marrow monocytes were collected on day 5. femur heads were incubated in serum free dmem high glucose which results in further tissue damage. in the present studies we found that mctr3 administration, using a therapeutic paradigm, potently limited clinical signs of joint inflammation in two distinct models of inflammatory arthritis. this reduction in joint inflammation was linked with the activation of joint protective mechanisms as demonstrated by a significant upregulation in both collagen 2 and collagen x expression, increased cartilage cover, increased bone volume and decreased callus size in joints from mice treated with mctr3. the anti-inflammatory activities of mctr3 are also in line with observations made with other spms, whereby at-rvd1, rvd3, mar1 and the rvd precursor 17-hdha were all found to display potent anti-inflammatory, antinociceptive and joint protective activities. 27, 40, 68, 69 trained immunity is now appreciated to play a significant role in both host protection from pathogenic infections as well as in the propagation of inflammation in chronic inflammatory conditions. 70 underpinning trained immunity is a change in the dna methylation status of the cell that leads to a shift in the cellular responses to subsequent inflammatory stimulus. studies investigating this process in ra demonstrated that circulating cd14+ monocytes from these patients expressed increased basal cd11b expression and produced higher concentrations of il-1b and il-6 when stimulated ex vivo. 71 notably, recent findings demonstrated that incubation of human monocytes with etanercept and adalimumab downregulated the trimethylation of h3k4, h3k27, h3k36 and h3k79 in the ccl2 promoter region by decreasing the expression of the related methyltransferases wdr5 and smyd2. 72 these finding suggest therapeutics that can modulate trained immunity to downregulate the expression of inflammation-initiating genes in monocytes and mdms may be attractive targets in the treatment of ra. notably, the process of trained immunity has to-date been primarily linked with the reprogramming of cells towards an activated, potentially pro-inflammatory status. 59, 71 our findings indicated that mctr3-reprogrammed monocytes exert both anti-inflammatory and tissue reparative activities as observed by a decrease in joint disease activity and upregulation in collagen 2 and collagen x expression. these protective activities of mctr3 on reprogramming monocyte responses were reversed when these cells were incubated with a dna methyltransferase inhibitor, underscoring a central role for epigenetic reprogramming in mediating the protective activities of mctr3 on monocytes. thus, the present findings suggest that mctr3 changes the epigenetic landscape of trained monocytes from arthritic mice to upregulate tissue protective and proresolving pathways in these cells. this hypothesis is supported by findings made in our transcriptomic and phosphor-proteomic analysis. whereby we found that mctr3 regulates the phosphorylation status of proteins involved in epigenetic and chromatin regulation in mononuclear phagocytes. furthermore, sc-rna seq analysis of synovial leukocytes from mice receiving mctr3-reprogrammed monocytes demonstrated a marked shift in the transcriptome of mdms with an upregulation of several immunoregulatory and host protective genes, including arg-1. this enzyme was recently found to exert anti-arthritic activities, whereby upregulation of arg-1 expression in macrophages in mice lacking the transcription factor fra-1, which containing 1% insulin-transferrin-selenium for 48 hours and then in dmem containing 10 % fbs and 10 ng/ml il1-b for a further 72 hours. bone marrow derived monocytes were incubated with vehicle (dpbs + 0.1 % dmso) or 10 µm rg108, a dnmt inhibitor, for 45 min and then with either vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 24 hours. cells were differentiated to monocytederived macrophages, then incubated with femur heads for 48 hours. the proteoglycan concentrations in the femur heads were assessed using safranin o staining. (left panels) representative images from safranin-o stained femur heads (right panel) quantification of safranin-o stained femur heads. results are mean § sem and expressed as percentage change from cells incubated with vehicle alone. n = 5-8 mice per group from two separate experiments. (statistical differences were evaluated using one-sample wilcoxon signed rank test). (b) mice were administered k/bxn serum on days 0, 2 and 9 then on day 12 they were treated 2£10 6 pkh67-labelled monocytes isolated from arthritic mice and incubated with either vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 90 min via i.v injection. after 10 days joints were harvested, cells were liberated and expression of arg-1 was evaluated in pkh67 + cd64 + cells using flow cytometry. results are from n=10 mice per group. (statistical differences were evaluated using a mann-whitney u test). (c) inflammatory arthritis was induced in c57bl/6 mice and bone marrow-derived monocytes were isolated and treated as in a and arg-1 expression was evaluated using flow cytometry. results are mean § sem and expressed as percentage change from cells incubated with vehicle alone. n = 8 per group from two separate experiments. (statistical differences were evaluated using one-sample wilcoxon signed rank test.) (d) mice were given k/bxn serum (via i.p. injection) on days 0, 2 and 9. on day 12 these were treated with 2£10 6 pkh67-labelled monocytes isolated from arthritic mice and incubated either with vehicle (dpbs + 0.1 % dmso) or 10 µm rg108, a dnmt inhibitor, for 15 min and then with a vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 90 min. on day 22, joints were collected and the expression of arg-1 in pkh67 + cd64 + cells was evaluated using flow cytometry. (a, b) femur heads and bone marrow-derived monocytes were collected 5 days following the induction of arthritis in c57bl/6 mice. femur heads were incubated in serum free dmem high glucose containing 1% insulin-transferrin-selenium for 48 hours and then in dmem containing 10 % fbs and 10 ng/ml il-1b for 4 days. monocytes were incubated with vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 24 hours, then differentiated to monocyte-derived macrophages. two days after the initiation of differentiation, cells were transfected with control sirna or sirna against arg-1. three days later these cells were incubated with arthritic femur heads for 48 hours. tissues were then collected and glycosaminoglycan content was evaluated using safranin-o staining. (a) representative images and (b) quantitation of safranin-o staining. results are mean § sem, n = 7-8 mice per group. (statistical differences negatively regulates arg-1 expression, was linked with an enhanced resistance to both joint inflammation and joint damage in experimental arthritis. 54 arg-1 activity in macrophages is also involved in regulating efferocytosis, a key pro-resolving activity of macrophages which was recently suggested to play an important role in tissue repair. 73 in this context, the arg-1 downstream metabolite putrescine regulates the ability of macrophages to uptake apoptotic cells, and disruptions in this pathway are linked with the propagation of tissue inflammation in atherosclerosis. 65 in the present studies we found that mctr3-reprogrammed monocytes yield mdms with elevated expression of arg-1 both in vivo and in vitro. this observation was linked with a downregulation of fra-1 expression in arthritic paws from these mice. notably, inhibition of arg-1 expression or activity reversed both the anti-inflammatory and the joint reparative activities of mctr3-reprogrammed monocytes. thus, these findings establish a central role of this enzyme in mediating the protective activities of mctr3 during inflammatory arthritis. over the past years there has been a steady increase in the development of cell-based therapeutics for the treatment of both chronic inflammatory conditions and cancers. many of these therapies involve complex and sometimes expensive gene editing approaches which have to some extent limited their application. in addition, these approaches have been primarily focused on the use of bone marrow-derived cells. harvest of these cells is a painful process for patients, it requires hospitalization, and can cause side effects that include bone marrow inflammation further complicating the application of cell-based therapies. another approach being developed is that of allogeneic cell therapies, which would in principle increase throughput and drive down costs. however, the application of this approach to patient treatment has been fraught with issues primarily linked with the activation of the recipient immune responses and rejection. results from the present studies indicate that circulating monocytes may represent a target population for cell-based therapeutics in patients with ra. a recent study supports the potential applicability of such an approach, whereby, moroni and colleagues in a phase i study collected peripheral blood monocytes from patients with liver cirrhosis. they then differentiated the cells to mdms ex vivo and re-injected these cells back to the patient, demonstrating that such an approach is both feasible and safe. 74 our findings from both in vivo studies and organ culture models suggest that mctr3-reprogrammed monocytes display enhanced protective activities over non-reprogrammed monocytes in facilitating both the resolution of joint inflammation and promoting the repair and regeneration of arthritic tissues. the present work leverages well established experimental methodologies for dissecting and evaluating mechanisms in tissue repair therefore they establish insights into the potential utility of mctr3 and mctr3-reprogrammed monocytes in both regulating inflammation and promoting joint repair. given that the evidence collected thus far is primarily based on experimental models, future studies will need to establish the translational potential of the present work to humans. they will also need to establish key pharmacological parameters as well as the targets that these potential spm-based therapeutics may regulate. in summation, the present studies demonstrate that mctr3 reprograms monocytes to differentiate to mdms with anti-inflammatory and joint reparative properties. whilst results from the present findings do not exclude a role for other cells in mediating the protective activities of mctr3 when administered in vivo, they provide compelling evidence for a central role of mdms in conferring both the anti-inflammatory and joint protective activities of this mediator. the results presented herein suggest that the protection observed in mice receiving mctr3-reprogrammed monocytes is mediated directly by the reprogrammed cells via the release of factors and/or molecules that exert joint reparative activities such as polyamines. the anti-arthritic propoertes of these cells are also likely propagated via the release of molecules, such as spm, that reprogram the tissue environment and resolve inflammation ( figure 5-7 ). together these mechanisms lead to the reduction of inflammation and repair of damaged tissues. thus, our findings elucidate potential therapeutic strategies in the treatment of patients with ra that target both the inflammatory component and promote joint repair. were evaluated using one sample wilcoxon signed rank test when assessing for differences vs vehicle group and using one-way anova and kruskal wallis post hoc test when evaluating differences vs mctr3 treated group). (c-f) mice were administered k/bxn serum on days 0, 2 and 9 then on day 12 they were treated 2£10 6 pkh67-labelled monocytes isolated from arthritic mice and incubated with either vehicle (dpbs + 0.1 % etoh) or 1 nm mctr3 for 90 min via i.v injection and 200 µg n v -hydroxy-nor-l-arginine (nor-noha), an arginase 1 inhibitor, or vehicle (dpbs) that were administered daily for a 10day period via i.p. injection. disease course was evaluated by assessing (c) clinical scores and (d) paw oedema. results are mean § sem, n = 8-10 per group from two distinct experiments and expressed as percent change from first day of treatment. (statistical differences were evaluated using two-way anova). (e-f) at the end of the experiments joints were collected and (e) dbl and (f) tgf-b1 expression was evaluated in pkh67 + cd64 + cells using flow cytometry. results are mean § sem and expressed as percentage change from vehicle group. n = 9-10 mice per group. (statistical differences were evaluated using one-sample wilcoxon signed rank test when assessing for differences vs vehicle group and using one-way anova and kruskal wallis post hoc test when evaluating differences vs mctr3 treated group). this approach may also be useful in other inflammatory conditions characterized by uncontrolled inflammation and tissue destruction. all data will be made available by the corresponding author upon reasonable request jd is an inventor on patents related to the composition of matter and/or use of pro-resolving mediators some of which are licensed by brigham and women's hospital or queen mary university of london for clinical development. jd is also scientific founder and director of resolomics ltd. k.p. and l.l. are inventors on patent applications related to the use of cell therapy relating the use of mctr3 reprogrammed monocytes to treat joint inflammation. other authors declare no relevant conflict of interest. towards a stratified targeted approach with biologic treatments in rheumatoid arthritis: role of synovial pathobiology leukocyte trafficking between stromal compartments: lessons from rheumatoid arthritis new pathogenic insights into rheumatoid arthritis pathogenetic insights from the treatment of rheumatoid arthritis cytokines as therapeutic targets in rheumatoid arthritis and other inflammatory diseases driving chronicity in rheumatoid arthritis: perpetuating role of myeloid cells neutrophil extracellular traps mediate articular cartilage damage and enhance cartilage component immunogenicity in rheumatoid arthritis synovial fibroblastneutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis non-classical monocytes as mediators of tissue destruction in arthritis origin and function of synovial macrophage subsets during inflammatory joint disease nonclassical ly6c(-) monocytes drive the development of inflammatory arthritis in mice hbegf(+) macrophages in rheumatoid arthritis induce fibroblast invasiveness distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis specialised pro-resolving mediators of inflammation in inflammatory arthritis inflammatory arthritis disrupts gut resolution mechanisms, promoting barrier breakdown by porphyromonas gingivalis identification of a third nuclear protein-coding gene required specifically for posttranscriptional expression of the mitochondrial cox3 gene is saccharomyces cerevisiae specialized pro-resolving mediator network: an update on production and actions identification and structure elucidation of the pro-resolving mediators provides novel leads for resolution pharmacology identification of 14-series sulfidoconjugated mediators that promote resolution of infection and organ protection identification and actions of a novel third maresin conjugate in tissue regeneration: mctr3 maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions cysteinyl maresins regulate the prophlogistic lung actions of cysteinyl leukotrienes synovial cellular and molecular signatures stratify clinical response to csdmard therapy and predict radiographic progression in early rheumatoid arthritis patients blood pro-resolving mediators are linked with synovial pathology and are predictive of dmard responsiveness in rheumatoid arthritis proresolving and cartilageprotective actions of resolvin d1 in inflammatory arthritis first total synthesis of pro-resolving and tissue-regenerative maresin sulfido-conjugates neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis integrated analysis of multimodal single-cell data networ-kanalyst 3.0: a visual analytics platform for comprehensive gene expression profiling and meta-analysis kegg: kyoto encyclopedia of genes and genomes string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets reconstructing kinase network topologies from phosphoproteomics data reveals cancer-associated rewiring cross-species proteomics reveals specific modulation of signaling in cancer and stromal cells by phosphoinositide 3-kinase (pi3k) inhibitors phosphoproteomic analysis of leukemia cells under basal and drug-treated conditions identifies markers of kinase pathway activation and mechanisms of resistance characterization of a tio(2) enrichment method for label-free quantitative phosphoproteomics highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns resolvin d3 is dysregulated in arthritis and reduces arthritic inflammation impaired production and diurnal regulation of vascular rvdn-3 dpa increase systemic inflammation and cardiovascular disease enriched marine oil supplements increase peripheral blood specialized pro-resolving mediators concentrations and reprogram host immune responses: a randomized double-blind placebo-controlled study dose-and time-dependent increase in circulating anti-inflammatory and pro-resolving lipid mediators following eicosapentaenoic acid supplementation in patients with major depressive disorder and chronic inflammation cutting edge: severe sars-cov-2 infection in humans is defined by a shift in the serum lipidome, resulting in dysregulation of eicosanoid immune mediators regression of human coronary artery plaque is associated with a high ratio of (18-hydroxy-eicosapentaenoic acid + resolvin e1) to leukotriene b4 from systemic t cell selfreactivity to organ-specific autoimmune disease via immunoglobulins apoe deficiency exacerbates the development and sustainment of a semi-chronic k/bxn serum transfer-induced arthritis model macrophages and skeletal health innate immune cell-epithelial crosstalk during wound repair immunization with glucose-6-phosphate isomerase induces t cell-dependent peripheral polyarthritis in genetically unaltered mice collagen of articular cartilage contribution of inflammation and bone destruction to pain in arthritis: a study in murine glucose-6-phosphate isomerase-induced arthritis p21cip1 is required for the development of monocytes and their response to serum transferinduced arthritis transcription factor fra-1 targets arginase-1 to enhance macrophage-mediated inflammation in arthritis wnt pathway in bone repair and regeneration -what do we know so far wnt signaling and cartilage: of mice and men lef1-mediated mmp13 gene expression is repressed by sirt1 in human chondrocytes expression and function of ezh2 in synovial fibroblasts: epigenetic repression of the wnt inhibitor sfrp1 in rheumatoid arthritis defining trained immunity and its role in health and disease bacille calmette-guerin induces nod2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes locally renewing resident synovial macrophages provide a protective barrier for the joint gm-csf expression and macrophage polarization in joints of undifferentiated arthritis patients evolving to rheumatoid arthritis or psoriatic arthritis the ap-1 transcription factor c-jun promotes arthritis by regulating cyclooxygenase-2 and arginase-1 expression in macrophages efferocytosis induces macrophage proliferation to help resolve tissue injury macrophage metabolism of apoptotic cell-derived arginine promotes continual efferocytosis and resolution of injury tgfbeta signaling in cartilage development and maintenance the role of omega-3 derived resolvins in arthritis the precursor of resolvin d series and aspirin-triggered resolvin d1 display anti-hyperalgesic properties in adjuvant-induced arthritis in rats imbalance of proresolving lipid mediators in persistent allodynia dissociated from signs of clinical arthritis the potential role of trained immunity in autoimmune and autoinflammatory disorders oligomeric s100a4 is associated with monocyte innate immune memory and bypass of tolerance to subsequent stimulation with lipopolysaccharides tumor necrosis factor-alpha inhibitors suppress ccl2 chemokine in monocytes via epigenetic modification efferocytosis fuels requirements of fatty acid oxidation and the electron transport chain to polarize macrophages for tissue repair safety profile of autologous macrophage therapy for liver cirrhosis we thank the patients and volunteers who donated blood. this work was supported by funding from the european research council ( key: cord-0073012-p2z3fs9r authors: wang, shaohui; hou, ya; li, xuanhao; meng, xianli; zhang, yi; wang, xiaobo title: practical implementation of artificial intelligence-based deep learning and cloud computing on the application of traditional medicine and western medicine in the diagnosis and treatment of rheumatoid arthritis date: 2021-12-23 journal: front pharmacol doi: 10.3389/fphar.2021.765435 sha: 7ebbc124de89a59786a6a0ecd5fbcbd164751922 doc_id: 73012 cord_uid: p2z3fs9r rheumatoid arthritis (ra), an autoimmune disease of unknown etiology, is a serious threat to the health of middle-aged and elderly people. although western medicine, traditional medicine such as traditional chinese medicine, tibetan medicine and other ethnic medicine have shown certain advantages in the diagnosis and treatment of ra, there are still some practical shortcomings, such as delayed diagnosis, improper treatment scheme and unclear drug mechanism. at present, the applications of artificial intelligence (ai)-based deep learning and cloud computing has aroused wide attention in the medical and health field, especially in screening potential active ingredients, targets and action pathways of single drugs or prescriptions in traditional medicine and optimizing disease diagnosis and treatment models. integrated information and analysis of ra patients based on ai and medical big data will unquestionably benefit more ra patients worldwide. in this review, we mainly elaborated the application status and prospect of ai-assisted deep learning and cloud computation-oriented western medicine and traditional medicine on the diagnosis and treatment of ra in different stages. it can be predicted that with the help of ai, more pharmacological mechanisms of effective ethnic drugs against ra will be elucidated and more accurate solutions will be provided for the treatment and diagnosis of ra in the future. rheumatoid arthritis (ra) is a non-fatal but currently incurable chronic symmetric autoimmune disease that involves multiple bones and joints, and their surrounding tissues, such as wrist joints, knuckles, metacarpophalangeal and metatarsophalangeal joints (sparks, 2019; scherer et al., 2020) . meanwhile, it can increase the incidence of venous thromboembolism (molander et al., 2021) , lung diseases (juge et al., 2018) , cardiovascular diseases (semb et al., 2020) , eye diseases (artifoni et al., 2014) , diabetes (westra et al., 2018; inamo et al., 2021) , periodontal diseases (courbon et al., 2019) and nonmelanoma skin cancer (assassi, 2016; raaschou et al., 2016) (supplementary figure s1 ). the epidemiological data show that the incidence of ra is positively correlated with age (the initial episode of ra is commonly of 40-60 years old) (allen et al., 2018) , with the characteristics of genetic tendency (webster et al., 2018; okada et al., 2019) , regional and ethnic differences (james, 1996; hughes et al., 2006) . less equally, women are three times more susceptible to ra than men, due to women's menstrual flow, pregnancy, delivery and breastfeeding behaviors that disrupt sex hormones and deplete qi and blood (favalli et al., 2019) . at present, the pathogenesis of ra has not been fully clarified and there is no specific radical cure for ra (croia et al., 2019) . traditional medicine has a long history in the treatment of ra. however, the unknown target and pharmacological mechanism of a single chinese medicine or preparation hinders the development of traditional medicine. in addition, the irregular diagnosis and treatment regimen of ra leads to uneven therapeutic effects. with the accumulation of big data for ra diagnosis and research, the improvement of computing network, machine learning and other models, ai will bring dawn to the breakthrough of ra diagnosis and treatment as well as drug discovery for ra. the subtle clinical signs of ra are easier to be confused with several other arthritis diseases (including osteoarthritis, psoriatic arthritis and gout) at onset. as the disease worsens, severe joint damage, undesirable and outrageous arthralgia and other troublesome complications are extremely distasteful to patients. so, it is pretty desirable to make timely and accurate early diagnosis, reasonable management and prospective prognosis for ra patients. however, there are obvious technical constraints (such as insensitivity, delayed diagnosis of diseases and obvious adverse reactions of biologics) in conventional physician check, joint imaging with diagnostic and treatment instruments, detection of various serum and synovial joint markers in ra patients (conigliaro et al., 2019; widdifield, 2019) . although western medicine, as well traditional medicine of various ethnic groups including unique tibetan medicine bath and other internationally recognized ra therapies has the potential role on ra, its mystery still needs to be uncovered to explore deeper pharmacological mechanisms. therefore, in addition to conventional approaches, clinicians should also consider the unknown etiology, complex pathologic process, unpredictable prognosis, and vast amounts of ethnic medicine therapy of ra when managing the main and/ or suspected symptoms of ra. in this review, we also confirmedly propose to weigh multiple parameters from data mining, aibased deep learning and digital pathology of ra-related medicine and disease database. and further reasonable introduction of computer algorithms is employed to integrate and analyze seemingly unrelated clinical signs of ra patients. this not only provides a reference for comprehensive management of ra patients, including disease status, optimal treatment and combination strategies, all possible prognostic and elusion tactics for ra. in addition, it is more important to promote the integration of ai and digital medical transformation, realize the integration of traditional medicine and western medicine under the vision of ai, enhance the clinical diagnosis and treatment of ra and drug development, and improve the quality and cost of healthcare. ai is considered a branch of engineering capable of realizing novel concepts and solutions to solve complex challenges. ai integrated medical big data processing is to give a relatively fast mining of massive existing data such as the cause of intractable diseases, disease-specific clinical examination images, related available drugs, and disease development, outcome and prognosis through multiple open source data analysis platforms, which eventually and accurately diagnose disease and provide individualized reasonable treatment recommendations by comparing clinical signals of patients with simulated artificial algorithms (torkamani et al., 2017; he et al., 2019; dzobo et al., 2020) . in recent years, clinical and scientific researchers have realized the importance and considerable prospect of ai application in medical fields, especially for complex diseases. the digitalization of medical image acquisition provides convenient conditions for the application of ai. lee et al. developed a computer-aided diagnosis system based on deep learning for diagnosing cervical lymph node metastasis of thyroid cancer by ct scanning, after eight modes of deep learning were used for detection, the recognition of lymph node metastasis reached 90.4% (lee et al., 2019a) . a trained deep learning algorithmconvolutional neural networks (cnns) has been developed for detecting diabetic retinopathy in retinal fundus photographs (wong and bressler, 2016) , and classifying age-related macular degeneration and diabetic macular edema, as well accurately distinguishing bacterial and viral pediatric pneumonia using retinal optical coherence tomography and chest x-rays images, respectively (kermany et al., 2018) . integrated platform of aibased deep learning, cnns and cloud computing has been developed for detection of congenital cataracts (long et al., 2017) . furthermore, ai has also achieved fine results in the application of pathological diagnosis. scientists have successfully used machine learning to distinguish fine-grained variability of different types of skin cancer using cnns (esteva et al., 2017) , dna methylation-based screening for central nervous system tumors (capper et al., 2018) , and identify clinical phenotypes of sepsis (seymour et al., 2019) . as well constructed an ai learning method that can automatically identify pathological images and clinically inherited non-small cell lung cancer gene mutations, providing a basis for personalized treatment of lung cancer (kumar et al., 2018) . early diagnosis and treatment of breast cancer are very important to the prognosis of patients, by evaluating and diagnosing breast cancer cell markers, ai can make more patient-appropriate treatment decisions (robertson et al., 2018) . in a word, ai-based deep learning and cloud computing have blossomed in fields such as digital pathological image diagnosis of diseases (niazi et al., 2019) , radiology (hosny et al., 2018; van assen et al., 2020) , oncology (bi et al., 2019) , cardiovascular disease (krittanawong et al., 2019) , genetics and genomics (langmead and nellore, 2018; zou et al., 2019) , proteomics (feng et al., 2017; vishnoi et al., 2020) , neurosciences (perkel, 2018; choi et al., 2020) , and discovery, optimization and verification of drug target and lead compounds in drug discovery (simm et al., 2018; raschka and kaufman, 2020) (figure 1 ). traditional medicine, especially tcm is a complete medical system with thousands of years of application history, its clinical practice mainly focuses on diagnosis and treatment. tcm has played a crucial role in the treatment of many diseases of chinese people, such as diabetes, cancer and ra, etc. wang et al., 2021b; wang et al., 2021d; . especially since the outbreak of covid-19, tcm has played its unique advantages and gradually gained recognition abroad (hou et al., 2021; zhao et al., 2021) . with the rapid development of science and technology, ai has attracted much attention in the field of tcm. the application of ai has not only observably improved the reliability and accuracy of diagnosis, but also provides favorable conditions for screening suitable tcm or program for disease treatment, as well as promoting the modernization of traditional medicine (wang et al., 2021d) . at present, the establishment of new methods such as systems biology, bioinformatics, integrated pharmacology and network pharmacology has brought new hope for the modernization of tcm research (ren et al., 2020; shen et al., 2020; wang et al., 2021c; fang et al., 2021) . for example, the establishment of databases such as integrative pharmacology-based research platform of traditional chinese medicine (tcmip), traditional chinese medicine database and analysis platform (tcmsp), the encyclopedia of traditional chinese medicine (etcm) and other databases based on ai has provided effective tool for revealing potential active ingredients, pharmacological mechanism and compatibility of tcm. integrated pharmacology based on traditional medicine is regarded as a paradigm shift in tcm (xu et al., 2021a) . some scholars demonstrated that the five compounds including baicalin, chlorogenic acid, sweroside, phillyrin and forsythoside a were the anti-influenza substances of shuang-huang-lian preparation, and the anti-influenza pharmacological mechanism was related to tnf signaling pathway by serum pharmaco-chemistry and network pharmacology . other studies have found that the key metabolites of xihuang pill enhancing the efficacy of anlotinib for lung cancer management are regulated by multicomponent and multi-target interaction networks through comprehensive metabolomics and network pharmacology . additionally, we can also use these ai techniques to bridge the relationship between the biological network of disease phenotype and patient genotype as a whole. in this way, we can not only understand the biological basis of tcm diagnosis, but also help us elucidate the mechanism of disease and therapeutic target to establish a new treatment method of targeted biological network therapy combining traditional medicine and western medicine. such as some scholars used the optimized support vector machine model to establish a diagnosis model of lung cancer based on serological indicators, and a molecular regulation model of wogonin in scutellaria baicalensis. they finally constructed the regulatory network of wogonin on cell apoptosis and serological susceptibility genes, which provided a new idea for the clinical diagnosis, treatment and prognosis of lung cancer . zhang et al. proposed a unified intelligent tcm framework based on the edge cloud computing system, which incorporated deep learning algorithm to establish the model for the syndrome differentiation and prescription recommendation through the differentiation of hypertension and cold . the channels and collaterals of tcm are closely related to diseases, which helps to improve the accuracy of clinical medication. wang et al. used machine learning to predict herbal active ingredients after labeling, manifesting that ai algorithm could explain the relationship between herbal medicines and meridians (wang et al., 2019b) . moreover, ai technology can be found in the disorderly data of each keyword potential link, and secondary sorting, deep learning and training for many times based on the existing data. by extracting and summarizing from the complex symptoms of tcm, potential association rules between symptoms, prescriptions and core drugs can be determined for clinical diagnosis and treatment . all in all, with the deepening of the integration of ai and the medical field, the digitalization of the four diagnostic methods of tcm, the intelligent decision system and the modernization of tcm theory will make great progress. it is not difficult to find that efficient and accurate medical image ai recognition is entering clinical practice. ai can not only facilitate medical diagnostic innovation and assist doctors in making diagnosis and treatment decisions, but also accelerate the discovery of innovative drugs with better efficacy (xu et al., 2021b) . currently, the application of ai in the medical field is mainly based on a certain module in the process of disease diagnosis and treatment. the establishment of ra auxiliary diagnosis and treatment system requires the combination of the examination and laboratory information of patients, medical record information and joint image data to make decisions, providing recommendations on prescription usage and dosage according to the diagnosis results. in this process, we need to integrate the existing information on the pathogenesis, diagnosis, treatment and patients of ra. besides, we also need to consider the accuracy of the obtained data and the security of the algorithm, as well as the supplement of new data. as a chronic autoimmune disease, t cells in the blood of ra patients enter the damaged joints, triggering an inflammatory cascade that causes joint synovium, cartilage, and bone damage weyand and goronzy, 2021) . the interleukin (il)-6, 12 and 23 released by dendritic cells stimulated antigen presenting cells to produce il-2 and 21, contributing to the maturation of t cells. on the one hand, the matured t cells differentiate into helper t (th)1, th17 and helper t follicular cells, and release ifn-γ and il-17 to activate macrophages (lai kwan lam et al., 2008; rao et al., 2017) . besides, the matured t cells stimulate b cells to differentiate into plasma cells and secrete a large number of autoantibodies such as anti-citrullinated protein antibodies, which formed massive immune complexes of autoantibodies and corresponding antigens in the synovium. after combining with rheumatoid factor (rf), the deposited immune complexes in damaged joints further promote its deterioration (blüml et al., 2014) . meanwhile, the immune complex and complement binding immune complex bind to fc and complement receptor of macrophages, respectively. consequently, the release of tumor necrosis factor (tnf), il-1 and il-6 by the activated macrophages stimulate fibroblasts and chondrocytes to release matrix metalloproteinases (mmp) to degrade cartilage (firestein and mcinnes, 2017) . in turn, it promotes the differentiation of t cells by releasing il-15, 18 and 32, forming a vicious inflammatory response cycle. in addition to t cells and macrophages, neutrophils and fibroblasts are also involved in the pathological deterioration of ra. neutrophils can proliferate in the joints of patients with ra and continue to damage the joints by secreting mmp and inducing inflammation. a new study confirms that the reduction in apoptosis and migration caused by the deletion of elmo1 gene in neutrophils can avoid the aggregation of neutrophils with lower migration ability and alleviate the progressive damage irritated by inflammation to joints (arandjelovic et al., 2019) . moreover, the receptor activator of nuclear factor kappa b (rank) released by activated macrophages can combine with fibroblasts-released rank-ligand (rankl) to promote the differentiation of osteoclasts and cause bone destruction and cartilage degeneration . evidence has also shown that fibroblasts located in the synovial lining and sublining can cause bone damage through different mechanisms. fibroblasts in the inner layer can directly destroy bone and cartilage by secreting fapα protein, causing bone erosion. while the fibroblasts of synovial sublining secrete a large number of cytokines, triggered by fapα and thy-1 proteins, which enhance the immune response of joints (croft et al., 2019; wynn, 2019; chu, 2020) (figure 2) . the pathological molecular mechanism of ra is complex and closely related to multiple signal pathways (catrina et al., 2016; smolen et al., 2016; mcinnes and schett, 2017; guo et al., 2018; smolen et al., 2018; simon et al., 2021) (figure 3 ). pi3k/akt frontiers in pharmacology | www.frontiersin.org december 2021 | volume 12 | article 765435 4 signaling pathway plays an important role in the proliferation and survival of many cells, especially in the regulation of leukocyte migration and fibroblast-evoked cartilage injury. at the same time, tnf-α, il-17 and other cytokines are stimulated to participate in osteoclast differentiation and generation, and promote osteoclast migration to destroy bone and articular cartilage. janus kinase/signal transducers and activators of transcription (jak/stat) signaling pathway is responsible for regulating the proliferation of fibroblast-like synoviocytes (fls), synovitis, cartilage and bone destruction. nf-κb is an important transcription factor involved in inflammation that is activated by many cytokines such as tnf-α, rankl, il-1 and il-6. excessive activation of nf-κb can lead to abnormal apoptosis of fls, stimulate the secretion of mmp, pro-inflammatory cytokines, inflammatory cells and mediators, and significantly promote bone destruction. fls and chondrocytes with the function of secreting mmps can degrade the components of joint extracellular matrix, leading to degeneration of joint cartilage. the activation of mammalian target of rapamycin (mtor) pathway in synovial cells of ra patients promotes the proliferation of il-17-induced fls and the generation of osteoclasts. wnt signaling pathway may be activated during the occurrence and development of ra, which controls the development of bone and the transformation of cartilage and bone, maintaining the dynamic balance of bone metabolism. thereby, cytokines secretion, osteoclasts activation, and proline-matrix metalloprotein up-regulation contribute to bone erosion by activated wnt signaling pathway. the toll-like family of receptors (tlrs) consists of myeloid differentiation factor (myd88) dependent and independent pathways. the former leads to the production of pro-inflammatory cytokines, while the latter is related to the upregulation of ifn and major histocompatibility complex-ⅱ. both of which cause the production of inflammatory cytokines by nf-κb, and thus enhance the inflammatory cascade reaction of ra joints. mitogen-activated protein kinases are important signaling pathways linking various cell surface proteins and inflammatory genes of the tnf family, chemokines, cytokines, and tlrs. the activation of p38, extracellular signal-regulated kinase (erk) and c-jun n-terminal kinase (jnk) in the families can regulate the expression of inflammatory factors, promote synovial inflammatory response, and mediate bone and cartilage destruction. in addition, it can activate macrophages and fls, and promote the expression of inflammatory cytokines. as an important molecular mechanism of ra bone resorption and joint destruction, rankl induces osteoclast differentiation by activating rank receptor on the surface of osteoclast precursor. osteoprotegerin is a natural bait of rankl and inhibits osteoclast differentiation and bone absorption by preventing the binding of rankl to rank. in addition to autoinflammatory immunity, ra is associated with genetics, such as hla-drb1, ccr6, and ccr5 gene loci mutations, as well as environmental factors such as smoking (ishigaki et al., 2017) . and again, ra can be triggered by periodontitis, intestinal, oral and pulmonary microorganisms, and viral infections du teil espina et al., 2019) . with the development of molecular biology, numerous emerging ra biomarkers have been gradually elucidated. correspondingly, there will be more novel and effective targeted therapies (isaacs and iqbal, 2019) . bi disease (痹病), first documented in the section of su wen (plain conversation)·bi theory of huangdi neijing, is caused by the interaction between different forms of exopathogens (wind, cold, dampness and heat) and internal pathogens (phlegm and blood stasis). tcm classifies ra as the scope of "bi disease". ancient and modern chinese medicine classics called ra li jie (历节), bai hu disease, gout, hexi wind (鹤膝风), wet bi, wan bi, bone bi and wang (尫) bi (zheng et al., 2017; liu and jiang, 2020) . modern and contemporary tcm masters declare that the pathogenesis of ra is essential empty and out solid (本虚标实), or asthenia of healthy qi and sthenia of pathogenic factors (正虚 邪实) (bu et al., 2019) . specifically, liver and kidney, spleen and stomach, and yang qi deficiency, or yingwei disharmony led to deficiency of healthy qi, which causes the external or internal evil to take advantage of the empty and retain tendons bones arthrosis to induce ra. the prolonged ra course thus causes physical weakness and damage to the five zang-organs. therefore, according to the characteristics of pathogenesis and the purpose of addressing both symptoms and root causes, the treatment of ra is mostly based on the principle of removing pathogenic factors and supporting the healthy. specifically, symptoms were alleviated by removing the pathogenic factors of internal and external wind, cold, dampness, heat, phlegm and blood stasis, while ra can be fundamentally annihilated by strengthening the physiological functions of the liver, kidneys, lungs, spleen and stomach. in addition, heat and toxin in tcm is equivalent to inflammation in western medicine, which can induce bi, stasis and pain in ra patients. therefore, heatclearing and detoxicating therapy is used throughout the entire course of ra (wang and ma, 2018) . ra is called "zhen bu disease" (真布病) in tibetan medicine, which also belongs to the category of bi disease, divided into six categories: rou bi, bone bi, mai bi, tendon bi, white bi and black bi. the understanding of ra by tibetan doctors can be traced back to the four-volume medical code written by the famous tibetan medical expert yutuo·yundan gongbu in the 8th century ad, and it is believed that the intolerance of qi and blood and the increase of yellow water caused by the disorder of long (隆), chi ba (赤巴) and pei gen (培根), induce ra (huang et al., 2021) . based on the theory of tibetan medicine, idiomatical external treatment of ra in the four-volume medical code includes tibetan fire moxibustion and tibetan medicine bath. fire moxibustion is to use the heat of moxa cone to apply drugs to the joints of patients, so as to achieve the purpose of treating ra. the type and dosage of medicinal herb in tibetan medicine bath can be adjusted according to the degree of joint pain, spasm, swelling, and blocked joint flexion and extension. it should be noted that the temperature of the tibetan medicine extract with liquor should be controlled at 39-42°c for 30 min sitz bath each time, and sheltered from wind for about 2 h after the bath . mongolian medicine classified ra as "tulai disease" (图赉), which is caused by the imbalance of the three roots (heyi-赫依, xila-希拉, and bada gan-巴达干), as well qisu (琪素), yellow water (黄水) and xila wusu (希拉乌素). according to the clinical symptoms, ra is divided into the following three types: "hari tulai" (哈日图赉). "zhagan tulai" (查干图赉). "alaga tulai" (阿拉嘎图赉). ra is caused by excessive intake of spicy and stimulating food, excessive daytime sleep and fatigue, little exercise after satiation, strenuous activities, or invaded by cold wind and damp, and working in cold water for a long time. the main pathogenesis is that increased xila wusu, and heyi as well qisu fight each other for dominance, which aggress the small joints of hands and feet, the large joints of the whole body and the surrounding tendons. for a long time, the dysfunction of white vein, bone heyi and qisu in the affected area resulted in joint deformity, often involving the skin and viscera guo, et al., 2021) . other ethnic medicine also has a unique understanding of ra. dai medicine regards ra as long-meng-sha-throat disease (拢蒙沙喉). affected by the person's constitution and seasonal environment, the discordance of the four towers and five yun in the body leads to external evil (wind, hot, cold and damp evil) shuttling back and forth through joints, causing local four-tower abnormality of the joint and inducing ra. therefore, the focus of dai medical treatment for ra is to remove the excessive external pathogenic factors in the local joints of the body, and restore the four-tower state of the joint lesions, so as to improve the symptoms of ra . in the theoretical system of zhuang medicine, ra is known as gun ke (滚克) and fa wang (发旺), which is mainly due to the patient's physical weakness makes avails the evil poison (wind, wet, cold and hot poison) of the opportunity to get in and block the three channels (grain, gas and water channel) and the two roads (dragon and fire road), thus obstructing the functions of viscera, qi, blood, and bone and flesh to cause ra (zhong et al., 2018) . ra is recorded in tujia medicine as zhongjie feng (肿节风), which was caused by dampness together with wind and cold invasion of three yuan bores (三元孔窍). its characteristic of the traditional external treatment to drive oil and fire is that the mixture of heated tung oil, ginger and shallot is used to thermally stimulate ra points. moreover, supplemented by ironing, wiping, kneading and pressing to relax the muscles and stimulate the blood circulation, thereby ameliorating blood circulation and peripheral tissue nutrition for anti-inflammatory and detumescence (ye et al., 2013; nan et al., 2019) . in addition, indoor buried in heated sand therapy also has a good effect on ra, known as "wajol mupasil" in uygur medicine . to sum up, different ethnic medicine has unique theoretical understanding of ra, which is gradually formed into a relatively complete theoretical system in long-term clinical practice with a long history ( supplementary figures s2 and s3 ). in addition to internal and external drugs, the emphasis on the wholeness and unity of the body enables the treatment strategies to engulf unique ethnic therapy, and take into account the importance of dietetic regulation, environment and patients' psychology to ra disease with less adverse reactions. therefore, we should recognize the irreplaceable advantages of different ethnic medicine in the treatment of ra and other chronic and stubborn diseases. in order to promote and develop distinctive ethnic therapy, it should be applied in clinical practice without hesitation. the updating of multiple disease surveillance methods has promoted the continuous in-depth understanding of ra, and the key diagnosis and treatment strategies have brought clinical benefits to more patients with ra. accordingly, the integration of multi-center data, the introduction of ai-based deep learning and cloud computing are forward-looking and wise choice. although it is just emerging, it is of great practical significance to guide the clinical treatment and basic research of ra. currently, the clinical diagnosis of ra still lacks the generally accepted gold standard. in addition to skilled rheumatologists making decisions about ra based on typical clinical signs and symptoms, it is also necessary to selectively detect various serological indicators related to ra, such as rf, c-reactive protein, erythrocyte sedimentation rate, glucose-6-phosphate isomerase, il-6, il-1, anti-keratin, anti-perinuclear factor, anticyclic citrullinated peptide, anti-ra33, anti-sa, and anti-p68 antibodies (kumar et al., 2016; malmström et al., 2017) . however, the inevitable reality is that although the detection of serum rf is simple, rapid and high sensitivity, rf is also highly expressed in the serum of patients with systemic lupus erythematosus, sjogren's syndrome, hepatitis, tuberculosis and bacterial endocarditis, so there is no specific serological ra detection index at present. we therefore propose the introduction of multiple visual joint imaging at different stages of the pathogenesis of ra, such as ultrasonography (us), magnetic resonance imaging (mri), x-ray plain film, computed tomography (ct), fluorescence optical imaging, and isotope radiography, which can more accurately track any further spread of the disease and develop a more rational therapeutic regimen (schäfer et al., 2013; d'agostino et al., 2016; kumar et al., 2016; allen et al., 2018; panayides et al., 2020) (figure 4) . the us, including gray-scale ultrasonography, high-frequency color doppler ultrasonography, power doppler ultrasonography, and superb microvascular imaging, mainly reflects the early synovial thickness, articular cavity effusion, cartilage and blood flow signal strength in ra patients. on a two-dimensional scale, us can intuitively locate the eroded cartilage tissue in ra patients roux et al., 2019) . mri can visualize the joint structure and histopathological changes of healthy persons and ra patients with multiple plane images, including articular cavity effusion, synovial hyperplasia, marrow edema, articular cartilage and bone destruction, ligament, tendon and inflammatory exudation, especially the imaging of pannus. mri is considered as the gold standard for clinical diagnosis of patients with early ra and plays an important role in follow-up, prognosis and efficacy evaluation (baker et al., 2017; hunt et al., 2017) . x-ray examination of the bones and joints in ra patients, including normal x-ray plain film, digital tomosynthesis and digital x-ray radiogrammetry, the main objective is to evaluate joint space stenosis and bone erosion (figueiredo et al., 2018) . three-dimensional reconstructed technology of ct can clearly display diverse angles of the articular surface and detailed bone. radionuclide imaging is not a routine examination of ra and is just used for the differential diagnosis of difficult miscellaneous diseases and the exclusion of malignant tumors, but it shows certain advantages in some aspects. in recent years, some researchers have proposed that single photon emission computed tomography, based on the specific radioactive concentration patterns of each substance, can be used for the detection of angiogenesis in early ra with high sensitivity and specificity (kubota et al., 2017) . to the best of our knowledge, some near infrared fluorescence probes developed and synthesized in recent years have shown promising prospects for application in vivo ra animal models. an investigation on ra mice have shown that hypochlorous acid produced during the pathogenesis of ra can significantly enhance the fluorescence activity of the probe by breaking the c n bond of the fluorescence probe, which may become a potentially sensitive monitoring method for the detection and evaluation of therapeutic effect and disease progression of ra (feng et al., 2018) . a novel near-infrared (nir)-ii fluorescent probe ch1055-wl may perform well in the detection of articular cartilage degeneration in early ra patients . irdye800cw (a safe, and either oral or subcutaneously injectable nir-ii fluorescence agent) has been shown to be able to detect the process of collagen-induced arthritis in ra mice in vivo and in real time (bhatnagar et al., 2019) . all the above methods for clinical or preclinical ra examination have their unique advantages. however, the high cost and complex procedures are unacceptable to most rheumatologists and patients. in summary, before conventional or emerging medical technologies can be approved for clinical diagnosis of ra disease activity, we need to conduct a cohort of clinical randomized trials to evaluate the clinical safety, effectiveness, accuracy, and range of application of each technique. in addition, and more importantly, reasonable statistical models for quantifying various ra images also play an important role in the identification of ra condition, prediction of progress trend and evaluation of therapeutic efficacy (antill et al., 2016; brown et al., 2017) . although the above imaging technologies have their own focus and advantages, available and valuable diagnostic information from images requires an experienced rheumatologist with time-consuming and laborious, and somewhat subjectivity. advanced algorithms-trained dl or ai has attracted the attention of more clinical rheumatologists in image processing, information acquisition, diagnosis and treatment of various diseases including ra with the merit of efficient, reasonable, accurate and comprehensive halo. at present, most western drugs for ra are mainly antiinflammatory, analgesic, immunosuppressive and hormone therapy, which aim to relieve patients' discomfort symptoms and control the development of the disease, protect the function of the affected joints to the greatest extent, and relieve joint deformity. the relevant drugs available are categorized into nonsteroidal anti-inflammatory drugs (nsaids), diseasemodifying anti-rheumatic drugs (dmards) and glucocorticoids (gcs). nsaids are primarily used to improve ra patients with severe joint pain and stiffness. but nsaids has relatively serious and high incidence of adverse reactions in the digestive system, mainly caused by cox-1 such as aspirin and indomethacin. dmards can prevent and delay the destruction of the articular bone structure of ra, including conventional synthetic dmards (cs-dmards) such as methotrexate (mtx), leflunomide, and sulphasalazine, as well as novel dmards (biologic dmards (b-dmards) and targeted synthetic dmards (ts-dmards) (hazlewood et al., 2016) . more and more clinical data have implied that novel dmards, including biologic dmards (b-dmards) and targeted synthetic dmards (ts-dmards), bring a new era to the treatment of ra. gcs is the most effective anti-inflammatory drug at present. the complex binding gcs with the corresponding receptors translocate into nucleus, reducing the transcriptional regulatory activity of nf-κb, and thus reducing the production of pro-inflammatory factors. therefore, it produces powerful anti-inflammatory and analgesic effects and enables patients to rapidly relieve symptoms. however, it is well known that gcs does not prevent the progressive deterioration of ra and joint destruction. in contrast, gcs can cause severe infections, hormonal osteoporosis, aseptic osteonecrosis, and iatrogenic adrenocortical dysfunction or hyperactivity (katz-talmor et al., 2018) . given the risk of fractures, the researchers suggested that the addition of statins and tnf-α inhibitors, and the limited use of opioid, gcs and psychotropic drugs may reduce the risk of vertebral fractures in ra patients with cardiovascular disease and other joint fractures of ra patients (ozen et al., 2019) . furthermore, although most rheumatologists prefer bdmards and gcs to control the inflammatory response of ra, they increase the risk of double infection after joint replacement or during routine treatment compared to csdmards and other ra drugs (george et al., 2019; mahase, 2019) . in conclusion, we suggest that rheumatologists should provide patients with a rational western drug treatment regimen for ra according to the latest official guidelines. every kind of ethnic medicine has taken their place in the era of contention of a hundred schools of thought. although the medical theories of various ethnic groups are obscure and difficult to understand, there is no shortage of effective drugs and prescriptions for some intractable and chronic diseases, which exact effects have been widely recognized internationally. ethnic drug interventions are inexpensive and generally affordable for patients in remission of ra. it can not only avoid the liver and kidney toxicity caused by western medicine, but also adjust the body immunity and improve the physique, so it is worth further study. however, when choosing the treatment regimen, rheumatologists should inquire the medical history of ra patients, and introduce related ethnic drugs under the guidance of ethnic medicine theory on the basis of existing western medicine treatment, or substitute ethnic drugs for western drugs with potential or serious adverse reactions. secondly, the patient's physique, applicable population, indications and contraindications of each drug should be mastered to achieve rational drug use. supplementary table s1 lists the representative ethnic drugs for ra. clinical investigation has confirmed that the efficacy of tripterygium wilfordii hook f (lv et al., 2015) or tripterygium glycosides (tgs) tablets (a commercially available chinese patent medicine containing tgs extract) for ra is similar to that of mtx, and is better when combined with mtx. a clinical parallel, randomized and non-blind controlled efficacy evaluation trial suggested that mtx combined with sinomenine (sin) may be an alternative to mtx with leflumide (huang et al., 2019) . although existing a concomitant histamine-releasing anaphylaxis of sin, its robust anti-inflammatory effects make it a good candidate for clinical treatment of ra (huang et al., 2017) . the n-butanol extraction of panax notoginseng (chang et al., 2007) , and sodium tanshinone iia (tiia) sulfonate (wang et al., 2019c) can play the role of anti-tnf-α and various inflammatory cytokines by inhibiting nf-κb and mapk signaling pathways, improving the symptoms of the experimental ra models in vitro and vivo. paeonia lactiflora pallas and total glucosides of paeony (tgp) exert antiinflammatory, analgesic, inhibiting synovial hyperplasia and angiogenesis, and therefore can be clinically applied in the treatment of ra (zhang and dai, 2012) . recent studies have shown that 12 weeks of tgp administration improves the symptoms of joint injury in ra rats by balancing intestinal microbes (peng et al., 2019) . surprisingly, long-term clinical evaluation has showed that additional tgp supplementation reduced hepatic toxicity in ra patients treated with mtx and leflumide (xiang et al., 2015) . quercetin, another anti-ra monomer, has been shown to act on anti-inflammatory, analgesic and anti-oxidant effects via the suppression of nf-κb and the activation of nuclear factor erythroid 2-related factor (nrf2)/heme oxygenase (ho-1) pathway (guazelli et al., 2018; yang et al., 2018) . in vivo and in vitro evidence also suggests that quercetin could maintain the th17/treg ratio, repress the activation of nlrp3 inflammasome (yang et al., 2018) , and downregulate the levels of inflammatory cytokines such as tnf-α, il-1β, il-6, and il-17, mmp and monocyte chemoattractant protein (mcp-1) in experimental ra models (haleagrahara et al., 2018) . as a splendid herbal source of immune modulators, panax ginseng and/or ginsenosides can improve the symptoms of ra by anti-tnf-α and anti-oxidant (lee ji. et al., 2019b; yi., 2019) , and there were almost no obvious clinical adverse reactions in ra patients (cho et al., 2018) . in ayurvedic medicine, a crude extract mixture of azadirachta indica-nimbidin, mainly composed of nimbin, nimbinin, nimbidinin, nimbolide and nimbidic acid, has potential antiinflammatory effects by inhibiting the functions of macrophages and neutrophils (kaur et al., 2004) , which partly explains the mechanisms of azadirachta indica for treating ra in the folk for a long time. curcuma longa and its main ingredient curcumin are also a traditional herbal treatment for ra in india (yang et al., 2019) . many evidence has confirmed that curcumin could inhibit ra-evoked inflammatory process, oxidative stress events (manca et al., 2019) , and synovial hyperplasia (dai et al., 2018) , by the inhibition of nf-κb signal transduction and induction of macrophage apoptosis (wang et al., 2019a) , as well targeting mtor pathway (dai et al., 2018) . in addition, the inhibition of the production of inflammatory cytokines and the increased antioxidant defense capacity reported by swertia chirayita (lad and bhatnagar, 2016) and nyctanthes arbortristis (rathore et al., 2007) also doomed its potential in the treatment of ra. in summary, most ethnic herbal medicines are being valued as treatments or alternative combinations for ra. the gradual frontiers in pharmacology | www.frontiersin.org december 2021 | volume 12 | article 765435 9 elucidation of the mechanisms of each drug and the positive clinical results have also subtly increased people's recognition and acceptance of ethnic drugs. somewhat imperfectly, due to the concept of different national medical culture and the traditional standards of drug utilization, the ethnic medicine inevitably has a gap with the understanding of diseases and the development of drugs in the modern medical system. however, the national medical law, which has been preserved for generations along the long evolution of human beings, is undoubtedly the precious property of all nations over the world. we should firmly believe that the preferential support of policies and the introduction of the emerging of modern advanced technology, such as multiple omics auxiliary disease biomarkers and drug targets screening, optimization of the extraction and separation technology, as well as the conspicuous advantages of explosive applications of ai in the medical field in recent years, the future will bring forth more ethnic medicine therapies to make up for the numerous helpless situations of western medicine in treating ra. considering that the above-described western or ethnic drugs can induce more or less reversible or irreversible adverse reactions, many rheumatologists and patients are gradually turning to nondrug therapies for ra treatment. among them, targetedacupoint-strategy (tas) can strengthen the body resistance to eliminate pathogenic factors by stimulating the acupoints in various parts of the body to dredge meridians, harmonize yin and yang. currently, tas clinical application includes electroacupuncture, moxibustion, gold thread acupuncture (joo and park, 2017) . the effectiveness of plastering, liniment, and drug injection on acupoint, as well other physical manipulation of acupoints (such as acupoint laser irradiation, pressure, and blockade) has also been demonstrated. similar therapies include fire needle, burning needle and picking needle therapy inherited and innovated by zhuang and yao nationality, as well as cautery and moxibustion therapy by hui nationality (qiao et al., 2011) . it is worth noting that the thread moxibustion therapy of zhuang medicine (huang, 1986) and sand therapy of uygur medicine (niyazi, 2002) have been proved to have a good therapeutic effect on ra for a long time, and they were listed as the third and fourth representative items of national intangible cultural heritage by china intangible cultural heritage protection center in 2011 and 2014 respectively. another interesting example is that the use of live bees to sting specific acupoints or lesions of ra patients, as well as the injection of bee venom (son et al., 2007) , which is also widely spread in chinese folk and has a good effect on ra. furthermore, scraping, wax therapy, tai chi, five animals play, aerobic exercise, resistance training, and joint gymnastics also have a definite effect on ra. different medical systems have also focused on the role of diet in regulating ra. low salt intake, moderate alcohol consumption, high quality balanced diet rather than a single diet, dietary fruits administration or short-term fasting may reduce the intensity of the autoimmune response by reducing the intake of dietary antigens (basu et al., 2018) . what's more, reducing the intake of n-glycolyl-neuraminic acid and increasing the intake of unsaturated fatty acids, polyphenols, glycosides and flavonoids can reduce the inflammatory response and improve clinical symptoms of ra. moreover, there are effective therapeutic strategies such as ozone self-blood therapy and personalized gene targeting therapy (adkar et al., 2017) , stem cell therapy, mineral spring bath, infrared and ultraviolet therapy. compared with conventional therapies that target inflammatory cytokine biologics, more promising may be the technologies with different sources of membrane-coated drugs, such as neutrophil-coated nanoparticles, have a broad spectrum of anti-inflammatory effects for ra without identifying which inflammatory cytokines are involved and largely depending on the function of the candidate cells . therefore, the clinical efficacy of non-drug therapy as a supplement to drug therapy in treating ra should not be ignored. however, most of which lack the support of solid evidence-based medical evidence, making it difficult to provide molecular explanations for their efficacy from a scientific perspective. besides, since non-drug therapy is largely dependent on the years of clinical experience of rheumatologists, we strongly recommend that ra patients choose an officially recognized institution for proper treatment. furthermore, surgical treatment is an effective method once patients are ineffective in the later stages of medical treatment and accompanied by joint pain, deformity, limited movement and loss of function. depending on the patient's complaint, rheumatologists may choose synovectomy, joint fusion or artificial joint replacement (maderbacher et al., 2018) . in view of a series of problems existing in ra diagnosis, identification, drug efficacy, prognosis and risk assessment, the application of ai involving multiple algorithms has shown irreplaceable advantages in many links of ra disease. active shape models combined with x-ray images segmentation of the joint have perfectly realized the diagnosis and disease progression assessment of osteoarthritis (seise et al., 2007) . fuzzy inference systems (singh et al., 2012) or an artificial neural network (ann) model of serum associated inflammatory factors training in patients with arthritis (heard et al., 2014) can be used for early identification and diagnosis of arthritis. the scoring system involving ann also improve the accuracy of arthritis risk prediction and assessment (yoo et al., 2016) . considering the similarity of various types of arthritis, ra has a higher misdiagnosis rate. fortunately, the image analysis based on 3d bone microstructure imaging combined with multiple intelligent algorithms can avoid ra misdiagnosis and with a higher precision of genetic algorithm (akgundogdu et al., 2010) . for articular cartilage injury that may occur in ra, mri images segmentation based on voxel classification approaches may have potential in assessing articular cartilage damage (shim et al., 2009) . further integration with ann will help assess changes in cartilage volume during ra progresses (hafezi-nejad et al., 2017) . by combining with other machine learning algorithms, such as support vector machine (svm), random forest and naïve bayes, it may be more helpful to obtain more information about frontiers in pharmacology | www.frontiersin.org december 2021 | volume 12 | article 765435 cartilage damage parameters in ra patients (du et al., 2018) . meanwhile, this computer-aided mri image segmentation method can be used to evaluate wrist bone erosion and edema in ra patients (crowley et al., 2011) . the first mention of ai in the diagnosis of ra, multi-center electronic health records should be attached importance to integrate the clinical characteristics of ra by using active or deep learning in machine learning-based electronic phenotype algorithms, providing valuable clinical references for the diagnosis and prognosis of ra (norgeot et al., 2019) . in the early 1990s, despite of few data sources and simple algorithms, scientists have tried to build a computer-aided medical decisionmaking systems for evaluation and management of ra patients through inductive learning (kern et al., 1993) . considering the complex anatomy of human joints, the anatomic localization of individual joints is very important for orthopedic surgeons to diagnose digitized radiographs of ra. as joints space width (jsw) is narrowed by destroyed articular cartilage, an artificial neural network-based algorithm was firstly developed to determine the anatomical landmarks and jsw in joints of the hand for quantitative arthritis assessment instead of semiquantitative scoring systems of radiography (duryea et al., 2002) . its sensitivity to progressive ra assessment remains to be proven, although it is fully automated and has very little user interaction. better still, the position and erosive contours of ra bone radiograph are determined by active shape models of computer aided algorithms (langs et al., 2009) . and it turns out that active appearance models (aam) can be used to calculate the modeled shape and texture variation of ra associated joints. however, it is a sterner challenge for aam to analyze medical images of contour disorder. while, robust aam method can process gross disturbed images of ra patient's joints based on gray-value appearance to some extent, but it cannot process the seriously disturbed joints (beichel et al., 2005) . in order to screen out novel and sensitive image-based biomarkers of ra, rigid and nonrigid image registration and analysis algorithms were employed to automatically quantify changes of ankle joints in ra rats by serial magnetic resonance imaging (leung et al., 2006) . instead of subjective evaluation of ra progression based on visual inspection, the histopathological changes were thus assessed in a three-dimensional perspective, which will contribute to the development of ra candidate therapeutic agents. similarly, the use of computer-aided diffuse optical tomography for feature extraction and image classification is helpful in the diagnosis of ra (montejo et al., 2013a; . dynamika-ra software exclude the unrelated motion disturbance and other noise artefacts, shown in mri images of ra patients, by using 2d and 3d registration algorithms, improving the sensibility to evaluate early efficacy and quantify synovial joint inflammation in ra patients based on automated voxel-by-voxel analysis algorithms (kubassova et al., 2010) . by introducing machine learning methods including kernel learning, neural network and linear discriminant algorithms, the electromyographic response (er) acquisition from healthy and ra individuals during gait was recorded and analyzed (nair et al., 2010) . while measuring er of ra patients during gait is of little significance for clinical diagnosis of ra, it can be used to partially assess the rehabilitative degree of ra patients after receiving any drug or non-drug therapy. secondly, ai also plays a strong function in exploring the complications and pathogenesis of ra. as one of the complications induced by ra, interstitial lung disease has been quantified by application of a computer-assisted mevis pulmo 3d software (marten et al., 2009) . although it saves time and has high reproducibility, due to fewer recruited volunteers, no pattern-based analysis of ct scan, signal attenuation of high cavity organs, such as respiratory tract and bronchus, and limited number of threshold value, therefore, longitudinal investigations and larger cohorts of ra patients with pulmonary disease are needed to further confirmed the effectiveness of this system, and reasonable algorithms should be added to enlarge the scope of the system. hierarchical learning algorithm (megasnphunter) was proposed to statistically identify and quantify the high-order local interactions of multiple single nucleotide polymorphisms (snps) in ra, which was simulated by wellcome trust case control consortium. shockingly, more interactive patterns among snps of ra were identified and quantified (wan et al., 2009) . in order to apply the results of whole genome data of ra to the disease diagnosis and related targeted drugs development, however, changes in global and false-positive interactions of ra-induced snps need to be further properly managed. in fact, the interaction between genes caused by ra at the chromosomal level that are far beyond the extent to which current technological means can reach. therefore, it is advisable to apply the supervised machine learning including multi-step logical progressive algorithms and multiple statistical methods to the screening and verification of candidate target genes related to ra disease in the early stage of experiment or new drug development (briggs et al., 2010) . however, the complexity of ra-evoked genetic changes determines the complexity of algorithms and statistical methods, which should be kept in mind when we incipiently construct computer-aided algorithm models. although the genetic variants will affect the phenotype of ra patients, it is not an exact match. hence, the complete machine learning platform should include risk assessment and prediction of faulty interacting genes, so that preventive measures can be taken before risks occur (kruppa et al., 2012) . amazingly, a divinable model of ra mortality and risk factors based on machine learning method-random survival forests provides the possibility of risk aversion at the early stage of ra diagnosis (lezcano-valverde et al., 2017) . third, ai is also attracting a lot of interest from pharmaceutical companies and pharmacologists around the world. tnf-α converting enzyme (tace) inhibitors have been proven to have the potential to treat ra by inhibiting excessive tnf-α production. cong et al. firstly develop four machine learning models, including svm, k-nearest neighbor (k-nn), back-propagation neural network and c4.5 decision tree, for screening the inhibitory efficiency of various candidate compounds on tace. after that, the authors evaluated the reliability of the established model by using two separate methods: 5-fold cross-validation and independent evaluation (cong et al., 2009) . we expect that the above updated multiple mechanical learning models can guide the discovery of more frontiers in pharmacology | www.frontiersin.org december 2021 | volume 12 | article 765435 candidate compounds or pharmacodynamic groups acting on ra related biomarkers in the future, and provide an efficient and non-blind pre-screening for the development of ra treatment drugs. recently, the combination of machine learning and glycomics has shown great potential for screening more available potential serum markers in ra patients (chocholova et al., 2018) , which will undoubtedly lead clinicians to a deeper understanding of ra. at the 2017 annual meeting of the american association of immunologists, twoxar, inc., an ai-driven biopharmaceutical company, announced that they have developed an integrative ai-driven bioinformatics drug discovery platform for rapid identification and validation of novel ra drug candidates, lead compounds, and a deeper understanding of ra pathophysiology, which could contribute to expand alternative treatment strategies for ra focusing on new targets. due to its powerful computing, analysis and induction functions, cloud computing attracts clinical and scientific researchers to deal with massive disease data obtained through proteomics, genomics, nucleic acid omics and intestinal microbial differences (feng et al., 2017; gibbs, 2017; langmead and nellore, 2018; koppad et al., 2021) . although cloud computing is still in the conceptual attempt stage in the medical field, we have reason to believe that the application of cloud computing in ra will bring new opportunities for ra diagnosis, treatment, drug development and disease prognosis. in general, in addition to conventional ra diagnosis methods and new drugs development processes, data mining, machine learning and cloud computing are still in the early stages of ra diagnosis and prognosis, biomarkers and lead compounds screening. figure 5 proposes a reasonable conceptual model of ai-assisted deep learning and cloud computing for ra diagnosis and treatment. whereas, in the figure 5 | ai-assisted deep learning and cloud computing conceptual model for ra diagnosis and treatment. frontiers in pharmacology | www.frontiersin.org december 2021 | volume 12 | article 765435 long run, these preclinical and clinical applications of computerassisted methods will renovate current medical practice over the next two or 3 decades. ai has laid a scientific and technological foundation for the diagnosis and treatment system of ra in traditional medicine and western medicine, and the ai algorithm based on cloud data sharing is an indispensable part. data acquisition and authenticity is our primary concern, these data mainly come from electronic medical record system of medical institutions, disease registration system, medical insurance system, public health survey and public health monitoring, birth/death registration database, genomics database, and other data generated in the process of disease prevention and health management zhu, 2020; sun et al., 2021) . although integrated medical information, acquired by data mining, deep learning and cloud computing have been emerging in the diagnosis and treatment decision making of ra, there are still aspects worth further improvement and optimization. the first thing to point out is that reliable information on the etiology, dynamic course and prognosis, as well as multiple and effective treatment strategies of ra, requires the selfless dedication and close cooperation of clinical and scientific researchers worldwide. primary clinical and scientific data on ra, including epidemiology, clinical and basic investigation, and traditional medical diagnostic methods, as well as potential targets and pharmacological mechanisms of various tcm/ethnic drug preparations or individual drugs should be selflessly uploaded and shared in the field in an officially authorized online database, so authenticity of the original data and timeliness of data sharing are critical. secondly, reasonable and efficient computer algorithms, which contribute to the intelligent and efficient integration of all information related to ra disease should be updated technically in a timely manner. interdisciplinary professionals in fields such as medicine and informatics or computer science, should be competent to leverage medical big data to facilitate ra. however, while narrowing the distance between patients with ra and therapists worldwide under the background of internet, it also increases the risk of patient privacy disclosure, which should not be ignored (craig, 2016; price and cohen, 2019) . in addition, we need to clearly recognize that ai can perform multifactor analysis of complex systems and find patterns in fragmented data to support processes such as disease diagnosis and drug discovery. however, ai cannot replace the diagnosis and treatment process of diseases, especially the spiritual comfort and deep communication for patients (car et al., 2019) . not every ra patient can be accurately diagnosed and treated according to conventional medical means. to achieve excellent values for integrated medical big data of ra, therefore, reasonable protocols should be developed to determine when and where to use data mining, deep learning, and cloud computing to assist ethnic medicine in ra diagnosis and treatment. in summary, considering the conclusive defects drawn by the three abovementioned methods in processes of data acquisition, processing and integration, the conclusions thus cannot effectively be used to guide and apply to clinical treatment of ra. we sincerely hope that in the era of medical big data, more detailed disease information for ra patients should be integrated together through reasonable of ai algorithms. relatively novel ra diagnosis and treatment strategies thus could further guide different ethnic medicine around the world to better serve the clinical ra patients, and improve the quality of the survival of ra patients. but by comparison, diverse data sources, reasonable computer algorithms and machine simulation training models can make ai-assisted deep learning and cloud computing have considerable applications in ra diagnosis and treatment. sw designed the research, conducted a literature search, and wrote the entire manuscript. yh and xl revised and edited the illustrations in the manuscript. yh and xm revised and edited the manuscript. xw and yz provided ideas for the manuscript and introduced the theme. all authors agreed with the current version of the manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.765435/ full#supplementary-material genome engineering for personalized arthritis therapeutics 3d image analysis and artificial intelligence for bone disease classification diagnosis and management of rheumatoid arthritis: a review frontiers in pharmacology | www.frontiersin.org diagnosis and management of rheumatoid arthritis in adults: summary of updated nice guidance micro-ct evaluation of rheumatoid arthritis mouse model disease progression: manual tracings versus semi-automated routines a noncanonical role for the engulfment gene elmo1 in neutrophils that promotes inflammatory arthritis ocular inflammatory diseases associated with rheumatoid arthritis rheumatoid arthritis, tnf inhibitors, and non-melanoma skin cancer relationship of patient-reported outcomes with mri measures in rheumatoid arthritis dietary fruits and arthritis robust active appearance models and their application to medical image analysis oral and subcutaneous administration of a near-infrared fluorescent molecular imaging agent detects inflammation in a mouse model of rheumatoid arthritis artificial intelligence in cancer imaging: clinical challenges and applications mechanisms of tissue damage in arthritis supervised machine learning and logistic regression identifies novel epistatic risk factors with ptpn22 for rheumatoid arthritis detection and characterisation of bone destruction in murine rheumatoid arthritis using statistical shape models research progress of traditional chinese medicine on rheumatoid arthritis dna methylation-based classification of central nervous system tumours beyond the hype of big data and artificial intelligence: building foundations for knowledge and wisdom mechanisms involved in triggering rheumatoid arthritis antiinflammatory effects of bt-201, an n-butanol extract of panax notoginseng, observed in vitro and in a collagen-induced arthritis model korean red ginseng exhibits no significant adverse effect on disease activity in patients with rheumatoid arthritis: a randomized, double-blind, crossover study glycomics meets artificial intelligence -potential of glycan analysis for identification of seropositive and seronegative rheumatoid arthritis patients revealed introduction to machine learning fibroblasts in rheumatoid arthritis prediction of novel and selective tnf-alpha converting enzyme (tace) inhibitors and characterization of correlative molecular descriptors by machine learning approaches challenges in the treatment of rheumatoid arthritis porphyromonas gingivalis experimentally induces periodontis and an anti-ccp2-associated arthritis in the rat understanding the links between privacy and public data sharing distinct fibroblast subsets drive inflammation and damage in arthritis one year in review 2019: pathogenesis of rheumatoid arthritis measuring bone erosion and edema in rheumatoid arthritis: a comparison of manual segmentation and ramris methods diagnosis and management of rheumatoid arthritis; what is the current role of established and new imaging techniques in clinical practice? curcumin alleviates rheumatoid arthritis-induced inflammation and synovial hyperplasia by targeting mtor pathway in rats talk to your gut: the oral-gut microbiome axis and its immunomodulatory role in the etiology of rheumatoid arthritis a novel method to predict knee osteoarthritis progression on mri using machine learning methods neural network based automated algorithm to identify joint locations on hand/wrist radiographs for arthritis assessment integrating artificial and human intelligence: a partnership for responsible innovation in dermatologist-level classification of skin cancer with deep neural networks herb: a high-throughput experiment-and reference-guided database of traditional chinese medicine sex and management of rheumatoid arthritis rapid response fluorescence probe enabled in vivo diagnosis and assessing treatment response of hypochlorous acid firmiana: towards a one-stop proteomic cloud platform for data processing and analysis methods for segmentation of rheumatoid arthritis bone erosions in high-resolution peripheral quantitative computed tomography (hr-pqct) immunopathogenesis of rheumatoid arthritis risk of biologics and glucocorticoids in patients with rheumatoid arthritis undergoing arthroplasty: a cohort study a test drive of a dna-analysis toolkit in the cloud quercetin attenuates zymosan-induced arthritis in mice research progress of mongolian medicine in treatment of rheumatoid arthritis rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies ultrasound in management of rheumatoid arthritis: arctic randomised controlled strategy trial prediction of medial tibiofemoral compartment joint space loss progression using volumetric cartilage measurements: data from the fnih oa biomarkers consortium flavonoid quercetin-methotrexate combination inhibits inflammatory mediators and matrix metalloproteinase expression, providing protection to joints in collagen-induced arthritis methotrexate monotherapy and methotrexate combination therapy with traditional and biologic disease modifying antirheumatic drugs for rheumatoid arthritis: abridged cochrane systematic review and network meta-analysis the practical implementation of artificial intelligence technologies in medicine a computational method to differentiate normal individuals, osteoarthritis and rheumatoid arthritis patients using serum biomarkers artificial intelligence in radiology highland mate: edible and functional foods in traditional medicine for the prevention and treatment of hypoxia-related symptoms zhuang medicine line point moxibustion therapy sinomenineinduced histamine release-like anaphylactoid reactions are blocked by tranilast via inhibiting nf-κb signaling comparison of combination therapy with methotrexate and sinomenine or leflunomide for active rheumatoid arthritis: a randomized controlled clinical trial systems pharmacology-based dissection of mechanisms of tibetan medicinal compound ruteng as an effective treatment for collagen-induced arthritis rats racial or ethnic differences in allele frequencies of single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene and their magnetic resonance imaging in individuals at risk of rheumatoid arthritis is type 2 diabetes mellitus an inverse risk factor for the development of rheumatoid arthritis? potential pharmacologic targets for the prevention of rheumatoid arthritis polygenic burdens on cell-specific pathways underlie the risk of rheumatoid arthritis racial variation in rheumatoid arthritis gold thread acupuncture for rheumatoid arthritis muc5b promoter variant and rheumatoid arthritis with interstitial lung disease cannabinoids for the treatment of rheumatic diseases -where do we stand? nimbidin suppresses functions of macrophages and neutrophils: relevance to its antiinflammatory mechanisms identifying medical diagnoses and treatable diseases by image-based deep learning medical decision making based on inductive learning method cloud computing enabled big multi-omics data analytics deep learning for cardiovascular medicine: a practical primer risk estimation and risk prediction using machine-learning methods a computer-aided detection system for rheumatoid arthritis mri data interpretation and quantification of synovial activity clinical value of fdg-pet/ct for the evaluation of rheumatic diseases: rheumatoid arthritis, polymyalgia rheumatica, and relapsing polychondritis advancement in contemporary diagnostic and therapeutic approaches for rheumatoid arthritis identifying associations between somatic mutations and clinicopathologic findings in lung cancer pathology reports amelioration of oxidative and inflammatory changes by swertia chirayita leaves in experimental arthritis local baff gene silencing suppresses th17-cell generation and ameliorates frontiers in pharmacology | www autoimmune arthritis cloud computing for genomic data analysis and collaboration automatic quantification of joint space narrowing and erosions in rheumatoid arthritis application of deep learning to the diagnosis of cervical lymph node metastasis from thyroid cancer with ct panax ginseng: a candidate herbal medicine for autoimmune disease automatic quantification of changes in bone in serial mr images of joints development and validation of a multivariate predictive model for rheumatoid arthritis mortality using a an integrative metabolomic and network pharmacology study revealing the regulating properties of xihuang pill that improves anlotinib effects in lung cancer traditional chinese medicine and lung cancer--from theory to practice exploration of the pathogenisis theory of "dampness-heat-stasis" in rheumatoid arthritis an artificial intelligence platform for the multihospital collaborative management of congenital cataracts artificial intelligence-biology: an important strategic resource and competitive hot spot in the future research of tcm modernization comparison of tripterygium wilfordii hook f with methotrexate in the treatment of active rheumatoid arthritis (trifra): a randomised, controlled clinical trial the knee joint in rheumatoid arthritis-current orthopaedic surgical treatment options rheumatoid arthritis: glucocorticoids are associated with nearly double infection risk post-surgery the immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting potential therapeutic effect of curcumin loaded hyalurosomes against inflammatory and oxidative processes involved in the pathogenesis of rheumatoid arthritis: the use of fibroblast-like synovial cells cultured in synovial fluid computer-assisted quantification of interstitial lung disease associated with rheumatoid arthritis: preliminary technical validation pathogenetic insights from the treatment of rheumatoid arthritis risk of venous thromboembolism in rheumatoid arthritis, and its association with disease activity: a nationwide cohort study from sweden computer-aided diagnosis of rheumatoid arthritis with optical tomography, part 1: feature extraction computer-aided diagnosis of rheumatoid arthritis with optical tomography, part 2: image classification the application of machine learning algorithms to the analysis of electromyographic patterns from arthritic patients general situation of tujia medical treatment of rheumatoid arthritis digital pathology and artificial intelligence study on treatment of rheumatic diseases with turpan sand in xinjiang assessment of a deep learning model based on electronic health record data to forecast clinical outcomes in patients with rheumatoid arthritis genetics of rheumatoid arthritis medications associated with fracture risk in patients with rheumatoid arthritis research status of dai medicine for treatment of rheumatoid arthritis ai in medical imaging informatics: current challenges and future directions dynamic alterations in the gut microbiota of collagen-induced arthritis rats following the prolonged administration of total glucosides of paeony web service makes big data available to neuroscientists privacy in the age of medical big data an analysis of moxibustion therapy in hui hui prescription rheumatoid arthritis, anti-tumour necrosis factor treatment, and risk of squamous cell and basal cell skin cancer: cohort study based on nationwide prospectively recorded data from sweden pathologically expanded peripheral t helper cell subset drives b cells in rheumatoid arthritis machine learning and ai-based approaches for bioactive ligand discovery and gpcr-ligand recognition comparative studies of different organs of nyctanthes arbortristis in frontiers in pharmacology | www modulation of cytokines in murine model of arthritis chinese herbal medicine for the treatment of small intestinal bacterial overgrowth (sibo): a protocol for systematic review and meta-analysis digital image analysis in breast pathology-from image processing techniques to artificial intelligence ultrasonographic criteria for the diagnosis of erosive rheumatoid arthritis using osteoarthritic patients as controls compared to validated radiographic criteria quantitative assessment of synovitis in patients with rheumatoid arthritis using fluorescence optical imaging the etiology of rheumatoid arthritis learning active shape models for bifurcating contours atherosclerotic cardiovascular disease prevention in rheumatoid arthritis derivation, validation, and potential treatment implications of novel clinical phenotypes for sepsis network pharmacology-based analysis of xiao-xu-ming decoction on the treatment of alzheimer's disease knee cartilage: efficient and reproducible segmentation on high-spatial-resolution mr images with the semiautomated graph-cut algorithm method repurposing high-throughput image assays enables biological activity prediction for drug discovery the jak/stat pathway: a focus on pain in rheumatoid arthritis diagnosis of arthritis through fuzzy inference system rheumatoid arthritis rheumatoid arthritis therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds rheumatoid arthritis progress and current considerations of artificial intelligence in the field of high-definition medicine artificial intelligence in cardiac radiology artificial intelligence and machine learning for protein toxicity prediction using proteomics data megasnphunter: a learning approach to detect disease predisposition snps and high level interactions in genome wide association study establishment of a lung cancer discriminative model based on an optimized support vector machine algorithm and study of key targets of wogonin in lung cancer treatment of rheumatoid arthritis using combination of methotrexate and tripterygium glycosides tablets-a quantitative plasma pharmacochemical and pseudotargeted metabolomic approach curcumin attenuates collagen-induced rat arthritis via anti-inflammatory and apoptotic effects big data requirements for artificial intelligence systems study on the antirheumatic mechanism of tibetan medicated-bath therapy using wuwei-ganlu-yaoyu-keli contemporary physicians' understanding and clinical experience summary of rheumatoid arthritis traditional herbal medicine: therapeutic potential in rheumatoid arthritis predicting meridian in chinese traditional medicine using machine learning approaches an update on potential biomarkers for diagnosing diabetic foot ulcer at early stage the impact of artificial intelligence on traditional chinese medicine sodium tanshinone iia sulfonate inhibits proliferation, migration, invasion and inflammation in rheumatoid arthritis fibroblast-like synoviocytes increased dna methylation variability in rheumatoid arthritis-discordant monozygotic twins fine-mapping and functional studies highlight potential causal variants for rheumatoid arthritis and type 1 diabetes the immunology of rheumatoid arthritis preventing rheumatoid arthritis: a global challenge artificial intelligence with deep learning technology looks into diabetic retinopathy screening mongolian medicine treatment of rheumatoid arthritis two types of fibroblast drive arthritis total glucosides of paeony can reduce the hepatotoxicity caused by methotrexate frontiers in pharmacology | www leflunomide combination treatment of active rheumatoid arthritis artificial intelligence-based diagnosis of diabetes mellitus: combining fundus photography with traditional chinese medicine diagnostic methodology a comprehensive review of integrative pharmacology-based investigation: a paradigm shift in traditional chinese medicine artificial intelligence: a powerful paradigm for scientific research. the innovation 2 curcumin in autoimmune and rheumatic diseases quercetin attenuates collagen-induced arthritis by restoration of th17/treg balance and activation of heme oxygenase 1-mediated anti-inflammatory effect a brief analysis of tujia medicine treating "swollen knot wind" with oil fire. guide china med a nir-ii fluorescent probe for articular cartilage degeneration imaging and osteoarthritis detection ameliorative effects of ginseng and ginsenosides on rheumatic diseases simple scoring system and artificial neural network for knee osteoarthritis risk prediction: a cross-sectional study defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry discovery of anti-flu substances and mechanism of shuang-huang-lian water extract based on serum pharmaco-chemistry and network pharmacology a unified smart chinese medicine framework for healthcare and medical services neutrophil membrane-coated nanoparticles inhibit synovial inflammation and alleviate joint damage in inflammatory arthritis mechanisms involved in the therapeutic effects of paeonia lactiflora pallas in rheumatoid arthritis the oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment prevention and treatment of covid-19 using traditional chinese medicine: a review research progress of tcm syndromes types of rheumatoid arthritis in recent ten years clinical observation of rheumatoid arthritis treatment with zhuang needle-pricking therapy progress on treatment of rheumatoid arthritis with ethnodrugs big data and artificial intelligence modeling for drug discovery a primer on deep learning in genomics the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-0075000-83k6qcir authors: wu, ning; yuan, taohua; yin, zhixin; yuan, xiaotian; sun, jianfei; wu, zunqiu; zhang, qilong; redshaw, carl; yang, shenggang; dai, xiaotian title: network pharmacology and molecular docking study of the chinese miao medicine sidaxue in the treatment of rheumatoid arthritis date: 2022-02-19 journal: drug des devel ther doi: 10.2147/dddt.s330947 sha: ae73d43adec52baa35ea717c6c4985c7c2306488 doc_id: 75000 cord_uid: 83k6qcir purpose: this study aimed to investigate the molecular mechanisms of compound sidaxue (sx), a prescription of chinese miao medicine, in treating rheumatoid arthritis (ra) using network pharmacology and in vivo experimental approaches. methods: network pharmacology was adopted to detect the active components of four traditional chinese herbal medicine (tcm) of sx, and the key targets and signaling pathways in the treatment of ra were predicted, and the key components and targets were screened for molecular docking. the predicted targets and pathways were validated in bovine type ii collagen and incomplete freund’s adjuvant emulsifier-induced rat ra model. results: in this study, we identified 33 active components from sx, predicted to act on 44 ra-associated targets by network pharmacology. ppi network demonstrated that tnf-α, vegf-a, il-2, il-6, akt, pi3k, stat1 may serve as the key targets of sx for the treatment of ra. the main functional pathways involving these key targets include pi3k-akt signaling pathway, tnf signaling pathway, nf-κb signaling pathway. molecular docking analysis found that the active components β-amyrin, cajanin, eleutheroside a have high affinity for tnf-α, vegfa, il-2, akt, and pi3k, etc. sx can improve joint swelling in collagen-induced arthritis (cia) rats, reduce inflammatory cell infiltration and angiogenesis in joint synovial tissue, and down-regulate il-2, il-6, tnf-α, vegf, pi3k, akt, p-akt, nf-κbp65, the expression of p-nf-κbp65, stat1, and ptgs2 are used to control the exacerbation of inflammation and alleviate the proliferation of synovial pannus, and at the same time play the role of cartilage protection to achieve the effect of treating ra. conclusion: through a network pharmacology approach and animal study, we predicted and validated the active compounds of sx and their potential targets for ra treatment. the results suggest that sx can markedly alleviate cia rat by modulating the vegf/pi3k/akt signaling pathway, tnf-α signaling pathway, il/nf-κb signaling pathway. rheumatoid arthritis (ra) is a common type of chronic inflammatory autoimmune disease that primarily affects joints, but in severe cases, also affects internal organs, such as the lung, heart, and kidney. ra is clinically characterized by a sudden onset of chronic and progressive joint swelling, pain, and joint deformities, which gradually cause joint dysfunction and eventually disability. 1-3 ra, as a refractory disease, has the highest disability rate among all joint diseases, affecting between 0.5% and 1% of the global population, and is most common amongst the elderly. further analysis reveals that the incidence for women is 2 to 3 times that of men, and is one of the main reasons for human loss of similar to that of positive drug tripterygium wilfordii, and furthermore sx can alleviate the joint swelling of freund's adjuvant-induced arthritis aa rats, but the specific mechanism of action is unknown. [13] [14] [15] traditional chinese medicine (tcm) has a long history of viewing an individual or patient as a system with varying status and has over the years accumulated numerous herbal formulae. the holistic philosophy of tcm shares much with the key ideas of emerging both network pharmacology and biology, and meets the requirements of overcoming complex diseases. network pharmacology is a technique used to systematically evaluate the pharmacological effects of drugs with multiple components and multiple targets by establishing links between targets, drugs, and diseases based on the principles of systems biology, with the latter sharing a similar holistic philosophy to tcm. [27] [28] [29] the components of tcm are complex and diverse, and the composition of the prescription is more complicated. there is an urgent need to establish research strategies and new methods that can reflect the overall characteristics of tcm. [27] [28] [29] in recent years, network pharmacology has integrated the three aspects of the active ingredients of traditional chinese medicine, related corresponding targets to corresponding diseases, and using them as three different types of nodes to form a "component-target-disease" network. network analysis methods are used to reveal the effective components and mechanism of action of chinese medicine in the treatment of related diseases. [27] [28] [29] therefore, network pharmacology is an important means to analyze the effective components and therapeutic targets of tcm. [27] [28] [29] as a widely used virtual screening method, molecular docking technology can study the interaction between small molecules and receptors, and thereby can predict binding modes and affinities. 27 at this stage, a number of studies have adopted network pharmacology and molecular docking technology to explore the material basis and mechanism of action of chinese medicine in treating certain diseases, and have determined the some of the underlying principles and feasibility of these technical methods. 30 in this study, we have applied network pharmacology and molecular docking through data mining and analysis, to screen and predict potential target molecules and signal pathways for sx active components in ra treatment. key candidate targets were further verified in animal models of ra. our results provide molecular insights into the mechanism of miao medicine prescription sx in the treatment of ra, and will inform further development of anti-ra drugs from sx. the following equipment was employed: a syngene™ gel imaging system (synoptics, usa), an abi steponeplus™ real-time fluorescent quantitative pcr instrument (thermo, usa), nanodrop2000 (thermo fisher scientific, waltham, ma, united states), microplate reader (thermo fisher, usa), electrophoresis apparatus(dyy-6d, china), sorvall™ st 8 small benchtop centrifuge(thermo fisherscientific, usa). the active compounds in sx were collected from the tcm systems pharmacology database and analysis platform (tcmsp;https://old.tcmsp-e.com/tcmsp.php), 31 chemistry database (http://www.organchem.csdb.cn/scdb/default.htm? ncount=13303008), 32 and pubmed (https://pubmed.ncbi.nlm.nih.gov/?term=). 32 the absorption, distribution, metabolism, and excretion (adme) profiles of these compounds were obtained via literature searches and used to facilitate the screening of effective ingredients. 33 the results from the three databases were integrated and duplicate output molecules were removed. to select for active compounds from the four herbal medicines, the raw results were further filtered using two important drug qualitative indicators, ie, oral bioavailability (ob, cut-off ≥ 30%) and drug-like properties (dl, cutoff ≥ 0.18). cytoscape version 3.6.1 software was used to construct the sx-active ingredient map. the corresponding targets of the putative compounds were searched in the tcmsp and the swiss target prediction database (http://www. swisstargetprediction.ch/). 35, 36 to expand the potential number of targets, the molecular similarity match tool, the simplified molecular input line entry system (smiles) in pubchem (https://pubchem.ncbi.nlm.nih.gov/#query= nevadensia) 35 was used. these two groups of targets were entered into the uniprot database with the organism defined as "homo sapiens" and were considered sx potential targets. uniprot (https://www.uniprot.org) was used to find gene names and uniprot numbers of the putative target genes. 35 the key word "rheumatoid arthritis" was used to search therapeutic target database (ttd) (http://db.idrblab.net/ttd/), 34 drugbank (https://www.drugbank.ca/), 34 and disgenet (https://www.disgenet.org/) 36 databases for ra-associated targets that have been experimentally verified. intersections of the targets were subjected to deduplication and their uniprot numbers were retrieved. 36 a venn diagram was drawn from the predicted sx active components and hypothetic ra-associated targets with venny2.1 software, 28 to identify potential sx therapeutic targets in ra. these targets were entered into the string version 10.5 database (https://string-db.org/) 28 to obtain the protein-protein interaction (ppi) diagram. the search was limited to human species, the protein interaction confidence score threshold was set to 0.4, and disconnected nodes were hidden in the network representation. 28 the ppi network was visualized using cytoscape version 3.6.1 software 28 to obtain the ppi diagram between the sx active components and ra-associated proteins. a network analyzer was used to analyze the network topology characteristics, including the degree centrality, betweenness centrality, and closeness centrality, and to identify core nodes of the ppi network with a degree value greater than 2-fold the average degree. active ingredients from the four herbal medicines that meet the criteria of ob ≥ 30% and dl ≥ 0.18 and their raassociated targets were selected and entered in cytoscape 3.6.1 to draw a network diagram of the sx active compounds and their therapeutic targets for ra. go enrichment and kegg pathway enrichment analyses of key targets were conducted using the david 6.8 database (http://www.david.niaid.nih.gov). 34 for go analysis, biological process (bp), cell composition (cc) and molecular function (mf) were selected. kegg pathway enrichment analysis used p < 0.05 as the cut-off value and the top 20 key pathways linked to arthritis were retained. 37 in silico molecular docking the pretreatment of ligands and target proteins was carried out by sybyl-x2.0. pretreatment of ligands consisted of gasteiger-huckel charges to small molecules, maximum number of optimization iterations was set at 10,000, with a gradient convergence value of 0.005 kcal/(mol*a), and with application of powell's energy optimization algorithm; all other values were set to default parameters. pre-treatment of target proteins consisted of removal of crystal water from all proteins, hydrogenation, removal of co-crystal ligand molecules occupying the binding sites, addition of missing amino acid residues and terminal residues, and application of kollman united/kollman all-atom force field and assisted model building with energy refinement (amber) charges to minimize the protein. autodocktools (adt) was used to https://doi.org/10.2147/dddt.s330947 drug design, development and therapy 2022:16 438 generate pdbqt files, and finally autodock-vina was used to calculate nine favored binding conformations. the binding energy between each ligand and its target with the highest score, ie, presenting with the conformation with the strongest binding capacity, was calculated. pymol software was used for visualization. 38 in this experiment, the molaical 39 program was used to calculate the lipinski rule of five parameters of these compounds. in order to meet the requirements of this program, all compounds should be converted to mol2.dat file format and all parameters were set to their default values prior to calculation. a total of 2 kg of the four herbal medicines constitutive of sx at a jxt:dxt:flzxgp:hgt weight ratio of 15:22:15:8 was boiled in 1000 ml of water and filtered with a gauze to obtain 500 ml of decoction. the herbal medicines were boiled again in 1000 ml of water and filtered with a gauze to obtain another 500 ml of decoction. the two 500-ml decoctions were combined and boiled again until the volume was reduced to 500 ml. this final solution was used as the test drug and stored in the refrigerator at 4°c for up to 1 week. [13] [14] [15] the final concentration of the drug corresponds to 4 kg of crude herbal medicine/l. all experimental procedures involving animals were approved by the institutional animal care and use committee (iacuc) (ethical number:1900868).the cia model rat was established: bovine type ii collagen at the concentration of 2 mg/ml was mixed at equal volume with incomplete freund's adjuvant on ice until uniformly and fully emulsified. 60 normal sd rats were injected with 200 μl of emulsifier subcutaneously at the base of the tail, and 10 blank control rats were injected with the same amount of normal saline at the same site. one week later, a second dose of 100 μl of emulsion was subcutaneously injected at the tail root to boost the immune response. the normal control group was injected with the same amount of normal saline subcutaneously at the tail root. the arthritis index (ai) score was determined to check for successful induction of arthritis. 40 fifty rats with successfully established cia were randomly assigned to the sx high-dose group (sx 40 g/kg group), sx medium-dose group (sx 20 g/kg group), sx low-dose group (sx 10 g/kg group), gtw-treated control group, and the model group (mod) according to the random number table method. in addition, the control group (nor) was established, which totaled 6 experimental groups, each with 10 animals. after the last injection, the hind foot joints of the rats were measured once per week for ai scoring, 13 which considered the redness, swelling, and joint deformities. 41 symptoms were given 0-4 scores as follows: 0 points (no redness or swelling), 1 point (red spots or mild joint swelling), 2 points (moderate redness and joint swelling), 3 points (moderate redness and joint swelling accompanied by mild dysfunction), and 4 points (severe swelling, stiffness and even joint deformity, and severe dysfunction). a score of 2 or more was considered successful ra development. the volume of the hind feet of the rats was measured by water displacement every week. an average value of 3 measurements was used to calculate the degree of toe swelling. the swelling degree (er) was calculated according to er (%) = (va − vb)/vb × 100% (where vb is the volume before immersion, and va is the volume after immersion). 42 two days after the successful establishment of cia, the drug was given by gavage once a day for 21 consecutive days. the sx 40 g/kg group, sx 20 g/kg group,sx 10 g/kg group were treated at different sx doses, gtw group was administered with gtw, and the blank and model groups were treated with saline. the cia rats were anesthetized with 10% chloral hydrate on the day following the last drug administration. when the rats were under final anesthesia, the blood was collected by cardiac puncture and centrifuged to obtain serum for elisa. the rats were killed by anesthetics overdose and placed on ice for collection of the inflamed hind legs. part of the leg joints were fixed in 4% paraformaldehyde for h&e staining and the remaining leg joints were stored in a −80°c freezer. rat hind leg ankle joints were fixed with 10% paraformaldehyde for 24 h, rinsed in 0.2 m phosphate buffer solution, and then decalcified in edta decalcification solution at room temperature. decalcification was checked every day and the decalcification solution was replaced once a week for one month. samples were then rinsed with distilled water for 25 min., dehydrated through a series of graded ethanol, cleared in xylenes, embedded in paraffin, cut into 3-5 μm tissue sections, and h&e stained. the histomorphological and pathological changes of the ankle synovium were analyzed under a microscope. 43 content of vegf-a, il-6, il-2, tnf-α protein after 24 hours, the final medication involved several steps, namely: 1) anesthesia with 10% chloral hydrate solution 0.4ml/100 g intraperitoneal injection; 2) taking blood from the abdominal aorta; 3) after 2 h at room temperature, 3000 r/min., 4°c centrifugation (radius 13.5 cm) for 10 min.; 4) the supernatant is kept in a −80°c refrigerator; 5) serum vegf-a, il-6, il-2, tnf-α levels in rats were measured by elisa method. the operation was carried out strictly according to the instructions of the kit. vegf-a, il-6, il-2, tnf-α levels in serum were calculated. 44 measurement of vegf-a, pi3k, akt, nf-κbp65, start1, ptgs2 mrna level in synovial tissues of cia rats by qrt-pcr trizol was used to extract the total rna from the rat synovial tissues. one microgram of the total rna was used for reverse transcription. primers were designed according to the sequence of rat vegf, pi3k, akt, nf-κb, stat1, ptgs2 and gapdh ( table 1 ). the pcr reactions were carried out according to the instructions provided in the used qpcr reaction kit (baori biotechnology, beijing, china). the reaction conditions were 5 min. of denaturation at 95°c, followed by 40 cycles of 95°c for 10 sec., and 60°c for 30 sec.; melting curves were obtained by heating the reaction at 95°c for 15 sec., 60°c for 60 sec., and 95°c for 15 sec. the results were calculated by the 2 −δδct method. 45 quantification of pi3k, akt, p-akt, nf-κbp65, p-nf-κbp65 protein levels in synovial tissues by western blot rat synovial tissues were ground in liquid nitrogen and the protein concentration was measured using a bicinchoninic acid assay (bca) kit. 20μl (40μg) of denatured proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and transferred to a pvdf membrane. the membrane was blocked overnight with 5% skimmed milk and incubated with the primary antibodies: anti-beta-actin diluted at 1:7000 at room temperature for 2 h, or anti-pi3k, akt, p-akt, nf-κbp65, p-nf-κbp65 antibodies diluted at 1:1000, at 4°c overnight. after 3 washes of 10 min. in tbst (ph 7.5, tris-hcl 10 mm, nacl 150 mm, tween-20 0.05%), the membrane was incubated with anti-rabbit (1:1000) or anti-mouse (1:5000) secondary antibodies at room temperature for 90 min. the membrane was then washed in 1 × tbst, developed and analyzed on a gel imager. 46 ob is an important parameter of drug absorption and metabolism. the higher the value, the better the biological activity and drug-like dl of the drug. therefore, a standard ob ≥ 30% and a dl ≥ 0.18 are commonly used in the literature as the selection criteria for active ingredient screening. tcmsp(https://old.tcmsp-e.com/tcmsp.php), traditional chinese medicine and chemical composition database(http://www.organchem.csdb.cn/scdb/default.htm?ncount=13303008), and pubmed(https://pubmed.ncbi.nlm.nih.gov/?term=) were searched for the chemical components of the four traditional chinese medicines in sx. after duplications were removed and the low confident hits eliminated by a cut-off score of 30% for ob and 0.18 for dl, a total of 33 active compounds were identified, including 5 compounds from jxt (15.15%), 11 from dxt (33.33%), 13 from flzxgp (39.39%), and 4 from hgt (12.12%) ( table 2 ).the network diagram for the active components of chinese herbal medicine was established (figure 1 ), and the triangle (or yellow) represents the drug, and the circle represents the active ingredient (blue individual drug ingredient, green represents the intersecting active ingredient). in the network diagram, according to the degree of value, jxt and flzxgp play a major role in the compound sx, and the first three active compounds were β-sitosterol (+)-catechin, ent-epicatechin ( figure 1 or supplementary table s1 ). in addition, a total of 457 putative protein targets of these compounds were identified using the tcmsp and the swiss target predication databases. the ra-related target genes were screened from the ttd (http://db.idrblab.net/ttd/), drugbank (https://www.drugbank. ca/), and disgene databases (https://www.disgenet.org/). using the keyword "rheumatoid arthritis" to search the databases, 306 associated genes were identified. from the comparative analysis between the 306 ra-related and 457 putative sx targets, 44 were predicted as sx targets for the treatment of ra ( figure 2 , supplementary table s2 ). 44 targets were entered into the string version 10.5 database, and then the cytoscape version 3.6.1 software was used for visualization processing to obtain the protein interaction (ppi) network diagram of the sx treatment of ra ( figure 3 ). the circle represents the target point. the color of the target point gradually changes according to the degree value. the larger the degree value, the larger the circle.the degree value is from large to small, and the color gradually changes from orange to green (at the same time, the graph is divided into 3 levels, the inner layer to the middle layer to the outer layer represents the degree value from large to small). the figure includes 48 nodes and 237 edges, where the node represents the target gene, and the edge represents the line connecting the node, the same node is passed by multiple lines, indicating that the more important. the average degree of freedom is 9.875. there are 6 targets that are more than twice the average degree of freedom. there are tnf, il6, vegf-a, cxcl8, ptgs2, il2 in descending order of degrees of freedom (table 3 , figure 3 ).(see supplementary table s3 for other target point degree values.) the sx-compounds-ra ppi network included 242 nodes and 743 edges ( figure 4 ). the inverted triangle represents the drug, the oblique square represents the component, and the circle represents the target. the color and size of the graph gradually changed from deep to shallow (or from orange to green) and large to small according to the degree of freedom. the larger the degree value was, the more important role the part played in the ppi. the average node degree was 6.14. according to the degree of freedom, it can be seen that the blood root bark of jxt and flzxgp are the main drugs, and the network construction for herbs-active ingredient. notes: in figure 1 , the triangle (or yellow) represents the drug, and the circle represents the active ingredient (blue individual drug ingredient, green represents the intersecting active ingredient). ingredient targets disease targets go biological processes and kegg pathways were further analyzed to infer potential biological functions to sx targets, with a threshold p value < 0.05. in the go function analysis ( figure 5 ), the top 10 items of biological function are listedon the vertical axis, including molecular function(mf), cell composition(cc), and biological process(bp), the horizontal axis in the figure represents the statistical p value of biological functions. in figure 5 , blue represents mf, green represents cc, and pink represents bp. among them, molecular function (mf) mainly includes lipid binding, protein dimerization activity, and protein homodimerization activity, endopeptidase activity, cytokine activity, peptidase activity, calcium ion binding, identical protein binding, identical protein binding, identical protein binding. from the mf-target map ( figure 6 ), we can see that the larger the degree value, the larger the circle.the degree value is from large to small, and the color gradually changes from orange to green (at the same time, the graph is divided into 3 levels, the inner layer to the middle layer to the outer layer represents the degree value from large to small). notes: the inverted triangle represents the drug, the oblique square represents the component, and the circle represents the target. the color and size of the graph gradually changed from deep to shallow (or from orange to green) and large to small according to the degree of freedom. the larger the degree value was, the more important role the part played in the ppi. target point, and the gray line represents the degree value of the node, the degree value from large to small follows a gradient from orange to green. the more lines and the greater the degree value, the greater its biological importance. according to the degree value, it can be seen that the key genes enriched include il2, tnf, adora2a, mmp14, ptgs2, pon1, ccr1, vegfa, alox5, mmp12, pde4b. the biological process (bp) is mainly the response to organic substances, the regulation of cell proliferation, the response to endogenous stimulus, the positive regulation of cell proliferation, the regulation of apoptosis, cell surface receptor linked signal transduction, and positive regulation of molecular function, regulation of programmed cell death, regulation of cell death, response to wounding. from the bp-target map (figure 8 ), we can see that the triangle represents the bp, the circle represents the target point, and the gray line represents the degree value of the node, the degree value from large to small follows a gradient from orange to green. the more lines, the greater the degree value indicates the greater its biological importance. according to the degree value, it can be seen that the key genes enriched include supplementary table s4) . kegg enrichment results in 22 kegg pathways, which are related to the immune response, inflammation, viral infection, cancer, etc. the top 20 kegg signaling pathways are selected for mapping ( figure 9 ). for the percentage, the area of the bubble represents the number of enriched genes in the pathway, and the color of the bubble represents the size of the p value. the signal pathways in the figure include sphingolipid signaling pathway, neuroactive ligand-receptor interaction, pi3k-akt signaling pathway, camp signaling pathway, tnf signaling pathway, nf-κb signaling pathway, tuberculosis, nod-like receptor signaling pathway, intestinal immune network for iga production, etc. in the keggtarget diagram (figure 10 ), the triangles represent pathway, the circle represents the target, the target in the figure is gradient from orange to green according to the degree value from large to small. the more lines, the greater the degree value indicates the greater its biological importance. according to the degree value, the key genes enriched include il6, tnf, and vegf-a, il2, ptgs2, pi3k, akt, nf-κb p65, casp1, adora3, ctsd, ptgs1, mcl1, alox5.(the remaining pathway-enriched genes are shown in supplementary table s5 ). to further document the potential effect of the 13 main active compounds of sx on the 9 key hypothetical targets, including tnf-α, vegf-a, vegfr-2, il-6, ptgs1, il-2, nf-κb, akt, and pi3k, we performed a molecular docking analysis in silico. the binding free energies are shown in table 5 . the free energy of binding between compound βamyrin, cajanin, (−)-catechin gallate, eleutheroside a, calycosin, β-sitosterol-3-o-β-d-glucopyranoside, and ormononetin, and the target proteins tnf-α, vegf-a, il-2, akt, and pi3k was less than the free energy of binding between these top 20 of pathway enrichment targets and their original ligands, suggesting that these compounds are the key pharmacological components of sx for ra treatment. the free energy of binding between the compounds β-sitosterol, eleutheroside a, medicarpin, and cajanin and the targets vegfr-2, ptgs, and nf-κb was close to the free energy of the binding between these targets and their original ligands. il-6 had a high compatibility with licochalcone a and cajanin. these results imply that treatment with sx may affect tnf-α, vegf-a, vegfr-2, il-6, nf-κb, akt, and pi3k in ra patients. the target proteins and the small molecules with strong binding affinity were visualized by pymol software (figure 11 ). docking results show that the binding between compounds and the target proteins is mainly mediated by hydrophobic interactions and especially hydrogen bonds.the lipinski rule calculation results show that most of the calculated molecules satisfy the ro5 criterion. however, there are still a few molecules that are beyond the limits of ro5. the relative molecular weight values of mol000087 and mol000357 are greater than 500, which exceeds the ideal range of ro5, furthermore, the number of hydrogen bond acceptors of these two molecules exceeds 5, and the number of rotating bonds is too large, causing them to be potentially overly flexible, which adversely affects their drugability. in addition, their excessive clogp values are also detrimental to the dissolution of these two drugs in the aqueous environment. (for lipinski rule data of compounds involved in molecular docking, see supplementary table s6 ). in order to determine whether the cia model was successfully established before treatment and assessment of drug efficacy, we established a cohort of cia rats and compared disease evolution in groups of these animal treated either with different doses of sx, with gtw, another herbal medicine used for its anti-arthritis properties, or left untreated. the ai score and foot swelling were determined. before treatment, the joints of hind limbs of cia rats all showed redness and swelling to varying degrees. the redness and swelling of the knees, ankles, and interdigital joints of the cytokine-cytokine receptor interaction hind limbs in each treatment group gradually decreased after drug administration. according to the results of the degree of foot swelling (table 6, figure 12a ) and ai score ( table 7 , figure 12b ), the swelling of the knees, ankles and interdigital joints of the hind limbs of the rats in each administration group gradually decreased after the administration. after 2 weeks of treatment, compared with the mod group, the sx 40 g/kg, sx 20g/kg and sx 10g/kg and gtw groups showed decreased foot swelling (p=0.000, p=0.003, p=0.000, p=0.000, p < 0.001, p < 0.01). after 3 weeks, all the sx groups and the gtw group showed decreased foot swelling, compared to the mod group, with the greatest decrease in the sx 40 g/kg and gtw groups (p=0.0014, p=0.000, p < 0.001) ( table 6 ). after 2 weeks of treatment (30 days after the beginning of cia induction), the ai score of sx 40 g/kg and gtw groups were lower than that of the mod group (p=0.018, p=0.003, p < 0.05, p < 0.01). this beneficial effect was table 7 ). (see supplementary table s7 for median data). it can be seen from the he slices that the black arrows represent the joint cavity and the blue arrows represent the blood vessels. the pictures are all magnified 100 times. there were no obvious inflammatory cell infiltrating into the toe synovial tissues of the nor group ( figure 13a ). in the mod group, a large number of inflammatory cells, fibrous tissue hyperplasia and edema were seen in the dermal papillary layers and reticular layers in the joints of cia rats. occasionally, the tissue structure of the different layers was disordered and the epithelial tissue was destroyed ( figure 13b ). in the gtw group, the inflammatory cell number and infiltration were significantly reduced ( figure 13c) . a very small amount of scattered inflammatory cells was occasionally noticed in tissues from the sx 40 g/kg group ( figure 13d ). in the sx 20 g/kg group, the inflammatory cell infiltration, the fibrous tissue proliferation and the edema were all reduced ( figure 13e ). in contrast, joints from the sx 10 g/kg group still showed more inflammatory cell infiltration and tissue edema than that of the nor group ( figure 13f ). the content of il-2 and tnf-α in the serum of rats was then changed. compared with the nor group, the mod group increased the content (il-2: p=0.0215, p<0.05;tnf-α: p=0.0064, p<0.01); the gtw group, sx 40g/kg and sx 20g/kg were all lower than the mod group (il-2: p=0.0324, 0.0412, 0.026, p< 0.05;tnf-α: p=0.0346, 0.0275, 0.0167, p<0.05); the content of the sx 10g/kg group was higher than that of the gtw group (il-2: p=0.0381, p<0.05;tnf-α: p=0.0219; p<0.05), and the rest of the sx group was not statistically significant compared with the gtw group (p>0.05) ( table 8 , figure 14a and b). the content of vegf-a and il-6 in the serum of rats was next changed. compared with the nor group, the mod group was significantly higher (vegf-a: p=0.0024, p<0.01; il-6: p=0.0078, p<0.01); the gtw treatment groups, sx 40g/kg and 20g/kg were all lower than the mod group (vegf-a: p=0.0062, 0.0039, 0.0273; p<0.01 or p<0.05;il-6: p=0.0043, 0.0059, p=0.0357, p<0.01 or p<0.05); the sx 40g/kg group and sx 20g/kg group when compared with the gtw group is not statistically significant (p>0.05); the sx 10g/kg group is higher than the gtw group (vegf-a: p=0.0318, p<0.05; il-6: p=0.0371, p<0.05). thus, serum vegf-a and il-6 level did not show linear relationship with sx doses (table 8, figure 14c and d). the elisa results show that sx can reduce the content of il-2, tnf-α, vegf-a, and il-6, thereby reducing the synovial inflammation and pannus, achieving therapeutic effects, and predicting the target analysis of sx treatment of ra by network pharmacology. compared with the nor group, the ptgs2 mrna expression level increased in the mod group (p=0.0081, p<0.01); compared with the mod group, the content in the sx 40g/kg group and the gtw group decreased (p=0.036, p=0.0067, p<0.05, p<0.01); sx 20g/kg there was no statistically significant difference between the sx 10g/kg and the mod groups (p>0.05); the sx 20g/kg group and the sx 10g/kg group were higher than the gtw group (p=0.023, p=0.0037, p<0.05, p<0.01). there was little difference between the sx 40g/kg group and the gtw group (p>0.05) ( figure 15c) . compared with the nor group, the expression of pi3k mrna in the mod group increased (p=0.0074, p<0.01); compared with the mod group, the expression of the sx 40g/kg group, the sx 20g/kg group and the gtw group decreased (p=0.024, p=0.0088, 0.0049, p<0.05, p< 0.01). there was no significant difference between the sx 10g/kg and the mod groups (p>0.05); the expression level of the sx 10g/kg group was higher than that of the gtw group (p=0.031, p<0.05), and the sx 40g/kg group. there was little difference between the sx 20g/kg group and the gtw group (p>0.05) ( figure 15d) . compared with the nor group, the akt mrna content in the mod group increased (p=0.0091, p<0.01); compared with the mod group, the akt mrna content in the gtw group, the sx 40g/kg group and the sx 20g/kg group decreased (p=0.0095, 0.0056, p=0.042, p<0.01, p<0.05) . among them, sx 40g/kg can significantly down-regulate the expression of akt mrna (p<0.01). compared with the gtw group, the content of the sx 20g/kg group and the sx 10g/kg group increased (p=0.0431, p=0.0075, p<0.05, p<0.01) ; compared with the gtw group, the sx 40g/kg group had little difference (p>0.05) ( figure 15e) . compared with the mod group, the vegf-a mrna content in the sx 40g/kg group, the sx 20g/kg group and the sx 10g/kg group all decreased (p=0.0075, p=0.039, 0.028, p<0.01, p<0.05) , and the sx 40g/kg could significantly down-regulate the expression of vegf-a mrna (p=0.0075, p<0.01). compared with the gtw group, the sx 40g/kg and the sx 10g/kg group revealed little difference (p>0.05); for the sx 20g/kg group compared with gtw, the difference was statistically significant (p=0.019, p<0.05) ( figure 15f ). network pharmacology analysis and molecular docking results suggest that nf-κbp65, stat1, ptgs2, pi3k, akt and vegf-a are important targets for sx treatment of ra. its mechanism of action is to control inflammation, reduce pannus, and achieve the effect of protecting joints. the qrt-pcr results indicate that sx can achieve the therapeutic effect of ra by reducing the gene content of nf-κbp65, stat1, ptgs2, pi3k, akt and vegf-a. in addition, the fold change of nf-κbp65, stat1, ptgs2, pi3k, akt and vegf-a mrna expression showed that compared with the nor group, the mod group had different changes, *p<0.05, **p<0.01; compared with the mod group, the gtw group and the sx 40g/kg group have different changes, # p<0.05, ## p<0.01; compared with the gtw group, the mod group and the sx 10g/kg group have different changes, ▲ p<0.05, ▲▲ p<0.01.(the fold change of gene expression is shown in supplementary table s8 ). effect of sx on the protein level of akt, p-akt, nf-κb p65, p-nf-κb p65, pi3k in synovial tissues of cia rats after administration for 21 days, western blot detected the protein expression of the cia rat synovial cells akt, p-akt, nf-κb p65, p-nf-κb p65, and pi3k ( figure 16a ). akt total protein expression: compared with the nor group, the mod group was significantly up-regulated (p=0.0015, p<0.01); compared with the mod group, the sx 40g/kg group, sx 20g/kg group, the gtw group was significantly down-regulated (p=0.0061, p=0.021, p=0.029, p<0.05) . there was no significant difference between the sx 10g/kg group and the mod group (p>0.05), while the difference between the sx 10g/kg group and the gtw group was statistically significant (p=0.0027, p<0.01) ( figure 16b ). akt phosphorylation level: compared with the nor group, the mod group was significantly up-regulated (p=0.0051, p<0.01); compared with the mod group, the sx 40g/kg group, the sx 20g/kg group, the gtw group was significantly down-regulated (p=0.022, p=0.038, p=0.0051, p<0.05); there was no statistically significant difference between the sx 10g/kg group and the mod group (p>0.05), while the difference between the sx40g/kg group, sx20g/kg group and gtw group was not statistically significant (p>0.05) ( figure 16c ). nf-κb p65 total protein expression: compared with the nor group, the mod group was significantly up-regulated (p=0.0042, p<0.01); compared with the mod group, the sx 40g/kg group, sx 20g/kg group, and gtw group were significantly down-regulated (p=0.042, 0.033, p=0.0076, p<0.05, p<0.01) . there was no significant difference between the sx 10g/kg group and the mod group (p>0.05), while the difference between the sx40g/kg group and the gtw group was not statistically significant (p>0.05) ( figure 16d ). nf-κb p65 phosphorylation level: compared with the nor group, the mod group was significantly up-regulated (p=0.0071, p<0.01); compared with the mod group, the sx 40g/kg group, the sx 20g/kg group, the gtw group was significantly down-regulated (p=0.013, p=0.032, p=0.0081, p<0.05, p<0.01). there was no significant difference between the sx 10g/kg group and the mod group (p>0.05), while the difference between the sx40g/kg group, sx20g/kg group and gtw group was not statistically significant (p>0.05) ( figure 16e ). pi3k protein expression: compared with the nor group, the mod group was significantly increased (p=0.0019, p<0.01); compared with the mod group, the sx 40g/kg group, sx 20g/kg group, and gtw group were significantly increased (p=0.0019, 0.0062, p=0.039, p<0.01, p<0.05). there was no significant difference between the sx 10g/kg group and the mod group (p>0.05), while the difference between the sx40g/kg group and the gtw group was not statistically significant (p>0.05) ( figure 16f ). through the network pharmacology analysis, the pi3k/akt signaling pathway and nf-kκb signaling pathway play an important role in the progression of ra disease, and western blot results confirmed that sx can reduce akt, p-akt, nf-κb p65, p-nf-κb p65. the protein content of pi3k may be related to slowing down joint inflammation and pannus, thereby achieving the effect of treating ra. ra is one of the most prevalent autoimmune disease with no cure. 1-3 synovial pannus and synovial inflammation play an important role in ra development. 1-3 a main feature of ra is the "tumor-like" infiltrating growth of new blood vessels. a large number of inflammatory factors in the inflamed joint cavity are known to induce excessive proliferation of synovial blood vessels and promote the formation of pannus, but the specific mechanism leading to this proliferation is currently unclear. 1-3 sx is a classic prescription of miao medicine for ra treatment used by the ethnic minority miao people in northern areas of guizhou province, china. this study combines network pharmacology and molecular docking to study the molecular mechanism of sx in the treatment of ra and our results provide more evidence that supports further use of sx in ra. [13] [14] [15] through research, obtained 33 active ingredients of the sx meeting the conditions at the same time, 457 potential targets, 44 targets overlapping with ra, involving 479 go biological processes and 22 kegg-related signal pathways (figures 1-10) . it shows that the sx treatment of ra has the characteristics of multiple components, multiple targets, and multiple pathways. in the network of herbs-active ingredient (figure 4) , the effective compounds were analyzed and arranged in descending order. jxt and flzxgp play a major role in the compound sx, and the first three active compounds were β-sitosterol (+)-catechin, ent-epicatechin. according to the sx-active ingredients-ra ppi network, the key ingredients include mol000358 (β-sitosterol), mol002565 (medicarpin), mol000469 (7-oxo-β-sitosterol), mol002663 (skimmianine), mol000392 (formononetin), mol000497 (licochalcone a). studies have shown that jxt mainly uses flavonoids in order to play an active role. jxt and flzxgp can inhibit the proliferation of ra synovial cells, has anti-inflammatory and immunomodulatory effects, and can inhibit the expression of tnf-α, il-1β and other factors in cia rats to exert its drug activity effect. 48 flzx ethanol total ointment and ethyl acetate can effectively inhibit the progression of arthritis in cia mice. 49 in addition, studies have shown that dxt and hgt also have therapeutic effects on ra. [13] [14] [15] the important pathological manifestations of ra patients are based on different stages of disease development, joint swelling, heat pain, joint deformity, and even disability may occur. according to the arthritis index score and the degree of foot swelling (figure 12 ), the sx can improve the pathological manifestations of joint swelling and deformity, and achieve the effect of treating ra. β-sitosterol and (+)-cajanin have good antiinflammatory and bacteriostatic effects, 50 among which the anti-inflammatory mechanism may inhibit the activation of inflammasome nlrp3 in epidermal cells and macrophages to inhibit cas1. the production and activation of the mapk signaling pathway reduces the expression levels of tnf-α, il-1β, il-6, and il-8 in cells, thereby playing an antiinflammatory effect. 51 in addition, β-sitosterol has an estrogen-like effect, and can combine with estrogen receptors to exert estrogen or anti-estrogen effects. we note that estrogen is one of the most important substances in regulating the balance of bone metabolism. 51 (+)-cajanin can inhibit the formation of actin loops in osteoclasts, thereby affecting the maintenance of normal morphology of osteoclasts, 52 and can also inhibit the expression of genes and proteins related to osteoclast differentiation such as c-fos, ctsk, acp5, and hinder the normal differentiation process of osteoclasts. there are many studies on the mechanism of ra bone destruction, of which inflammation-mediated enhancement of osteoclast differentiation is the most important. 52, 53 during ra disease activity, inflammatory mediators such as tnf-α, il-1, il-6 induce the expression of rankl, etc., which can increase the secretion of the osteoclast activating factor and reduce the secretion of the osteoblast activating factor, so that the synovial membrane around the joint can be formed. the differentiation of fibroblasts and osteoclasts is accelerated, and the differentiation of osteoblasts is inhibited, leading to the destruction of local articular cartilage and bone and joint dysfunction. 52, 53 in this study ( figure 14b and d), the sx can reduce the expression of tnf-α and il-6, and play a role in the treatment of ra, but the specific mechanism of which active ingredient in sx treats bone destruction needs to be further studied. medicarpin is a flavonoid component, which has anti-inflammatory, anti-tumor and anti-oxidant characteristics. 54 skimmianine is effective in the treatment of rheumatoid arthritis. the histopathological features of ra are: moderate narrowing of the articular cavity, moderate to severe hyperplasia of articular cartilage, severe hyperplasia of synovial tissue and visible pannus formation, while skimmianine can relieve bone and cartilage erosion, inhibit inflammatory cell infiltration, synovial tissue proliferation, pannus formation and other effects. 55, 56 licochalcone a has a wide range of pharmacological activities, including antitumor, anti-inflammatory, anti-bacterial, anti-parasitic and so on. 57, 58 formononetin can regulate il-6, il-3 and other inflammatory factors to promote the proliferation of hematopoietic cells. 59 a chinese herbal compound is a complex prescription, and the efficacy of a single component on ra remains to be further studied. the immune cells that develop ra mainly include synovial b cells, synovial macrophages, neutrophils and t cells. studies have shown that there are a large number of macrophages in the cartilage tissue and synovial cells of ra patients. 60 when the synovial macrophages are activated, they can make the histocompatibility complex class ii molecules mhc ii molecules, chemokines and inflammatory factors. such overexpression, which in turn leads to synovial tissue hyperplasia, promotes inflammation and aggravates the pathological changes of pannus. neutrophils account for 60% of human peripheral blood and are the most important immune cells in the human body. 60 they have important functions such as sterilization, phagocytosis, and division. their expression is elevated in the synovial fluid of ra patients. a large number of t cells accumulate in the synovial fluid and synovial tissues of ra. after they bind to mhc ii and antigenic polypeptides, they will activate macrophages, etc., and promote the release of inflammatory cytokines such as tnf-α and il-1, thereby activating the synovial fluid. membrane fibroblasts and chondrocytes secrete a variety of enzymes that degrade glycoproteins and collagen, thereby destroying tissues and aggravating the condition of ra. 61, 62 in this experiment ( figure 13a-f) , the pathological section of the joint synovial tissue showed that after the sx treatments, the infiltration of lymphocytes, monocytes and other inflammatory cells in the synovial tissue and the proliferation of blood vessels and fibrous tissues in the cia rats alleviate, and the destruction of cartilage cells is reduced, but the specific mechanism of action needs to be further studied. according to the sx-active ingredients-ra ppi network ( figure 4 ) and the protein interaction (ppi) network of the sx in the treatment of ra (figure 3) shows that the key targets include esr1, ca2, ptpn1, tnf, il-6, vegfa, cxcl8, ptgs2, il-2. studies have shown that tnf, il6, ptgs2, il2, and vegf-a are highly expressed in ra patients. among them, tnf, il6, ptgs2, and il2 mainly mediate the inflammation of joint synovium, and vegf-a is an important factor involved in the growth of blood vessels in the body. the pathological manifestations of ra not only show repeated and continuous synovitis, but also show vascular proliferation. the interaction between the two causes the ra condition to become worse. 63 including il-1, il-6, il-17, il-2, tnf-α, mmps, etc. when these cytokines are overexpressed, it will promote the ra joint cavity. the internal inflammation continues repeatedly, and inflammation will promote vascular proliferation, promote the expression of vegf-a, and cause ra synovial pannus. 63, 64 this study shows that sx can reduce the expression of vegf-a, inhibit inflammation and vascular proliferation, and achieve the effect of treating ra ( figure 14c and f). the repeated action of the two is one of the most important pathogenesis of ra. 63, 64 prostaglandin endoperoxide synthase 2 (ptgs2), also known as cox-2, is a critical enzyme in prostaglandin biosynthesis and plays an essential role in the inflammatory response. the level of inflammatory factors caused by ra increases can cause overexpression of ptgs2. the overexpression of ptgs2 induces the activation of the nf-κb signaling pathway and tnf-α signaling pathway, which leads to more severe inflammation in the body. [65] [66] [67] the etiology of ra is complex. in this study (figures 15 and 16) , sx can reduce the expression of tnf-α, nf-κb p65, inhibit the expression of ptgs2, and improve ra inflammation, but the intermodulation relationship between tnf-α, nf-κb p65 and ptgs2 needs to be further studied. in addition to inflammatory cells and infection factors, it also includes changes in hormone levels, genetic and environmental factors, diet and living habits, and intestinal flora. studies have shown that changes in hormone levels also have certain incentives. as far as sex hormones are concerned, epidemiological surveys show that male patients with ra have about three times that of women, and the premenopausal incidence of women has increased significantly, surpassing men of the same age. 68, 69 the incidence rate tends to be the same after the age of 80, so sex hormones are involved in the pathogenesis and development of ra. 68,69 esr1 gene polymorphism has a certain relationship with the pathogenesis of knee osteoarthritis. esr1 gene mutation may increase the risk of knee osteoarthritis, but its exact mechanism still needs further study. 68 the analysis of go biological process ( figure 5 ) and kegg pathway ( figure 9 ) shows that go biological process includes cell proliferation, apoptosis regulation, cytokine regulation, inflammatory response, cell signal transduction regulation7. the key genes (figures 6-8 and 10 ) enriched include mmp14, mmp2, mmp12, stat1, etc. il2, tnf-α, ptgs2, adora2a7. matrix metalloproteinase (mmp) is an important proteolytic enzyme in the organism. under the stimulation of cytokines, synovial cells and vascular endothelial cells produce a variety of mmps, breaking the gap between the production and degradation of extracellular matrix. balance, causing arthritis, is one of the important pathogenesis modes of ra. 69 studies have shown that cd147 can promote synovial cells to secrete a variety of matrix metalloproteinases such as: mmp1, mmp3, mmp9 and so on. vascular endothelial cell growth factor (vegf) is a multifunctional cytokine that specifically acts on vascular endothelial cells. it is one of the main factors that promote angiogenesis and is currently known to have the strongest effect on blood vessels. 70 vegf can bind to many types of receptors, the most important of which is the tyrosine kinase receptor containing 7 globulin-like domains: type i receptor, type ii receptor and type iii receptor. 70, 71 vegf binds to these three receptors to induce the migration of vascular endothelial cells and the maintenance of tubular structure. the angiogenesis effect is mainly the result of vegf activation of vegfr-2. high expression of vegf will break the stability of blood vessels, cause basement membrane damage and increase blood vessels. permeability and inflammation lead to vascular disease, which leads to vascular functional diseases. 72, 73 in ra patients, the high expression of vegf can make mmp3 and mmp9 in an activated state, and they can then participate in the degradation of extracellular matrix (ecm) and promote the destruction of ra bone. mmp2 can theoretically degrade the important articular cartilage of type i, iii, iv, v, vii and xi collagen, fibronectin, laminin and proteoglycan in ecm. it is speculated that mmp2 plays an important role in the degradation of ra cartilage. 74 however, some studies have found that the levels and activity of mmp2 remain unchanged in ra animal models, and mmp2 knockout mice will develop more severe arthritis, so the mechanism of mmp2 in ra is very complicated. it may have a dual mechanism of protection and disease. the activity of mmp is regulated by the nf-κb signal transduction pathway. when the body is stimulated by external factors, the nf-κb signal pathway is activated, which leads to the overexpression of downstream mmp, and this then participates in the inflammation response together with other inflammatory factors, and accelerates the joints. one result is destruction of cartilage. 75 among them, tnf-α has been shown to play a key role in tissue inflammation, autoimmunity, and is a strong inflammatory cytokine. targeted biologics agent for tnf-α have been used in the clinic. 76 in this study, sx can reduce the expression of vegf-a, tnf-α, and nf-κbp65, but its effect on mmp-related genes needs to be studied in subsequent experiments.the janus kinase-signal transducer and activator of transcription (jak/stat) signaling pathway plays a specific and pleiotropic biological function in the process of immune function regulation and other processes, and is an important way of cell signal transduction. a large number of studies have confirmed that the jak/ stat pathway is involved in regulating the production and activation of cytokines in the synovium of ra joints, and is involved in the pathophysiological process of the disease. 77 by activating jak1, the activation is mainly responsible for ra immune-mediated inflammation. jak is a non-receptor tyrosine protein kinase in the cytoplasm. the main substrate of jak is the stat family. many cytokines, such as tnf-α, il-1, etc., activate stat through the jak/ stat signal pathway and induce ra inflammation and bone destruction. studies have shown that stat1 and stat6 are expressed in ra synovial tissue, and stat1 can regulate ra inflammation, bone formation and bone destruction under the action of inf-γ, il-6, il-10 and other cytokines. 78 the results of this study show that sx can reduce the expression of stat1 to reduce inflammation and protect bones and joints. its mechanism of action may be related to sx's ability to down-regulate tnf-α and il-6. the pathogenesis of ra is complex, and the related molecular mechanisms of action are also diverse [79] [80] [81] [82] [83] (figure 15 ). ra synovial vascular proliferation and inflammation are important causes of joint destruction, which are regulated by cytokines, signal pathways and other factors. among them, vegf/pi3k/akt signal and tnf-α signal and il/nf-κb signal pathway are closely related to many signal transduction pathways. 84, 85 in recent years, it has been found that there are abundant expressions of angiogenic factors during the evolution of ra synovial lesions. the vegf/pi3k/akt signaling pathway exists in a variety of cells and participates in cell proliferation, differentiation, apoptosis, and blood vessel growth process. phosphatidylinositol 3 kinase (pi3k) is a heterodimer composed of two subunits: a catalytic subunit d (pll0) with a molecular weight of 10kd and a regulatory subunit (p85) with a molecular weight of 85kd. akt is a downstream signal molecule of pi3k and also has serine/threonine protein kinase activity. the amino acid sequence of its catalytic active center is similar to that of protein kinase a (proteinkinase a, pka) and protein kinase c (protein kinase c, pkc). the sequence homology is very high, so it is also called pkb. after pi3k is activated, it binds to the intracellular phosphoinositide dependent kinase (pdkl), prompting pdkl to phosphorylate the ser308 site of akt protein to activate akt. the phosphorylation site in the process of akt activation is located ser473 and thr308 in the active center domain. 86, 87 akt activation can initiate downstream signaling molecules, the most important of which is mammalian rapamycin harrowing protein (mtor), which can continue to transmit downstream signals and produce biological effects such as gene transcription and cell proliferation. 84 studies have shown that pi3k/akt can promote the synthesis of vegf at the gene level, which is gradually phosphorylated by activating the kinase system, thereby activating the pathway to regulate cytokines, such as vegf and hif-α. 83 in the pathological changes of ra, inflammation of the synovium interacts with pannus, causing the disease to be repeated. the nuclear transcription factor nf-κb signaling pathway mainly includes nuclear factor-κb (nf-κb), nf-κb inhibitory protein (iκb), and nf-κb inhibitory protein kinase complex (ikks), which play a role in the regulation of inflammation. this is an important role, and is closely related to the occurrence and development of rheumatoid arthritis. 88, 89 nf-κb is a protein factor that specifically binds to the enhancer κb sequence of the immunoglobulin κ light chain gene. the dimeric protein composed of p50/p65 is the main component of the active form of nf-κb in most cells. high levels of tnf-α in the synovial fluid of patients with rheumatoid arthritis can induce the expression of nf-κb ligand activation genes and activate a large number of cytokines, leading to repeated inflammation. 88, 89 the molecular docking results ( figure 11 ) suggest that β-amyrin, cajanin, (-)-catechin gallate, eleutheroside a, calycosin, β-sitosterol-3-o-β-d-glucopyranoside, ormononetin may act on tnf-α, vegf-a, il-2. targets such as akt, pi3k, etc. play a role in the treatment of ra, but the specific role needs to be further studied. through network pharmacological analysis and animal experiments, it is confirmed that the mechanism of sx treatment of ra may be related to the inhibition of the expression of key factors in the vegf/pi3k/akt signaling pathway, tnf-α signaling pathway, and il/nf-κb signaling pathway. in summary, the sx treatment of ra has the characteristics of multiple components, multiple targets, and multiple pathways. sx can improve joint swelling in cia rats, reduce inflammatory cell infiltration and angiogenesis in joint synovial tissue, and by down-regulating the expression of il-2, il-6, tnf-α, vegf, pi3k, akt, p-akt, nf-κbp65, p-nf-κbp65, stat1 and ptgs2are used to control the exacerbation of inflammation and alleviate the proliferation of https://doi.org/10.2147/dddt.s330947 drug design, development and therapy 2022:16 462 synovial pannus, and at the same time play the role of cartilage protection to achieve the effect of treating ra. the results of this experiment provide a new way to probe deeper into the molecular mechanism of the treatment of ra by the sx and provide new ideas for the study of target therapeutic mechanisms. however, the mechanism of action of specific monomer components and target effects still need to be further studied. ethics approval: the experimental scheme was approved by the experimental animal ethics committee of guizhou medical university (ethical number: 1900868) and followed the guidelines for the ethical review of laboratory animal welfare people's republic of china national standard gb/t 35892-2018. the experimental process is strictly in accordance with the national institutes of health published guidelines for the care and use of experimental animals (nih publication 85-23, revised in 1996) and china's guidelines for the ethical review of laboratory animal welfare. current trends in theranostics for rheumatoid arthritis rheumatoid arthritis the risk of deliberate self-harm following a diagnosis of rheumatoid arthritis or ankylosing spondylitis: a population-based cohort study comparison of efficacy of ketoprofen and ibuprofen in treating pain in patients with rheumatoid arthritis: a systematic review and meta-analysis research progress of miao medicine in the treatment of rheumatoid arthritis immunometabolism in early and late stages of rheumatoid arthritis novel dmard monotherapy in rheumatoid arthritis effect of filgotinib vs placebo on clinical response in patients with moderate to severe rheumatoid arthritis refractory to disease-modifying antirheumatic drug therapy: the finch 2 randomized clinical trial hplc-ms/ms method for the determination and pharmacokinetic study of six compounds against rheumatoid arthritis in rat plasma after oral administration of the extract of caulophyllum robustum maxim baihu jia guizhi decoction improves rheumatoid arthritis inflammation by regulating succinate/sucnr1 metabolic signaling pathway identification of characteristic autoantibodies associated with deficiency pattern in traditional chinese medicine of rheumatoid arthritis using protein chips is use of traditional chinese medicine associated with non-adherence to prescribed western rheumatic medications among chinese-american patients? a cross-sectional survey anti-inflammatory effects of miao drug sidaxue on rheumatoid arthritis the effects of sidaxue, a kind of miao national herb, on tnf-α and il-1β in adjuvant rheumatoid arthritis rats effects of miao national herbs sidaxue on rheumatoid arthritis in rats review on research progress of chemical constituents and pharmacological activities of spatholobi caulis drug design observation on the efficacy of self made ningleg soup in the treatment of hemodialysis patients with restless legs syndrome study on quality standard of jixueteng granules clinical observation on the effect of external divergent shock wave combined with danggui jixiteng decoction in the treatment of inflammatory heel pain of plantar fascia recent advances in chemistry and bioactivity of sargentodoxa cuneata study on the chemical constituents of sargentodoxa cuneata chemical constituents from the roots of toddalia asiatica(l.) lam a systematic review on traditional medicine toddalia asiatica (l.) lam.: chemistry and medicinal potential a review on traditional usages, chemical constituents and pharmacological activities of periploca forrestii schltr research progress on miao medicine periploca forrestii schltr and predictive analysis on quality markers the shape characteristics and indication scope of the "sidaxue" of miao medicine prescription investigation of the mechanism of action of porana sinensis hemsl. against gout arthritis using network pharmacology and experimental validation modern traditional chinese medicine: identifying, defining and usage of tcm components yatcm: yet another traditional chinese medicine database for drug discovery the stability of r-spine defines raf inhibitor resistance: a comprehensive analysis of oncogenic braf mutants with in-frame insertion of αc-β4 loop tcmsp: a database of systems pharmacology for drug discovery from herbal medicines exploring the mechanism of action xianlingubao prescription in the treatment of osteoporosis by network pharmacology traditional chinese medicine network pharmacology: theory, methodology and application mechanism of action of xiaoyao san in treatment of ischemic stroke is related to anti-apoptosis and activation of pi3k/akt pathway proteogenomic analysis of pitaya reveals cold stress-related molecular signature cdk1 and cdc20 overexpression in patients with colorectal cancer are associated with poor prognosis: evidence from integrated bioinformatics analysis a molecular docking study repurposes fda approved iron oxide nanoparticles to treat and control covid-19 infection molaical: a soft tool for 3d drug design of protein targets by artificial intelligence and classical algorithm application of gc/ms-based metabonomic profiling in studying the therapeutic effects of aconitum carmichaeli with ampelopsis japonica extract on collagen-induced arthritis in rats quantifying spatial morphology and connectivity of urban heat islands in a megacity: a radius approach mir-125 regulates pi3k/akt/mtor signaling pathway in rheumatoid arthritis rats via parp2 histological evaluation of lumbar spine changes in rats with collagen-induced arthritis il-17a and il-17f expression and functional responses in rheumatoid arthritis and peripheral spondyloarthritis vegf/vegf-r/runx2 upregulation in human periodontal ligament stem cells seeded on dual acid etched titanium disk all-trans-retinoid acid induces the differentiation of p19 cells into neurons involved in the pi3k/akt/gsk3β signaling pathway effect of moxibustion on hif-1α and vegf levels in patients with rheumatoid arthritis foundation and activity of ra the study on of the analgesic, anti-inflammatory and anti-rheumatoid arthritis activities of extracts form radix toddaliae asiaticae β-sitosterol attenuates the intracranial aneurysm growth by suppressing tnf-α-mediated mechanism progress in the pharmacological action of β-glusterol research of the effect and mechanism of cajaninstilbene acid in the prevention and treatment of glucocorticoid wang yue experience in rheumatoid arthritis medicarpin prevents arthritis in post-menopausal conditions by arresting the expansion of th17 cells and pro-inflammatory cytokines ipso-substitution in derivatives of the quinoline alkaloid skimmianine research progress on chemical constituents and pharmacological effects of yinyu licochalcone a inhibits proliferation and promotes apoptosis of colon cancer cell by targeting programmed cell death-ligand 1 via the nf-κb and ras/raf/mek pathways the effect of licorice chalcone a on osteoarthritis in rats and its relationship with p38-mapk inflammatory signaling pathway formononetin attenuates h2o2-induced cell death through decreasing ros level by pi3k/akt-nrf2-activated antioxidant gene expression and suppressing mapk-regulated apoptosis in neuronal sh-sy5y cells pathogenesis of rheumatoid arthritis s100a11 (calgizzarin) is released via netosis in rheumatoid arthritis (ra) and stimulates il-6 and tnf secretion by neutrophils protectin dx restores treg/t17 cell balance in rheumatoid arthritis by inhibiting nlrp3 inflammasome via mir-20a sclerostin inhibition promotes tnf-dependent inflammatory joint destruction rasgrp2 (caldag-gefi) expression in rheumatoid synovium contributes to the development of destructive arthritis the pre-clinical phase of rheumatoid arthritis: from risk factors to prevention of arthritis paternal exposure to immunosuppressive and/or biologic agents and birth outcomes in patients with immune-mediated inflammatory diseases fcer1g and ptgs2 serve as potential diagnostic biomarkers of acute myocardial infarction based on integrated bioinformatics analyses study on the correlation between esr1 gene polymorphism and the risk of knee osteoarthritis progress in mmp-2/mmp-9 studies in inflammation chemical profiling and the potential active constituents responsible for wound healing in periploca forrestii schltr calycosin alleviates allergic contact dermatitis by repairing epithelial tight junctions via down-regulating hif-1α effect of angiotensin ii on bone erosion and systemic bone loss in mice with tumor necrosis factor-mediated arthritis renin-angiotensin system molecules are associated with subclinical atherosclerosis and disease activity in rheumatoid arthritis effects of fulingwan modified and subtracted recipe on mmp-3, mmp-13 and timp-1 expression in rabbit knee osteoarthritis model mmp2 and tlrs modulate immune responses in the tumor microenvironment clinical effect of tnf-α antagonist in the treatment of rheumatoid arthritis based on jak/stat pathway to explore the anti-inflammatory effect of guizhi shaoyao zhimu decoction on mh7a cells induced by tnf-α rheumatoid pannus presenting as a large epidural mass in the subaxial cervical spine: a case report dendrobium huoshanense stem polysaccharide ameliorates rheumatoid arthritis in mice via inhibition of inflammatory signaling pathways repurposing of pirfenidone (anti-pulmonary fibrosis drug) for treatment of rheumatoid arthritis umbelliferone ameliorates complete freund adjuvant-induced arthritis via reduction of nf-κb signaling pathway in osteoclast differentiation panax japonicuscomputational prediction of antiangiogenesis synergistic mechanisms of total saponins of against rheumatoid arthritis integrated serum pharmacochemistry and network pharmacological analysis used to explore possible anti-rheumatoid arthritis mechanisms of the shentong-zhuyu decoction rheumatoid arthritis and cloves syndrome: a tricky diagnosis anti-angiogenic effect of shikonin in rheumatoid arthritis by downregulating pi3k/akt and mapks signaling pathways drug design sin1/mip1 maintains rictor-mtor complex integrity and regulates akt phosphorylation and substrate specificity circrna_09505 aggravates inflammation and joint damage in collagen-induced arthritis mice via mir-6089/akt1/nf-κb axis tnf-induced inflammatory genes escape repression in fibroblast-like synoviocytes: transcriptomic and epigenomic analysis peptide-sirna nanocomplexes targeting nf-κb subunit p65 suppress nascent experimental arthritis drug design, development and therapy drug design, development and therapy is an international, peer-reviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are a feature of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use the authors report no conflicts of interest in this work. case�report abstract a 65 year old female patient suffering from rheumatoid arthritis reported with multiple joint pains, malaise, fatigue, difficulty in breathing, difficulty in opening jaw, difficulty in holding objects and menopausal symptoms. she had been taking multiple medications including indomethacin, aspirin, prednisolone, methotrexate etc. treatment at al-sayed hospital rawalpindi included autologous bone marrow derived stem cell transplantation and exercise training program. specific exercises were given four weeks before and eight weeks after stem cell transplant at our center. after one year of treatment, the patient had improvement in all of her signs and symptoms, which enabled her to discontinue several medications. there was marked improvement in her joint pain, range of motion, muscle power, grip function, functional activities and activities of daily living (adls). key words: bone marrow derived stem cells, exercise training, rheumatoid arthritis. available for public use. it was only then that these pioneering endeavors saw their first introduction, thus dramatically changing the lives of rheumatoid a r t h r i t i s p a t i e n t s w i t h h i g h l y s e l e c t i v e 5 immunotherapy. in a vast majority of patients, who were given anti-tumor necrosis factor (tnf) therapy, significant improvement was noted clinically with minimal adverse effects right after the treatment. of late, the first antiinter leukin1 (il-1) therapy has been approved for use clinically as an il6 1 receptor antagonist (anakinra). bone marrow derived stem cell transplant is a promising treatment choice for patients suffering from ra. it is suggested that hematopoietic stem cell transplantation (hsct) is a useful therapy for severe ra on the basis of animal models and case reports of patients undergoing the procedure for other indications. case report a sixty five year old female came to us with the history of multiple joint pains since she was 19 years old and was diagnosed with rheumatoid arthritis 01 year later. she had taken multiple medicines including indomethacin, aspirin, prednisone, methotrexate, and gold. she was complaining of severe pain, decreased range of motion (rom) decreased mostly in lower limbs, muscle tightness, deformities, functional limitations, unable to change posture from lying to sitting and sitting to standing, muscle wasting, pulmonary problems, difficulty in opening jaw, difficulty in holding objects. muscle introduction “rheumatoid arthritis is a chronic progressive inflammatory disease of the joints resulting in painful deformities and immobility, especially in the small joints”. it decreases red blood cell count and causes inflammation around lungs and heart. it also 1 presents with fever and low energy. middle age women are affected 2.5 times more than men. the diagnosis is made mostly on the basis of a patient's 2,3 signs and symptoms. the main objective of the treatment is to reduce pain and inflammation, and improve a person's overall daily activities. this may be assisted by ensuring proper balance between rest and exercise, by using splints and assistive devices. the progression of the disease may be delayed by the use of a class of medicines known as disease-modifying antirheumatic drugs (dmards). in certain cases surgery to repair, replace, or fuse joints is opted for and 4 proves useful. alternative medicine and related treatments are not yet supported by evidence. before the year 1999 biological treatments were not effects of autologous bone marrow derived stem cell transplant and exercise training program on rheumatoid arthritis 1 2 3 4 syed salman naeem gilani , muhammad naeem , tauseef bukhari , muhammad asad ullah correspondence: dr. syed salman naeem gilani hod, stem cell al sayed hospital, rawalpindi e-mail: alsayedstemcell@gmail.com 1 2 3 department of stem cell/ physiotherapy / director hospital al sayed hospital, rawalpindi 4 department of physiotherapy funding source: nil; conflict of interest: nil received: jan 17, 2017; revised: apr 15, 2017 accepted: aug 27, 2017 effects of stem cells and exercise on rheumatoid arthritisjiimc 2017 vol. 12, no.3 157 power is given in table i. lab investigations showed: hemoglobin: 11.1 g/dl, esr: 84, ra factor and anti-ccp (cyclic citrullinated peptide) antibodies: positive, hba1c: 9.2. exercise training program included active and passive range of motion, stretching of tight musculoskeletal structures, isometrics, deep breathing exercises, cycle ergometry for upper and lower limbs to improve endurance and aerobics was given at our hospital for four weeks before and eight weeks after stem cell transplant. this session was given twice a day with twenty to thirty repetitions of each activity. patient was then transitioned to home exercise management and followed up at three, six and twelve months after the procedure. stem cell extraction and transplant procedure: patient was given injection filgrastim (granulocyte colony stimulating factor analog) 10 µg per kg body w e i g h t s u b c u t a n e o u s l y w i t h o u t m e t h y l prednisolone or cyclophosphamide and baseline cbc with hpc (hematopoietic progenitor cells) was performed. two days later cbc with hpc was repeated and patient's bone marrow stem cells were harvested by apheresis. a total of 100cc of autologous bone marrow stem cells were harvested. cbc with hpc was repeated at the end of apheresis to confirm the total number of hematopoietic stem cells. harvested stem cells were t h e n i n j e c t e d i n t o h e r k n e e , s h o u l d e r, metacarpophalyngeal and inter-phalyngeal joints bilaterally in the procedure room under aseptic technique. afterwards stem cells were transfused to the patient intravenously. outcome measurements patient showed marked improvement in muscle power one year after the stem cell transplant. a comparison of muscle power before starting the treatment at our center and one year after stem cell transplant is given in table i. patient's pain was reduced from severe to pain free. range of motion (rom) significantly increased. range of motion (rom) was measured by goniometry. grip function was measured by the “sollerman test”. the results of sollerman test are given in table ii. modified hss (hospital for special surgery) score for both knees were measured that improved from 57 to 90 for her left knee and 57 to 87 for her right knee. discussion bone marrow contains hematopoietic and nonhematopoietic (mesenchymal) stem cells, the latter being with immunoregulator properties. it is suggested that adult mesenchymal stem cells (msc) are useful as cellular therapy in several inflammatory 7 diseases, including ra. as msc may transfer to sites of injury in vivo, it is suggested that inflamed joints are targeted by cells which might have a therapeutic effect o n a rt h rit is t h ro u gh m sc med iated 8 immunosuppression. leeds group presented a study in 1997 in which g-csf was used at a dose of 5µg/kg/day to mobilize stem cells in five patients. in peripheral blood cd34+ count was checked to establish good efficacy. patients remained stable after the treatment but we observed that administering methylprednisolone (median 80mg, table i: muscle power before and one year a�er bone marrow derived stem cell transplant table ii. “sollerman test” result. performance is graded from 4 (best) to 0 (worst). grade 04 is used for correct grip and performance of the ac�vity within 20 seconds. grip strength was measured in newton's by means of an electronic hand dynamometer effects of stem cells and exercise on rheumatoid arthritisjiimc 2017 vol. 12, no.3 158 range 40–120mg) intramuscular or intra-articular diminished any pro-inflammatory effects of filgrastim. a phase i placebo controlled 1110 study was conducted in australia to investigate the efficacy of g-csf in patients suffering from active form of ra 9 for the use of stem cell collection. in paris, four patients had their stem cells mobilized with cyclophosphamide 4g/m2 followed by g-csf 5µg/kg/day. probably, in three of the patients cd34+ cell yields were higher with g-csf along with cyclophosphamide than with g-csf alone. arthritis and extra-articular manifestations improved markedly in these patients. to some extent the disease activity persisted in three patients, although it never went back to the same level even two years 10 after the procedure. in this case, the patient presented with inflammatory arthritis of almost forty five years' duration. she described the pain in her hands, wrists, elbows, shoulders and knees as frequently unbearable. she had significant muscle wasting, decreased muscle power, deformities, and weak grip strength. one year after autologous bone marrow stem cell transplant, the patient felt significant decrease in all subjective symptoms and discontinued her medications. esr had normalized, indicative of clinical reduction of arthritis. she also felt improvement in adls. in our study autologous bone marrow derived stem cells combined with exercises showed that it is a safe and effective treatment option for the patient suffering from rheumatoid arthritis and warrants a larger scale phase i / ii clinical study. references 1. tugwell p, pincus t, yocum d, stein m, gluck o, kraag g, et al. combination therapy with cyclosporine and methotrexate in severe rheumatoid arthritis. new england journal of medicine. 1995; 333: 137-42. 2. de punder ym, hendrikx j, den broeder aa, pascual ev, van riel pl, fransen j. should we redefine treatment targets in rheumatoid arthritis? low disease activity is sufficiently strict for patients who are anticitrullinated protein antibody-negative. the journal of rheumatology. 2013; 40: 1268-74. 3. van nies ja, krabben a, schoones jw, huizinga tw, kloppenburg m, van der helm-van mil ah. what is the evidence for the presence of a therapeutic window of opportunity in rheumatoid arthritis? a systematic literature review. annals of the rheumatic diseases. 2014; 73: 861-70. 4. smolen js, landewé r, breedveld fc, dougados m, emery p, gaujoux-viala c, et al. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs. annals of the rheumatic diseases. 2010; 69: 964-75. 5. maini r, st clair ew, breedveld f, furst d, kalden j, weisman m, et al. infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase iii trial. lancet. 1999; 354: 1932–9. 6. snowden ja, brooks pm, biggs jc. haemopoietic stem cell transplantation for autoimmune diseases. british journal of haematology. 1997; 99: 9-22. 7. wang y, chen x, cao w, shi y. plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. nature immunology. 2014; 15: 1009-16. 8. bouffi c, bony c, courties g, jorgensen c, noel d. il-6dependent pge2 secretion by mesenchymal stem cells inhibits local inflammation in experimental arthritis. plos one. 2010; 5: e14247. 9. snowden ja, biggs jc, milliken st, fuller a, staniforth d, passuello f, et al. a randomised, blinded, placebocontrolled, dose escalation study of the tolerability and efficacy of filgrastim for haemopoietic stem cell mobilisation in patients with severe active rheumatoid arthritis. bone marrow transplantation. 1998; 22: 1035-41. 10. moore j, brooks p, milliken s, biggs j, ma d, handel m, et al. a pilot randomized trial comparing cd34-selected versus unmanipulated hemopoietic stem cell transplantation for severe, refractory rheumatoid arthritis. arthritis & rheumatism. 2002; 46: 2301-9. effects of stem cells and exercise on rheumatoid arthritisjiimc 2017 vol. 12, no.3 159 page 40 page 41 page 42 key: cord-0037182-w2tz1bdt authors: junker, anna; kokornaczyk, artur kamil; strunz, ann kathrin; wünsch, bernhard title: selective and dual targeting of ccr2 and ccr5 receptors: a current overview date: 2014-03-05 journal: chemokines doi: 10.1007/7355_2014_40 sha: c6c2b70378ceb955eef5dcbb10bd65f91e32c8dd doc_id: 37182 cord_uid: w2tz1bdt the chemokine receptor 2 (ccr2) and chemokine receptor 5 (ccr5) are important mediators of leukocyte trafficking in inflammatory processes. the emerging evidence for a role of ccr2 and ccr5 receptors in human inflammatory diseases led to a growing interest in ccr2and ccr5-selective antagonists. in this review, we focus on the recent development of selective ccr2/ccr5 receptor ligands and dual antagonists. several compounds targeting ccr2, e.g., incb8761 and mk0812, were developed as promising candidates for clinical trials, but failed to show clinical efficacy as presumed from preclinical models. the role of ccr5 receptors as the second co-receptor for the hiv-host cell fusion led to the development of various ccr5-selective ligands. maraviroc is the first ccr5-targeting drug for the treatment of hiv-1 infections on the market. the role of ccr5 receptors in the progression of inflammatory processes fueled the use of ccr5 antagonists for the treatment of rheumatoid arthritis. unfortunately, the use of maraviroc for the treatment of rheumatoid arthritis failed due to its inefficacy. some of the ligands, e.g., tak-779 and tak-652, were also found to be dual antagonists of ccr2 and ccr5 receptors. the fact that ccr2 and ccr5 receptor antagonists contribute to the treatment of inflammatory diseases renders the development of dual antagonists as promising novel therapeutic strategy. in the last 25 years, chemokines and their receptors have become promising targets in many fields of research. because the chemokine receptors 2 (ccr2) and 5 (ccr5) represent highly interesting candidates of the chemokine receptor family, much of investigations have recently been carried out in the development of ligands for these receptors. maraviroc (1) is the first and to date the only fda-and emeaapproved drug on the market, targeting the ccr5 receptor (see fig. 1 ) [1, 2] . the intention of this review is to elucidate the structure activity relationships of various small-molecule ccr2 and ccr5 ligands. the focus will be on the receptor binding affinity, antiviral activity for the treatment of hiv, and chemotactic activity for the treatment of atherosclerosis. in addition to the receptor binding affinity, many further aspects, which play a crucial role in drug development, will be discussed, e.g., physicochemical properties, lipophilicity, and the affinity to the human ether-à-go-go-related gene (herg), a k + -channel which would lead to severe side effects induced by the compounds. the ccr2 receptor has become a promising target in the therapy of atherosclerosis. the concerted action of the chemokine ligand ccl2 and the ccr2 receptor plays an important role in the recruitment of monocytes from the bone marrow into the arterial wall, which is known to be an early key step in atherosclerotic plaque formation. lesions of the arterial endothelium are caused by mechanic injury or toxins and lead to migration of monocytes into the subendothelium that is mediated by adhesion molecules and chemokine receptors. in the artery wall, monocytes differentiate into macrophages, which develop to foam cells by taking up blood lipids [3] [4] [5] . advanced plaques become unstable and can suddenly rupture. they expose their content to the blood, leading to platelet aggregation and occlusion of the blood vessel. thrombosis, stroke, and myocardial infarction result as serious long-term complications. with regard to the increasing number of patients, the use of small-molecule ccr2 antagonists in atherosclerosis has attracted significant attention in the last years [6] . the ccr2 receptor is linked also to progression and development of other inflammatory diseases like multiple sclerosis (ms) and rheumatoid arthritis (ra) [7] . the ccr5 and the cxcr4 receptors are mainly known as co-receptors required for the development of the hiv-1 infection [8] . the binding of gp120 of the virus to cd4 receptors on t-lymphocytes and macrophages leads to a conformational change of gp120 and enables the interaction with the ccr5 co-receptor. this triggers conformational changes in gp41, which leads to the fusion of the virus with the host cell [9] . at the beginning of an infection, the vast majority of the transmitted virus strains is m-or r5-tropic and uses the ccr5 receptor as a co-receptor. the t-or x4-tropic virus strains, which use the cxcr4 receptor as a co-receptor, are associated with an advanced disease progression [10] . a genetic polymorphism of the ccr5 receptor, characterized by a deletion of 32 bp in the gene segment encoding the receptor (ccr5δ32 bp), results in a lack of function of the ccr5 receptor in homozygote individuals. these ccr5δ32 bp mutations are found in 1-5% among uninfected caucasian population and is exceedingly rare in infected patients (<0.1%), which indicates that ccr5δ32 bp homozygotes are strongly resistant against hiv-1 infection [11] [12] [13] . the ccr5δ32 bp mutant has also been linked to reduced susceptibility to coronary artery diseases and myocardial infarction [14, 15] . ccr5δ32 bp polymorphism, as well as the function of the ccr5 receptor as a co-receptor for the hiv-host cell fusion, stimulated the beginning of several drug development programs by different pharmaceutical companies since the early 2000s. in this review, we will focus on selective ccr2 (sect. 2) and ccr5 (sect. 3) receptor ligands, which can be used in the treatment of several immunological diseases including arthritis [16] , asthma [17] , multiple sclerosis [18] , vascular diseases [19, 20] , and hiv-1 infection [7, 21] . the fact that the antagonism of both ccr2 and ccr5 receptors may contribute to the treatment of inflammatory diseases makes the development of dual antagonists attractive. dual ccr2 and ccr5 antagonists will be discussed in sect. 4. the ccr2 receptor plays an important role in an inflammatory response and is involved in several diseases of the immune system including atherosclerosis. the interaction of the chemokine ccl2 with the ccr2 receptor is responsible for the recruitment of blood monocytes to the site of inflammation and is also an early key step in the pathogenesis of atherosclerosis. the ccr2 receptor represents a promising therapeutic target for the treatment of atherosclerosis and is discussed as potential pet (positron emission tomography) target for diagnostic use [7] . current atherosclerosis treatments are restricted to manipulation of indirect mechanisms, e.g., the modulation of cholesterol or triglyceride concentration, control of homoeostasis, or reduction of other risk factors associated with the metabolic syndrome. with regard to millions of patients (and numbers rising) who suffer under this chronic inflammation, ccr2 antagonists have attracted substantial attention during the past years [20] . early developments of ccr2 antagonists in the late 1990s have been already reviewed elsewhere [6, [22] [23] [24] . this chapter will analyze structural features from different classes of ccr2 antagonists that were published until august 2013. we will compare them with regard to their structure activity relationships (sars), explain strategies that led to increasing ccr2 affinity and selectivity, and elucidate the influence on the cyp system and herg inhibition as well. the affinity of ligands to the ccr2 receptor is usually determined in the radioligand displacement assays, where the chemokine [ 125 i] ccl2 is used as the radioligand [25] . chemotactic assays are generally used to determine the compounds' ability to inhibit the ccl2-stimulated chemotaxis in human peripheral blood monocytes [26] . several series of ccr2 antagonists from different structural classes have been described in patents and publications. the majority of known ccr2 antagonists consist of a basic center flanked by two lipophilic residues as aromatic rings or one aromatic and one aliphatic moiety as demonstrated in the pharmacophore model in fig. 2 . the basic amine or quaternary ammonium ion of the ligand essentially anchors a small-molecule ligand to glu291 (in the transmembrane domain 7 (tm7)) of the ccr2 receptor by a salt-bridge formation [28] . the aryl or heteroaryl motif r2 on side 2 is another essential feature of this pharmacophore model. the substitution pattern of this aromatic system greatly influences the ccr2 receptor affinity. the linker l2 of 6-9 atoms with lipophilic or polar, peptidic or saturated, and unsaturated or aromatic structural elements is well tolerated by the receptor and determines the specificity of binding. the left part of the molecules tolerates more variations: the moiety r1 can be an aliphatic or aromatic ring. the linker l1 can be short and aliphatic (1-4 atoms) , can be part of a ring system, or it can represent an aliphatic system in case of absence of a ring system [27] . compounds with an aromatic ring in the r1 position show interactions with the herg channel [29] . most of the ccr2 ligands, discussed in this chapter, correspond to the described pharmacophore model. the report of the crystal structure of the cxcr4 receptor [30] , which has a high sequence homology with the ccr2 receptor, initiated various structure-based approaches in the ccr2 ligand design and the investigations of the ligand-receptor interactions. predominantly, lipophilic amines that contain a central basic amine flanked by two lipophilic moieties according to the ccr2 pharmacophore model (fig. 2) show high affinity for the human ether-a-go-go-related gene (herg) k + -channel [29, 31] . the aromatic moieties of a drug form π-π-stacking and hydrophobic interactions with the residues phe656 and tyr652 from the large cavity of the herg channel [32] . the herg gene encodes the α-subunit of the inwardly rectifying k + -channel, which is highly expressed in the human heart. this channel is involved in the repolarization of the cell. the mutation of the herg gene or a channel blockade by drugs can lead to prolongation of the qt interval in the electrocardiogram in severe arrhythmia. high sensitivity of the herg channel to a blockage by many drugs and with the resulting cardiovascular adverse events like torsades de pointes (tdp), which can degenerate into ventricular fibrillation, led to the requirement of regulatory agencies that the effect of novel drugs on the herg channel has to be investigated and reported [33] . hence, considering herg channel blockade is essential to improve cardiovascular safety of novel ccr2 antagonists. the increase of overall polarity of the drugs by introduction of hydrophilic ring systems and substituents on side 2 has been a successful strategy to eliminate herg inhibition [31] . further, successful approaches are the attenuation of the pk a value of the basic amine, modification of its steric environment, and the formation of zwitterions [32, 34] . incyte's incb3344 (2), one of the first potent pyrrolidine derivatives with a 3,4-methylenedioxyphenyl residue (table 1) , was well investigated in receptor binding and chemotaxis assays with human (hccr2) and murine (mccr2) receptors [35] . despite the fact that human and murine ccr2 receptors show high sequence homology, binding affinities of ligands differ considerably among species [36] . incb3344 (2) showed the ic 50 values of 5.1 nm in hccr2-binding and 3.8 nm in chemotaxis assay. 2 has been used as a tool in rodent in vivo efficacy models for multiple sclerosis, arthritis, and obesity and was effective in lowering macrophage levels in the targeted tissue [25, 26, 37] . despite a high selectivity over other chemokine receptors, incb3344 (2) was not a suitable clinical candidate due to its moderate herg binding activity (ic 50 ¼ 13 μm) and inhibition of cyp 3a4 [6, 35] . this data led to further structural modifications of 2. the removal of the ethoxy group at the quaternary carbon at position 3 of the pyrrolidine ring led to a loss of the mccr2 affinity but retained the hccr2 affinity. previous sar studies proved that the trifluoromethylphenyl residue on side 2 was crucial for the ccr2 binding affinity. the (r)-configuration at the position 3 of the pyrrolidine ring was also known to be important for the ccr2 affinity [38, 39] . the optimization involved the replacement of one phenyl ring by a heteroaromatic ring to reduce hydrophobicity (logp) as in incb3284 (3). 3 includes a 6-methoxy-3-pyridyl moiety on side 1 and is a selective and potent ccr2 antagonist with ic 50 values of 3.7 nm in hccr2 binding and 4.7 nm in chemotaxis assay. in contrast to incb3344 (2), incb3284 (3) was a substrate for cyp 3a4 and cyp 2d6, but had no inhibitory or inducing effects on the cyp system. the inhibition of the herg-associated potassium channel was rather low (ic 50 ¼ 84 μm) [35] . the balanced profile and safety data made incb3284 (3) a promising candidate for phase i and phase ii clinical trials, which were unfortunately terminated ahead of schedule [40] . a further clinical candidate from this series, which was tested in osteoarthritis and liver fibrosis, was incb8761 (a.k.a. pf-4136309) (4) , which belongs to the group of "inverse" pyrrolidines. molecular modeling led to a new series of compounds, in which the contacts to the (r)-3-aminopyrrolidine as the main functional group on side 1 and 3-trifluoromethylbenzoyl aminoacetyl moiety on side 2 in the incn3284 (3) series were reversed [39] . by analogy to the incb3344 and incb3284 series, the stereochemistry at the cyclohexyl ring required to be cis. because previous studies demonstrated that the hydroxyl group and heteroaryl moiety at position 4 of the cyclohexyl ring led to weak herg blockade and low intrinsic clearance, both structural elements were retained. the 6-methoxy-3-pyridyl moiety, previously present in the lead compound 3, was replaced by a (pyrimidin-2-yl)-pyridin-2-yl residue in the potent analog incb8761 (4) (ic 50 ¼ 5.2 nm). compound 4 demonstrated no significant inhibition of other chemokine receptors or any influence on the cyp system. in contrast to the incb3284 series, (s)-configuration in position 3 of the pyrrolidine ring is preferred over the (r)-enantiomer [27, 39] . the length of both linkers in compounds 2, 3, and 4 corresponds well to the pharmacophore model, where the linker l1 contains 4 carbon atoms and the linker l2 7 carbon atoms. merck has disclosed a variety of ccr2 antagonists, which contain a piperidine ring and a cyclopentanecarboxamide substructure. they identified a series of pyridoannulated piperidines like mk0812 (5) that has a tetrahydro-3-trifluoromethyl-1, 6-naphthyridine substructure ( table 2 ) [41] . this compound contains four chiral centers and is a potent ccr2 antagonist with an ic 50 value of 5.0 nm and inhibits the chemotaxis with an ic 50 value of 0.2 nm. mk0812 (5) became a clinical candidate in arthritis and multiple sclerosis, but failed in the phase ii due to lack of efficacy and was therefore discontinued from the company's pipeline [37, 40] . mk 0483 (6) is another potent clinical candidate derived by merck with an ic 50 value of 4.0 nm in the ccr2 binding [27] . 6 contains a piperidine and a 1,3-phenoxazine system instead of the tetrahydro-1,6-naphthyridine moiety as in 5 [24] . further structural variations led to compound 7 not only with a binding affinity of 1.3 nm but also with a potent herg inhibition (ic 50 ¼ 54 nm). to minimize effects on the herg channel, the 4-fluorophenyl substituent in the r1 moiety was replaced by diverse more polar aryl and heteroaryl residues. the lowest inhibition of the herg channel was observed after the introduction of a carboxylic acid in position 3 of the phenyl substituent, unfortunately the ccr2 binding also decreased in similar range [42] . a benzylamide incorporated in 7 is also a promising common structural element of merck's spirocyclic ccr2 antagonists 13 and 14 (see sect. 2.6). further development of piperidine-based ccr2 ligands led to a series exemplified by compound 8. the potent ccr2 antagonist 8 includes a heteroaromatic system of an indole, representing r1, a central core with a cyclohexyl and a piperidine ring and a trans-configured cinnamide instead of the benzamide as in 7. in respect to cyclohexane stereochemistry, the trans-substituted compounds were more active in the ccr2 binding (ic 50 (hccr2) ¼ 12 nm) than the cis-configured derivatives (ic 50 (hccr2) ¼ 240 nm). it was shown that two substituents in meta or para position provided the highest ccr2 affinity. although the in vitro herg binding of 8 was rather high (ic 50 ¼ 8 μm), an influence on hemodynamic parameters in a guinea pig model was not observed. compound 8 also reached animal studies in an inflammation model (thioglycollate-induced peritonitis) [43] . the replacement of the trifluoromethyl naphthyridine group in mk0812 (5) by a (trifluoromethyl pyridazinyl)piperazine moiety led to a new series of piperazinebased ccr2 antagonists. pf-4254196 (9) is a potent ligand of the ccr2 receptor (ic 50 ¼ 8.1 nm) without any cardiovascular liabilities (ic 50 (herg) ¼ 31.3 μm) ( table 3 ) [34] . similar to merck's piperidines mk0812 (5) and mk0483 (6), piperazines 9 and 10 also include a cyclopentane core with an amino substituent in position 3 and a carboxamide and isopropyl substituent in position 1. the development of pf-4254196 (9) started with modifications of the spacer length between the cyclopentane carboxamide and the trifluoromethyl containing aryl residues in existing series of ccr2 ligands. prior compounds included a trifluoromethyl-substituted pyridine but showed a significant herg inhibition. to eliminate the cardiovascular risk modifications of both, the side 1 tetrahydropyran ring and side 2 heterocycle were explored. based on former sar studies, a substitution of side 2 with more polar and/or potential π-π-stacking residues was expected to be well tolerated [34] . the introduction of a pyridazine ring led to pf-4254196 (9), which displayed a significantly better ccr2/herg index and selectivity than the corresponding pyridine-containing compound. pyridine-based compounds were also reported to be dual ccr2 and ccr5 antagonists [38] . further modifications led to a methano-bridged piperazine derivative 10 (2,5-diazobicyclo[2.2.1]heptane). although 10 contains a boc (tert-butoxycarbonyl) moiety instead of an aryl or heteroaryl ring, it showed improved ccr2 affinity (ic 50 ¼ 2.9 nm) [44] . a further series of potent ccr2 ligands contains an aromatic ring on side 1 and a second terminal piperazine ring on side 2 connected via a carbonyl linker. the prototype of this series 11 demonstrated a high binding affinity to the ccr2 receptor (ic 50 ¼ 3.5 nm) and a significant reduction of herg activity compared to methylene-linked subseries and other heterocycles at the side 2. in the herg assay, a 10,000-fold selectivity for the ccr2 receptor over herg was observed. compound 11 did not interact with other chemokine receptors except with the ccr5 receptor (ic 50 ¼ 22 nm) and can therefore be considered as a dual antagonist with the ccr2 receptor preference [45] [46] [47] . a prominent example of the spiropiperidine class of ccr2 inhibitors, rs504393 (12) , was reported by roche/iconix. the central structural element of this class included a tertiary amine in a benzannulated piperidine ring system and an orthogonal relationship between the 3,1-benzoxazin-2-one and the piperidine ring, caused by the spirocyclic connection of the rings (table 4 ). another important aspect is the hydrogen binding potential of the urethane moiety and the restriction to small substituents at the benzoxazine heterocycle. sar studies led to rs504393 (12) (ic 50 ¼ 89 nm) as the most affine compound of this benzoxazine class. the sars of spiropiperidines were extensively investigated. in these spirobenzoxazine systems, the position 4 of the piperidine is disubstituted by a spiro-phenyl urethane system. only piperidines or linear alkyl chains were accepted by the ccr2 receptor, other substituents were inactive or revealed reduced affinity. this group of compounds is highly selective for the ccr2 receptor [48] . site-directed mutagenesis of acidic residues glu291 and asp284 in the ccr2 receptor to ala, asn, or gln showed the importance of both glu291 and asp284 for ligand interactions via hydrogen bonding towards the tertiary amine of the piperidine ring. for this reason, spiropiperidines show an affinity to receptors in which a glutamic acid residue is in a similar position as in the ccr2 receptor [49] . it was also known that spiropiperidines prevent ccl2 binding by occupying the same region in the inter-helical bundle on the extracellular side [48] . in contrast, the ccr2 receptor binding of ccr2 antagonists without a basic amino moiety was not affected by glu291 mutations [22] . merck's compound 13 was claimed as a ccr2 antagonist for the potential treatment of inflammatory and rheumatic diseases [49] . this spiro[indenepiperidine] 13 showed ic 50 values of 1.3 and 0.45 nm in the ccr2 binding and the chemotactic assay, respectively. 13 possessed a high selectivity over other chemokine receptors including the ccr5 receptor (%500-fold). the tested isomer 13, shown in table 4 , was found to be the only active stereoisomer of the four possible stereoisomers. the series of spiropiperidines similar to 13 demonstrated that the presence of methyl groups in position 3 of the 1,3-disubstituted cyclopentane ring and in the piperidine ring is crucial for the high affinity at the human and mouse ccr2 receptors. the introduction of an additional methyl group in position 4 of cyclopentane instead of the methyl group in position 3 led to a total loss of the ccr2 activity, as the removal of the methyl group in position 3 of the piperidine ring eliminated ccr2 activity in the assay with both human and murine ccr2 receptors [6, 50] . compared to analogs without a methyl group in position 1 of the cyclopentane ring, the ccr2 binding affinity increased twofold [51] . compound 13 also belongs to the series of cyclopentyl and cyclobutyl constrained analogs, in which a quaternary carbon substitution of the central cyclopentane ring was preferred for ccr2 binding [37] . the spiropiperidine 14 from merck contains a tertiary carbon in side 2. the side 1 is represented by a spiro[indene-piperidine] in compounds 13 and 14, but the cyclopentyl ring of 13 is replaced by an open chain, which includes an additional secondary amine and a cyclopropylmethyl moiety leading to a tertiary carbon atom. the substituent of the tertiary carbon atom was also varied. a p-fluorophenyl substituent at that position led to merck's "compound 26", a dual ccr2 and ccr5 receptor antagonist, which is, apart from this substituent, identical to the cyclopropylmethyl derivative 14. in contrast to the p-fluorophenyl derivative, the cyclopropyl derivative 14 was selective for the ccr2 receptor, showing an ic 50 value of 4 nm and promising pharmacokinetic properties [22, 23] . in both 13 and 14, the aromatic residue r1 is represented by an indene and linked to the central tertiary amine via a 2-carbon linker. the size of linker l2 is broadly based, including 2 atoms in 12, 6 in 13, and 7 in 14. potent ccr2 receptor antagonists that contain a quaternary ammonium salt have also been reported ( table 5 ). the quaternary ammonium moiety of these compounds is expected to form an ionic interaction with glu291 in the binding pocket of the ccr2 receptor [27] . developments of these types of ccr2 antagonists started from tak-779 (15, see also sect. 3.1), first developed as a ccr5 antagonist and later found to have also significant ccr2 affinity (ic 50 ¼ 27 nm) [40, 52] . compounds 16 and 17, both showing promising binding affinities in a 125 i-labeled ccl2 assay using a thp-1 cell line, resulted from the systematic modifications of side 2 in 15 [53] . jnj171668 (16) , developed by johnson&johnson, is a potent candidate with a binding affinity of 20 nm. in line with its quaternary ammonium structure, jnj171668 (16) had poor oral bioavailability, but entered clinical trials for the treatment of allergic rhinitis as a nasal application [40] . the 3,4-dichloro phenyl ring of 16 led to higher binding affinity at the ccr2 receptor than its analogs with other substitution patterns. from a series of different biphenyl-containing compounds, it was evident that the presence of a chloro or bromo group leads to improved binding affinities compared to electrondonating (ome, me) and electron-withdrawing groups (cn, cf 3 ). structural modifications on the side 2 were also found to be responsible for selectivity related to the interactions with the ccr2 and ccr5 receptor. a different modification on side 2 led to compound 17 with a linker l2 consisting of 9 carbon atoms. 17 contains a biphenyl moiety attached to the 3-position of acrylamide displaying the binding affinity of 10 nm [53] . in comparison with the pharmacophore model, the linker l1 is missing in 15, 16, and 17, whereas r1 is represented by a tetrahydropyran moiety. in recent years, new structural classes of ccr2 ligands appeared in the literature, mostly including only small series of compounds. here, we want to highlight three promising classes, each exemplified by a typical ligand: (1) sulfonamides are represented by 18, (2) azetidines by 19, and (3) bicyclic compounds by 20. chemocentryx and glaxosmithkline started the development of sulfonamides as ccr2 ligands [6] . the efforts resulted in the triazolyl-substituted compound as a promising example. modifications of the substitution pattern led to the trichlorosubstituted n-phenylbenzenesulfonamide 18 as a potent ccr2 ligand (the gtpγs accumulation assay, ic 50 ¼ 10 nm). 18 inhibited monocyte recruitment but also showed inhibitory effects on the cyp2c19 and cyp2c9 activity ( table 6 ). the investigation in a thioglycollate-induced peritonitis model for inflammation in a mouse strain with the human ccr2 receptor knocked-in (hccr2ki mouse) verified the dose-dependent and strain-specific inhibition of monocyte recruitment by 18 [54] . in an analogy to piperidines (see sect. 2.4) and pyrrolidines (see sect. 2.3), azetidine-based ccr2 ligands were described. in order to eliminate the zwitterionic character of a piperidine-based amino acid derivative developed by johnson&johnson and to increase solubility, a cyclohexylazetidine system was prepared first [31, 55] . different six-and five-membered heterocycles were introduced in the 4-position of the cyclohexane ring, which reduced the herg channel activity. finally, the thiazole derivative 19 was identified as the most potent candidate. a cis orientation of the thiazolyl and the azetidinyl substituents on the cyclohexane ring was found to be essential for the high ccr2 binding affinity [55] . compound 19 revealed an ic 50 value of 37 nm in the ccr2 binding assay and did not interact with the herg channel (ic 50 > 50 μm). a promising cardiovascular safety profile was confirmed in an anesthetized dog safety study. a high selectivity against other chemokine receptors was found, but the pharmacological profile remains to be reported [56] . in 2013, further variations of the heteroaromatic substituents at the n-acylglycine moiety were published, which started from 19 as a lead compound. although various compounds with the high ccr2 affinity, promising functional activity and low herg channel affinity were identified, none of these compounds displayed a promising pharmacokinetic profile [57] . in 2013, cai et al. published a novel series of ccr2 antagonists with a bicyclo [3.3.0]octane or bicyclo[4.3.0]nonane scaffold. this class of ligands was designed according to the ccr2 pharmacophore model mentioned above. the first generation was based on a 7-aminobicyclo[3.3.0]octane system [58] . replacement of the methylene moiety in 3-position by an amino moiety led to the closely related class of 7-amino-3-azabicyclo[3.3.0]octanes. a systematic evaluation of the substituents on the exocyclic amino moiety resulted in 20, the most promising compound of this series. 20 displayed the high ccr2 affinity (ic 50 ¼ 31 nm) and low cardiovascular risk (herg ic 50 > 50 μm). the clinical potential of this new candidate will be evaluated after investigation of its in vivo properties [59] . as predicted by computational-based homology modeling studies that included few potent ccr2 ligands, different amino acids were postulated to be essential for the ligand binding to the ccr2 receptor. the most recent studies were based on the homology modeling, where in 2010 published crystal structure of the closely related cxcr4 receptor served as a template. the cxcr4 structure shows a higher sequence homology as well as a larger binding pocket than the structure of bovine rhodopsin. the binding pocket of the ccr2 receptor is formed by transmembrane domains tm2, tm3, tm5, tm6, and tm7 [28] . glu291, located on the transmembrane region 7 (tm7), was proposed to be an important anchor residue of various ccr2 ligands, including spiropiperidines [48, 60, 61] . the central basic amine, present in most ccr2 receptor ligands, forms a salt bridge to this conserved acidic residue. mutagenesis studies also implied that tyr120 and his121 might be crucial because of their ability to form hydrogen bonds with endogenous ligands or synthetic molecules. hydrophobic interactions of the ligands were observed with aromatic residues tyr49 and trp98 [28] . the predicted binding site of tak-779 (15) was studied best. the residues of the ccr2 receptor that strongly interact with tak-779 are shown in fig. 3 . the most important interaction is the electrostatic interaction of the carboxylate of glu291 with the quaternary ammonium group of tak-779. the tetrahydropyran oxygen forms hydrogen bonds with both tyr49 in tm1 and thr292 in tm7. the biaryl system on the other site is fixed between the hydroxyphenyl moieties of tyr120 and tyr259 and to a lesser extent by his121 via π-π-stacking interactions. his121 also interacts with arg206 (tm 5), which results in a weaker ligandhistidine interaction. in the ccr5 receptor, his121 is replaced by phenylalanine (see fig. 5 ), which cannot form interaction with arginine and therefore adopts an alternative rotameric conformation increasing the π-π-interactions. in case of tak779, the residues tyr49, trp98, tyr120, and his121 are discussed to form an aromatic cluster contributing to the ccr2 receptor binding [60, 62] . for the binding of the spirocyclic antagonist rs504393 (12), glu291 and asp284 were identified as hydrogen bond partners for the tertiary amine within the piperidine (15), modified according to [28, 60, 62] 202 a. junker et al. ring [48, 49] . expectedly, the binding of the ccr2 antagonists devoid of basic amine was not affected by glu291 mutations [22, 23] . altogether diverse compounds with high affinity to the ccr2 receptor have been identified. a few ccr2 antagonists were investigated in clinical trials. up to now, ccr2 antagonists have not shown promising clinical efficacy in inflammatory diseases as presumed from preclinical models. whether this failure is a result of wrong target selection, off-target effects or poor drug-like properties of the small-molecule antagonist remains to be elucidated [27, 37, 41 ]. the unique opportunity to study the impact of ccr5 receptor antagonists by exploiting the ccr5δ32 bp polymorphism as well as its function as a co-receptor for the hiv-host cell fusion brought the ccr5 receptor into focus of many pharmaceutical companies. there are various ccr5 antagonists reported in the literature so far. the ccr5 receptor ligand maraviroc (1, celsentri ® , uk-427,827) ( fig. 1 ) developed by pfizer is the only ccr5 ligand approved for the treatment of confirmed r5-tropic hiv-1 infection by the fda and emea [2] . on account of this, many further investigations have already been undertaken, starting from maraviroc (1) as a lead compound. the intention of this chapter is to summarize the sars of various ccr5 antagonists focusing on ccr5 antagonists derived from tak compounds (takeda), maraviroc, and related tropane-based ccr5 ligands. several different aspects such as antiviral activity, cyp inhibition, leading to several drug-drug interactions and severe adverse effects will be discussed [65, 66] . inhibition of the herg k + -channel is a common challenge in developing ccr5 selective ligands due to the basic amine, which is required for the interaction with glu283 of ccr5 receptor [29, 65, 66] . therefore, the affinity to the herg k + -channel [67] and the oral bioavailability, which significantly influence the ccr5 ligand development, will be discussed. takeda pharmaceutical company has set themselves the task of creating a new class of antihuman immunodeficiency virus 1 (hiv-1) entry inhibitors. one way to inhibit hiv-1 replication is to prevent the viral entry into the target cell. the potential of this approach is shown by t20, a peptide that prevents the conformational change in the viral glycoprotein gp41 that drives membrane fusion [68] . therefore, takeda has designed several compounds which were based on hits of a high-throughput screening (hts). the most promising compound resulting from these hits was tak-779 (15) (fig. 4) . tak-779 (15) antagonizes the binding of ccl5 to ccr5-expressing chinese hamster ovary (cho) cells completely at a concentration of 100 nm and showed an ic 50 value of 1.4 nm. moreover, 15 was shown to block membrane fusion of hiv-1 at nanomolar concentrations. the binding of ccl3 and ccl4 to the ccr5-expressing cells was also blocked with ic 50 values around 1.0 nm. although tak-779 inhibited the binding of [ 125 i]-ccl2 to ccr2 in cho/ccr2 cells, its ic 50 value for ccr2 receptor (ic 50 ¼ 25 nm) was approximately 20-fold higher than that for ccr5 receptor [52] . the sequence homology between ccr5 and ccr2 receptors is 76% [69] , which might explain the dual antagonistic character. molecular modeling and mutagenesis studies have shown that the active site of the ccr5 receptor is very hydrophobic with multiple aromatic residues forming a tight binding pocket [70] . the benzene ring of the benzo [7] annulene moiety of 15 was observed to interact with aromatic side chains of tyr108 and trp248 via a t-shaped π-π-stacking. additionally tyr108 forms a hydrogen-bond interaction between the phenolic oh group and the carbonyl moiety of 15. strong hydrophobic interaction between the p-tolyl group and ile198 on tm5 accompanied by some weaker interactions of 15 with thr195, ile198, phe109, trp248, and tyr251 were also found. the limited ionic interaction between the quaternary ammonium moiety of 15 and glu283 was caused by the steric shielding of the positively charged center (fig. 5 ) [63] . due to the fact that 15 inhibits the ccr5 receptor, an anti-r5 hiv-1 assay was performed. the measured effective concentrations in the anti-fusion assay were 1.2 nm (ec 50 ) and 5.7 nm (ec 90 ) [71] . it was additionally shown that tak-779 did not interact with ccr1, ccr3, or ccr4 receptors. the quaternary ammonium moiety of tak-779 led to a good binding affinity, but poor oral bioavailability, which required further optimization. in order to develop an active ccr5 antagonist with a reasonable oral bioavailability, derivatives of tertiary amines were investigated. the tertiary amine 21, derived from tak-779 by removing one ch 3 group, resulted in decrease of ccr5 affinity but increased oral bioavailability. in order to enhance the ccr5, affinity modifications of the [7] annulene ring were undertaken [72] . the exchange at the 5-ch 2 -moiety of the benzo [7] annulene of tak-779 (15) by a s-atom (22) did not significantly increase ccr5 receptor affinity [73] . the introduction of a sulfoxide (23) or a sulfone (24) led to a slightly increased affinity [74] . the exchange of the 5-ch 2 -group by an o-atom resulted in the benzoxepine 25 with high ccr5 affinity, which could not be retained in the tertiary amine 26. the highest affinity of the tertiary amines was found for the 1-methyl-1-benzazepine 27 (ic 50 ¼ 130 nm) [72] . because the compounds 24 and 27 possess high oral bioavailability (>50%) in rats [73] , further variations of the benzothiepine-1,1-dioxide and the 1-benzazepine cores were envisaged (table 7) . in order to enhance the ccr5 receptor affinity, modifications of the p-methyl group of 24 were investigated ( table 8 ). replacement of the methyl group by an ethyl substituent (28) led to a 3-fold increased ccr5 receptor affinity, which was further increased by introduction of alkoxy groups (compounds 29 and 30) [75] . the butoxyethoxy derivative 30 was chosen for further optimization. the combination of the butoxyethoxy group with a 1-benzazepine scaffold led to the next series of ccr5 antagonists. homologation of the n-methyl group to an n ethyl group (31) resulted in an increased ccr5 affinity, but low inhibition of hiv-1 envelope-mediated membrane fusion ( table 9 ). introduction of propyl (32), isobutyl (33) , benzyl (35) , and methylpyrazolyl (36) residues increased the inhibitory activity, whereas the cyclopropylmethyl group (34) resulted in a remarkable drop of inhibitory activity [75] . compounds 37-42 were synthesized to examine the effect of various sulfoxides on the ccr5 affinity (table 10 ). the methylimidazolyl-sulfinyl derivative 40 led to increased ccr5 receptor affinity. elongation of the alkyl substituent to an ethyl (41) or propyl (42) moiety led to increased ccr5 affinity. the enantiomer (s)-42 was found to be more potent than the (r)-enantiomer (r)-42 [72] . expansion of the seven-membered azepine ring of (s)-42 to an azocine ((s)-43), azonine ((s)-44), and azecine ring ((s)-45) led to a series of potent compounds (table 11 ) [72] . due to the high ccr5 affinity, virus fusion inhibition, and oral bioavailability, tak-652 ((s)-43) became a promising hiv-1 entry inhibitor for clinical studies. it was shown that tak-652 inhibited the binding of ccl5 (ic 50 ¼ 3.1 nm), ccl3, compounds 46-48 represent ccr5 antagonists with entirely different structures. the core structure of 46-48 is characterized by an n-(3-piperidinopropyl) carboxamide. the propandiamine substructure has become an important pharmacophore element for the development of ccr5 antagonists (table 12 ). the hts of the takeda's compound library led to the discovery of n-(piperidinopropyl)carboxamide 46 with low micromolar ccr5 binding affinity. subsequent optimization resulted in a series of piperidine-4-carboxamides, exemplified by 47, which had low nanomolar affinity for ccr5 receptors and exhibited high anti-hiv-1 activity [77] . the fast metabolism of 47 stimulated further optimization, which led to the most promising derivative 48 (tak-220) of this new series (table 12) . it demonstrated high inhibition of the [ 125 i]-ccl5 binding to ccr5expressing cho cells (ic 50 ¼ 3.5 nm), high inhibition for hiv-1 membrane fusion (ic 50 ¼ 0.42 nm), and also high metabolic stability upon incubation with human hepatic microsomes [78] . a comparison of binding affinities and antiviral activity of tak-779 (15), tak-652 (43) , and tak-220 (48) is summarized in table 13 . the conformational flexibility of tak-220 is higher than that of tak-779 due to the higher number of rotatable bonds. docking into a 3-d homology model of the ccr5 receptor showed that tak-220 forms a strong salt bridge with glu283. mutagenesis studies indicated that the residues trp86, tyr108, trp248, tyr251, and met287 (see fig. 5 ) are important for tak-779 binding, but have little effects on tak-220 binding. however the hydrophobic interaction of tak-220 with ile198 is as strong as in case of tak-779 binding (fig. 6) . the 3-chloro-4methylphenyl group of tak-220 is placed in the similar region within the helical bundle between phe109, trp248, and tyr251 as the phenyl group of maraviroc (1) [63, 79] . tropane-based ccr5 ligands the first 1-(3,3-diphenylpropyl)piperidine-based ccr5 antagonists were found by astrazeneca and pfizer by an hts of their compound libraries [45, 80] . astrazeneca's screen resulted in two closely related hits, 49 and 50, and pfizer's screen in the hits 51 (uk-107,543) and 52 (table 14) . astrazeneca's approach focused on the development of ccr5-selective antagonists for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis [81] and inflammatory bowel disease [82] . compounds 49 and 50 demonstrated similar ccr5 binding affinities in low micromolar range, indicating no advantage of the cyclized n-substituent of 49. the sar investigations, in which the substituent r 2 of the acyl group was varied, revealed that neither (hetero)aromatic nor aliphatic groups increased the ccr5 receptor affinity (table 15 ). the phenylacetyl derivative 53 was the only compound with potency in the high sub-micromolar range. introduction of substituents at the oand m-position of the phenylacetyl group did not significantly affect the binding affinity. however, an increase in affinity was observed after introduction of polar electron-withdrawing substituents in p-position. in particular, sulfamoyl (55) , n,n-dimethylsulfamoyl (56, 57) , and methylsulfonyl (58) groups showed nanomolar ccr5 affinities. replacing the methyl (56) with the ethyl group (57) at the amide n-atom slightly increased the ccr5 affinity (table 15 ). replacement of the amide substructure by a sulfonamide moiety was shown to be detrimental, whereas the introduction of a urea moiety instead of the amide retained the ccr5 affinity. compounds fig. 6 schematic presentation of interactions between the ccr5 receptor and tak-220 (48), modified according to [79] 210 a. junker et al. and 57 showed no affinity towards ccr1, ccr2b, ccr3, cxcr1, and cxcr2 receptors. the n,n-dimethylsulfonamide 57 displayed micromolar affinity to muscarinic and serotonergic receptors as well as the herg channel [45] . therefore, the methyl sulfone 58 was chosen as the new lead compound for further ligand development. next sar studies around the benzhydryl structure were undertaken. introduction of one 60 or two 59 fluorine atoms into the p-position of the phenyl rings resulted in decreased ccr5 affinity. in contrast, one chlorine atom in p-position (61) was highly beneficial. therefore, further substituents in p-position of one compounds with strongly electron-withdrawing substituents such as trifluoromethyl (62), cyano (64) , and methylsulfonyl (66) were highly potent, but also the methoxy derivative 65 demonstrated high ccr5 affinity (table 16) . electronic effects alone are not sufficient to explain the sar. the weakly potent difluoro (59) and 4-fluoro (60) compounds as well as the methylsulfonyl (66) derivative demonstrated sufficient metabolic stability and a good pharmacokinetic (pk) profile. the potential of the fluorine atoms to reduce oxidative metabolism on the phenyl rings of the diphenylpropyl moiety indicated a possible approach to improve oral bioavailability in this series of ccr5 ligands [47] . the (s)-enantiomer of the methylsulfonyl derivative (s)-66 was found to be twice as potent as the racemate 66. moreover the enantioselective synthesis of (s)-66 was used to prepare several analogs with various substituents at the second phenyl ring. since fluorine atom in p-position of the second phenyl moiety led to dramatic loss of the ccr5 affinity, the m-position was addressed for the introduction of the fluoro substituent (67). 67 displayed increased ccr5 affinity, but also fast clearance and short half-life. this could be improved by introduction of halogen atoms at both m-positions (table 17 ). the 3,5-difluoro (69), 3-fluoro (67), and 5-chloro (68) analogs showed favorable pharmacokinetic profiles. the 3,5-difluoro derivative 69 displayed no affinity towards ccr1, ccr2b, ccr3, cxcr1, and cxcr2 receptors and other human g-protein-coupled receptors (human m 1 , m 2 , and 5-ht 2a receptor). unfortunately, an inhibition of cyp 2d6 (1.6 μm) and herg ion channel binding (7.3 μm) were detected [46] . in order to reduce cardiotoxicity, the benzhydryl part of the molecule was further modified. sar investigation clearly indicated the requirement of one phenyl substituent, whereas the replacement of the second phenyl ring by other substituents was tolerated. introduction of a piperazine and c-atom-linked piperidine ring led to compounds with reduced lipophilicity, promising ccr5 affinity and decreased herg ion channel binding (table 18 ) [83] . the piperazine derivative 71 showed only moderate bioavailability in dogs and very fast plasma clearance in rats, whereas the c-linked piperidine with the methylsulfonyl substituent at the n-atom demonstrated good bioavailability in both species with high selectivity over cyp 1a1, 2c9, 2c19, 2d6, and 3a4 enzymes. therefore 72 (azd5672) was selected as drug candidate for the treatment of rheumatoid arthritis (ra). the development of azd5672 (72) was terminated in a phase iib study with ra [84, 85] . compounds 51 and 52 showed weak ccr5 binding affinity, antiviral activity could not be detected, and, moreover, high affinity to the cyp 2d6 enzyme was found (tables 14 and 19 ) [80] . the replacement of the imidazopyridine structure, responsible for the interaction with cyp 2d6 by coordination of the pyridine n-atom to the heme iron, led to benzimidazole 73. compound 73 showed potent inhibition of ccl4 binding and much weaker cyp 2d6 inhibition, but still no antiviral activity (table 19 ). in order to increase the polarity, one of the phenyl groups of the diphenylmethyl moiety was replaced by an amide bearing substructure already found in compound 52. amides 74-77 inhibited the ccl4 binding but, more interestingly, moderate levels of antiviral activity determined against hiv-bal in pm-1 cells [80, 86] were found (table 19 ). the benzamide 75, the isobutyramide 76, and the cyclobutanecarboxamide 77 were found to be the most active antiviral compounds in this series. the data of ccl4 inhibition and antiviral activity do not correlate, indicating that the binding domains of hiv gp120 and ccl4 are distinct and separate. in order to determine the eutomers within the amide series, the benzamide 75 and the cyclobutanecarboxamide 77 were synthesized stereoselectively. the (s)-enantiomers (s)-75 and (s)-77 had higher ccr5 affinity and antiviral activity than the (r)-enantiomers. compound uk-347,503 ((s)-77) also showed decreased affinity to the cyp 2d6 enzyme and was chosen as a lead compound for further ccr5 antagonist development [80] . compounds with affinity to the cyp 2d6 enzyme have a basic amino group in 5-7 å distance to a possible site of oxidation. the basic amine is interacting with asp301 of the enzyme [87, 88] . in order to avoid cyp 2d6 affinity a series of analogs of 77 with a modified piperidine ring was designed. the benzimidazole derivatives exo-78 and endo-78 demonstrated increased inhibition of viral replication (table 20) combined with reduced cyp 2d6 affinity. the similar activity of the exoand the endo-isomers is caused by different orientations of the bridged piperidine rings. the benzimidazole forces the isomer endo-78 into a boat conformation, well overlapping with the chair conformation of exo-78 [1] . all compounds 78-80 demonstrated potent antiviral activity against clinically relevant ccr5-tropic viruses, but failed in safety screenings due to high inhibition of the herg ion channel [89] . therefore the next aim was to obtain selectivity against the herg channel. the first approach to overcome herg affinity was driven by the exploration of the prodrug concept of compound 81. compound 81 demonstrated high bioavailability and high herg binding but was rapidly oxidized to the highly selective primary metabolites tetrahydropyran s-oxide 82 and s,s-dioxide 83, 82 and 83 displayed high inhibition of cell-cell fusion without any binding to the herg channel at 10 μm in in vitro assays (table 21) . however, the bioavailability of metabolites 82 and 83 after p.o. administration of 81 to rats was lower than 10%. gut wall metabolism and excretion by the liver were suggested to be responsible to the failure of 81 as oxidizable prodrug [74] . because the first strategy to overcome herg affinity by a prodrug concept failed, the second strategy focused on the modification of the basicity and steric environment of the central amino moiety and alteration of the orientation and substitution patterns of the aromatic rings in lead compound 78. in the oxagranatane exo-80 (table 20) , the basicity of the central amino group is reduced to pk a 6.0 compared to pk a 7.8 of exo-78. however, the herg channel affinity was not reduced, which suggested that the basic center itself is not essential for herg binding [90] . docking of 78 into a herg channel model indicated a lipophilic interaction of the phenyl ring of the benzimidazole moiety with the herg channel residues. in order to inhibit this overlap, a triazole moiety (84) instead of the benzimidazole ring (78) was introduced, which dramatically decreased the herg affinity. the introduction of an isopropyl side chain at the triazole motif (85) increased the antiviral activity, but the added lipophilicity increased the affinity to the herg ion channel as well. the cyclobutyl group of the amide 78 was shown to overlap nicely with the lipophilic binding pocket of the herg channel. in order to interrupt this interaction, polar fluorinated groups were introduced (86, 1). the 4,4-difluorocyclohexyl derivative 1 does not show any binding to the herg channel, even at a concentration of 1,000 nm. combined with low nanomolar antiviral potency (table 22 ) [89] , a broad anti-r5 hiv-1 spectrum and no inhibition of cyp 1a2, 2c9, 2c19, 3a4, and 2d6 enzymes compound 1 were characterized as an inverse agonist of ccr5 receptors, stabilizing the receptor in the inactive conformation [91] . maraviroc (1, uk-427,857) was the product of a long optimization process leading to the first ccr5 ligand approved for the treatment of confirmed r5-tropic hiv-1 infection on the market [2] . the use of maraviroc for the hiv-1 therapy is currently complicated by the growing number of maraviroc-resistant hi-virus strains (mvc res ) [92] [93] [94] , which makes the ligands with an improved resistance profile desirable. the crystal structure of the ccr5-maraviroc complex, reported in 2013, shows the binding of the ligand at the bottom of a pocket formed by residues from helices tm1, 2, 3, 5, 6, and 7. the tropane n-atom is protonated and forms a salt bridge with glu283. the nh moiety of the amide forms a hydrogen bond with the phenolic group of tyr251 (fig. 7) . the length of the propyl chain between the two n-atoms correlates with the positions of glu283 and tyr251 in the receptor. the fluorine atoms in the cyclohexane ring form two hydrogen bonds with thr195 and thr245. the phenyl group interacts with five aromatic residues, tyr108, phe109, phe112, trp248, and tyr 251 in the binding pocket. the interaction of the benzene ring with trp248 is believed to prevent the activation-related motion of the receptor, which underlines the inverse agonist character of maraviroc (see fig. 7 ) [95] . compared to the cxcr4/it1t structure [30] , the binding site of maraviroc (1) was found to be deeper, without any contact to extracellular loops. the availability of the x-ray crystal structure of the ccr5 receptor will help to promote the development of novel potent ccr5 ligands with optimized properties. fig. 7 schematic representation of interactions between the ccr5 receptor and maraviroc (1), modified according to [95] selective and dual targeting of ccr2 and ccr5 receptors: a current overview long and coworkers from the shanghai institute of materia medica developed the lead compound 1 in more detail by applying lead deconstruction strategy. this approach combines privileged structures of a lead compound with new motifs. replacement of the difluorocyclohexyl moiety of maraviroc by a phenoxy group and the introduction of the trifluoromethyl group at the p-position of the phenyl ring resulted in the moderate ccr5 ligand 87 (td0444, table 23 ). further improvement of the ccr5 affinity was achieved by introduction of an exo-oriented 2-methyl-3h-imidazo [4,5-b] pyridine-3-yl residue instead of the triazolyl moiety and inversion of the amide substructure, which led to the potent ccr5 ligand 88, whereas the corresponding endo-isomer of 88 is inactive (table 23) [96, 97] . pf-232798 (90c , table 24 ) is the follow-up clinical candidate of maraviroc (1), currently in phase ii clinical studies, evolved from the efforts to increase the absorption and improving the pharmacokinetic profile (pk) of maraviroc (1). the structure of pf-232798 (90c) resulted from an alternative approach which intended to circumvent the cyp 2d6 and herg activity of the hts lead uk-107,543 (51) . the introduction of the tropane substructure instead of the piperidine moiety was previously proven to reduce cyp inhibition [1] and was therefore incorporated into the new lead compound. the lipophilic imidazopyridine and benzimidazole substructures of 51 and 78 were shown to be responsible for the inhibition of cyp 2d6 and high herg binding [80] . in order to prevent lipophilic interactions with the herg ion channel, the imidazopyridine substructure was replaced by more polar 1,4,6,7-tetrahydro-imidazo [4,5-c] pyridine, which led to the 3-substituted (89a-c) and 1-substituted (90a-d) series of compounds. the methyl carbamates 89a and 89b demonstrated high herg inhibition. reducing the size of the amide substituent to an acetyl group (89c) significantly increased the selectivity for herg ion channel within the series 89. also the herg affinity was reduced by incorporation of a m-fluoro substituent into the phenyl ring (89b). switching the substitution position of the 1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine slightly improved the gp160 inhibition from 89c to 90a, but was detrimental in terms of herg binding. the lowest herg inhibition (ic 50 ¼ 12 μm) could be achieved by introduction of an isopropoxycarbonyl substituent 90c (table 24) . compound 90c demonstrated complete oral absorption in rat and dog that was accompanied by improved metabolic stability compared to maraviroc (1) and other compounds in this series. moreover, pf-232798 (90c) displayed antiviral activity against maraviroc-resistant viruses and was therefore chosen as the follow-up clinical candidate of maraviroc (1) [98] . the growing number of reports on maraviroc-resistant hi-viruses [93, 99, 100] underlines the need for development of a next generation of ligands with different resistance profile. the tropane-core represents the central structural motif of all previously successfully developed ccr5 ligands. the key features of the tropane moiety are an increased steric hindrance around the basic amino group and restricted conformational flexibility of the molecule. in order to retain the steric table 24 3-substituted (89a-c) and 1-substituted (90a-d) 1,4,6,7-tetrahydro-imidazo [4,5-c] pyridines hindrance, the introduction of an additional methyl moiety into the propyl chain was envisaged. several heterocycles as well as different amido substituents were screened for their antiviral activity, oral bioavailability, and low propensity towards herg ion channel inhibition and interaction with a range of cyp enzymes. a large series of piperidines 91-94, substituted with a n-heterocycle, bearing an α-methyl moiety were prepared (table 25 ) [101, 102] . the cyclobutyl derivate 91 reveals high antiviral activity combined with the low herg inhibition. introduction of two additional fluorine atoms at the alkyl substituent (92) was shown to be beneficial in terms of antiviral activity, as it was already observed during the optimization of maraviroc [89] . increasing the size of the alkyl substituent to a cyclohexyl group (93) channel leading to an affinity higher than 10 μm. enantioselective synthesis of 93 allowed the determination of (r)-93 as eutomer, with a 100-fold higher antiviral activity than the (s)-enantiomer. bioisosteric replacement of the 1,3,4-triazolyl residue of (r)-93 by a 1,2,4-triazol-1-yl ring in (r)-94 resulted in a highly metabolically stable and potent ccr5 ligand. the 1,2,4-triazole (r)-94 displays excellent whole-cell antiviral activity (ic 90 ¼ 2.6 nm), complete oral absorption in rat, and does not inhibit human cyp 1a2, 2c9, and 2d6 enzymes. moderate affinity towards cyp 3a4 (ic 50 ¼ 3.4 μm) was also found [101, 102] . therefore, compound (r)-94 represents the new lead compound for the development of novel ccr5 ligands with new resistance profile. glaxosmithkline's approach combining in-house hts with computer-assisted drug design resulted in identification of two 4,4-disubstituted piperidines 95 and 96 (table 26) . compounds 95 and 96 show high ccr5 affinity and high antiviral activity combined with promising pharmacokinetic profile in rodents. unfortunately, 95 and 96 showed also moderate herg affinity of ic 50 of 2 μm and 10 nm, respectively, which required further optimization [103] . the optimization focused on the substitution pattern of the phenyl rings, which led to the discovery of several potent ccr5 ligands with decreased inhibition of herg. introduction of a sulfonamide and two halogen substituents at the phenyl ring (97) turned out to decrease herg affinity and retained antiviral activity. the exchange of the positions of the fluoro and chloro atoms from 97 to 98 decreased the antiviral activity. the reverse sulfonamide 100 is less hydrophobic than the forward sulfonamide 99 and was found to be very potent in anti-hiv assays (table 27 ) [104] . however, compound 99 (gsk163929) was favored for further investigation as a clinical candidate, due to the potential aniline metabolite formation from reverse sulfonamide 100. gsk163929 (99) revealed high oral bioavailability and good pharmacokinetic profile in rats and dogs. seven-day safety studies in both species did not show any adverse effects at therapeutic doses. the high antiviral activity, favorable pharmacokinetic profile, and safety data support further development of 99 in phase i clinical studies [104] . investigations of the glaxosmithkline research laboratories revealed a new class of ccr5 ligands with a 2-phenylbutane-1,4-diamine core structure. the sulfonamide substituent turned out to produce superior antiviral activity over all other amides tested. removal of the methyl substituent at the amide n-atom of the benzimidazole derivative 101 led to an 8-fold loss in the antiviral activity (102) . introduction of a second methyl moiety at position 3 of the butanediamine linker (103) increased the activity (table 28 ). compounds bearing other heterocycles than benzimidazole were less sensitive to the effect of n-methyl substitution [105] . compounds 102 to 104 exhibit moderate to fast clearance, only 101 and 105 showed measurable bioavailability. all derivatives were rapidly metabolized and therefore further optimization is required in order to increase metabolic stability and improve the pharmacokinetic profile [105] . the development of tak-779 (15), maraviroc (1), and their follow-up compounds exemplifies the sars of ccr5 receptor ligands and the existing hurdles. the ccr5 receptor antagonist maraviroc (1) developed by pfizer has already been approved for the treatment of confirmed r5-tropic hiv-1 infection [2] . however, the increasing number of maraviroc-resistant hi-virus strains makes the development of ligands with a distinct resistance profile highly desirable [93] . the role of ccr5 receptors in the development and progression of inflammatory diseases led to an increased interest of using ccr5 antagonists for the treatment of rheumatoid arthritis [106] . unfortunately, the use of 1 for the treatment of rheumatoid arthritis failed due to low efficacy [107] . the reasons for this failure remain to be elucidated. according to the involvement of both ccr2 and ccr5 receptors in the pathogenesis of inflammatory diseases [108] , these receptors have become attractive targets for the pharmaceutical industry. the chemokine system is very complex, and the ccr2 receptor binds multiple endogenous ligands including ccl2, which binds exclusively to the ccr2 receptor, as well as ccl8, ccl7, and ccl13 [109] which are rather unselective. the ccr2 receptor is abundantly expressed on blood monocytes and regulates their migration from the bone marrow into inflamed tissue, whereas the ccr5 receptor is expressed on macrophages. the in vivo function of the ccr5 receptor is less well defined than that of the ccr2 receptor, but has been shown to be related to the activation, survival, and retention of macrophages in the core of inflammation and is associated with th1 cell recruitment and activation. the ccr5 receptor also binds various ligands, including ccl3, ccl4, ccl5, ccl8, and ccl3l1. ccl4 and ccl3 are known to be selective for the ccr5 receptor. during the differentiation of monocytes, a reciprocal pattern of expression and function of the ccr2 and ccr5 receptor was observed, showing a downregulation of ccr2 and an upregulation of the ccr5 receptor expression [110, 111] . ccr2 and ccr5 receptors are expressed on different cells but in a complementary manner. both receptors are important in the mediation of leukocyte trafficking in case of inflammation, e.g., during the pathogenesis of cardiovascular (atherosclerosis) and immunological diseases (rheumatoid arthritis, crohn's 226 a. junker et al. disease, transplant rejection). thus targeting both receptors with dual antagonists appears to have therapeutic potential [112] . the clinical efficacy of compounds acting selectively with a particular chemokine receptor remains to be shown. clinical trials with various chemokine ligands failed because a benefit at a critical endpoint was not shown. the complex pharmacology of chemokines and their receptors most likely contributed to the failures. more than 50 different chemokines have been identified that interact with more than 20 classical and atypical chemokine receptors. a few chemokines show a one-to-one specificity as ccl2 is specific for the ccr2 receptor, while other chemokines are promiscuous and bind to different receptors [109, 113, 114] . also, various chemokine receptors have more than one chemokine ligand, which usually leads to differential functional response mediated by the same chemokine receptor. due to the fact that multiple chemokine receptors are involved in the pathophysiology of a disease, dual antagonists that target and inhibit two most prominent receptors could be a possibility to enhance the therapeutic effect. a successful example for a dual antagonism in the field of gpcrs is ps433540, which inhibits the at 1 receptor and the et a receptor. ps433540 appears to be successful as antihypertensive in rats and reached phase iia clinical trials. therefore, promiscuous compounds that target several receptors are suggested to be particularly effective for the treatment of complex diseases, for example, multiple sclerosis or rheumatoid arthritis, in which both ccr2 and ccr5 receptors are involved [41] . as ccr2 and ccr5 receptors belong to the same subfamily (cc) of chemokine receptors their amino acid sequences are highly homologous, mainly in the transmembrane (tm) domains [62, 115] . both receptors contain two conserved disulfide bridges cys32-cys277 and cys113-cys190 in the ccr2 receptor and cys20-cys269 and cys101-cys178 in the ccr5 receptor. comparative analysis revealed 66 % sequence identity in general between ccr2 and ccr5 receptors and 82 % identity in the active site. receptor homology modeling studies predicted a ligand binding pocket of the ccr2 receptor formed by tm2, 3, 5, 6, and 7. in the ccr5 receptor tm1, 2, 3, 5, 6, and 7 form the binding pocket for ccr5 inhibitors, which is located at the extracellular region and is partly covered by the extracellular loop. both ccr2 and ccr5 receptors possess glutamic acid in the anchor site in tm 7, glu291 in ccr2 receptors, and glu283 in ccr5 receptors. glu291/glu283 is essential for the interaction with protonated tertiary amines or quaternary ammonium ions. superposition of ccr2/ccr5 binding sites revealed that all residues are identical except three: ser101/tyr89, his121/phe109, and arg206/ile198, which differ considerably in their electronic and hydrophobic properties. contemporary modeling studies performed on the basis of the 2010 crystallized cxcr4 receptor show higher sequence homology to ccr2/ccr5 than prior used templates based on bovine rhodopsin or the β2-adrenergic receptor [28] . the recently reported x-ray crystal structure of the ccr5 receptor/maraviroc complex [95] will allow a deep insight in the binding site and sophisticated modeling studies. the dual ccr2/ccr5 antagonist tak-779 (15) (table 29) is the most extensively investigated compound regarding binding site experiments and computational predictions. using computational calculations, low-energy three-dimensional receptor conformations of human ccr2 and ccr5 receptors were created, and the binding sites of 15 within the ccr2 and ccr5 receptor were predicted. mutation experiments in which single amino acids were replaced within the receptor structure were performed, and after transient expression in the l1.2 cells chemotactic and competitive binding experiments to ccr2 and ccr5 receptors were carried out. based on these data, it was postulated that trp98/thr292 in the ccr2 receptor (fig. 3) and trp86/tyr108 in the ccr5 receptor (fig. 5 ) were significantly associated with the efficacy of tak-779. his121 in the ccr2 receptor was also important for antagonistic efficacy and was replaced by tyr108 in the ccr5 receptor. an altered rotational orientation of tm3 is responsible for a different positioning of these aromatic residues in both ccr2 and ccr5 receptors. a comparison of quaternary ammonium salt tak-779 (15) with antagonists including a tertiary amine showed differences in binding of these interacting residues. depending on glu291 in the ccr2 receptor and glu283 in the ccr5 receptor facing either tm1 and tm2 or tm3 and tm6, different orientations of tm7 are possible: the first receptor conformation leads to a receptor activation network formed between tm 1, 2, 3, and 7, which is supposed to be required for the receptor activation by the chemokine. the second conformation is expected to be stabilized by antagonist binding [62] . the interaction of tak-779 (15) with the respective binding sites of ccr2 and ccr5 receptors resulted in ic 50 values of 27 nm for the ccr2 receptor and 1.4 nm for the ccr5 receptor. 15 also inhibited the binding of ccl3 and ccl4 to the cells expressing the ccr5 receptor with an ic 50 value of 1.0 nm. no interaction was found between tak-779 and ccr1, ccr3, ccr4, or cxcr4 receptors [71] . tak652 (43) (table 29) , a benzazocine compound, was developed by takeda inc. as a ccr5 antagonist for anti-hiv-1 therapy in order to improve the poor oral bioavailability of the quaternary ammonium salt tak-779. in addition to the high ccr5 affinity (ic 50 ¼ 3.1 nm in [ 125 i]-ccl5 assay), tak-652 was also found to be a potent ccr2 antagonist with binding affinity of 5.9 nm [116] . this effect was neither observed for tak-220 (48) and maraviroc (1) nor any other ccr5 ligand [117] . as described in sect. 2, compounds with an aminocyclopentanecarboxamide scaffold were developed as ccr2 antagonists with promising receptor affinity but also significant herg inhibition. mk0483 (6) ( table 30) showed high ccr2 affinity (ic 50 ¼ 4 nm, measured as inhibition of [ 125 i]-ccl2 binding) and displayed an ic 50 value of 0.3 nm in chemotactic assays. mk0483 inhibited [ 125 i]-ccl3 binding to ccr5 receptors with an ic 50 value of 25 nm. additionally a low erg affinity (ic 50 ¼ 33 μm) was found. the improved lack of herg inhibition with regard to merck's previous compounds was shown to be associated with the 3-carboxyphenyl in position 4 of the piperidine of 6 [118] . a broad screening against different receptors, cyp enzymes and ion channels displayed high selectivity for ccr2 and ccr5 and weak interaction with muscarinic receptors m 2 and m 4 without any interaction with cyp enzymes, including cyp 3a4, 2c9, 2d6, 1a2, and 2c19. efficacy of 6 was also evaluated in different rhesus blood experiments, suggesting that subnanomolar potency can be achieved in vivo [118] . the indolyltropane skb3380732 (106) (table 30) , developed as a potent ccr2 ligand, displayed an ic 50 value of 40 nm for the ccr2 receptor. the development of 106 started from a potent ccr2 antagonist with an indolylpiperidine scaffold. it showed selectivity over the ccr5 receptor, but due to its high structural similarity with serotonin (5-hydroxytryptamine), this indolylpiperidine was not selective and interacted with several serotonergic and dopaminergic receptors. in order to improve the selectivity, a conformational restriction of the indolylpiperidine via a tropane moiety was performed, and the steric bulk around the basic amine was increased. moreover, the flexible pentyl chain was exchanged by a methylcyclohexyl linker. the conformational constraint of both the piperidine ring and the pentyl alkyl chain led to 1,000-fold increased ccr2 selectivity over a number of serotonin and dopamine receptors but retained high ccr2 affinity. in contrast to previous compounds, 106 showed moderate ccr5 affinity (ic 50 ¼ 4,000 nm) (table 30 ) [41, 119] . incyte and pfizer discovered a series of dual ccr2/ccr5 antagonists, leading to incb10820/pf-4178903 (107) (table 30 ) as the most potent compound. sar studies on both the left and right part of the molecule, containing an aminocyclopentanecarboxamide with an isopropyl moiety, resulted in 107 as most promising dual antagonist. compound 107 displayed a ccr2 affinity of ic 50 ¼ 3.0 nm in [ 125 i]-ccl2 assay and ic 50 ¼ 5.3 nm to the ccr5 receptor in [ 125 i]-ccl4 binding assay. compared the analog bearing a 3-trifluoromethylphenyl instead of the 3-trifluoromethylpyridin-2-yl moiety (107), the ccr5 affinity increased 4-fold. regarding to chemotactic activity, 103 showed similar ic 50 values in both ccr2 (ic 50 ¼ 3.2) and ccr5 (ic 50 ¼ 4.3 nm) binding assays. a further replacement of the 3-trifluoromethylpyridin-2-yl moiety by the 4-trifluoromethylpyrimidin-2-yl residue provided a less active analog. screening of the affinity towards various receptors, enzymes, and ion channels (>50) indicated 107 to be a selective and dual ccr2 and ccr5 antagonist. 107 did not inhibit cyp 3a4 and 2d6 enzymes, but inhibited the herg channel (ic 50 ¼ 1.7 μm). due to the promising in vivo properties with oral bioavailability of 84% in rats and 57% in monkeys and high metabolic stability (t 1/2 ¼ 93 min), 107 became a candidate for clinical studies [120] . the binding of 107 was also analyzed by docking into the binding site of the ccr2 and ccr5 receptor. the tertiary amine of 107 forms a salt bridge with the acidic residues glu291 (ccr2) and glu283 (ccr5). the trifluoromethyl substituent was also identified to interact with arg206 of the ccr2 receptor and ile 198 of the ccr5 receptor [28] . as detailed in sect. 2, several ccr2 antagonists from various chemical classes have already been reported. merck developed further a new class of promising ccr2 antagonists based on the γ-aminobutyramide core. the screening of merck's sample collection led to some lead compounds, which upon further optimization resulted in ligands 108, 109, and 110 (table 31) with both ccr2 and ccr5 antagonistic properties. a high structural similarity to known ccr5 antagonists was achieved by incorporation of a substituted piperidine ring. compound 108 with the 4-phenylpiperidine moiety demonstrated moderate binding affinity to the ccr2 receptor (ic 50 ¼ 150 nm) as well as to the ccr5 receptor (ic 50 ¼ 72 nm). 108 showed improved potency compared to previously characterized unsubstituted piperidine analogs. further variations of the phenylpiperidine moiety by 3-phenylazetidine, 3-phenylpyrrolidine, and 4-phenylazepane showed that the piperidine ring was best in terms of ccr2 and ccr5 affinity. the spiro[indenepiperidine] 109 was about 2-fold more active with ic 50 values of 80 nm (the ccr2 receptor binding affinity) and 30 nm (the ccr5 receptor binding affinity), whereas its closely related saturated spiro[indane-piperidine] analog was less potent. the introduction of a methyl group in various positions of the γ-aminobutyramide backbone and the piperidine ring decreased potency with the exception of a methyl moiety in the 3-position of the piperidine ring. however, the ccr2 and ccr5 affinity is strongly dependent on the relative and absolute configuration of the piperidine derivatives. compound 110 with (r,r,s)-configuration was the most potent ligand from this series with the ic 50 value of 59 nm in the ccr2 binding and an 26% inhibition at 10 nm in ccr5 binding. in chemotactic assays, progression from 108 to 109 and 110 was observed: 108 inhibited the ccl2 stimulated monocyte chemotaxis by only 40% at a concentration of 1 μm, while 109 with an ic 50 value of 176 nm and 110 with an ic 50 value of 41 nm. in selectivity screenings, compounds 108, 109, and 110 were found to be highly selective against ccr1, ccr3, cxcr3, ccr4, cxcr4, and ccr8 receptors. only the pharmacokinetic properties of 109 were evaluated in a rat model, which showed good pharmacokinetic parameters and proper oral bioavailability at 3 mg/kg body weight [121] . the emerging evidence for the role of ccr2 and ccr5 receptors in human inflammatory diseases led to a growing interest in selective and dual ccr2/ ccr5 antagonists. the availability of potent ccr2 and ccr5 antagonists allows the selective targeting of these receptors and the development of novel concepts for the therapy of inflammatory diseases (e.g., multiple sclerosis and atherosclerosis). maraviroc (1) is up to now the only commercially available ccr5 antagonist for the treatment of hiv-1 infections, but the growing number of reports on maravirocresistant viruses underlines the need of new drugs with improved resistance profile. during the development of new lead compounds, many issues like herg channel affinity and metabolic stability had to be considered. the most promising ccr5 antagonists for the treatment of hiv infections are gsk163929 (99) and pf-232798 (90c), which will enter clinical studies. the development of clinical candidates targeting the ccr2 receptor is also associated with the optimization of several aspects. although the potent ccr2 antagonist mk0812 (5) has reached phase ii clinical trials, the further development was terminated due to no significant improvement compared with placebo. the ccr2 antagonist jnj17166864 (16) was tested in clinical trials for the local treatment of allergic rhinitis. however, the study was terminated due to lack of efficacy. in contrast to promising results in preclinical animal models of inflammation, ccr2 antagonists do not show sufficient efficacy in clinical trials of inflammatory diseases so far. the clinical trials performed with selective ccr2 and ccr5 antagonists suggest that targeting a single receptor might not be sufficient for high efficacy. the fact that both receptors are important in the pathogenesis of cardiovascular and/or immunological diseases indicates great therapeutic potential of dual antagonists. many compounds that were originally developed as selective antagonists of ccr2 or ccr5 receptors have shown later to address both subtypes. the systematic development of dual ccr2/ccr5 antagonists resulted in incb10820 (107) as the most promising antagonist. in addition to the important central amine, 107 contains a fluoro substituent which interacts with arg206 of the ccr2 receptor and ile198 of the ccr5 receptor. the recently published x-ray crystal structures of the cxcr4 and ccr5 receptors represent the basis for docking studies and virtual screening campaigns, which might lead to discovery of innovative ligands and the generation of novel selective and dual antagonists with desired pharmacological properties. the discovery of tropane-derived ccr5 receptor antagonists selective and dual targeting of ccr2 and ccr5 receptors: a current overview 233 maraviroc (uk-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor ccr5 with broad-spectrum anti-human immunodeficiency virus type 1 activity immune and inflammatory mechanisms of atherosclerosis immune and inflammatory mechanisms in the development of atherosclerosis immune and inflammatory mechanisms in the pathogenesis of atherosclerosis recent developments in ccr2 antagonists the ccr2 receptor as a therapeutic target chemokines and hiv-1 second receptors gulliver's travels in hivland hiv biological variability unveiled: frequent isolations and chimeric receptors reveal unprecedented variation of coreceptor use concordance of ccr5 genotypes that influence cell-mediated immunity and hiv-1 disease progression rates the black death and aids: ccr5-delta32 in genetics and history involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (cad). coincidence of elevated lp(a) and mcp-1-2518 g/g genotype in cad patients polymorphisms in the cc-chemokine receptor-2 (ccr2) and à5 (ccr5) genes and risk of coronary heart disease among us women chemokine receptor 5 (ccr5) deletion polymorphism in north indian patients with coronary artery disease association of rheumatoid arthritis with a functional chemokine receptor, ccr5 association of ccr5-delta32 mutation with reduced risk of nonatopic asthma in slovenian children influence of ccr5 delta32 polymorphism on multiple sclerosis susceptibility and disease course the chemokine system as therapeutic target in cardiovascular disease therapeutic targeting of chemokine interactions in atherosclerosis hiv and the ccr5-delta32 resistance allele chemokine receptor antagonists: part 1 chemokine receptor antagonists: part 2 ccr2 antagonists discovery of incb3344, a potent, selective and orally bioavailable antagonist of human and murine ccr2 pharmacological characterization of incb3344, a small molecule antagonist of human ccr2 chapter 12 ccr2 antagonists for the treatment of diseases associated with inflammation structural insights from binding poses of ccr2 and ccr5 with clinically important antagonists: a combined in silico study overcoming undesirable herg potency of chemokine receptor antagonists using baseline lipophilicity relationships structures of the cxcr4 chemokine gpcr with small-molecule and cyclic peptide antagonists overcoming herg activity in the discovery of a series of 4-azetidinyl-1-aryl-cyclohexanes as ccr2 antagonists medicinal chemistry of herg optimizations: highlights and hang-ups rb+ flux through herg channels affects the potency of channel blocking drugs: correlation with data obtained using a high-throughput rb + efflux assay discovery of ((1s,3r)-1-isopropyl-3-((3s,4s)-3-methoxy-tetrahydro-2h-pyran-4-ylamino)cyclopent yl) (4-(5-(trifluoro-methyl)pyridazin-3-yl)piperazin-1-yl)methanone, pf-4254196, a ccr2 antagonist with an improved cardiovascular profile discovery of incb3284, a potent, selective, and orally bioavailable hccr2 antagonist cloning and functional characterization of the rabbit c-c chemokine receptor 2 chapter 14 advances in the discovery of cc chemokine receptor 2 antagonists therapeutic targeting of chemokine receptors by small molecules selective and dual targeting of ccr2 and ccr5 receptors: a current overview 235 discovery of incb8761/pf-4136309, a potent, selective, and orally bioavailable ccr2 antagonist chemokine receptor antagonists chemokine receptor antagonists: overcoming developmental hurdles potent heteroarylpiperidine and carboxyphenylpiperidine 1-alkylcyclopentane carboxamide ccr2 antagonists the discovery of novel cyclohexylamide ccr2 antagonists design and synthesis of novel ccr2 antagonists: investigation of non-aryl/heteroaryl binding motifs modulators of the human ccr5 receptor. part 1: discovery and initial sar of 1-(3,3-diphenylpropyl)-piperidinyl amides and ureas modulators of the human ccr5 receptor. part 3: sar of substituted 1-[3-(4-methanesulfonylphenyl)-3-phenylpropyl]-piperidinyl phenylacetamides modulators of the human ccr5 receptor. part 2: sar of substituted 1-(3,3-diphenylpropyl)-piperidinyl phenylacetamides identification of the binding site for a novel class of ccr2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle unraveling the chemistry of chemokine receptor ligands discovery of 3-piperidinyl-1-cyclopentanecarboxamide as a novel scaffold for highly potent cc chemokine receptor 2 antagonists 3-amino-1-alkyl-cyclopentane carboxamides as small molecule antagonists of the human and murine cc chemokine receptor 2 discovery of novel, potent, and selective smallmolecule ccr5 antagonists as anti-hiv-1 agents: synthesis and biological evaluation of anilide derivatives with a quaternary ammonium moiety potent and selective cc-chemokine receptor-2 (ccr2) antagonists as a potential treatment for asthma in vivo activity of an azole series of ccr2 antagonists design, synthesis and sar of indazole and benzoisoxazole containing 4-azetidinyl-1-aryl-cyclohexanes as ccr2 antagonists discovery of a 4-azetidinyl-1-thiazoyl-cyclohexane ccr2 antagonist as a development candidate a novel series of n-(azetidin-3-yl)-2-(heteroarylamino)acetamide ccr2 antagonists novel 2-aminooctahydrocyclopentalene-3a-carboxamides as potent ccr2 antagonists discovery and sar of 5-aminooctahydrocyclopentapyrrole-3a-carboxamides as potent ccr2 antagonists ccr2: characterization of the antagonist binding site from a combined receptor modeling/mutagenesis approach prediction of binding mode between chemokine receptor ccr2 and its known antagonists using ligand supported homology modeling elucidation of binding sites of dual antagonists in the human chemokine receptors ccr2 and ccr5 molecular interactions of ccr5 with major classes of small-molecule anti-hiv ccr5 antagonists structural and molecular interactions of ccr5 inhibitors with ccr5 the effect of cytochrome p450 metabolism on drug response, interactions, and adverse effects review: pharmacogenetic aspects of the effect of cytochrome p450 polymorphisms on serotonergic drug metabolism, response, interactions, and adverse effects drug-and non-drug-associated qt interval prolongation hiv-1 clade c envelopes obtained from late stage symptomatic indian patients varied in their ability towards relative cd4 usages and sensitivity to ccr5 antagonist tak-779 molecular cloning and functional expression of a new human cc-chemokine receptor gene a binding pocket for a small molecule inhibitor of hiv-1 entry within the transmembrane helices of ccr5 a small-molecule, nonpeptide ccr5 antagonist with highly potent and selective anti-hiv-1 activity selective and dual targeting of ccr2 and ccr5 receptors: a current overview 237 orally active ccr5 antagonists as anti-hiv-1 agents. part 3: synthesis and biological activities of 1-benzazepine derivatives containing a sulfoxide moiety synthesis of 1-benzothiepine and 1-benzazepine derivatives as orally active ccr5 antagonists an in situ oxidation strategy towards overcoming herg affinity orally active ccr5 antagonists as anti-hiv-1 agents: synthesis and biological activity of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives containing a tertiary amine moiety tak-652 inhibits ccr5-mediated human immunodeficiency virus type 1 infection in vitro and has favorable pharmacokinetics in humans ccr5 antagonists as anti-hiv-1 agents. part 3: synthesis and biological evaluation of piperidine-4-carboxamide derivatives discovery of a piperidine-4-carboxamide ccr5 antagonist (tak-220) with highly potent anti-hiv-1 activity analysis of binding sites for the new small-molecule ccr5 antagonist tak-220 on human ccr5 the discovery of ccr5 receptor antagonists for the treatment of hiv infection: hit-to-lead studies role of ccr5 delta32 bp deletion in ra and sle mice with a selective deletion of the cc chemokine receptors 5 or 2 are protected from dextran sodium sulfatemediated colitis: lack of cc chemokine receptor 5 expression results in a nk1.1+ lymphocyte-associated th2-type immune response in the intestine balancing herg affinity and absorption in the discovery of azd5672, an orally active ccr5 antagonist for the treatment of rheumatoid arthritis hiv-1-resistant individuals may lack hiv-1 coreceptor identification of a major co-receptor for primary isolates of hiv-1 cloning and functional expression of a human eosinophil cc chemokine receptor novel approach to predicting p450-mediated drug metabolism: development of a combined protein and pharmacophore model for cyp2d6 a novel approach to predicting p450 mediated drug metabolism. cyp2d6 catalyzed n-dealkylation reactions and qualitative metabolite predictions using a combined protein and pharmacophore model for cyp2d6 overcoming herg affinity in the discovery of the ccr5 antagonist maraviroc new p-methylsulfonamido phenylethylamine analogues as class iii antiarrhythmic agents: design, synthesis, biological assay, and 3d-qsar analysis new insights into the mechanisms whereby low molecular weight ccr5 ligands inhibit hiv-1 infection hiv-1 resistance to maraviroc conferred by a cd4 binding site mutation in the envelope glycoprotein gp120 a maraviroc-resistant hiv-1 with narrow cross-resistance to other ccr5 antagonists depends on both n-terminal and extracellular loop domains of drug-bound ccr5 reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the ccr5 antagonist maraviroc utilize inhibitorbound receptor for entry structure of the ccr5 chemokine receptor-hiv entry inhibitor maraviroc complex efficient synthesis and identification of novel propane-1,3-diamino bridged ccr5 antagonists with variation on the basic center carrier discovery of novel (s)-α-phenyl-γ-amino butanamide containing ccr5 antagonists via functionality inversion approach an imidazopiperidine series of ccr5 antagonists for the treatment of hiv: the discovery of n-{(1s)-1-(3-fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-1h-imidazo different selection patterns of resistance and cross-resistance to hiv-1 agents targeting ccr5 evaluation of genotypic tropism prediction tests compared with in vitro co-receptor usage in hiv-1 primary isolates of diverse subtypes 1-amido-1-phenyl-3-piperidinylbutanes -ccr5 antagonists for the treatment of hiv: part 2 1-amido-1-phenyl-3-piperidinylbutanes -ccr5 antagonists for the treatment of hiv. part 1 selective and dual targeting of ccr2 and ccr5 receptors: a current overview 239 discovery of bioavailable 4,4-disubstituted piperidines as potent ligands of the chemokine receptor 5 and inhibitors of the human immunodeficiency virus-1 novel 4,4-disubstituted piperidine-based c-c chemokine receptor-5 inhibitors with high potency against human immunodeficiency virus-1 and an improved human ether-a-go-go related gene (herg) profile synthesis and evaluation of 2-phenyl-1,4-butanediamine-based ccr5 antagonists for the treatment of hiv-1 what is the future of ccr5 antagonists in rheumatoid arthritis? maraviroc, a chemokine receptor-5 antagonist, fails to demonstrate efficacy in the treatment of patients with rheumatoid arthritis in a randomized, double-blind placebo-controlled trial the many roles of chemokines and chemokine receptors in inflammation ccr5: a novel player in the adipose tissue inflammation and insulin resistance? loss of ccr2 expression and functional response to monocyte chemotactic protein (mcp-1) during the differentiation of human monocytes: role of secreted mcp-1 in the regulation of the chemotactic response increase of ccr1 and ccr5 expression and enhanced functional response to mip-1 alpha during differentiation of human monocytes to macrophages benzimidazoles as benzamide replacements within cyclohexane-based cc chemokine receptor 2 (ccr2) antagonists the chemokine system in diverse forms of macrophage activation and polarization chemokine receptors: multifaceted therapeutic targets molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails highly potent and orally active ccr5 antagonists as anti-hiv-1 agents: synthesis and biological activities of 1-benzazocine derivatives containing a sulfoxide moiety isolation and characterization of human immunodeficiency virus type 1 resistant to the small-molecule ccr5 antagonist tak-652 discovery of a potent and orally bioavailable ccr2 and ccr5 dual antagonist conformationally restricted indolopiperidine derivatives as potent ccr2b receptor antagonists discovery of incb10820/pf-4178903, a potent, selective, and orally bioavailable dual ccr2 and ccr5 antagonist novel, orally bioavailable gamma-aminoamide cc chemokine receptor 2 (ccr2) antagonists selective and dual targeting of ccr2 and ccr5 receptors: a current overview 241 key: cord-0046741-szb45jm2 authors: reza khorramizadeh, m.; saadat, farshid title: animal models for human disease date: 2020-06-26 journal: animal biotechnology doi: 10.1016/b978-0-12-811710-1.00008-2 sha: bbf2bf8dfbda39fa161c3d4ed5208445d0e6ec02 doc_id: 46741 cord_uid: szb45jm2 this chapter introduces some types of animal models which are used for better understanding the disease mechanisms and its treatment. these experimental models fall into two categories: spontaneous models and induced models. among the diseases, rheumatoid arthritis (ra) as an autoimmune disease was considered. to study the pathogenesis of ra, we explained collagen-induced arthritis as an animal model that reflects a characteristic feature of ra patients. in addition, experimental allergic encephalomyelitis (eae) as an experimental model for multiple sclerosis (ms) was explained in detail to represent a standard method to investigate in its mechanism, finding the way for the amelioration of this incurable neurological disorder. the architecture of human body is comprised in such a manner that cells cannot be considered as a separate entity. physiologically, homeostasis is the reason that these components live and perform their functions within that environment. disruption of this process leads to fatal conditions and is considered a disease. to investigate the mechanism of disease and to find the means to reverse adverse conditions, various strategies are used including cell-based assays and tissue culture studies. although these models can provide useful information, they fail to address various physiological conditions and the complex interactions among different cell types of tissues and organs. ideally a useful animal model for any disease has to have pathology similar to the disease conditions in humans. use of animals in research has a long history that dates back to the fourth century b.c. in the 1600s, william harvey used animals to describe the blood circulatory system. many scientists, such as louis pasteur and emil von behring, have used animal models for experimental purposes to prove their hypotheses. animal models are good for understanding disease mechanisms and treatment and for overcoming the limitations of clinical trials that use human subjects. for example, experimental animal models for diseases like rheumatoid arthritis or multiple sclerosis have been successfully employed to screen new bioengineered, chemical, or herbal therapeutics that might have the potential for the treatment of human patients. so far, more than 550,000 studies have been reported in the ncbi database; they use animal models for different diseases. animal model studies have been the main reason for a better understanding of disease mechanisms. animal models of disease can be divided into two categories (kurkó et al., 2013) : spontaneous disease models and ( van heemst et al., 2014) induced disease models. in the case of induced disease models, induction can occur by various agents, both chemical and biological. this chapter discusses some of the most important animal models. rheumatoid arthritis (ra) is an autoimmune disorder with progressive occurrence that preferentially affects peripheral joints. in spite of the fact that ra is severe and crippling and affects large numbers of people, very little knowledge about its etiology and pathogenesis is available in the literature. rheumatoid arthritis affects about 1% of the population. the ratio of the prevalence of ra in males and females is 1à2.5. ra can occur at any age, but it is mainly reported to affect the 40-to 70-year-old age group. no doubt the incidence has been reported to increase with age. the etiology of ra is unknown, but it has been predicted that genetic and environmental factors play an important role in the onset of ra. recent advances have identified genetic susceptibility markers both within and outside of the major histocompatibility complex (mhc). human leukocyte antigen (hla) genes located on chromosome 6p have been found to have a strong association with rheumatoid arthritis. the contribution of hla to heritability of ra has been estimated to be 11%à37%. individuals carrying hla-dr4 and hla-dr1 alleles have been shown to have a higher risk of ra. apart from the known shared epitope alleles (hla-drb1 ã 01, drb1 ã 04), other hla alleles, such as hla-drb1 ã 13 and drb1 ã 15, have been linked to ra susceptibility (kurkó et al., 2013) . the hla class ii locus is the most important risk factor for anticitrullinated protein antibodies (acpa)-positive ra (acpa 1 ra) (van heemst et al., 2014) . a positive correlation has been suggested for the role of hla in terms of the severity of ra rather than the onset of the disease. the most relevant non-hla gene single nucleotide polymorphisms (snps) associated with ra include ptpn22, il23r, traf1, ctla4, irf5, stat4, ccr6, and padi4 (kurkó et al., 2013; suzuki and yamamoto, 2015; stanford and bottini, 2014) . although the data regarding this conclusion are inconsistent, some of the studies have shown associations between tumor necrosis factor (tnf) alleles and rheumatoid arthritis. other genes like those for corticotrophin-releasing hormone, interferon (ifn)-γ, and interleukin-10 (il-10) have also been implied for ra. it can be concluded that the role of genetic components in ra is modest at the best (viatte et al., 2013) . epigenetics is another important factor that contributes to ra. in the case of identical twins, ra has not been shown to have 100% concordance; therefore, the role of nongenetic factors has also been implicated in the etiology of ra (meda et al., 2011) . throughout the world, rheumatoid arthritis is more common in women than in men. this indicates that hormones may play an important role in the development of the disease. pregnancy has also been considered as a risk factor for rheumatoid arthritis. studies show that the onset of ra is rare during pregnancy, but the risk increases after delivery. smoking is associated with increased incidences of ra, especially in men. on the contrary, populations that consume a diet high in omega-3 fatty acids have been reported to be protected from rheumatoid arthritis. from experimental models in animals, a large number of infectious agents such as viruses and bacteria have also been suggested to trigger or contribute to the development of rheumatoid arthritis. however, no relationship between infectious agents and the development of ra has been found. tissue edema and fibrin deposition are prominent and can manifest clinically as joint swelling and pain. within a short period, the synovial lining becomes hyperplastic, commonly becoming ten or more cells deep and consisting of type a (macrophage-like) and type b (fibroblast-like) synoviocytes that produce glycosaminoglycans (e.g., hyaluronan, as reported to be present in synovial tissue and synovial fluid). the sublining also undergoes alterations for its cellularity, both in cell type and in cell numbers, with prominent infiltration of mononuclear cells, including t cells, b cells, macrophages, and plasma cells. the abundance and activation of macrophages at the inflamed synovial membrane correlates significantly with the severity of the disease. activated macrophages over-express major histocompatibility complex (mhc) class ii molecules and produce pro-inflammatory or regulatory cytokines and growth factors [il-1, il-6, il-10, il-13, il-15, il-18, tnf-α, and granulocyte macrophage colony stimulating factor (gm-csf)], chemokines [il-8, macrophage inflammatory protein 1 (mip-1), monocyte chemoattractant protein 1 (mcp-1)], metalloproteinases, and neopterin. these biomolecules are routinely detected in inflamed joints. most of the t cells infiltrating the rheumatoid synovium express cd45ro and cd4, which is an indication that the t-cell subset present in the synovium is memory helper t cells. surprisingly, 10%à15% of the t cells present in the case of the synovium have granzymes a and perforins. this 10%à15% of cells present in the synovium represents cytotoxic t-cell subsets. therefore, it can be concluded that cd8-expressing cells are infrequent in the synovium. in the synovial fluid of rheumatoid arthritis patients, cd4 and cd8 t cells are equally represented. tcrα/tcrß is expressed on most of the t cells while only a minority of cells show tcrγ/tcrδ expression. it has, however, been found that the expression of tcrγ/tcrδ is increased in the synovium of patients with active ra. synovial-vessel endothelial cells transform into high endothelial venules early during the course of disease. high endothelial venules are specialized post-capillary venules usually present in secondary lymphoid tissue or inflamed nonlymphoid tissues; these venules facilitate the transit of leukocytes from the bloodstream into tissues. the cytokine-mediated events have conventionally been viewed in the milieu of the cd4 1 th1/th2 paradigm. nowadays, newer cytokines of the il-17/il-23 axis and others have changed investigations into the immunopathogenesis of arthritis. both the cd4 1 th17 and γδ-t cells secreted il-17 which is a chemotactic for neutrophils and its response inhibited by il-27 due to ifn-γ induction. the roles of other cytokines such as il-18 and il-33 in arthritis have been clarified further with inhibitors of them (veenbergen et al., 2010; palmer et al., 2009) . recent studies in arthritis models have revealed new aspects toward regulatory t cell (treg) activity. in the cia model, treatment with il-35 induced the regression of arthritis via expansion of regulatory t cells (kochetkova et al., 2010) . the formation of locally invasive synovial tissue (i.e. pannus) is a characteristic feature of rheumatoid arthritis. pannus is involved in the erosion of joints in rheumatoid arthritis. pannus is histologically distinct from other regions of the synovium and shows phases of progression. initially, there is penetration of cartilage by synovial pannus, which is composed of mononuclear cells and fibroblasts, with a high-level expression of matrix metalloproteinases (mmps) by synovial lining cells. in later phases of the disease, cellular pannus can be replaced by fibrous pannus comprised of a minimally vascularized layer of pannus cells and collagen overlying cartilage. the tissue derivation of pannus cells has not been fully elucidated, although they are thought to arise from fibroblast-like cells (type-b synoviocytes). in vitro work shows that these fibroblast-like synoviocytes have anchorageindependent proliferation and loss of contact inhibition, which a phenotype is usually found in transformed cells. however, the molecular pathogenic mechanisms driving pannus formation still remains poorly understood. the range of presentations of rheumatoid arthritis is broad, but the disease onset is insidious in most cases, and several months can elapse before a firm diagnosis can be ascertained. the predominant symptoms are pain, stiffness, and swelling of peripheral joints. although articular symptoms are often dominant, rheumatoid arthritis is a systemic disease. active rheumatoid arthritis is associated with a number of extraarticular manifestations, including fever, weight loss, malaise, anemia, osteoporosis, and lymphadenopathy. the clinical course of the disorder is extremely variable, ranging from mild, self-limiting arthritis to rapidly progressive multisystem inflammation with a profound morbidity and mortality. analyses of clinical course and laboratory and radiological abnormalities have been defined as negative prognostic factors for progressive joint destruction; unfortunately, none of these are reliable enough to allow therapeutic decisionmaking. frequent assessment of disease symptoms and responses to therapy is crucial for a successful and long-term management of rheumatoid arthritis. joint destruction from synovitis can occur rapidly and early in the course of the disorder; radiographic evidence is present in more than 70% of patients within the initial 2 years. more sensitive techniques such as magnetic resonance imaging (mri) can identify substantial synovial hypertrophy, bone edema, and early erosive changes as early as 4 months after the onset of disease. these radiographic changes predate misalignment and functional disability by years; by the time physical deformity is evident, substantial irreversible articular damage has commonly occurred. furthermore, the biopsy analysis of clinically symptomless knee joints in patients with early rheumatoid arthritis shows active synovitis, highlighting the poor correlation between clinical assessment and disease progression, and the rapid development of polyarticular synovitis. the main goal of ra treatment is to stop inflammation, relieve symptoms, prevent joint damage, and reduce long-term complications. the past decade has seen a major transformation in the treatment of rheumatoid arthritis in terms of approach and choice of drugs. the previous therapeutic approach generally involved initial conservative management with nonsteroidal antiinflammatory drugs (nsaids) for several years; disease-modifying antirheumatic drugs (dmards) were withheld until a clear evidence of erosion was seen. dmards were then added individually in slow succession as the disease progressed. this form of treatment has been supplanted by early initiation of dmards and combination dmard therapy in patients with the potential for progressive disease. the idea of early intervention with the conventional disease-modifying antirheumatic drugs (cdmard) has been validated in several randomized trials. cdmards contain medications from different classes of drugs including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards are often partly effective and poorly tolerated for long-term therapy. in metaanalyses of dropout rates from clinical trials, 20%à40% of patients discontinued the use of dmards assessed as monotherapy during the duration of the trial; even in clinical practice, the median duration of dmard monotherapy was less than 2 years for nonmethotrexate agents. although there are many reasons for the lack of long-term adherence to treatment, poor efficacy, delayed onset of action, and toxic effects are major limitations. additionally, dmards therapy requires patients to undergo frequent monitoring of blood and physical examinations for toxic effects of treatment protocol. results from clinical trials showed that dmard therapy decreased markers of inflammation such as erythrocyte sedimentation rate and swollen joint counts, and that improved symptoms in a selected subset of patients; however, most patients continued to show progression of irreversible joint destruction on radiography. cdmards is increasingly burdened by side effects or clinical inefficacy, so other immunosuppressive drugs such as tacrolimus that blocks t-cell activation by specifically inhibiting calcineurin pathway and leflunomide have been developed. a new synthetic dmard, iguratimod, which exerts its action by the inhibition of the inflammatory cytokines (tnf-α, interleukin (il)-1 β, il-6, il-8, and il-17), is recently developed. the findings illustrate the consequences of progressive disease and have shown the need for the development of new and more effective therapies based on the therapeutic principles used for oncology; it means that treatment protocols for ra patients require the use of several therapeutic agents from different classes to be used in combination. recent studies have shown that combination therapy of biological dmards like tnfα inhibitors with methotrexate has clear-cut benefits with tolerable toxic effects. treatment with agents that can block tnf-α function has proved to be highly effective against ra. further studies reported downregulation of synovial gm-csf, il-6, and il-8, suggesting that tnf-α supports the production of other pro-inflammatory cytokines. however, the mechanisms behind the clinical effect of the tnf-α-blocking treatment are not fully understood. in an animal model, tnf-α-blocking agents such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody) reduce the expression of vascular adhesion molecules and inhibit the spontaneous production of il-1 and il-6. patients with a new onset of symptoms and those with diseases of several years' duration and who had failed previous dmard therapy all benefited. these results suggest that patients in many stages of disease progression can benefit from combination therapy (chiu et al., 2012) . with the approval of tnf-α inhibitors (infliximab, etanercept, adalimumab, certolizumab, and golimumab), non-tnf biologic agents (rituximab, abatacept, tocilizumab, and anakinra), and other biologic agents, determining advances in treatment options of ra were made. rituximab (chimeric monoclonal antibody targeted against cd 20) is a selective b-cell depleting agent for treating refractory rheumatoid arthritis. abatacept selectively modulates t-cell co-stimulation and has shown efficacy in several clinical trials. tocilizumab, a humanized monoclonal antiinterleukin-6 receptor antibody, has proven to be efficacious in patients who did not respond to methotrexate or other synthetic dmards. recently, several clinical trials have focused on a new class of drug: the janus kinase (jak) inhibitors. jaks are a family of nonreceptor tyrosine kinases 156 8. animal models for human disease (jak1, jak2, jak3, and tyk2) involved in the intracellular signal transduction of many cytokines. tofacitinib is a pan-jak inhibitor that primarily inhibits jak1 and jak3. in addition to tofacitinib, other jak inhibitor molecules including baricitinib, peficitinib, and decernotinib have also been studied in rcts. finally, filgotinib is a selective jak1 inhibitor which is currently in clinical development for the treatment of ra (calabrò et al., 2016) . as mentioned before, pro-inflammatory/regulatory cytokines and growth factors play important roles in the pathogenesis of ra. therefore, each of them or their pathway represents an attractive therapeutic target for ra. tocilizumab, a humanized monoclonal antibody targeting il-6 receptor, has already been approved for the treatment of ra in patients who failed to achieve remission with cdmards. another cytokine that plays an important role in the pathogenesis of ra is il-17 in which ixekizumab and brodalumab as humanized monoclonal antibody were developed against il17a and its receptor. a new possible therapeutic target for the treatment of ra is the gm-csf pathway. the efficacy and safety of mavrilimumab (an anti-gm-csf receptor monoclonal antibody) in patients with moderate-to-severe ra has been investigated (takeuchi et al., 2015) . as an increased activation of osteoclasts contributes to bone erosions in ra, the inhibition of rankl that is essential for the osteoclast activation by denosumab (human monoclonal antibody against rankl) can reduce joint destruction in ra patients. takeuchi et al. (2016) , finally, do not forget that certain nutritional components interfere in the pathological inflammatory process, so that they should be considered as coadjuvant in the treatment of ra. it has been mentioned that flavonoids reduce cytokine expression and secretion. in this regard, flavonoids may have a therapeutical potential in the treatment of inflammationrelated diseases as cytokine modulators (rosillo et al., 2016; leyva-lópez et al., 2016) . in order to study the pathogenesis of ra, one can use different animal models. there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease, there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate a particular aspect of the disease. despite the fact that ra is not a spontaneously developing disease, spontaneously developing models for arthritis may be useful to study the role of genetics in the development of the disease. an activated immune response was reported in models such as the human tumor necrosis factor-α transgenic (htnftg), interleukin 1receptor-α (il-1ra) knockout, il-6ractivating mutation knockin, or skg mouse which bears the primary inflammatory response in joints (keffer et al., 1991) . in addition, arthritis can be rapidly induced with an adoptive transfer of t-helper 17 cells in the il-6r knockin mouse. transgenic mice expressing a tcr specific for bovine pancreas ribonuclease develop spontaneous arthritis that is mediated by antibodies (korganow et al., 1999) . this model is particularly interesting because it demonstrates that t cells specific for a ubiquitous antigen may induce an organspecific autoimmune disease. the expression of the gene product causes an upregulation of several cytokines (il-1, il-6, tgf-β1, ifn-γ, and il-2) and subsequent development of arthritis (iwakura et al., 1995) . there are some other spontaneous models for arthritis in nontransgenic mice (bouvet et al., 1990) . arthritis can be induced by complete freunds' adjuvant (cfa). pearson (pearson, 1956) described this model for the first time. subsequently, it was demonstrated that other adjuvants, such as ifa, pristane, or squalene, could also induce arthritis (carlson et al., 2000) . microbially derived products such as lipopolysaccharide (lps), muramyle dipeptide (mdp), and trehalosedimycolate (tdm) can also induce arthritis when given with mineral oil (lorentzen, 1999; kohashi et al., 1980) . collagen-induced arthritis (cia) is normally induced by the immunization of susceptible mouse (e.g., dba/1) or rat (da, lewis) strains at the base of the tail. the inoculum used for immunization contains both adjuvant and collagen type ii. the adjuvant has to be sufficiently strong to cause tissue destruction as well as induction of a strong pro-inflammatory immune response (holmdahl and kvick, 1992; kleinau et al., 1995) . susceptibility to cia is dependent on both mhc (class ii region) and non-mhc genes (lorentzen and klareskog, 1996) . antibodies against collagen ii are essential for the development of cia. this fact has been demonstrated by the passive transfer of anti-cii antibodies, which results in synovitis (svensson et al., 1998) . t cells are also important for cia development during early stages of disease progression. the dependence of both t-and b-cell responses has also been demonstrated in the same model (seki et al., 1988) . in cia, an immune response is being directed against a joint collagen type ii (cii) antigen. inflamed joints in cia are infiltrated by inflammatory cells that accumulate in the synovial membrane and fluid, similar to ra. the most frequent cell type in the synovial fluid is granulocyte. there is also a great infiltration of leukocytes into the synovial membrane. these cells have signs of an activated phenotype of ra since mhc class ii molecules are expressed (klareskog and johnell, 1988 ). in addition, there is an intense production of macrophage-derived cytokines in inflamed joints (e.g., tnf-α and il-1β) (mussener et al., 1997; ulfgren et al., 2000) . a small number of t cells are encountered, and some of these t cells have il-2 receptor α chain upregulated. the disease shows a thickened synovial membrane that subsequently forms a pannus on the cartilage surface (holmdahl et al., 1988; holmdahl et al., 1991) . in both cia and ra, cartilage and bone destruction occurs mainly at the carti-lageàpannus junction. there are some features of the pathology of cia that differ from what is usually observed in ra (e.g., extra-articular manifestation). although the compatibility of the cia model to human ra has been argued, many pathological features of cia are similar to those of rheumatoid arthritis. currently, collagen type ii-induced arthritis in mice and rats is one of the most widely used arthritis models in academia and industry. experimental collagen-induced arthritis was initiated by injecting bovine collagen type ii at the base portion of the tail of the animal (saadat et al., 2005) . male lewis rats weighing about 160à180 g were used. after the induction of cia, animals were divided randomly into four or more groups based on the experimental design. at least four different groups were needed, including a control group without arthritis, animals with collagen-induced arthritis, cia animals with treatment, and cia animals treated with methotrexate as a positive control. sample preparation: bovine collagen type ii (cii) was dissolved in 0.1 m acetic acid at a concentration of 2 mg/ml by stirring overnight at 4 c (the dissolved cii can be stored at 270 c if it has to be used at a later time). before injecting the animals, cii was emulsified with an equal volume of complete freund's adjuvant (cfa). for the induction of cia, on day 1 rats were injected intradermally at the base of the tail with 100 μl of emulsion (containing 100 μg of cii). after 12à16 days, animals showed the development of inflammation at peripheral joints ( fig. 8.1 ). on day 21, a booster injection of cii in cfa was administered. this model was used to evaluate the anti-ra effect by giving intraperitoneally injections of test materials (e.g., chemical or herbal extracts). methotrexate was a control used to evaluate the effect of the test compound and to compare the efficacy of the new compound with methotrexate. in this model, the test compound was given from day 25, where the frequency, route of administration, and dose could be selected as needed. the end point and days for the evaluation of different parameters were selected; one of the most common points was day 35 (flow chart 8.1). the paws and knees were then removed for the histopathological assay. the visual observation can be done by using the macroscopic system as given in table 8 .1. moreover, rats immunized with cfa should be checked for weight gain from the first to the end of experiment at least every other day. the decline in body weight that followed on the onset of arthritis was proportional to the disease severity and, hence, can be used as a measure of disease activity. the scaling to record the observation should be from 0 to 4 for each paw (szabó et al., 1998) . on day 35, animals were anesthetized with sodium pentobarbital (45 mg/kg intraperitoneally) and euthanized. blood was collected by intracardiac puncture, and paws and knees were removed, trimmed, and fixed in 10% buffered formalin, decalcified, and then embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin for the histological examination. joint damage was assessed based on synovial hypertrophy, pannus formation, inflammatory cell infiltration, and cartilage and subchondral bone destruction. joint erosion was graded on a scale of 0à3 for each limb (table 8. 2), according to the severity of damage ( fig. 8.2 ). radiological scoring was performed by an investigator who was blind to the treatment protocol (on day 35). radiographical analysis of affected joints in control rats typically showed soft tissue swelling, joint space narrowing, reduced lucency due to demineralization, and areas of recalcification indicative of new bone formation. a score was assigned to each joint on the basis of the information as listed in table 8 .3. scores were 0à3 per joint (0, normal; 3, maximum joint destruction). multiple sclerosis (ms) is a chronic inflammatory demyelinating disorder of the central nervous system (cns) that affects over 2.3 million individuals worldwide. similar to the affected population in other autoimmune diseases, twice as many women as men have ms. multiple sclerosis, like many other diseases, has existed as long as human life. in the 1860s, the first report by dr. jean-martin charcot certified ms as a disease. a patient of him who suffered an unusual symptom died. after dissection, brain lesions were discovered. he called the disease scleroseen plaques. myelin was subsequently discovered, although its exact role was not recognized. about one century of research resulted in the discovery multiple sclerosis is the most common inflammatory demyelinating disease of the cns in europe and north america. the prevalence of ms in north america and europe is b 80à100 per 100,000 people. however, the prevalence is not globally uniform, geographically decreases in latitudes, and has been observed in only b1à2 per 100,000 individuals in africa and asia. the etiology of ms is unknown. both genetic involvement and environmental factors have been indicated in ms. the only consistent correlation of involvement of the mhc locus is the mhc class ii allele hla-dr2, which reflects a linkage with ms. in addition to associations within the major histocompatibility complex (mhc) region, other non-mhc loci reached a genome-wide significance. they map to the genes l3mbtl3, maz, erg, and shmt1. products of the genes l3mbtl3, maz, and erg play important roles in immune cell regulation. shmt1 encodes a serine hydroxymethyl transferase catalyzing the transfer of a carbon unit to the folate cycle, which is important for establishment and maintenance of epigenetic signatures (andlauer et al., 2016) . some other factors like dietary components (e.g., milk), pathogens like human herpes virus 6 (hhv-6), measles virus, epsteinàbarr virus, and chlamydia have been implied as etiological factors. however, the association between any of these agents with ms is debatable. the presence of cns inflammation is a hallmark of ms. this inflammatory process greatly increases in the cns by the activation and deregulation of different cell types of the immune system. activation and entry of myelin-specific lymphocytes into the cns cause damage to oligodendrocytes, leading to demyelination. most of the cells from the immune system can contribute toward demyelination, but the main process of demylation is mediated by antibody and complement. so far, it has been noticed that antibody and complement are responsible for lesions in 40%à50% of ms patients. myeloid cells may cause axonal damage by releasing molecules, such as glutamate, reactive oxy-gen species, and reactive nitrogen species. besides, these cells decreased the expression of glutamate clearance. as a result of increased glutamate in the cerebrospinal fluid, ms patients would be vulnerable to degeneration (yandamuri and lane, 2016) . in addition, cd8 1 t cells play an important role in ms pathogenesis. cd8 1 t cells make up the largest percentage of lymphocytes found in the brain of ms patients. all neuroectodermal cells in ms lesions express mhc class i molecules, making them an excellent target for cd8 1 t cells. in addition to their proinflammatory properties, cd8 1 t cells can also suppress the immune system and down-regulate inflammation. however, experiments with perforin, an important regulator of cytotoxic damage to immune cells, have made it clear that cd8 1 t cells present at ms lesions cause cytotoxicity, which could be the main source for demyelination and axonal damage (sinha et al., 2015) . bystander cd4 1 t cells do not contribute to the demyelinating process, but once cd4 1 t cells move into the cns and become activated against myelin antigen, these cd4 1 t cells could be contributing directly toward the demyelination of cns (basdeo et al., 2016) . moreover, cd4 th17 effector t cells are postulated to play a crucial role in the pathogenesis of ms (bettelli et al., 2006) . in addition to autoreactive immune cells against myelin and nerves, a progressive loss of the structure and function of neurons occurs. it has been reported that the alterations in the expression of mirnas may play a crucial role in ms pathogenesis (huang et al., 2016) . after demyelination, remyelination is possible, which could further damage the cns. the ratio of demyelination to remyelination determines whether a patient will develop secondary progressive ms (spms) or relapse remitting ms (rrms). if remyelination occurs before axonal damage, irreversible physiological damage can be prevented. none of the fda-approved therapies target oligodendrocytes to stimulate remyelination, but it is a very interesting possibility for future therapeutic intervention (huang et al., 2016) . the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. clinical motor manifestations include weakness, stiffness, and/or pain in arms or legs, abnormal reflex activity, and spasticity. often, the earliest symptoms of ms are somatosensory, including numbness and tingling. in ms, cerebral involvement is often accompanied by symptoms such as ataxia and intention tremor. many individuals with ms complain of increased urinary frequency, urgency, and incontinence. bladder and bowel disturbances remain among the most disabling and embarrassing symptoms experienced by ms patients. sexual symptoms are also very common among both men and women. fatigue, sleep disturbances, depression, and deficits in cognitive functioning are also common. the clinical course of ms is often highly variable and is generally characterized by relapses or exacerbations and deterioration of neurologic function, which entitle relapsing remitting ms. the features of relapsing remitting ms are defined as "episodes of acute worsening of neurologic function followed by a variable degree of recovery, with a stable course between attacks." approximately 80%à85% of patients are initially diagnosed with rrms that evolves from an isolated demyelinating attack, which is characterized by multifocal inflammation along with varying degrees of axonal injury. a patient may experience disease progression with or without relapses and minor remissions; that clinical condition is defined as secondary progressive ms. it has been seen that 75% of rrma patients will eventually develop the spms state. primary progressive ms (ppms) affects b10%à15% of ms patients. ppms is defined as "disease progression from onset, with occasional plateaus and temporary minor improvements in clinical condition." the duration of ms varies significantly among ms patients. some patients will live with ms for several decades, while about 10% will develop an acute, fulminant form of ms. patients with an acute and fulminant form of ms show a rapid deterioration in their clinical signs and symptoms that have fatal consequences; these patients usually die within 1à3 years after the onset of disease. in general, the clinical spectrums among ms patients represent a benign disease and a low relapse rate, and these may never develop into secondary progressive disease. the heterogeneity of the clinical course of ms is shown to have a similar variation in its pathology. multiple sclerosis lesions were recently segregated into four distinct subtypes. the general pathology of ms, the formation of demyelinating lesions in the cns associated with infiltrating cd3 1 t cells, activated macrophages, and microglia-containing myelin debris, and infiltrating b cells, is common to all forms of the disease. it is thought that ms lesions are mediated by soluble factors such as tnf-α and immunoglobulin deposition on the myelin sheath, and the local activation of the complement cascade. the diagnostic criteria for clinically definite ms (cdms) include factors such as clinical history, mri imaging, and csf abnormalities. at present, there are no identifiable biomarkers that can predict the clinical subtype of ms. similarly, there are no factors that can assist in predicting whether a patient diagnosed with ms will develop either a progressive or a benign version of the disease. the clinical and pathological heterogeneity in ms has made it important to either develop or identify reliable biomarkers. several cytokines, immunoglobulins, mmps, markers of axonal/neuronal injury, and apoptotic markers have been suggested to have potential as biomarkers, but these biomarkers need validation by rigorous durability trials. in ms, treatment strategies can be either acute or long term. during a relapse, the goal of acute treatment is to reverse neurological disability as well as to delay further neurological dysfunction, so that the normal function can be restored. this type of treatment for ms patients is in contrast to the goals of long-term treatments. the main objective of long-term treatment for ms patients is to decrease relapses (both severity and frequency), which could lend support to stopping the progression of disability. patients experiencing a relapse, such as optic neuritis or transverse myelitis, are often administered high-dose corticosteroid firstline therapy. during progressive phases of the disease, patients may be prescribed immunosuppressive agents such as cyclophosphamide or mitoxantrone because the progressive phase is often accompanied by worsening inflammatory demyelination and axonal degeneration (rommer et al., 2019) . in 1993, ifn-β was the first agent to demonstrate the significant clinical efficacy among patients suffering with rrms. although the exact disease-modifying effects of ifn-β in ms are unknown, several immunomodulatory mechanisms have been suggested. presently, two forms of ifn-β, including ifn-βla (avonex and rebif) and ifn-βlb (betaseron and extavia), have been prescribed. glatiramer acetate (copaxone) is a synthetic mixture of polypeptides that has been approved to treat rrms. similar to ifn-β, glatiramer acetate is found to be not effective for progressive forms of ms. natalizumab (tysabri) is an alpha-4 integrin antagonist and is the first drug of an entirely new class of immune-directed therapies that has been approved by the fda to treat relapsing ms. natalizumab is a humanized recombinant monoclonal antibody that blocks leukocyte migration into the cns by binding to α-4 integrins; these are components of the very late antigen-4 (vla-4) complex constitutively expressed on the leukocyte surface. in monotherapy trials, natalizumab has been reported to reduce the risk for sustained progression of disability as well as decrease the frequency of relapses. based on the current literature, natalizumab appears to be one of the most effective agents to prevent relapses as well as to stop disease progression. other monoclonal antibodies administered by intravenous (iv) infusion include lemtrada (alemtuzumab) and novantrone (mitoxantrone). currently, numerous other monoclonal antibodies are under investigation as potential therapies for ms; for example, anti-cd25 (daclizumab), anti-cd 20 (rituximab), and so on. a number of other agents are under investigation for possible future use in ms including secukinumab (a humanized monoclonal antibody to il-17), rtl1000 (inhibitor of the activation of myelin-reactive t cells), firategrast (affect on the vla-4 system) and aimspro (neuropeptide stabilizer). moreover, stem cell-based therapy might be considered as another approach for attenuating ms through regulating the immune system, although several challenges should be resolved. more investigations and clinical trials should be designed to assess the effectiveness of several drugs and approaches that can target both inflammatory and degenerative components of ms. these kinds of approaches may offer hope for individuals who are suffering from this debilitating disease (mansoor et al., 2019; agrawal and yong, 2007; hart and bainbridge, 2016) . to gain ideas about ms mechanisms, a number of models have been developed. these experimental models fall into two categories: spontaneous models and induced models. each model reflects characteristic features of ms patients and has its own merits and demerits. myelin basic protein mutant (taiep rat), proteolipid protein mutants (rumpshaker and jimpy mice), as well as gene-knockout animals (the myelin-associated glycoprotein (mag) knockout, thy1-eb3-yfp mice, and thy1-xfp mice) show dysmyelination, altered neurotransmission and, in some instances, clinical disease. these models have frequently been used to study myelination. with chemically induced lesions, viral and autoimmune models are developed to show some evidence of demyelination, which is considered a pathological hallmark of ms. direct injection of ethidium bromide or lysolecithin into the cns produces demyelination. these induced models are usually effectively repaired once macrophages clear the myelin debris. for this reason, these models are rarely used at the present time. besides, local administration of glutamate or nitric oxide donors induces axonopathy in mice and have also been used to understand mechanisms of axonal degeneration and regeneration (luchtman et al., 2016) . a number of viruses, including semliki forest virus, theiler's murine encephalomyelitis virus, and a murine coronavirus have been found to induce disease by neurotrophic infection of the cns, specifically oligodendrocytes (lane and hosking, 2010) . moreover, studies using immunodeficient rag1 2 / 2 mice have indicated that cd4 1 and cd8 1 t lymphocytes as well as macrophages are key contributors to demyelination in coronavirus-infected mice (dandekar et al., 2001) . finally, experimental allergic encephalomyelitis (eae) has received the most attention as a model for ms; this animal model is routinely used for testing different therapeutic strategies. today, eae as the most commonly used preclinical murine model of ms induced actively by the injection of defined encephalitogenic myelin protein epitopes plus cfa, or passively by the transfer of encephalitogenic myelin-sensitized t lymphocytes. some of these eae models also require the administration of the microbial-based immunologic adjuvant pertussis toxin (pt) (yandamuri and lane, 2016) . eae exhibits many clinical and histological features of ms and is caused by autoimmunity induced against antigens that are expressed either naturally or artificially in cns (denic et al., 2011) . the method for eae induction and preparation of antigens to induce eae in c57bl/6 mice was adapted from the method described by kafami et al. (2010) . it is important for the successful induction of eae to follow standard precautions for the use of animals. female c57bl/6 mice that are 4-to 6-week old are used for the induction of eae. animals must adhere to the normal laboratory animal maintenance guide. animals were immunized with the hooke kits (hooke labs, ek-0115, lawrence, ma, usa). it is recommended to follow the manufacturer's instructions. a mesh was dampened in ether and put in a desiccator. the mouse was kept in the desiccator and observed until breathing slowed down to ascertain whether the mouse had been anesthetized. the mouse was removed from the anesthetic chamber and laid on its side. two syringes were filled with 1 ml of myelin oligodendrocyte glycoprotein (mog) emulsion with complete freund's adjuvant. each animal was given an injection of 200 μl. the needle was gently inserted into the subcutaneous space at the base of the tail, and 200 μl of emulsion was injected into the site. since it was difficult to give the mouse a 200-μl injection, every mouse was given a 100-μl injection at two different sites on the same day. immediately, and after 24 hours from the first injection, each mouse was given an intraperitoneal injection of pertussis toxin (100 μl/ animal). the animal was observed until complete recovery, and it could move without a floppy gate. this procedure was repeated for all animals. after 2à3 days, the flanks were bulging in response to the subcutaneous injection (flow chart 8.2). one day before immunization, and from the 7th to the 35th day post-immunization, the animals were evaluated on a daily basis for signs of eae following the 10-point score system (table 8. three different clinical parameters were analyzed to compare the course of eae (fig. 8.3) : (1) severity of disease as the cumulative disease index (cdi) was the mean of the clinical scores of the animals; (2) disease onset, calculated as the mean of the first-day animals showed the signs of the disease in experimental animals; and (3) peak of disease score, which represented the mean of the highest clinical score of disease for all animals in each group. tonicity of the tail and the distal part of the tail was ascertained by touching the tip of the tail. if the distal part of the tail was flaccid, the animal was removed from the base and observed to see if its tail remained erect or fell down (examined with the touch of the finger). after ascertaining tonicity of the tail, the gate of the animal was observed by keeping it in an open area (like a tabletop) and allowing it to walk. after checking the gate, the hind limb was observed by grabbing its tail. after that, the paralysis score was recorded for unilateral paralysis. by holding the animal in the palm of the hand, it was easy to evaluate the type of paralysis (unilateral or bilateral). it was noted whether the mouse rolled spontaneously in its cage or was dead with complete paralysis. after 35 days, animals that had an eae score of 5 and did not change for 3 more days were euthanized by chloral hydrate injection (0.3 ml, ip). for histopathlogical evaluations, different tissues were harvested after dissecting the animals. animals were placed appropriately in the dissection tray. a midline incision was made on the abdomen; the diaphragm was opened while ribbons were cut to expose the beating heart. the needle was inserted into the left ventricle of the heart while a phosphate-buffered saline (pbs) tap was allowed to fill the heart for 2 seconds. the right aorta was cut with small scissors to allow the pbs and pfa to circulate to exit. pbs allowed perfusion until the liver turned from red to yellow (b2à3 minutes). the best sign was when the liquid flowed out of the incised left aorta and turned from red to clear. another indicator was when pbs entered the pulmonary system and emerged through the nose of the animal. then, the pbs tap was closed and the tap was turned on for 4% paraformaldehyde (pfa, ph 7.4 at 37 c) to allow pfa to flow and perfuse the circulatory system for 3 minutes. perfusion was evaluated by involuntary hind limb movement and tail shivering. when the mouse became stiff, it was time to stop pfa perfusion. after the perfusion was complete with pfa, the system was washed with pbs to remove residual pfa. after perfusion, the various tissues of interest were harvested and stored in fresh 4% pfa for 3 days at 4 c. then, these tissues were washed with pbs and the pfa-fixed tissue could be stored in pbs for a few months. these tissues were then available for sectioning and staining (fig. 8.4) . for immunohistochemistry, the three sections showing the highest infiltrations were studied. an area $ 1.5 3 107 μm 2 from the brain/spinal cord was selected and analyzed under 200 3 magnification to assess the average number of positive cells per millimeter square and to quantify it on a computerized imaging system [bx51 microscope (olympus, hamburg, germany) with analysis software (special sis docu; soft imagingsystem)] by planimetry. the inflammatory index had to be calculated as a percentage determined by dividing the number of visual fields with .10 cd3 t cells by the total number of visual fields examined. detection of amyloid precursor protein (app) was performed for acute axonal damage. to assess the content of circulating proinflammatory cytokines like il-6, il-4, il-12, il-10, tnf-α, and ifn-γ, enzyme-linked immunosorbent assay (elisa) was employed. to evaluate the levels of different cytokines, blood was collected into tubes by a retro-orbital plexus method. the collected blood was kept in the tube to clot. after the clotting of the blood serum, it was separated and stored at 220 c. these serum samples were then used for the evaluation of different cytokines using the elisa. in order to quantify the mrna of different proinflammatory cytokines such as tnf-α and ifn-γ, antiinflammatory cytokines like il-10, myelin-deteriorating matrix metalloproteinase mmp-9, and the content of 164 8. animal models for human disease myelin basic protein (mbp 3à4), samples from animals had to be analyzed by real-time pcr. animals were sacrificed with lethal injection and perfused with cold pbs. then, the limbs and muscles were removed with scissors and the skin removed from these organs. a transverse cut was made at the base of the skull and vertebral column to separate them. the nasal bridge was broken with a small scalpel and the eyeballs removed. very thin forceps were used under the skull bones to break it into pieces from the frontal to occipital lobes. the bony connection under the cerebellum was broken to expose the cerebellum. the broken bones of the skull needed to be removed. the nerve root connection with the brain was cut. the brain was removed and stored in liquid nitrogen. for the removal of the spinal cord, an oblique cut was made from the lateral side of the spinal cord (started from the cervical part) to the furthest part of the vertebral column (both sides). the spinal cord was then exposed by cutting the boney flap. the steps above were repeated to get to the coda aquina. the spinal cord was taken out by cutting its adhesion to the base. it was then stored in liquid nitrogen. the frozen tissue sample was used for rna extraction. first, the sample was homogenized by pushing and rotating it with a sterile glass homogenizer. next, the homogenate sample was left on the bench top at room temperature (15 cà25 c) for 5 minutes to promote the dissociation of nucleoprotein complexes. then, 200 μl of chloroform was added to the tube and the tube was shaken vigorously for 15 seconds. the tube containing the homogenate was placed on the bench top at room temperature for 2à3 minutes and then centrifuged again at 15,000 rpm for 15 minutes at 4 c. after centrifugation, the sample separated into three phases: an upper, colorless, aqueous phase containing rna; a white interphase; and a lower, red, organic phase. the upper, aqueous phase was transferred to a new sterile eppendorf tube. one volume (usually 600 μl) of 70% ethanol was added to the tube containing the aqueous phase and mixed thoroughly by vortexing. visible precipitates after the addition of ethanol could then be noticed. up to 700 μl of the sample was processed for total rna extraction by using an rneasy mini spin column (roche germany) according to the kit instructions. after rna extraction, rna was quantified spectrophotometrically and the purity of rna was ascertained by taking out a ration between the od at 260 and 280 nm. a quantitative real-time reverse transcriptase pcr was performed to analyze the levels of mrna of different cytokines using cytokine-specific primers. the first step was to perform cdna synthesis by using a cdna synthesis kit (takara, japan), which was followed by a syber green i real-time pcr master mix kit (takara, japan). a house-keeping gene (like the β-actin gene) was included in the study to compare the results. the use of laboratory animals in research is of major ethical concern. much of the argument revolves around moral values. today, there is a wide spectrum of views on animal rights. this has prompted the establishment of guidelines on the care and use of experimental animal models. the guidelines endorse some essential principles for the care and use of animals for scientific projects. the basis of these principles is to replace animals with other methods such as mathematical models, computer simulations, and in vitro biological systems, thus reducing the number of animals used in order to obtain valid results without unnecessary duplication, and finally, refining projects by selecting appropriate species and techniques to minimize pain or distress to animals using appropriate sedation or anesthesia. as a researcher, one must always assume that procedures that cause pain to humans will cause pain in such situations in animals. surgical procedures should be performed on anaesthetized animals. it should be kept in mind that if the animal would suffer severe pain during a procedure, or if at the end point cannot be alleviated swiftly, the animals must be killed humanely. the transportation, housing, feeding, and handling of animals are also important. housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care. the place should be designed to facilitate control of environmental factors. cages should be comfortable and should fulfill behavioral requirements such as free movement and activity, bedding, contact with others of the same species, lighting, temperature, air quality, appropriate day/night cycles, and protection from excessive noise. the population density of animals within cages should also be considered from an ethical standpoint. this statement refers to the need for the reader to operate in accordance with the guidelines at her/his academy. the concept of translational research is to try to convert the results derived in animal models into a new understanding of disease mechanisms and therapeutics in human beings. it is a bridge from experimental models to clinical medicine. over recent years, the importance of this kind or research has progressively increased. consequently, translational research is considered a key component to finding practical applications, especially within medicine. with the improvement of technologies, significant progress has been made in producing various types of engineered experimental animal models based on a better understanding of the molecular and genetic principles of disease. as a result, any interventions in experimental models are more practical and repeatable when compared to patient-oriented research. various risk factors that are linked to, or even responsible for, differences in clinical results should also be considered as significant for the development of experimental models; this will enhance the translational value of experimental models. these risk factors can be categorized into genetic factors, acquired factors, and health conditions, which can be studied in models in a controlled manner. in medicine, the performance of successful translational research requires data from hospitals. as we mentioned before, rheumatoid arthritis as a progressive debilitating disease is characterized by hyperplasia of synoviocytes leading to joint destruction and permanent deformity. although the definite pathophysiology of ra is ambiguous, some evidence suggests that telomerase is also involved in the pathogenesis of this disease. nobel laureates in physiology/ medicine in 2009, elizabeth blackburn, jack szostak and carol greider, have solved a major problem of the chromosomal protection against degradation during cell divisions. they identified telomerase and a unique dna sequence in the telomeres. telomerase is a ribonucleoprotein enzyme that adds repeated units of ttaggg to the ends of chromosomes. this enzyme is composed of an rna component, called htert which serves as a template for addition of telomeric repeats. although it is now known that the dna sequence in the telomere attracts proteins that form a protective cap around the fragile ends of the dna strands, a number of reports have mentioned a link between the increased telomerase activity of human tumor samples and degree of invasiveness. in patients with ra, an impaired telomerase enzyme and premature cellular ageing (senescence) of thymic naïve and memory t cells was reported. moreover, transfection of rheumatoid arthritis synovial fibroblasts with vectors expressing antisense oligonucleotide against the htert component of telomerase enzyme has led to cytolysis of these cells that exhibit high telomerase activity. taken together, their discoveries have shed light on disease mechanisms and stimulated the development of potential new therapies in experimental models. there are many methods to evaluate telomerase activity, but we measured it by telomere repeat amplification protocol using trapeze telomerase detection kit (intergen, inc., usa) in animals treated with camellia sinensis stew. in detailed, biopsies of synovial tissue were obtained aseptically from the knee joints of rat after the induction of cia. synovial tissue specimens were rinsed, minced, and digested with 0.2% collagenase in high-glucose dmem containing 10% fbs and antibiotics. following overnight incubation at 37 c, cells were collected, plated in culture flask, and allowed to reach confluency at 37 c in a humidified atmosphere of 5% co2. after the lysis of equal number of cells which harvested from synovial tissue with the chaps lysis buffer, the telomerase was first extended for 30 minutes at 30 % oc and then amplified by30 cycles of pcr. the products of pcr were detected by polyacrylamide gels and revealed by silver nitrate staining. telomerase activity was calculated as the ratio of the intensity of telomerase ladders to the intensity of the 36-bp internal standard. in conclusion, we show that c. sinensis stew effectively suppresses collagen arthritis and a potent inhibitory effect on telomerase activity. so, natural products should continue to provide innovative lead compounds currently entering clinical trials. recently, the circular plant peptide kalata b1 (cyclotide) was investigated by thell et al. using the ms mouse model experimental autoimmune encephalomyelitis. according to their findings, treatment of mice with the cyclotide resulted in a significant delay and diminished symptoms of eae by oral administration. taken together, natural product should be considered as a candidate for the future investigations to possible implication for human health. using these above-mentioned models associated with other experimental models gives us such an opportunity to accomplish many findings in human medicine. until now, many progresses in medical sciences have been achieved. the discovery of numerous types of antibiotics for controlling infectious disease and elimination some viral disease like smallpox might be considered as one of researcher and indeed experimental animals honor. also, blood transfusions, open heart surgery, and other life-saving techniques have all been developed. nevertheless, there are many unsolved subjects included cancer, aging, alzheimer's disease, and acquired immunodeficiency syndrome in front of the society. without no doubt, until to find another means for answering human beings dilemma, the use of living animals in scientific research would be the best and applicable procedure. with all those valuable function, it is pivotal to consider ethical concerns over the quality of life of animals when you as a young researcher start to write a proposal. medlineplus is the national institutes of health (nih) site for patients and their families and friends. produced by the national library of medicine, it brings you information about diseases, conditions, and wellness issues in easy-to-understand language. medlineplus offers reliable, up-to-date health information, anytime, anywhere, for free. http://www.ebi.ac.uk/ipd/imgt/hla/ the imgt/hla database provides a specialist database for sequences of the human major histocompatibility complex (hla) and includes the official sequences for the who nomenclature committee for factors of the hla system. the imgt/hla database is part of the international immunogenetics project. adjuvant a substance such as complete freund's adjuvant (cfa) that enhances t-and b-cell activation, mainly by promoting the accumulation and activation of antigen-presenting cells at the site of antigen exposure. adjuvants stimulate the expression of t-cellactivating co-stimulators and cytokines by antigen-presenting cells and may also prolong the expression of peptideàmhc complexes on the surface of these cells. autoimmune disease a disease caused by a breakdown of selftolerance such that the adaptive immune system responds to selfantigens and mediates cell and tissue damage. autoimmune diseases can be organ specific (e.g., thyroiditis or diabetes) or systemic (e.g., systemic lupus erythematosus). cd molecules cell surface molecules expressed on various cell types in the immune system that are designated by the "cluster of differentiation (cd) number." disease-modifying antirheumatic drugs (dmards) they contain medications from different classes including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards were often only partly effective and poorly tolerated in long-term therapy of autoimmune diseases. enzyme-linked immunosorbent assay (elisa) a method of quantifying an antigen immobilized on a solid surface by use of a specific antibody with a covalently coupled enzyme. the amount of antibody that binds the antigen is proportional to the amount of antigen present and is determined by spectrophotometrically measuring the conversion of a clear substrate to a colored product by the coupled enzyme. experimental autoimmune encephalomyelitis (eae) this is an animal model of multiple sclerosis, an autoimmune demyelinating disease of the central nervous system. eae is induced in rodents by immunization with components of the myelin sheath (e.g., myelin basic protein) of nerves, mixed with an adjuvant. the disease is mediated in large part by cytokine-secreting cd4 1 t cells specific for the myelin sheath proteins. granulocyte-monocyte colony-stimulating factor (gm-csf) a cytokine made by activated t cells, macrophages, endothelial cells, and stromal fibroblasts that acts on bone marrow to increase the production of neutrophils and monocytes. gm-csf is also a macrophage-activating factor and promotes the differentiation of langerhans cells into mature dendritic cells. granuloma a nodule of inflammatory tissue composed of clusters of activated macrophages and t lymphocytes, often associated with necrosis and fibrosis. granulomatous inflammation is a form of chronic delayed-type hypersensitivity, often in response to persistent microbes or to particulate antigens that are not readily phagocytosed. granzyme a serine protease enzyme found in the granules of ctls and nk cells is released by exocytosis, enters target cells, and proteolytically cleaves and activates caspases and induces target cell apoptosis. homeostasis in the adaptive immune system, the maintenance of a constant number and diverse repertoire of lymphocytes, despite the emergence of new lymphocytes and the tremendous expansion of individual clones that may occur during responses to immunogenic antigens. homeostasis is achieved by several regulated pathways of lymphocyte death and inactivation. human leukocyte antigens (hla) mhc molecules expressed on the surface of human cells. human mhc molecules were first identified as alloantigens on the surface of white blood cells (leukocytes) that bound serum antibodies from individuals previously exposed to other individuals' cells. interferons a subgroup of cytokines originally named for their ability to interfere with viral infections, but that have other important immunomodulatory functions. type i interferons include interferon-α and interferon-β, whose main functions are antiviral; type ii interferon, also called interferon-γ, activates macrophages and various other cell types. interleukins any of a large number of cytokines named with a numerical suffix roughly sequentially in order of discovery or molecular characterization (e.g., interleukin-1 and interleukin-2). some cytokines were originally named for their biological activities and do not have an interleukin designation. lipopolysaccharide (lps) a component of the cell wall of gramnegative bacteria that is released from dying bacteria and stimulates many innate immune responses, including the secretion of cytokines, induction of microbicidal activities of macrophages, and expression of leukocyte adhesion molecules on endothelium. lps contains both lipid components and carbohydrate moieties. major histocompatibility complex (mhc) molecule a heterodimeric membrane protein encoded in the mhc locus that serves as a peptide display molecule for recognition by t lymphocytes. two structurally distinct types of mhc molecules exist. class i mhc molecules are present on most nucleated cells, bind peptides derived from cytosolic proteins, and are recognized by cd8 1 t cells. class ii mhc molecules are restricted largely to dendritic cells, macrophages, and b lymphocytes, bind peptides derived from endocytosed proteins, and are recognized by cd4 1 t cells. matrix metalloproteinase (mmp) mmps are a family of highly conserved endopeptidases dependent on zn2 1 ions for activity. mmps can collectively cleave most extracellular matrix. at present, 25 vertebrate mmps and 22 human homologs have been identified and characterized. mmps participate in many physiological processes, such as embryonic development, organ morphogenesis, blastocyst implantation, ovulation, nerve growth, cervical dilatation, postpartum uterine involution, mammary development, endometrial cycling, hair follicle cycling, angiogenesis, inflammatory cell function, apoptosis, tooth eruption, bone remodeling, and wound healing. myelin oligodendrocyte glycoprotein (mog) mog is a cnsspecific type i membrane glycoprotein of the immunoglobulin superfamily expressed mainly on the outermost layer of the myelin sheath, making it an ideal target for antibody-mediated demyelination. it is highly immunogenic, and unlike other myelin proteins used to induce eae, is unique in inducing both an encephalitogenic t-cell response and a demyelinating response in eae. multiple sclerosis (ms) a chronic inflammatory demyelinating disorder of the central nervous system. the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. nitric oxide (no) a biologic effector molecule with a broad range of activities that in macrophages functions as a potent microbicidal agent to kill ingested organisms. pannus formation of locally invasive synovial tissue is a characteristic feature of rheumatoid arthritis. perforin a protein that is homologous to the c9 complement protein and is present in the granules of ctls and nk cells. when perforin is released from the granules of activated ctls or nk cells, it promotes the entry of granzymes into the target cell, leading to apoptotic death of the cell. rheumatoid arthritis (ra) an autoimmune disease characterized primarily by inflammatory damage to joints and sometimes inflammation of blood vessels, lungs, and other tissues. cd4 1 t cells, activated b lymphocytes, and plasma cells are found in the inflamed joint lining (synovium), and numerous proinflammatory cytokines, including il-1 and tnf, are present in the synovial (joint) fluid. reverse transcriptase (rt) an enzyme encoded by retroviruses, such as hiv, that synthesizes a dna copy of the viral genome from the rna genomic template. purified reverse transcriptase is used widely in molecular biology research for purposes of cloning complementary dnas encoding a gene of interest from messenger rna. th1 cells subset of cd4 1 helper t cells whose principal function is to stimulate phagocyte-mediated defense against infections via secretion of a group of cytokines, including ifn-γ. th2 cells subset of cd4 1 helper t cells whose principal functions are to stimulate ige and eosinophil/mast cell-mediated immune reactions via a particular set of cytokines, including il-4 and il-5. th17 cells subset of cd4 1 helper t cells that are protective against certain bacterial infections and also mediate pathogenic responses in autoimmune diseases. tumor necrosis factor (tnf) a cytokine produced mainly by activated mononuclear phagocytes that stimulates the recruitment of neutrophils to sites of inflammation. tnf-α blocking agents a group of biological disease-modifying antirheumatic drugs such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody). very late antigen (vla) the set of integrins that shares a common beta-1 chain. long-answer questions immunopathogenesis of multiple sclerosis novel multiple sclerosis susceptibility loci implicated in epigenetic regulation immunology increased expression of tbet in cd4 1 t cells from clinically isolated syndrome patients at high risk of conversion to clinically definite ms reciprocal developmental pathways for the generation of pathogenic effector th17 and regulatory t cells spontaneous rheumatoid-like arthritis in a line of mice sensitive to collagen-induced arthritis one year in review 2016: novelties in the treatment of rheumatoid arthritis the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats access to the next wave of biologic therapies (abatacept and tocilizumab) for the treatment of rheumatoid arthritis in england and wales axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus the relevance of animal models in multiple sclerosis research current and emerging treatment of multiple sclerosis hla and rheumatoid arthritis: how do they connect? vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type ii collagen-induced arthritis in the same rat strain are different diseases early appearance of activated cd4 1 t lymphocytes and class ii antigen-expressing cells in joints of dba/1 mice immunized with type ii collagen involvement of macrophages and dendritic cells in synovial inflammation of collagen induced arthritis in dba/1 mice and spontaneous arthritis in mrl/lpr mice micrornas associated with the pathogenesis of multiple sclerosis autoimmunity induction by human t cell leukemia virus type 1 in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in c57bl/6 mice transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis induced expression of class ii transplantation antigens in the cartilage-pannus junction in ra: chronic synovitis as a model system for aberrant t-lymphocyte activation role of adjuvants in turning autoimmunity into autoimmune disease il-35 stimulation of cd39 1 regulatory t cells confers protection against collagen ii-induced arthritis via the production ofil-10 arthritis-inducing ability of a synthetic adjuvant, n-acetylmuramyl peptides, and bacterial disaccharide peptides related to different oil vehicles and their composition from systemic t cell self-reactivity to organ-specific autoimmune disease via immunoglobulins genetics of rheumatoid arthritis -a comprehensive review the pathogenesis of murine coronavirus infection of the central nervous system flavonoids as cytokine modulators: a possible therapy for inflammation-related diseases identification of arthritogenic adjuvants of self and foreign origin susceptibility of da rats to arthritis induced with adjuvant oil or rat collagen is determined by genes both within and outside the major histocompatibility complex in vivo and in vitro effects of multiple sclerosis immunomodulatory ther-apeutics on glutamatergic excitotoxicity the potential use of mesenchymal stem cells for the treatment of multiple sclerosis the epigenetics of autoimmunity cytokine production in synovial tissue of mice with collagen-induced arthritis (cia) inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis development of arthritis, periarthritis and periostitis in rats given adjuvants an update on dietary phenolic compounds in the prevention and management of rheumatoid arthritis effect of pyrimethamine in experimental rheumatoid arthritis type ii collagen-induced murine arthritis. i. induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity cd8(1) t-cells as immune regulators of multiple sclerosis ptpn22: the archetypal non-hla autoimmunity gene from genetics to functional insights into rheumatoid arthritis b celldeficient mice do not develop type ii collagen-induced arthritis (cia) protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly (adpribose) synthetase efficacy and safety of mavrilimumab in japanese subjects with rheumatoid arthritis: findings from a phase iia study effect of denosumab on japanese patients with rheumatoid arthritis: a doseresponse study of amg 162 (denosumab) in patients with rheumatoid arthritis on methotrexate to validate inhibitory effect on bone erosion (drive)-a 12-month, multicentre, randomized, double-blind, placebo-controlled, phase ii clinical trial interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment the natural soluble form of il-18 receptor beta exacerbates collagen-induced arthritis via modulation of t-cell immune responses genetics and epigenetics of rheumatoid arthritis imaging axonal degeneration and repair in preclinical animal models of multiple sclerosis use of animals in scientific research. indian council of medical research ministry of health & family welfare new delhi animal models of rheumatoid arthritis and their relevance to human disease animal models of multiple sclerosis-potentials and limitations the rights of animals animal models of multiple sclerosis. neuroinflammation 55à79. glossary adhesion molecule a cell surface molecule (e.g., selectin, integrin, and member of the ig superfamily) whose function is to promote adhesive interactions with other cells or the extracellular matrix describe the significance of animal modeling in biotechnology? how cia is induced and how the ability of medications is evaluated in mice? discuss about different types of animal model to study the pathogenesis of rheumatoid arthritis? why the presence of inflammation in the cns is considered as a hallmark of multiple sclerosis? explain the various methods for evaluation of experimental models of multiple sclerosis? short answer questions what is the reason of reportedly experiencing different animal models for studying the pathogenesis of rheumatoid arthritis? give an example which shows the impact of the epigenetics in the initiation of ra? which types of evaluation should be performed after "collagen-induced arthritis" aroused? 4. what are the "intervening factors after the activation of the immune system, which type of lymphocytes enters into central nervous system answers to short answer questions 1. there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate particular aspect of the disease.2. the epigenetics of ra have also been responsible in the initiation of ra. since the concordance of rheumatoid arthritis in identical twins is not 100% other nongenetic factors also play a role in the disease etiology. 3. daily clinical assessment according to a macroscopic scoring system, histological processing and assessment of arthritis damage, radiographic evaluation by an investigator blinded to the treatment protocol on day 35. 4. age, weight, and possible infectious disease in animals should be considered as the intervening factors. 5. following activation, myelin-specific lymphocytes enter into the cns and oligodendrocytes are damaged.yes/no type questions 8. natalizumab blocks leukocyte migration into the cns by binding to icam. 9. eae induced actively by injection of the microbialbased immunologic adjuvant pertussis toxin. 10. housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care.answers to yes/no type questions 1. yes-the most important risk factor for acpa 1 ra is the hla class ii locus. default normal template original article possible role of interleukin-6 in rheumatoid arthritis batool a. al-haidary *ph.d summary: background: rheumatoid arthritis (ra) is an autoimmune disease. many etiological agents are proposed to play a role in its pathogenecity. one of these factors is cytokines such as interleukin6. material & methods: elisa method has been used for il-6 estimation in 75 ra patients in comparison with 61 sle as patient controls and 39 apparently healthy controls. j fac med baghdad 2007; vol. 49, no.2 received july 2006 accepted jan. 2007 results: this study showed that there was an elevation of il-6 in the sera of ra patients with high significant differences between ra patients and controls (p< 0.001). moreover a good correlation between il-6 level & rf titer were observed. however, for most patients with high il-6 were shown to be hla-dr4. conclusions: interleukin-6 play a crucial role in the disease which may be participate in the severity of ra & subsequently its treatment. key words: ra, il-6, rf, hla-dr4, elisa, microlymphocytotoxicity __________________________________________________________________________________________ introduction:______________________________ the best knowledge of ra treatment depends on the studying the cellular & humoral factors that involved in the disease pathogenecity. on the tope of these factors are the cytokines, including il-6 as an inflammatory factor. recent study showed that there is a cross talk between il-1 and il-6 signaling pathways in ra synovial fibroblasts [1]. while a number of interleukins such as interleukin-1 (il-1) and (il-10) seem to be pleiotrophic in their effects, (il-6) may be considered the prototrophic cytokine. this is reflected in the variety of names originally assigned to 11-6 based on function, including interferon (32, il-1 inducible 26kd protein, hepatocyte stimulating factor, cytotoxic tcell differentiation factor, b cell differentiation factor (bcdf) and / or b cell stimulating factor 2 (bcsf2). all these activities associated with inflammatory process [2]. so il-6 is considered as a pleiotrophic inflammatory cytokine produced by t cells, monocytes, macrophages and synovial fibroblasts [3]. originally identified as a factor that induces the final maturation of b cells into plasma cells. recently an interesting studies involving the il-1 and il-6 in initiating the significant pathway in ra symptoms. interleukin-6 was observed to be involved in diverse _________________________________________ * clinical immunology/ ass. prof./ college of medical & health technology. biologic processes, such as the activation of t cells, induction of the acute-phase response, stimulation of the growth and differentiation of hematopoietic precursor cells, and proliferation of synovial fibroblasts results in synovial inflammation and ultimately joints damage [4].. the current study is a trail to estimate il-6 concentration in the patients' sera in comparison with patient controls and apparent healthy controls. this , however, might open a gate for entrance into the treatment of this disease. subjects & methods: seventy-five rheumatoid arthritis (ra) patients' sera samples have been studied for il-6 in comparison with 61 sle patients as patient controls besides 39 apparent healthy individuals. il-6 has been estimated by using elisa technique [diaclone, france lot. no. 1006-24]. all patients were diagnosed according to the modified criteria [revised criteria of the american rheumatism association [5].. in this study hlatyping using micro-lymphocytotoxicity by were assessed [6]. statistical analysis: all the data have been analyzed statistically using kriiskalallis test and mann-whitney analysis for measuring the differences between the studying groups [7].. results:: i. interleukin-6 level in the sera of ra patients and control groups: the results of il-6 estimation in the sera of ra patients, patient controls and apparent healthy individuals are listed in the table-l. this table shows that there is high significant differences between il-6 levels in the patients sera and controls (p < 0.001 for each comparison), with a median of 195, 50, and 8 pg / ml for ra patients, patient controls and healthy controls respectively. j fac med baghdad vol. 49 , no. 2 , 2007 249 simpo pdf merge and split unregistered version http://www.simpopdf.com possible role of interleukin-6 in rheumatoid arthritis batool a. al-haidary table -1 il-6 concentrations in the ra atients sera and control rou s interleukin-6 (il-6) concentration in pg / ml sporadic ra (75) * patient control [pc] (61) healthy control [hc] (39) range 21-576 pg. / ml 28-570 pg. / ml 1-157 pg. / ml mean ± sd 216.2 ± 159.3 137.7 ± 166.58 16 ± 2 6.54 median 195 50 8 p (kruskal-wallis) for the three groups differences = <0.001 p (mann-whitney) for the differences between two groups ra vs. pc = < 0.001, ra vs. hc = < 0.001 * = no. between brackets represents the no. of patients. the above figure reveals the distribution of il6 concentration in ra patient's sera according to its frequencies. this figure reveals that there was a normal distribution for the frequencies of il-6 concentrations among ra patients sera, though the highest frequencies are observed between 25-125 pg / ml. ii. the correlation between il-6 and rf: an interesting finding that elevation of il-6 accompanied by high rf titer which is clearly shown in figure 3. this figure shows that the majority of patients [65 out of 75] with a high il-6 level [extends to about 600 pg/ml] are sero-positive for rf [i.e. > 20 iu/ml] while the minority of patients [10 out of 75] with very low il-6 concentration are sero-negative for rf . j fac med baghdad 250 vol. 49 , no. 2 , 2007 simpo pdf merge and split unregistered version http://www.simpopdf.com possible role of interleukin-6 in rheumatoid arthritis batool a. al-haidary iii. correlation between il-6 and hladr4: the current study shows that there is a prominent correlation between hla-dr4 molecules and il-6 concentration in the patient's sera as shown in the figure -4. this figure shows that the frequency of hla-dr4 molecules are higher among patients with high il-6 level [42] characterized by presence of hla-dr4 molecules on the surfaces of their b cells while the others [33] whose sera apparent with low il6 concentration posses other dr molecules rather than dr-4. j fac med baghdad 251 vol. 49 , no. 2 , 2007 simpo pdf merge and split unregistered version http://www.simpopdf.com possible role of interleukin-6 in rheumatoid arthritis batool a. al-haidary discussion: studies are denoted that il-6 was elevated in rheumatoid arthritis patients' synovial fluids as well as their sera [8-9]. the results of the current study have revealed an increasing in il-6 level in ra patients sera in comparison with the control groups which is reckoned as an inflammatory mediator particularly among those patients with disease flare up. this result was comparable to [9-11]. the observed median level is 195 pg./ml which is higher than that of abroad (106 'p g:/ml).'[12]. this variation may be related to the variation in the duration of the disease and its activity particularly most the patients included in this study are at the acute stage of the disease flare up time [13-14]. treated ra patients had displayed significantly reduced serum il-6 (mean, 9.9 pg/ml) [15]. moreover this study showed that there was a good correlation between il-6 and rf levels, in which the later usually enhances the disease severity and may act in synergetic fashion with il-6. these findings were observed to be in consistent with that of [16], which revealed an association between il-6 and other humoral inflammatory, disease activity parameters as crp and esr. rheumatoid factor is considered as a prominent feature of ra and play a crucial role in ra pathogenecity [17]. the current study revealed a high correlation between rf and il-6 which explains the role of rf in ics formation, complement activation and finally cellular infiltration resulting in cytokine production [17]. it was denoted that the disease becomes worse in the presence of rf, hla-dr4 molecules [signs of high disease activity], especially when they accompanied with elevation of il-6 as shown by this study and other [18]. sporadic patients are those with no familial history for ra , however, the frequency of hladr4 was still higher than that for apparently healthy individuals [42 out of 75 ra patients]. this study demonstrated that (il-6) is a protein overproduced in the sera of people with rheumatoid arthritis, where it's believed to be responsible for joint damage and swelling. interleukin-6 may also be a cause of fever and excess blood platelets (thrombocytosis) in people with rheumatoid arthritis. researchers hope that blocking il6 can reduce the damage it does [19]. references: 1. deon d, ahmed s, tai k, scaletta n, herrero c, lee i-h, krause a & ivashkiv lb " cross-talk between il-1 and il-6 signaling pathways in rheumatoid arthritis ." the j. immunol. , (2001) 167: 5395-5403 2. arthritis research reagents : in scope " interleukin=6" ' http://\vww.,c / rnd systems.com october (2002). 3. van snick j "interleukin-6: an overview." ann.rev. immunol. (1990) 8: 253-78. 4. choy , e.h.s. & panayi , g.s. : cytokine pathways & joint inflammation in rheumatoid arthritis . educational series ; medical sciences, update (2001) 2(7) july. 5. arnett fc, edworthy sm, bloch da, et. al., "the american association 1987 revised criteria for the classification of rheumatoid arthritis." arthritis rheum. (1988) 31: 315-24 . 6. terasaki p and mcclelland j, "micro-droplet assay of human serum cytotoxins" nature (1964), 204: 9981000. 7. sorlie de "medical biostatistics and epidemiology: examination and broad review." 1st ed; (1995) norwalk connecticut appleton and lange com: 74-88. 8. bertazzolo n, punzil l, stefani mp , et al., "interrelationships between interleukin(il)-1, il-6 and il-8 in synovial fluid of various arthropathies." agents actions (1994) mar; 41(1-2): 90-2. 9. lacki jk, samborski w & mackiewicz sh "interleukin-l0 and interleukin-6 in lupus erthymatosus and rheumatoid arthritis, correlations with acute phase proteins." clin. rheumatol. (1997) may; 16(3): 275-8. 10. robak t, gladalska a, stepien h, &.robak k. " serum levels of interleukin-6 type cytokines and soluble interleukin-6 receptor in patients with rheumatoid arthritis. "mediators-inflamm. (1998) 7(5): 347-53 11. oelzner p, franke s, muller a, hein g & stein g "relationship between soluble markers of immune activation and bone turnover in post-menopausal women with rheumatoid arthritis." rheumatologyoxford (1999) sep; 38(9): 841-7 12. al-awadhi a, olusi s, al-zaid n, & prabha k. " serum concentration of interleukin-6, osteocalcin, intact parathyroid hormone, and marker of bone resorption in patients with rheumatoid arthritis." j. immunol. (1999) jun; 26(6): 1250-6 13. roony m, david j, symons j, di-giovine f, varsani h & woo p " inflammatory cytokine responses in juvenile chronic arthritis," br. j. rheurnatol. (1995) may ; 34(5): 454-60. 14. lacki jk, klama k, mackewicz sh, mackiewicz h & muller w. "circulating interleukin-10 and interleukin-6 serum levels in rheumatoid arthritis patients treated with methotrexate, gold salts: preliminary report." inflamm.res. (1995 ) jan 44(1): 24-6. 15. wellby ml; kennedy ja; pile k; true bs; barreau p. "serum interleukin-6 and thyroid hormones in rheumatoid arthritis." metabolism (2001) 50(4):4637 (issn: 0026-0495) 16. boss b & neek g, " correlation pf il-6 with the classical humoral disease activity parameters esr and crp and with cortisol, reflecting the activity of the_ hpa axis in active rheumatoid arthritis. " zrheumatol. (2000) 59 suppl. 211:624 17. al-haidary ba, mousawy km, & al-khafaji jt,. "hla-typing for rheumatoid arthritis patients (familial profile) " a thesis submittecf''totlie.. college of medicine and committee of graduated studies of university of baghdad in partial fulfillment of requirement for the degree of doctorate philosophy in medical microbiology/ clinical immunology, (2004). 18. kloppenburg m, dijkmans ba, verwej cl, & breedveld fc." infalammatory and immunological parameters of disease activity in rheumatoid arthritis patients treated with minocycline" immunopharmacology ,1996 mar 31(2-3): 163-9 19. van-zeben and breedveld, "prognostic factors in rheumatoid arthritis" j. rheumatol. suppl. (1996) mar; 44: 31-3 j fac med baghdad 252 vol. 49 , no. 2 , 2007 simpo pdf merge and split unregistered version http://www.simpopdf.com http://systems.com/ 1.doc 2.doc references list all authors when six or less; when seven or more, list only first three and add et al. tables submission of manuscripts mail the required number of manuscript copies in a heavy-paper envelope, enclosing the manuscript copies and figures in cardboard. manuscripts should be accompanied by a covering letter from the author who will be responsible for correspondence regarding the manuscript. the covering letter should contain a statement that the manuscript has been seen and approved by all authors. 3.doc modified sugiura operation for portal hypertension and bleeding esophageal varices 172 omar s. khattab,samie b. safar post-surgical loco regional recurrence of breast carcinoma in iraq 193 ali m. al-saiegh 4.doc 5.doc modified sugiura operation for portal hypertension and bleeding esophageal varices omar s. khattab*, m.b.ch.b , h.d.s , h.d.l.m , f.i.c.m.s, c.a.b.s samie b. safar, m.b.ch.b , f.r.c.p, f.r.c.s, f.a.c.s 6.doc introduction:______________________________ types of incisional hernias: _________________________________________ clinical types of incisional hernias: patients and methods: discussion conclusions references 7.doc 8.doc 9.doc post-surgical loco regional recurrence of breast carcinoma in iraq ali m. al-saiegh * m.b.ch.b. d.s f.i.c.m.s. summary conclusion: carcinoma of the breast affecting iraqi females at younger ages in a high & increasing rate than other studies with a higher loco-regional recurrence rate. significant association were found regarding latency period, staging, histopathology & grading of primary tumour . aims of study: 1. to assess the incidence of post operative loco regional recurrence of breast carcinoma in iraqi female patients. 2. to determine the significance of certain variables that may affect the loco regional recurrence rate . introduction:______________________________ * head of department of surgerymedical college kufa university results: discussion: conclusion references 10.doc 11.doc 12.doc 13.doc 14.doc 15.doc 16.doc back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. __________________________________________________________________________________________ introduction:_______________________________ **lecturer, college of dentistry – university of mosul patients and method: results: 17.doc 18.doc 19.doc hla -a hla-b total increased frequency study groups blood groups 20.doc subject and methods: 21.doc 22.doc 23.doc 24.doc 25.doc 26.doc rabab .n. al-saadi.* ficms back ground: psoriasis is a chronic relapsing disorder with no life long cure, many systemic and topical modalities are available, one of these topical modalities is the vitamin d analogue (calcipotriene) which is widely used recently to treat psoriasis and many other skin problems. 27.doc 28.doc 29.doc 30.doc 31.doc 16.pdf back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. __________________________________________________________________________________________ introduction:_______________________________ **lecturer, college of dentistry – university of mosul patients and method: results: 166.pdf back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. __________________________________________________________________________________________ introduction:_______________________________ **lecturer, college of dentistry – university of mosul patients and method: results: iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 40 formulation and evaluation of optimized zaltoprofen lyophilized tablets by zydis technique suray a. hazzaa *,1 and shaimaa n. abd-alhameed ** * department of pharmacy, al-anbar health directorate, ministry of health, anbar, iraq ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract “orodispersible tablet” a tablet that is to be placed in oral cavity where it disperses rapidly by saliva with no need for water before swallowing. zaltoprofen (zlp) is one of nsaids which is used in the treatment of rheumatoid arthritis and osteoarthritis as well as to relieve inflammation and pain after surgery, injury and tooth extraction. the present study was aimed to prepare rapidly dissolved lyophilized zaltoprofen tablet with different pharmaceutical excipients and studying the factors affecting pharmaceutical properties like (solubility, disintegration time dt, dissolution, etc.) of tablets. the lyophilized disintegrating tablets (ldts) were prepared using zydis technique by lyophilization an aqueous dispersion of zaltoprofen with a matrix forming agent, gelatin, and a collapse protectant, glycine. in addition to many excipients like pvpk30 was used to improve the in vitro, in vivo disintegration time and dissolution rate, mannitol as bulk forming agent. fourteen formulations were prepared to inspect the variables that affect the disintegration time and dissolution rate. all the formulations were evaluated for their physical appearance, mechanical strength, x-ray diffraction, ftir, dt, and in vitro drug release. the prepared tablets were optimized and formula was subjected to different measured parameters such as disintegration time, drug content, and in-vitro drug release. results obtained from dissolution studies and dt showed that lyophilized disintegrating tablets (ldts) (f8,f10,f12,f13 was 45,37,21 and 17 sec.) respectively ,while(f14) displayed considerably faster in vitro dissolution rate of (zaltoprofen) 3 min. and dt 9 sec. the (lyophilized disintegrating tablets) were also evaluated showing the transformation into amorphous state and absence of interaction of zaltoprofen with the components of the tablets. from visual inspection ,physical strength ,dt and release behavior obtained ,one can conclude that the formulas(f14) which contains zaltoprofen 3.2% ,gelatin3%, mannitol 3%, glycine 1.5%, pvp k30 1.5% was the most suitable one. keywords : zaltoprofen, lyophilization, pvpk30 . " zydis حبوب انسانتوبرفين انمحسنة وانمجففة بانتجميد باستخداو تقنية ال" تصييغ وتقيى سري عبد هساع *،1 و شيماء نسار عبد انحميد ** * طحت االٗبار ، وسارة اُظحت، اُؼزام دائزةهسْ اُظٍذُت . ** كزع اُظٍذالٍٗاث ، ًٍِت اُظٍذُت ، صآؼت بـذاد ، بـذاد اُؼزام. الخالصة ائبت كٔىٌا باٗها اُحبىب اُخً ٓا إ حىضغ بخضىٌق اُلْ كاٗها حخلٌي وح٘خشز بسزػت بىصىد اُِؼاب دوٕ ذٌٌٖٔ حؼزٌق اُحبىب اُ اُحاصت ُِٔاء هبَ إ حبِغ.وٌؼخبز اُشاُخىبزوكٍٖ ٖٓ ادوٌت ٓضٔىػت ٓضاداث االُخهاب اُال سخٍزوٌذٌت أُسخخذٓت ُؼالس اُزوٓاحٍشّ االطاباث وهِغ االس٘إ. اُذراست اُحاٍُت حهذف ُخحضٍز حبىب اُشاُخىبزوكٍٖ سزٌؼت وًذُي ػالس االّ ٓا بؼذ اُؼٍِٔاث اُضزاحٍت و اُذوبإ بطزٌوت اُخضلٍذ باسخخذاّ)سىاؽ طٍذالٍٗت(ٓخخِلت ودراست اُؼىآَ أُؤرزة ٓزَ اُذوباٍٗت,ٓؼذٍ اُخلٌي وححزر اُذواء ػِى ُخضٍٔذ حٍذ حخْ ػٍِٔت اُخضلٍذ ُِٔحِىٍ أُائً ُِشاُخىبزكٍٖ ٓغ اُخىاص اُظٍذالٍٗت ُِحبىب.هذ حْ ححضٍز اُحبىب بطزٌوت اُخضلٍق با ًٔادة ٓحس٘ه ُخلٌي اُحبت وًذُي ٓؼذٍ ػٍِٔت اُذوبإ 63اُضٍالحٍٖ وٓضاد االٗهٍار اٌُالٌسٍٖ باالضاكت اُى اُبىًُ كٍَ٘ باٌزوُذٌٖ ى ُخوٍٍْ ُِؼىآَ أُؤرزةػِى حلٌي اُحبت و ٓؼذٍ اُذوباٍٗت. وأُاٍٗخىٍ ًٔادة ٌٓىٗت ُِشٌَ. اربؼت ػشز طٍـت حْ ححضٍزها ٓغ أُؼاٌ٘ه وا وهذ حْ حوٍٍْ ًَ اُظٍؾ كً ٓا ٌخض شٌِها اُلٍشٌاوي وهىحها أٌٍُاٌٍٍٗه وحلٌي اُحبت وححزٌز اُذواء. اُ٘خائش بؼذ ححضٍز اُحبىب بخوٍ٘ت (zydis) ُحبت ٖٓ اُذواء وٓؼذٍ ححزٌز اُذواء وًاٗج وححسٍٖ اُظٍؾ حْ اخضاػها ُؼذة حؤٍٍاث ٓزَ وهج اُخلٌي وٗسبت احخىاء ا ( اظهزث ُ٘ا ٓؼذٍ سزٌغ ُخحزٌز اُذواء خالٍ اُذهائن 47اُ٘خائش حخضٖٔ إ اُحبىب أُضللت باُخضٍٔذ ُِشاُخىبزوكٍٖ ُِظٍـت رهْ ) ضللت باُخضٍٔذ باسخخذاّ ( رىاًٗ .حْ حوٍٍْ وكحض اُحبت سزٌؼت اُذوبإ ا9ُٔ% ووهج اُخلٌي ُِحبت بِؾ )433اُزالد االوُى وطِج اُى ( واظهزث ححىٍ اُذواء اُى اُظٍـت اُالبِىرٌت اُالٓ٘خظٔت ٓغ ؿٍاب كزطت اُخذاخَ بٍٖ ٌٓىٗاث اُحبت ftirحوٍ٘ت االشؼت اُسٍٍ٘ت و) % 4.8%, ًالٌسٍٖ 6% ,ٓاٗخىٍ 6%, اُضٍالحٍٖ 6.5واُشاُخىبزوكٍٖ.ٗسخ٘خش ٖٓ اُذراست إ اُظٍـت اُخً ححخىي ػِى اُشاُخىبزوكٍٖ % ًاٗج اُظٍـت االكضَ .4.8 63وبىًُ كٍَ٘ باٌزوُذوٕ ى .03نتوبرفين ,تجفيف بانتجميد ,بوني فنيم بايروندين كزا انكهمات انمفتاحية: 1 corresponding author e-mail: suraymaster336@gmail.com received: 21/5/2017 accepted: 22/6/2017 mailto:suraymaster336@gmail.com iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 41 introduction a fast disintegrating or dissolving system or tablet can be defined as a solid dosage form that can disintegrate or dissolve within 30 seconds, in the oral cavity resulting in a solution or suspension without administration of water (1) .the technologies used for manufacturing fast dissolving tablets are freeze-drying, spray-drying, tablet molding, sublimation, tablet compression, and disintegration addition (2) . lyophilization (freeze-drying) is a process in which water is sublimated from the product after freezing at a specific temperature and pressure. lyophilization technique is used in order to improve the dissolution of the given substance and improve the oral bioavailability of the drugs with poor solubility and high permeability (3) . zaltoprofen is bcs class-ii having low solubility and high permeability (4) . zaltoprofen, chemically it is 2-(10,11-dihydro10-oxodibenzo(b, f)thiepin-2-yl) propionic acid, is a derivative of 2-arylpropionic acids(2apa), is one of non-steroidal antiinflammatory drugs (nsaids) and has potent inhibitory effects on acute and chronic inflammation, according to their chemical structure or their selective inhibition of cyclooxygenase cox-1 and cox-2, zaltoprofen is cox-2 inhibitor and selectively inhibits prostaglandin e2 (pge2) production at the sites of inflammation with less adverse reactions on the gastrointestinal tract than other nsaids (5) . figure (1): chemical structure of zaltoprofen (5) materials and methods: materials zaltoprofen was purchased from hyperchemical china, gelatin, was kindly gifted from baghdad college of pharmacy, mannitol, sorbitol ,glycine ,pvp k30 were purchased from m/s provizer pharma company india . the water used was distilled de-ionized water. all other chemicals were reagent grade. determination of melting point the melting point of zaltoprofen powder was measured according to usp method using capillary tube method using electrical melting point apparatus. the tube was dipped in the drug powder closed from one end and placed inside the melting point apparatus, the temperature was increased gradually. the temperature at which the powder liquefied was recorded as the melting point (6) . determination of absorption maxima (λ max) a solution of (10 μg/ml) of zaltoprofen in 0.1 n hcl (ph 1.2), phosphate buffer (ph 6.8), and distilled water were scanned by a uv spectrophotometer from 400-200nm, and λ max of zaltoprofen was indicated for each solution. preparation of standard curves of zaltoprofen: calibration curves of zaltoprofen were constructed in distilled water, 0.1n hc1 (ph 1.2), and phosphate buffer (ph 6.8), separately using uv spectrophotometer (uv-6100 pc). serial dilutions were prepared with different concentrations (4,8,10,12, and 14 μg/ml) from stock solution containing (20μg/ml) zaltoprofen in 0.1n hcl (ph 1.2), phosphate buffer (ph 6.8) and(2,4,5,8,10,12,14 µg/ml) from stock solution containing (20μg/ml) zaltoprofen in distilled water, samples were then analyzed spectrophotometerically for zaltoprofen at its λ max. the determined absorbance was recorded and plotted versus concentration to get a calibration curve. estimation of zaltoprofen saturation solubility solubility studies of zaltoprofen were carried out in distilled water; simulated gastric fluids (sgf) ph 1.2 and simulated intestinal fluids (sif) ph 6.8 were used to study solubility behavior of zaltoprofen. using shake -flask method .saturated solutions were prepared by adding excess of zaltoprofen to the all mentioned liquids in 10 ml tube which were placed in a shaker water bath at 40 rpm for 48 hours at 25 °c. then the samples were filtered through a 0.45 μm millipore filter. the solutions were diluted suitably, analyzed by uv-spectrophotometer at λ max of the drug. three determinations were carried out for each sample to calculate the solubility of zaltoprofen (7) . formulation of zaltoprofen fdts by lyophilization techniques in this study, zaltoprofen, fdts containing gelatin as matrix forming agent, glycine as collapse protectant, sorbitol and mannitol as bulk forming agent and pvpk30 as matrix supporting agent, were prepared by lyophilization technique according to formulae given in table 1.the percentage of excipients used was optimized during the formulation iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 42 process to obtain a withstand and elegant tablet that could be handled with ease. to prepare different batches, all ingredients according to the formula were accurately weighed. gelatin was first dissolved in distilled water at about 40°c stirred on a magnetic stirrer (stuart, u.k.) until a clear phase was obtained, then glycine, pvpk30, mannitol were added separately with continuous stirring until homogenous mixture was obtained then the required zaltoprofen amount was added exactly (0.5 ml) of the resultant solution was poured into each of the pockets of tablet blister pack .the tablets blister pack each contained 10 tablets , then they were then transferred to a freezer at about -22 °c and kept in the freezer over night until complete freezing was established .the frozen tablets were placed in a lyophilizer for 24hr utilizing a vacuum freeze dryer (techsupport, korea) with a condenser temperature of 45°c and pressure (8.6) pascal. the lyophilized tablets were kept away from moisture at room temperature until further investigation was performed (8) . table (1) :composition of different zaltoprofen lyophilized tablets formulas tablets formula material zlp ( g ) gelatin %w/v sorbitol %w/v mannitol %w/v glycine %w/v pvpk30 %w/v f1 3.2 2 f2 3.2 3 f3 3.2 4 f4 3.2 3 2 f5 3.2 3 3 f6 3.2 3 1 f7 3.2 3 2 f8 3.2 3 3 f9 3.2 3 3 1 f10 3.2 3 3 1.5 f11 3.2 3 3 2 f12 3.2 3 3 1.5 0.5 f13 3.2 3 3 1.5 1 f14 3.2 3 3 1.5 1.5 total to make 20ml preliminary screening for optimizing lyophilized tablets: the effect of concentration of matrix forming agent formulas 1-3 were used to study the effect of concentration of the matrix forming agent (gelatin 1%,2%,3%) on the mechanical properties and other parameters on the prepared tablets table 1. the effect of bulking agent type and concentration formulas 4-8 were used to study the effect of the bulking agent type and concentration (sorbitol 2%, 3% and mannitol 1%, 2%, 3%,) on the mechanical properties and other parameters on the prepared tablets table 1. the effect of collapse protectant concentration (glycine) formulas 9-11 were used to study the effect of collapse protectant concentration (glycine 1%, 1.5%, 2%) on the mechanical properties and other parameters on the prepared lyophilized tablets table 1. the effect of matrix supporting agent formulas 12-14 were used to study the effect of matrix supporting agent on the mechanical properties and other parameters on the prepared lyophilized tablets table1. compatibility study of drug with excipients fourier transform infrared (ftir) spectroscopy ftir spectroscopy analysis was done in the range of 4000-500cm -1 (fourier transform infrared system ftir8400 s shimadzu, japan) by mixing the optimum formula ( f14 ) and drug alone, separately with small amount of dry kbr powder , compressed into transparent disc and spectra was recorded .these spectra were determined to ensure there was no interaction between the drug and the excipients which may occur throughout the process (9) . powder x-ray diffraction (xrd) analysis x‐ray diffraction experiments were performed in an x‐ray diffractometer using xray powder diffraction analyzer (6000 xrd, shimadzu, japan), operated with cu k α x iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 43 radiation at 40 k v and 30 ma. the scans were conducted 5◦ to 35◦. diffraction patterns for zaltoprofen powder and for the optimized orally lyophilized tablet were obtained and identification of the samples was carried out (10) . evaluation of lyophilized tablets weight variation twenty zaltoprofen tablets were randomly taken from each batch and the weight of their average weight was determined. then individual tablet was taken and its weight was calculated. that individual weight was compared with average weight. the weights were measured using weighing balance (sartorius balance werke gmbh, germany) (11) . content uniformity three zaltoprofen lyophilized tablets were powdered weighed and transferred into a 100 ml volumetric flask. initially, 10 ml of methanol was added and shaken for 10 minutes. then, the volume was made up to 100 ml with buffer 6.8. subsequently, the solution in the volumetric flask was filtered, 1 ml of the filtrate was suitably diluted and analyzed for drug content at 243 nm using uvspectrophotometer (uv-6100 pc) (12) . in vivo disintegration test the time needed for each prepared tablet to be totally disintegrated in the oral cavity was estimated from six healthy people .all subjects had been notified about the purpose behind the test. the subjects washed their mouth with purified water with distilled water. tablets were put on the tongue and instantly the tongue softly moved and the interval for complete disintegration with -out residue was recorded (13) . in-vitro disintegration test disintegration time was measured in 900 ml artificial saliva (ph 6.8) according to the usp 24 method without disc at 37 ± 0.5°c temperature. the disintegration time of 6 individual tablets were recorded and the average disintegration time was reported (14) . in vitro dissolution test this test is intended to determine compliance with the dissolution request for solid dosage forms taken orally. in vitro dissolution studies of fd tablets were studied using usp xxiii tablet dissolution test apparatus employing a paddle stirrer. 900 ml of ph 6.8 phosphate buffer with 2% brij35 was used as a dissolution medium. the temperature of the dissolution medium is maintained to 37 ± 0.5 ° c .one tablet from each batch was used in each test. 5 ml of the sample of dissolution medium was withdrawn by means of pipette at known intervals of time and the sample was filtered using the whattman filter paper. the volume withdrawn at each interval was replaced with same quantity of fresh dissolution medium. each sample was analyzed for drug release, spectrophotometerically using uv-visible spectrophotometer (uv-6100 pc) after suitable dilutions (15) . results and discussion determination of melting point the measured melting point for zaltoprofen was found to be 139 °c. this result was the same to the data reported ,which reflects the purity of the powder used in the study (16) . determination of λ max of zaltoprofen: the uv scan (10 μg/ml) of zaltoprofen in three different media 0.1n hcl solution, phosphate buffer (ph 6.8) solutions, and distilled water as shown in the figures (1), (2), and (3), respectively in each media the drug showed maximum absorbance peaks at (243 nm) in the all above media as reported by the reference (17) . figure ( 1) uv scan of zaltoprofen in 0.1n hcl solution figure (2): uv scan of zaltoprofen in phosphate buffer (ph 6.8 ) solution iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 44 .. figure (3): uv scan of zaltoprofen in distilled water determination of calibration curves of zaltoprofen: figures ( 4 ), ( 5 ), and ( 6 ) show the calibration curves of zaltoprofen in 0.1n hcl, phosphate buffer (ph 6.8) and distilled water respectively. a straight line was obtained as a result of plotting the absorbance versus concentration. for each media which indicates that it follows beerlambert's law within the concentrations used (18) . figure (4): calibration curve of zaltoprofen in 0.1n hcl figure (5) :calibration curve of zaltoprofen in buffer 6.8 figure (6) :calibration curve of zaltoprofen in deionized water solubility studies the solubility profile of zaltoprofen was determined in various solvents as shown in table (2). the results show that zaltoprofen showed significantly (p˂0.05) higher solubility in phosphate buffer ph 6.8 than in 0.1 hcl and in dw .in addition to that it's known that zaltoprofen is acidic compound with pka of 5 (19) . therefore; the solubility of acidic compound increases as the ph is above pka value, this increase in ph may alter the ratio of ionized to non-ionized species which can be predicted by application of henderson-hassel balch equation (20) . table ( 2 ): solubility of zaltoprofen in various solvents solvents solubility (%w/w) mean ± s.d.* distilled water 0.01346 ± 0.0015 hcl 0.1 n 0.01216 ± 0.0015 buffer 6.8 3.1155 ± 0.264 *s.d. standard deviation from mean. n=3 the effect of concentration of matrix forming agent (gelatin) suitable selection of polymer is the corner stone to get a freeze dried tablets with acceptable mechanical properties and rapid release rates (21) .it was found that the prepared lyophilized tablets based on gelatin as binding agent showed better physical inspections compared with the tablets made from pvp k30 alone which were found to be unacceptable during the manual handling process. so, gelatin had been further optimized as matrix forming agent using 3% w/v with pvp k30 1.5%. in parallel to most of the other dosage forms, even in case of fast disintegrating tablets, the overall description of the mechanism of disintegration include iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 45 weakening of the intermolecular bonds upon penetration of the disintegration medium in the tablet’s excipients resulting in complete disintegration of the tablet (22) .with increase in gelatin concentration (f34%) ,the so formed network is anticipated to become more stable and extensive owing to increase in the fiber cross links and inter chain h-bonds (23) , so consolidated a pernicious effect on the disintegration time of the tablets due to increase in intermolecular attraction between the binder molecules resulting in retardation in disintegration time profile .from other side, beyond a certain concentration, the gelatin matrix becomes so extensive and synonymously less porous that the interaction with disintegration medium resulted in formation of thick cohesive gels that were difficult to disintegrate . this oversight could be more expressed by documented phenomenon of formation of rough three dimensional (3d) gel network of gelatin at high concentration (24) . the decrement in mechanical properties of fdts prepared with 2% gelatin (f1) could be assigned to the fewer number of crosslinks formed between the gelatin strands as the concentration decreases non porous tablet will form. the use of 3% w/w gelatin as a binder showed enhanced mechanical properties of the prepared tablets .thus, incorporation of matrix supporting agent pvp k30 was as a reasonable solution to improve dt as shown in table 3. the effect of type and concentration of bulking agent sorbitol used in (f4,f5) showed unsuitable visual inspection (shape) with hard surface texture of prepared tablets this may explain the combination of gelatin–mannitol was found to be better over gelatin–sorbitol (isomer of mannitol) in terms of tablet shape, appearance, surface texture and disintegration time and it could be attributed to superior hydrophilicity of mannitol over others(sorbitol) (25) . the formulas ( f6,f7,f8) were prepared with different mannitol concentrations ( 1% , 2% , 3%) , according to the study of their physical properties ,the results showed that (f8) have a good physical handling and appearance while (f6 ,f7) failed to pull off from blister packs because of fragility ,then after optimization applied on the (f8) to improve the dt and manual handling to progress forward until reaching a suitable formula . excipients used in lyophilization are materials used to ease freeze drying of various biological components, which are usually inert ingredients like sugars , they are also used to preserve the solid matrix against collapse .freezing system also influences the extent of crystallization and polymorphism of important formulation adjuvant drug, such as mannitol (26) . crystallization is favored in a formulation when mannitol functions as a bulking agent. mannitol can gain some advantages like lyophilization at high temperatures and thus shorten freeze drying cycles, and elegant product without defects caused by material collapse. mannitol used as a stabilizer leads to a system that behaves like a physical mixture, allowing interactions only at the phase boundary. bulking agents are used to give product elegance (i.e., acceptable appearance) as well as adequate cake, mechanical strength to avoid product blow-out. bulking agents simply function as fillers to increase the density of the product cake (27,28) . the effect of collapse protectant concentration (glycine) on the mechanical properties glycine has a polar surface and therefore has a high affinity to water which generate aqueous channels that make the diffusion of the dissolution media into the tablet more quickly ,which enhance the in vivo disintegration of the prepared tablets and subsequently promote the dissolution rate .these results are documented for some amino acids which act as disintegration accelerators .the addition of glycine especially at concentration 1.5% (f10) improved the wetting time in comparison with formula(f9,f11),this due to its polar surface free energy which composed of about 75 % of its component. the polar character of glycine has a power affinity to water and generate aqueous channels that enhancing the tablets wettability (29) . also glycine represents highly water soluble amino acid (25 g /100 ml), the solubility of amino acid in water affects the wetting of tablet, which increased the ability of water penetration into capillary of tablet matrix. a significant increase in dt as the concentration of glycine increased (direct relationship) (f11). so the increment in glycine concentration will deteriorate the dt (145 second) in comparison with formula of lower glycine concentration (f9,f10) 68, 37 seconds respectively, this due to low water holding capacity (lower and slower swelling nature of the powder) (30) . iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 46 table(3):physical appearance ,in vitro , in vivo d.t. and content uniformity for lyophilized prepared tablets formula pull off uniformity physical handling in-vitro d.t. in vivo d.t. content uniformity (%) f1 -ve -ve brittle f2 -ve +ve good 96.7±0.85 f3 -ve -ve hard f4 -ve -ve brittle f5 -ve -ve brittle f6 -ve +ve brittle f7 -ve +ve brittle f8 +ve +ve good 45 57 99.5±0.78 f9 +ve +ve accepted 68 79 97.7±0.84 f10 +ve +ve good 37 42 96.4±0.59 f11 +ve +ve hard 145 f12 +ve +ve good 21 26 99.8±1 f13 +ve +ve good 17 23 100.5±0.89 f14 +ve +ve good 9 12 100.1±0.97 note:pull off means remove the tablet from blister. +ve means easy to pull off the tablet from blister pack, uniform tablet shape. -ve means tablet stick to blister pack(difficult to pull off) , not uniform tablet shape. fourier transformed infrared spectroscopy (ftir) the most widely reported spectroscopic techniques for solid state characterization is ftir. the spectra of pure zaltoprofen powder shown in figure (7 ) and that of the selected lyophilized tablet (f14 ) spectra figure(8) display no significant shifting in the position of the characteristics peaks of the main functional groups ,so these results indicated the absence of the probabilities for interaction between the zaltoprofen and the polymers used in the preparation of lyophilized tablets (31) . figure (7) : ftir spectrum of pure zaltoprofen iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 47 figure (8) : ftir spectrum of lyophilized tablet (f14 ) powder x-ray diffraction analysis (pxrd) of pure zaltoprofen and selected formula (f14) the x-ray diffraction pattern of pure zaltoprofen powder shows characteristics zaltoprofen diffraction peaks at 2θ diffraction angles of 4°, 13.5°, and 20.2° referring to presence of crystalline structure as in figure (9). the diffraction study of optimized zaltoprofen oral lyophilized formula (f14) showed absence of main drug diffraction peaks at (4°, 13.5°, and 20.2° ) with decrement in the intensities of sharp peaks referring to present amorphous form as in figure (10 ) . figure ( 9 ) :x-ray diffraction pattern of pure zaltoprofen powder figure ( 10 ): x-ray diffraction pattern of selected formula (f14) iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 48 in-vitro dissolution study the in-vitro drug release characteristics of zaltoprofen lyophilized tablets (f14) were performed in phosphate buffer 6.8 (2% brij 35) as a medium for dissolution .the cumulative ratio of zaltoprofen release in 2 minute (d2) was 81.6% and t80% was 1.96 minute as shown in figure (11). figure ( 11 ) : in vitro drug release profile of( f14) in phosphate buffer ph 6.8 with 2% (brij 35) at 37 ± 0.5°c (results are expressed as mean , n=3) conclusion based on results obtained from the study one can conclude that, zaltoprofen was successfully formulated as lyophilized flash tablet using zydis technique by incorporation of gelatin 3% as a matrix forming agent which give a suitable manual handling, mannitol 3% was the best bulk forming agent compared with the others (sorbitol) ,so gelatin–mannitol combination offered proper manual handling and retained the intactness of odts . in addition to the polar character of glycine has a high affinity to water and generate aqueous channels that enhance the tablets wettability .from all above, f14 was the best formula to prepare zaltoprofen lyophilized tablet. references 1. abdelbary g, eouani c, prinderre p, joachim j, reynier j, piccerelle p. determination of the in vitro disintegration profile of rapidly disintegrating tablets and correlation with oral disintegration. international journal of pharmaceutics. 2005;29-41. 2. deshmukh keshav ram, patel vidyanand,varma shekhar,pandy alok kumar, dewangan pramod.a review on mouth dissolving tablet techniques.international journal of research in ayurveda & pharmacy.2011;66-76. 3. raguia ali shoukri a, iman saad ahmed b, rehab n. shamma .in vitro and in vivo evaluation of nimesulide lyophilized orally disintegrating tablets .a european journal of pharmaceutics and biopharmaceutics .2009;73:162–171. 4. k. chatap, gajanan m. marathe, abhishek r. maurya, nandkishor d. patil amruta papdiwal, vishal pande and kishor sagar .design and characterization of zaltoprofen nanosuspension by precipitation method .der pharma chemica.2014; 6(3):161-168. 5. pramod d. mahajan, dr. surajj m. sarode, prof. bhushankumar s. sathe, dr. parag v. jain, bharat. v. jain, dr. gautam.p. vadnere. formulation and evaluation of colon specific drug delivery system of zaltoprofen. world journal of pharmacy and pharmaceutical sciences. 2014; 3( 3): 933-952. 6. united states pharmacopoeia. the usp convention. 2007, cd. 7. prasanna ri, anitha p, chetty cm. formulation and evaluation of buccoadhesive tablets of sumatriptan succinate. international journal of pharmaceutical investigation. 2011;1(3):182-191. 8. raguia ali shoukri a, iman saad ahmed b, rehab n. shamma a. in vitro and in vivo evaluation of nimesulide lyophilized orally disintegrating tablets .journal of pharmaceutics and biopharmaceutics. 2009;73: 162–171. 9. vivek dave, renu bala yadav, richa ahuja, sachdev yadav.formulation design and optimization of novel fast dissolving tablet of chlorpheniramine maleate by using lyophilization techniques .bulletin of faculty of pharmacy, cairo university.2016. 10. roula safarab, wassim abdelwaheda, mustafa f. chehnaa , ghania degobertb, hatem fessib .preparation and characterization of new oral lyophilizates containing a non steroidal anti inflammatory drug . int j pharm pharm sci. 2011; 3( 3): 108-114. 11. a. kalia, s. khurana, n. bedi .formulation and evaluation of mouth dissolving tablets of oxcarbazepine. int. j. pharm. sci. 2009;1(1): 12–23. 12. kalpesh gaur a , lalit k. tyagi a, m. l. kori a, c. s. sharmab, r. k. nemac. formulation and characterization of fast disintegrating tablet of aceclofenac by using sublimation method . international journal of pharmaceutical sciences and drug research. 2011; 3(1): 19-22 13. tizkam hh, rassol aa, hussain aa. preparation of a new dosage form of metoclopramide hydrochloride as iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 49 orodispersible tablet . iraqi j pharm sci. 2009;18 (1):38-48. 14. c.p. jain1 and p.s. naruka . formulation and evaluation of fast dissolving tablets of valsartan .international journal of pharmacy and pharmaceutical sciences.2009;1(1). 15. sanjay k. sharma*, md. nawaz alam, manish jaimini, arindam chatterjee, shailender mohan .formulation and invitro evaluation of fast disintegrating tablets of zaltoprofen .international journal of current trends in pharmaceutical research ijctpr. .2014; 2(2): 391-399. 16. m. rahil g. bhura, patel niharikaben bhulabhai and dr. samir k. shah. comparative study of different solubility enhancement techniques on dissolution rate of zaltoprofen .world j pharm sci .2015; 3(8): 1706-1712 . 17. tank snehal k, sen dhanya b, sen ashim k, seth. ak.analytical method development and validation of zaltoprofen in bulk and pharmaceutical dosage form . sept2013 ;4 (4). 18. watson d g. pharmaceutical analysis. elsevier churchill livingstone 3rd edition; 2012: p.94. 19. dr. rakesh p. patel miss hirva a. shah .formulation and evaluation of chitosan based nano formulations of zaltoprofen .the faculty of pharmacy ,pharmaceutics and pharmaceutical technology , ganpat university, kherva. may, 2013. 20. po hn, senozan nm. the hendersonhasselbalch equation: its history and limitations. j. chem. educ. 2001;78(11):1499-1503. 21. low aqj, parmentier j, khong ym, et al. effect of type and ratio of solubilising polymer on characteristics of hot-melt extruded orodispersible films. int j pharm .2013;47:138–455. 22. alhusban f, perrie y, mohammed ar. formulation and characterization of lyophilized rapid disintegrating tablets using amino acids as matrix forming agents. eur j pharm biopharm .2010;75: 254–62. 23. akbari b, valaki b, maradiya v, et al. optimization of super disintegrants and subliming agent on dissolution rate of rosuvastatin orodispersible tablets by using a 32 factorial design .int j compare pharm. 2011;1:1–6. 24. basu b, bagadiya a, makwana s, et al. formulation and evaluation of fast dissolving table of cinnarazine using superdisintegrant blend and subliming agent.j adv pharm technol res .2011;2:266–73. 25. mizumoto t, masuda y, yamamoto t, et al. formulation design of a novel fastdisintegrating tablet . int j pharm .2005;306:83–90. 26. hsu, c.c., walsh, a.j., nguyen, h.m., overcashier, d.e.,koning-bastiaan, h., bailey, r.c., nail, s.l. design and application of a low-temperature peltiercooling microscope stage . journal of pharmaceutical sciences. 1996; 85 (1): 70-74 . 27. pikal, m.j. freeze drying . in encyclopedia of pharmaceutical technology, marcel dekker, new york.2002;1299-1326 . 28. tang, x., pikal, m.j. design of freezedrying processes for pharmaceuticals practical advice . pharmaceutical research .2004;21 (2):191-200 . 29. goel h, kaur g, tiwary ak, rana v. formulation development of stronger and quick disintegrating tablets. a crucial effect of chitin. the pharmaceutical society of japan. 2010;130(5):729-735. 30. dakhil ea, maraie nk. formulation and evaluation of trifluoperazine hydrochloride orodispersible tablets. international journal of pharmacy and pharmaceutical sciences.2014;6(1):294399. 31. tugba g, reyhan n, levent o. design and characterization of nanocrystal formulations containing ezetimibe .chem pharm bull .2011;59: 41-5 . v30n2salazar-et-al3.dvi revista colombiana de estadística volumen 30 no. 2. pp. 213 a 229. diciembre 2007 modelo de markov de tres estados: comparación de parametrizaciones de la tasa de intensidad de transición. aplicación a datos de artritis reumatoidea three state markov model: comparing three parameterizations of the transition intensity rate. application to rheumatoid arthritis data juan carlos salazar1,a, rené iral palomino1,b, enrique calvo5,c, adriana rojas2,d, maría eugenia hincapié2,e, juan manuel anaya2,3,4,f, francisco javier díaz1,g 1escuela de estadística, universidad nacional de colombia, medellín, colombia 2corporación para investigaciones biológicas, cib, medellín, colombia 3universidad del rosario, bogotá, colombia 4clínica universitaria bolivariana, medellín, colombia 5departamento imágenes diagnósticas, facultad de medicina, universidad nacional de colombia, bogotá, colombia resumen se considera un modelo múltiple de tres estados donde uno de ellos es absorbente. se asume que la dependencia entre las observaciones registradas para un mismo sujeto sigue un proceso de markov. se comparan, vía simulación, tres diferentes parametrizaciones de la tasa de intensidad de transición: la primera está basada en el modelo de hazard multiplicativo de andersengill (andersen et al. 1993), la segunda, en el modelo logístico, y la tercera depende del modelo log-log complementario. el método de estimación de parámetros se basa en la función de verosimilitud la cual se optimiza usando las soluciones exactas de un sistema de ecuaciones de kolmogorov hacia adelante junto con el algoritmo de newton-raphson (abramowitz & stegun 1972). usando el sesgo relativo, se selecciona el mejor método de parametrización y se ilustra usando datos recopilados en la corporación para investigaciones aprofesor asistente. e-mail: jcsalaza@unalmed.edu.co bprofesor asociado. e-mail: riral@unalmed.edu.co cprofesor asociado. e-mail: ecalvopa@yahoo.com dmédica reumatóloga. e-mail: arojas@cib.org.co einvestigadora asistente. e-mail: mehincapie@cib.org.co fprofesor adjunto. e-mail: janaya@cib.org.co gprofesor asociado. e-mail: fjdiaz@unalmed.edu.co 213 214 juan carlos salazar, et al. biológicas, cib1, acerca de pacientes con artritis reumatoidea. palabras clave: procesos estocásticos, tasas de intensidad, datos longitudinales, artritis reumatoidea. abstract we consider a three state model with an absorbing state assuming an underlying markov process to explain the dependence among observations within subjects. we compare, using a simulation study, three different parameterizations of the transition intensity rate: the first one is based on the andersen-gill’s multiplicative hazard model (andersen et al. 1993), the second one is based on the logistic model, and the third one depends on the complementary log-log model. the method to estimate the effect of the parameters is based on the likelihood function which can be optimized using the exact solutions of a kolmogorov forward differential equations system in conjunction with the newton-raphson algorithm (abramowitz & stegun 1972). we use the relative bias to select the best estimation estrategy. the methodology is ilustrated using longitudinal data about rheumatoid arthritis (ra) from the corporación para investigaciones biológicas, cib. key words: stochastic processes, intensity rates, longitudinal data, rheumatoid arthritis. 1. introducción los modelos de estados múltiples2 conforman una importante familia de herramientas estadísticas que son apropiadas para el análisis de datos longitudinales con respuesta categórica; por ejemplo, la progresión de una enfermedad incurable, tal como la enfermedad de alzheimer, artritis reumatoidea o la preferencia de un usuario de telefonía celular por un plan específico. los modelos de estados múltiples han recibido gran atención en la literatura en años recientes. con los notables avances en estadística, algunos autores han podido aplicar estos modelos en áreas tales como el estudio de datos longitudinales con valores perdidos y en el campo de los modelos lineales mixtos; ver por ejemplo, gao (2004), ten et al. (2000) y (aitkin & alfó 1998). este tipo de modelos de estados múltiples han tenido una exitosa acogida en campos de la ciencia tan diversos como biología, física, farmacia, epidemiología, ciencias sociales y medicina. este trabajo está motivado, básicamente, por los trabajos realizados por kay (1986), marshall et al. (1995), frydman (1995), joly & commenges (1999) y salazar et al. (2007). la información acerca de la progresión de un fenómeno, tal como una enfermedad incurable, usualmente se recolecta por medio de un proceso de mediciones repetidas tomadas en diferentes ocasiones en el tiempo (datos longitudinales, diggle et al. (2002)). con esto se busca registrar el cambio en el tiempo de una respuesta de interés. es por esto que determinar la función de intensidad de transición3 1www.cib.org.co/ 2también conocidos como modelos de compartimientos (jacquez 1972). 3conocida también como tasa de intensidad de transición. se define formalmente en la sección 2. revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 215 (bhat 1994) que se asocia con cada uno de estos cambios, resulta importante para entender e identificar que factores se relacionan con el riesgo que un paciente o una unidad experimental particular tiene de transitar a través de diferentes estados de un proceso. por tanto, es necesario contar con herramientas que permitan estimar estas tasas con un grado de precisión aceptable. puesto que la manera de formular las tasas de intensidad en términos de las covariables no es única, es pertinente preguntarse cuál de esas formas resulta más adecuada para el proceso de estimación. un proceso cuidadoso de identificación de factores de riesgo debe incluir modelos estadísticos que permitan detectar características que se relacionan con los cambios de estado que una persona puede experimentar en el tiempo y medir su grado de asociación con la respuesta de interés (ver woodward (1999)). adicionalmente, debe tener en cuenta el proceso de recolección de datos, la estructura de asociación de las medidas repetidas y la naturaleza de la respuesta. los modelos de markov basados en parametrizaciones de las tasas de intensidad son de gran utilidad ya que tienen en cuenta estos elementos con la ventaja adicional que permiten cuantificar el papel de las covariables en las distintas transiciones. debido a que existen diferentes métodos para incorporar el efecto de las covariables sobre las tasas de intensidad de transición en este tipo de modelos es importante identificar cuál de ellos proporciona respuestas más razonables. el interés específico de este trabajo es estudiar metodologías para evaluar las tasas de intensidad de transición cuando el fenómeno bajo estudio se puede idealizar por medio de un modelo de tres estados con un estado absorbente como el que se ilustra en la figura 1. figura 1: modelo de tres estados con un estado absorbente. como objetivo general se pretende evaluar diferentes metodologías para estimar el conjunto de tasas de intensidad de transición (por brevedad se denominarán de ahora en adelante tasas de intensidad) que gobiernan las transiciones de los sujetos dentro de un modelo múltiple de tres estados con un estado absorbente, y decidir cuál de ellas es más adecuada. específicamente, se quiere: 1. cuantificar el cambio, en términos del sesgo relativo, que experimentan los estimadores de máxima verosimilitud de las tasas de intensidad en presencia de distintas parametrizaciones. revista colombiana de estadística 30 (2007) 213–229 216 juan carlos salazar, et al. 2. identificar condiciones con las cuales se pueden obtener estimadores de máxima verosimilitud de las tasas de intensidad más precisos dentro del modelo múltiple de tres estados con un estado absorbente. este trabajo está organizado de la siguiente manera: en la sección 2 se presenta el modelo junto con el sistema de ecuaciones de kolmogorov hacia adelante; luego, en la sección 3, se detallan las distintas parametrizaciones que se van a comparar; posteriormente, en la sección 4, se exponen los tipos de datos junto con la función de verosimilitud. la sección 5 está dedicada a un estudio de simulación, mientras que en la sección 6 se ilustra el modelo usando datos longitudinales de artritis reumatoidea. finalmente, en la sección 7, se analizan los méritos y las limitaciones de los modelos. 2. el modelo considere un proceso estocástico {y (t) : t ≥ 0} con espacio de estados finito s = {1, 2, . . . , k}, (y (t) ∈ s con probabilidad 1). se asume que este proceso satisface la propiedad de markov, esto es: p ( y (tn) = in | y (t1) = i1, y (t2) = i2, . . . , y (tm) = im ) = p ( y (tn) = in | y (tm) = im ) donde t1 < t2 < · · · < tm < tn. esto implica que el proceso es homogéneo. adicionalmente, se asume que el estado k es absorbente y que t representa el tiempo transcurrido desde la primera visita. por ejemplo, en el modelo de tres estados, el espacio de estados es s = {1, 2, 3} y solo se admiten las transiciones 1 → 2, 1 → 3 y 2 → 3. los estados de este proceso se pueden describir por medio de una matriz de probabilidades de transición dependientes del tiempo (bhat 1994). suponga que se toman n historias independientes de este proceso y que cada una de ellas se organiza de acuerdo con su patrón de transición. este patrón de transición se observa a través de k estados previamente definidos y varía de historia a historia. el interés es estimar las tasas de intensidad asociadas con los distintos estados del proceso. formalmente hablando, una tasa de intensidad para una transición de un estado i a un estado j se define como: αij (t) = ĺım ∆t→0 p ( y (t + ∆t) = j | y (t) = i ) ∆t donde, i, j = 1, . . . , k y αij (t) ≥ 0, i, j = 1, . . . , k. asumir la propiedad de markov implica que las funciones de intensidad son funciones constantes del tiempo, y por esto resulta apropiado llamarlas tasas de intensidad. además, esta propiedad sirve para vincular en el modelo la correlación entre las mediciones repetidas de una misma unidad. la relación entre estas tasas revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 217 de intensidad y las probabilidades de transición se establece a partir de un sistema de ecuaciones de kolmogorov hacia adelante (bhat 1994), d dt p(t) = p(t)q, p(0) = ik+1, con q = [αij ] donde: p(t) es la matriz de probabilidades de transición, αij la tasa de intensidad del estado i al estado j y q la matriz de tasas de intensidad. cuando se considera el modelo de tres estados (ver figura 1) este sistema tiene una solución exacta dada por p11(t) = exp ( − (α12 + α13)t ) p12(t) = α12 α∗∗ [ 1 − exp(−α∗∗t) ] exp(−α23t) p13(t) = 1 − p11(t) − p12(t) p22(t) = exp(−α23t) p23(t) = 1 − exp(−α23t) α∗∗ = α12 + α13 − α23 en la práctica, las tasas de intensidad podrían estar influidas por covariables, y por esta razón se deben incorporar dentro del siguiente conjunto: a = {αi,j (θ; z) | i, j = 1, . . . , k} donde θ es un vector de parámetros y zt = [z1, . . . , zp] t es un vector de covariables. la forma en que estas covariables se vinculan con las tasas de intensidad no es única. el plan de trabajo consiste en evaluar, vía simulación, tres diferentes metodologías para estimar el conjunto de tasas de intensidad que gobiernan las transiciones de los sujetos de un estado a otro. el objetivo de este trabajo es evaluar cuál de esas tres parametrizaciones consideradas resulta ser la más recomendable. a continuación se detallan las parametrizaciones estudiadas. 3. parametrizaciones de la tasa de intensidad con el fin de estudiar el comportamiento de los estimadores de máxima verosimilitud de las tasas de intensidad se hace una comparación, por medio de un estudio de simulación, de tres diferentes parametrizaciones de la tasa de intensidad. modelo de andersen-gill: la primera parametrización está basada en el modelo de hazard multiplicativo de andersen-gill (andersen et al. 1993): αi,j (θ; z) = α ∗ ij e β t ij z aquí, α∗ij es un número positivo no especificado por estimar y βij es un vector que representa los efectos desconocidos de las covariables en una transición del estado i al estado j, i, j = 1, . . . , k. revista colombiana de estadística 30 (2007) 213–229 218 juan carlos salazar, et al. modelo logístico: la segunda parametrización está basada en la distribución logística y se define como: αi,j (θ; z) = α∗ij e β t ij z 1 + α∗ij e βt ij z donde α∗ij es un número positivo no especificado por estimar y βij es un vector que representa los efectos desconocidos de las covariables en una transición del estado i al estado j, i, j = 1, . . . , k. modelo log-log complementario: la tercera parametrización está basada en la transformación log-log complementario: αi,j (θ; z) = 1 − e −α ∗ ij e βt ij z donde α∗ij es un número positivo no especificado por estimar y βij es un vector que representa los efectos desconocidos de las covariables en una transición del estado i al estado j, i, j = 1, . . . , k. estos tres métodos se comparan con el método no paramétrico (naive) para obtener estimaciones de las funciones de intensidad descrito en kay (1986); básicamente, este método propone estimar las tasas de intensidad usando la siguiente relación: α̂ij = mij ti donde mij es el total de transiciones del estado i al j y ti es el total del tiempo que las unidades permanecieron en el estado i. 4. función de verosimilitud el método de estimación de las tasas de intensidad dentro de cada parametrización se basa en la función de verosimilitud. por tratarse de un proceso homogéneo en el tiempo, las probabilidades de transición de un estado a otro para los individuos solo dependen de la diferencia de tiempos entre visitas sucesivas. la tabla 1 muestra el esquema general de la información recopilada longitudinalmente para un individuo particular. tabla 1: esquema de recolección de datos para un participante con historia de transición: 1 → 2, 2 → 2, y 2 → 3. visita v1 v2 v3 v4 tiempos de transición t1 t2 t3 t4 estado 1 2 2 3 probabilidad de transición p12(t2 − t1) p22(t3 − t2) p23(t4 − t3) para este ejemplo, la contribución de este individuo a la verosimilitud será: p12(t2 − t1) ∗ p22(t3 − t2) ∗ p23(t4 − t3) revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 219 en general, si s (k) i representa el estado del k-ésimo participante en la i-ésima visita, n el número de participantes bajo estudio, mk número de visitas para el k-ésimo participante y p (k) si,si+1 la probabilidad de que el k-ésimo participante pase del estado si al estado si+1 en el intervalo de tiempo (ti, ti+1), entonces la contribución del k-ésimo participante a la verosimilitud está dada por mk∏ i=1 p (k) si,si+1 (ti+1 − ti) note que para obtener estimaciones de las tasas de intensidad en el modelo markoviano no es necesario conocer el momento exacto en el que el participante emigra de un estado a otro. la función de verosimilitud para los n participantes está dada por n∏ k=1 mk∏ i=1 p (k) si,si+1 (ti+1 − ti) para estimar las tasas de intensidad, es necesario derivar esta última función de verosimilitud con respecto a los parámetros del modelo. la dificultad radica en que es muy complicado obtener una solución explícita para la ecuación de verosimilitud. esto es especialmente cierto para modelos con más de tres estados, en donde la solución de las ecuaciones de kolmogorov hacia adelante se obtienen en términos de la descomposición espectral de la matriz de tasas de intensidad, la cual puede generar soluciones que no son números reales (ver kalbfleish et al. 1983). es por esto que se debe usar un método numérico en asocio con las ecuaciones diferenciales para obtener estas estimaciones. 5. estudio de simulación para llevar a cabo el estudio de simulación se asume un proceso estocástico de markov de tres estados que se denotarán 1, 2 y 3, y donde el estado 3 es absorbente; las únicas transiciones que se admiten son 1 → 2, 1 → 3 y 2 → 3. las condiciones en las cuales se ejecutaron las simulaciones se describen a continuación. primero, se simularon 1000 muestras de tamaños n = 200, 300, 400, 500 y 600 unidades, respectivamente, que contenían historias aleatorias de transiciones en el modelo de tres estados para los n participantes; luego para cada tamaño muestral se generaron un máximo de cinco o seis medidas repetidas por unidad y una covariable dicotómica, que para fines prácticos puede indicar la presencia o ausencia de una característica genética; la variable edad se incorpora en el modelo con tres categorías: ≤ 75 años, 75 − 85 años y ≥ 85 años. para calcular los valores de referencia se usaron estimaciones de las tasas de intensidad reportadas en un artículo de harezlak et al. (2003) obtenidas a partir de datos del indianapolis-ibadan dementia project (hendrie et al. 2001), el cual es un estudio longitudinal acerca del envejecimiento y demencia realizado con participantes de revista colombiana de estadística 30 (2007) 213–229 220 juan carlos salazar, et al. dos poblaciones ubicadas una en indianapolis, estados unidos, y la otra en nigeria, áfrica. estos valores se modificaron ligeramente para incorporar la dependencia de las transiciones en la variable dicotómica generada. cada ronda de simulación estimaba los efectos de las covariables y con estos se obtenían estimadores de α12, α13 y α23 para cada grupo de edad. todas las simulaciones se llevaron a cabo usando el software sas iml c© (sas institute inc 1990). el proceso de selección del mejor modelo de parametrización se basa en el sesgo relativo (sr), que se define como: sr = α̂ij − αij αij , i, j = 1, 2, 3 el modelo asociado con los menores sesgos relativos se considera el más recomendable para obtener los estimadores de máxima verosimilitud de las tasas de intensidad. en el proceso de simulación, la función de verosimilitud se optimizó iterativamente usando las soluciones exactas de un sistema de ecuaciones de kolmogorov hacia adelante (asociado con el modelo de tres estados) junto con el método de newton-raphson (abramowitz & stegun 1972). la tabla 2 muestra los resultados correspondientes a los tres métodos cuando se simuló con tamaños muestrales de 200, 300, 400, 500 y 600 y un número máximo de visitas de 5, mientras que la tabla 3 muestra los resultados con los mismos tamaños muestrales pero un número máximo de visitas por participante de 6. en ambas tablas se nota que el método no paramétrico (naive) está asociado con las peores estimaciones y que, consistentemente, tiende a subestimar el valor de referencia. también, el modelo de andersen-gill subestima consistentemente los valores de referencia, pero en general los sr están dentro de un rango razonablemente cercano a los valores de referencia. en la tabla 2 se observa una sobreestimación de la tasa α12, excepto para el método no paramétrico, en el grupo de edad de ≤ 75 años. a medida que se aumenta el tamaño muestral, las estimaciones de α12 se acercan más a los valores de referencia. con n = 200 y usando el modelo logístico se observa el sr más alto (0.758), mientras que el modelo de andersen-gill generó el sr más bajo (0.053). para el grupo de edad de 75 − 85 años también se registran valores estimados cercanos a los de referencia siendo el modelo logístico el que produce mejores estimaciones. en este mismo grupo y el de los ≥ 85 el desempeño del modelo logístico y el log-log complementario es mejor que el del modelo de andersen-gill. para el caso de los ≤ 75, los tres métodos producen estimaciones similares. en el grupo de edad de los ≥ 85 se observan estimaciones cercanas a los valores de referencia; de hecho, el sr oscila entre −0.292 y 0.136 con valores tan pequeños como 0.003 y 0.008. cuando el número de visitas máximo se incrementa a seis (ver tabla 3) y se aumentan los tamaños muestrales, se observa una mejora en las estimaciones, pero las tendencias son las mismas que las observadas en el caso de cinco visitas. revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 221 tabla 2: estimación (sesgo relativo). número máximo de visitas = 5. grupo de edad n método ≤ 75 75 − 85 ≥ 85 α12 α13 α23 α12 α13 α23 α12 α13 α23 200 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.015 0.043 0.054 0.031 0.062 0.068 0.040 0.087 0.083 (-0.201) (-0.146) (-0.315) (-0.401) (-0.201) (-0.459) (-0.660) (-0.333) (-0.599) andersen-gill 0.030 0.051 0.069 0.053 0.077 0.111 0.107 0.124 0.148 (0.589) (0.020) (-0.126) (0.033) (-0.014) (-0.114) (-0.101) (-0.050) (-0.287) logístico 0.033 0.056 0.076 0.058 0.088 0.128 0.120 0.148 0.175 (0.758) (0.123) (-0.034) (0.136) (0.128) (0.026) (0.008) (0.136) (-0.158) log-log 0.032 0.052 0.072 0.057 0.082 0.117 0.113 0.134 0.196 (0.695) (0.043) (-0.084) (0.110) (0.045) (-0.062) (-0.047) (0.033) (-0.059) 300 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.013 0.041 0.056 0.024 0.059 0.074 0.033 0.085 0.094 (-0.342) (-0.180) (-0.295) (-0.522) (-0.249) (-0.408) (-0.719) (-0.348) (-0.550) andersen-gill 0.025 0.047 0.072 0.045 0.072 0.110 0.093 0.116 0.161 (0.292) (-0.051) (-0.093) (-0.111) (-0.073) (-0.121) (-0.222) (-0.109) (-0.228) logístico 0.026 0.051 0.079 0.051 0.079 0.127 0.109 0.130 0.197 (0.384) (0.012) (-0.003) (-0.009) (0.008) (0.015) (-0.084) (0.003) (-0.055) log-log 0.026 0.049 0.074 0.048 0.076 0.117 0.100 0.124 0.189 (0.353) (-0.028) (-0.059) (-0.055) (-0.032) (-0.064) (-0.156) (-0.046) (-0.093) 400 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.012 0.041 0.057 0.022 0.058 0.075 0.030 0.085 0.098 (-0.392) (-0.189) (-0.283) (-0.571) (-0.251) (-0.398) (-0.749) (-0.350) (-0.531) andersen-gill 0.022 0.047 0.073 0.043 0.072 0.110 0.089 0.115 0.166 (0.178) (-0.067) (-0.072) (-0.160) (-0.082) (-0.117) (-0.248) (-0.118) (-0.203) logístico 0.024 0.049 0.080 0.046 0.078 0.126 0.098 0.129 0.201 (0.255) (-0.016) (0.016) (-0.099) (-0.004) (0.012) (-0.178) (-0.007) (-0.035) log-log 0.023 0.047 0.076 0.045 0.074 0.119 0.095 0.122 0.189 (0.232) (-0.063) (-0.033) (-0.116) (-0.052) (-0.049) (-0.201) (-0.063) (-0.092) 500 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.011 0.040 0.056 0.021 0.057 0.076 0.029 0.083 0.099 (-0.412) (-0.195) (-0.288) (-0.587) (-0.275) (-0.393) (-0.758) (-0.358) (-0.524) andersen-gill 0.021 0.046 0.072 0.041 0.070 0.109 0.084 0.111 0.166 (0.105) (-0.085) (-0.091) (-0.205) (-0.101) (-0.129) (-0.292) (-0.145) (-0.202) logístico 0.023 0.048 0.079 0.045 0.076 0.125 0.095 0.126 0.203 (0.186) (-0.048) (-0.005) (-0.125) (-0.028) (0.004) (-0.201) (-0.033) (-0.024) log-log 0.022 0.047 0.074 0.043 0.074 0.116 0.090 0.121 0.185 (0.144) (-0.063) (-0.065) (-0.162) (-0.054) (-0.071) (-0.244) (-0.072) (-0.110) 600 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.011 0.040 0.057 0.021 0.057 0.076 0.029 0.084 0.099 (-0.435) (-0.204) (-0.281) (-0.587) (-0.269) (-0.393) (-0.758) (-0.355) (-0.525) andersen-gill 0.020 0.045 0.073 0.040 0.070 0.109 0.085 0.114 0.165 (0.053) (-0.104) (-0.079) (-0.208) (-0.097) (-0.127) (-0.287) (-0.126) (-0.205) logístico 0.023 0.048 0.080 0.046 0.077 0.126 0.099 0.128 0.202 (0.186) (-0.038) (0.010) (-0.105) (-0.013) (0.007) (-0.164) (-0.018) (-0.031) log-log 0.021 0.046 0.075 0.042 0.074 0.116 0.090 0.121 0.184 (0.120) (-0.077) (-0.054) (-0.167) (-0.057) (-0.069) (-0.245) (-0.069) (-0.117) revista colombiana de estadística 30 (2007) 213–229 222 juan carlos salazar, et al. tabla 3: estimación (sesgo relativo). número máximo de visitas = 6. grupo de edad n método ≤ 75 75 − 85 ≥ 85 α12 α13 α23 α12 α13 α23 α12 α13 α23 200 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.015 0.044 0.056 0.031 0.064 0.072 0.038 0.088 0.090 (-0.235) (-0.128) (-0.288) (-0.389) (-0.181) (-0.421) (-0.682) (-0.320) (-0.566) andersen-gill 0.031 0.053 0.073 0.055 0.080 0.118 0.109 0.126 0.167 (0.616) (0.066) (-0.072) (0.070) (0.022) (-0.053) (-0.080) (-0.031) (-0.195) logístico 0.033 0.063 0.082 0.060 0.093 0.138 0.125 0.150 0.211 (0.741) (0.268) (0.038) (0.174) (0.196) (0.108) (0.049) (0.157) (0.013) log-log 0.033 0.056 0.076 0.058 0.086 0.126 0.117 0.141 0.217 (0.726) (0.116) (-0.037) (0.146) (0.106) (0.011) (-0.019) (0.088) (0.042) 300 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.013 0.041 0.058 0.024 0.060 0.076 0.032 0.087 0.099 (-0.340) (-0.178) (-0.267) (-0.521) (-0.226) (-0.391) (-0.734) (-0.332) (-0.525) andersen-gill 0.024 0.048 0.076 0.046 0.074 0.118 0.097 0.120 0.176 (0.269) (-0.047) (-0.042) (-0.096) (-0.054) (-0.059) (-0.182) (-0.080) (-0.153) logístico 0.026 0.051 0.083 0.050 0.081 0.135 0.108 0.136 0.215 (0.347) (0.023) (0.045) (-0.024) (0.042) (0.078) (-0.091) (0.049) (0.034) log-log 0.026 0.048 0.079 0.049 0.076 0.127 0.103 0.127 0.207 (0.355) (-0.040) (-0.003) (-0.037) (-0.025) (0.016) (-0.136) (-0.023) (-0.006) 400 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.012 0.041 0.058 0.022 0.060 0.078 0.030 0.085 0.101 (-0.371) (-0.183) (-0.264) (-0.559) (-0.235) (-0.374) (-0.748) (-0.343) (-0.512) andersen-gill 0.023 0.046 0.076 0.047 0.070 0.117 0.100 0.112 0.180 (0.225) (-0.080) (-0.040) (-0.085) (-0.097) (-0.068) (-0.156) (-0.138) (-0.134) logístico 0.024 0.049 0.082 0.048 0.078 0.133 0.106 0.129 0.216 (0.271) (-0.016) (0.042) (-0.052) (-0.005) (0.060) (-0.113) (-0.007) (0.036) log-log 0.024 0.047 0.078 0.047 0.074 0.124 0.102 0.123 0.201 (0.247) (-0.056) (-0.011) (-0.072) (-0.046) (-0.004) (-0.144) (-0.056) (-0.033) 500 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.011 0.041 0.059 0.022 0.059 0.079 0.029 0.086 0.102 (-0.396) (-0.180) (-0.257) (-0.578) (-0.238) (-0.371) (-0.755) (-0.341) (-0.510) andersen-gill 0.022 0.046 0.077 0.044 0.071 0.117 0.092 0.113 0.180 (0.159) (-0.082) (-0.026) (-0.142) (-0.090) (-0.061) (-0.223) (-0.129) (-0.132) logístico 0.024 0.050 0.085 0.047 0.079 0.136 0.102 0.130 0.220 (0.237) (-0.007) (0.070) (-0.076) (0.008) (0.085) (-0.142) (0.004) (0.059) log-log 0.023 0.046 0.080 0.047 0.073 0.126 0.101 0.120 0.203 (0.217) (-0.071) (0.010) (-0.081) (-0.059) (-0.010) (-0.154) (-0.074) (-0.023) 600 referencia 0.019 0.050 0.079 0.051 0.078 0.125 0.119 0.130 0.208 naive 0.011 0.041 0.058 0.021 0.059 0.079 0.029 0.085 0.102 (-0.404) (-0.181) (-0.264) (-0.583) (-0.245) (-0.369) (-0.754) (-0.345) (-0.508) andersen-gill 0.022 0.045 0.076 0.044 0.071 0.116 0.094 0.112 0.179 (0.155) (-0.090) (-0.033) (-0.135) (-0.095) (-0.069) (-0.209) (-0.135) (-0.138) logístico 0.024 0.048 0.083 0.049 0.077 0.135 0.108 0.127 0.222 (0.243) (-0.033) (0.049) (-0.044) (-0.015) (0.078) (-0.094) (-0.024) (0.065) log-log 0.023 0.047 0.079 0.046 0.074 0.126 0.100 0.122 0.202 (0.185) (-0.062) (-0.002) (-0.097) (-0.045) (0.005) (-0.160) (-0.065) (-0.026) revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 223 a medida que el tamaño muestral aumenta, se evidencia una reducción en los sesgos relativos de la función α12 para el grupo de edad de menores de 75 años. los sesgos relativos asociados con los otros grupos de edad y las otras funciones se mantienen en un rango razonablemente bajo (−29.2 % y 14.8 %). de acuerdo con este estudio de simulación, tamaños muestrales cercanos a 200 y un seguimiento de aproximadamente cinco visitas producen estimadores dentro de un rango razonablemente cercano a los valores de referencia. el desempeño de los tres métodos es similar en todas las combinaciones de tamaños muestrales y número de visitas. esto implica que cualquiera de ellos se puede recomendar en la práctica notándose una ligera ventaja en el modelo de andersen-gill por ser éste un estimador no acotado por encima; además, este método es más estable computacionalmente como se puede observar en las tablas 4 y 5 donde se reportan las tasas de convergencia de cada simulación asociadas a cada modelo. según este estudio de simulación, el cambio que experimentan los estimadores de máxima verosimilitud de las funciones de intensidad bajo distintas parametrizaciones no es muy significativo. tabla 4: tasas de convergencia asociadas a los tres métodos de estimación. número máximo de visitas = 5. tamaño muestral modelo 200 300 400 500 600 andersen-gill 97.1 % 99.4 % 99.5 % 99.6 % 99.6 % logístico 94.8 % 98.1 % 98.1 % 98.8 % 98.7 % log-log 75.1 % 90.5 % 95.0 % 97.5 % 97.7 % tabla 5: tasas de convergencia asociadas a los tres métodos de estimación. número máximo de visitas = 6. tamaño muestral modelo 200 300 400 500 600 andersen-gill 98.1 % 99.0 % 98.9 % 99.6 % 98.9 % logístico 96.1 % 97.3 % 98.1 % 99.1 % 97.5 % log-log 81.3 % 91.6 % 96.2 % 97.7 % 96.7 % 6. ejemplo: progresión radiográfica en artritis reumatoidea el registro radiográfico de daño en las articulaciones de manos y pies de un paciente con artritis reumatoidea (ar) es de considerable interés para entender esta enfermedad. puesto que los patrones de daño radiográfico varían dentro del mismo paciente y también de paciente a paciente, es razonable modelar esta evolución asumiendo la existencia de estados predefinidos a través de los cuales la enfermedad progresa. una pregunta de interés se relaciona con la identificación de revista colombiana de estadística 30 (2007) 213–229 224 juan carlos salazar, et al. factores que pueden incidir en el tránsito por los distintos estados de severidad de la enfermedad. los datos utilizados para ilustrar incluyen 464 radiografías de 146 pacientes diagnosticados con artritis reumatoidea, 84.9 % mujeres, edad promedio 47.1±13.4 años, un promedio de tres radiografías de manos y pies. la artritis reumatoidea (ar) es una enfermedad crónica autoinmune e inflamatoria que compromete las articulaciones que tienen movimiento (anaya et al. 2006). afecta principalmente a las mujeres entre la cuarta y quinta décadas de la vida. con frecuencia compromete otros órganos distintos a las articulaciones. dadas las características mencionadas, la ar tiene un impacto adverso en la esfera biopsicosocial y su costo es alto (anaya et al. 2006). estos pacientes fueron monitoreados durante un promedio de 4.2±1.6 años por el mismo equipo médico y siguiendo un régimen terapéutico estándar. las radiografías de cada paciente fueron leídas y evaluadas por dos profesionales calificados siguiendo el método de sharp van der heijde (van der heijde 1999). el objetivo consistía en registrar el promedio de erosiones en manos y pies. las lecturas se hicieron de manera independiente y ninguno de los dos lectores conocía el nombre del paciente al cual pertenecía una radiografía específica (ver figura 2). el acuerdo en sus lecturas fue evaluado posteriormente haciendo uso del coeficiente de correlación intraclase (icc) y se observó un acuerdo notable entre ambas lecturas; de hecho, el icc para las lecturas de manos fue de 0.95 mientras que para pies fue de 0.80. el puntaje de erosión, correspondiente al promedio de manos y pies de acuerdo al método de sharp van der heijde, fue categorizado en tres niveles de acuerdo con su severidad: 1 = leve (≤ 4), 2 = moderado (> 4 y ≤ 16) y 3 = severo (> 16). figura 2: radiografía de manos comparativas en proyección oblicua (1) y anteroposterior (2) con compromiso moderado, según puntaje sharp van der heijde. se destacan erosiones a nivel de las articulaciones metacarpofalángicas (flecha blanca). debido a la naturaleza progresiva de la enfermedad, la transición del estado leve al severo no es posible, y por tanto esta ilustración es un caso particular del modelo expuesto en el estudio de simulación (ver figura 1). treinta pacientes que fueron observados en el estado severo al momento de la primera radiografía fueron revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 225 excluidos del análisis ya que no aportaban información relevante para el proceso de estimaciones de las funciones de intensidad. para la estimación de las funciones de intensidad (αlm , αms ) se incluyó la covariable ausencia o presencia de la secuencia del share epitope (se): 0 = no, 1 = sí. esta característica genética ha sido reportada en la literatura como un factor de riesgo para la artritis reumatoidea (delgado et al. 2006). la historia familiar no se incluyó en el análisis puesto que se contaba con muy poca información. la función de verosimilitud se optimizó usando el algoritmo de newton-raphson implementado en el sas iml c© (sas institute inc 1990) usando las tres parametrizaciones. en la tabla 6 se observan los valores estimados de las funciones de intensidad para los grupos con presencia y ausencia de la secuencia genética del se. tabla 6: estimación de las funciones de intensidad (error estándar) αlm y αm s usando las tres parametrizaciones (n = 146). función de intensidad estimada ausencia de presencia de estadístico usando el modelo de andersen-gill se se z valor-p leve → m oderado, (bαlm ) 0.117 (0.028) 0.110 (0.029) −0.174 0.862 m oderado → severo, (bαm s ) 0.097 (0.042) 0.264 (0.078) 1.885 0.059 función de intensidad estimada ausencia de presencia de estadístico usando transformación logit se se z valor-p leve → m oderado, (bαlm ) 0.132 (0.028) 0.124 (0.029) −0.198 0.843 m oderado → severo, (bαm s ) 0.107 (0.043) 0.358 (0.122) 1.940 0.052 función de intensidad estimada ausencia de presencia de estadístico usando transformación log-log se se z valor-p leve → m oderado, (bαlm ) 0.124 (0.028) 0.117 (0.029) −0.174 0.862 m oderado → severo, (bαm s ) 0.102 (0.043) 0.307 (0.135) 1.447 0.148 la tabla 6 muestra los valores estimados de las tasas de intensidad junto con el valor del estadístico z y su correspondiente valor-p. los errores estándar se calcularon usando un método reportado en iral & salazar (2006) que está basado en el método delta. en el modelo de andersen-gill no se observa una diferencia significativa en las tasas de intensidad de leve a moderado en ausencia y presencia del se (valor-p = 0.862); sin embargo, se observa una diferencia importante en las tasas de intensidad de moderado a severo en ausencia y presencia del se (valor-p = 0.059). en el modelo logístico no se observa una diferencia significativa en las tasas de intensidad de leve a moderado en ausencia y presencia del se (valor-p = 0.843) pero el modelo sí detecta una diferencia importante en las tasas de intensidad de moderado a severo en ausencia y presencia del se (valor-p = 0.052). finalmente, en el modelo log-log complementario no se detecta ninguna diferencia importante entre las tasas de intensidad de leve a moderado y de moderado a severo (valor-p = 0.862 y valor-p = 0.148, respectivamente). según estos resultados, la influencia del se es más importante en transiciones del estado moderado a severo. revista colombiana de estadística 30 (2007) 213–229 226 juan carlos salazar, et al. 7. conclusiones y recomendaciones en este trabajo se ha discutido el problema de estimar las tasas de intensidad en un proceso de markov de múltiples estados al comparar tres métodos distintos para parametrizar la tasa de intensidad. por medio de un estudio de simulación intensivo se evaluó el comportamiento de cada una de estas parametrizaciones según diferentes especificaciones de tamaño muestral y número máximo de visitas. se recomienda la parametrización basada en el modelo de andersen-gill ya que no está acotada por encima y es más estable computacionalmente; esto la hace más apropiada en la práctica. sin embargo, las otras parametrizaciones se pueden usar en situaciones donde se sospecha que las tasas de intensidad son pequeñas. una de las desventajas más notables del modelamiento discutido en este trabajo es que requiere una gran cantidad de datos. otros autores han propuesto modelos que tienen en cuenta censura arbitraria, pero la complejidad en su implementación los hacen poco prácticos (joly & commenges 1999). no obstante, para respuestas categóricas recolectadas longitudinalmente este tipo de modelamiento ha demostrado efectividad siempre y cuando se cuente con un número apropiado de datos y visitas. agradecimientos agradecimiento especial a todos los pacientes que hicieron parte del estudio radiográfico sobre progresión en artritis reumatoidea. su aporte es esencial para avanzar en el entendimiento de este mal que afecta a una cantidad considerable de personas en la actualidad. proyecto patrocinado por unc-dime número 30802921. recibido: mayo de 2007 aceptado: octubre de 2007 referencias abramowitz, m. & stegun, i. a. (1972), handbook of mathematical functions with formulas, graphs, and mathematical tables, dover publications, inc., new york. aitkin, m. & alfó, m. (1998), ‘regression models for binary longitudinal responses’, statistics and computing 8, 289–307. anaya, j. m., pineda, r., gómez, l. m., galarza, c., rojas, a. & martín, j. (2006), artritis reumatoide: bases moleculares, clínicas y terapéuticas, cib, universidad del rosario, funpar, medellín, colombia. andersen, p. k., borgan, o., gill, r. d. & keiding, n. (1993), statistical models based on counting processes, springer-verlag, new york. revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 227 bhat, u. n. (1994), elements of applied stochastic processes, second edn, wiley, new york. delgado, a. m., martín, j., granados, j. & anaya, j. m. (2006), ‘epidemiología genética de la artritis reumatoide: ¿qué esperar en américa latina?’, biomédica 26, 562–584. diggle, p., heagerty, p., liang, k. y. & zeger, s. (2002), analysis of longitudinal data, oxford statistical science series 25, second edn, oxford university press inc., new york. frydman, h. (1995), ‘semiparametric estimation in a three-state duration dependent markov model from interval-censored observations with application to aids data’, biometrics 51, 502–511. gao, s. (2004), ‘a shared random effect parameter approach for longitudinal dementia data with non-ignorable missing data’, statist. med. 23, 211–219. harezlak, j., gao, s. & l., h. s. (2003), ‘an illness-death scholastic model in the analysis of longitudinal dementia data’, statistics in medicine 22, 1465– 1475. hendrie, h. c., ogunniyi, a., hall, k. s., baiyewu, o., unverzagt, f. w. & gureje, o. (2001), ‘incidence of dementia and alzheimer disease in 2 communities: yoruba residing in ibadan, nigeria, and african americans residing in indianapolis’, jama 285(6), 739–747. iral, r. & salazar, j. c. (2006), efecto de las covariables en la estimación de intervalos de confianza para las tasas de transición en un modelo de markov de tres estados, in ‘memorias xvi simposio de estadística 2006: estadística en la industria. iii encuentro colombia-venezuela de estadística’, universidad nacional de colombia, bucaramanga, colombia. jacquez, j. a. (1972), compartmental analysis in biology and medicine: kinetics of distribution of tracer-labeled materials, elsevier pub. co., amsterdam, new york. joly, p. & commenges, d. (1999), ‘a penalized likelihood approach for a progressive three-state model with censored and truncated data: application to aids’, biometrics 55, 887–890. kalbfleish, j. d., lawless, j. f. & vollmer, w. m. (1983), ‘estimation in markov models from aggregate data’, biometrics 39, 907–919. kay, r. (1986), ‘a markov model for analyzing cancer markers and disease states in survival studies’, biometrics 42(4), 855–865. marshall, g., guo, w. & jones, r. h. (1995), ‘markov: a computer program for multi-state markov models with covariables’, computer methods and programs in biomedicine 47, 147–156. revista colombiana de estadística 30 (2007) 213–229 228 juan carlos salazar, et al. salazar, j. c., schmitt, f. a., yu, l., mendiondo, m. & kryscio, r. j. (2007), ‘shared random effects analysis of multi-state markov models: application to a longitudinal study of transitions to dementia’, statist. med. 26, 568– 580. sas institute inc (1990), sas/iml software: usage and reference, version 6, 1st edn, sas institute inc., cary, nc. ten, t. r., miller, m. e., reboussin, b. a. & james, m. k. (2000), ‘mixed effects logistic regression models for longitudinal ordinal functional response data with multiple-cause drop-out from the longitudinal study of aging’, biometrics 56, 279–287. van der heijde, d. (1999), ‘how to read radiographs according to the sharp/van der heijde method’, j rheumatol 26, 743–745. woodward, m. (1999), epidemiology: study design and data analysis, chapman and hall/crc, new york. apéndice a. solución del sistema de ecuaciones de kolmogorov hacia adelante en el modelo de tres estados en el modelo de tres estados, el sistema de ecuaciones de kolmogorov hacia adelante resultante es: p ′ 11(t) = −(λ12 + λ13)p11(t) p ′ 12(t) = λ12p11(t) − λ23p12(t) p ′ 13(t) = λ13p11(t) + λ23p12(t) p ′ 22(t) = −λ23p22(t) p ′ 23(t) = λ23p22(t) = −λ23p23(t) λ∗∗ = λ12 + λ13 − λ23 para la primera ecuación, observe que: p ′ 11(t) = −(λ12 + λ13)p11(t) ⇐⇒ p′11(t) p11(t) = exp ( − (λ12 + λ13)t ) integrando a ambos lados respecto a t se tiene: log p11(t) = c − (λ12 + λ13)t ⇐⇒ p11(t) = exp(c) exp ( − (λ12 + λ13)t ) por la condición inicial, se tiene que p11(t) = 1 y así c = 0. con esto la solución a esta primera ecuación es p11(t) = exp ( − (λ12 + λ13)t ) . para la segunda ecuación observe que: p ′ 12(t) = λ12p11(t) − λ23p12(t) ⇐⇒ p ′ 12(t) + λ23p12(t) = λ12p11(t) revista colombiana de estadística 30 (2007) 213–229 modelo de markov de tres estados 229 multiplicando por exp(λ23t) se tiene que exp(λ23t)p ′ 12(t) + λ23 exp(λ23t)p12(t) = λ12 exp(λ23t)p11(t) ⇐⇒ d dt [ exp(λ23t)p12(t) ] = λ12 exp(λ23t)p11(t) remplazando p11(t) e integrando respecto a t en ambos lados se tiene que: exp(λ23t)p12(t) = − λ12 λ∗∗ exp(−λ∗∗t) + c, con λ∗∗ = λ12 + λ13 − λ23 como p12(t) = 0, entonces exp(λ23t)p12(t) = λ12 λ∗∗ [ 1−exp(−λ∗∗t) ] ⇐⇒ p12(t) = λ12 λ∗∗ [ 1−exp(−λ∗∗t) ] exp(−λ23t) dado que p11(t) + p12(t) + p13(t) = 1, entonces p13(t) = 1 − p11(t) − p12(t). para la cuarta ecuación, se tiene que: p ′ 22(t) = −λ23p22(t) ⇐⇒ p22(t) = exp(c) exp(−λ23t) por la condición inicial p22(0) = 1, con esto se obtiene que p22(t) = exp(−λ23t) como p22(t) + p23(t) = 1, entonces p23(t) = 1 − p22(t) = 1 − exp(−λ23t). revista colombiana de estadística 30 (2007) 213–229 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 74 design, synthesis, characterization and preliminary anticancer study for methotrexate silibinin conjugates shams a. nadhum * and mohammed h. mohammed *,1 * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract the spectrum of clinical efficacy of methotrexate (mtx) is broad in that mtx is used in the treatment of certain cancers, severe psoriasis and rheumatoid arthritis.various mechanisms by which cancer cells grown in tissue culture become resistant to anticancer drugs. the use of multiple drugs with different mechanisms of entry into cells and different cellular targets allows for effective chemotherapy and high cure rates. in an efforts to develop effective strategies that increase the therapeutic potential of anticancer drugs with less systemic toxicity ,are being directed towards the investigation of dietary supplements and other phytotherapeutic agents for their synergistic efficacy in combination with anticancer drugs. a promising approach to improve the cancer cell selectivity of methotrexate is the chemical transformation into reversible derivatives which convert the conjugate to the parent drug by virtue of enzyme within cancer tissue. the present study includes the synthesis of two derivatives of methotrexate which are :-schiff base methotrexate-silibinin conjugate ( compound 5), and methotrexate-silibinin conjugate (compound 6).the synthesis of the target compounds was accomplished following multistep reaction procedures. the chemical reactions were followed up and purity of the products was checked by tlc. the structures of the final compounds and their intermediates were characterized and identified by their melting points, infrared spectroscopy, 1 hnmr and elemental microanalysis(c h n s).the anticancer activity of these compounds was investigated by hep-2 cell line(larynx carcinoma), which showed that compounds 5 and 6 have the higher activity than methotrexate or silibinin alone.these are promising data for the discovery of new anticancer agents in future. these compounds may deliver the parent drug selectively into the cancer cells to be hydrolyzed by enzymes that are elevated in tumor tissues compared with normal tissues . keywords: methotrexate, silibinin, cancer treatment resistance, folate receptor, cancer cell targeting. جصميم وجصىيع وجشخيص ودراسة اولية لمضادية السرطان لمقحرن سليبيىيه –ميثىجركسيث واظم شمس عىاد * و محمذ حسه محمذ *،1 .تغذاد، اٌعشاق ،تغذاد اٌصُذٌح، خاِعح اٌصُذالُٔح، وٍُحفشع اٌىُُّاء * الخالصة فٍ عالج أىاع ِٓ اِشاض اٌسشطاْ،واٌصذفُح واٌرهاب السرعّاٌها ذرُّز ِادج اٌّثىذشَىسُد تطُف ِٓ اٌفعاٌُح اٌسشَشَح ورٌه ج اٌّفاصً اٌشوِاذُذٌ. هٕان عذج اٌُاخ اٌرٍ تّىخثها ذّٕى اٌخالَا اٌسشطأُح فٍ اٌزساعح إٌسُدُح واٌرٍ ذىىْ ِماوِح ٌالدوَح اٌّضاد ِخرٍفح َسّح ٌعالج وُُّائٍ ِؤثش وِعذالخ ادوَح ِرعذدج ِع اٌُاخ ِخرٍفح ٌٍذخىي اًٌ اٌخالَا واهذاف خٍىَح اسرعّايٌٍسشطاْ. اْ وٌغشض ذطىَش سرشاذُدُاخ فعاٌح فاْ خهىدا ذثزي ٌزَادج اٌفعاٌُح اٌعالخُح ٌالدوَح اٌّضادج ٌٍسشطاْ ِع الً سُّح ِٓ شفاء عاٌُح تاالذحاد ِع فعاٌُح اوثش ٌرصثح راخخالي ذىخُهها ٔاحُح اٌرحشٌ عٓ اٌّىّالخ اٌغزائُح وعىاًِ االدوَح إٌثاذُح )االعشاب اٌطثُح( االدوَح اٌّضادج ٌٍسشطاْ.وِٓ االهذاف اٌىاعذج ٌرحسُٓ أرمائُح اٌخالَا اٌسشطأُح ذداٖ عماس اٌُّثىذشَىسُد هى اٌرحىي اٌىُُّائٍ ك ِشرمُٓ ٌٍّمذِاخ اٌذوائُح اًٌ اٌعماس االَ )االصً( تحىُ االٔزَُ اٌّىخىد فٍ إٌسُح اٌسشطأٍ. ذرضّٓ اٌذساسح اٌحاٌُح ذخٍُ ٌٍّثىذشَىسُد وهّا : (.5سٍُثُٕٓ )ِشوة –لاعذج شف ي ِمرشْ اٌّثىذشَىسُد (.6سٍُثُٕٓ )ِشوة –ِمرشْ ُِثىذشَىسُد ذمُٕح عّاياٌّشوثاخ اٌرٍ َهذف اٌُها اٌثحث تاذثاع طشائك ِرعذدج اٌخطىاخ وذُ اٌراوذ ِٓ ٔماوج هزٖ اٌّشوثاخ ورٌه تاسر ذحضُشذُ وشوِاذىغشافُااٌصفائح اٌشلُمح.وزٌه ذُ ذشخُص وذىصُف إٌرىاذح إٌهائُح وِىادها اٌىسطُح ِٓ خالي ذعُُٓ دسخاخ أصهاسها واطُاف االشعح ذحد اٌحّشاء وطُف اٌشُٔٓ إٌىوٌ اٌّغٕاطُسٍ ٌٍثشذىْ،واٌرحًٍُ اٌذلُك ٌٍعٕاصش. وهٍ خالَا سشطاْ 2اٌّشوثاخ تاسرخذاَ اٌخٍُح اٌسشطأُح اي هة ٌمذ اثثرد دساسح اٌفعاٌُح اٌّضادج ٌٍسشطاْ ٌهزٖ َّرٍىىْ فعاٌُح اعًٍ ِٓ اٌُّثىذشَىسُد واٌسٍثُُٕٓ و هزٖ إٌرائح ذعرثش واعذج 6واٌّشوة 5اٌحٕدشج واٌرٍ اظهشخ تاْ اٌّشوة عالٖ َرضح تاْ هزٖ اٌّشوثاخ ٌها اٌمذسج عًٍ اَصاي الورشاف ِضاداخ سشطأُح خذَذج فٍ اٌّسرمثً. ووفما ٌهزٖ إٌرائح اٌّثُٕح ا سرىي عاٌٍ فٍ االٔسدح اٌسشطأُح ِماسٔح ِع ّاالدوَح تأرمائىح ٌٍخالَا اٌسشطأُح وتاٌُح ذحشَش ذرضّٓ ذاثُش االٔزَّاخ اٌّىخىدج ت .االٔسدح اٌطثُعُح ةالت الفىليث،اسحهذاف الخاليا السرطاويالكلمات المفحاحية:ميثىجركسيث ،سيلبىيه،مقاومة عالج السرطان،مسحقب 1 corresponding author e-mail: dr.mhassanm666666@yahoo.com received: 17 /1/2015 accepted: 23/5/2015 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 75 introduction the design of cancer chemotherapy has become increasingly sophisticated,yet there is no cancer treatment that is 100% effective against disseminated cancer. resistance to treatment with anticancer drugs results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumors, even those from the same tissue of origin (1) . researchers have been tabulating the various mechanisms by which cancer cells grown in tissue culture become resistant to anticancer drugs. some of these mechanisms, such as loss of a cell surface receptor or transporter for a drug, specific metabolism of a drug, or alteration by mutation of the specific target of a drug, all of which occur for antifolates such as methotrexate (2) . mtx competitively inhibits dihydrofolate reductase (3) . folate receptor a (fra) is a membrane-bound protein with high affinity for binding and transporting physiologic levels of folate into cells. folate is a basic component of cell metabolism and dna synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain dna synthesis, an observation supported by the wide spread use of antifolates in cancer chemotherapy (4) . endocytosis is the general mechanism of ( fra) mediated folate uptake. one specialized route, potocytosis , was proposed from the observation that( fra) recycled between an acid-resistant (intracellular) and acid-sensitive (extracellular) pool. proposed that (fra )was concentrated in clusters in invaginations of the cell membrane surface called caveolae, whereby the membrane would transiently close and internalize the folate-bound receptor complex. increased acidification of the internal compartment would dissociate folate from the receptor and move it across the membrane into the cytoplasm of the cell using the energy generated by the acidic gradient (5) . folate receptor-targeted cancer therapies constitute a promising treatment for the approximately one third of human cancers that over express the folate receptor (fr). however, the potencies of all folate-receptor targeted therapies depend on the rate of folate-linked drug conjugate binding to the cancer cell surface, the dose of folate conjugate that will saturate tumor cell surface fr in vivo, the rate of fr internalization, un loading,and recycling back to the tumor cell surface for another round of conjugate uptake, and the residence time of the folate conjugate before its metabolism or release from the cell (6) .linkage of a drug to folic acid can generate a molecular “trojan horse” that can enable tumor-specific delivery of both imaging and therapeutic agents into cancer cells. molecules that have been targeted to tumors by conjugation to folic acid include radiopharmaceuticals (7) , enzymes for prodrug activation (8) ,nanoparticles (9) , peptides, toxic proteins (10) , immunologically potent haptens (11) , antisense oligonucleotides (12) , chemotherapeutic agent , gene therapy vectors (13) , viruses (14) ,and polymeric drug carriers and liposomes (15) ,for example conjugation of methotrexate to poly(l-lysine) increases drug transport and overcomes drug resistance (16) and conjugation of methotrexate with 5fluorouracil (17) . in an effort to develop effective strategies that increase the therapeutic potential of anticancer drugs with less systemic toxicity, more strategies are being directed towards the investigation of dietary supplements and other phytotherapeutic agents (18) .silibinin has shown promising chemo preventive and anticancer effects in various in vitro and in vivo studies (19) .several studies have shown that silibinin and silymarin have anticancer activity against breast, skin, androgen-dependent and independent prostate, cervical, bladder, hepatocellular,colon, ovarian, and lung cancer cells in culture and several in vivo animal cancer model systems (20) . the efficacy of silibinin in inhibiting the growth of different cancer lines is quite different. such differences in the potency of the drug in arresting cell growth may be due to differences in the experimental conditions used, the cell type and potential carcinogenicity of the cell lines (21) . in view of this observation, two derivatives of methotrexate are designed, synthesized and characterized. experimental section general chemicals used in the synthesis were of analytical grade(methotrexate and p-nitro phenyl chloroformate from(hongmao,china)and silibinin(tolbiac s.r.l ,argentina). the melting points of the compounds and their intermediates (uncorrected) were determined by capillary tube method on barnstead electrothermal (usa) and ft-ir spectra were recorded on ft-ir spectrophotometer shimadzu (japan) at the college of pharmacy /university of baghdad. the chns analysis was carried out using euro-vector ea3000a(italy).ascending thin layer chromatography(tlc)was run on silica gel 60 f254 pre-coated aluminum sheets, merck (germany) to check the purity and the reactions progress, 1 h-nmr analysis was performed on 1h-nmr spectroscopy (shimadzu,japan) at university of al-bayt,jordan, and the iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 76 chromatograms were eluted by three solvent systems: a-aceton:chloroform (3:1). baceton:chloroform :acetic acid(3:1: 0.5) (22). the evaluation of cytotoxic activity was done at al-naharin biotechnology research center using rosswell park memorial institute (rpmi1640)which consists from hepes buffer and l-glutamin( sodium bicarbonate ,streptomycin ,benzyl penicillin g,mycostatin ,fetal calf serum) . chemistry the synthetic procedures for the designed target compounds 5 and 6 are illustrated in schemes (1 and 2) . the starting material for the generation of the target compounds 5 and 6 was methotrexate which was refluxed with 2 equivalent benzaldehyde in the presence of 10% sodium hydroxide to give the imines derivative of methotrexate (compound 2). compound 2 was converted to acylchloride derivative by useing thionyl chloride and triethylamine in which one carboxylate group was blocked. the acylchloride derivative was allowed to react with nh2 group of dimethyl ester derivative of cystine in the presence of triethylamine to give the imine-methotrexate cystine derivative (compound 3) which was reacted with paranitrophenylchloroformate to give compound 4. then (compound 4 ) was stirred with silibinin in the presence of sodium hydroxide to give iminemethotrexate silibinin conjugate (compound 5).the methotrexate silibinin conjugate (compound 6 )was obtained by hydrolysis of compound 5.the physical properties of the synthesized compounds are showed in table (1). the structures of the final compounds were characterized by elemental microanalysis (table2) .infrared spectroscopy (table3) and 1 hnmr done for compound 5(table4). methanol socl2 s s oo oo o n n n nh2n nh2 n o h n oh o oho compound1 10% naoh n n n n n nn n h ho o oho o compound 2 2 reflux methotrexatebenzyldehyde l-cystine (sch2ch(nh2)co2h)2 nh3cl nh3cl scheme (1): synthesis of compounds 1and 2. nh3cl nh3cl socl2 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 77 n n n n n nn n h h n o oho o s o o so o hn o o n o o compound 4 n n n n n nn n h ho o oho o s s oo nh3cl nh3cl oo n n n n n nn n h h n o oho o s o o so o compound 3 compound 2 10°c /tea socl2 / -15 °c stirring/tea 1. 2. + reflux o c cl o no2 p-nitrophenylchloroformate nh2 socl2 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 78 silibinin 1n hcl compound 5 2. 5%hcl 1. 5%naoh acetonitrile schiff base hydrolysis compound 6 scheme( 2): synthesis of compounds 3 – 6 . chemical methods: synthesis of 3,3'-disulfanediylbis(1-methoxy1-oxopropan-2-aminium) chloride.(compound 1). a suspension of cystine (41.6mmole,10g) in (150 ml)of absolute methanol,was cooled to -15 ᵒ c then thionyl chloride(99.8 mmole,7.4 ml) was added drop wise ,(the temperature should be keep below -10 ᵒ c) and the reaction mixture was left at 40 ᵒ c for 1hr,then reflux for 4 hr and left at room temperture over night,the excess solvent was evaporated to dryness under vacuum;recrystallize the product from methanol-diethyl ether and collected as hcl salt (23) . synthesis of 2 ( 4 ( ( ( 2 , 4 -bis ( benzylideneamino ) pteridin 6 -yl) methyl ) (methyl) amino) benzamido) pentanedioic acid.(compound2). in 500 ml round bottom flask attached with a reflux condenser, mixture of benzaldehyde (44 mmole,4.47 ml ), methotrexate (11mmole,5 g), and 10% sodium hydroxide (22mmole,0.88 g). add 200 ml of absolute ethanol to the mixture and the final mixture is refluxed for 3 hours, cooled and acidified to ph 5with 5% hcl . the mixture was filtered,the product was washed with cold ethanol and dried (24) . 1n hcl hcl iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 79 synthesis of 4 [ 2 ( 2 amino 2 methoxycarbonyl ethyldisulfanyl ) 1 methoxycarbonyl ethylcarbamoyl ] 2 (4 { [ 2 , 4 bis ( benzylidene amino ) pteridin 6 ylmethyl-methyl amino } benzoylamino ) buty ric acid .(compound 3). a suspension of compound 2 (7.93mmol,5 g) and triethylamine (7.93mmole ,1.10ml ) in acetonitrile (50ml),was coolded down to-15 ᵒ c then thionyl chloride ( 7.93mmol,0.59ml) was added slowly, and the mixture was refluxed for 4hr at( 60-70) ᵒ c with continuous stirring.then evaporate the excess solvent by using rotary evaporater .the viscous liquid was slowly added drop wise into beaker contain compound 1(7.93mmol,2.7g) and triethylamine (15.86mmol, 2.2ml) dissolved in acetonitrile(50ml) for 1 hr in an ice bath ,then stirring over night at room temperature. the obtained suspension was filtered and the filterate was washed with distilled water (20ml),dried over anhydrous magnesium sulfate and the solvent was evaporated by vacuum. the obtained compound was recrystallized from diethyl ether-petroleum ether(4:6)to give compound 3. (25) synthesis of 2-(4-(((2,4bis(benzylideneamino)pteridin-6yl)methyl)(methyl) amino)benzamido)-5-((1methoxy-3-((3-methoxy-2-(((4nitrophenoxy)carbonyl) amino)-3oxopropyldisulfanyl)-1-oxopropan-2yl)amino)-5-oxopentanoic acid.(compound 4). to a stirred solution of compound 3 ( 2.17 mmol,2 g) in toluene(100ml) at -10°c was added triethylamine ( 2.17 mmol,0.61ml). after 5 min, a cooled mixture of paranitrophenylchloroformate (2.17 mmol,0.443 g) in toluene (5 ml)was added drop wise . the mixture was stirred for 2 hr at 10°c and for 15 hr at room temperature, filtered,and evaporated to dryness under reduced pressure.the residual oil was crystallized twice from diethyl ether (150ml) to give a yellow powder (26) . synthesis of15-(4-(((4-benzylideneamino)-2((z)-benzylidene amino ) pteridin 6 -yl ) methyl ) ( methyl ) amino )phenyl ) 1 (4-(3(hydroxylmethyl)-7-(3,5,7-trihydroxy-4oxochroman 2 yl ) -2 , 3 dihydrobenzo [b][1,4]dioxin-2-yl)-2-methoxyphenoxy)-1,10, 15-trioxo-5,6-dithia-2,9,14triazapentadecane-3,8,13-tricarboxylic acid .( compound 5) . to a mixture of silibinin(0.477mmol,0.23 g) and compound 4 (0.477 mmol ,0.5 g)in 30ml distalled water ,add 5%naoh drop wise until the solution become basic at ph 10 and left stirred for 48hr.the mixture was then acidified with hydrochloric acid 5% drop wise to give a precipitate.this precipitate was washed with distalled water thoroughly ,then with ethanol and dried in oven at 50°c,to provide compound 5. (27-29) synthesis of15-(4-(((2,4-diaminopteridin-6yl)methyl)(methyl)amino) phenyl)-1-(4-(3-( hydroxymethyl ) -7 ( 3 , 5 , 7 trihydroxy -4 oxochroman 2 – yl ) 2 , 3 -dihydrobenzo [b][1,4]dioxin-2-yl)-2-methoxyphenoxy)-1 , 10 , 15 – trioxo 5,6-dithia-2,9,14-triaza penta decane-3,8,13-tricarboxylic acid. (compound 6). compound (5) (0.36 mmole, 0.5 g) was dissolved in 2.5 ml of acetonitrile. the ph of the solution was adjusted to 2 with 1n hcl and it was stirred for 1hr at (0-5) ᵒ c. the precipitate was filtered,washed with ether and dried to give compound 6 (30) . cell culture and culture conditions hep2 cell line(passage number( 75) were isolated from human epidermoid larynx carcinoma . this type of cells were maintained in rpmi-1640 medium with 30% bovine calf serum and 10% dmso , then the cell treated with trypsin/versin mixture in order to pursue subculture process . evaluation of cytotoxic activity the in vitro cytotoxic activity (cell viability assay) of these compounds(mtx,silibinin, compounds 5,6) were evaluated by neutral red dye assay; a nonradioactive ,fast assay widely used to quantify cell viability and proliferation . a set of 3 concentrations (0.5,1,2) μg/ml was made for each product and the exposure time of the assay was 24 hours,48hours. the cytotoxicity assay done according to a reported method (31) where the cell exposed to different concentrations of different compounds (0.5,1,2) μg /ml respectively, each compound was added to the cell in triplicate form of each concentration ,rpmi media and cell added as positive control ,only cells incubated with culture media represented the negative control. results and discussion the prepared compounds were characterized by means of physical properties, ftir,chns analysis and 1 h-nmr.the results are illustrated in table 1,2,3 and4,respectively . mechanism of action the compounds(5 and 6) can be considered as prodrugs, when inter the cancer cell can be hydrolysed by reductase enzyme which elevated in cancer cell and lead to hydrolysis of disulfide, and production of two moieties (32) . iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 80 table(1): physical data of the synthesized compounds. compound physical appearance %yield melting point observed ( o c) rf value 1 white powder 59.8 105-107 a= 0.65 b=0.4 2 dark yellow powder 90.9 185 (decomposed) a=0.166 b=0.2 3 yellow powder 52.2 115-118 a=0.71 b=0.78 4 yellow powder 54.28 145-148 a=0.85 b=0.82 5 yellow powder 30 203 (decomposed) a=0.72 b=0.78 6 yellow powder 58.6 170 (decomposed) a=0.78 b=0.9 table (2): elemental microanalysis of the final compounds. elemental microanalysis % empirical formula molecular weight compound 58.15 58.2 c c66h60n10o19s2 1360 5 4.649 4.41 h 10.688 10.29 n 4.48 4.7 s 51.984 52.7 c c52h52n10o19s2 1184 6 4.582 4.38 h 12.141 11.8 n 5.192 5.4 s table (3): the characteristic ir bands of synthesized compounds(1-6). compound characteristic i.r. bands (cm -1 ) 1 (3404-2800broad band n-h stretching vibration of nh3cl,(1745c=o stretching vibration of ester ). 2 (3346n-h stretching vibration of amide), (1668c=o stretching of carboxylic acid),(1655n-h bending of amide),(1600c=n stretching of imine 3 (3346n-h stretching vibration of amide), (1745c=o stretching vibration of ester),(1643c=o stretching vibration of amide),(1602c=n stretching of imine). 4 (3331n-h stretching vibration of amide),(1745 c=o stretching vibration of ester),(1716c=o stretching vibration of carbamate),(1643c=o stretching vibration of amide),(1604c=n stretching of imine),(1521 asymmetric stretching vibration of no2),,(1346symmetric stretching vibration of no2). 5 (3354n-h stretching vibration of amide),(1735c=o stretching vibration of cooh),(1637c=o stretching vibration of amide),(1604c=n stretching of imine). 6 (3346n-h stretching vibration of amide) (1735c=o stretching vibration of cooh),(1637c=o stretching vibration of amide). 1 h-nmr analysis: 1 h-nmr analysis was performed on 1 hnmr spectroscopy (shimadzu,japan) at university of al al-bayt,jordan,using dimethyl sulfoxide(dmso) as a solvent and the results are listed on table (4). iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 81 table (4): 1 h-nmr interpretation of compound (5). n h s s h n n h o oh oho oho o o o o o o o o o oh oh ho ho n n n n nn n 1 3 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 24 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 h chemical shift (ppm)  functional group 1,3 5.56 aromatic oh 2,4 5.99 1-benzen 6 2.94 alcoholic oh 5,7 5.59 methine 8,9,10,16 6.87 1-benzen 15,17 7.17 1-benzen 13 4 methylene 14 3.7 alcoholic oh 18 3.83 methyl 19,26 8.16 secondary amide 20,25,30 11.47 carboxylic acid 21,24,29 4.68 methine 22,23 2.94 methylene 32,34 7.58 1-benzen 33,35 6.97 1-benzen 36 3.13 methyl 37 4.54 methylene 38 8.63 2-pyrazine 39,45 8.42 ar.protons, benzylidenimin 40,44,46,50 7.79 ar.protons, benzylidenimin 41,42,43,45,47,48,49 7.51 ar.protons, benzylidenimin 1 h-nmr spectrum of compound 5 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 82 results of cytotoxicity assay the cytotoxic study was done on hep2 cell line(passage number( 75 ) were isolated from human epidermoid larynx carcinoma inhibition rate% the inhibition rate percent (i.r. %)(33) or inhibitory concentration percent (ic %) was estimated, and the result was varied among samples as shown in figure (1,2). figure(1):the cytotoxic activity represented by inhibition rate percent of compounds mtx,silibinin,compound 5,6 at different concentrations (0.5 ,1,2) μg/ml in 24 hrs time of exposure . figure(2): the cytotoxic activity represented by inhibition rate percent of compounds mtx,silibinin,compound 5,6 at different concentrations(2,1,0.5) μg/ml in 48 hrs time of exposure. one cell lines was studied (hep-2) at two times of exposure 24 and 48hr , using a 3 fold dilutions to get concentrations from ( 2 to 0.5 ) μg / ml of our compounds. the produced effect was explained as the following: the cytotoxic effect of mtx on hep – 2 (passage no.75), fig.1 revealed that the high concentration 2 μg/ml gave a significantly high inhibition rate of cells while being low gradually with low concentration 0.5 μg/ml during 24 hr of exposure( fig.1) , while during 48hr the cytotoxic effect high at high concentration 2 μg/ml (figure 2 ) and proliferation develop during lo w concentration due to develop resistant of cell cancer to mtx. the cytotoxic effect of silibinin gave high inhibition rate (33.1%) at high concentration 2 μg/ml and low with low concentration 0.5 μg/ml during 24 hr of exposure (fig.1) while low inhibition rate at low concentration 0.5 μg/ml and proliferation develop as the concentration increase((1,2) μg/ml) during 48 hr of exposure (figure 2). the compound 5 shows high inhibition rate at high concentration 2 μg/ml , in both times and decrease as the concentration decreases( fig.2) , but shows higher inhibition rate at 1μg/ml during 24hr of exposure (fig 1). the compound 6 also shows high inhibition rate (41.2%) at high concentration 2 μg/ml and at 1μg/ml shows higher inhibition rate 44.2% during 24 hr( fig.1), while the inhibition rate decrease during 48 hr( fig.2)and proliferation was develop. an explanation for this behaviour ,that in the design of cell culture experiment , it was important to be aware of growth state of the culture, as well as the quantitative characteristic of cell stain or cell line. culture will vary significantly in many of their properties between exponential growth and stationary phase (34) . also the differences in hep-2 responding to word different treatment might indicate a presence or absence of specific cellular receptor in each type of cell line, making the cell interact at same concentration in different manners. moreover the metabolic pathways in response to each treatment differed from one cell line to another .this fact was mentioned in different studies which investigated at different plant extracts in treating several types of cell line (35,36) . hep-2 resistance might be explained by over expression phenomena through genes responsible for bindingreceptor blockage that prevent the cytotoxic effect of any treatment (37) .from the result we can conclude that the activity of compounds 5,6 better than the activity of mtx or silibinin alone,and these may be attributed to the reduction in the resistance development. conclusions 1. the synthetic procedure for the designed target compounds was successfully achieved and the structural formula for the synthetic compound was characterized using ft-ir spectroscopy, elemental microanalysis, melting points and rf values and 1 h-nmr analysis done for compound 5. 2.the activity of compounds 5 and 6 were studied as anticancer drugs.they were more iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 83 effective drugs compared with mtx and silibinin. references 1. gottesman m.m., mechanisms of cancer drug resistance, annu. rev. med., 2002; 53:615–27. 2. longo-sorbello g.s. and bertino j.r., current understanding of methotrexate pharmacology and efficacy in acute leukemias, use of newer antifolates in clinical trials, haematologica,2001 ;86:121–27. 3. smith s. the ny state poison centers toxicology letter. methotrexate poisoning,october 2007;12 ( 4): 1-4. 4. kelemen l. e., the role of folate receptor a in cancer development, progression and treatment,cause, consequence or innocent bystander, int. j. cancer, 2006;119:243– 250 . 5. kamen b.a., wang m.t., streckfuss a.j., peryea x andanderson r. delivery of folates to the cytoplasm of ma104 cells is mediated by a surface membrane receptor that recycles, j. biol. chem. ,1988;263:13602–9. 6. paulos c. m., reddy j. a. , leamon c . p. , turk m. j. , and low p. s. , ligand binding and kinetics of folate receptor recycling in vivo: impact on receptor-mediated drug delivery, mol .pharmacol .,2004; 66:1406–1414. 7. leamon c.p., parker m.a., vlahov i.r., xu l.c., reddy j.a., vetzel m., and douglas n synthesis and biological evaluation of ec20: a new folate-derived, 99mtcbased radiopharmaceutical , bioconjug . chem.,2002; 13:1200–1210. 8. lu y., lowe d.a., kennedy m.d., and low p.s., folate-targeted enzyme prodrug cancer therapy utilizing penicillin-v amidase and a doxorubicin prodrug, j. drug target ,1999; 7:43–53. 9. oyewumi m.o. and mumper r.j., influence of formulation parameters on gad gadolinium entrapment and tumor cell uptake using folate-coated nanoparticles, int. j. pharm., 2003; 251:85–97. 10. leamon c.p. and low p.s. ,cytotoxicity of momordin-folate conjugates in cultured human cells. j. biol. chem 1992; 267:24966–24971. 11. 11.lu y. and low p.s. ,folate targeting of haptens to cancer cell surfaces mediates immunotherapy of syngeneic murine tumors. cancer immunol immun. ,2002; 51: 153–162. 12. citro g, szczylik c, ginobbi p, zupi g, and calabretta b, inhibition of leukaemia cell proliferation by folic acid-polylysinemediated introduction of c-myb antisense oligodeoxynucleotides into hl-60 cells. br. j. cancer,1994; 69:463–467. 13. leamon c..p and low p.s. ,folatemediated targeting: from diagnostics to drug and gene delivery. drug discov today 6:44–51 reddy ja and low ps (2000) enhanced folate receptor mediated gene therapy using a novel ph-sensitive lipid formulation, j. control release,2001;64:27–37 14. reddy j.a. and low p.s. ,enhanced folate receptor mediated gene therapy using a novel ph-sensitive lipid formulation. j .control release,2000; 64:27–37. 15. gabizon a., horowitz a.t., goren d., tzemach d., mandelbaum-shavit f., qazen m.m., and zalipsky s. , targeting folate receptor with folate linked to extremities of poly(ethylene glycol)grafted liposomes: in vitro studies, bioconjug. chem.,1999; 10:289–298. 16. hugues j. ryser p. and shen w., conjugation of methotrexate to poly(llysine) increases drug transport and overcomes drug resistance in cultured cells, proc. natl. acad. sci. usa,1978; 75( 8):3867-3870. 17. daniel j., fernandes and bertino j., 5fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyuridylate binding to thymidylate synthase by dihydropteroylpolyglutamates, proc. natl. acad. sci. usa 1980; 77(10): 5663-5667. 18. singh rp.and agarwal r., prostate cancer chemoprevention by silibinin: bench to bedside, mol. carcinog ,2006; 45: 436–42. 19. kaur m.and agarwal r., silymarin and epithelial cancer chemoprevention :how close we are to bedside? toxicol appl. pharmacol., 2007 nov 1 ;224(3):350-9. 20. noh em ., yi ms., youn hj., lee bk., lee yr., han jh., yu hn., kim js. and jung sh. ,silibinin enhances ultravioletb induced apoptosis in mcf-7 human breast cancer cells, j. breast cancer ,2011; 14: 8-13. 21. kelloff gj. , perspectives on cancer chemoprevention research and drug development, adv. cancer res., 2000; 78: 199–334. 22. hershy a.d.,dixon j.,chase m., nucleic acid economy in bacteria infected with ,bacteriophage t2, j gen physiol, 1953 ; 36(6): 777–789. 23. deodhar m., sable p., bhosale a., juvale k., dumbare r. andsakpal p., iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 84 synthesis and evaluation of phenytoin derivatives as anticonvulsant agents ,turk. j. chem.,2009;33:367-373. 24. rao v. k. , reddyi s. s. , krishna b. s. , naidu k. r., raju c. n. and ghosh s. k., synthesis of schiff’s bases in aqueous medium: a green alternative approach with effective mass yield and high reaction rates, green chemistry letters and reviews, september 2010 ; 3 ( 3):, 217-223. 25. mishra a., veerasamy r. , jain p. k. , dixit v. k. and agrawal r. k. ,synthesis ,characterization and pharmacological evaluation of amide prodrug of flurbiprofen ,j .braz. chem. soc.,2008; 19 (1):89-100. 26. meienhofer j. and trzeciak a., solidphase synthesis with attachment of peptide to resin through an amino acid side chain: [8-lysine]vasopressin, proc. nat. acad. sci. usa, may 1971 ; 68 ( 5): 1006-1009. 27. hegarty a.f.,forst l.n. and coy j.h., j.org.chem,1974;39:1089 28. hsi r.s., brown l.w., kagan f.,hauze a.r., stafford j.f. and forsit a.a., ibid,1972; 37:3427. 29. taft r.w. jr., newman m.s. and verhoek f.h. j., am. chem. soc. 1950;72: 4511 30. meltem cű, nurten a .and rahmiye e., synthesis and antimicrobial activities of some new flavonyl prodrug esters of ampicillin , turk. j. chem., 2005;29:187191. 31. freshney r.i.; culture of animal cells: a manual for basic technique, 5 th ed., 2005, john wiley &sons, inc. publication, new york. 32. dosio, f.; milla, p.; cattel, l. ec-145, a folate-targeted vinca alkaloid conjugate for the potential treatment of folate receptor-expressing cancers. curr. opin. invest. drugs 2010; 11:1424–1433. 33. chumchalova j. and smarda j.,folia microbiol (praha),2003;48 (1):111-5 34. fershney r. i., culture of animal cells, 3 th ed.,wiley-liss,newyork,1994. 35. chen f. d.,wu m.,wang h. e., hawang j.j. and yen s. h., sensitization of tumor but not normal tissue to the cytotoxic effect of ionization radittion using panax notoginseng extract,am.j. chem.med.,2001;16:234-242. 36. li y. m. ,ohmo y.,minatoguchi s.,fukuda k. and fujiwora h., extracts from the roots of lindera strychifolia induces apoptosis in lung cancer cell and prolongs survival of tumor,wearing mice ,am.j. clin. med.,2003;31:857869. 37. kim s. e.,kim y. h. and lee j.j., a new sequiterpene ester from calastrus orbiculatus reversing multi-drug resistance in cancer cells ,j.nat.prod., 1998 ;61: 108-111. 19© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) abstract objective: a budget impact analysis was performed to evaluate cost implications for the italian national health service (nhs) of the introduction of rituximab (rtx) in the treatment of rheumatoid arthritis (ra). methods: ra patients eligible to treatment with a second-line biologic dmard (disease modifying antirheumatic drugs) were identified and quantified and available strategies for their management were explored. costs associated with the different alternatives were estimated, and the impact on the nhs budget was estimated using a cohort simulation based on a markov chain with a time horizon of 5 years and 1-year cycles. seven alternative strategies were analyzed, each of them starting after the failure of a first anti-tnfα: 1) the use of a second and a third anti-tnfα; 2) the use of a second anti-tnfα followed by rtx; 3) the use of a second anti-tnfα followed by abatacept (abat); 4) the use of rtx as a second biological line, followed by an anti-tnfα; 5) the use of abat as a second biological line, followed by an anti-tnfα; 6) the use of rtx as a second biological line, followed by abat; 7) the use of abat as a second biological line, followed by rtx. only direct medical costs were considered: drug acquisition, administration, incidental premedication and monitoring exams. results: italian patients eligible to second biological line therapies (ra patients refractory or intolerant to at least one anti-tnfα therapy) were estimated in about 650 per year. the adoption of rtx as a second line therapy produced a substantial saving in total costs (-33% at the fifth year) with respect to the strategy with rtx as third line and the one with only anti-tnfα (the last two resulting substantially cost-equivalent). the number of patients in active treatment (biologic dmard) per unit cost resulted of about 8.1 patient-years/100,000 € with the strategy based only on anti-tnfα and increased of 10% with rtx as a third line. the strategy of rtx as a second line provided a further 41% increase (with respect to rtx as a third line). conclusions: the introduction of rtx in the treatment of italian ra patients represents a valuable new therapeutic option for this population especially if anticipated after a first anti-tnfα failure; it can also induce a reduction in health resources consumption for the nhs. keywords: rituximab, rheumatoid arthritis, analytic model, budget impact farmeconomia e percorsi terapeutici 2009; 10(1): 19-31 analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia maurizio benucci (1), sergio iannazzo (2), luciano sabadini (3) corresponding author sergio iannazzo s.iannazzo@adreshe.com analisi economica (1)uos reumatologia, nuovo ospedale s. giovanni di dio, firenze (2)adres, health economics & outcomes research, torino (3)responsabile di sezione dipartimentale reumatologia, dipartimento di medicina interna, usl8, arezzo introduzione l’artrite reumatoide (ar) è una patologia autoimmune sistemica caratterizzata da infiammazione cronica delle membrane sinoviali delle articolazioni interessate (prevalentemente quelle diartroidali), accompagnata da dolore e impotenza funzionale. la maggior parte dei pazienti presenta deterioramento della cartilagine e dell’osso, che può portare a disabilità permanente con perdita parziale o totale dell’indipendenza [1]. l’ar comporta considerevoli conseguenze dal punto di vista economico, non solo a causa dell’ammontare dei costi sanitari diretti, ma anche di quelli indiretti (correlati al calo di produttività dei pazienti) e di quelli intangibili, legati al deterioramento della qualità di vita [2]. sia i costi diretti sia quelli indiretti aumentano al peggiorare della malattia [2]. da ciò deriva l’importanza, anche dal punto di vista economico, di utilizzare precocemente le opzioni terapeutiche in grado di modificare l’andamento della patologia a breve ma soprattutto a lungo termine, limitando così l’evoluzione del danno strutturale. il trattamento farmacologico dell’ar si avvale di due categorie di farmaci: molecole a effetto esclusivamente sintomatico (fans e corticosteroidi) e principi attivi volti ad arrestare la progressione della malattia (disease modifying antirheumatic drugs, dmard) [3]. tra i dmard è possibile distinguere i dmard tradizionali dai più recenti dmard biologici; questi ultimi possono ulteriormente essere suddivisi in base al target molecolare in anti-tnfα (anti-tumour necrosis factor alpha) e antilinfocitari (rituximab, rtx; abatacept, abat). i dmard tradizionali come metotressato, ciclosporina e leflunomide sono i primi farmaci 20 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2009; 10(1) analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia utilizzati per la cura della malattia; si tratta di agenti immunosoppressivi con azione citotossica o antiproliferativa in grado di prevenire e ridurre la distruzione articolare comportata dalla patologia. negli ultimi 10 anni sono stati introdotti sul mercato i tre dmard biologici che inibiscono le azioni biologiche del tnfα, citochina che ricopre un ruolo chiave nella regolazione dell’infiammazione sinoviale [3]. attualmente questi farmaci (etanercept, etan; infliximab, infl; adalimumab, adal) vengono utilizzati in caso di risposta inadeguata al trattamento con un dmard tradizionale [4]. i farmaci di più recente introduzione sono rtx, un anticorpo monoclonale in grado di provocare la lisi delle cellule b grazie al legame specifico del dominio cd20 [5] e abat, una proteina di fusione capace di attenuare l’attivazione dei linfociti t umani inibendo uno specifico segnale di costimolazione [6]. essi sono stati specificatamente sviluppati per il trattamento dei pazienti con ar di grado severo con dimostrata resistenza o intolleranza all’uso di almeno un anti-tnfα [5,6]. l’inserimento nell’arsenale terapeutico di rtx rappresenta un sostanziale progresso nella terapia dell’ar; tuttavia le conseguenze economiche di tale inserimento per l’italia non sono al momento note. obiettivo di questo lavoro è calcolare, attraverso l’implementazione di un modello analitico decisionale, l’impatto sul budget del ssn comportato dall’impiego di rtx nel trattamento dei pazienti affetti da ar di grado severo rispetto alle altre strategie disponibili. materiali e metodi lo studio è stato condotto tramite un modello analitico basato su una catena di markov, sviluppato in ms excel®. la simulazione è di tipo coorte, ossia tutti i pazienti vengono fatti transitare simultaneamente attraverso il modello. il ciclo della simulazione è di un anno e l’orizzonte temporale dell’analisi è di 5 anni. le fasi di analisi condotte possono essere sintetizzate nella maniera seguente: stima del numero di pazienti affetti da artrite reumatoide, in base ai dati epidemiologici nazionali; individuazione della frazione di pazienti potenzialmente trattabili con rtx, sulla base delle indicazioni approvate; identificazione delle strategie terapeutiche, definite come mix di trattamenti utilizzati, da porre in confronto; analisi dei costi diretti sanitari a carico del ssn; valutazione comparativa dell’impatto sul budget del ssn delle strategie terapeutiche considerate. descrizione del modello il modello utilizzato per il calcolo del budget impact è un modello analitico di progressione di stati e di trattamenti (catena di markov) (figura 1). dal punto di vista della popolazione simulata, il modello è aperto (incidente con accumulo): questa impostazione è ottimale per sviluppare analisi su orizzonti temporali medio-lunghi con patologie croniche, anche secondo le linee guida internazionali per le analisi di budget impact [7]. secondo questo sistema ogni anno vengono inseriti nel modello i nuovi pazienti che vi rimangono fino al decesso. il modello così creato è in grado di analizzare e confrontare l’impatto economico di strategie alternative per la gestione dei pazienti con ar. le strategie terapeutiche valutate consistono in sequenze di trattamenti che vengono intrapresi in seguito al fallimento di un primo anti-tnfα (prima linea biologica). per ogni strategia, al termine della sequenza di trattamenti attivi (dmard biologici) si suppone che il paziente venga indirizzato a una terapia di prosecuzione con finalità palliative. nella pratica corrente questa è basata su un anti-tnfα (nonostante l’insuccesso) o un altro dmard tradizionale non utilizzato in precedenza [8]. in questa analisi si è supposto che la terapia di prosecuzione fosse costituita da metotressato in monoterapia. nel modello essa rimane fissata per tutti i pazienti e non costituisce un differenziale tra le strategie alternative. le strategie considerate sono sette: solo anti-tnfα: dopo il fallimento del primo anti-tnfα, viene intrapreso il trattamento con un secondo e con un terzo. quando anche il terzo trattamento con anti-tnfα viene interrotto per inefficacia o intolleranza, il paziente passa alla terapia di prosecuzione fino al decesso. prima dell’introduzione di rtx e abat questa strategia rappresentava l’unica possibilità, oggi però è poco praticata perché scarsamente efficace; rtx in 3° linea (biologica): dopo il fallimento del primo anti-tnfα, viene intrapreso il trattamento con un secondo. al fallimento di questo si passa a rtx e in seguito alla terapia di prosecuzione. questa strategia è largamente attuata nella pratica clinica italiana; abat in 3° linea (biologica): come sopra, ma al fallimento del secondo anti-tnfα viene prescritto abat anziché rtx; rtx in 2° linea (biologica): il trattamento con rtx viene anticipato alla seconda linea biologica, ossia dopo il fallimento con il primo anti-tnfα. se rtx viene interrotto per mancanza di efficacia, si passa 21© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) m. benucci, s. iannazzo, l. sabadini solo atnf (atnf-atnf-atnf-tp) infl etan adal etan adal etan metotressato monoterapia adal infl infl modello rtx 3° linea (atnf-atnf-rtx-tp) infl etan adal etan adal etan metotressato monoterapia rtx modello abat 3° linea (atnf-atnf-abat-tp) infl etan adal etan adal etan metotressato monoterapia abat modello abatrtx rtx 2° linea (atnf-rtx-atnf-tp) infl etan adal etan adal etan metotressato monoterapia modello abat 2° linea (atnf-abat-atnf-tp) infl etan adal etan adal etan metotressato monoterapia modello abatrtx inn rtx (atnf-rtx-abat-tp) infl etan adal metotressato monoterapia modello inn abat (atnf-abat-rtx-tp) infl etan adal rtx metotressato monoterapia modello abat figura 1 riepilogo schematico dei flussi dei pazienti nel modello. ciascuna strategia è composta dai trattamenti di seconda e terza linea biologica. ogni sequenza terapeutica si chiude, all’esaurimento delle opzioni disponibili, col ricorso alla terapia di prosecuzione abat = abatacept; adal = adalimumab; atnf = anti-tumour necrosis factor alpha; etan = etanercept; infl = infliximab; inn rtx = strategia innovativa rituximab; inn abat = strategia innovativa abatacept; rtx = rituximab; tp = terapia di prosecuzione 22 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2009; 10(1) analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia a un anti-tnfα, seguito dalla terapia di prosecuzione; abat in 2° linea: come sopra, ma al fallimento del primo anti-tnfα viene prescritto abat anziché rtx; innovativa rtx: il trattamento con rtx è alla seconda linea biologica; al suo fallimento si passa all’altro farmaco innovativo (abat) e successivamente alla terapia di prosecuzione. questa strategia si sta affermando in alcune realtà cliniche italiane; innovativa abat: come sopra ma l’ordine tra rtx e abat è invertito ossia abat è seconda linea biologica, rtx terza, seguito dalla terapia di prosecuzione. la sequenza dei tre anti-tnfα è stata definita, sulla base della pratica clinica, supponendo che: i pazienti che hanno utilizzato infl come primo anti-tnfα, se la strategia lo prevede, usano etan (secondo anti-tnfα) ed eventualmente adal (terzo anti-tnfα); i pazienti che hanno utilizzato etan come primo anti-tnfα, se la strategia lo prevede, usano adal (secondo anti-tnfα) ed eventualmente infl (terzo anti-tnfα); i pazienti che hanno utilizzato adal come primo anti-tnfα, se la strategia lo prevede, usano etan (secondo anti-tnfα) ed eventualmente infl (terzo anti-tnfα). per stimare la permanenza in trattamento con ciascun dmard biologico è stata condotta una ricerca bibliografica finalizzata a individuare i percorsi terapeutici di pazienti italiani con ar trattati con farmaci biologici. in italia, a differenza di molti altri paesi, manca un registro nazionale di pazienti con ar e anche l’annunciato database lorhen, nato dal coordinamento di quattro grandi centri ospedalieri lombardi, al momento di questa analisi non ha ancora reso pubblici i suoi dati. l’unico studio al momento pubblicato è relativo a 711 pazienti reclutati tra il 2002 e il 2004 in 23 centri reumatologici italiani e ha presentato parzialmente i propri risultati all’11° congresso europeo dell’ispor (international society for pharmacoeconomics and outcomes research) [9]. la permanenza in trattamento con ciascun farmaco anti-tnfα è stata determinata sulla base dei dati italiani di sopravvivenza in terapia elaborati e combinati con i dati presentati nel registro nazionale spagnolo (biobadaser) di pazienti con varie forme di artrite cronica in trattamento con farmaci biologici [10]. per rtx e abat non sono ancora disponibili dati relativi ai tempi di permanenza in terapia per cui sono stati assunti valori corrispondenti alla media degli anti-tnfα utilizzati come primo farmaco biologico. popolazione simulata pazienti con ar in italia il numero di pazienti affetti da ar residenti in italia è stato stimato applicando il tasso nazionale di prevalenza della patologia alla popolazione residente nel nostro paese [11]. tale tasso risulta essere, per quanto riguarda i soggetti maggiorenni, pari allo 0,46% [12,13]. le caratteristiche demografiche della popolazione affetta da ar sono state dedotte da uno studio osservazionale italiano condotto su circa 200 pazienti affetti da questa patologia [2]; la loro età media risulta essere pari a 55,3 anni e il rapporto tra sesso maschile e femminile è di 1:4. la mortalità nella coorte simulata è stata calcolata applicando alla mortalità per età e sesso della popolazione generale italiana [14] un tasso specifico per la patologia (2,03), ottenuto da un vasto studio svedese di popolazione che ha reclutato, tra il 1964 e il 1994 , circa 47.000 pazienti con ar [15]. pazienti in ingresso nel modello per definire il numero di pazienti che annualmente entrano nel modello è stato necessario determinare: il numero di pazienti attualmente in cura con farmaci biologici in italia; la frazione di questi che ha interrotto la terapia a base di anti-tnfα. sulla base dei dati di vendita dei farmaci biologici per ar nel 2008 [16] è possibile stimare che circa 7.000 pazienti (il 3,1%) siano attualmente in trattamento con un primo antitnfα. di questi, sempre in base a dati di mercato, si stima che il 22,4% sia trattato con infl, il 43,9% con etan e il 33,7% con adal. per determinare la frequenza di interruzione di un primo trattamento con anti-tnfα è stato fatto riferimento ai dati relativi alla sopravvivenza in terapia presentati nel registro nazionale spagnolo biobadaser [10]; in base a questi dati è stato dedotto che i tassi annuali di interruzione sono all’incirca l’11,2%, 7,4% e 10,4% nei pazienti in cura, rispettivamente, con infl, etan e adal. queste percentuali, applicate alla coorte di pazienti italiani con ar e in terapia con anti-tnfα, determinano il numero di pazienti eligibili a una seconda linea biologica e dunque il numero di pazienti che entrano annualmente nel modello. analisi dei costi i costi sono calcolati nella prospettiva del servizio sanitario, tenendo quindi conto esclusivamente dei costi diretti sanitari. i costi considerati comprendono: costo di acquisizione dei farmaci; costo di somministrazione dei farmaci; 23© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) m. benucci, s. iannazzo, l. sabadini costo dei trattamenti profilattici per ridurre il rischio di tossicità; costo di test/esami di monitoraggio. tutti i farmaci biologici sono considerati in associazione con metotressato. costo di acquisizione dei farmaci per ogni farmaco, il costo di terapia è stato calcolato sulla base della confezione presente in commercio con costo/mg inferiore; nei casi in cui diverse confezioni presentano uguale costo/mg, è stata considerata la confezione più coerente con il dosaggio raccomandato. il costo d’acquisizione della maggior parte dei farmaci considerati nell’analisi è stato calcolato sulla base del prezzo ex-factory al netto di eventuali sconti obbligatori, trattandosi di farmaci ospedalieri inseriti nel prontuario h. per metotressato, farmaco distribuito nelle farmacie territoriali (prontuario territoriale), è stato invece considerato il prezzo al pubblico. i prezzi utilizzati sono aggiornati a febbraio 2009 [17] e sono stati calcolati al netto degli sconti obbligatori riportati in gazzetta ufficiale. il costo di acquisizione dei farmaci è stato valutato sulla base degli schemi posologici raccomandati in rcp per pazienti con ar non appartenenti a popolazioni particolari. nei casi di rcp con diverse posologie, ugualmente indicate, la valorizzazione del farmaco è stata basata sulle raccomandazioni riportate dalle linee guida italiane [8] o dei dati di letteratura. sono state considerate le seguenti posologie: per rtx un ciclo di trattamento ripetuto ogni 9 mesi, formato da 2 infusioni da 1 g distanziate di 15 giorni [5]; la distanza temporale di 9 mesi è stata assunta come valore intermedio dei dati emersi nello studio reflex (ritrattamento dopo 6-12 mesi) [18]; per abat un’infusione di 750 mg (corrispondente a un paziente medio con un peso tra 60 e 100 kg) a 2 settimane dalla prima e successivamente ogni 4 settimane [6]; per infl un’infusione di 4 mg/kg alla prima, seconda e sesta settimana e successivamente ogni 7 settimane [19,20]; per etan un’iniezione da 25 mg 2 volte a settimana [8,21]; per adal un’iniezione da 40 mg ogni 2 settimane [8,22]; per metotressato, che viene somministrato in associazione a ciascuno dei trattamenti e costituisce, da solo, la terapia di prosecuzione, è stata considerata una posologia di 15 mg a settimana somministrati per via orale [8,23]. per le posologie espresse in mg per kg di peso corporeo (infl), in assenza di dati pubblicati sulla popolazione di pazienti con ar severa, la dose è stata calcolata sulla base di una stima del peso medio della popolazione italiana (62 kg per le donne e 78 kg per gli uomini) [24] ponderato per la prevalenza della patologia nei due sessi. tale posologia non sempre prevede l’utilizzo completo del contenuto della fiala; tuttavia l’eccesso di farmaco non utilizzato non è stato considerato perso. costo della somministrazione rtx, abat e infl sono farmaci somministrati per via endovenosa (ev), la cui preparazione e somministrazione avvengono in ambiente ospedaliero. le risorse sanitarie consumate per questi procedimenti comprendono sia il materiale utilizzato che il tempo di lavoro del personale sanitario. il materiale utilizzato per la ricostituzione e la somministrazione dei farmaci ev è stato stimato sulla base delle indicazioni riportate in rcp; i costi unitari derivano dal prezzo di acquisto dei dispositivi medici da parte delle aziende sanitarie della regione piemonte [25], mentre il costo della soluzione per preparazioni iniettabili (sodio cloruro 0,9%) è stato calcolato sulla base del prezzo ex-factory, aggiornato a febbraio 2009, della confezione con costo/mg inferiore [17]. il tempo di lavoro del personale ospedaliero è stato stimato sulla base dei dati raccolti da uno studio osservazionale retrospettivo italiano che ha analizzato il consumo di risorse comportato dal trattamento di circa un centinaio di pazienti con artrite reumatoide in terapia con infl [20]; secondo tale studio il tempo medio dedicato dal reumatologo per ogni somministrazione di infl è pari a 15 minuti, mentre l’infermiere viene occupato per 20 minuti su 180 di infusione. per rtx e abat è stato mantenuto lo stesso tempo di lavoro del reumatologo, mentre il tempo dell’infermiere per ogni infusione è stato calcolato applicando a rtx e ad abat lo stesso rapporto con la durata dell’infusione misurato nello studio per infl. il costo per minuto di lavoro del personale infermieristico e del medico reumatologo è stato stimato dagli autori dello studio pari rispettivamente a € 0,43 e € 0,97 [20]. etan e adal sono farmaci somministrati mediante iniezione sottocutanea (sc); per essi, non essendo gestiti in ambito ospedaliero, non è stato considerato alcun costo né di materiale né di lavoro da parte del medico reumatologo. tuttavia, secondo uno studio osservazionale retrospettivo spagnolo condotto su 1.111 pazienti in terapia con farmaci anti-tnf [26], nel 15% dei casi il paziente sottoposto a questo genere di terapia necessita di una visita domiciliare da parte di un infermiere. il tempo di tale visita, comprensivo di trasporto al domicilio, è stato da noi stimato in 30 minuti, con un costo al minuto pari a quello attribuito all’infermiere ospedaliero. infine per metotressato, farmaco somministrato oralmente, non è stato considerato alcun costo di somministrazione. 24 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2009; 10(1) analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia costo delle premedicazioni la somministrazione di alcuni farmaci, in particolare quelli somministrati per via endovenosa, richiede di essere preceduta da quella di un altro farmaco al fine di ridurre il tasso e la gravità di eventuali reazioni avverse. la maggior parte dei farmaci considerati nell’analisi non necessita di alcuna premedicazione; solamente rtx richiede la somministrazione di prednisolone 100 mg ev 30 minuti prima di ogni infusione del farmaco [5] al fine di ridurre il tasso e la gravità delle reazioni infusionali acute. costo degli esami clinici nello studio sono stati considerati i costi degli esami di laboratorio necessari al monitoraggio della terapia per ciascuno dei farmaci antireumatici analizzati. tali esami sono: emocromo con indicatori di flogosi (ves e proteina c reattiva); esame di funzionalità renale (comprensivo di creatinina, sodio, ferro, calcio, potassio e urea plasmatici); esame di funzionalità epatica (con alt, ast, ggt, albumina e bilirubina); esame di rilevazione degli anticorpi antidna. il costo unitario di tali esami è stato calcolato in base alle tariffe riportate dal nomenclatore tariffario nazionale [27], mentre la frequenza di esecuzione è derivata dalle rcp dei farmaci. pazienti eligibili (n°) anno 1 anno 2 anno 3 anno 4 anno 5 solo atnf (atnf-atnf-atnf-tp) nuovi pazienti 648 648 648 648 648 rtx abat infl 124 219 303 374 etan 421 782 1.105 1.394 1.652 adal 227 387 512 614 698 tot pazienti in trattamento attivo 648 1.293 1.836 2.311 2.724 terapia di prosecuzione 96 254 467 pazienti deceduti 4 14 29 50 rtx 3° linea (atnf-atnf-rtx-tp) nuovi pazienti 648 648 648 648 648 rtx 148 341 566 811 abat infl etan 421 782 1.105 1.394 1.652 adal 227 363 464 539 594 tot pazienti in trattamento attivo 648 1.293 1.910 2.499 3.057 terapia di prosecuzione 22 66 134 pazienti deceduti 4 14 29 50 abat 3° linea (atnf-atnf-abat-tp) nuovi pazienti 648 648 648 648 648 rtx abat 148 341 566 811 infl etan 421 782 1.105 1.394 1.652 adal 227 363 464 539 594 tot pazienti in trattamento attivo 648 1.293 1.910 2.499 3.057 terapia di prosecuzione 22 66 134 pazienti deceduti 4 14 29 50 continua > 25© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) m. benucci, s. iannazzo, l. sabadini tabella i suddivisione annuale dei pazienti eligibili al trattamento biologico di seconda linea (pazienti che ogni anno interrompono il primo farmaco anti-tnfα) fra nuovi farmaci biologici (rtx, rituximab; abat, abatacept) e anti-tnfα (infl, infliximab; etan, etanercept; adal, adalimumab) per ciascuna delle sette strategie di gestione dei pazienti con artrite reumatoide atnf = anti-tumour necrosis factor alpha; inn abat = strategia innovativa abatacept; inn rtx = strategia innovativa rituximab; tp = terapia di prosecuzione pazienti eligibili (n°) anno 1 anno 2 anno 3 anno 4 anno 5 rtx 2° linea (atnf-rtx-atnf-tp) nuovi pazienti 648 648 648 648 648 rtx 648 1.196 1.687 2.127 2.520 abat infl etan 63 119 177 235 adal 34 63 92 119 tot pazienti in trattamento attivo 648 1.293 1.869 2.396 2.874 terapia di prosecuzione 62 168 317 pazienti deceduti 4 14 29 50 abat 2° linea (atnf-abat-atnf-tp) nuovi pazienti 648 648 648 648 648 rtx abat 648 1.196 1.687 2.127 2.520 infl etan 63 119 177 235 adal 34 63 92 119 tot pazienti in trattamento attivo 648 1.293 1.869 2.396 2.874 terapia di prosecuzione 62 168 317 pazienti deceduti 4 14 29 50 inn rtx (atnf-rtx-abat-tp) nuovi pazienti 648 648 648 648 648 rtx 648 1.196 1.687 2.127 2.521 abat 96 230 394 580 infl etan adal tot pazienti in trattamento attivo 648 1.292 1.917 2.521 3.101 terapia di prosecuzione 14 44 92 pazienti deceduti 4 14 29 50 inn abat (atnf-abat-rtx-tp) nuovi pazienti 648 648 648 648 648 rtx 96 230 394 580 abat 648 1.196 1.687 2.127 2.521 infl etan adal tot pazienti in trattamento attivo 648 1.292 1.917 2.521 3.101 terapia di prosecuzione 14 44 92 pazienti deceduti 4 14 29 50 26 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2009; 10(1) analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia solamente per quanto riguarda lo svolgimento dell’esame per la rilevazione degli anticorpi antidna (richiesto solo per il monitoraggio della terapia con tutti i farmaci anti-tnfα) è stata utilizzata la frequenza di esecuzione riportata dallo studio osservazionale spagnolo illustrato in precedenza [26]. è stato assunto che tutti i pazienti, prima di iniziare una nuova terapia, indipendentemente dal tipo di farmaco, siano sottoposti a esami di laboratorio routinari; questo consumo di risorse non è stato quindi considerato differenziale ai fini dell’analisi, come anche il numero annuo di visite specialistiche (reumatologo), di visite di medicina generale e di indagini radiografiche, che non sono stati fatti dipendere dal tipo di trattamento somministrato. risultati pazienti in trattamento il numero di pazienti affetti da ar in italia è stato stimato pari a 227.560 casi; fra questi, 7.000 risultano essere in cura con un primo farmaco anti-tnfα, di cui circa 1.570 trattati con infl, 3.070 con etan e 2.360 con adal. il numero di pazienti che ogni anno interrompono la prima linea biologica e che, quindi, vengono considerati nel modello è pari a 648. la distribuzione dei pazienti con i vari dmard biologici per la terapia di seconda e terza linea biologica è stata analizzata separatamente per ciascuna delle sette strategie di gestione dei pazienti con ar nell’arco dei 5 anni di analisi. dai risultati, riportati in tabella i, emerge come le strategie non presentino differenze per il numero annuale di pazienti deceduti (non è, infatti, stato modellizzato alcun effetto farmaco anno costo farmaci (€) costo somministrazione (€) costo premedicazioni (€) costo monitoraggio (€) costo totale/anno (€) rtx + metotressato primo 7.102,13 74,79 5,74 154,98 7.337,64 successivi 7.102,13 74,79 5,74 154,98 7.337,64 abat + metotressato primo 13.530,68 264,77 154,98 13.950,43 successivi 12.568,65 245,85 154,98 12.969,49 infl + metotressato primo 10.462,86 252,40 182,78 10.898,03 successivi 8.134,37 195,89 182,78 8.513,04 etan + metotressato primo 12.510,48 201,24 174,23 12.885,95 successivi 12.510,48 201,24 174,23 12.885,95 adal + metotressato primo 12.175,42 50,31 172,09 12.397,82 successivi 12.175,42 50,31 172,09 12.397,82 metotressato primo 62,28 154,98 217,26 successivi 62,28 154,98 217,26 tabella ii riepilogo dei costi annui per ciascun trattamento. nella tabella è stato valorizzato anche metotressato in monoterapia, necessario alla terapia di prosecuzione (tp) abat = abatacept; adal = adalimumab; etan = etanercept; infl = infliximab; rtx = rituximab dei trattamenti sulla mortalità), mentre appare significativamente diverso il numero di pazienti che ogni anno viene mantenuto in trattamento attivo oppure indirizzato alla terapia di prosecuzione. in particolare, nella strategia basata solo su anti-tnfα, i pazienti in trattamento attivo al quinto anno sono oltre 2.700, nelle strategie con rtx (o abat) utilizzato come terza linea biologica sono oltre 3.000, mentre risultano circa 2.900 nelle strategie con rtx (o abat) utilizzati come seconda linea. infine nelle due strategie che fanno esclusivamente uso di farmaci innovativi rtx e abat in seconda e terza linea biologica (e viceversa) i pazienti in trattamento attivo al quinto anno risultano 3.100. costi il costo annuo di acquisizione dei farmaci antireumatici varia approssimativamente in un range compreso fra circa € 7.100 (rtx) e € 13.530 (abat nel primo anno di trattamento). i costi di somministrazione per ogni anno sono compresi fra € 50 di adal (somministrato per via sottocutanea) e € 267 di abat (somministrato tramite infusione endovenosa), e sono correlati principalmente al numero di infusioni annue necessarie alla terapia. il costo delle premedicazioni (necessarie solo nel caso di rtx) ammonta a € 5,7 l’anno; per quanto riguarda i test e gli esami necessari, il monitoraggio della terapia con abat + metotressato e di quella con rtx + metotressato richiede la stessa cifra (€ 155), mentre il monitoraggio della cura con farmaci anti-tnfα (infl, etan e adal, tutti in associazione a metotressato) comporta un costo annuo oscillante tra € 183 e € 172, causa la necessità di eseguire per essi l’esame degli 27© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) m. benucci, s. iannazzo, l. sabadini voci di costo (€ x 1.000) anno 1 anno 2 anno 3 anno 4 anno 5 solo atnf (atnf-atnf-atnf-tp) acquisizione 8.305 15.784 22.253 27.904 32.835 somministrazione 96 208 301 383 455 premedicazioni monitoraggio 112 225 335 443 549 totale 8.243 16.217 22.889 28.730 33.839 rtx 3° linea (atnf-atnf-rtx-tp) acquisizione 8.035 15.246 21.891 28.026 33.679 somministrazione 96 187 271 350 423 premedicazioni 1 2 3 5 monitoraggio 112 222 329 434 537 totale 8.243 15.656 22.493 28.813 34.644 abat 3° linea (atnf-atnf-abat-tp) acquisizione 8.035 16.197 23.962 31.383 38.420 somministrazione 96 215 334 452 568 premedicazioni monitoraggio 112 222 329 434 537 totale 8.243 16.634 24.625 32.269 39.525 rtx 2° linea (atnf-rtx-atnf-tp) acquisizione 4.605 9.688 14.247 18.456 22.310 somministrazione 48 104 153 199 242 premedicazioni 4 7 10 12 14 monitoraggio 100 202 303 402 501 totale 4.757 10.001 14.713 19.069 23.067 abat 2° linea (atnf-abat-atnf-tp) acquisizione 8.773 16.850 24.094 30.709 36.712 somministrazione 172 321 454 576 685 premedicazioni monitoraggio 100 202 303 402 501 totale 9.045 17.373 24.851 31.687 37.898 inn rtx (atnf-rtx-abat-tp) acquisizione 4.605 9.798 15.016 20.246 25.420 somministrazione 48 115 186 260 336 premedicazioni 4 7 10 12 14 monitoraggio 100 200 299 398 495 totale 4.757 10.120 15.511 20.915 26.265 inn abat (atnf-abat-rtx-tp) acquisizione 8.773 16.340 23.464 30.159 36.426 somministrazione 172 314 444 565 675 premedicazioni 1 1 2 3 monitoraggio 100 200 299 398 495 totale 9.045 16.854 24.209 31.123 37.599 tabella iii costo annuo totale (per l’intera popolazione esaminata) suddiviso per voci di costo (acquisizione, somministrazione, premedicazione e monitoraggio) relativo alle strategie di gestione dei pazienti con artrite reumatoride abat = abatacept; atnf = anti-tumour necrosis factor alpha; rtx = rituximab; inn abat = strategia innovativa abatacept; inn rtx = strategia innovativa rituximab; tp = terapia di prosecuzione 28 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2009; 10(1) analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia anticorpi anti-dna. il riepilogo dei costi totali annui comportati da ciascun trattamento è presentato in tabella ii. da essa emerge come il trattamento meno costoso sia quello con rtx (che comporta una spesa annua di circa € 7.300) seguito dagli anti-tnfα (il cui costo annuale si aggira attorno a € 13.000 e risulta leggermente inferiore, sul lungo termine, nel caso di infl) e infine da abat, che richiede una spesa di quasi € 14.000 nel primo anno e poco meno di € 13.000 in quelli successivi. impatto sul budget del ssn generalmente l’inclusione di rtx negli schemi terapeutici di gestione dei pazienti con ar determina una riduzione del costo totale dovuta ai minori costi di acquisto e a un numero di somministrazioni annue inferiore rispetto agli altri farmaci. nonostante, quindi, la presenza per rtx di una voce di costo aggiuntiva relativa alle premedicazioni (non necessarie per le altre terapie) il risultato globale è un abbassamento dei costi totali di terapia. questo effetto è visibile nella tabella dei costi totali (tabella iii) se ad esempio si confronta la strategia che fa uso di rtx come terza linea biologica rispetto a quella basata su soli anti-tnfα (differenze in tabella iv). l’effetto è, tuttavia, controbilanciato dall’aumento del numero dei pazienti in trattamento attivo, per cui il bilancio di costo totale, che dal secondo al quarto anno è a favore della strategia a base di rtx, al quinto anno risulta ribaltato, a favore della strategia con soli anti-tnfα, portando a una conclusione di sostanziale equivalenza di costo fra le due. particolare attenzione merita la strategia che fa uso di rtx come seconda linea biologica. in essa si anticipa, estendendolo di conseguenza a un numero maggiore di pazienti, il trattamento più economico e questo consente di massimizzare il risparmio di costi (-33% al quinto anno rispetto alla strategia con rtx come terza linea) (tabella iv). le strategie basate sull’utilizzo di abat invece, pur comportando una riduzione dei costi di monitoraggio rispetto agli anti-tnfα (sia rtx che abat non necessitano dell’esame di rilevazione degli anticorpi anti-dna, richiesto invece da tutte le terapie anti-tnfα), determinano un innalzamento complessivo delle spese budget impact (€ x 1.000) anno 1 anno 2 anno 3 anno 4 anno 5 rtx 3° linea vs solo atnf variazione (%) 0 0,0 -563 -3,5 -397 -1,7 +83 +0,3 +805 +2,4 rtx 2° linea vs rtx 3° linea variazione (%) -3.486 -42,3 -5.654 -36,1 -7.780 -34,6 -9.742 -33,8 -11.576 -33,4 inn rtx vs rtx 2° linea variazione (%) 0 0,0 +119 +1,2 +798 +5,4 +1.845 +9,7 +3.198 +13,9 tabella iv impatto budgetario annuale comportato dall’applicazione della strategia che fa uso di rituximab (rtx) come terza linea biologica rispetto a quella basata solo su anti-tnfα (atnf) e della strategia con rtx come seconda linea rispetto a rtx come terza. infine è valutato l’impatto di una strategia basata su rtx in seconda linea e seguita da abatacept (abat) rispetto alla stessa ma con rtx seguito da anti-tnfα (rtx 2° linea). una differenza negativa indica un risparmio, mentre una differenza positiva indica un aggravio di spesa a carico del ssn inn rtx = strategia innovativa rituximab pazienti per unità di costo (pz-anno/100.000 €) anno 1 anno 2 anno 3 anno 4 anno 5 rtx 3° linea vs solo atnf rtx 3° linea (atnf-atnf-rtx-tp) 7,86 8,25 8,49 8,67 8,83 solo atnf (atnf-atnf-atnf-tp) 7,86 7,97 8,02 8,04 8,05 differenze 0,00 +0,29 +0,47 +0,63 +0,78 variazione (%) 0,0 +3,6 +5,9 +7,9 +9,6 rtx 2° linea vs rtx 3° linea rtx 2° linea (atnf-rtx-atnf-tp) 13,63 12,92 12,71 12,57 12,46 rtx 3° linea (atnf-atnf-rtx-tp) 7,86 8,25 8,49 8,67 8,83 differenze +5,76 +4,67 +4,22 +3,89 +3,63 variazione (%) +73,3 +56,5 +49,7 +44,9 +41,2 inn rtx vs rtx 2° linea inn rtx (atnf-rtx-abat-tp) 13,63 12,77 12,36 12,05 11,80 rtx 2° linea (atnf-rtx-atnf-tp) 13,63 12,92 12,71 12,57 12,46 differenze 0,00 -0,15 -0,35 -0,51 -0,66 variazione (%) 0,0 -1,2 -2,7 -4,1 -5,3 tabella v numero di pazienti in trattamento attivo (intesi come i pazienti per cui non è ancora stato necessario ricorrere alla terapia di prosecuzione per esaurimento delle opzioni terapeutiche biologiche) per unità di costo (€ 100.000) e variazione percentuale di questo dato rispetto alle alternative abat = abatacept; atnf = anti-tumour necrosis factor alpha; inn rtx = strategia innovativa rituximab; rtx = rituximab; tp = terapia di prosecuzione 29© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) m. benucci, s. iannazzo, l. sabadini dovuto principalmente all’elevato costo di acquisizione e somministrazione del farmaco. dal punto di vista del mero costo, le due strategie basate esclusivamente su farmaci innovativi si collocano in una posizione intermedia fra le strategie precedenti, mescolando il trattamento con rtx, meno costoso della media, con il trattamento con abat, più costoso della media (+14% al quinto anno della strategia inn rtx rispetto a quella con rtx come terza linea seguita da anti-tnfα) (tabella iv). un ulteriore aspetto utile per la valutazione della performance economica delle alternative considerate è l’elaborazione del numero di pazienti in trattamento attivo per unità di costo (tabella v). questo indicatore permette di effettuare un confronto tra le strategie in termini di efficienza allocativa delle risorse (limitate) del ssn. infatti esso, sulla base dell’ipotesi che viene fatta in questo studio di budget impact di equivalenza terapeutica dei trattamenti, permette di stimare il numero di pazienti che il ssn può mantenere in trattamento attivo dato un ammontare di risorse economiche. risulta che la strategia che fa uso di rtx come terza linea biologica, pur avendo un costo complessivo sostanzialmente analogo a quella basata su soli anti-tnfα, permette di incrementare rispetto alla stessa il numero di pazienti trattati per unità di costo di circa il 10% al quinto anno. ancora migliore risulta, in questi termini, il dato relativo alla strategia con rtx come seconda linea, che permette di incrementare, rispetto a rtx come terza linea, i pazienti trattati a parità di costo di circa il 41% al quinto anno. infine, rispetto a quest’ultima, non pare portare significativi vantaggi la strategia con solo farmaci innovativi (inn rtx) in cui, in pratica, abat sostituisce gli anti-tnfα (riduzione di circa il 5% al quinto anno del numero di pazienti trattati per unità di costo). conclusioni l’obiettivo principale di questo lavoro è stato valutare l’impatto economico sul budget del servizio sanitario nazionale, causato dall’inserimento di un farmaco innovativo come rtx, nelle sequenze terapeutiche dei pazienti affetti da ar di grado severo che hanno mostrato resistenza o intolleranza all’uso di almeno un anti-tnfα. il razionale di quest’analisi è rappresentato dal fatto che tale farmaco può costituire per i pazienti affetti da questa patobibliografia edwards j. an exploration of patients’ experiences of anti-tnf therapy. 1. musculoskeletal care 2004; 2: 40-50 leardini g, salaffi f, montanelli r, gerzeli s, canesi b. a multicenter cost-of-illness study on rheumatoid arthritis 2. in italy. clin exp rheumatol 2002; 20: 505-15 logia una nuova e valida alternativa terapeutica capace di ritardare il momento in cui, esauriti i trattamenti efficaci, risulta necessario ricorrere a una terapia di prosecuzione. in letteratura sono numerosi gli studi di costo-efficacia e costo-utilità che attestano come l’aggiunta di rtx alle sequenze terapeutiche dei pazienti con ar provochi, a fronte di un lieve aumento dei costi, un miglioramento della qualità di vita espressa come qaly guadagnati, con un icer (incremental cost-effectiveness ratio) che rientra ampiamente nei limiti definiti accettabili dai paesi industrializzati [28-34]; se rtx viene utilizzato in sostituzione di un’altra opzione terapeutica al fallimento di un primo biologico, i costi medi annuali di trattamento risultano ridotti [28,31-35]. inoltre vi sono indicazioni che il rapporto costi-benefici di rtx sia tanto migliore quanto più precoce la sua introduzione nella sequenza terapeutica [36]. nel contesto europeo, l’adozione di rtx per la cura dell’ar comporta un costo annuo medio inferiore a quello di qualunque trattamento a base di anti-tnfα, e un costo per qaly guadagnato che oscilla fra 18 e 23.000 € a seconda del sistema sanitario considerato [33]. l’analisi di budget impact non prende in considerazione alcun parametro relativo all’efficacia e alla qualità di vita e non è dunque in grado di fornire un giudizio sistematico sull’efficienza allocativa delle risorse stanziate, ma fornisce un’indicazione sui costi complessivi delle scelte sanitarie intraprese. il nostro studio ha mostrato che la terapia con rtx per l’ar può offrire vantaggi anche in termini di consumo di risorse sanitarie, se confrontata con le terapie a base di anti-tnfα. secondo l’analisi presentata, l’uso di rtx implica una diminuzione dei costi totali di terapia rispetto alle terapie a base di anti-tnfα, in particolar modo se utilizzato come seconda linea biologica, ossia immediatamente in seguito al fallimento di un primo anti-tnfα, piuttosto che come terza linea, come frequentemente accade nella prassi clinica. rtx permette altresì, a parità di risorse investite, la gestione di un maggior numero di pazienti in trattamento attivo rispetto alle altre strategie, assicurando dunque un miglioramento della qualità di cura di tali pazienti. disclosure il presente lavoro è stato realizzato con il supporto di roche spa. 30 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2009; 10(1) analisi di budget impact dell’utilizzo di rituximab nel trattamento dell’artrite reumatoide in italia filippini m, bazzani c, zingarelli s, figlioli t, nuzzo m, vinelli m et al. anti-tnfalpha agents in elderly patients 3. with rheumatoid arthritis: a study of a group of 105 over sixty five years old patients. reumatismo 2008; 60: 41-9 leardini g, bernardi c, vaccaro e. farmacoeconomic impact of anti-tnf-alpha. 4. reumatismo 2004; 56: 80-6 mabthera5. ® – riassunto delle caratteristiche di prodotto orencia6. ® – riassunto delle caratteristiche di prodotto mauskopf ja, sullivan sd, annemans l, caro j, mullins cd, nuijten m et al. principles of good practice for 7. budget impact analysis: report of the ispor task force on good research practices – budget impact analysis. value health 2007; 10: 336-47 linee guida per la diagnosi precoce e la terapia dell’artrite reumatoide. a cura del comitato esecutivo sir (società 8. italiana reumatologia) per lo sviluppo delle linee guida. febbraio 2004 punzi l, cantini f, matucci cerinic m, ferri c, fiocco u, intorcia m et al. discontinuation rate of the 19. st and 2nd anti-tumor necrosis factor theraphies in patients with rheumatoid arthritis in italy. ispor eleventh annual european congress, athens 2008 gomez-reino jj, carmona l, biobadaser group. switching tnf antagonists in patients with chronic arthritis: 10. an observational study of 488 patients over a four-year period. arthritis res ther 2006; 8: r29 popolazione residente al 1 gennaio 2008 per età sesso e stato civile. disponibile on line all’indirizzo www.demo.11. istat.it marotto d, nieddu me, cossu a, carcassi a. prevalenza dell’artrite reumatoide nel nord della sardegna: lo studio 12. di tempio pausania. reumatismo 2005: 57: 273-6 salaffi f, deangelis r, grassi w. prevalence of musculoskeletal conditions in an italian population sample: results 13. of a regional community-based study. the mapping study. clin exp rheumatol 2005; 23: 819-28 tavola 2.16 tavola di mortalità per sesso ed età anno 2004. annuario statistico italiano, istat 200714. bjornadal l, baecklund e, yin l, granath f, klareskog l, ekbom a. decreasing mortality in patients with rheu-15. matoid arthritis: results from a large population based cohort in sweden, 1964-95. j rheumatol 2002; 29: 906-12 analisi di mercato sui farmaci biologici. roche, 2008 [dati non pubblicati]16. informatore farmaceutico – 69a edizione. milano: ed. elsevier masson, 200917. cohen sb, emery p, greenwald mw, dougados m, furie ra, genovese mc et al. rituximab for rheumatoid ar-18. thritis refractory to anti-tumor necrosis factor therapy: results of a multicenter, randomized, double-blind, placebocontrolled, phase iii trial evaluating primary efficacy and safety at twenty-four weeks. arthritis rheum 2006; 54: 2793-806 remicade19. ® – riassunto delle caratteristiche di prodotto favalli eg, marchesoni a, colombo gl, sinigaglia l. pattern of use, economic burden and vial optimization of 20. infliximab for rheumatoid arthritis in italy. clin exp rheumatol 2008; 26: 45-51 enbrel21. ® – riassunto delle caratteristiche di prodotto humira22. ® – riassunto delle caratteristiche di prodotto methotrexate23. ® – riassunto delle caratteristiche di prodotto comune di ferrara servizio statistica del: indagine campionaria triennale sulle condizioni di vita a ferrara anno 24. 2003. disponibile on line all’indirizzo http://servizi.comune.fe.it/attach/statistica/docs/informanumeri7_04abitudine_ al_fumo_e_obesita.pdf dispositivi medici acquisiti dalle aziende sanitarie della regione piemonte (art. 57 comma 5 legge 289/2002). 25. disponibile on line all’indirizzo www.regione.piemonte.it/sanita/opt/opr/dwd/articolar1.xls rubio-terrés c, ordovás baines jp, pla poblador r, martínez nieto c, sánchez garre mj, rosado souvirón ma 26. et al. use and cost of biological disease-modifying anti-rheumatic drugs in spain (praxis study). farm hosp 2007; 31: 78-92 nomenclatore tariffario nazionale dm 22 luglio 199627. giuliani g, de vita s, diamantopoulos a, brown b, kielhorn a. modelling of the cost-effectiveness of rituximab 28. for treatment of rheumatoid arthritis in italy. ispor eleventh annual international meeting, philadelphia 2006. code: par7 value health 2006; 9 31© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2009; 10(1) m. benucci, s. iannazzo, l. sabadini pompen m, diamantopoulos a, moers r, kielhorn a. economic consequences of providing rituximab as a treat-29. ment alternative for rheumatoid arthritis in the netherlands ispor tenth annual european congress, dublin 2007. code: md2 value health 2007; 10 mistry b, mccormick j, diamantopoulos a, kielhorn a. cost-utility analysis of rituximab as a new therapeutic 30. option for rheumatoid arthritis patients in canada. ispor ninth annual european congress, copenhagen 2006. code: par6 value health 2006; 9 kielhorn a, rubbert a, porter md, de vita s, brown b, aristides m et al. cost-effectiveness of rituximab therapy 31. for rheumatoid arthritis: a pan-european analysis. ispor eleventh annual international meeting, philadelphia 2006. code: par10 value health 2006; 9 porter d, lewis g, brown b, diamantopoulos a, kielhorn a. cost-effectiveness analysis of rituximab as a new 32. therapeutic option for rheumatoid arthritis in the uk. ispor eleventh annual international meeting, philadelphia 2006. code: par4 value health 2006; 9 rubbert a, kielhorn a, schach s , brown b, aristides m. cost-effectiveness of rituximab as a new treatment 33. modality for rheumatoid arthritis in germany ispor eleventh annual international meeting, philadelphia 2006. code: par5 value health 2006; 9 kielhorn a, porter d, diamantopulos a, lewis g. uk cost-utility analysis of rituximab in patients with rheumatoid 34. arthritis that failed to respond adequately to a biologic disease-modifying antirheumatic drug. curr med res op 2008; 24: 2639-50 saggia mg, santos ea, nasciben v. cost minimization analysis of rituximab versus infliximab, adalimumab and 35. etanercept for rheumatoid arthritis from a payer perspective in brazil. ispor tenth annual european congress, dublin 2007. code: par14 value health 2007; 10 solé jp, ortega gp, kielhorn a. cost-benefit of rituximab for rheumatoid arthritis patients in argentina. ispor 36. tenth annual european congress, dublin 2007. code: par3 value health 2007; 10 role of proand anti-inflammatory cytokines in rheumatoid arthritis: ahmed a. h. al-hassan correlation with disease activity j fac med baghdad vol.52, no.3, 2010 286 role of proand anti-inflammatory cytokines in rheumatoid arthritis: correlation with disease activity ahmed a. h. al-hassan* vmbchb, msc, phd clinical immunology summary: background: rheumatoid arthritis (ra) is a chronic and debilitating autoimmune disease characterized by chronic inflammation with subsequent cartilage and bone destruction. cytokines are key mediators of inflammation and can be found in abundance both in the joint and blood of patients. this study was designed to evaluate the role of proand anti-inflammatory cytokines in pathogenesis of ra, as well as study the correlation among these cytokines. patients and methods: forty patients with ra and thirty age-matched healthy controls were included in this study. serum cytokines were measured by enzyme-linked immunosorbent assay. results: the serum levels of pro-inflammatory cytokines (il-1α, il-2, il-6, il-8, il-12 and tnf-α) were significantly higher in ra patients than in healthy controls (p<0.01, p<0.05). moreover, these levels were significantly increased in active ra patients than in inactive ra (p<0.01, p<0.05). on the other hand, the serum levels of ifn-γ and anti-inflammatory cytokines (il-4, il-10) showed no significant differences between ra patients and healthy controls and neither between active ra patients and inactive ra (p>0.05). interestingly strong positive correlation was found among each of (il-1α, il-2, il-6, il-8, il-12 and tnf-α), p<0.05.while strong negative correlation was noticed between il-6 and (il4 and il-0) and also between tnf-α and (il4 and il-0), p<0.05. conclusion: the current study suggests that serum levels of pro-inflammatory cytokines (il-1α, il-2, il-6, il-8, il-12 and tnf-α) may play an important role in ra and may be used as a marker of disease activity. moreover imbalance between proand anti-inflammatory cytokines may yield effective therapeutic targets in this inflammatory disease. key words: rheumatoid arthritis, pro-inflammatory cytokines, antiinflammatory cytokines. introduction: rheumatoid arthritis (ra) is a systemic autoimmune disease characterized by chronic joint inflammation and subsequent joint destruction. the chronic inflammatory process is mediated through a complex cytokine network. the release of specific cytokines into the systemic circulation has been observed in a variety of inflammatory disease including ra. their concentration levels usually reflect disease severity and prognosis (1, 2). cytokines are differentiated into two groups on the basis of their action, pro-inflammatory cytokines (tnf-α, ifn-γ, il-1, il-2, il-6, il-8, il-12, il-15, il-17 and il-18) and anti-inflammatory cytokines (il-4, il-10). in ra, the balance between pro-and anti-inflammatory cytokines determines the degree and extent of inflammation, and thus can lead to different clinical effects (2). il-1, il-2 and tnf-α are among the many cytokines that can act alone or in synergy as mediators of tissue damage or chronic inflammation and which have been implicated in the pathogenesis of arthritis (3, 4). il-1α and tnf-α are able to act locally to induce bone and cartilage resorption, both cytokines, independently or together with ifn-γ, can induce the proliferation of synoviocytes. the main stimuli for il-8 production are il-1 and tnf-α. il-8 as a neutrophile chemoattractant is responsible for the increased number of neutrophils in ra joints, and therefore for *dept. of clinical immunology, medical college/ al-nahrain university. the clinical manifestation of joint and pain. in vivo, it has been shown that inoculation of only one intraarticular injection of il-8 induces synovial hyperplasia similar to the human ra (5). in ra excess levels of il-6 are produced in the joints, particularly in the thin tissue layer covering the joint. it may also cause permanent damage of bone and cartilage, as it encourages the body to break down bones and blocks the formation of bones (6,7). another important pro-inflammatory cytokine is il12, produced by different antigen presenting cells. it has been shown critical role in inducing th1 phenotype, thus initiating cellmediated immune response. it was suggested that il-12, modulating cellular and humoral immune response, is involved in the pathogenesis of immune rheumatic diseases (8). consequently, the potential beneficial effects of anti-inflammatory cytokines such as il-4 and il-10 in ra are of great interest. il-4 inhibit the production of il-1 α, tnf-α and il-6, it does not merely inhibit many proinflammatory cytokines; but also up-regulates the expression of antiinflammatory mediators such as il-1 receptor antagonist and the il-1 type ii receptor, indicating that il-4 is an anti-inflammatory cytokine (9). il 10 can effectively block the production of the proinflammatory cytokines (tnf-α, il-1, and il-8) by synovial macrophages and synoviocytes. based on its immunomodulating functions, il-10 has been considered an attractive candidate for therapeutic applications for treatment of acute and chronic original article fac med baghdad 2010; vol. 52, no. 3 received apr. 2010 accepted may 2010 role of proand anti-inflammatory cytokines in rheumatoid arthritis: ahmed a. h. al-hassan correlation with disease activity j fac med baghdad vol.52, no.3, 2010 287 inflammation, autoimmunity, cancer and infectious disease (10). this study was designed to evaluate the role of proand anti-inflammatory cytokines in pathogenesis of ra, as well as study the correlation among these cytokines. patients and methods: patients: forty patients with ra, 36 females and 4 males, their age range from 25-66 years, were included in this prospective study. they are attendants of rheumatology and rehabilitation center at al-kadhumyia teaching hospital in baghdad, compared with 30 age and sex matched apparently healthy individuals. patient’s group was divided in to two groups according to the disease activity (31with active ra and 9 with inactive ra). determination of serum pro-and anti-inflammatory cytokines: the serum obtained from ra patients and healthy controls were analyzed for, il-1α, il-2, il-4, il-6, il-8, il-10, il-12, tnf-α and ifn-γ using commercially available elisa kits following the manufactures’ instructions (biosource europe s.a. company, belgium). statistical analysis: comparison of serum cytokines levels among groups were calculated by kruskal-wallis-test and mann-whitney-test. correlation between the different parameters was calculated by the spearman test and p values of p<0.01and p<0.05 were considered significant. results: forty iraqi patients with ra (36 females and 4 males) were recruited for the present study; their mean age was 48±12 years (range 25-66 years). clinical presentation demonstrated that 31 (77%) of patients were with active ra (ara) and 9 (23%) were with inactive ra (iara). thirty (75%) of patients were positive for rheumatoid factor (rf) and 10 (25%) patients had negative rf. cytokines profiles: the serum cytokines profiles were analyzed both in ra patients and control individuals. the median serum levels of proinflammatory cytokines il-1α, il-2, il-6, il-8, il12 and tnf-α were significantly elevated in ra patients compared to controls (p<0.01, p<0.05). interestingly, the median serum level of ifn-γ was not elevated in patients and there was no significant differences between patients and controls (p>0.05), table-1. on the other hand, the present results were not observe any differences in the median serum levels of anti-inflammatory cytokines (il-4 and il10) between patients and controls (p>0.05), table-2. when the ra patients were categorized according to the disease activity, the following cytokines il-1α, il-2, il-6, il-8, il-12 and tnf-α were noted to be significantly elevated in active ra patients as compared to inactive patients (p<0.01, p<0.05). conversely, the median serum levels of ifn-γ, il-4 and il-10 was not observed any difference between active and in inactive ra patients (p>0.05), table-1 and table-2. correlation among pro-and antiinflammatory cytokines: the present study revealed significant strong positive correlation among each of (il-1α, il-2, il-6, il-8, il-12 and tnf-α) p<0.05.while strong negative correlation was noticed between il-6 and each of (il4, il-0) and also between tnf-α and each of (il4, il-0), p<0.05, as shown in table-3. role of proand anti-inflammatory cytokines in rheumatoid arthritis: ahmed a. h. al-hassan correlation with disease activity j fac med baghdad vol.52, no.3, 2010 288 table 1: the difference in median levels of serum pro-inflammatory cytokines il-1α, il-6, il8, il-12, tnf-α (pg/ml) and il-2, ifn-γ (iu/ml) among the three studied groups. ara cases iara cases healthy control p (kruskalwallis) serum il-1α minimum 3 0 0 maximum 73 30 10 median 21 1.5 4.00 p<0.01 no. 31 9 30 p (mann-whitney) ara x iara p<0.05 arax healthy control p<0.01 serum il-2 minimum 0.00 0.00 0.00 maximum 77.2 30.00 50.00 median 33.00 0.00 7.5 p<0.01 no. 31 9 30 p (mann-whitney) ara x iara p<0.05 arax healthy control p<0.01 serum il-6 minimum 1.8 1.8 1.5 maximum 161.0 60.0 30.0 median 19.0 1.8 4.0 p<0.01 no. 31 9 30 p (mann-whitney) ara x iara p<0.05 arax healthy control p<0.01 serum il-8 minimum 7 7 2 maximum 296 21 44 median 24.0 7.0 2.0 p<0.01 no. 31 9 30 p (mann-whitney) ara x iara p<0.01 arax healthy control p<0.01 serum il-12 minimum 10 10 6 maximum 292 65 37 median 70.0 11 8.5 p<0.01 no. 31 9 30 p (mann-whitney) ara x iara p<0.05 arax healthy control p<0.01 serum tnf-α minimum 3.2 3 1.5 maximum 256 63.2 32 median 40.1 4 4 p<0.01 no. 31 9 30 p (mann-whitney) ara x iara p<0.01 arax healthy control p<0.01 serum ifn-γ minimum 0.00 0.00 0.00 maximum 10 6 6 median 0.00 0.00 1.00 p>0.05 no. 31 9 30 p (mann-whitney) ara x iara p>0.05 arax healthy control p>0.05 role of proand anti-inflammatory cytokines in rheumatoid arthritis: ahmed a. h. al-hassan correlation with disease activity j fac med baghdad vol.52, no.3, 2010 289 table 2: the difference in median levels of serum anti-inflammatory cytokines il-4 and il-10 )pg/ml) among the three studied groups. ara cases iara cases healthy control p (kruskalwallis serum il-4 minimum 0 0 0 maximum 1 4 9 median 2.00 0.00 3.50 p>0.05 no. 31 9 30 p (mann-whitney) ara x iara p>0.05 arax healthy control p>0.05 serum il-10 minimum 0.00 0.00 0.00 maximum 47.2 10 41.0 median 2.0 5.00 1.00 p>0.05 no. 31 9 30 p (mann-whitney) ara x iara p>0.05 arax healthy control p>0.05 table 3: correlation among pro-and anti-inflammatory cytokines spearman’s correlation il-1α il-2 il-4 il-6 il-8 il-10 il-12 tnf-α ifn-γ il-1α il-2 il-4 il-6 il-8 il-10 il-12 tnf-α ifn-γ 0 0.62 0.47 0.75* 0.85* 0.52 0.74* 0.80* 0.61 0 0.57 0.58 0.49 0.59 0.35 0.84* 0.41 0 -0.78* 0.55 0.65 0.41 -0.87* 0.53 0 0.74* -0.71* 0.55 0.85* 0.66 0 0.66 0.87* 0.74* 0.89* 0 0.67 -0.85* 0.44 0 0.51 0.58 0 0.84* 0 *: strong significant if p>0.70 discussion: several studies in recent years were conducted to confirm the role of cytokines in the pathogenesis of ra. disequilibrium between stimulatory and inhibitory factors has a fundamental role in pathogenesis of this disease (11, 12, and 13). in line with present findings, high levels of tnf-α, il-1α, il-2, il-6, il-8 and il-12 have also been reported by other studies (14, 15). rostamian et al., found that serum levels of tnf-α and il-1α in active patients with bone erosion were higher than that in inactive patients and healthy control. so they concluded that serum levels of inflammatory factors such as tnf-α and il-1α could be associated with ra pathogenesis. however; anti-inflammatory drugs against these cytokines would be a useful treatment mode (12). high concentration of il-2 in active ra patients than inactive patients and healthy control were observed in current study, and was consistent with manhal (16), but at variance with altomonte and colleagues, who reported that il-2 significantly reduce in ra patients compared to control and explained that the reduce levels of il-2 may be an expression of deficiency of t-cells to produce il-2 in the active phases of ra or may be due to a possible absorption of il-2 by lymphocyte receptors (17). it is well known that il-6 required for the development of autoimmune arthritis; il-6 deficient mice are resistant to the induction of autoimmune disease (18). tchorzewski and associates mentioned that slightly diminished il-6 production by non stimulated lymphocytes from ra patients and enhanced il-6 production after pha stimulation has proved the possibility that il-6 can play a key role in the development of arthritis in these patients. moreover reduced severity of ra was observed in il-6 deficient mice (19). il-8 was detected in the role of proand anti-inflammatory cytokines in rheumatoid arthritis: ahmed a. h. al-hassan correlation with disease activity j fac med baghdad vol.52, no.3, 2010 290 sera of most our ra patients, correspondingly peichl et al, pointed out to the important role of il-8 and its auto-antibodies in inflammatory processes of ra, and may provide a clinically useful marker for the diagnosis of disease activity (20). on the other hand the present result was in contrast to other studies (21, 22). similar to published findings, the current study was found increase in serum levels of il-12 in ra patients when compared to controls (23, 24). ebrahimi and colleagues study 43 iranian patients with ra, and observed that serum il-12 was significantly high in those patients when compared to healthy controls, thus the authors concluded that serum il-12 may be more important and predictive factor in ra course and in the active form of the disease (23), as we confirmed in the present study. regarding the serum levels of ifn-γ, previous studies showed that serum ifn-γ was increased in ra patients as compared to controls (24, 16). on the other hand sakito et al, observed that serum ifn-γ correlated well with the number of peripheral lymphocytes but not reflect the activity of ra (25). however; our findings failed to show any significant increase or correlation with disease activity in ra patients. conversely, serum levels of anti-inflammatory cytokines (il-4 and il-10) were not increase in our patients as reported by many papers (2, 14, and 15). normal levels of antiinflammatory cytokines may support the result in the present study regarding the negative correlation between pro-inflammatory (tnf-α, il-6) and antiinflammatory cytokines because one of the normal biological activities of il-4 and il-10 is down regulates of pro-inflammatory cytokines production by monocyte (9, 26). similarly lacki and colleagues observed inverse correlation between il-10 and il6, and conducted that il-10 decrease il-6 production (27). finally, this variation among different cytokines in the same sample reflects the intricate cytokine network and its regulatory functions. the balance between pro-inflammatory and anti-inflammatory cytokines in ra determines the degree and extent of inflammation which can lead to major clinical effects (14). in conclusion the current study suggests that serum levels of proinflammatory cytokines (il-1α, il-2, il-6, il-8, il12 and tnf-α) may play an important role in ra and may be used as a marker of disease activity. moreover imbalance between proand antiinflammatory cytokines may yield effective therapeutic targets in this inflammatory disease. references: 1.feldmann m, brennan fm, maini rn. rheumatoid arthritis. cell.1999;85:307-10. 2. agarwal v and malaviya an. cytokines network and its manipulation in rheumatoid arthritis. j.indian.rheumatol.assoc.2005;13:86-91. 3. feldmann m, brennan fm, maini rn. role of cytokines in rheumatoid arthritis. annu.rev.immunol.1996;38:151-60. 4. altomonte l, zoli a, mirone l, scolieri p. serum levels of il-1β, tnf-α and il-2 in rheumatoid arthritis: correlation with disease activity. clin.rhuematol.1992;11(2):202-8. 5. national rheumatoid arthritis society website: what is ra? http://www.rheumatoid.org.uk/article.php?article_id =224 last accessed 21st may 2009. 6. spadaro a, scrivo r, rinaldi t, riccieri v, siliscavalli a. the role of il-12 in immune-mediated rheumatic disease. rhuematismo.2002;54(2):11321. 7. sebba a. tocilizumab: the first interleukin-6receptor inhibitor. ameri.j. health-system pharmacy. 2008;65(15):1413-18. 8. salvic v, stankovic a, kamenov b. the role of il8 and monocyte chemotactic protein-in rheumatoid arthritis. med.bio.2005;12(1):19-22. 9. cicuttini f, byron k, maher d, muirden k, hamilton j. serum il-4, il-10 and il-6 levels in inflammatory arthritiss. rheumatology. international.2004;14(5):201-6. 10. hart ph, hunt ek, bonder c, watson cj, jones jj. regulation of surface and soluble tnf receptor expression on human monocytes and synovial fluidmacrophages by il-4 and il10.j.immunol.1996;157:3672–80. 11. christodoulou c and choy eh. joint inflammation and cytokine inhibition in rheumatoid arthritis. clin. experi. med.2006;6(1):13-19. 12. rostamian ar, naji ah, gharibdoost f,khalvat a, saraf lg. evaluation of il-1α and tnfα serum levels in rheumatoid arthritis patients with active and inactive, with or without bone erosion. acta. med. iranica.2007;45(6):487-92. 13. monari c, bevilacqua s, piccioni m, pericolini e, perito s, calvitti m. a microbial polysaccharide reduces the severity of rheumatoid arthritis by influencing th17 differentiation and proinflammatory cytokines production. j. immunol.2009;183:191-200. 14. paramalinam ss, thumboo j, vasoo s, thio st, tse c, fong ky. in vivo proand anti-inflammatory cytokines in normal and patients with rheumatoid arthritis. ann. acad. med. singapore.2007;36:96-9. 15. ozaki m, kawabe y, nakamura h, migita k, tsukazaki k. elevated serum cytokine levels in a rheumatoid arthritis patients with large granular lymphocyte syndrome. rheumatology.2001;40:59293. 16. manhal fs. cytokines profile in patients with rheumatoid arthritis. j. fac. med. baghdad.2009;51(4);433-36. 17. altomonte l, zoli a, mirone l, scolieri p, magaro m. serum levels of il-1β, tnfα and il-2 in rheumatoid arthritis: correlation with disease activity. clin. rheumatol. 1992;11(2):202-5. 18. drakesmith h, chain b, beverly p. how can dendritic cells cause autoimmune disease? immunol. today.2000;21:214-7. 19. tchorzewski h, krasomski g, biesiada l, glowacka e, lewkowicz p. il-12, il-6 and ifn-γ role of proand anti-inflammatory cytokines in rheumatoid arthritis: ahmed a. h. al-hassan correlation with disease activity j fac med baghdad vol.52, no.3, 2010 291 production lymphocytes of pregnant women with rheumatoid arthritis remission during pregnancy. mediators of inflammation. 2000;9:289-93. 20. peichl p, ceska m, broell h, effenberger f, lindley ij. human neutrophil activating peptide\ il8 acts as an autoantigen in rheumatoid arthritis.ann. rheum. dis.1992;51:19-22. 21. troughton pr, platt r, bird h, el-manzalawi e, bassiouni m, wright v. synovial fluid il-8 and neutrophil function in rheumatoid arthritis and sernegative polyarthritis. bri. j. rheumatol.1996;35:1244-51. 22. boiardi l, macchioni p, meliconi r, pulsatelli l, facchini a, salvarani c. relationship between serum rantes levels and radiological progression in rheumatoid arthritis patients treated with methotrexate. clin. exppri. rheumatology.1999;17:419-25. 23. ebrahimi as, noshad h, sadreddini s, hejazi ms, ghojazadeh m. serum levels of tnfα, tnf αri, tnfαrii and il12 in rheumatoid arthritis patients. iran. j. immunol.2009;6(3).abstract. 24. vandenbroeck k, alloza i, gadina m, matthys p. inhibition cytokines of the il-12 family: recent advances and novel challenges. j. pharm. pharmacol.2004;56:145-60. 25. sakito s, ueki y, eguchi k, kawabe y, nagataki s. serum cytokines in patients with rheumatoid arthritis.rheumatology.international.1995;15(1):3137. 26. mattal ga and joshi vr. il-10 in early rheumatoid arthritis. j. indian.rheumatol. assoc. 2002;10:59-60. 27. lacki jk, klama k, macckiewicz sh, macckiewicz u, muller w. circulating il10 and il6 serum levels in rheumatoid arthritis patients treated with methotrexate or gold salts: preliminary report. inflamm.resear.1995; 44(1):24-26. iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 38 lithotripsy of different urinary tract stones by using seeds of carum copticum ahmed g. sabar* ,1 * department of community health ,college of health and medical technology, baghdad,iraq . abstract it has been a well-known practice to use seeds and the essential oil of carum copticum as a strongly antiseptic , antispasmodic , aromatic , bitter , diaphoretic , digestive , diuretic , expectorant and tonic. also used for cure influenza, asthma, and rheumatoid arthritis. to our knowledge it will be the first time to use the seeds of this herb as a urinary tract stone lithotripsy.this research aimed to the use of these seeds as a lithotripsian against different types of urinary stones and determine the efficiency of these preparation against which types of stone.a liquid solution was prepared from dissolving the seeds powder in cow milk and then concentration this preparation was done by boiling at 100 ° c to reduce the volume of solution to the half.the treatment was given via oral administration for successive 9 days before breakfast. 350 patients with urinary stone of different type took part in this research. all patients were subjected to ultrasonography and intravenous pyelography examinations to localized the position and detect diameter of stone. the above examination and also biochemical tests for diagnosis of stones ingredients were repeated after the administration of treatment and excretion of stone fragments in urine. the results were so promising especially against pure caoxalate stone. key words: carum copticum; lithotripsy; ca-oxalate stone; mixed stone الخالصة اسزخذِذ ثذٚر ٔجبد إٌخٛح )وّْٛ اٌٍّٛوٟ( ٚوذٌه اٌش٠ٛد اٌط١برح اٌّسزخٍصخ ِٓ اٌجذٚر وّٛاد ِضبدح ٌٍّغص ,ِذرح, ِسبعذح ٌٍٙضُ, ِمشعخ , ٚاسزعٍّذ ا٠ضب ٌعالج اٌجزد ٚاٌسعبي ٚاٌزثٛ ٚاالسٙبي ٚاٌزٙبة اٌّفبصً اٌزِٚبرشِٟ. ٚاسزخذِٕب ثذٚر ٘ذا ٝ اٌّجبرٞ اٌج١ٌٛخ حست ِعٍِٛبرٕب الٚي ِزح فٟ ٘ذا اٌّجبي . ٠ٙذف ٘ذا اٌجحث اٌٝ اسزخذاَ ثذٚر ٔجبد إٌجبد وعالج ٌزفز١ذ حص إٌخٛح إٌٙذ٠خ وعالج ٌزفز١ذ حصٝ اٌّجبرٞ اٌج١ٌٛخ ثبٔٛاعٙب اٌّخزٍفخ, ٚرحذ٠ذ اٌفعب١ٌخ االوجز رجبٖ اٞ ٔٛع ِٓ أٛاع اٌحصٝ . رُ درجخ ِئ٠ٛخ الخزشاي اٌى١ّخ اٌٝ 011اَ ح١ٍت االثمبر ٚرُ رزو١ش إٌم١ع ثغ١ٍبٔٗ اٌٝ رحض١ز ٔم١ع ِٓ ِسحٛق ثذٚر إٌجبد ثبسزخذ ِز٠ضب ِّٓ ٠عبْٔٛ ِٓ ٚجٛد 051ا٠بَ ِززب١ٌخ لجً االفطبر .شبرن فٟ ٘ذٖ اٌذراسخ 9إٌصف . اعطٟ اٌخ١ٍظ عٓ طز٠ك اٌفُ ٌّذح فٛق اٌصٛر١خ( ,ٚاالشعخ اٌٍّٛٔخ ٌزحذ٠ذ ِٛلع ٚحجُ اٌحصٝ , ثُ اٌحصٝ فٟ اٌّجزٜ اٌجٌٟٛ اخضعٛا اٌٝ فحٛصبد اٌسٛٔبر )اٌّٛجبد اجزٞ ٌىبفخ ع١ٕبد اٌجحث فحص االدرار اٌعبَ .اع١ذد ٘ذٖ اٌفحٛصبد ثعذ اسزخذاَ اٌعالج وذٌه اجز٠ذ اٌفحٛصبد اٌجب٠ٛو١ّ١ب٠ٚخ الج فعبي ثىفبءح عب١ٌخ رجبٖ حصبح اٚوشالد عٍٝ اٌحص١بد اٌّزفززخ ٚإٌبسٌخ فٟ االدرار ٌزشخ١ص ِىٛٔبرٙب ح١ث رج١ٓ اْ ٘ذا اٌع اٌجٛربس١َٛ إٌم١خ ٚثذرجخ الً ٌٍحصٛاد ِٓ االٔٛاع االخزٜ . introduction renal stones (nephrolithiasis) are concretion composed of crystalline components and organic matrix [1]. although the symptomatic presentations may be similar ;the disorder is heterogeneous as to composition and etiology.today, most urinary stones in patients in most countries are renal stones. about 1-4% of the population is believed to have kidney stones every year in usa and europe. about 2-5% of population in asia, 815% in europe and north america and 20% in saudia arabia develop kidney stone in their lifetime [2, 3, and 6] . renal stones tend to recur, and the rate of recurrence is about 75% during 20 years environmental and genetic factors [4-5] . a specific diagnosis for every patient with kidney stones, may give very important information about the stone-formation mechanism and the pharmaceutical manner to prevent recurrent stone formation [5-6] .carum copticum with herbarium number 2930303-1 is a plant in umbelliferae family with a white flower and small, brownish seeds. this plant is commonly grows in india,iran,egypt and europe [7] .the seeds and especially the essential oil are strongly antiseptic, antispasmodic, diuretic, and used in the treatment of so many diseases.the seeds contains about 4-6% essential oil, of which 4555% is thymol [8] , while the essential oil in the dried fruits (2.5-5% ) is dominated by thymol (35-60%) [9] .from south india carum copticum fruits, almost pure thymol has been isolated (98%) , but the leaf oil was found to be composed of monoterpenoids and sesquiterpenoids: 43%cadinene, 11% longifolene, 5% thymol, 3% camphor and others [9] . the effective components of this 1corresponding author email : ahmedalqaicy@yahoo.com , ahmed@hotmail.com received : 16/11/2009 accepted : 1/8/2010 mailto:ahmedalqaicy@yahoo.com mailto:ahmed@hotmail.com iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 39 plant , responsible for the observed bronchodialatory effect [6] ,despite the availability of modern medication the propensity towards the traditional medications is growing through out the word [7,8] which needs scientific investigation for evaluating the therapeutic effects and their mechanism of action. indeed no acute toxicity data were available for carum copticum ,although animal studies of putative beneficial effect of this plants seeds involved the use of a dose of 500mg/kg body weight in mice and rat without morbidity or mortality ascribable to the herb, suggesting that the oral ld50 of carum copticum (fruit/seeds) is likely to be higher than that figure [9] . the present study was carried out to determine the role of carum copticum seeds as a herbal medication for treatment urinary tract stones. materials and methods the study of effects of carum copticum seeds as a lithotripsian agent was carried out in 2008 on (350) patients diagnosed by specialist physician in private clinic from different sites of iraq (baghdad,diala,mousol) and the experimental part was undertaken during the period (2001-2008). materials seeds of carum copticum (other latin name : trachyspermum ammi ), were collected from local market in baghdad, identified under expert guidance ,taxonomy was performed by national herbarium of iraq at 1997,depended on ayurvedic pharmacopoeia of india (api) [10] , and kew herbarium [11] , liquid milk, sugar. the dose normally recommended in traditional ayurvedic use is 3-6 gm / day , presumably being 3 gm once or twice a day [12] . also this dose was documented in arabic antique manuscript (tathkarat daood al-antaky) [13] . methods before started giving the preparation we obtained the written consent of the patients who included in the study; (15gm) of seeds were ground to a very fine powder.(total dose for each patients taken with in 9 days) ;(5gm) of grounded seeds were boiled with 150 ml of liquid cow milk and 25 gm sugar, until half of the volume was obtained; the preparation was kept cool [13] ;this preparation divided to ( 3 ) equal doses, each patients was given single dose a day before breakfast for a period of 3 days ; this procedure was repeated for remainder (10gm) of grounded seeds ; ultrasonography (u /s) and intravenous pyelography (ivp), were performed pre-, and post treatment to be sure of curing urinary tract from any stone; urinary tract stones were of different sizes ranging from 5mm and 1.2 cm and they were seen in the kidney (renal stone) at upper pole calyx, mid renal part and lower pole calyx, also they were seen in the ureter (uretral stones) , and finally they were seen inside urinary bladder (vesical stones) . in addition general urine examination (gue) was done for all patients ; qualitative analysis of stone / fragments passed after herbal treatment, a procedure described by hodgkinson [14] , was employed to figure out the chemical constituent of urinary stone results the present study included (350) patients with different urinary tract stones were treated with liquid extract of carum copticum seeds. table 1 : chemical constituent of stones patients age range(years) % no. of patients type of stone (20-45) 48,57 170 pure ca-oxalate 20-50)) 28.57 100 (ca-oxalate/uric acid) mixed stone (22-50) 22,86 80 ca-oxalate/) hydroxyapatite) mixed stone 100% 350 total table 2 : lithotripsy events table 3 : duration of lithotripsy % no. of lithotripsed stone no. of patients type of stone 100 170 170 pure ca-oxalate 53 53 100 (ca-oxalate./ uric acid) mixed stone 31.25 25 80 ( ca-oxalate./ hydroxyapatite) mixed stone 350 total time (days) type of stone 2-7 pure ca-oxalate 7-12 ca-oxa./uric acid))mixed stone. 7-15 ca-oxalate./hydroxyapatite) mixed stone. iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 40 table 4 : response the types of stones to the treatment with carum copticum * h.s: highly significant discussion the results of this study including the investigation of the efficiency of carum copticum seeds (liquid solution), locally prepared on urinary stone among (350) patients.the results present in table (1) showed that pure caoxalate consist the larger percentage among types of stones. since a (170) patients out of (350) patients (48.57%) have a pure ca-oxalate urinary stone.results of table (1) revealed that (28.57), (22.86) of patients got mixed stone (caoxalate\uric acid) and (ca-oxalate \hydroxyapatite) respectively.the results cleared out in table (2) indicated that the local preparation of carum copticum seeds have a good affectivity on ca-oxalate stones , since a hundred percent of this type of urinary stone had been lithotripsed , comparing to (53%) for mixed (ca-oxalate\uric acid) and (31.25%) for mixed (ca-oxalate\hydroxyapatite) . recently in india had successfully purified an anticalcifying protein from the seeds of carum copticum using oxalate depletion assay and deciphered its inhibitory activity against ca-oxalate crystals growth .the antilithiatic potential of carum copticum was confirmed by its ability to maintain renal functioning, reduce renal injury and decrease crystal excretion in urine and retention in renal tissue [15] .it was obvious from the results presented in table (3) that pure ca-oxalate required the minimum duration to complete lithotripsy. while mixed stone of both types required a longer time to complete lithotripsy. the response of types of stone was indicated in table (4) which showed that pure ca-oxalate was the most affected types of stones by the treatment with carum copticum seeds compared with other two types of urinary stones mainly (caoxalate\uric acid) mixed stone and (caoxalate\hydroxyapatite) mixed stone. on the other hand type two of stone (ca-oxalate\uric acid) mixed stone was affected by treatment with carum copticum seeds more than the mixed stone (ca-oxalate\hydroxyapatite). our results confirmed the antilithiatic properties of carum copticum seeds that authorized by many researchers [16-19] . biostatistical analysis (binomial-test and z-test) confirmed these results. it could be argued for that the effect of herb extract (crude extraction) is depending totally on chemical structure of urinary tract stones, also it is clear from the results presented in table (4) that ca-oxalate whenever existed as one of the constituent of urinary tract stones it will provoke or stimulate the action of herbs seeds extract, in other word ca-oxalate is decisive component of urinary tract stone that encourage lithotripsy whenever carum copticum seeds extract are available.in this view, mixed stones which are either (ca-oxalate\uric acid) or (caoxalate\hydroxyapatite) showed different response to the treatment with carum copticum seeds extraction and that in our view is corresponding to the amount of ca-oxalate present in the mixture .in general, the lithotripsic effects of carum copticum seeds against urinary tract stones were mentioned by arabic scientist sheikh dawood antaki (about 1008 a. h) [13] . conclusion thus, the present study suggests the potential of carum copticum seeds in lithotripsy of urinary stone, especially calcium oxalate and forms the basis for the development of antilithiatic drug interventions against urolithiasis. acknowledgment the author is grateful to dr. nabeel fadel , dr. nasseer abed agha, and dr. akram mohd al-mahdawy, for help in the diagnosis of the caseas. thank also to dr. zuhair nouman al-ani a professor of pharmacogenetic for his scientific advices. references 1. pak cyc. kidney stones. williams textbook of endocrinology 1992; 1519-36. 2. pak cyc.kidney stones. lancet 1998; 351: 1797-801. 3. kamoun a, daudon m, abdelmoula j, hamzaoui m,chouachi b, houissa t, zghal a, ben ammar s, belkahia c, lakhoua r. urolithiasis in tunisian children. pediater nephrol 1999; 13(9):920-5. 4. gault mh, chafe l. relationship of frequency, age, sex, stone weight and composition in 15 624 stones. j urol 2000; 64 (2): 302-7. 5. kourambas j, aslan p, the cl, mathias bj, preminger m. role of stone analysis in metabolic evaluation and medical treatment of c.s (p-value) between each pairs of types ca-oxa.×mixed(1) ca-oxa.×mixed(2) mixed(1)×mixed(2) response (%) type of stone 0.003 h.s 0.000 h.s 0.000 *h.s 170 (100%) pure ca-oxalate. 53 (53%) ca-oxalate./uric acid mixed stone. (1) 25 (31.25%) caoxa./hydroxyapatite mixed stone. (2) iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 41 nephrolithiasis. j endocrinol 2001; 15 (2): 181-6. 6. thomas se, stapleton fb. leave no (stone) untreated; understanding the genetic bases of calcium-containing urinary stones in childhood. adv pediatr 2000; 47: 199221. 7. seyed hassan hejazian; mohd hossein ,and mohd dashti,.(antinociceptive effect of carum copticum extract in mice using formalin test).,world appl.sci.j. 2008,3(2):215-219 8. chopra. r. n., nayar. s. l. and chopra. i. c. glossary of indian medicinal plants (including the supplement). council of scientific and industrial research, new delhi. 1986. 9. versita,warsaw. fumigant toxicity of essential oil from carum copticum against indian meal moth,journal of plant protection research,dec. 2008, 48(4);14274345. 10. ayurvedic pharmacopoeia of india,a jamoda.government of india.part 1980; vol.i,first edition:2-3. 11. bull.of miscellaneous information(royal garden,kew), 1931 ; 1931(6); 299 344. 12. the ayurvedic pharmacopoeia of india,appendix.,government of india.part i,volume ii,first edition: 1999; 189-93. 13. shaikh dawood antaki. tadhkirat uli al albab wal jamia lil ajab alujab. medieval medical book. 10 th century, product code : 1568. 14. hodgkinson a. a combined qualitative and quantitative procedure for the chemical analysis of urinary tract calculi. j.clin.path. 1971, 24,147-151. 15. tan zeer kaur , rakesh, et al., in vivo efficacy of trachyspermum ammi anticalcifying protein in urolithiatic rat model. jour. of ethnopharmacology .2009, 126(3) 459-462. 16. a k pathak, n nainwal et al., pharmacological activity of trachyspermum ammi : a review. jour.of pharmacy research, 2010; 3(4); 895899. 17. gurinder j and daljit s.a., bioactive potential of anethum graveolens,foeniculum vulgare and trachyspermum ammi belonging to the family umbelliferae-current status. jor. of medicinal plant research 2010; 4(2), 87-94. 18. shazia s. ,mir a.k, mushtaq a. and muhammed z., indigenous knowledge of folk medicines by the women of district chakwal, pakistan . jor.ethnobotanical leaflets 2006; 10: 243-253. 19. muhammed h.,sumera a.,mir ajab k, ethnopharmacology, indigenous collection and preservative technique of some frequently used medicinal plants of utror and gabral,district swat,pakistan. afric.jor.of tradition. ,complem.,and altenet. med. 2006 ; 3(2) , 57-73. kameda et al.indd drug target insights 2007:2 239–247 239 review correspondence: hideto kameda, m.d./ph.d., division of rheumatology/clinical immunology, department of internal medicine, saitama medical center, saitama medical university, 1981 kamoda, tsujido-machi, kawagoe, saitama 350-8550, japan. tel/fax: +81-49-228-3574; email: kamehide@saitama-med.ac.jp copyright in this article, its metadata, and any supplementary data is held by its author or authors. it is published under the creative commons attribution by licence. for further information go to: http://creativecommons.org/licenses/by/3.0/. platelet-derived growth factor as a therapeutic target for systemic autoimmune diseases hideto kameda, miyuki suzuki and tsutomu takeuchi division of rheumatology/clinical immunology, department of internal medicine, saitama medical center, saitama medical university, kawagoe, saitama, japan. abstract: some systemic rheumatic diseases and disorders, especially fi brotic and vascular disorders, are often refractory to corticosteroid therapy. recently, ever accumulating evidence suggests that platelet-derived growth factor (pdgf) is involved in those refractory diseases. imatinib mesylate inhibits the activation of pdgf receptor as well as c-abl, bcr-abl and c-kit tyrosine kinases. it has therefore been widely used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. imatinib effectively suppresses the activation and proliferation of fi broblasts, mesangial cells and smooth muscle cells both in vitro and in vivo. additionally, it has recently been reported that some patients with rheumatoid arthritis or idiopathic pulmonary arterial hypertension demonstrated a good clinical response to imatinib therapy. imatinib may therefore overcome the limitations of current therapeutic strategy with corticosteroids and immunosuppressive agents for refractory diseases, such as systemic sclerosis and interstitial lung diseases, without clinical intolerability. keywords: imatinib mesylate, rheumatoid arthritis, fi broblast, rheumatic diseases, interstitial lung disease introduction the developmental process of systemic autoimmune diseases has been an unsolved mystery for decades. despite the identifi cation of autoreactive t lymphocytes or autoantibodies, both of which may participate in the pathogenesis of the diseases, any precise insight into “immunodistortion” present in each patient is lacking. therefore, overall immunosuppression, instead of a specifi c correction of “immunodistortion,” has been a common choice in clinical practice. corticosteroids and immunosuppressive agents have been the mainstay of the therapeutic strategy of systemic rheumatic diseases. however, the presence of refractory diseases/patients to such approaches, and various adverse effects such as increased risk of severe infections, limits their effi cacy. recent progress in our general understanding of molecular pathophysiology of diseases possibly downstream of the “immunodistortion” has led us to develop molecular targeted therapies in which the function of the key molecule(s) of the diseases is manipulated in order to control disease activity. we have then an excellent safety profi le in terms of an eventual lack of lethal dosage enabled most of the biologic response modifi ers (biologics) that can be applied at the dosage with which they suffi ciently inhibit the function of the targeted molecule. for example, tumor necrosis factor α (tnfα) inhibitors dramatically improved the outcome of patients with rheumatic diseases such as rheumatoid arthritis (ra) and seronegative spondyloarthropathies [takeuchi, 2005]. those agents include a chimeric antitnfα monoclonal antibody (infl iximab), a humanized anti-tnfα monoclonal antibody (adalimumab), and a p75 tnf-receptor/igg-fc fusion protein (etanercept). in addition, a chimeric anti-cd20 monoclonal antibody (rituximab), anti-interleukin-6 receptor monoclonal antibody (tocilizumab), and cytotoxic t-lymphocyte antigen 4 (ctla-4; cd152)/igg-fc fusion protein (abatacept) have been also successful in ra. on the other hand, low molecular weight chemical agents developed as molecular targeted therapies do have some drawbacks compared to biologics in terms of selectivity and limitations in dosage. nonetheless, their intracellular activity which large-molecular agents such as biologics do not possess, although slightly less selective, is still indispensable in the treatment of diseases in which various cytoplasmic/nuclear molecules play important roles. in diseases refractory to glucocorticoids and conventional immunosuppressants such as cyclophosphamide—both of which are chiefl y targeted to leukocytes—, cells of mesenchymal origin http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 240 kameda et al drug target insights 2007:2 such as fi broblast and vascular cells play a pivotal role. novel molecular targeted therapies should therefore include growth factors participating in the remodeling of mesenchymal tissues. among various growth factors, platelet-derived growth factor (pdgf) is an excellent candidate due to its multipotent roles in many rheumatic diseases [östman and heldin, 2001; paniagua and robinson, 2007]. it can be interfered with monoclonal antibodies against pdgf or its receptor (pdgf-r). another approach in inhibiting pdgf signaling is the application of tyrosine kinase inhibitors of pdgf-r. in contrast to non-selective tyrosine kinase inhibitors such as genestein, imatinib mesylate (sti571; gleevec® or glivec®) has been widely used for the treatment of chronic myeloid leukemia (cml) [druker et al. 2001a; druker et al. 2001b] and gastrointestinal stromal tumors (gist) [demetri et al. 2002]. therefore, in order to speculate on the clinical importance of pdgf-targeted therapy in refractory diseases and patients, this review article summarizes the involvement of pdgf in systemic autoimmune diseases, as well as preclinical or clinical results of imatinib or other pdgf-targeted therapies. signaling from pdgf-r and its inhibition by imatinib pdgf is a family of homoor hetero-dimeric molecules of disulfi de-bonded polypeptide chains with a conserved sequence of approximately 100 amino acid residues containing a characteristic motif of 8 cystein residues [östman and heldin, 2001; heldin and westermark, 1999; pietras et al. 2003]. combinations of subunits aa, ab, bb, cc and dd have been identifi ed to date. pdgf is synthesized by many cell types such as platelets, endothelial cells, fi broblasts, macrophages, and vascular smooth muscle cells. the pdgf-rs occur as α (~170 kda) and β (~180 kda) homodimers or heterodimers. the receptor sequences have 5 immunoglobulin-like domains in their extracellular parts and tyrosine kinase domains intracellularly into which is inserted an interrupting sequence of about 100 amino acids. pdgf-aa binds to pdgf-rαα, ab and cc to αα and αβ, bb binds to all 3 combinations of receptors, and dd binds to αβ and ββ [pietras et al. 2003]. the dimerization and activation of receptor tyrosine kinases lead to cellular activation of targeted cells including fibroblasts, smooth muscle cells and mesangial cells. a large number of sh2 domain-containing protein kinases/phosphatases and adaptor proteins, such as phosphatidylinositol 3-kinase (pi3k), phospholipase cγ (plcγ), sh2containing protein tyrosine phosphatase shp-2, grb2, nck and shc, link pdgf-r with downstream signaling molecules and lead to upregulated gene expression and proliferation, or transformation (fig. 1) [östman and heldin, 2001; heldin and westermark, 1999]. originally, imatinib focused on attempts to inhibit pdgf-r kinase with an ic50 of 0.12– 0.15 µm. however, it was soon noted that imatinib effectively inhibited c-abl (ic50 0.1–0.3 µm), bcr-abl (ic50 0.25 µm) and c-kit (ic50 � 1 µm) [carroll et al. 1997; druker et al. 1996; capdeville et al. 2002]. imatinib was also reported to be well tolerated and have signifi cant antileukemic activity in patients with cml with whom treatment with interferon α had failed, as well as those with blast crisis of cml or bcr-abl-positive acute lymphoblastic leukemia [druker et al. 2001a; druker et al. 2001b]. imatinib was later revealed to induce a sustained objective response in more than half of patients with an advanced unresectable or metastatic gist [demetri et al. 2002]. imatinib has now been widely approved for the treatment of cml and unresectable and/or metastatic c-kit (cd117)-positive gist. systemic sclerosis systemic sclerosis (ssc; scleroderma) is characterized by vascular damage and excessive fi brotic response on the basis of immunologic abnormalities (“immunodistortion”). the progression of organ damage is usually insidious in ssc, with the exception of scleroderma renal crisis which can be successfully treated with angiotensinconverting enzyme inhibitors. therefore, overall disappointing response to corticosteroids has been attributed to the paucity of involvement of active infl ammation, characterized by massive leukocyte infi ltration. for this reason, disease-modifying drugs for ssc have not been available to date [wollheim, 2007]. as for vascular damage, inhibitors targeting endothelin receptors, such as bosentan, represent a breakthrough in the treatment of pulmonary arterial hypertension (pah), a complication with one of the worst prognosis in ssc, systemic lupus erythematosus (sle) or mixed connective tissue 241 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 disease (mctd) [denton et al. 2006; kondo, 2001]. bosentan may also reduce the fibrotic process via its inhibitory effect on fibroblasts [braun-moscovici et al. 2004], although these effects have not yet been proven in clinical trials. at the same time, a recent report describing a stimulatory autoantibody against pdgf-r is of great interest in view of the pathogenesis and management of fi brosis in ssc [baroni et al. 2006]. although the determination of autoantibodies recognizing nuclear components have been useful in defi ning clinical subgroup of ssc, antinuclear antibodies (anas) have not been shown to be directly pathogenic. in contrast to this, stimulatory anti-pdgf-r antibodies exclusively found in sera from ssc patients were demonstrated to activate pdgf-r and induce the upregulation of the ha-ras-erk1/2 (external signal regulated kinases 1 and 2) cascade. this stimulated the expression of type i collagen gene expression and α-smooth muscle actin (α-sma) [baroni et al. 2006] which resembles the case of anti-thyroid stimulating hormone antibodies developing hyperthyroidism. early in 2007, distler et al. reported that imatinib reduced basal and pdgfor tgf-β-stimulated synthesis of extracellular matrix (ecm) proteins such as collagen and fibronectin; and that a 50–150 mg/kg/day intraperitoneal administration of imatinib effectively inhibited dermal thickness, the number of myofi broblasts and synthesis of ecm proteins in bleomycin-induced dermal fi brosis model—all without evidence of toxic adverse effects [distler et al. 2007]. a signifi cant increase in activated pdgf-rβ expression in idiopathic pah lungs compared with healthy donor lungs, and successful reversal of pulmonary vascular remodeling, in both monocrotaline-induced rat model and hypoxia-induced mouse model of pah [schermuly et al. 2005], led to the compassionate treatment of a patient with idiopathic pah with a daily administration of 200 mg of oral imatinib [ghofrani et al. 2005]. the patient’s condition imatinib gene regulation figure 1. signaling through the pdgf-r. the binding of pdgf dimer (pdgf-bb, for example) to pdgf-r results in the tyrosine phosphorylation of its cytoplasmic domain, where many adapter proteins and kinases/phosphatases assemble and augment the activation signaling leading to upregulation of gene expressions. imatinib inhibits the activation of pdgf-r tyrosine kinase. 242 kameda et al drug target insights 2007:2 improved impressively over 3 months in pulmonary vascular resistance (from 1056 dyn.sec.cm−5 to 815 dyn.sec.cm−5), six-minute walk distance (from 260 m to 383 m), and new york heart association functional class (from iv to ii) [ghofrani et al. 2005]. these reports provide the possibility that imatinib may be a disease-modifying drug for ssc. on the other hand, recombinant human anti-tgfβ1 antibody (cat-192) showed no evidence of effi cacy in a multicenter, randomized, placebocontrolled phase i/ii trial for early-stage diffuse cutaneous ssc [denton et al. 2007]. the blockade of multiple isoforms of tgfβ may be required for an effective inhibition of fi brosis. in this context, the pan-isoform-specifi c anti-tgfβ antibody 1d11 could be potentially effective for fi brotic diseases. nonetheless, imatinib has the advantage of inhibiting both pdgf and tgfβ signaling via pdgf-r and c-abl, respectively. interstitial lung disease (ild) in ssc will be discussed in the following section. ild in systemic rheumatic diseases the lung is one of the vital organs involved most frequently in systemic rheumatic diseases, and the presence of ild is a signifi cant prognostic factor of polymyositis/dermatomyositis (pm/dm), rheumatoid arthritis (ra), systemic vasculitis syndromes and ssc. the clinical courses of patients with ild associated with those diseases can be categorized into 4 groups: 1) a/sip with rapid deterioration within a month (acute) or within 2–3 months (subacute); 2) chronic progression of pulmonary fibrosis causing non-productive coughing, breath-shortening upon exertion, and occasionally leading to respiratory failure after more than 6 months; 3) acute or subacute exacerbation of chronic ild that is recurrent in some cases; and 4) asymptomatic ild detected in a milder form by radiographic examination or pulmonary function tests in the absence of clinically apparent signs and symptoms throughout the observation period [kameda and takeuchi, 2006]. the american thoracic society/european respiratory society (ats/ers) international multidisciplinary consensus classifi cation is usually used for the classifi cation of idiopathic interstitial pneumonias (iip) [american thoracic society; european respiratory society 2002]. diffuse alveolar damage (dad) and organizing pneumonia (op) usually develop acutely or subacutely, while non-specifi c organizing pneumonia (nsip) shows a subacute course, and usual interstitial pneumonia (uip) represents a chronic form of ild called pulmonary fi brosis. however, whether ild associated with systemic rheumatic diseases resembles iip remains debatable: the histological fi ndings may consist of the overlapping features of two or more patterns, nsip and op, or nsip and uip. moreover, patients having idiopathic uip (clinically idiopathic pulmonary fi brosis) show poorer prognosis compared to patients with systemic rheumatic diseases, such as ssc demonstrating uip in lung histology [bouros et al. 2002]. nevertheless, patients with ild associated with systemic rheumatic diseases also tend to show a favorable response to corticosteroids when the lung histology reveals op or cellular nsip, while they demonstrate a relatively poor response when the lung histology shows dad or uip; fi brotic nsip stands for an intermediate response. the addition of a single immunosuppressive agent or the combination of 2 or more improved the mortality and morbidity of rapidly or slowly progressive ild associated with pm/dm or ssc [white et al. 2000; kameda et al. 2005]. despite the intensifi ed immunosuppressive strategies, a signifi cant number of ild patients resulted in fatal outcomes. because the pathological examination of ild with poor prognosis, dad and uip, shares an excessive fibrosis, another therapeutic strategy such as anti-fi brotic agents is necessary. pirfenidone, nacetylcysteine and interferon-γ have been shown to be potentially effective for the prevention of lung fi brosis [american thoracic society 2000]. at the end of the last century, it was reported that the inhibition of the autophosporylation of pdgf-r by ag1296 effectively prevented v2o5stimulated proliferation of alveolar epithelial/ mesenchymal cells as well as hydroxyproline accumulation [rice et al. 1999]. in that model, a specific inhibition of epidermal growth factor (egf) receptor activation by ag1478 also showed a preventive efficacy, although slightly less compared to ag1296. another approach using an expression plasmid of the extracellular domain of pdgf-rβ for bleomycin-treated c57bl/6 mice also ameliorated the increases in the wet weight, hydroxyproline content and the histologic changes in the lung [yoshida et al. 1999]. daniel et al. proved that imatinib dramatically reduced hydroxyproline content and prevented 243 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 histopathologic changes in bleomycin-treated lungs of 129tsvems mice [daniel et al. 2004]. they also suggested that the effi cacy of imatinib is mediated, in part, by the inhibition of serine/threonine tgf-β receptor kinase signaling through c-abl tyrosine kinase. aono et al. also showed that imatinib (50 mg/kg) signifi cantly attenuated bleomycin-induced pulmonary fi brosis in terms of histology and collagen deposition in c57bl/6 mice. interestingly, the early treatment with imatinib (from day 0 to 14), but not the late treatment (from day 15 to 28) significantly inhibited bleomycin-induced lung fi brosis in that model [aono et al. 2005]. however, another bleomycininduced lung fi brosis model using rats showed that imatinib (50 mg/kg) treatment commenced at day 10 was still effective, while prednisolone was effective exclusively in the preventive model started at day 1 [chaudhary et al. 2006]. in the radiation (20 gy)-induced lung fi brosis model using c57bl/6 mice, treatment with either of 3 pdgf-r inhibitors (su9518, su11657 and imatinib) markedly attenuated the development of pulmonary fi brosis in excellent correlation with clinical, histological, ct results, and life span. in addition, no decline in their effi cacy was observed when they were started after thoracic irradiation compared to a prevention schedule [abdollahi et al. 2005]. these results may provide a possibility that imatinib could be chosen for the treatment of corticosteroid(/immunosuppresants)-refractory ild. in contrast to the above, a recent report failed to demonstrate the effi cacy of imatinib (10 mg/kg, intraperitoneal or oral administration) in the protection against bleomycin-induced pulmonary fi brosis in c57bl/6 mice [vittal et al. 2007]. lupus nephritis systemic lupus erythematosus (sle) is a prototype of systemic autoimmune diseases and its renal involvement has been posing major challenges for physicians. renal accumulation of immune complexes followed by complement activation, and antibody-effector-cell interactions through the fc receptor have been implicated in the pathogenesis of lupus nephritis. high-dose corticosteroids combined with immunosuppressive agents such as cyclophosphamide or mycophenolate mofetil are the mainstay in current strategy for the induction of response in lupus nephritis. some patients however do not respond favorably to the above regimens or show repeated relapses, and ultimately progress to end-stage renal disease. renal expression of pdgf, which is synthesized by mesangial cells, endothelial cells, macrophages, and smooth muscle cells in the kidney, and of pdgf-r was signifi cantly increased in the glomeruli of patients with mesangial proliferative glomerulonephritis (iga nephropathy, henochschönlein purpura nephritis, and lupus nephritis) compared with normal glomeruli [matsuda et al. 1997]. thus, pdgf/pdgf-r axis is considered a novel therapeutic target of lupus nephritis and other forms of glomerulonephritis. indeed, administration of neutralizing anti-pdgf igg [johnson et al. 1992] or oligonucleotide aptamer [floege et al. 1999], which specifi cally binds to the pdgf-b chain and inhibits its activity to anti-thy-1 glomerulonephritis rats resulted in a signifi cant reduction in mesangial cell proliferation and largely prevented the increased deposition of ecm. imatinib (0.013–2.0 µm) inhibited pdgfstimulated, but not fgf-stimulated, mesangial cell proliferation in a dose-dependent fashion in vitro [gilbert et al. 2001]. moreover, the effi cacy of imatinib in vivo has been proven in 3 different models of (lupus) nephritis. first, pdgf-r tyrosine kinase blockade by imatinib (50 mg/kg/day) was associated with a signifi cant reduction in mesangial cell proliferation, the number of α-sma-positive mesangial cells, and glomerular type iv collagen deposition in male wistar rats with anti-thy-1.1 glomerulonephritis [gilbert et al. 2001]. second, 50 mg/kg imatinib inhibited proliferation of glomerular cells and crescent formation, and also prolonged the life span of mrl/lpr female mice [sadanaga et al. 2005]. intriguingly, attenuation of lymphadenopathy and salivary gland infl ammation, as well as reduction in serum anti-doublestranded dna antibodies, was also observed in the imatinib-treated mice. third, (nzb/w)f1 mice treated with imatinib (50 mg/kg b.i.d./day) showed ameliorated survival, delayed onset of proteinuria, and preserved renal function [zoja et al. 2006]. histologic examination provided evidence of reduced glomerular hypercellularity, deposits, tubulointerstitial damage, and accumulation of α-sma-positive myofi broblasts. rheumatoid arthritis the synovial membrane in patients with ra is characterized by hyperplasia, angiogenesis, and an 244 kameda et al drug target insights 2007:2 infi ltrate of infl ammatory cells including cd4+ t lymphocytes [choy and panayi, 2001]. synovial fi broblast-like (sfl) cells from ra patients show transformed characteristics [firestein and zvaifl er, 2002; müller-ladner et al. 2000; yamanishi and firestein, 2001]: altered morphology, anchorageindependent growth [lafyatis et al. 1989], loss of contact inhibition, oncogene activation [müllerladner et al. 1995], monoclonal or oligoclonal expansion [imamura et al. 1998], cartilage invasion in severe combined immunodefi cient mice [müllerladner et al. 1996], etc. pdgf-rs are abundantly expressed on the surface of ra-sfl cells, and stimulation with pdgf enhances both the anchorage-dependent and -independent growth of ra-sfl cells and thus implicate pdgf in the activation and transformation of ra-sfl cells [lafyatis et al. 1989; rubin et al. 1998; remmers et al. 1991; sano et al. 1993; watanabe et al.]. indeed, pdgf immunostaining of ra synovia is more extensive and intense than that of osteoarthritis (oa) or normal synovia. also, pdgf-r expression is elevated in ra synovia compared with oa and normal synovia [remmers et al. 1991]. moreover, pdgf, together with tnfα, were identified as the major growth factors of ra-sfl cells [thornton et al. 1991]. furthermore, thrombin activity in synovial fl uid is signifi cantly higher in the patients with ra than with oa, and the mitogenic activity of thrombin toward ra-sfl cells is associated with an increase in the expression of mrna of pdgf-rs [ohba et al. 1996]. we demonstrated that 1 µm of imatinib effectively inhibited the pdgf-stimulated tyrosinephosphorylation of pdgf-r in ra-sfl cells, as well as pdgf-enhanced anchorage-dependent and -independent ra-sfl cell proliferation in vitro [kameda et al. 2006]. inhibition of pdgf-r signaling by 1 µm of imatinib did not induce apoptosis in cultured ra-sfl cells [sandler et al. 2006]. the effi cacy of imatinib in vivo has been almost simultaneously reported. imatinib (33– 100 mg/kg) effectively prevented and treated collagen-induced arthritis model of dba/1 mice in terms of synovitis, pannus formation and joint erosion, although preventive administration was more effi cacious than therapeutic administration [paniagua et al. 2006]. notably, imatinib inhibited mast cell c-kit activation, proinflammatory cytokine production, immunoglobulin production from b cells, and t cell response. however, collagen-induced arthritis in lewis rats were resistant to imatinib treatment: only high-dose (150 mg/kg, not 50 mg/kg) imatinib showed a signifi cant inhibition of osteoclast formation and joint destruction, and failed to reduce paw swelling [ando et al. 2006]. although the downstream signaling pathway from pdgf-r in ra-sfl cells has not been clarifi ed, many adaptor proteins are likely to be involved. for example, gab1 and gab2 were expressed in ra-sfl cells, and both adaptor proteins were rapidly tyrosine-phosphorylated after the stimulation of ra-sfl cells with 10 ng/ml of pdgf [kameda et al. 2006]. the fact that the expression of gab1 lacking the pleckstrin homology domain is associated with the enhanced anchorage-independent growth of syrian hamster embryo fi broblasts under growth factor stimulation suggests that similar alteration in signaling proteins might be involved in the acquisition of the transformed phenotype of ra-sfl cells [kameda et al. 2001]. this is supported by the fact that pdgf stimulation enhanced anchorage-independent growth of ra-sfl cells [kameda et al. 2006]. recent reports on ra patients successfully treated with imatinib were more encouraging than above in vitro and in vivo results. miyachi et al. reported a case with ra and chronic myeloid leukemia, both of which were successfully treated using imatinib [miyachi et al. 2003]. moreover, eklund et al. described three patients with severe ra who were treated for 12 weeks with escalating daily doses (from 100 to 400 mg) of imatinib [eklund and joensuu, 2003]. all three patients had failed to respond to prior anti-rheumatic medications, including methotrexate and infl iximab. all reported less pain and disease activity, and their health assessment questionnaire scores improved subsequent to the imatinib treatment. therefore, the addition of, or the switching to, imatinib may be benefi cial to patients with ra who failed in conventional therapies. perspective and conclusion besides those described above, pdgf is a fascinating molecular target in various other systemic rheumatic diseases such as spondyloarthropathy and systemic vasculitis. the strategies of pdgf signaling inhibition include antibodies against pdgf or pdgf-r, soluble recombinant fusion proteins of pdgf-r and igg, oligonucleotide 245 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 figure 2. predicted molecular mechanisms and possible therapeutic targets of rheumatic infl ammation and fi brosis. various cell types shown here are likely to be involved in the development and progression of rheumatic infl ammation. autoimmune responses and the subsequent infi ltration of neutrophils and lymphocytes are known to occur. resident macrophage activation, increased apoptosis of epithelial cells and endothelial dysfuction may result in the formation of fi brin clots and a provisional matrix, initiating the proliferation of fi broblasts. any of those cells could serve as therapeutic targets in the treatment of systemic rheumatic diseases, and a simultaneous control of them seems to be essential in overwhelming the persistent infl ammation. table 1. summary of preclinical fi ndings positively supporting the application of imatinib for systemic rheumatic diseases. ssc: inhibition of the differentiation of fi broblast into myofi broblast, proliferation, and ecm production [distler et al. 2007, in vitro and in vivo] pah: inhibition of medical thickening of pulmonary arteries leading to the improvement in right ventricular pressure, cardiac output, and arterial pressure of oxygen [schermyuly et al. 2005, in vivo] ild: inhibition of the differentiation of fi broblast into myofi broblast, proliferation, and ecm production, leading to histologic effi cacy [daniels et al. 2004; aono et al. 2005; in vitro and in vivo; abdollahi et al. 2005; chaudhary et al. 2006, in vivo] lupus nephritis: inhibition of mesangial proliferation, ecm production, and crescentic formation leading to decrease in proteinuria and improvement in renal failure [gilbert et al. 2001; sadanaga et al. 2005; zoja et al. 2006, in vivo] ra: inhibition of transformation/proliferation of synovial fi broblasts [kameda et al. 2006; sandler et al. 2006, in vitro], delay in joint destruction [paniagua et al. 2006; ando et al. 2006, in vivo] endothelial cell # anti-thrombosis epithelial cell # antigen presentation # apoptosis # cytokine production alveolar macrophage # tnf α production # chemokine production neutrophil # superoxide production # proteinase secretion lymphocyte # lymphokine production # autoantibody production # complement activation fibroblast # myofibroblast differentiation # migration and proliferation # provisional ecm secretion # angiotensin ii secretion 246 kameda et al drug target insights 2007:2 aptamer interfering pdgf-r signaling, and i n h i b i t o r s o f p d g f r t y r o s i n e k i n a s e o r downstream signaling molecules. among many candidates, imatinib possesses some advantages in: 1) clinical use with well acceptable tolerability; 2) additional inhibition of abl and c-kit, which leads to the suppression of tgf-β and mast cell activity, respectively. thus, imatinib is likely to play a multipotent role in the treatment of various systemic rheumatic diseases (table 1). and indeed, several clinical trials examining the effi cacy of imatinib for skin sclerosis, ild and pah are ongoing. to date, the principal of the treatment for systemic rheumatic diseases has been chiefl y targeting leukocytes. thus, future therapeutic strategies should focus on other cell types, including fi broblasts, epithelial cells and endothelial cells (fig. 2). combined therapy of antiinfl ammatory and anti-fi brotic agents may shed light on the treatment of refractory diseases described in this review and further improve the long-term survival of patients with systemic rheumatic diseases. references abdollahi, a., li, m., ping, g. et al. 2005. inhibition of platelet-derived growth factor signaling attenuate pulmonary fi brosis. j. exp. med., 201:925–35. american thoracic society. 2000. idiopathic pulmonary fi brosis: diagnosis and treatment. international consensus statement. am. j. respir. crit. care med., 161:646–64. american thoracic society; european respiratory society. 2002. american thoracic society/european respiratory society interstitial multidisciplinary consensus classifi cation of the idiopathic interstitial pneumonias. am. j. respir. crit. care med., 165:277–304. ando, w., hashimoto, j., nampei, a. et al. 2006. imatinib mesylate inhibits osteoclastogenesis and joint destruction in rats with collageninduced arthritis (cia). j. bone miner. metab., 24:274–82. aono, y., nishioka, y., inamiya, m. et al. 2005. imatinib as a novel antifi brotic agent in bleomycin-induced pulmonary fi brosis in mice. am. j. respir. crit. care med., 171:1279–85. baroni, s.s., santillo, m., bevilacqua, f. et al. 2006. stimulatory autoantibodies to the pdgf receptor in systemic sclerosis. n. engl. j. med., 354:2667–76. bouros, d., wells, a.u., nicholson, a.g. et al. 2002. histopathologic subsets of fi brosing alveolitis in patients with systemic sclerosis and their relationship to outcome. am. j. respir. crit. care med., 165:1581–6. braun-moscovici, y., nahir, a.m. and balbir-gurman, a. 2004. endothelin and pulmonary arterial hypertension. semin. arthritis rheum., 34:442–53. capdeville, r., buchdunger, e., zimmermann, j. and matter, a. 2002. glivec (sti571, imatinib), a rationally developed, targeted anticancer drug. nat. rev. drug discov., 1:493–502. carroll, m., ohno-jones, s., tamura, s. et al. 1997. cgp57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing bcr-abl, tel-abl, and tel-pdgfr fusion proteins. blood, 90:4947–52. chaudhary, n.i., schnapp, a. and park, j.e. 2006. pharmacologic differentiation of infl ammation and fi brosis in the rat bleomycin model. am. j. respir. crit. care med., 173:769–76. choy, e.h. and panayi, g.s. 2001. cytokine pathways and joint infl ammation in rheumatoid arthritis. n. eng j. med., 344:907–16. daniels, c.e., wikes, m.c., edens, m. et al. 2004. imatinib mesylate inhibits the profi brogenic activity of tgf-β and prevents bleomycin-mediated lung fi brosis. j. clin. invest., 114:1308–16. demetri, g.d., von mehren, m., blanke, c.d. et al. 2002. effi cacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. n. engl. j. med., 347:472–80. denton, c.p., humbert, m., rubin, l. and black, c.m. 2006. bosentan treatment for pulmonary arterial hypertension related to connective tissue disease: a subgroup analysis of the pivotal clinical trials and their open-label extensions. ann. rheum. dis., 65:1336–40. denton, c.r., merkel, p.a., furst, d.e. et al. 2007. recombinant human anti-transforming growth factor β1 antibody therapy in systemic sclerosis. a multicenter, randomized, placebo-controlled phase i/ii trial of cat-192. arthritis rheum., 56:323–33. distler, j.h.w., jüngel, a., huber, l.c. et al. 2007. imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fi brosis. arthritis rheum., 56:311–22. druker, b.j., tamura, s., buchdunger, e. et al. 1996. effects of a selective inhibitor of the abl tyrosine kinase on the growth of bcr-abl positive cells. nat. med., 2:561–6. druker, b.j., talpaz, m., resta, d.j. et al. 2001a. effi cacy and safety of a specifi c inhibitor of the bcr-abl tyrosine kinase in chronic myeloid leukemia. n. engl. j. med., 344:1031–7. druker, b.j., sawyers, c.l., kantarjian, h. et al. 2001b. activity of a specifi c inhibitor of the bcr-abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome. n. engl. j. med., 344:1038–42. eklund, k.k. and joensuu, h. 2003. treatment of rheumatoid arthritis with imatinib mesylate: clinical improvement in three refractory cases. ann. med., 35:362–7. firestein, g.s. and zvaifl er, n.j. 2002. how important are t cells in chronic rheumatoid synovitis? ii. t cell-independent mechanisms from beginning to end. arthritis rheum., 46:298–308. floege, j., ostendorf, t., janssen, u. et al. 1999. novel approach to specifi c growth factor inhibition in vivo. antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. am. j. pathol., 154:169–79. ghofrani, h.a., seeger, w. and grimminger, f. 2005. imatinib for the treatment of pulmonary arterial hypertension. n. engl. j. med., 353:1412–3. gilbert, r.e., kelly, d.j., mckay, t. et al. 2001. pdgf signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis. kidney int., 59:1324–32. heldin, c-h. and westermark, b. 1999. mechanism of action and in vivo role of platelet-derived growth factor. physiol. rev., 79:1283–316. imamura, f., aono, h., hasunuma, t. et al. 1998. monoclonal expansion of synoviocytes in rheumatoid arthritis. arthritis rheum., 41:1979–86. johnson, r.j., raines, e.w., floege, j. et al. 1992. inhibition of mesangial cell proliferation and matrix expression in glomerulonephritis in the rat by antibody to platelet-derived growth factor. j. exp. med., 175:1413–6. kameda, h., risinger, j.i., han, b-b. et al. 2001. expression of gab1 lacking the preckstrin homology domain is associated with neoplastic progression. mol. cell. biol., 21:6895–905. kameda, h., nagasawa, h., ogawa, h. et al. 2005. combination therapy with corticosteroids, cyclosporine a, and intravenous pulse cyclophosphamide for acute/subacute interstitial pneumonia in patients with dermatomyositis. j. rheumatol., 32:1719–26. kameda, h. and takeuchi, t. 2006. recent advances in the treatment of interstitial lung disease in patients with polymyositis/dermatomyositis. endocr. metab. immune. disord. drug targets, 6:409–15. 247 pdgf as a target for systemic autoimmune diseases drug target insights 2007:2 kameda, h., ishigami, h., suzuki, m. et al. 2006. imatinib mesylate inhibits proliferation of rheumatoid synovial fibroblast-like cells and phosphorylation of gab adapter proteins activated by platelet-derived growth factor. clin. exp. immunol., 144:335–41. kondo, h. 2001. vascular disease in mixed connective tissue disease (mctd). intern. med., 40:1176. lafyatis, r., remmers, e.f., roberts, a.b. et al. 1989. anchorageindependent growth of synoviocytes from arthritic and normal joints. stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids. j. clin. invest., 83:1267–76. matsuda, m., shikata, k., makino, h. et al. 1997. gene expression of pdgf and pdgf receptor in various forms of glomerulonephritis. am. j. nephrol., 17:25–31. miyachi, k., ihara, a., hankins, r.w. et al. 2003. effi cacy of imatinib mesylate (sti571) treatment for a patient with rheumatoid arthritis developing chronic myelogenous leukemia. clin. rheumatol., 22:329–32. müller-ladner, u., kriegsmann, j., gay, r.e. et al. 1995. oncogenes in rheumatoid arthritis. rheum dis. clin. north am., 21:675–90. müller-ladner, u., kriegsmann, j., franklin, b.n. et al. 1996. synovial fi broblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into scid mice. am. j. pathol., 149:1607–15. müller-ladner, u., gay, r.e. and gay, s. 2000. activation of synoviocytes. curr. opin. rheumatol., 12:186–94. ohba, t., takase, y., ohhara, m. et al. 1996. thrombin in the synovial fl uid of patients with rheumatoid arthritis mediates proliferation of synovial fi broblast-like cells by induction of platelet-derived growth factor. j. rheumatol., 23:1505–11. östman, a. and heldin, c-h. 2001. involvement of platelet-derived growth factor in disease: development of specifi c antagonists. adv cancer res., 80:1–38. paniagua, r.t., sharpe, o., ho, p.p. et al. 2006. selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis. j. clin. invest., 116:2633–42. paniagua, r.t. and robinson, w.h. 2007. imatinib for the treatment of rheumatic diseases. nat. clin. pract. rheumatol., 3:190–1. pietras, k., sjöblom, t., rubin, k. et al. 2003. pdgf receptors as cancer drug targets. cancer cell., 3:439–43. remmers, e.f., sano, h. and wilder, r.l. 1991. platelet-derived growth factors and heparin-binding (fi broblast) growth factors in the synovial tissue pathology of rheumatoid arthritis. semin. arthritis rheum., 21:191–9. rice, a.b., moomaw, c.r., morgan, d.l. and bonner, j.c. 1999. specifi c inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fi brosis in rats. am. j. pathol., 155:213–21. rubin, k., terracio, l., ronnstrand, l. et al. 1988. expression of plateletderived growth factor receptors is induced on connective tissue cells during chronic synovial infl ammation. scand. j. immunol., 27:285–94. sadanaga, a., nakashima, h., matsutani, k. et al. 2005. amelioration of autoimmune nephritis by imatinib in mrl/lpr mice. arthritis rheum., 52:3987–96. sandler, c., joutsiniemi, s., lindstedt, k.a. et al. 2006. imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fi broblasts. biochem. biophys. res. commun., 347:31–5. sano, h., engleka, k., mathern, p. et al. 1993. coexpression of phosphotyrosine-containing proteins, platelet-derived growth factor-b, and fi broblast growth factor-1 in situ in synovial tissues of patients with rheumatoid arthritis and lewis rats with adjuvant or streptococcal cell wall arthritis. j. clin. invest., 91:553–65. schermuly, r.t., dony, e., ghofrani, h.a. et al. 2005. reversal of experimental pulmonary hypertension by pdgf inhibition. j. clin. invest., 115:2811–21. takeuchi, t., amano, k., kameda, h. and abe, t. 2005. anti-tnf biological agents in rheumatoid arthritis and other infl ammatory diseases. allergology international, 54:191–202. thornton, s.c., por, s.b., penny, r., richter, m., shelley, l. and breit, s.n. 1991. identifi cation of the major fi broblast growth factors released spontaneously in infl ammatory arthritis as platelet-derived growth factor and tumor necrosis factor-alpha. clin. exp. imunol., 86:79–86. vittal, r., zhang, h., han, m.k., moore, b.b., horowitz, j.c. and thannickal, v.j. 2007. effects of the protein kinase inhibitor, imatinib mesylate, on epithelial/mesenchymal phenotypes: implications for treatment of fi brotic diseases. j. pharmacol. exp. ther., 321:35–44. watanabe, n., ando, k., yoshida, s. et al. 2002. gene expression profi le analysis of rheumatoid synovial fi broblast cultures revealing the overexpression of genes responsible for tumor-like growth of rheumatoid synovium. biochem. biophys. res. commun., 294:1121–9. white, b., moore, w.c., wigly, f.m., xiao, h.q. and wise, r.a. 2000. cyclophosphamide is associated with pulmonary function and survival benefi t in patients with scleroderma and alveolitis. ann. intern. med., 132:947–54. wollheim, f.a. 2007. treatment of pulmonary fi brosis in systemic sclerosis: light at the end of the tunnel? arthritis rheum., 56:9–12. yamanishi, y. and firestein, g.s. 2001. pathogenesis of rheumatoid arthritis: the role of synoviocytes. rheum dis. clin. north am., 27:355–71. yoshida, m., sakuma-mochizuki, j., abe, k. et al. 1999. in vivo gene transfer of an extracellular domain of platelet-derived growth factor β receptor by the hvj-liposome method ameliorates bleomycininduced pulmonary fi brosis. biochem. biophys. res. commun., 265:503–8. zoja, c., corna, d., rottoli, d., zanchi, c., abbate, m. and remuzzi, g. 2006. imatinib ameliorates renal disease and survival in murine lupus autoimmune disease. kidney int., 70:97–103. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preserveepsinfo true /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /apply /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 300 /colorimagedepth -1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasgrayimages false /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 300 /grayimagedepth -1 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasmonoimages false /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 1200 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects false /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /unknown /description << /jpn <feff3053306e8a2d5b9a306f30019ad889e350cf5ea6753b50cf3092542b308030d730ea30d730ec30b9537052377528306e00200050004400460020658766f830924f5c62103059308b3068304d306b4f7f75283057307e305930023053306e8a2d5b9a30674f5c62103057305f00200050004400460020658766f8306f0020004100630072006f0062006100740020304a30883073002000520065006100640065007200200035002e003000204ee5964d30678868793a3067304d307e305930023053306e8a2d5b9a306b306f30d530a930f330c8306e57cb30818fbc307f304c5fc59808306730593002> /fra <feff004f007000740069006f006e007300200070006f0075007200200063007200e900650072002000640065007300200064006f00630075006d0065006e00740073002000500044004600200064006f007400e900730020006400270075006e00650020007200e90073006f006c007500740069006f006e002000e9006c0065007600e9006500200070006f0075007200200075006e00650020007100750061006c0069007400e90020006400270069006d007000720065007300730069006f006e00200070007200e9007000720065007300730065002e0020005500740069006c006900730065007a0020004100630072006f0062006100740020006f00750020005200650061006400650072002c002000760065007200730069006f006e00200035002e00300020006f007500200075006c007400e9007200690065007500720065002c00200070006f007500720020006c006500730020006f00750076007200690072002e0020004c00270069006e0063006f00720070006f0072006100740069006f006e002000640065007300200070006f006c0069006300650073002000650073007400200072006500710075006900730065002e> /deu <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> /ptb <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> /dan <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> /nld <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> /esp <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> /suo <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> /ita <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> /nor <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> /sve <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> /enu <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> >> >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice the role of resistin in patients with primary fibromyalgia mohammed h. alosami  j fac med baghdad 152                                                                  vol.53, no.2, 2011  the role of resistin in patients with primary fibromyalgia mohammed h. alosami * cabm, ficms (med), ficms (rheum) summary: background: fibromyalgia syndrome (fms) is a common rheumatologic syndrome with multiple manifestations and associated with many diseases, it characterized by chronic wide spread muscular pain and tenderness. resistin is an adipokine discovered in 2001 and considered as a link between obesity and type 2 diabetes by antagonizing and resisting the action of insulin as it name encompass ;resistin (resist insulin) . it is secreted mainly from adipose tissue in mice and from white blood cells besides the adipose tissue in humans. research reviled that resistin has a role in some inflammatory diseases like rheumatoid arthritis (ra). objective: the objective of this study is to measure resistin concentration and to evaluate its role in the pathogenesis of primary fms. patients and methods: fifty patients with primary fms were included in this study (37 females and 13 males) the age range of (17-65) years (mean ± sd) (40.13 ± 12.0) years, and thirty healthy individuals volunteers (21 females and 9 males), whose age and sex matching with fms patients, age ranging (18 63) years, (mean ± sd) (36.1 ±10.0) years. resistin concentration was measured in sera of patients and controls by elisa kit. anthropometric measurements like body mass index (bmi) and waist circumference (wcr) were taken, besides other features like sleep disturbance, emotional distress, and fatigue were reported. results: resistin concentration was higher in fms patients than controls (2.15±0.9) ng /ml vs. (1.57±0.6) ng /ml. this elevation was highly significant statistically (p=0.003).other measurements in patients group like bmi, wcr were (28.39±5.0) kg/m2 and (100.34±13.21) cm respectively .these values were highly significant when compared to their control group (p=0.002) and (p=0.008) respectively . serum resistin level was positively correlated with both waist circumference (r=0.411,p=0.003) and age (r=0.405 ,p=0.004) . waist circumference was positively correlated with age (r=0.604, p=0.000).clinical features like sleep disturbance, emotional distress ,and fatigue showed highly significant difference between the two groups . no significant differences were reported with respect to age and sex. conclusion: the result of the current study suggest that resistin might have a role in the pathogenesis of fms. keywords: resistin, fibromyalgia. introduction: according to the criteria of the american college of rheumatology (acr) ,fibromyalgia (fms) is characterized by chronic widespread pain in all 4 quadrants of the body at least 3 months duration associated with tender points and associated with constitutional symptoms of fatigue, aching, and non restorative sleep(1). the etiology of fms remains elusive. resistin is an adipokine, a member of secretary protein family known as resistin like molecules (relms) (2), the family is characterized by highly conserved cystienes – rich c terminus in which the spacing of cystienes is invariant, four members of this family are found in mice which these are (resistin, relmα, relmβ, and relmγ), only two counterparts were found in human (resistin and relmβ) (3). resistin is 114 amino acid peptide with a molecular weight of (12.5 kda) secreted as disulfide-link homodimer(4) . many hormones and molecules can regulate resistin gene expression, such as thiozolidindiones (tzds), insulin, glucose, glucocorticoid, growth hormone and others (5,6). *dept. of medicine, college of medicine, university of baghdad although resistin was firstly postulated to contribute to insulin resistance more evidence indicated that it may also be involved in inflammatory process, some proinflammatory agents ,such as (tumor necrosis factorα),(interleukin-6) and lipopolysaccharide (lps), can regulate resistin gene expression in human peripheral blood mononuclear cells in vivo and in vitro potentially mediated via the integration of nuclear factor kappa – light chain –enhancer of activated b cells (nf-kb) signaling pathway (7). nf-kb is a protein complex that controls the transcription of dna and is found in almost all cell types and involved in cellular response to stimuli such as stress ,cytokines ,free radicals ,oxidized ldl-c ,and bacterial or viral antigens and play a key role in regulating the immune response to infection (8) .some researchers investigated the role of resistin in inflammation related diseases such as rheumatoid arthritis and found that the synovial fluid from patient with(ra) showed significantly higher level of resistin compared with control samples and resistin level in ra synovial fluid was positively correlated with synovial leukocyte count fac med baghdad 2011; vol. 53, no. 2 received dec. 2010 accepted may. 2011 original article the role of resistin in patients with primary fibromyalgia mohammed h. alosami  j fac med baghdad 153                                                                  vol.53, no.2, 2011  and il-6 level(9) ,resistin found to be accumulated in inflamed joints of ra patient (10).in the present study a trail was made to investigate the role of resistin in patients with primary fms and its association with the pathogenesis of the syndrome and if there is association between circulating resistin and body mass index , waist circumference ,age and gender in fms. patients and methods: subjects: the study has included 50 patients with primary fms (37 females and 13 males) the age range of (17-65) years (mean ± sd) (40.13 ± 12.0) years. the clinical diagnosis of these patients was confirmed by consultant rheumatologists of the baghdad teaching hospital according to the acr 1990 criteria for the diagnosis of fms. patients with primary fibromyalgia were included in this study. thirty healthy individuals volunteers (21 females and 9 males), whose age and sex matching with fms patients, age ranging (18 63) years, (mean ± sd) (36.1 ±10.0) years.they had no musculoskeletal complaints or lower back pain and did not seek any medical help for pain. a pretested questionnaire was designed to obtain information from both patients and control group about past medical and drug history. inclusion criteria:known cases of fms approved by clinical, laboratory, and radiological diagnosis. patients on medical treatments that never affect the laboratory tests. eexxcclluussiioonn ccrriitteerriiaa:: diabetes mellitus (dm) ,rheumatoid arthritis (ra),systemic lupus erythematosis (sle), sjögren's syndrome (ss), osteoarthritis (oa),sleep apnea ,patients on steroid therapy, chronic renal failure, chronic liver disease, previous breast surgery, inflammatory systemic disease or infection, serious cardiopulmonary, vascular or other internal medical condition. medication that may influence with the results (e.g. local corticosteroids, biological agents). blood collection: after overnight fasting venous blood samples (10 ml ) were aspirated from each patient and control at 9.00 am 12.00 pm using disposable plastic syringes . seven ml of the blood samples were allowed to clot in plane tubes at room temperature for (20-30) minutes. sera were separated by centrifugation at 3000 rpm for 10 minutes. for each sample, the serum was transferred into plastic plane tubes and kept frozen at (-20 ْ◌c) until the time of assay. the rest 3 ml blood samples were collected in edta tubes for esr, pcv, and wbc tested by routine work to exclude inflammatory reasons. methods: serum resistin was determined by drg elisa kit, usa which is solid phase enzymelinked immunosorbent assay based on the sandwich principle .kit used was from international inc-usa. pain was measured using visual analogue scale (vas). statistical analysis: to compare the significant of the difference in the means values at any two patients and controls, spss (social process statistical system) was used. student t-test was applied (p < 0.05) was considered statistically significant, and the correlation coefficient ((rr)) tteesstt iiss uusseedd ttoo ddeessccrriibbee tthhee aassssoocciiaattiioonn bbeettwweeeenn ddiiffffeerreenntt ppaarraammeetteerrss ssttuuddiieedd results: as shown in table-1, there was significantly high concentration of resistin in patients with fms than healthy controls (p=0.003). patients and controls were age, sex match (p=0.124), (p=0.698) respectively.there was a statistically significant difference in bmi and wcr between patients and controls (p=0.002), (p=0.008) respectively. table(1):shows all mean values of the parameters measured for patients and control group, (mean± sd) values for age ,bmi, resistin ,wcr , in fms group (n=50), and control group (n=30) . figure (1):mean value for serum resistin level in fms patients and control group serum resistin level was positively correlated with both wcr (r = 0.411) , (p = 0.003) , figure (2) and age ( r = 0.405 , p =0.004 ) figure (3) .waist circumference was positively correlated with age in this study ( r = 0.604 ),( p < 0.000 ) figure (4). in this study there is significant positive correlation between bmi (kg/m2) and age (year),(r=0.397) ,(p=0.004) , figure (5). characteristic fms patients n=50 mean ± sd hc n =30 mean ± sd pvalue sig resistin (ng/ml) 2.15 ± 0.90 1.57 ± 0.60 0.003 hs bmi ( kg/m2) 28.39 ± 5.0 25.14 ± 3.19 0.002 hs wcr (cm) 100.34 ± 13.21 92.63 ± 10.76 0.008 hs age (year) 40.18 ± 12.08 36.10 ± 10.02 0.124 ns the role of resistin in patients with primary fibromyalgia mohammed h. alosami  j fac med baghdad 154                                                                  vol.53, no.2, 2011  figure (2) correlation between serum resistin and wcr in fms patients(r = 0.411) , (p =0.003), (n =50) . figure (3) : correlation between resistin and age in fms patients (r= 0.405), (p= 0.004 ) , (n=50) . figure(4): correlation between waist circumference and age in patients with fms ( r=0.604 , ( p< 0.000 ) (n = 50 ). figure (5) : correlation between bmi (kg/m2) and age (year),(r=0.397), (p=0.004),(n=50) . discussion: the role of resistin in fms was not assessed by previous studies to the best of our knowledge. a significant positive correlation between resistin and waist circumference is found in this study figure (2). the significant positive correlation between resistin and wcr comes in agreement with other previous studies (11,12) , since resistin expression was found to be higher in human abdominal adipose tissue than other adipose tissue depots .resistin mrna concentration were similar in both abdominal and omental depots while its concentration was lower in thighs leading to the conclusion that abdominal fat could explain the increased risk of type 2 diabetes associated with central obesity (13) .in this study we found a significant positive correlation between resistin and age figure (3), a significant positive between correlation between waist circumference and age figure (4), and a significant positive correlation between bmi and age figure (5). only two studies mentioned that resistin correlates with age but without any explanation (12,14).the correlation between waist circumference and age determined with several factors: 1. the effect of sex hormones : since the adipocytes show receptors attenuated to the androgen and estrogen , testosterone inhibits the activity of lipase lipoprotein and stimulates mobilization of triglyceride (tgs) in the adipocytes , which leads to lower accumulation of this tissue in males (15) , estrogen , like testosterone , has control on the lipase lipoprotein and reduce adipocyte growth in females , this effect of sex hormones suggests that a decrease in their secretion may promote accumulation of intraabdominal adipose tissue (iaat) once menopause occurs in women and andropause in men (16). as adipocytes grow larger they become dysfunctional and large adipocytes are insulin resistance , hyperlipolytic and resistant to the antilipolytic effect of insulin .visceral adipose tissue contains greater number of large adipocytes while subcutaneous adipose tissue contain small adipocytes which are more insulin-sensitive and have high avidity for free fatty acids and tgs uptake preventing their deposition in non-adipose tissue (17,18). 2. the effect of the hypothalamic –pituitaryadrenal axis : the increased fat in iaat is also related to the action of the (hpa –axis), and stimulation of the hypothalamus produces corticotropin-releasing hormone which stimulates the secretion of cortisol in adrenal glands which favors the accumulation of tgs in the iaat and inhibits their mobilization (15). in cases with chronic stress leads to elevated cortisol levels that leads to accumulation of visceral adipose tissue (19) .its noteworthy that among the anthropometric measurements, wcr has been promoted as a tool to establish distribution of adipose tissue in the central area and the risk of developing metabolic syndrome in adults (20).plasma level of resistin , produced by adipose tissue, might be present at high circulating level in subject with high fat content (12). in the present the role of resistin in patients with primary fibromyalgia mohammed h. alosami  j fac med baghdad 155                                                                  vol.53, no.2, 2011  study patients are predominantly overweight (40%) and obese (36%) and their waist circumference is higher than the healthy controls (mean ±sd) , (100.34 ± 13.21) cm vs. (92.63 ± 10.67) cm table (1) .in human , evidence was accumulated that resistin expression is high in mononuclear peripheral blood cells and other non adipocytes of adipose tissue than in adipose tissue themselves (21,22) .therefore absolute plasma levels of resistin were increased with generalized obesity and its relationship with clinical parameter of visceral obesity was not linear. this could explain some of the inconsistent finding in human studies (22) .in this study resistin did not correlate with pain intensity measured by vas or any clinical manifestation or any of the symptoms of fms .in conclusion resistin has been found to be significantly higher in fms patients than healthy subjects and its concentration correlate strongly with waist circumference , for further investigation detection of the expression of resistin, peripheral blood mononuclear cells, muscle biopsies, and muscle biopsies of tender points (if possible) in patients with primary fms this should add more information on the role of this adipokin in the pathogenesis of fibromyalgia. references: 1. wolfe f, smythe h, yunus m, et al. the american college of rheumatology 1990 criteria for the classification of fibromyalgia. report of the multicenter criteria committee . arthritis rheum .1990 ; 33:160–172. 2 . steppan cm, brown ej, wright cm, bhat c, banerjee r, gregory h lazar ma, et al. a family of tissue-specific resistin-like molecules. pnas. 2001; 98(2):501-506. 3 steppan cm, lazar ma. the current biology of resistin. j intern med. 2004; 255:439-447. 4. steppan cm, bailey st, bhat s, et al. the hormone resistin links obesity to diabetes. nature. 2001; 409:307-312. 5. way jm, gorgun cz, tong q, et al. adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferatoractivated receptor gamma agonists. j biol chem.2001 ;276 :25651-25653. 6. andrews rc, walker br. glucocorticoids and insulin resistance: old hormones, new targets. clin sci (lond). 1999;96:513-523. 7. kusminski cm, dasilva nf, creely sj, et al. the in vitro effects of resistin on the innate immune signaling pathway in isolated human subcutaneous adipocytes. the j clin endocrinol & metabol.2007; 92(1): 270-276. 8. perkins nd. integration cell-signalling pathways with nf-kb and ikk function .nat .rev mol cell biol. 2007; 8(1):49-62. 9. otero m, lago r, gomez r, lago f, dieguez c, gómez-reino j and gualillo o. changes in plasma levels of fat-derived hormones adiponectin, leptin, resistin and visfatin in patients with rheumatoid arthritis. ann rheum dis .2006;65;1198-1201. 10. canoruç n, kale e, turhanoglu a, özmen ş, ogün c, kaplan a. plasma resistin and leptin levels in overweight and lean patients with rheumatoid arthritis.turk j med sci.2009; 39(3):447-451. 11. al-shamma z. the role of obesity related resistin in type 2 diabetes mellitus via biochemical and molecular genetic study .university of baghdad .msc . thesis 2009. 12. won jc, park cy, lee wy, lee es, oh sw, park sw. association of plasma levels of resistin with subcutaneous fat mass and markers of inflammation but not with metabolic determinants or insulin resistance j korean med sci .2009;24;695-700. 13. mcternan cl, mcternan pg, harte a l, levick pl, barnett ah and kumar s. resistin, central obesity, and type2 diabetes.lancet.2002;359, 46–47. 14 al-harithy rn. strong association between serum levels of leptin and resistin in non-diabetic saudi adult women. kuwait med. j.2007; 39(1):3135. 15. björntrop p. do stress reactions cause abdominal obesity and comorbidies? .obes rev.2001;2:73-86. 16. ramos –ibanez n. intra-abdominal adipose tissue :growth assessment and association with the development of metabolic alterations in children and adolescent. biol med hosp infant mex .2009;66:492-501. 17. marin p, anderson b, ottosson m, olbe l, chowdhurg b, kvist h, holm g, sjöström l, björntorp p. the morphology and metabolism of intra-abdominal adipose tissue in men. metaolism.1992; 41:1241-1248. 18. hisra a, vikram nk. clinical and pathophysiological consequences of abdominal adiposity and abdominal adipose tissue depots. nutrition . 2003; 19:457-466. 19. world health organization. obesity: prevalence and managing for the global epidemic. report of a who consultation .3-5 jun 1997. geneva: who; 1998.p.12. 20. lehrke m, reilly mp, millington sc, iqbal n, rader dj, lazar ma. an inflammatory cascade leading to hyperresistinemia in humans. plos med.2004; 1(2):161-168. 21. savage db, sewter cp, kv, klenk es, segal dg, vidal-puig a, considinse , o'rahilliy s. resistin /fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-gamma in humans. diabetes .2001; 50: 2199-2202. 22. patel l, buckels ac, kinghom ij, murdock pr, holbrook jd, pulmpton c, macphee ch, smith sa. resistin is expressed in human macrophage and directly regulated by ppar gamma activators. biochem . biophy res. commun.2003; 300:472-476. iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 63 preparation and evaluation of oral disintegrating tablets of ketoprofen by dirct compression saddam j.nasser *,1 ,laith h. sameen * , mowafaq m.ghareeb * *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract ketoprofen is a non-steroidal anti-inflammatory (nsaid) drug with analgesic, antiinflammatory, and antipyretic effects. it is widely used in the treatment of inflammation and pain associated with rheumatic disorders such as rheumatoid arthritis, osteoarthritis, and in soft tissue injury. the purpose of this study was to prepare an oral disintegrating tablets of ketoprofen by simple method. the tablets were prepared by direct compression method and different ratios of various subliming agents or superdisintegrants were incorporated. then these tablets were evaluated for hardness, friability, weight variation, water absorption ratio, disintegrating time and dissolution time. the results showed that formula f11 batch had short disintegration time (18.0 sec) with good physical properties. this formula demonstrated a promised potentials for rapid absorption, improved bioavailability, effective therapy and patient compliance. key words: rapid disintegrating tablets, ketoprofen, direct compression, superdisintegrants. تحضير وتقييم حبت الكتوبروفين سريعت التفكك بالفم بطريقت الكبس المباشر صدام جمعت ناصر ،*1 * ، موفق محمد غريب*ليث حمزة سمين و ، بغذاد ، انؼراقبغذاد تجايؼ ، تانصٍذنتكهٍ ، انصٍذالٍَاثفرع * الخالصة افط نهحرارة . اَه شائغ االسخؼًال بؼالج دواء انكٍخىبروفٍٍ هى يضاد انخهاباث غٍر سخٍروٌذي يغ اثر يسكٍ وخ االنخهاباث واالالو انًصاحبت اليراض انًفاصم كانرثىي وانخهاب انؼظاو وجروح االَسجت انرخىة . هذف انذراست هى ححضٍر يىاد وحراكٍس حبىب سرٌؼت انخفخج بانفى بذوٌ اسخؼًال انًاء بطرٌقت انكبس انًباشر انبسٍطت وباسخؼًال ػذة ػهى شكم انكٍخىبروفٍٍ خضؼج انحبىب انًحضرة انى ػذة اخخباراث يُها انصالبت وانهشاشت واخخالف يخخهفت يٍ انًىاد انًخسايٍت ويسبباث انخفخج انًخطىرة. w/w( انًخكىَه يٍ حركٍس ) 11انىزٌ وَسبت ايخصاص انًاء ووقج انخفخج وانخحهم . أثبخج انُخائج اٌ انخركٍبت )ف 11%(crospovidone ٍس )وحركw/w 11% (microcrystaline cellulose ph(102)( ثاٍَت يغ 1..1نها اقصر وقج حفخج) .خىاص فٍسٌاوٌت جٍذة . وهً حركٍبت واػذة نسٌادة االيخصاص وححسٍ انخىافر انحٍىي وفؼانٍت انذواء وحقبم انًرٌط سريعة التفكك .حبوب سريعة التفكك ، كيتوبروفين ، الكبس المباشر ، مواد الكلمات المفتاحية : introduction the conventional oral dosage forms (tablet and capsule) have wide acceptance up to 50-60 % of the total dosage forms (1) . tablet is still most popular dosage form because of its convenience in terms of self administration, compactness and ease in manufacturing. however, geriatric and pediatric patients experience difficulty in swallowing conventional tablets, which leads to poor patient compliance (1) . orally disintegrating tablets are also called as orodispersible tablets (odts), quick disintegrating tablets, mouth dissolving tablets, fast disintegrating tablets, fast dissolving tablets, rapid dissolving tablets, porous tablets, and rap melts. however, of all the above terms, united states pharmacopoeia (usp) approved these dosage forms as odts. recently, european pharmacopoeia has used the term orodispersible tablet for tablets that disperses readily and within 3 min in mouth before swallowing (2) .to overcome this problem, scientists have developed innovative drug delivery systems known as mouth dissolving tablets. their characteristic advantages such as administration without water, anywhere, anytime which lead to their suitability to geriatric and pediatric patients. they are also suitable for the mentally ill, the bedridden and patients who do not have easy access to water. the benefits, in terms of patient compliance, rapid onset of action, increased bioavailability and good stability made these tablets popular as a dosage form in the current market (3,4) . a non-steroidal antiinflammatory (nsaid) has been indicated for various painful indications (5) and proved as effective as other nsaids with lower indications of gastro-intestinal adverse effects and thus, resulted in a greater compliance with treatment (6) . ketoprofen is propionic acid derivative anti-rheumatic drug with wellknown anti-inflammatory, antipyretic and analgesic properties to treat mild to moderate pain and dysmenorrhea the structure of ketoprofen as shown in (fig.1) (7, 8) . 1 corresponding author email :saddamjumaa @ yahoo.com received : 20/11/2011 accepted : 27/6/2012 iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 64 also it is an inhibitor of prostaglandin synthetase (9) . however, it is not freely soluble in water and causes systemic disturbances in gastrointestinal tract (10) . in the present study, an attempt has been made to develop mouth dissolving tablets of ketoprofen using different subliming agents on the disintegration of orodispersible tablets with good physical properties and acceptance. figure 1: ketoprofen chemical structure material and methods materials ketoprofen powder, crospovidone (cp), and croscarmellose sodium (ccs) were purchased from 3b pharmaceutical (wuhan) international co. ltd, china. microcrystalline cellulose (avicel ph 102)&(avicel ph 101) were purchased from hekma drug industry, jordan. magnesium stearate, mannitol, ammonium bicarbonate were purchased from riedel-de-haen ag seelze, germany. camphor was purchased from evans medical ltd, liverpool, england. all other materials were of analytical grade. methods formulation of orodispersible tablets of ketoprofen the orodispersible tablets of ketoprofen were prepared using the camphor and ammonium bicarbonate as subliming agent, menthol and crospovidone (cp), and croscarmellose sodium (ccs), and sodium starch glycolate (ssg) as superdisintegrant, microcrystalline cellulose (mcc) and mannitol as diluent, sodium saccharin as sweetening agent, and talc as flow promoter and magnesium stearate as lubricant, the composition of each batch is shown in table 1. the ketoprofen (50mg) and excipients were passed through sieve (#80) to ensure the better mixing. the powder was compressed using manesty, type f3 compression machine equipped with 8 mm round punch by direct compression technique. sublimation was performed from tablets contain subliming agents at 60ºc. a minimum of 50 tablets was prepared for each batch. table 1: composition of different batches of orodispersible tablets of ketoprofen material /mg formula no. f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 ketoprofen 50 50 50 50 50 50 50 50 50 50 50 50 camphor 4.5 (2.5%) w/w ammonium bicarbonate 4.5 (2.5%) w/w 9 (5%) w/w ccs 9 (5%) w/w 18 (10%) w/w 27 (15%) w/w ssg 9 (5%) 18 (10%) w/w 27 (15%) w/w cp 9 (5%) w/w 18 (10%) w/w 27 (15%) w/w (mcc102) 18 18 18 18 18 18 18 18 18 18 18 18 talc 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 sodium saccharin 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 mg stearate 1 1 1 1 1 1 1 1 1 1 1 1 mannitol q.s. to 180 180 180 180 180 180 180 180 180 180 180 180 iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 65 pre compression parameters angle of repose angle of repose was determined using funnel method (11) . the blend was poured through funnel that can be raised vertically until a maximum cone height (h) was obtained. radius of the heap (r) was measured and angle of repose was calculated using the formula; tan θ = h/r where, θ is the angle of repose, h is height of pile; r is radius of the base of pile. compressibility (carr’s) index an accurate weight of formula granules was poured into a volumetric cylinder to occupy a volume (v◦) and then subjected to a standard tapping procedure onto a solid surface until a constant volume was achieved (vf). the carr's index was calculated using following equation (11) . evaluation of the prepared orodispersible ketoprofen tablets weight variation randomly, twenty tablets were selected after compression and the mean weight was determined. none of the tablets deviated from the average weight by more than ±7.5%. uniformity of content the content of the prepared ketoprofen orodispersible tablets was determined. ten tablets were assayed individually. each tablet was crushed individually and dissolved in 100ml of methanol .the filtrated solution was diluted appropriately and the drug content was measured spectrophotometrically at 260nm using (carry uvvisible spectrophotometer) (11) . the requirement for this test is met if the amount of ingredient in each of the ten tablets lies within the range of (85-115) % of the drug present in each tablet. wetting time a piece of tissue paper (12cm x10.75cm) folded twice was placed in a petri dish (internal diameter=9cm) containing 10ml of buffer solution simulating saliva ph 6.8 and amaranth. a tablet was placed on the paper and the time taken for complete wetting was noted. three tablets from each formulation were randomly selected and the average wetting time was recorded (12) . hardness the crushing strength of the tablets was measured using a monsanto hardness tester. three tablets from each formulation batch were tested randomly and the average reading ± sd was recorded. friability twenty tablets were weighed and placed in a roche friabilator and the equipment was rotated at 25 rpm for 4 min. the tablets were taken out, dedusted and reweighed. the percentage friability of the tablets was calculated using following equation (13) . in vitro disintegration time the disintegration time was defined as the time in seconds taken for complete disintegration of the tablet with no palpable mass remaining in the apparatus was measure in second using artificial saliva as disintegration medium. six tablets were placed individually in each tube of disintegration test apparatus. the values reported are mean ± standard deviation (14, 15) . in vitro drug dissolution test dissolution rate was studied by using usp xxiii type-ii dissolution apparatus employing a paddle stirrer at 50 rpm the two experiments were done for dissolution using two different dissolution media 900 ml of ph( 6.8 ) phosphate buffer and 0.1 n hcl at 37±0.5ºc.one tablet was used in each test. a (5 ml) sample from dissolution medium was withdrawn at different time intervals (0, 1, 2, 4, 6, 8, 10, 15 and 20 min). the withdrawn sample was replaced by same amount of fresh dissolution medium to maintain sink conditions. the samples were filtered through 0.22 µm membrane filter and analyzed for drug content by measuring the absorbance at 260 nm using uv spectrophotometer (16) . statistical analysis the mean ± standard deviation of the experiments results were analyzed using one way analysis of variance (anova). iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 66 results and discussion ketoprofen tablets were prepared by direct compression method. twelve formulations were prepared with two different subliming agents or three different superdisinegrants. each superdisinegrants was used in three different concentrations (5% (w/w), 10% (w/w) and 15% (w/w)). table 2 shows the data obtained from the precompression evaluation of tablets. all batches of the tablets were evaluated for various pre and post compression parameters. precompression parameters like angle of repose, and compressibility index while post compression parameters such as hardness, friability, drug content, wetting time, water absorption ratio, and disintegration time were evaluated. post compression parameters are reported in table 3. all the above properties and value were near to boundary of standard limit except formula (f1) produced mechanically weak tablets with hardness and friability (2 kg/cm² and 1.21%) (13) .all the tablets maintained hardness in the range 3.17– 6.08 kg/cm 2 . the loss in total weight of the tablets due to friability was in the range of 0.26-0.88%. the drug content in different formulation was highly uniform and in the range of 99.60-99.92%. (13) .wetting time is used as an indicator of the ease of tablet disintegration and found to be 8.9-41sec. water absorption ratio ranged from 6.66106.1. the result in vitro disintegration time indicated that formula (f1) has shortest disintegration time this decrease in the disintegration time may be attributed to the increasing porosity of the tablet so that the porous hydrophilic matrix will easily pick up the disintegration medium and thus facilitating the wicking action of the superdisintegrant and bringing about rapid tablet disintegration and shorter wetting time (17) . but unfortunately with unacceptable hardness (2 kg/cm 2 ) and in general formulas based on subliming agent produce longer disintegration time than formulas used superdisintegrant thus for formulas (f4-f12) the shortest disintegration time was 18.04 for formula f11 which contain 10% crospovidone as superdisintegrant . an increasing in concentration of superdisintegrants over an optimum lead to increase in the disintegration time of orodispersible tablet. the reason behind this increase may be due to formulation of a viscous gel layer on the surface of tablet which prevents the further penetration of disintegration medium and hinder the disintegration (18) . this prevents the penetration of water to the core of the tablet. in contrast, cp has little tendency to form a gel (19) .the formula (f11) contain 10% of cp resulted in an improvement of both disintegration time and wetting time due to the property of cp and a significant difference (p<0.05) in the dt value of tablets in presence of the cp, ccs and ssg superdisintegrants (20) . from this can concluded that the superdisintegrant efficiency is in following descending order; crospovidone > sodium starch glycolate > croscaramellose, and 10% is the optimum percent for good disintegration.the formula (f11) was selected as the best formula that subjected for dissolution studies in comparison to conventional (toprec® tablet ) formula as shown in figure (2) .the results of dissolution study indicated that the selected formula and the conventional one shows release 100, 32.8% respectively at 10 minutes. table 2.: precompression parameters of ketoprofen orodispersible formulas formula code carr’s index (%) angle of repose(°) flow characters f1 10.00±0.50 28.75±0.39 good f2 30.29±0.41 29.41±1.55 good f3 30.83±0.76 29.41±1.55 good f4 31.71±0.62 29.87±0.76 good f5 29.83±0.28 31.60±1.27 passable f6 27.83±0.28 32.86±1.24 passable f7 33.20±0.14 30.73±1.51 passable f8 33.21±0.18 32.45±0.72 passable f9 33.61±0.34 29.42±0.76 good f10 35.70±0.81 32.00±1.92 passable f11 35.12±0.82 26.45±0.40 good f12 37.43±0.98 28.00±3.43 good iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 67 table 3.:post compression parameters of prepared ketoprofen orodispersible tablets formula code hardness (kg/cm²) friability (%) weight variation wetting times (sec.) water absorption ratio (%) in vitro disintegration times (sec.) f1 2.00 ± 0.10 1.21± 0.010 179 ± 1.14 12.25 ± 0.90 42.06 ± 27.86 14.51 ± 1.101 f2 4.00 ± 0.50 0.26± 0.008 179 ± 0.85 14.47 ± 2.54 42.88± 28.28 24.33 ± 1.506 f3 4.33 ± 0.76 0.61± 0.009 179 ± 1.14 41.00 ± 3.606 6.660 ± 3.360 51.50 ±1.761 f4 4.33 ± 0.58 0.88 ± 0.023 178 ± 1.48 21.90 ± 7.36 67.74 ± 11.87 23.95 ± 1.268 f5 3 .17 ± 0.29 0.78± 0.005 179 ± 0.94 21.01 ± 2.66 74.99 ± 0.638 34.09 ± 1.170 f6 3.33 ± 0.29 0.88± 0.013 179 ± 1.70 19.26 ± 1.58 98.68 ± 3.650 34.79 ± 0.944 f7 3.42 ± 0.38 0.84± 0.003 178 ± 1.41 21.00 ± 2.00 106.1 ± 0.630 25.06 ± 1.202 f8 3.50 ± 0.50 0.84± 0.003 178 ± 1.52 19.85 ± 2.55 104.9± 0.630 22.47 ± 3.224 f9 3.17 ± 0.29 0.85± 0.010 178 ± 1.41 22.00 ± 4.35 221.5 ± 8.146 29.17 ± 2.563 f10 6.08 ± 0.52 0.66± 0.010 178 ± 1.66 13.72 ± 2.29 92.55 ± 4.046 20.50 ± 0.548 f11 5.50 ± 0.50 0.75± 0.005 180 ± 1.79 8.950 ± 0.08 89.40 ± 1.189 18.04± 0.093 f12 5.17 ± 0.76 0.53± 0.028 179 ± 1.32 14.57 ± 2.34 98.48 ± 14.80 19.83 ± 3.267 figure 2: dissolution profile of ketoprofen odts from selected formula (f11) and conventional formula in phosphate buffer,( ph=6.8 )at 37°c ± 0.5°c and 50 r. p.m. references 1. habib w, khankari rk, hontz j. fast dissolve drug delivery systems. crit rev ther drug carrier syst 2000; 17:61-72. 2. sastry sv, nyshadham jr , fix ja . recent technological advances in oral drug delivery. a review.pharm sci technol today 2000; 3: 138-45. 3. chang r, guo x, burnside b, couch r. a review of fast dissolving tablets. pharm tech 2000; 12:52-8. 4. bi y, sunada h, yonezawa y, danjo k, iida k. preparation and evaluation of compressed tablets rapidly disintegrating in the oral cavity. chem pharm bull 1996; 44:2121-7. 5. british national formulary. 54 rd , 2007. 6. legrand e. aceclofenac in the management of inflammatory pain. exp opin pharmacother 2004; 5:134757. 7. ralph niiokaitettey-amlalo;invitro release of ketoprofen from proprietary and extemporaneously manufactured gels. thesis submitted to rhodes university in fulfilment of the requirements for the degree of master of science (pharmacy), 2005. 8. ahmed i,nafadi m, fatahalla f. formulation of a fast-dissolving ketoprofen tablet using freeze-drying in blisters technique. drug devind pharm 2006; 32:437–442. 9. methaqhamadsabar; preparation and in vitro evaluation of ketoprofen as a sustained release tablet using polyelectrolyte complex as a matrix former.thesis ,2011. 10. bhupendra g prajapati and nayan ratnakar; a review on recent patents on fast dissolving drug delivery system. international journal of pharmtech research, 2009; 1(3):790-798. 11. british pharmacopoeia commission. powder flow. london, uk: british pharmacopoeia commission; 2007, appendix xvii n. 12. hisakadzu s, yunxia b. preparation, evaluation and optimization of rapidly disintegrating tablets. powder technology. 2002; 122:188-198. 13. british pharmacopoeia 2009. 14. mohsin a.a; nimbalakr n.e; sanaullah s; aejaz a; formulation and evaluation of mouth dissolving tablets of amitriptyline hydrochloride by direct compression technique. international journal of pharmacy and pharmaceutical sciences, 2010; 2(1):204 -210. 15. eouani c., prinderre p., joachim j., determination of the in vitro disintegration profile of rapidly disintegrating tablets and iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 68 correlation with oral disintegration. international journal of pharmaceutics. 2004; 292:29-41. 16. nirav v patel; narendra p chotai1; mayur p patel; formulation design of oxcarbazepine fast-release tablets prepared by melt granulation technique. asian journal of pharmaceutics.2008,2225. 17. patel d., patel m., optimization of fast dissolving etoricoxib tablets prepared by sublimation technique. indian j. of pharmaceutical sciences. 2008; 70(1): 621625. 18. sunita a., chaudhary a., excipients updates for orally disintegrating dosage forms. int. j. pharm. sci. 2010; vol-1, issue-2, 103-107. 19. patel d., patel m., shah r., jogani l., balapatel i., studies in formulation of orodispersible tablets of rofecoxib. indian j. pharm. sci. 2004; 66: 621-625. 20. mallikarjuna settee; d. v. k. prasad; v. r.m. gupta and b. sa; development of fast dispersible aceclofenac tablets: effect of functionality of superdisintegrants. indian journal of pharmaceutical sciences, 2008; 180-185. key: cord-0035681-0wyl2h62 authors: appanna, vasu d. title: dysbiosis, probiotics, and prebiotics: in diseases and health date: 2018-02-06 journal: human microbes the power within doi: 10.1007/978-981-10-7684-8_3 sha: 42150f8e237816e954dd33e0b899d2593c521729 doc_id: 35681 cord_uid: 0wyl2h62 the microbiome like any other components of the body undergoes numerous challenges during the life-span of a human being. these complications may involve injuries, aggression by pathogens, pollution, hormonal variations, genetic pre-disposition, unbalanced nutrition and onset of diseases. although the microbial reconfiguration provoked by these stressors are not immediately evident as in the case of an afflicted visible organ where the abnormality is readily observable, the biological perturbations induced manifest themselves in form of various illnesses. the disruption of a working microbiome is referred to as dysbiosis and is a condition whereby the fine balance between the microbial communities and the host is distressed. diseases such as cancer, irritable bowel syndrome, rheumatoid arthritis, acne, gastric ulcers, obesity and hypertension can ensue. the pathogeneses of some pulmonary disorders, digestive complications and neurological abnormalities can be traced to the imbalance in the constituents of the microbiome. however, rebiosis, the re-establishment of the native microbiota is proving to be an excellent remedy against this condition. probiotics, prebiotics, and synbiotics are potent therapeutic tools designed to rectify this situation. probiotics such as lactobacillus spp are more or less like stem cells utilized to replenish and rejuvenate the microbiome while prebiotics like fructose oligosaccharides (fos) are microbiome fertilizers akin to mineral supplements or energy nutrients aimed at promoting the proliferation of select microbes in the invisible organ. synbiotics is a combination of both probiotics and prebiotics in a proper dosage aimed at remedying dysbiosis. the molecular understanding of dysbiosis and rebiosis will offer a very effective non-invasive means in preventing and curing diseases with probiotics and prebiotics. this will have a dramatic impact on our well-being. abnormalities can be traced to the imbalance in the constituents of the microbiome. however, rebiosis, the re-establishment of the native microbiota is proving to be an excellent remedy against this condition. probiotics, prebiotics, and synbiotics are potent therapeutic tools designed to rectify this situation. probiotics such as lactobacillus spp are more or less like stem cells utilized to replenish and rejuvenate the microbiome while prebiotics like fructose oligosaccharides (fos) are microbiome fertilizers akin to mineral supplements or energy nutrients aimed at promoting the proliferation of select microbes in the invisible organ. synbiotics is a combination of both probiotics and prebiotics in a proper dosage aimed at remedying dysbiosis. the molecular understanding of dysbiosis and rebiosis will offer a very effective non-invasive means in preventing and curing diseases with probiotics and prebiotics. this will have a dramatic impact on our well-being. keywords microbial imbalance · antibiotics · cancer · obesity · anti-oxidant · drug activation as our microbiome accomplishes a variety of critical tasks for us, it is clear that we will not be the way we are without the microbes inhabiting in and on our body. without this microscopic support, our anatomical constituents and our diet would have been completely different. the diets of carnivores and herbivores are also dependent on the microbial helpers these animals possess. the cows we see grazing would not be engaging in such an activity if it was not for the microbeladen stomach they have. indeed, humans also have evolved to be reliant on microbes to carry numerous functions they are not able to execute without the assistance of their traditional organs. without the presence of these microbes, human anatomy and physiology would have been entirely different; we would not have been biologically the way we are. thus, it is not surprising that if this harmonious relationship is perturbed, major complications may arise with disastrous consequences to the bodily operation. in fact, the delicate balance among the microbial ecosystems needs to be preserved if the body is to function normally. the metabolically compatible members of this community work in harmony and as they are assigned a specific job that enables the community to function properly. this synergy breeds inter-dependence and a specialization of functions. the community forges a collective work ethic amongst all the stake-holders. constant interspecies communication ensures that their activity is controlled and excessive growth is under check. however, perturbation of this fine microbial-balancing act can result in debilitating impact on the host. the increase in some bacterial species with the concomitant decrease of others triggers abnormal signals that can be the harbingers of diseases. this situation is similar to the operation of our social communities. for instance, if there is an excessive amount of doctors and limited number of nurses and other health professionals, the medical delivery system becomes sick. the disruption in the microbial community is bound to create a shift in the families, genera or species of the resident microbes. this can be fatal or make the business of living unpleasant. numerous diseases are known to be triggered by the disturbance of microbial population within the body. the imbalance of the microbiome is referred to as dysbiosis (fig. 3.1) . diet, physical stress, exercise, psychological distress, radiation, flying, humidity, geographical location, age and medications including antibiotics have been shown to exert demonstrable change in the microbial landscape in the body. for instance, ampicillin utilization leads to a sharp decrease in the gut microbiome, while the antibiotic, cefoxilin perturbs the fine balance by promoting the proliferation of clostridium difficile at the expense of other microbes. the amount of some lactobacillus species is sharply diminished during flight and may lead to anxiety associated with air travel. the increased levels of catecholamine produced during stress trigger the growth of pathogenic microbes like e.coli and impede the proliferation of good microbes like lactobacillus and bifidobacteria. farm workers who are exposed to pesticides suffer a similar diminution in lactobacillus and bifidobacteria. exposure to metal pollution like cadmium limits the amount of micro-organisms belonging to the family of bacteriodes that in turn is reflected in reduction of metabolites like scfa (shortchain fatty acids), contributors to numerous pivotal functions in the body as we have seen before. these changes lead to various abnormalities ( fig. 3 .2). the skin is the primary contact with the external environment and is constantly exposed to a variety of fluctuating conditions that affect the microbial communities residing on it. humidity, temperature, clothing, cosmetics, soaps, age, and personal hygiene are some of disruptors of the skin microbiome leading to the onset of various abnormalities (box. 3.1). acne is a common skin complication that afflicts primarily adolescents. during puberty there is a major change in the landscape of the skin with the increase of hair follicles and the maturation of the pilosebaceous glands. these oil-producing vesicles enrich the nutritional content of the skin and trigger the proliferation of oil-loving microbe like the propionibacterium acnes. these microbes secrete lipases and proteins that enable them to gobble oil. unfortunately, these molecular scissors also bruise the tissues adjoining the oily glands. such an unintended assault compels the body to unleash its own guards to defend itself, with devastating outcome. this counter response to the shift in microbial population results in the formation of those unwelcomed blemishes on the face. hormonal imbalance may also aggravate this problem (fig. 3. 3). the dermal microbiota is prone to disruption by a wide variety of factors as it is permanently exposed to the external environment. geography, pollution, ultraviolet radiation (uv-r), occupation and the host biology are major contributors to the skin microbiome. injury, chronic conditions, infection, cosmetics and the use of hygiene products may result in a shift in the bacterial communities. uv-r promotes the release of anti-microbial peptides that may block the body's immune system and creates an environment for the cancer-causing viruses to flourish. chemicals like triclosan, a common ingredient in household products like soap and toothpaste can perturb the fine microbial balance. this is further compounded by the inherent diverse landscape that the skin offers; the face is oily, hairy and exposed to environment while the axilla is occluded, moist and laden with microbial nutrients. the dysbiosis triggered by the host of environmental and microbial-driven features are at the origin of numerous skin diseases. for instance, the atopic dermatitis that is a chronic pruritic inflammatory disorder is characterized by an overwhelming increase in staphylococcus aureus. a decrease in filaggrin, a microbial sensing protein coupled with a decrease in dermicidin, a peptide defending bacterial invasion results in this lack of microbial diversity and the proliferation of a select few microbes. prebiotics like fucosylated oligosaccharides derived from chicory roots and probiotics like staphylococcus epidermis are being introduced in cream to fight dermal dysbiosis. eczema is another common skin ailment that finds its origin in the imbalance of the dermal microbial community. it is a chronic, recurring skin disorder that is more prevalent in children compared to adults. this disease is characterized by a major shift in the microbial population resulting in diminished biodiversity. such a situation promotes the unchecked growth of the microbe, staphylococcus aureus. increased colonization by this organism provides a fertile landscape for the nasty streptococcus to proliferate. the tight-knit functional community is unraveled. the new uncontrolled colonizers secrete toxins that induce the degranulation of the dermal mast cells and force the body's immune system to respond. this change in landscape is partly responsible for the dry red spots that are characteristic of eczema. these patches are sometimes accompanied by local inflammation. in the case of psoriasis, a disease that is prevalent world-wide, similar conditions conspire to create a dramatic shift in the harmonious microbial population. the ensuing diminished biodiversity propelled by the over-representation of the genera staphylococcus belonging to the firmicutes family automatically leads to a reduction in other family members that had bonded into this dermal community and learnt to live in harmony. in this instance, the territorial presence of members belonging to the acetinobacteria family is restrained. thus, begins the initiation of psoriasis. environmental factors like temperature, humidity or presence of cosmetics on our skin may hasten the onset and the severity of this malady. the establishment of the unbalanced microbial community residing in the psoriatic lesions generate antimicrobial peptides and other modulators that hyper-activate the body's immune system. this reaction modifies the life-cycle of the skin cells that respond by growing rapidly, a situation culminating in itchy, dry, red plaques. these become the hub of other uncontrolled microbial activity and can aggravate into psoriatic arthritis. wound healing in diabetic patients is a problem as cuts on the skin of these individuals take unusually longer time to repair. here again the inability to effectively recover from wounds is again due to the uncontrolled proliferation of certain microbes at the expense of others. the sugary environment provided by the diabetic blood and the presence of other nutrients create an ideal ecosystem for the multiplication of staphylococcus. this nutrient-rich landscape on the skin-cut acts as a magnet for some bacteria and disrupts the harmony among the otherwise orderly community members. this microbial imbalance is a recipe for disaster and opens the body for invasion as some of our trusted partners are either too small in numbers to make any difference or are completely wipe-out of our intimate landscape by the opportunistic intruders. and wound healing becomes a very abnormal exercise for the body to partake in the absence of some of the invisible allies. the mouth is another part of the body that lends itself to constant contact with the invisible world. as it is an extension of our environment, the microbial traffic is high. anytime we open our mouth to eat, drink, laugh, smile, yawn or cry, we are enabling millions of microbes to access our body. however, due to the vigilant watch of resident oral microbes and their anti-microbial components, only a select few can contemplate to occupy a piece of the real estate either in our mouth or lungs. the microbial communities composed of diverse constituents that set foot are scrupulously and carefully interrogated by our molecular sentinels. interactions with prospective residents are permitted only if they work for a common purpose. only when a member can contribute to a specific function that the community depends on, the microbial member is accepted and a shelter is made available. the contribution of each of the member is critical for the functioning of the community. however, this situation can rapidly change specially in the mouth where there is a constant flux of diverse exchanges occurring with respect to microorganisms. furthermore the buccal chemical landscape also undergoes tremendous fluctuation due to the regular intake of foods and drinks. this situation can be complicated if one allows any food component to remain in the mouth for too long. carbohydrate-rich nutrients become easy prey on which some opportunistic microbes can thrive. despite the vulnerability of the mouth to possible invisible intruders, the microbial population is kept in check by the watchful guard of other community members including the host. for instance, in the establishment of dental caries, the streptococcus mutans utilizes these sugary goodies stuck on the tooth to produce lactic acid, an event that increases the acidity of the buccal ecosystem. this presents an opportune situation for other microbes like the veillonella and lactobacillus salivarus/acidophilus to colonize this territory and further aggravate the affliction on the tooth (fig. 3.4) . the fine microbial balance that is perturbed as a result of ensuing chemical change in the mouth is an ongoing concern for people especially children with poor dental hygiene. this disruption and the resulting medical complication can be averted by controlling the chemical landscape in the mouth. halitosis is also caused by microbial disruption that results in the increase in h 2 s, a molecule responsible for smelly breath. this may also be aggravated by the genetic make-up of the host (fig. 3 .5). the perturbation of controlled microbial growth in the oral cavity is known to trigger the gum disease referred to as periodontics. the homeostatic equilibrium in the microbial community for a healthy gum is maintained partly by the body's own immune system. this is upset by the invasion and colonization of porphyromonas gingivalis, a microbe responsible for the destruction of gum tissues. these degraded components create a nutrient-rich environment that becomes a breeding ground for other opportunistic organisms that are usually kept at bay by our oral microbiome and the lack of appropriate nourishments they can thrive on. this feeding frenzy results in the overpopulation of some species compared to others and give rise to a misfit community of microbes. bacteria such as prevotella and desulfobulbus are two culprits that exploit this situation and assist in the task of invading the gum. thus, dysbiosis an uncontrolled microbial proliferation resulting from the temporary nutritional change in the oral ecosystem is at the root cause of numerous complications that ache both the hard and soft components belonging to this part of the body. the lack of microbial biodiversity in the crypts of the soft tissue like the tongue is at the origin of halitosis, an affliction that emanates unpleasant smell that most of us find repulsive (box 3.2). the mouth has the second most diverse microbiota in the body with over 700 species. the chemical landscape of the saliva plays an important role in the establishment of the microbial communities that reside in the buccal (continued) box 3.2 salivary microbiome and its health impact (continued) cavity. nearly 10 8 microorganisms are found in one milliliter of saliva, a fluid rich in proteins, lipids, carbohydrates and enzymes. the presence of lactoferrin, lysozyme and lactoperoxidase act in concert to dissuade the colonization by fortuitous bacteria. hydrogen peroxide, hypothiocyanite and nitric oxide derived from nitrate in foods are potent antimicrobial and are part of the defense armoury deployed to fend against cariogenic intruders. however, poor hygiene, nutritional habits, smoking and diseases like diabetes tend to weaken this defense. the presence of increased glucose level generates acidic metabolites that drop the ph of the mouth from 7.0 to 4.0. this promotes the acid tolerant microbe like staphylococcus mutans and results in an increase in the firmicutes family coupled with a decrease in the bacteriodetes family. the solubilization of calcium and phosphate is preceded by a variety of dental diseases. furthermore, the dysbiosis created by this situation leads to reduction of nitrate reducing bacteria, a situation restricting the output of nitric oxide, no. this has major health impact including cardiovascular complications. the use of chlorhexidine containing mouthwash also has a negative influence on no-producing bacteria. hence, the saliva and its microbial residents are an important contributor to a healthy outcome. the mouth and the nostrils are constantly providing the lungs the air we need to live. without this obligatory and mundane routine we will not be able to produce energy (atp) that keeps our body machine active. this process is hard-wired in our brain that we keep on doing until we are no more alive. thus we are constantly exposed to the microbes and chemicals in the environment where we are studying, sleeping, playing, working or just relaxing. the lungs are permanently bombarded with the myriad of micro-organisms our surroundings have to offer. although the microbial populations in the lungs are well-maintained, these communities can be harassed by medications we ingest and by the chemicals in the air we are surrounded by. farmer workers, employees in factories and other industrial organizations are at high risk in this regard as they are surrounded by chemical constituents that affect the microbial composition of the lungs. the development of asthma is triggered by the decrease in the diversity of the pulmonary microbial ecosystem. the release of nutrient-rich mucus during lung infection aimed at arresting the proliferation of intruders can also inadvertently act as a magnet for some opportunistic microbes that further compound the problem. a shift in the microbial community favouring a group of lactobacillus is observed during the onset of chronic obstructive pulmonary disease (copd). during cystic fibrosis (cf) the viscous sputum allows the rapid growth of pseudomonas aeruginosa, a microbe that is controlled in non-cf lungs. this microbial imbalance generates chemicals like alginate that further aggravates the situation. the nearly unimpeded pathway of the air tract between the mouth, lungs and the environment render these organs an easy target of dysbiosis. these body parts are challenged on an ongoing basis with changing microbial population and environmental factors such as chemicals and gaseous pollutants that make them a fertile ground for microbial disharmony. hence, it is critical that the crevices in the mouth do not have nutrients stuck for long as these are perfect ingredients that can dislodge the well-established functional microbial communities and inflict us with diseases. the mouth and nose gears are a common sight in cities around the world with high level of pollution. in beijing, new-delhi and mexico city, citizens are often seen wearing these protective screens in an effort to limit the intake of the polluted air in order to diminish its influence on the lung microbiome. during outbreaks of microbial diseases such as severe acute respiratory syndrome (sars) or influenza, this phenomenon is observed around the globe in order to ensure that the lungs are not further burdened by air loaded with viruses as these will undoubtedly promote dysbiosis. the mouth and lungs are the first line of defence to combat the growth of opportunistic microbes lurking around in the air. thus, oral hygiene and the quality of air one breathes in will go a long way in helping maintain the proper microbial balance required for a healthy body. however, if these conditions are not met, microbial disruption resulting from the reduction of biodiversity in the pulmonary and oral landscape can be a cause of concern. it is not surprising that the deteriorating quality of the air across the planet has led to sharp rise in asthma and other lung-related illnesses ( fig. 3 .6, box 3.3). rheumatoid arthritis (ra) is a debilitating autoimmune disorder associated with the inflammation of the joints that can lead to bone erosion and deformity. the hyperactive immune response can also damage other parts of the body like the eyes, lungs and skin. this dysregulation of the immune system triggers pro-inflammatory tendencies with an increase in t-lymphocytes and self-reactive antibodies. although genetic factors may contribute to this disorder, a major shift in the constituents of the microbiome is observed in ra patients. there is an overrepresentation in the provetella species including provetella copri, an increase in clostridium spp and a reduction in bacteriodes spp. this dysbiosis evokes the elevated production of homocysteine, hydrogen sulphide and lipopolyssacharide (lps) that all known to promote inflammation, a feature culminating in the attack of one's own body part. probiotics like lactobacillus casei has been shown to mitigate symptoms associated with ra. asthma is another inflammatory disease where the respiratory airways are challenged by the aggressive action of the immune system. there is an overproduction of mucus and an intense remodeling of the airway wall ensues. the presence of dust, pollen and spores tend to aggravate this situation. however, this disease is characterized by a marked change in the lung microbiota. in children there is reduction in such microbes as the faecalcibacteria, veillonella and rothia while in adults an increase in proteobacteria and a decrease in bacteriodetes and firmicutes are common. hence, restoration of the microbiome with probiotics and prebiotics can be an alternative therapy. the digestive tract is the home of most bacteria in the body. although the vast majority is housed in the intestine, some do find refuge in the stomach. despite the inhospitable environment presented by this acidic organ, the microbes belonging to the helicobacter spp have set-up their home here. they have done so by learning how to tame the low ph these environs are immersed in. they cling to the mucus of the cell-wall where the acidity tends to a bit more manageable. by neutralising this unfriendly territory, these microbes are able to develop a friendly habitat where they can reside. even with these territorial adjustments, the stomach is the organ that harbours the least amounts of bacteria. however, the perturbation of this microclimate can quickly change this microbial landscape. the intake of medications like the proton pump inhibitors and antibiotics tend to promote a shift in this microbial population to the upper confines of the gastrointestinal tract. once, this uncontrolled colony is established, it produces ammonia that compels an empty stomach to generate acid. it is critical that this digestive compartment is acidic only when it has food in its midst. any change in the acidity in the absence of food distresses this organ. it is more or less like the stomach is chewing itself. this becomes the genesis of aches and heartburns that result into peptic ulcers. there is a rise in this microbially-induced disease around the world and the disruption of the fine working arrangement struck among the invisible residents may be at play. it is not surprising that the majority of ulcers can be cured by antibiotics and not by drugs aimed at diminishing the acidity of stomach, a finding that has dismayed many pharmaceutical companies as they have invested heavily on the incorrect cause of the disease. the understanding of how the disruption of these gastric microbial communities will pave the way for better remedies ( fig. 3 .7). the majority of the constituents of our microbiome is lodged in the small and large intestines with the latter harbouring the bulk of the invisible partners. once the colonies are established in these locations, the stability of the residents is constantly being questioned due to the passage of foods and other edibles that transit through. this environment is under constant flux due to our daily intake of foods that can come in all shape, size, content, well-done, rare, or raw. the colonizers have to adapt. low carbohydrate, high fat, high protein, dietary fibres, medications, antibiotics, sugary drinks all have major impact on the ecosystem. this constant pounding by these diverse ingredients, roughly around 60 tons during our life span is bound to take its toll on these miniscule residents within. such a situation is akin to us changing our clothes conatantly in response to temperature changes in the environment. how would you feel if you have to face snow, rain, sun and fog on an ongoing basis and keep modifying your outerwear continually ( fig. 3 .8). whether the food is starchy, oily or meaty; the microbes have to adjust. a shift from a high fat/high sugar diet to a regime of low-fat/plant-based polysaccharides can change the gastrointestinal microbial ecosystem in a matter of days. for instance, while bacteriodes spp thrive in people who consume high fat foods, prevotella spp dominates the guts of individuals who eat more carbohydrate-rich products; refined sugar intake on the other hand favours clostridium spp. the lovers of vegetarian diets are host to less pathogenic bacteria while protein-rich nutrients tend to provoke increased activity of enzymes like nitroreductases derived from microbial sources. this situation contributes to the weakening of the inflammatory response and prevents the metabolism of short-chain fatty acids (scfa). remember the oxidation of scfa like butyrate provides the intestines with an important source of energy and perturbation of this process can result in abnormal physiological functions. preserved foods, dehydrated vegetables and alcoholic drinks with their high content in sulphate provide a fertile ecosystem for suphate-reducing bacteria like desulfovibrio spp to thrive and distress the body. these micro-organisms generate metabolites that restrict the growth of probiotics that help us execute numerous tasks. medications like opioids and the popular diabetic drug metformin impair the mobility of the gut. these are also responsible for shifting and deranging the community-like environment that good bacteria nurture. this disruption provides an opportune situation for bad microbe like the clostridium difficile to multiply. even psychological stress that is known to trigger the production of signaling chemicals like catecholamines has an impact on the microbiome. well-established microbes like lactobacilli and bifidobacteria that are known to confer a number of healthy attributes in the body tend to diminish (box 3.4). box 3.4 food additives, dysbiosis and health impact numerous chemicals are introduced into foods to improve taste, to extend shelf-life, to add volume, to enhance appearance and to impart a variety of other properties. some of these are known to interact with the microbial communities in the gut and perturb the fine communal balance. dietary emulsifiers like carboxymethyl cellulose (cmc) is one of the additives that is widely utilized in foods, cosmetics, and hygiene products. ice-cream, cookies, toothpaste and laxatives are some of the products where this polysaccharide provides texture, viscosity and structural integrity. despite being mostly composed of glucose, cmc cannot be digested either by the visible or the invisible organs. however, it creates a major ecological change in the gut by interfering with mucin, a natural barrier lining the gut. mucin is heavily glycosylated and act as a potent fence against any opportunistic pathogens to access the inner walls of the intestine. it inhibits the absorption of dietary flavonoids and also promotes the proliferation of microbial flora desired by the body. these functions are perturbed by cmc as it shares some chemical and physical features with mucin. this disruption results in dysbiosis leading to the multiplication of pathogens and their translocation into the intestinal tissues. there is an increase in proteobacteria spp and inflammation ensues. chronic intestinal complications have been attributed to these additives. hence, it is not surprising that these chemical, environmental and behavioural changes that the gut microbiome has to contend with invariably lead to the disturbance of the delicate balance cementing the microbial communities and the host in an effective functional relationship. this flux in the gut ecosystem creates conditions for the abnormal proliferation of some members at the expense of others. the subsequent activation of enzymes and production of disease-causing factors can contribute to such maladies as irritable bowel syndrome (ibs), colorectal cancer, rheumatoid arthritis (ra), type 2 diabetes, obesity, cardiovascular complications and neurological disorders. some of the metabolic diseases like diabetes, obesity and heart ailments have reached an epidemic proportion globally. a recent report revealed there are more obese people than underweight individuals, a phenomenon that is a first for human history. an average person has become 1.5 kg heavier over each decade. thus, if your grand-father was say 50 kg, your dad is 65 kg and you will be 80 kg at the same age of course. this scenario will be true if nothing else changes. the study on body mass index that was done over 40 years in 186 countries is alarming indeed. policies on food and the quality of food we have access to will help to some extent. it is not surprising to see various north american cities are imposing an obesity tax aimed at sugar-laden soft drinks. examining the role of our microbial ecosystem is also pivotal if this issue is to be remedied. the gut microbiome is an important participant in the manner we digest food, extract the maximal energy and nourish the rest of the organs. a disruption in the microbial landscape can trigger metabolic diseases. for instance, during irritable bowel syndrome (ibs) microbes belonging to the firmicutes family decrease while an increase in members of the bacteroidetes family is observed. this disorder in the intricate microbial balance propels a concomitant rise in facultative bacteria like the enterobacteriae spp. the unregulated establishment of the microbial community tends to impair the integrity of the mucus lining of the intestine. this shift perturbs the ability of the resident intestinal microbes to properly communicate with the mucosal barrier; such a situation results in the inability of the body to tolerate the harmless bacteria and neutralize the invading squatters. following this distress imposed on this natural fence, conditions are perfect for the release of signals and chemical shuttles by the microbes to enable their migration across the intestinal defensive wall. this sequence of events forces the host to activate its guards with no clear mandate on the proper target and the result is an unintended inflammation giving rise to crohn's disease and ulcerative colitis. these are the two most prevalent forms of ibs that can be traced to the perturbation of microbes residing in the gut. in fact, these diseases can be mitigated by increasing the presence of firmicutes in the gastro-intestinal tract. it is clear that the conditions promoting the opportunistic microbes and the colonizing factors they produce are at the root cause of these disorders and re-establishing the original microbial communities goes a long in alleviating the pain of individuals suffering from these complications. obesity is a major problem afflicting the world. the rise in overweight individuals has been staggering and this malady attacks regardless of any economic and geographical boundaries. almost all countries surveyed are experiencing the illeffects of this metabolic disease. a variety of elements such as diet rich in simple carbohydrates, low in vegetables and fibres; life-style and genetic factors may be at play. however, the disturbance of the microbial harmony in the body cannot be discounted as a potential trigger responsible for the soaring rate of obesity around the globe. there is a clear distinction in the microbial landscape associated with obese people compared to underweight individuals. lean individuals tend to have higher amounts of microbes belonging to the bacteriodetes family while obese subjects and those suffering from metabolic syndrome are home to more members from the firmicutes family. although the specific species of these two microbial families responsible for the lean and obese traits have yet be catalogued, it is clear that obesity is characterized with increased levels of toxins like lipopolysaccharide (lps) in the blood. symptoms associated with weight gain and decreased sensitivity to insulin have been attributed to the presence of microbial toxins. hence, modulating the nature of the gut microbiome and restoring the proper microbiota have to be included as part of the strategy dedicated to combating these disorders. the food we eat is a major instigator of the establishment and fluctuation of the microbiome we possess. in fact, the adage we are what we eat applies aptly to the microbiota that constitute our body. one can even add that our physiological processes are the way they are because of the microbes we own. these invisible partners that accompany us throughout our live-journey are part of the elaborate communities responsible for our well-being. however, this is not a permanent relationship as it is susceptible to evolution and disruption by what we eat. this intimate relationship that we have forged with our invisible partners and all of our other bodily organs faces lots of twists and turns as we navigate through our lifelong journey. in the case of our microbial ecosystem these changes are non-stop as it is extremely prone to the vagaries of what we put in the mouth. and this is a daily activity that is most relentless and continual and commences the day we are born and ceased only upon our death. indeed it is important to remember that every component of body is in constant flux, we are never exactly with the same body parts that we are born with. these are evolving every second. for instance, we get a new skeletal system every 40 years and this happens on an ongoing basis day by day. otherwise you will be soft like jelly one day and then wake up the next morning with a brand new skeleton. thus, depending on our eating habits, we may be saddled with a microbial ecosystem that can promote or impede our wellness in the same way we have toned body if we exercise or we are stuck with a flabby belly if we are a potato couch. a fat-rich diet together with a combo of a sugary drink is sure a concoction to upset the fine balance of our invisible organ. such a nutritional habit is responsible for the non-alcoholic fatty acid disease (nafld) and cardiovascular complications. these greasy foods are known to promote the proliferation of some bacteria and arrest the growth of others. the establishment of this unbalanced ecosystem is a perfect brewing ground for the genesis of liver and heart diseases. these oilladen nutrients contain copious amounts of choline, a chemical responsible for the yellow colour in the egg yolk. it plays an important role in the development of the brain in children. however, its excessive consumption via the food we intake specifically in adults can be problematic and the unwelcomed microbial residents aggravate our overindulgence in these choline-containing goodies. in reality our gut microbiota transform this into a product known as trimethylamine (tma). this diversion of choline into tma prevents the body from making some good lipids and subsequently leads to the accumulation of the bad lipid known as triglycerides. remember the number 200 associated with triglycerides in the blood of adults. this magic number if higher is a relatively precise predictor of potential coronary abnormalities. these triglycerides have nowhere to go but to accumulate in the liver where they become the precursor of fatty acid disease. this is not all that can come out of this greasy food, it can become worst. if you harbour microbes that can metabolize tma i.e. process it into the oxygenated variety known as trimethylamine oxide (tmao), this can spell danger for your heart. the benign tma falls prey to microbial manipulation in our gut that renders this relatively innocuous agent into a toxin with devastating impact on our well-being. tmao is an important tell-tale sign of cardiovascular diseases. there are other intestinally derived toxins fuelled by microbial activities that can cause havoc to the normal functioning of our body. sulphate derivatives of aromatic chemicals originating from microbial intervention in the intestine can be a major burden on the kidney and may lead to renal abnormalities. controlling the microbes that are in the business of transforming harmless chemicals into noxious ones is critical strategy that need to be pursued in order not to succumb to the microbial imbalance promoted by fatty foods (fig. 3.9 ). even though we may not be completely aware there is an important communication corridor between the gut and the brain that is nourished by numerous signals emanating from the microbial activities in the intestine. the gut-brain axis relies on the messengers and/or their precursors generated by the microbes we harbour. they produce short-chain fatty acid (scfa) like butyrate and neurotransmitters like γ-aminobutyric acid (gaba), dopamine and serotonin. whenever you have a rewarding feeling due to some activity like eating that you are engaged in, blame it on dopamine. these instruction-laden chemicals that are also produced by other organs in our body are central to numerous tasks executed by the brain and can stir our behaviour in one direction or the other depending on the concentration of a specific neurotransmitter. hence, factors that modulate the flux of the microbial communities and their propensity to dish out these neuro-active commanders have tremendous influence on our health and well-being. for instance the lack of microbial diversity associated with ageing impedes the ability of the body to generate scfa such as acetate, propionate and butyrate that supply the brain cells with energy. the loss and/or the rationing of the nutrients fuelling the brain result in a reduction of cognitive and memory power. this attribute is a common characteristic reminiscent of ageing. hence, the propensity of people in retirement homes not to remember or retain mundane facts (fig. 3.10) . the microbiome definitely has its imprint on the functioning of the brain, a feature that has yet to be fully deciphered. for instance, the amino-acid tryptophan is the precursor of numerous neurotransmitters that dictate a variety of our behavioural responses that are usually tailored to the stimuli we receive. this is an essential nutrient that humans cannot make with their own traditional organs and have to acquire it from the food we eat. however, depending on our microbial communities we can easily have ample supply as numerous gut microbes are capable of synthesizing this nutrient; but with caveat that the amounts of tryptophan we have access to need to be modulated if our response is to be commensurate to its presence or its derivatives. any abnormal variation of this commodity in our body can become a harbinger of neurological complications. a shift in microbial communities induced by a myriad of factors can create havoc with blood tryptophan levels. this can then results in depression, mood swings and neurological ailments like parkinson's or alzheimer's diseases. keeping a careful watch on our fig. 3 .10 dysbiosis, abnormal metabolite production and neurological disorders microbial partners and their ability to generate these brain responsive signals will lead to better health outcomes (box 3.5). the ability of the microbiome to produce a variety of neuroactive chemicals makes the invisible organ a potent modulator of human behavior and an instigator of neurological diseases. for instance, parkinson's disease is an incurable adult neurodegenerative disorder characterized by abnormal movements due to defective motor control. dopamine production is impeded and there is an increased formation of aggregated neurotoxic protein known as α-synuclein. these two biochemical manifestations may be shaped by the nature of the microbial residents by limiting precursors of dopamine and activating the immune system with the release of pro-inflammatory chemicals like lps. the gut microbiome in parkinson's patients is usually characterized by a decrease in prevotella spp and an increase in enterobacteria spp. a diminution of mucin synthesis increases intestinal permeability and leads to an acute dysbiosis that contributes to the abdominal discomfort, bloating and premature satiety reported in these patients. autism spectrum disorders (asd) are another set of neurological perturbations leading to social and behavioural impairments including repetitive behaviours and nonstandard communications. here again, the microbiome may be a contributing factor. a decrease in veillonella spp coupled with a rise in clostridium spp and campylobacter spp have been reported. microbial products like pcresol has been observed in elevated amounts in autistic children and 4-ethyl sulfate (4-eps) has been shown to trigger anxiety like behavior. this microbial link is prompting the search of probiotics (e.g. bifidobacterium fragilis) that may help in curing these diseases. cancer is a multi-factorial disease that is a major global concern. almost everyone knows of someone who has been affected by this disease. numerous governments have recognized this disease as a national priority and have decided to provide all the support necessary to fight cancer. although life-style, genetic disposition and environmental factors are important contributors to the spread of this disease, it is becoming amply clear that our microbial landscape and cancer proliferation are interconnected. it is being increasing recognized that the microbiome plays a key role in preventing and promoting cancer. the gut microbial ecology provides an important cover against invading pathogens that contribute to the onset of tumour formation. however, a shift in microbial communities induced by diet and pollution creates a favourable environment for opportunistic microbes to thrive and neutralise the protection offered by the finely tuned original microbial guards. the invading microbes produce virulence elements responsible for unimpeded cellular growth, a key feature of all cancers. they also put in motion processes that result in the instability of the dna and furthering the progress of cancerous cells. understanding the communal work of the intestinal microbes is pivotal if this problem is to be tackled. indeed it has been shown that africans from rural areas exhibit lower risk of colon and rectal cancers due to the increased abundance of the microbes known as bacteroides spp. these are avid producers of short-chain fatty acid like butyrate, an important energy-producer and a promoter of cellular growth. this is a critical signaling molecule that can goad the body to synthesize sentinels that stop cancer cells on their tract. on the other hand, the group of individuals whose colon is home to higher amounts of prevotella spp tend to have higher levels of bile acid and lower amounts of butyrate. this switch in colonic microbial ecology is suspected to be responsible for the colorectal cancers ( fig. 3.11) . the prevotella-rich individuals are programmed to secrete bile acids in the feces. these compounds are known to promote the proliferation of cancerous cells. the interplay between these two microbial communities and their ability to produce these metabolites afforded wellness to one group and colorectal cancer to the other. hence, the promotion and nurturing of functional microbial communities and limiting the presence of unregulated microbial ecosystem in the gut are pivotal to maintaining our wellbeing. this invisible organ has to be tended in the same manner like any other visible organ; one cannot expect to have working lungs supplying the furthest nooks in the body with oxygen if one indulges in smoking or toiling in occupation with toxic air. this cardinal rule also applies to the microbiome even if its anatomical features do not readily pop out to our eyes. otherwise we run the risk of turning this organ that we almost never see but hear rumblings of once in a while into a cancer-causing entity. the critical attributes of these microbes need to be tapped as cancer preventers and fighters. the microbiome is an essential component of humans if not of all multicellular organisms. this invisible component of the body that quite often makes itself heard with a rumbling sound is involved in a variety of biological tasks that are essential for the proper functioning of any human. although these microbes residing in and on us have their genetic information they are bound by the communal existence they have adopted. the role and behaviour of each member are dictated by the host and other members of the collectivity. this harmony and synchronicity of purpose force everyone to comply with the rules set-up during the establishment of the community. however, these communities evolve in relation to the host and the changing landscape they have to deal with. they accompany us from the very beginning when we are developing till death. it is becoming more apparent that these microbes contribute to our development and eventually mould us the way we are both anatomically and physiologically. they have a say virtually in all aspects of our life and prod us as we go about our daily living. from providing essential nutrients like vitamin k that we are dependent on but incapable to make on our own to protecting us against opportunistic organisms and helping heal our wounds, these microbes partake in a myriad of tasks just as visible organs do. however, their constituents and interactions like all other organs in the body are modulated to large extent by the changes in environment, food, stress, and hormonal fluctuations we are subjected to. the invisibility of this organ and its dispersed anatomy have contributed to the relegation of the microbiome to a low esteem in the hierarchy of our body parts. but this view is being discarded at a rapid pace as intriguing information about its role is being revealed. its intimate link to our existence was only visualized very recently. what you cannot see, you cannot appreciate. this dictum fits perfectly the microbiome. the advent of next generation sequencing (ngs) coupled with bioinformatics tools is laying bare the indispensability of the microbial communities roaming within and on us to our very existence. our life will not be the same without our intricate network of microbial communities. hence, understanding how this invisible communal habitat is constituted, how it evolves and how the various partners communicate amongst themselves is crucial for our being. the host will not exist without the microbiome. it is as crucial as the heart and any other body part. this mission is as critical as our desire to unravel the workings of the brain or to venture in the outer galaxy. its systematic functional identification will be akin to the discovery of a new organ as in occurred in the sixteenth century when human was first beginning to comprehend the workings of various body parts. just imagine the excitement when the italian surgeon, realdo columbo identified how the four valves of the heart permitted the flow of blood in only one direction i.e. from the right ventricle to lungs and back to the left ventricle and on to the aorta. we are on the brink of tantalizing discovery on a part of our body that has long been ignored due to the lack of proper tools to decipher the microbial communities. it is indeed more exciting as this invisible organ has at least 1,000 times more genes than our own visible body. the microbiota we possess must play an important role in our development and we are physically and physiologically the way we are because of them. we would be entirely different if it was not for the microbiome and this holds true to most if not all organisms wandering on this planet and most probably beyond. the proper functioning of this community with multiple partners depends on the finely tuned relationship among all the constituents. every member operates in a mode that is beneficial to this cooperative life-style where interdependence is the modus operandi. however, this harmony can be perturbed by a variety interfering influences and can result in a multitude of ailments. the intrusion of opportunistic organisms fuelled by a change in the ecosystem gives rise to dysfunctional communities that are the precursors of debilitating diseases like cancers, ibs, and vaginosis. the latter is a disease arising from the uncontrolled proliferation of microbes such as gardnernella vaginalis, veillonella spp, and bacteriodes spp. that promotes an increase in ph, fatty acids and polyamines (fig. 3.12) . cadaverine, a polyamine secreted during this dysbiosis is the cause of the malodor characteristic of the disease. once the fine balance involving the constituents and the signals that are responsible for a proper microbial community is uncovered, we will be in a better position to predict what makes us tick and what ails us. this knowledge is just now beginning to emerge and ignoring this important aspect of our body will be to our peril; it would be akin to not wanting to know how the brain functions (fig. 3.12; fig. 3 .13). the microbial communities that constitute our microbiome are dependent on a myriad of factors such as the genes we inherit, our mother's milk, the environment we live in, our hobbies, the seasonal changes we are subjected to, the food we eat, the life-style we lead and the medications we take. despite the ability of our microbiota to respond and adjust to these situations, the invisible organ can be influenced by either taking in select beneficial microbes with known functional attributes or by consuming foods and plant products that promote the proliferation of specific microorganisms. this microbiome-rearing strategy is akin to feeding our brain with the books we read or the educational programs we watch or the games we play. remember some eager mothers keen on giving their children a head-start in life, read to them or play music even when babies are in the womb. to promote a healthy life-style, seniors are being encouraged to partake in neuron-stimulating games on a regular basis. this brain exercise has becoming a common place in residences for the elderly. thus probiotics, prebiotics and synbiotics can be utilized to guide the microbiome to perform optimally and ameliorate our well-being (fig. 3.14) . probiotics are live microbes administered as foods or even in capsules that confer health benefits on the body. microbial cultures have been consumed by humans since the dawn of civilization. mongolian women sprayed fermented milk on horsemen and their horses in belief that this provided strength and health on the recipients, while in some civilizations fermented milk was utilized to treat a variety of ailments. dahi, a fermented milk is widely consumed in the indian sub-continent and is known to impart numerous healthy outcomes. however, the first clinic trials on the health claims of these bacteria-rich fermented foods were not performed until the twentieth century. eli metchnikoff who obtained a nobel laureate in medicine was first to correlate the longevity of some bulgarian citizens to the prolific consumption of fermented milk that was rich in lactobacillus bulgaricus and streptococcus thermophilus. in 1930, the japanese scientist shirota isolated microbes with probiotic properties from healthy human subjects. these were later utilized in the development of milk products that were commercialized as yakult. subsequently, a french pediatrician reported the lack of bifidobacteria in stool of infants suffering from diarrhea. in 1965 the term probiotics was coined by lilly and stillwell to describe products that stimulate the growth of microorganisms beneficial to the body. it was in 1984 the first probiotic species, lactobacillus acidophilus was introduced followed subsequently by bifidobacterium spp. their use in food products is widespread. the significance of probiotics in fortifying the immune system has officially been recognized fig. 3 .14 nurturing of the microbiome, the brain and the muscle. (activities and nutrients involved in fortifying these organs) fig. 3 .15 consumption of probiotic-rich foods around the globe by the world health organization (who). currently a wide variety of microorganisms such as e. coli, propionobacterium, enterococcus, streptococcus, leucomostoc, and bacillus cereus are being consumed in order to regulate and adjust the body's microbiome (fig. 3.15, box 3.6) . box 3.6 probiotics: occurrence and uses worldwide humans have been consuming microbe-laden foods unknowingly since the dawn of civilization. fermented milk, vegetables, meat and fish were and are a regular part of the daily diet for most people around the globe. recently probiotics enriched foods like yogurt, yakult, kefir, dahi and cheese have become a regular repertoire of natural products that nutritionists are eager to extoll the health virtues of. lactobacillus spp, bifidobacterium spp and bacillus spp. are the more prominent probiotics even though other microbes are being added to this list. they are also being used in non-consumable items like oral care gel and anti-ageing serum. their ability to produce acid, enzymes, scfa, immune responsive factors and to help establish functional microbial flora have made these probiotics excellent candidates to cure a variety of diseases that are provoked by dysbiosis. while e.coli nissle provides relief to patients suffering from ulcerative colitis, a disease like vaginitis can be remedied with lactobacillus rhamnosus gg. irritable bowel syndrome victims can find comfort with the intake of bifidobacterium animalis and lactobacillus acidophilus. the role lactic-acid microbes like bifidobacterium breve has been recognized in diminishing high blood pressure due to their ability to produce vitamin d, anti-hypertensive factors and interrupting cholesterol absorption. although influence of probiotics in promoting wellness and as therapeutic agents aimed at numerous illnesses are becoming more prominent, their usage will become universal once the dosage and the intake frequency of these microbial supplements have been properly evaluated. probiotics not only help the body to fine-tune the microbiome, they can also perform some very specific functions. depending on the probiotics, these microbes act as a barrier and prevent colonization by opportunistic bacteria. they do so by re-enforcing the protective function of the gut mucosa and by generating signals that arrest the invasion by infectious organisms. they produce antibiotics, antioxidants and improve the body's immune system. probiotics are also known for their ability to synthesize scfa and vitamins, ingredients essential for numerous metabolic activities. they secrete enzymes that help in digestion and in the elimination of wastes. a good probiotic should be able to impart health benefits to the host and be bestowed with no pathogenic properties. its presence in the microbial community should enhance some biological functions and/or curtail any negative influence on an individual's well-being. these can preferably be taken orally and must survive the harsh environment of the digestive system, especially the stomach where the ph can be unforgiving. the low ph of the stomach can be avoided if the probiotics are mixed with food such as milk, dietary fibres and yogurt. they can be designed to generate specific biomolecules that can be of benefit to the host. for example, lactose-intolerant individuals can consume probiotics with the ability to synthesize lactase or galactosidase that helps in metabolizing the milk sugar, lactose. probiotics have virtues almost like stem cells. stem cells i.e. cells that have not yet made up their mind what they will become or which organ they will develop into can be utilized as a therapy to fortify ailing components of the body. for instance, they can be injected in the cornea or the liver with the proper commands that enable them to mature into corneal or hepatic cells that become part of the rejuvenated organs with optimal biological functions. probiotics act in a similar manner in regard to our microbial communities. they can be consumed with the aim of enhancing a specific function like in the case of a select targeted fortification that is desired in the body. individuals suffering from the inability to digest milk are readily relieved of their aversion to milk or milk products by the consumption of lactobacillus. this microbe secretes lactase, an enzyme that can clip the milk sugar lactose into glucose and galactose which are then utilized by the body. probiotics such as aspergillus oryzae found in fermented foods like soy sauce, and sake secretes amylase and lipases that help in the digestion of starchy and oily meals. in instances where the microbiome has been infiltrated by opportunistic microorganisms that prevent the invisible organ from performing its regular task, an intake of the probiotics can shift the microbial balance toward a more fruitful one for the body. one case in point is the colonization of the dental space by streptococcus, a microbe responsible for carving cavities on the tooth. food enriched with various species of lactobacillus can rectify this situation by creating an unfavourable situation for the invading bacteria to survive. among other various biological weapons they have in their armoury, lactobacillus group of probiotics can generate an acidic environment. this situation is known to impede the growth of the occupying bacteria and arrest their assault on the teeth. unlike the stem cells that are mainly involved in regenerating desired tissue or organ, probiotics can tilt the balance of a non-functional microbiome towards a functional one by quashing undesirable elements in the community. they can also be engaged in a targeted task designed to thwart a discomfort an individual is experiencing as in the case of intolerance to lactose or abnormal movement of the digestive system. world-wide people have been consuming fermented products laden with probiotics. these foods have evolved through centuries and each region has its own speciality of delicacies full of microbes. these probiotics have become part of the culinary culture and it will not be surprising if we learn that our palates have evolved accordingly. yogurt, a diary product supplemented with microbes lactobacillus, bifidobacteria and lactococcus is part of the grocery shelves in almost all countries. in india dahi, a fermented milk product rich in lactococcus lactis and lactobacillus acidophilus is taken with nearly all foods and its curative powers are readily touted. in the caucus region, kefir with its bountiful of lactobacillus, lactococcus, leuconostoc and bifidobacteria is a daily staple. this is derived from goat's milk and contains yellowish grains resembling cauliflower. these grains are in fact polysaccharide capsules full of probiotics. in japan the miso soup prepared from barley, rice, beans and rye is king while in africa uji the probiotics charged maize or sorghum or millet is widely cherished. although not currently available probiotics with the ability to metabolize ethanol or cure obesity will be a welcome help to those individuals who are susceptible to adverse reaction upon intake of alcoholic drinks or have difficulty controlling their body mass index (fig. 3.16 ). the mouth is literally a continuation of our external environment and is being constantly challenged by the microbes we are exposed to in our surroundings. even under this threat, the soft and hard components of the mouth harbour a good number of microbial communities that allow them to fulfill various essential tasks. the change in the chemical milieu in the mouth triggered by the intake of foods provides a fertile landscape for foreign organisms to set foot, proliferate and disturb the working microbial harmony. this happens at the onset of dental cavities when streptococcus rapidly colonizes the tooth. in this instance, a probiotic like lactobacillus rhamnosus can spring in action and create an acidic environment by the secretion of lactic acid. its ability to produce antimicrobial factors administers another debilitating punch thwarting the streptococcus-driven assault. this probiotic also helps train the immune system to fend the noxious microbe. bile, a product that is produced in the liver has been shown to be involved in a variety of tasks that make the body ticks. its acid derivatives are emerging as important signals that allow us to control our weight, regulate the level of cholesterol in the blood and determine how much fat we are going to store. the key enzyme that facilitates this formation of the bile-derived acids is bile salt hydrolase. a number of probiotics like lactobacillus, bifidobacteria, bacteriodes and enterococcus spp is known to secrete this enzyme. these probiotics can be designed to produce this enzyme that will be an excellent tool to control weight gain. this role of probiotics is akin to the use of lactase-producing lactobacillus. intake of food enriched with lactobacillus is a common therapy given in order to easy the misery of people suffering from the aversion to milk. in fact, lactase derived from probiotics is the most popular form of therapy for this ailment. nearly 90-100% adults in east asia cease to produce lactase as they reach adulthood. although it is totally natural not to have lactase after the weaning years, the omnipresence of dairy products significantly raises the importance of this enzyme beyond our childhood. hence, the remedy provided by lactobacillus-supplemented foods or capsules is a boon to the adult population world-wide who wants to partake in their dairy-laden delicacies. the curing of enzyme deficient diseases or conditions with the aid of probiotics will become a common practice as our understanding of the microbiome becomes clearer (fig. 3.17) . in diabetic patients a microorganism referred to as akkermansia muciniphilia that shelters in the mucus in the intestine can be an excellent candidate to promote carbohydrate metabolism. these microbes constitute 3-5% of the gut microflora and are known to stimulate glucagonlike peptide (glp), an important modulator of blood sugar. glucagon and insulin are the sugar police in the blood. glucagon becomes active at night during asleep. this is when the blood sugar is low. the sugar sentinel ensures that we wake up healthy by maintaining the right sugar balance. insulin on other the hand, protects against high blood sugar. for instance, when you indulge in a big bar of chocolate, it preserves the proper blood sugar and directs the extra to be stored. hence, people with a hardy dose of a. muciniphilia are less insulin resistance, a major contributing factor to obesity. the abundance of these microbes decreases with age and the intake of high fat diet. the magic of a.muciniphilia is in the production copious amount of mucin, a slimy lining for intestine that promotes a diverse microbial ecosystem and fends off opportunistic microbes. hence, the population of this blood-sugar balancing microbe can achieved by either taking it as a probiotic or by consuming nutritional fertilizer like fibers aimed at stimulating its proliferation. bifidobacteria is another group of microorganisms that dissuade infectious microbes to colonize the gut by blocking the anchoring sites and stimulating the immune system. it is not surprising that they constitute almost 80% of the cultivable microbes in the stool of infants as opposed to the meagre 20% found in adults. they contribute to the defense power in babies as they are vulnerable at this tender age (fig. 3.18a) . infants especially those who are born premature can also suffer from damaged intestinal tissues that begin to die. this disease known as necrotizing enterocolitis is characterized by bloating and swollen abdomen responds favourably to probiotics. use of the probiotic lactobacillus is quite effective in mitigating this disease. on the other hand, the intervention with a concoction of bifidobacterium, propionobacterium and lactobacillus significantly reduces the risk of allergy in infants delivered by c-section. in fact these c-section babies are devoid of the microbes that mothers impart on them if they are not born naturally. recent studies involving the sprucing the babies with the mums' microbiota are showing positive results. this kind of probiotic treatment where someone else or one's own microbe is used as probiotics to rectify aberrant biological activities is becoming more prevalent. transfer of microbes from fecal matter, ear wax, skin and other body parts is gaining medical traction and are being implemented in health centers world-wide. microbial transplant undoubtedly impart numerous health benefits (fig. 3.18b ; 3.18c). elderly individuals are another segment of the population who can benefit tremendously from the enrichment of their microbiome with probiotics. ageing is characterized with a sharp decline in microbial diversity and density. this phenomenon is the leading cause of the decrease of metabolic activities and in some cases of physiological abnormalities observed in seniors. the lower abundance of bifidobacteria coupled with the increased presence of enterobacteriaceae is a common predictor of the ageing process and such a shift in microbial population is responsible for the diminished production of enzymes and essential vitamins like vitamin b that are critical in maintaining a constant supply of energy in the body. there is also a build-up of toxic products as the proper microbial communities involved in their decomposition are severely hampered. this situation is analogous to someone not going to the toilet on regular basis. the build-up of noxious elements in the colon can be the cause of a variety of ailments including head aches and digestive discomfort. the advanced age of centenarians is attributed to a diversity of microbial populations that are a hallmark of all individuals living beyond the ripe of age 100 years. the mission of these microbes may be compared to the role stem cells play in rejuvenating the organs and extending their functional life span (fig 3.19 ). in this instance the diversity of the microbes observed in centenarians may be allowing these microbiota to supplement some of the functions their visible organs cannot perform or to generate chemical ingredients triggering the proper physiological responses from these ageing organs. hence, harnessing the power of probiotics in reconfiguring the dysfunctional microbiome characterized by the ageing process can be an important gamechanger in the life-style of seniors. this will be critical in seniors who are usually on microbiome-distressing medications. that is why it should not come as a surprise if elderly individuals take more time to heal and are invaded at a rapid rate by opportunistic bacteria. in fact microbial infection is a major concern when elderly patients are admitted to health facilities. they may be in for a hip replacement but unfortunately they run a higher risk of being contaminated by hospital dwelling-microbes. the prevalence of hard to eliminate microbes like clostridium difficile in elderly patients is a problem world-wide. and often infectious outbreaks occur in these places. unbalanced nutrition and erratic eating habits induced by reduced sensation in the olfactory and taste systems add to the dilemma facing seniors. these conditions create a nutritional environment deficient in essential nutrients like calcium and vitamins that further exacerbate the microbial ecosystem. intake of the probiotic bifidobacterium longum stimulates the proliferation of other bifidobacterium spp. administration of lactobacillus acidophilus is known to increase the synthesis of antioxidants like glutathione and oxidant bursting enzymes like catalase. while the regular consumption of bifidobacterium animalis helps reduce the time food transits in the gut, the probiotics lactobacillus rhamnonus and a propionibacterium spp contribute to the improved defecation frequency. hence the implementation of a nutritional regime rich in probiotics will go a long way in easing the discomfort and some of the ailments that are associated with old age. evidence-based research is revealing how probiotics can help reverse ailments caused by the disruption of the microbial communities in our body. irritable bowel syndrome (ibs) is one such disease where a perturbation in microbial population is the main cause of the disease. this shift in the ecosystem of the microbiota is punctuated by a loss in immune tolerance and a rise in inflammatory response. to mitigate this situation and to remedy this ailment, the administration of probiotics like e.coli nissle has been relatively more effective than the use of antibiotics. in this instance, the probiotics appear to deliver a three-pronged attack on the invasive and crafty microbes. they improve the fence lining the intestine, they out-compete the bad bacteria and they secrete molecular soldiers like hydrogen peroxide and lactate that stop invaders right on their tract. the significance of probiotics as therapeutic agents is only now beginning to be appreciated and is slowly challenging the aggressive use of antibiotics in treating these conditions. unfortunately, antibiotic-based remedies tend to seriously interfere with the workings of our microbiome as their action against toxic microbes also eliminates some of our trusted invisible partners. the uncontrolled proliferation of the stomach residing helicobacter pylori is a cause of concern as it is a risk factor for gastroduodenal ulcers and lymphomas. the intake of probiotics such as lactobacillus and streptococcus thermophilus has been demonstrated to correct this imbalance. the ability of these probiotics to secrete scfa like acetate and butyrate is known to be a key factor in impeding the march of the over-reactive h. pylori. wound healing and cancer prevention are other two activities that probiotics are also involved in. lactobacillus acidophilus that is an excellent candidate with the power to heal injuries is known to arrest the colonization driven by the scheming pseudomonas aeruginosa. the acidic environment nurtured by the lactobacillus renders the ability to conquer the tear on the skin almost impossible. the propensity of probiotics to bind to mutagenic compounds and inhibit the production of carcinogens by opportunistic pathogens is central for their anti-tumour activity. the lactic acid producing bacteria such as lactobacillus acidophilus, bifidobacterium bifidum and streptococcus lactis are routinely being recommended for the prevention of cancers. some select tumours like in colorectal cancers are being treated with the aid of these life-microbial concoctions. hence it comes as no surprise that the use of probiotics as a prevention of diseases and a therapy against some specific disorders is on the rise. the commercial value of this industry is into hundreds of billions of dollars, a figure that is further expected to increase as probiotics are safe and well-tolerated in children, premature infants and in the general population as a whole. furthermore, unlike pharmaceutical products, probiotics are very easily administered mostly as components of foods and the lingering effects of these chemicals are quickly becoming a relic of the past. the microbiome can also be nurtured and programmed with the assistance of prebiotics. these are natural products that foster an environment propelling the proliferation of some desirable microbes. in the same manner as we are always eager to give our visible organs a boost when they are not working well or when they are sluggish, we can revitalize our invisible organ. our more traditional visible organs can be invigorated by taking supplements like vitamins or minerals that provide ammunition to various metabolic activities in the muscle, brain, heart or liver. for instance, athletes take creatine, a product responsible for energy stabilization to improve their performance. creatine and creatine kinase duo enables us to engage in physical activities quickly and fast. without the intervention of this dynamic couple, daily chores will happen in a relatively sluggish manner. individuals with excess body mass consume carnitine with the hope of inducing the various body parts in burning fats. well, the constituents of the invisible organ can be strengthened with the intake of prebiotics. these are like fertilizers that create the proper environment for the desired microbial ecosystem to flourish. they tilt the microbial community in a manner favouring some specific tasks to be performed. for instance, a prebiotic promoting the growth of lactase-secreting microbes will help lactose intolerant people while a prebiotic inhibiting the competitors of vitamin k-producing bacteria will be of immense assistance to patients having difficulty with blood coagulation. prebiotics are excellent nutritional supplements involved in modulating and adjusting the microbiome that can undergo disruption due to ageing, infection, climate change and a sleuth of other factors one may be subjected to as one goes through one's daily activities (fig. 3.20) . what are these magical prebiotics? they are usually plant derived fibers and complex carbohydrates contained in milk that help promote the growth of specific microbial ecology in our body especially in our digestive system. they are mostly resistant to digestion by the enzymes produced by the gut. they are metabolized only by our invisible organ and/or promote proper microbial communities that impart health benefits to the host. the production of short-chain fatty acids (scfa-acetate, butyrate) in response to the intake of prebiotics can contribute to the energy budget of the muscle, can regulate cholesterol biosynthesis and can provide fuel to the colon. stimulation of calcium absorption, reduction in infections and repression of allergic symptoms are some of the other activities prebiotic intake is known confer on the host. these physiological processes are of course a major boon to our well-being as these are like lubricants oiling our body machines. the prebiotics prime the microbiome and this invisible organ is reflective of the prebiotics consumed. as a toned muscle system is indicative of the time spent at the gymnasium, specificity and diversity of the invisible organ is a tell-tale sign of our dietary fiber-eating habit (fig. 3.21) . prebiotics or dietary fibers have been part of human nutrition since the dawn of civilization. in 450 bc hippocrates had noted the laxative attribute of coarse wheat compared to the refined variety. in the 1920s kellogg sang the praise of bran as a nutrient that increased stool weight, prevented diseases and acted as a laxative. all health gurus extoll the praise of the nutritional quality of fiber-rich foods. after a relative quiet epoch in the health benefits of the dietary fibers, their involvement in mitigating ailments like obesity, cardiovascular disorders and diabetes were widely revived in the 1970s and has now become a staple of balanced nutrition. the main prebiotics are the galacto-oligosaccharides (gos) obtained from milk, inulin associated-fructo-sugar (fos) found in chicory roots and xylooligosaccharides (xos) present in plant products like palm oil and corn plants. they also occur naturally in milk, asparagus, garlic, onion, leeks, wheat, oats and soya beans. they are soluble, resistant to the acidic environment in the stomach, fermentable and stimulate growth or the activity of intestinal microbes responsible for the well-being of the host. prebiotics tend to be relatively selective to microorganisms like bifidobacteria and bacteroides. they are converted into simpler derivatives before they reach the colon. here they act as an anchor for the establishment of a unique ecosystem that is aimed at aiding the host in accomplishing a variety of tasks. they generate energy that fuels the muscles and the colon. they are also involved in repressing allergic symptoms. the reduction of infection and promoting the proliferation of good microbes like akkermansia are some other positive features prebiotics contribute to. they are also known to decrease glucose absorption, activate cholesterol excretion, and promote laxation. one of the most intriguing influence of prebiotics in dictating our microbial ecosystem and promoting our well-being has been observed in infants just immediately after birth. babies who are fed their mothers' milk have much better health outcomes than those fed formulated milk. they are less susceptible to allergic reactions and kids nourished by natural milk tend to be less prone to asthma. the gos, a natural ingredient in the mother's milk cannot be digested by the babies' developing guts. they are designed to promote the growth of microbes that are crucial for a variety of tasks at this critical juncture in their lives. one can safely conclude that mother-nature is making these goodies not for the babies but for the microbes feeding on them. in fact, the presence of a unique ingredient known as sialic acid in the milk of some malawian mothers result in well-nourished infants as opposed to infants who are fed by mothers without this magical tonic in the milk. this prebiotic is responsible for the growth of a unique set of microbes that generates simple sugars utilized by infants for their development. even from such an early stage of human life, the body is programmed to depend on these invisible partners. it is not surprising to learn that various milk products are being formulated with oligosaccaharides in an effort to generate increased microbial population in growing infants. the elderly can also benefit from the intake of prebiotics as their microbiota tend to undergo a drastic change and there is a significant reduction in the diversity and abundance of the microbial ecosystem as age progresses. the fructooligosaccharide (fos)-based prebiotics have an excellent bifidogenic activity while resistant starch like sprouted legumes improves bowel movement by promoting the growth of lactobaccilli and bifidobacteria. these microbial fertilizers can be given to relieve seniors of constipation, a condition quite acute in this segment of the population. prebiotics not only promote a healthy life, their intake can be tailored to remedy a number of ailments that are due to dysbiosis i.e the perturbation of microbiota in the body. their ability to nurture a select group of bacteria that possess a range of tools to combat the opportunistic invisible invaders can be put to good use. obesity is a disease that is characterized by low grade inflammation and is punctuated by a decrease in the diversity of gut microbiome, a situation resulting in the ineffective harvesting of calories from what we eat. the inadequate energy extraction from food intake automatically triggers the hunger hormone, ghrelin which stimulates the desire to eat more food. this vicious circle causes the body to gain weight. the utilization of fos prebiotics like inulin-laced pasta decreases the bacterial lps, suppresses the hunger signal, promotes satiety and increases microbial diversity events aimed at countering obesity. a prebiotics is an effective means in controlling weight gain. cardiovascular diseases do also respond well to prebiotics therapy. water soluble fibers such as pectin found in fruits and guar gum from guar beans are known to decrease the bad cholesterol (ldl low density lipoprotein) without affecting the good cholesterol (hdl high density lipoprotein). they also help lower the blood pressure. the positive influence of these prebiotics on the functioning of the heart has been widely documented and accepted as a means of averting cardiovascular diseases. mitigating constipation and prompting proper bowel mobility are the gold standard treatment fuelled by the intake of prebiotics. cereal fibers like wheat bran are religiously utilized to counter any abnormality associated with fecal bulking. prebiotics can also be a potent remedy against cancers. enhanced micronutrient absorption and stimulation of microbes with cancer fighting enzymes like glutathione transferase are some of the mechanisms that these nutritional elements confer to the body. the ability of prebiotics to enrich the diversity of the microbiome is central to its curative power (fig. 3.22; fig. 3.23; fig. 3 .24). prebiotics is almost like a dietary manure that transforms the microbial landscape in the gut. a similar strategy is utilized to help the visible body parts if something goes amiss. during anemia when the level of hemoglobin is low, iron sulfate pills enables the blood system to stimulate the production of this oxygen carrier. in the case of keratoconus, an eye condition where the cornea loses its focusing power, the surgical administration of vitamin b2 allows the eye to function normally. in this instance, the infusion of vitamin b2 promotes the crosslinking of collagen, a biological event that fortifies the cornea and enables it to contribute to visual sensation. the same can be said of lipoic acid and calcium supplements. the former helps fortify the workings of the brain while the latter enable the proper functioning of the skeletal system. as these ingredients assist the target organs optimize their biological roles, the intake of prebiotics provides a proper nutritional environment for the right microbial ecosystem to flourish in order to maximize the output of the invisible organ in promoting a healthy body. however, this is a non-invasive exercise and usually corresponds to an accompaniment of the food we consume unlike the surgical intervention needed to feed the cornea with vitamin b2. hence, prebiotics are natural fertilizers for the microbiome and can remedy the microbial perturbations that the body has to often undergo due to age, environmental change or intake of medications. one can also utilize a two-pronged approach comprising probiotics and prebiotics to regulate an unbalanced microbial landscape. this is termed as synbiotic and can be equated to a multi-drug regime to cure a disease. a combination of these two stimulators can have a more vigorous impact on the invisible organ and can provide a very effective therapy against ailments mediated by uncontrolled proliferation of some opportunistic microbes. the blending of live microbes and the microbial fertilizers that enable them to thrive is a very powerful tool to goad the constituents of the invisible organ in executing a dedicated task. for instance, muscle wasting and inflammation that characterize cancer cachexia has been shown to be mitigated with a concoction of a synbiotic consisting of lactobacillus reuteri and inulin-based fructans (fos). the intake of this synbiotic reduces the population of enterobacteriacae, replenishes the lactobacillus colonies and prolongs the life of the patients. the therapeutic value of the fos and lactobacilli taken together in combatting hemorrhage triggered by bacterial infection has also been recognized. in this instance, a sharp decrease in e.coli counts coupled with an increase in the population of lactobacillus spp contribute to bringing relief to these patients. quite often a cocktail of drugs is prescribed to combat a viral infection; this can be equated to synbiotic therapy. the aids virus, can only be eliminated from the blood system of infected individuals by the use of anti-proteases and medications inhibiting the replication of the viral genetic code. hence, this double punch aimed at aiding the microbiome naturally is of immense benefit to the body. the human body is made of cells that are in constant flux due a variety of factors including wear and tear as age progresses. these can be repaired or rejuvenated with the aid of medical intervention. one can seek the help of a cosmetic surgeon to remove ageing spots or modify the shape of the nose. in case of the microbiome, any perturbations (dysbiosis) that the invisible organ experiences can be modulated by the programmed intake of probiotics and prebiotics. hence, the microbiome behaves in manner that is comparable to the organs composed of visible cells. it is clear that the body is made up of visible and invisible cells. most visible cells assemble as various organs we can see; there are few exceptions like the blood system where the cells are mobile and interact directly and indirectly with all the organs. just like dysbiosis that robs the microbiome of its natural functions, the organs can also be subjected to a number of challenges over body's life span. the inherent repair machinery cannot remedy the situations the organs are facing in a timely manner, intervention in form of medication or surgery is needed to bring them back as contributing members of the body. for example if some oily clutters are impeding the flow of blood, the arteries are unclogged by surgical procedure. if organs of the body are insulted by overuse of some products or by accidental intake of toxic compounds, antidotes are utilized to bring back these anatomical structures in tip-top shape. for instance, fomepizole can give reprieve to a liver challenged by alcohol abuse while high levels of cholesterol in the blood that contribute to plaque build-up in the arteries can be rectified by statin family of cholesterol-busters (fig. 3.25 ). in the same manner any disturbance in the microbial communities constituting the microbiome can be adjusted with the intake of probiotics, prebiotics or synbiotics. these 'therapies' are akin to the medications, the surgery, antidotes or stem cells that are deployed to tweak the visible organs in an effort to bring them to a normal working condition (fig. 3.26) . the fact that the microbiome is invisible and its molecular operation is still not fully understood compared its visible counterparts like the liver should not preclude it from being an important component of the cellular collectivity responsible for the body's well-being. the microbial system spans a wide surface area within and on the body just like the blood system as it imparts extremely valuable functions. this microbial ecosystem is to some extent responsible for the human anatomy and makes it work the way it does. understanding how the invisible organ operates, how the harmony amongst its cells are disrupted and how the fine-balance is reconstituted with the aid of probiotics and prebiotics are essential if we are to understand the intimate details hence, dysbiosis and rebiosis propelled by the infusion of live seeds (probiotics) and the fertilizers (prebiotics) will go a long way toward promoting wellness and will bestow upon us a healthy life probiotics, prebiotics and colorectal cancer prevention immune system stimulation by probiotic microorganisms interactions between the microbiota and pathogenic bacteria in the gut individual diet has sex-dependent effects on vertebrate gut microbiota microbiota and healthy ageing: observational and nutritional intervention studies a gastroenterologist's guide to probiotics the impact of the gut microbiota on human health: an integrative view probiotics and gastrointestinal disease: successes, problems and future prospects probiotics and prebiotics in ulcerative colitis microbiome-wide association studies link dynamic microbial consortia to disease prebiotics: why definitions matter the oral microbiome -an update for oral healthcare professionals probiotics in prevention and treatment of obesity: a critical view comparison of the immunomodulatory properties of three probiotic strains of lactobacilli using complex culture systems: prediction for in vivo efficacy the microbiome in asthma the skin microbiome: is it affected by uv-induced immune suppression? front microbiol 7:1235 the role of bile acids in reducing the metabolic complications of obesity after bariatric surgery: a systematic review recent developments in prebiotics to selectively impact beneficial microbes and promote intestinal health regulation of the immune system by biodiversity from the natural environment: an ecosystem service essential to health functions of the skin microbiota in health and disease psychobiotics and the manipulation of bacteria-gutbrain signals cancer-promoting effects of microbial dysbiosis default normal template original article oxidative stress & c – reactive protein in patients with arthritis imad a. thanoon*ph.d nahla o. tawfik** farhad n. hussin*** summary: back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. j fac med baghdad 2007; vol. 49, no.2 received jan.2007 accepted april 2007 method: serum tas , mda and crp levels were measured in 16 ra and 24 oa patients and compare with those obtained from 25 healthy controls. results: serum tas were significantly lower in ra group than in the oa and control groups (p < 0.05). serum mda and crp levels were significantly higher in patients with ra than in those with oa and healthy subjects (p< 0.05). there were significant negative correlations between tas and mda, crp levels (r = -0.850; p < 0.001) and ( r = -0.498; p < 0.05) respectively and a positive correlation between mda and crp levels in the ra group (r = 0.686; p < 0.01) . in oa group, the level of crp was significantly increased (p< 0.05) and there was significant positive correlation between age and mda level (r = 0.553; p < 0.01). conclusion: our results demonstrated that levels of lipid peroxidation are increased in patients with ra compared to controls and patients with oa , in addition serum tas levels were decreased in ra. serum tas levels may be used as a routine and rapid test to verify the levels of oxidative stress in ra. furthermore correlating tas and mda levels with a cute phase reactants such as crp may give some clues about disease activity in ra . __________________________________________________________________________________________ introduction:_______________________________ there has been great interest among researchers in the past 20 years for the role of oxidative stress in the development of arthritis(1). evans and halliwell stated that the damaging oxidative species (reactive oxygen, nitrogen and others) arise as byproducts of metabolism and as a physiological mediators and singling molecules (2). the levels of these oxidative intermediates are held in cheek by the antioxidant defense system, the component of this defense system are micronutrients like vitamin c and e(3). a deficiency in these micronutrients leads to oxidative stress which leaves the body tissue open to the damaging effects of the oxidative intermediates which may accompany inflammatory and immunological process seen in ra patients(4,5). in addition many studies have suggested the major role of these intermediates in altering chondrocyte (cartilage cells) function in oa(6). _________________________________________ *assistant professor, college of medicineuniversity of mosul **lecturer, college of dentistry – university of mosul *** lecturer, college of medicineuniversity of saladin oxygen free radicals have been suggested to exert their cytotoxic effect by causing oxidation of membrane phospholipids ( i.e. lipid peroxidation)(7). many products of lipid peroxidation are not overtly toxic or are minor products of major toxicological interest one of them is malondialdehyde (mda)(8),a major reactive aldehyde and is used as an indicator of tissue damage(9). elevated levels of serum mda were observed in ra patients which suggested that there is an increase in oxidant stress in those patients(10,11,12). it is now widely accepted that inflammation and oxidative stress are two important processes integrally involved in the development and progression of arthritis(13), c–reactive protein (crp) is a well established marker of inflammation and is classified as an acute phase reactant(14) often showing coordinated response with interleukin 6(15), crp measurement served mostly in diagnostic, albeit non – specific one , and in monitoring role in such fields of infections and rheumatology(16). kumon et. al.(17) found that serum crp was significantly higher in ra than in oa, and correlated with erythrocyte sedimentation rate (esr) in arthritis patients, he concluded that higher levels of crp seems to reflect greater degree of joint inflammation in ra and oa patients. j fac med baghdad vol. 49 , no. 2 , 2007 227 simpo pdf merge and split unregistered version http://www.simpopdf.com oxidative stress & c – reactive protein in patients with arthritis imad nahla farhad the purpose of the present study was to examine the levels of lipid peroxidation (mda), crp, and tas in patients with ra and oa and to compared that with healthy control subjects. j fac med baghdad 228 vol. 49 , no. 2 , 2007 patients and method: sixteen ra patients all fulfilling the american rheumatism criteria for ra(18) and, twenty four oa patients of the same age group were included . all patients had early active disease and were on non steroidal anti– inflammatory drugs as maintenance therapy. no prior treatment with second line anti rheumatic medication . the data collected include age, sex , weight and height. body mass index (bmi) was calculated with patient clothed without shoes and expressed in kg/m2 . the control group consisted of twenty five healthy individuals of the same age group . all of the subjects in this study were non – smoker. blood samples were collected from the antecubital vein into plain tubes. mda levels were assayed in serum as described by ohkawa (19). tas level were estimated using commercial assay kit obtained from randox(20). serum crp concentration were measured by nephlometric immunoassay(21). results were expressed as mean ± standard deviation (sd) . differences in mean values of serum variables between healthy controls and arthritis patients were analyzed with the students – t – test. p value of < 0.05 was considered significant. pearson correlation coefficient was done to calculate the r value between the parameters analyzed in the serum. results: patients characteristics are given in table (1), the levels of antioxidant and markers of oxidative stress are given in table (2). table (1) base line characteristics of arthritis and control groups characteristics control n= 25 ra group n=16 oa group n= 24 male : female 11 : 14 7 : 9 10 : 14 age (years) 49.12 ± 4.25 49 ± 3.72 52.08 ± 3.32 bmi kg/m2 24.36 ± 1.32 22.24 ± 2.18 23.06 ± 2.09 n= number of patients data are presented as mean ± sd. table (2) serum tas, mda and crp levels in ra, oa and control groups subject croup n tas (mmol/l) mda (μ mol/l) crp (mg/l) control group 25 1.44±0.19 1.08±0.21 2.875±0.52 ra group 16 1.06±0.21* 2.01±0.26** 23.61±10.43*** oa group 24 1.43±0.20 1.19±0.36 5.50±0.83* n= number of patients data are presented as mean ± sd. * p<0.05, ** p<0.01, *** p<0.001 vs. the control group. in ra patients, mean values of tas were significantly lower by – 25.35% (p< 0.05) from the control group while mda and crp mean levels are significantly increased by 85.32% (p< 0.01) and 723.0 % (p< 0.001) respectively when compared with health control subject. serum levels of mda and crp where significantly higher (p< 0.05) in patients with ra that those with oa while the tas level was significantly lower (p< 0.05). pearson correlation showed that there were significant positive correlation between mda and crp level (r = 0.686 ; p< 0.01) (fig. 1). while there were significant negative correlation between tas and crp (r = -0.498; p < 0.05) (fig. 2) and between tas and mda levels (r = -0.850; p < 0.001) as shown in (fig. 3). in oa group, the level of crp was significantly increase (p< 0.05) and the percentage of increment was 91.64 % when compared with control group, no significant differences were observed between tas and mda levels on comparison with control. there were no significant correlation between these parameters in oa patients, the only significant correlation was between age and mda level (r = 0.553 ; p < 0.05) as shown in (fig. 4). figure (1): correlaion betwee n m da &crp le ve ls in rheumatoid arthritis patients y = 0.0174x + 1.6046 r = 0.686(sig.)** 0 0.5 1 1.5 2 2.5 3 0 10 20 30 40 50 60 crp (m g/l) m d a ( µ m o l/l ) simpo pdf merge and split unregistered version http://www.simpopdf.com oxidative stress & c – reactive protein in patients with arthritis imad nahla farhad j fac med baghdad 229 vol. 49 , no. 2 , 2007 discussion: rheumatoid arthritis and osteoarthritis are the most common inflammatory diseases worldwide. in the present study serum tas levels were significantly lower in the ra group than the oa and control groups, while serum mda and crp concentrations were significantly higher in patients with ra than those with oa and healthy subjects. these results are consisted with many studies (5,11,13), were an increased levels of lipid peroxidation reaction and lower tas level in ra patients as compared to healthy subjects were reported. figure (2): corre lation be twe e n tas & crp le v e ls in rhe umatoid arthritis patie nts y = -24.51x + 49.737 r = -0.498 (sig.)* 0 10 20 30 40 50 60 0 0.5 1 1 tas ( mmol/l ) c r p our previous study (12) had shown a decreased antioxidant level (glutathione) and an increase in the mda level in erythrocyte and plasma of patients with active ra compared with healthy subjects. antioxidants hypothesized to provide protection against ra, was 400mg of vitamin e and 100mg vitamin c given twice daily and were significantly effective in reducing duration of morning stiffness, ritchie joint index, ( patient and physician global assessment of disease activity parameters in ra patients). wang et. al.(4) found that dietary supplementation with vitamin e alone reduces the base line inflammatory status, that is indicated by crp concentration in healthy baboon. .5 ( m g/ l ) figure (3): corre lation be twe e n tas & m da le v e ls in rhe umatoid arthritis patie nts y = -0.68x + 2.4362 r = -0.850(sig.)** 0 0.5 1 1.5 0 1 2 3 tas( m m ol/l) m d a ( µ m o l/l ) a significant increase in mda and a decrease in tas levels in the present study reveals that there is an increase in the oxidative stress in ra. the negative correlation between mda, crp and tas parameters can be explained by the impairment of the antioxidant defense mechanisms due to excess utilization by the inflamed tissue to scavenge the excessive lipid peroxide that are generated at the inflammatory site or to scavenge accumulated lipid peroxides in the plasma(22). increase in the in vivo generation of oxidants and lipid peroxidation product was demonstrated in plasma of ra patients which correlated with the antioxidant level (14). this may indicate evidence of oxidative stress in those patients and that it plays an important role in the pathogenesis of the disease(1). crp was positively associated with mda and this could give a clue the extent and rate of tissue damage due to increase oxidative stress and increased lipid peroxidation. sukkar and rossi (25) had supposed that oxidative stress may trigger inflammatory activity and therefore it may induce a flare of the disease. figure (4): correlation between age & mda levels in osteo arthritis patients y = 0.0605x 1.9584 r= 0.553(sig.)** 0 0.5 1 1.5 2 0 20 40 60 80 age ( year ) m d a m o l/l ) µ many studies have reported higher oxidative stress in oa (23,24) and this is in accordance with our results, where the only significant increase was in the crp level. this increase combined with haemostatic and haemodynamic reaction to the stress task could give an indication to the disease activity in oa patients(6). kumon et.al. (17) found ( simpo pdf merge and split unregistered version http://www.simpopdf.com oxidative stress & c – reactive protein in patients with arthritis imad nahla farhad j fac med baghdad 230 vol. 49 , no. 2 , 2007 that serum and synovial crp levels were significantly higher in ra than oa patients, and it correlate with esr in all arthritis patients. in this study mda levels were significantly correlated with age in oa patients. our results are consisted with the hypothesis that with age the prevalence of oa increases and the efficacy of articular cartilage repair decrease(26). it was suggested that in vivo chondrocyte essences contributes to the age related increase in the prevalence of oa and decrease in the efficacy of cartilage repair. kehan and archer(6) found that articular cartilage is susceptible to many forms of injury some of which may lead to secondary arthritis at much later time. numerous medical studies (22,25) concluded that oxidative stress is the root cause of arthritis when the antioxidant defense system is over whelmed, this oxidative stress within the joint causes damage to the surrounding cartilage (4). in oa and ra, there is a focal loss of cartilage resulting from catabolic pathway in the joint over production of free radicals and lack of oxygen processing enzymes and free radical scavenging molecules can lead to inflammation. inflammatory products like phagocytosis from neutrophils create even more free radicals (2).it is this viscious low grade inflammatory response that leads to the destruction f the joint and this may contribute to the pathogenesis of the disease (24,25). increasing numbers of health care recognizes the need for diagnostic laboratory tests that measure oxidative damage and the status of the individuals antioxidant defenses. given the multiplicity of antioxidant and the influences of life style and nutritional supplements on an individuals antioxidant capacity, it is important to be able to quantitively measure the total antioxidant capacity of the antioxidant power within biological specimens and use it as a routine work to detect the level and to correlate it with the disease activity for patients at various stages of the disease. references: 1aaseth j, haugen m, forre o. rheumatoid arthritis and metal compounds. perspective on the role of oxygen radical detoxification. analys 1998;123:36-44. 2evans p, halliwell b. micronutrient: oxidant/ antioxidant stuatus. br j nutr 2001;85: 67-74. 3hamilton jmj, gilmore ws, benzie iff, mulholland cw. interactions between vitamins c and e in human subjects. british journal of nutrition 2000;84: 261-267. 4wang l, rainwater dl, mohaney mc, stocker r. co supplementation with vitamin e and coenzyme q10 reduces circulating markers of inflammation in baboons. am j clin. nutr. 2004; 80: 649-655. 5vansanthi p, ganesan n, hariprasad c, rajesekhan g, meera s. plasma lipophilic antioxidant and pro-oxidant levels in rheumatoid arthritis. j indian rheumatol assoc. 2004:12:40-42. 6khan im, archer cw. oxidative stress induce expression of markers of early osteoarthritis and chondrocyte terminal differentiation in cultured bovine explants. european cell and mineral 2006; 12(3),36. 7sarban s, kocyigita, yazar m, isikan ue. plasma total antioxiddnt capacity, lipid peroxidation and erythrocyte antioxidant enzyme activities in patients with rheumatoid arthritis and osteoarthritis. clin. biochem. 2005; 38: 981986. 8mayes pa. structure and function of lipid-soluble vitamins in: harpers biochemistry, 25th ed., murray rk, granner dk, mayes pa, rodwell vw (eds). appleton and lange, standford, connecticut :219-223. 9dezwart ll, neerman jhn, commandeur inm, vermeulen npe. biomarkers of free radicals damage applications in experimental animals and in humans. free radic biol. med. 1999; 26: 202-226. 10stock j, dormandy tl. the autoxidation of human red cell lipids induced by hydrogen peroxide. br j haem 1971; 20-95-111. 11gambhir jk, lali p, jain ak. correlation between blood antioxidant levels and lipid peroxidation in rheumatoid arthritis. clin biochem. 1997; 30: 200-204. 12tawfik nom. the role of antioxidant and non steroidal anti inflammatory drugs in the management of patients with rheumatoid arthritis ph. d. thesis college of medicineuniversity of mosul 2004. 13vijaya kumar d, suresh k, monaharan s. lipid peroxidation and antioxidant status in blood of rheumatoid arthritis patients. indian j of clinical biochemistry 2005; 21: 104-108. 14nagler rm, salameh f, reznick az, livshits v, nahir am. salivary gland involvement in rheumatoid arthritis and its relationship to induced oxidative stress. rheumatology 2003; 42: 1234-1241. 15sac ke. evaluation of patients c: laboratory assessment. in: primer on the rheumatoid disease, 12th ed., klipped jh, crofford lj. ston jh, weyand cm (eds.). arthritis foundation, atlanta georgia, 2001; 218-225. 16crockson ra, crockson ap. relationship of the erythrocyte sedimentation rate to viscosity and plasma proteins in rheumatoid arthritis. ann rheum. dis. 1974; 33:53-56. 17kumon y, suehiro t, nishia k, hashimoto k nakatani k, sipe jd. ferritin accelerates with c-reactive protein and acute phase serum amyloid. amyloid 1999; 6:130135. 18arnett fc, edworthy sm, bloch da, et. al. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis. rheum 1988; 31: 315-325. 19ohkawa h, ohishi n, yagi k. assay for lipid peroxides in animals tissues by thiobarituric acid reaction. analytic biochem. 1979; 95:351-358. 20miller nj, rice evance c, duvies mj, gopinathan v, milner a. total antioxidant status by colorimetric methodclinical science. 1993; 84:407-412. 21wener mh, daum pr, mcquillan c. the influence of age, sex and race on the upper reference limit of serum c-reactive protein concentration. j rheumat 2000; 27:2351-2359. 22halliwell b, hoult hr, blake dr. oxidants inflammation and anti inflammatory drugs asebj1988; 2: 2867-2873. 23maneesh m, jayalekshmi h, sama t, chatterjee s, chakrabati a, sigh ta. evidence for oxidative stress in osteoarthritis. indian j of clinical biochemistry 2005; 20 :129130. 24yudoh k, trieu nv, nakamura h, kato kt and nishioka k. potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis. arthritis res ther 2005; 7: 380-383. 25suckker yg, rossi e. oxidative stress and nutritional prevention in autoimmune rheumatic disease. autoimmunity review 2004; 3:199-206. 26mortin ja, buckwalter ja. the role of chondrocyte senescence in the pathogenesis of osteoarthritis and in limiting cartilage repair. j of bone and joint surgery 2003; 85: 106-110. simpo pdf merge and split unregistered version http://www.simpopdf.com 1.doc 2.doc references list all authors when six or less; when seven or more, list only first three and add et al. tables submission of manuscripts mail the required number of manuscript copies in a heavy-paper envelope, enclosing the manuscript copies and figures in cardboard. manuscripts should be accompanied by a covering letter from the author who will be responsible for correspondence regarding the manuscript. the covering letter should contain a statement that the manuscript has been seen and approved by all authors. 3.doc modified sugiura operation for portal hypertension and bleeding esophageal varices 172 omar s. khattab,samie b. safar post-surgical loco regional recurrence of breast carcinoma in iraq 193 ali m. al-saiegh 4.doc 5.doc modified sugiura operation for portal hypertension and bleeding esophageal varices omar s. khattab*, m.b.ch.b , h.d.s , h.d.l.m , f.i.c.m.s, c.a.b.s samie b. safar, m.b.ch.b , f.r.c.p, f.r.c.s, f.a.c.s 6.doc introduction:______________________________ types of incisional hernias: _________________________________________ clinical types of incisional hernias: patients and methods: discussion conclusions references 7.doc 8.doc 9.doc post-surgical loco regional recurrence of breast carcinoma in iraq ali m. al-saiegh * m.b.ch.b. d.s f.i.c.m.s. summary conclusion: carcinoma of the breast affecting iraqi females at younger ages in a high & increasing rate than other studies with a higher loco-regional recurrence rate. significant association were found regarding latency period, staging, histopathology & grading of primary tumour . aims of study: 1. to assess the incidence of post operative loco regional recurrence of breast carcinoma in iraqi female patients. 2. to determine the significance of certain variables that may affect the loco regional recurrence rate . introduction:______________________________ * head of department of surgerymedical college kufa university results: discussion: conclusion references 10.doc 11.doc 12.doc 13.doc 14.doc 15.doc 16.doc back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. __________________________________________________________________________________________ introduction:_______________________________ **lecturer, college of dentistry – university of mosul patients and method: results: 17.doc 18.doc 19.doc hla -a hla-b total increased frequency study groups blood groups 20.doc subject and methods: 21.doc 22.doc 23.doc 24.doc 25.doc 26.doc rabab .n. al-saadi.* ficms back ground: psoriasis is a chronic relapsing disorder with no life long cure, many systemic and topical modalities are available, one of these topical modalities is the vitamin d analogue (calcipotriene) which is widely used recently to treat psoriasis and many other skin problems. 27.doc 28.doc 29.doc 30.doc 31.doc 16.pdf back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. __________________________________________________________________________________________ introduction:_______________________________ **lecturer, college of dentistry – university of mosul patients and method: results: 166.pdf back ground: oxidative damage has been suggested to play a key role in accelerating inflammation and to be involved in the pathogenesis of rheumatoid arthritis (ra) and osteoarthritis (oa). many studies had shown that those patients have low antioxidants level and are at risk of increased oxidative stress. objective: this study was designed to examine the levels of serum total antioxidant status (tas). malondialdehyde (mda) as index of lipid peroxidation and c–reactive protein (crp) as a marker of oxidative stress in patients with ra and oa and compared them with healthy control. __________________________________________________________________________________________ introduction:_______________________________ **lecturer, college of dentistry – university of mosul patients and method: results: dermatology: practical and conceptual observation | dermatol pract concept 2016;6(4):5 23 dermatology practical & conceptual www.derm101.com case report a female in her seventies, skin phototype ii, presented to her gynecologist with a two-month history of a lesion on the inside of her right labia minora. her past medical history was significant for rheumatoid arthritis requiring ongoing treatment with methotrexate for 20 years and adalimumab for 10 years. her gynecologist prescribed, based on the suspicion of a herpes simplex infection, acyclovir 5% cream and, after some days, a hydrocortisone 1% / clotrimazole 1% cream for two weeks. because no change was visible at the followup examination, a biopsy was taken of a part of the nodule. based on the histopathological diagnosis of an amelanotic melanoma (breslow thickness of 1.3 mm), the patient was dermoscopic appearance of an amelanotic mucosal melanoma andreas blum1, ulrike beck-zoul2, laura held3, sylvie haase1 1 public, private and teaching practice of dermatology, augustinerplatz 7, 78462 konstanz, germany 2 public and private practice of gynecology, rosgartenstrasse 27, 78462 konstanz, germany 3 dermatopathology, siemensstraße 6/1, 88048 friedrichshafen, germany key words: melanoma, mucosal melanoma, dermoscopy citation: blum a, beck-zoul u, held l, haase s. dermoscopic appearance of an amelanotic mucosal melanoma. dermatol pract concept 2016;6(4):5 .doi: 10.5826/dpc.0604a05 received: august 24, 2016; accepted: august 31, 2016; published: october 31, 2016 copyright: ©2016 blum et al. this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. funding: none. competing interests: the authors have no conflicts of interest to disclose. all authors have contributed significantly to this publication. corresponding author: andreas blum, md, msc, public, private and teaching practice of dermatology, augustinerplatz 7, 78462 konstanz, germany. tel. +49 7531 643 11; fax. +49 7531 600 54. email: a.blum@derma.de background: hypomelanotic or amelanotic melanomas are challenging to identify, especially at mucosal sites. the dermoscopic clues to the diagnosis of mucosal melanomas have been reported to be structureless zones with the presence of blue, gray, or white colors. case: a female in her seventies noted a new lesion on the inside of her right labia that first appeared two months prior. her past medical history was significant for rheumatoid arthritis requiring ongoing treatment with methotrexate for 20 years and adalimumab for 10 years. after no response to two weeks of local treatment for suspected herpes simplex infection, her gynecologist performed a skin biopsy. based on the histopathological diagnosis of an amelanotic melanoma (breslow thickness of 1.3 mm) the patient was referred to dermatology for further assessment. polarized dermoscopy revealed a distinct asymmetric, sharply demarcated homogenous white papule (4 x 5 mm) as well as polymorphous vessels. conclusion: dermoscopy may aid in the diagnosis of amelanotic mucosal melanomas. our case revealed a structureless white area and polymorphous vessels. additional clues to the diagnosis were the advanced age of the patient and the clinical presentation of a new lesion. abstract 24 observation | dermatol pract concept 2016;6(4):5 the correct diagnosis is often delayed, leading to a poor prognosis of malignant tumors, especially mucosal melanomas [6]. possible differential diagnoses of our case include herpes infection, condylomata acuminata, condyloma lata of syphilis, inflamed cysts or glands, and squamous cell carcinoma. referred to dermatology for closer examination. clinically a whitish papule on the inside of her right labia minora was visible (figure 1). under polarized dermoscopy a distinct asymmetric, sharp demarcated homogenous white papule (4 x 5 mm) with polymorphous vessels was present (figure 2). histologic examination revealed malignant melanoma mucosal type with densely packed atypical spindly-shaped melanocytes with hyperchromatic nuclei, confirmed by positivity of anti-mela antibody (figure 3). conclusion this case suggests that amelanotic mucosal melanoma may reveal diagnostic clues upon dermoscopic examination. a multicenter study that examined the dermoscopic appearance of mucosal lesions revealed that the combination of structureless zones with blue, gray or white color(s) is useful for the diagnosis of malignant lesions [1]. the highest diagnostic sensitivity was achieved when considering only the presence of blue, gray, or white color [1-3]. furthermore, polymorphous vessels is also a hint for malignancy, particularly melanoma [4,5]. additional clues to the diagnosis of our case included the age of the patient and the presentation of a new lesion [1]. pigmented and non-pigmented lesions at the mucosa are difficult to examine clinically and dermoscopically. therefore, figure 1. clinical appearance of a non-pigmented lesion of the right labia. [copyright: ©2016 blum et al.] figure 2. polarized dermoscopy showing a distinct asymmetric, sharp demarcated homogenous white papule (4 x 5 mm) with polymorphous vessels (e.g., linear, curved, hairpin-like with different diameter) (handyscope, fotofinder, bad birnbach, germany; iphone 5, apple inc., cupertino, usa). [copyright: ©2016 blum et al.] figure 3. histologic appearance: histology revealed mucosal melanoma on hematoxylin and eosin: densely packed atypical spindlyshaped melanocytes with hyperchromatic nuclei, confirmed by positivity with anti-mela antibody (top right inset). [copyright: ©2016 blum et al.] observation | dermatol pract concept 2016;6(4):5 25 3. lin j, koga h, takata m, saida t. dermoscopy of pigmented lesions on mucocutaneous junction and mucous membrane. br j dermatol 2009;161(6):1255-61. pmid:19673880. doi: 10.1111/j.1365-2133.2009.09251.x. 4. menzies sw, kreusch j, byth k, et al. dermoscopic evaluation of amelanotic and hypomelanotic melanoma. arch dermatol 2008;144:1120-7. pmid:18794455. doi: 10.1001/archderm.144. 9.1120. 5. menzies sw, moloney fj, byth k, et al. dermoscopic evaluation of nodular melanoma. jama dermatol 2013;149:699-709. pmid:23553375. doi:10.1001/jamadermatol.2013.2466. 6. mehra t, grözinger g, mann s, et al. primary localization and tumor thickness as prognostic factors of survival in patients with mucosal melanoma. plos one 2014;10;9(11):e112535. pmid: 25383553. doi:10.1371/journal.pone.0112535. this observation encourages the use of the dermoscope at the mucosa even in non-pigmented lesions to diagnose a malignant process as soon as possible. all suspicious lesions should undergo prompt biopsy for definitive diagnosis. references 1. blum a, simionescu o, argenziano g, et al. dermoscopy of pigmented lesions of the mucosa and the mucocutaneous junction: results of a multicenter study by the international dermoscopy society (ids). arch dermatol 2011;147(10):1181-7. pmid:21680757. doi: 10.1001/archdermatol.2011.155. 2. de giorgi v, massi d, salvini c, et al. thin melanoma of the vulva: a clinical, dermoscopic-pathologic case study. arch dermatol 2005;141(8):1046-7. pmid:16103344. doi: 10.1001/ archderm.141.8.1046. http://www.ncbi.nlm.nih.gov/pubmed/19673880 http://dx.doi.org/10.1111/j.1365-2133.2009.09251.x http://www.ncbi.nlm.nih.gov/pubmed/18794455 http://dx.doi.org/10.1001/archderm.144.9.1120 http://dx.doi.org/10.1001/archderm.144.9.1120 http://www.ncbi.nlm.nih.gov/pubmed/23553375 http://dx.doi.org/10.1001/jamadermatol.2013.2466 http://www.ncbi.nlm.nih.gov/pubmed/25383553 http://dx.doi.org/10.1371/journal.pone.0112535 http://dx.doi.org/10.1001/archderm.141.8.1046 http://dx.doi.org/10.1001/archderm.141.8.1046 key: cord-0066109-g7ncbf9p authors: bogunia-kubik, katarzyna; wojtowicz, wojciech; swierkot, jerzy; mielko, karolina anna; qasem, badr; wielińska, joanna; sokolik, renata; pruss, łukasz; młynarz, piotr title: disease differentiation and monitoring of anti-tnf treatment in rheumatoid arthritis and spondyloarthropathies date: 2021-07-09 journal: int j mol sci doi: 10.3390/ijms22147389 sha: da7440fe0b17e52ef9fc20c8e3c4db08358102b5 doc_id: 66109 cord_uid: g7ncbf9p rheumatoid arthritis (ra), ankylosing spondylitis (as), and psoriatic arthritis (psa) are comprehensive immunological disorders. the treatment of these disorders is limited to ameliorating the symptoms and improving the quality of life of patients. in this study, serum samples from ra, as, and psa patients were analyzed with metabolomic tools employing the 1h nmr method in combination with univariate and multivariate analyses. the results obtained in this study showed that the changes in metabolites were the highest for as > ra > psa. the study demonstrated that the time until remission or until low disease activity is achieved is shortest (approximately three months) for as, longer for ra and longest for psa. the statistically common metabolite that was found to be negatively correlated with the healing processes of these disorders is ethanol, which may indicate the involvement of the gut microflora and/or the breakdown of malondialdehyde as a cell membrane lipid peroxide product. a complex of factors may induce immune responses and lead to the development of autoimmune diseases. long-term inflammation contributes to the pathological state and is associated with organ-specific and systemic disorders, such as rheumatoid arthritis (ra), ankylosing spondylitis (as), and psoriatic arthritis (psa), which are of considerable interest to researchers at present [1] . ra, as, and psa are autoimmune diseases associated with changes in the joints and spine due to long-term inflammation, and these diseases are associated with significant reductions in patients' quality of life. rheumatoid arthritis is a chronic, inflammatory autoimmune disease that affects approximately 1% of the population. ra is described as synovial inflammation and joint destruction that leads to significant disability and early mortality. the etiology of ra is multifarious and has not been fully elucidated to date, although genetic and environmental factors have been implicated in disease development [2] . many inflammatory processes involving various immune cells, cytokines, chemokines, proteases, and matrix metalloproteinases play critical roles in the inflammatory cascade of the joint environment, leading to clinical impairment and ra [3] . even though therapy with tnf (tumor necrosis factor)-alpha inhibitors constitute a breakthrough in ra management, this treatment results in no improvement in approximately 30% of cases. the reasons moreover, takahashi et al., using capillary electrophoresis-time-of-flight mass spectrometry (ce-tofms), identified betonicine, glycerol 3-phosphate, n-acetylalanine, hexanoic acid and taurine as serum biomarkers able to predict a response in ra patients receiving anti-tnf therapy [24] . recently, ou et al. proved that serum metabolites were also correlated with as and tnf inhibitor treatment [25] . these results motivated us to analyze the metabolomic profiles of rheumatic disorders. during the present study, metabolomics was used to characterize and compare unique disease-associated metabolites in the serum of ra, as, and psa patients treated with anti-tnf drugs. the metabolites of ra, as, and psa patients were analyzed before the induction of biological treatment and at subsequent time points-i.e., 3 and 6 months after therapy initialization. in the end, our main goal was to identify potential biomarkers that may be employed in the future to elucidate the pathophysiology and treatment outcomes of these diseases. among patients with ra, after 3 months of treatment, low disease activity was achieved in three (11%) patients, and disease remission was achieved in five patients (19%). after 6 months of therapy, these results were 13 (50%) and nine patients (35%), respectively. no response according to eular was observed in four patients (15%) after 6 months (table s1 ). in the group treated for as, 13 patients (45%) and one patient (3%) achieved low disease activity (basdai between 3 and 4) in the third and sixth month of therapy, respectively. in the same time frame, 13 (45%) and 26 patients (90%) achieved remission, respectively. no response was observed in three patients (10%) after 3 months and two patients (7%) after 6 months of therapy. among patients with psa after 3 months of treatment, seven patients (30%) had low disease activity, and three (13%) had remission. after 6 months of treatment, these results were four (17%) and 11 (48%), respectively. no response was observed in 13 patients (57%) after 3 months and in eight patients (35%) after 6 months. metabolomic profiles were analyzed and compared in serum samples collected from ra patients before anti-tnf treatment (bf) and 3 months (3m) and 6 months (6m) after initialization of the treatment (figure 1 and figure s1 ). the first multivariate analysis ( figure 1 ) performed by pca (principal component analysis) exhibited large scattering of patients without any grouping trends among pc1 and pc2. the patients' comparison between each time period by pls-da (partial least-squares discriminant analysis) showed the differences between bt vs. 3m and bt vs. 6m; however, the model 3m vs. 6m did not pass the validation by cv-anova (cross-validated residuals of analysis of variance) test ( figure s1 , table 1 ). the vip (variable importance in projection) score comparison between each analyzed time point demonstrates the differences between each state by the importance of single metabolites in the model variance explanation (table s2 ). the quantified resonance signals were tested by anova, and their dispersions are shown in figure 2 . the evaluation of changes during whole treatment progression identified eight metabolites in which seven metabolites-2-oxoisocapoate, 3 methyl-2-oxovalerate, alanine, glutamine, propylene glycol, tryptophan, and tyrosine-were increasing, and only one-ethanol-was decreasing ( figure 2 ). model type pc/lv n = r 2 x (cum) r 2 y (cum) q 2 (cum) cv-anova figure 2 . boxplots for metabolites with vip scores above 1.00 and statistical importance after p value adjustment (q < 0.05). red bars-ra patients before treatment; blue bars-3 months after initialization of anti-tnf treatment; green bars-6 months after treatment with tnfi. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). green line-average positive response for treatment; yellow line-average neutral response for treatment. pink circle-data point for before treatment; red circle-data point for no information about response; green circle-data point for positive response for treatment; yellow circle-neutral response for treatment. * q value < 0.05 (precise values in table s2 ). boxplots for metabolites with vip scores above 1.00 and statistical importance after p value adjustment (q < 0.05). red bars-ra patients before treatment; blue bars-3 months after initialization of anti-tnf treatment; green bars-6 months after treatment with tnfi. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). green line-average positive response for treatment; yellow line-average neutral response for treatment. pink circle-data point for before treatment; red circle-data point for no information about response; green circle-data point for positive response for treatment; yellow circle-neutral response for treatment. * q value < 0.05 (precise values in table s2 ). the multivariate analysis showed the same trend between treatment progressions in the studied as patient groups, as was observed for ra (figures 3 and s2, table 2 ). significant differences were observed between bt vs. 3m and bt vs. 6m. nonetheless, each time period was characterized by different metabolites (table 2, figure 4) , which may reflect a considerably more altered metabolism in as patients than in ra patients during the treatment. several metabolites exhibit similar variability, such as 2-oxoisocaproate, 3-methyl-2-oxovaletarte, ethanol, glutamine, propylene glycol, and tryptophan (table s3 ). an increasing trend between bt and two other treatment time points was observed for creatine, histidine, leucine, phenylalanine, and unk_11 (unknown). four metabolitescitrate, formate, sn-g3p, and unk_15-first increased and later decreased through treatment progression. six metabolites-acetone, ethanol, isobutyrate, unk_10, unk_2, and unk_8-distinctly decreased over the course of the treatment (table s3, figure 4 ). the multivariate analysis showed the same trend between treatment progressions in the studied as patient groups, as was observed for ra ( figure 3 and figure s2 , table 2 ). significant differences were observed between bt vs. 3m and bt vs. 6m. nonetheless, each time period was characterized by different metabolites (table 2, figure 4) , which may reflect a considerably more altered metabolism in as patients than in ra patients during the treatment. several metabolites exhibit similar variability, such as 2-oxoisocaproate, 3methyl-2-oxovaletarte, ethanol, glutamine, propylene glycol, and tryptophan (table s3 ). an increasing trend between bt and two other treatment time points was observed for creatine, histidine, leucine, phenylalanine, and unk_11 (unknown). four metabolitescitrate, formate, sn-g3p, and unk_15-first increased and later decreased through treatment progression. six metabolites-acetone, ethanol, isobutyrate, unk_10, unk_2, and unk_8-distinctly decreased over the course of the treatment (table s3, figure 4 ). anti-tnf therapy did not reflect the greatest changes in psa treatment. the mva did not show any significant grouping trends (pca) ( figure 5 ), and none of the calculated pls-da models passed the cv-anova test ( figure s3 , table 3 ). nonetheless, the univariate analysis exhibited decreased levels of four metabolites-acetate, ethanol, unk_2, and unk_8 (table s4, figure 6 ). . boxplots for metabolites with vip scores above 1.00 and statistical importance after p value adjustment (q < 0.05). red bars-as patients before treatment; blue bars-3 months after initialization of anti-tnf treatment; green bars-6 months after treatment with tnfi. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). green line-average positive response for treatment; yellow line-average neutral response for treatment. pink circle-data point for before treatment; red circle-data point for no information about response; green circle-data point for positive response for treatment; yellow circle-neutral response for treatment. * q value < 0.05 (precise values in table s3 ). anti-tnf therapy did not reflect the greatest changes in psa treatment. the mva did not show any significant grouping trends (pca) (figure 5 ), and none of the calculated pls-da models passed the cv-anova test ( figure s3 , table 3 ). nonetheless, the univariate analysis exhibited decreased levels of four metabolites-acetate, ethanol, unk_2, and unk_8 (table s4, figure 6 ). . boxplots for metabolites with vip scores above 1.00 and statistical importance after p value adjustment (q < 0.05). red bars-as patients before treatment; blue bars-3 months after initialization of anti-tnf treatment; green bars-6 months after treatment with tnfi. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). green line-average positive response for treatment; yellow line-average neutral response for treatment. pink circle-data point for before treatment; red circle-data point for no information about response; green circle-data point for positive response for treatment; yellow circle-neutral response for treatment. * q value < 0.05 (precise values in table s3 ). . boxplots for metabolites with vip scores above 1.00 and statistical importance after p-value adjustment (q < 0.05). red bars-psa patients before treatment; blue bars-3 months after initialization of anti-tnf treatment; green bars-6 months after treatment with tnfi. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). green line-average positive response for treatment; yellow line-average neutral response for treatment. pink circle-data point for before treatment; red circle-data point for no information about response; green circle-data point for positive response for treatment; yellow circle-neutral response for treatment. * q value < 0.05 (precise values in table s4 ). this analysis showed that ra, as, and psa were characterized by different levels of metabolites before treatment ( figure s4 and s5). however, the unsupervised multivariate analysis did not show any grouping trends among pc1 and pc2. a significant pls-da model was obtained in the ra vs. as individual case comparison (table 4, figure 7 ). the ra vs. psa and as vs. psa comparisons did not pass the cv-anova validation test. figure 6 . boxplots for metabolites with vip scores above 1.00 and statistical importance after p-value adjustment (q < 0.05). red bars-psa patients before treatment; blue bars-3 months after initialization of anti-tnf treatment; green bars-6 months after treatment with tnfi. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). green line-average positive response for treatment; yellow line-average neutral response for treatment. pink circle-data point for before treatment; red circle-data point for no information about response; green circle-data point for positive response for treatment; yellow circle-neutral response for treatment. * q value < 0.05 (precise values in table s4 ). metabolomic profiles of patients with various rheumatic diseases were compared by analyzing serum samples collected from 26 patients with ra, 29 patients with as and 23 patients with psa before initialization of biological therapy. all patients presented with active, advanced disease. this analysis showed that ra, as, and psa were characterized by different levels of metabolites before treatment ( figures s4 and s5 ). however, the unsupervised multivariate analysis did not show any grouping trends among pc1 and pc2. a significant pls-da model was obtained in the ra vs. as individual case comparison (table 4, figure 7 ). the ra vs. psa and as vs. psa comparisons did not pass the cv-anova validation test. the univariate analysis revealed 12 resonance signal relative integrals that were common for all three pathological units: ethanol, isoleucine, leucine, unk_7, valine, proline, alanine, histidine, unk_4, sn-g3pc, unk_8, and unk_3 (table 5 ). these resonance signals were compared for the studied groups at the bt time point. the univariate analysis revealed 12 resonance signal relative integrals that were common for all three pathological units: ethanol, isoleucine, leucine, unk_7, valine, proline, alanine, histidine, unk_4, sn-g3pc, unk_8, and unk_3 (table 5 ). these resonance signals were compared for the studied groups at the bt time point. to verify possible dissimilarities between threatened patients of the studied groups, the comparison between serum samples originating from patients after 6 months of treatment outcome was evaluated. subsequently, anova or kruskal-wallis tests identified seven metabolites, and two unknown signals were determined to be significant for the comparison of ra, as, and psa after 6m (table 6, figure 8 ). assessment of important changes in metabolite levels highlighted the visible relative integral decrease commonly observed in the psa group. most decreased levels were observed in creatine, unk_16, lysine, sn-3gp, unk_14 and acetate. the as entity exhibited the most prominent increase in the levels of ethanol and glutamate. moreover, acetate and sn-3gp exhibited differentiating possibilities only between as and psa diseases. analogs were visible between ra and psa in lysine, creatine, unk_14, and unk_16. the possibility of monitoring treatment progression and the quality of response in terms of patient quality of life seems to be vital for future treatment improvement. at present, metabolomics is a well-defined and well-utilized scientific discipline. this approach enables quantitative and qualitative assessment of the composition of a variety of different biological materials. with the use of modern chemical analytical platforms-that is, mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) together with statistical and chemometrics analysis-metabolomics became a powerful tool to collect multivariate biological data from a single biosample for cohort studies. together with current data analysis methods, this approach enables us to investigate the "needle in a haystack" approach to detect even the slightest variation in the studied biological material. in our previous study, this approach was employed to analyze and compare the metabolomic profiles of females with ra and female controls [17] . this analysis enabled us to identify 12 metabolites important for the discrimination of both patient and control groups. in this study, the metabolomic serum approach was also employed for monitoring rheumatoid arthritis treatment. we observed that after treatment, patients did not move toward the healthy controls but rather formed a separate group. moreover, the differences in metabolites between patients with various il-17 genotypes (rs2275913 and g-197a) were determined to affect ra progression and response to anti-tnf-α treatment [26] . in the present study, metabolomics analyses were employed to collect and verify changes among low-molecular-weight compounds in 78 patients suffering from ra, as, and psa. patients were analyzed at three time points: before and 3 and 6 months after initialization of treatment with a biologic agent. there are important differences between both previous and present studies. the former study focused on 20 female ra patients analyzed at two time points (before and 3 months after tnfi induction) compared with the control group, whereas in the current study, both male and female patients with three (1, (4) (5) (6) (7) (9) (10) (11) test for ra, as, and psa groups of patients before treatment. whiskers-1.5 × interquartile range (iqr); bar-average; box-range between first quartile (q1) and third quartile (q3). * metabolites important in tukey's hsd (2, 3, 8, 12) or dunn-sidak test (1, (4) (5) (6) (7) (9) (10) (11) . the possibility of monitoring treatment progression and the quality of response in terms of patient quality of life seems to be vital for future treatment improvement. at present, metabolomics is a well-defined and well-utilized scientific discipline. this approach enables quantitative and qualitative assessment of the composition of a variety of different biological materials. with the use of modern chemical analytical platforms-that is, mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) together with statistical and chemometrics analysis-metabolomics became a powerful tool to collect multivariate biological data from a single biosample for cohort studies. together with current data analysis methods, this approach enables us to investigate the "needle in a haystack" approach to detect even the slightest variation in the studied biological material. in our previous study, this approach was employed to analyze and compare the metabolomic profiles of females with ra and female controls [17] . this analysis enabled us to identify 12 metabolites important for the discrimination of both patient and control groups. in this study, the metabolomic serum approach was also employed for monitoring rheumatoid arthritis treatment. we observed that after treatment, patients did not move toward the healthy controls but rather formed a separate group. moreover, the differences in metabolites between patients with various il-17 genotypes (rs2275913 and g-197a) were determined to affect ra progression and response to anti-tnf-α treatment [26] . in the present study, metabolomics analyses were employed to collect and verify changes among low-molecular-weight compounds in 78 patients suffering from ra, as, and psa. patients were analyzed at three time points: before and 3 and 6 months after initialization of treatment with a biologic agent. there are important differences between both previous and present studies. the former study focused on 20 female ra patients analyzed at two time points (before and 3 months after tnfi induction) compared with the control group, whereas in the current study, both male and female patients with three rheumatic diseases were included. moreover, different sample preparations were applied based on methanol protein precipitation to maximize the biological information obtained from 1d 1 h nmr spectra. furthermore, mva models were calculated based on relative integrals and not on binned spectra. among all multivariate analyses, only one ra vs. as comparison passed the validation test, showing the differences in metabolite contents between these two disease units. this result could suggest that these differences between ra and as may be caused by either the differences in molecular basis between these two rheumatic disorders or, most likely, the different contribution to the variables from all time points, which reflects the treatment impact on the metabolic fingerprint of the patients. anova or the kruskal-wallis test demonstrated that in most cases, the level of metabolites between studied units was at the lowest value in ra. these results suggest that the most significant differences between ra and as metabolomics profiles might also be associated with the clinical pictures of these diseases. as and psa belong to the same group of so-called spondyloarthropathies. psa may be clinically oligoarthritis, may resemble ra, or may have an axial form that resembles as. among the studied patients, the most common psa was diagnosed with oligoarthritis or polyarticular forms (87%). the picture of changes in the synovial membrane of inflamed joints may often be similar in patients with ra and psa, but on the other hand, changes in bones are different. in ra patients, joint destruction (erosions) is observed, while in psa, destruction and new bone formation (erosions and enthesophytes) are observed. patients suffering from ra and psa before starting biological drugs were previously treated with classic dmards, mainly methotrexate (65%), and patients with as were treated primarily with nsaids (only nine were treated with mtx). ra does not involve the spine but the peripheral joints, while as is an axial spondyloarthropathy that primarily affects the spine and sacroiliac joints. moreover, both groups of patients (ra and as) differed with respect to the percentage of males and females. ra primarily affects women, while as primarily affects men, and this relationship is also clearly visible in our analyzed groups (table s1 ). nevertheless, it appears that ra and as can be distinguished based on the observed differences in metabolic profiles. these results may suggest that metabolomics could serve as a severe diagnostic tool. analyzing the calculated pls-da models clearly showed that each of the treatment time intervals exhibited alterations in reference to the before treatment group. however, the period 3m vs. 6m seems to be on the marginal value of significance, which highlighted the clear need for further expanding the size of the studied group. this finding may suggest that each period of therapy significantly changes the subject's metabolism, and monitoring this change is possible. the treatment is distinctly reflected by the changes in metabolite levels (table s2) , as they are important at different time points. regarding the paired samples comparison (figure 2 ), the amino acid (ala, trp, tyr, and glu) levels are increasing, which is in agreement with previously published data and reflects the overall trend that the amino acid pool is low in ra [27] . 2-oxoisocaproate and 3-methyl-2-oxovalerate are two ketoanalogs of ketoleucine and ketoisoleucine. both of these metabolites are neurotoxins and metabotoxins; therefore, the finding that their levels increase with the patient's improving condition is difficult to explain. however, these metabolites were recognized to be downregulated in ra patients [28] , where they were associated with cartilage destruction caused by decreasing of amino acids levels [29] . the increase in these metabolites may be caused by increasing leucine and isoleucine levels and turnover, which, according to previously published data, are decreased in comparison to healthy people [30, 31] . additionally, in this study, both amino acids were increased (table 3) . these amino acids can balance the energy demand during increased energy consumption and lack of energy from other sources [3] . propylene glycol is a known "concomitant" to originate from cosmetics and was found to be a component of the serum metabolome [32] . however, the extraction of propylene glycol during sample preparation cannot be completely excluded. notably, the level of this compound increases with time, which may indicate that the equilibrium between its metabolization release and storage is changed during therapy. however, the increasing level of this compound may be associated with less need for energy substrates, as propylene glycol can be converted to lactate [33] . this hypothesis can be supported by increasing the levels of alanine and glutamine, which can both funnel the tricarboxylic acid cycle (tca) pathway, alanine to pyruvate and glutamine by glutamate to a-ketoglutarate. however, alanine and glutamine metabolism are mutually related [34] . increased glutamine was determined to be a marker between responders and non-responders to etanercept treatment [23] . notably, tca production can be accompanied by increases in citrate and lactate together. this phenomenon can be caused by glycolysis/gluconeogenesis energy sources (amino acids) shifting toward high pyruvate biosynthesis (table 3) . however, high lactate production has a completely opposite trend in the published data [35] . notably, the ethanol level was decreased, and this metabolite can be associated with the consumption of alcoholic beverages, but in our opinion, this association may be a very "rough" assumption. similarly, the microbial activity in serum samples can be excluded due to sample storage at −80 • c [36] . therefore, the presence of autogenerated endogenous ethanol should be considered. this finding could be closely related to the activity of the gut metabolome, where the presence of the filamentous fungus [37] [38] [39] candida albicans can modulate autoethanol production. on the other hand, ethanol is closely related to acetaldehyde generated from pyruvate, threonine, deoxyribose-5phosphate, phosphoethanolamine, and alanine [40] . acetaldehyde can also be the product of malondialdehyde breakdown as the product of cell disintegration and accompanying membrane lipid peroxidation [41] . interpretation of pls-da models vip scores (>1.00) and univariate statistical analysis highlights the metabolites set with high impact in discrimination possibilities. the pls-da models showed that the bt time point differs from 3m and 6m, while 3m vs. 6m did not exhibit significant changes, most likely due to continuously occurring healing processes and/or more rapid replies to drug intervention. table s3 shows the changes in metabolite levels, while considering the metabolites of paired samples, it is clear that treatment with as caused considerably more metabolic changes than treatment with ra, where only a few of the same metabolites were changed. among these metabolites, gln and trp increased, but the stabilization effect was already seen after 3m (trp) or showed a slight drop (6m-gln). in addition, tryptophan was found to be downregulated in the plasma of as and ra patients, which can be strongly associated with the activity of indoleamine 2,3dioxygenase (ido), which transforms tryptophan to kynurenine [24, 35] and was suggested to be an indicator of disease progression. according to a prior hypothesis, two other amino acids-his and leu-were increasing, and their levels after 3m were already equilibrated. observed earlier than amino acids, keto analogs first demonstrated an increasing trend after 3m, and a stabilization effect was subsequently observed. the reverse trend in phe was reported in the literature data in comparison to the obtained results, where this metabolite was upregulated in as patients, whereas its dipeptide phe-phe was downregulated [42] . however, phe was found to be statistically important in ra and showed the same trend as in this study [31] . propylene glycol and ethanol showed the same trend as that observed for ra. the increasing trend of citrate can be associated with an accelerated tca cycle, which reversibly correlates with keto body acetone levels, which may be a sign of decreased ketoacidosis. formic acid is a naturally occurring metabolite in the serum metabolome; in this study, its level first increased and then decreased. the presence of formic acid can be modulated by internal metabolism or by gut microbiota [17] . in our previous study, the level of formic acid was decreased in female ra patients undergoing anti-tnf therapy [17, 26] . the next metabolite that can be associated with the human microbiome is isobutyrate, which decreased significantly with the sampling time. this serum short-chain amino acid is the product of gut valine degradation [43, 44] and can be related to the lipid profile [45] . creatine metabolism is associated with ser, gly thr, arg, and pro metabolism; however, none of these compounds were observed to be significantly changed [kegg] . however, supplementation with creatine can attenuate muscle loss [46] . glycerol 3-phosphate is a product of glucose breakdown, and the intermediate substrate for lipid metabolism can also be obtained by the reduction of dihydroxyacetone-3-phosphate [47] . therefore, the increase in g3p levels may be caused by accelerated lipolysis, rather than glycolysis. the observed changes in the abovementioned metabolite levels confirm that, at least in part, the same biochemical pathways are unblocked and that some new pathways are also activated. these results suggest that the keto analogs, amino acids, ethanol, creatine, and isobutyrate may reflect the treatment outcome and serve as potential biomarkers. the metabolic analysis clearly demonstrated that this disease entity requires long-term treatment. the positive therapy results were visible only after 6 months ( figure s3 , table 3 ); however, the smallest changes between metabolites were observed among ra and as patients. the univariate analysis also identified only two metabolites that were changed, namely, acetate and ethanol. both metabolites exhibited decreasing levels. ethanol is a biomarker that appears to be associated with rheumatoid-based inflammation in all investigated entities, while short-chain fatty acid acetate has been determined to be significant for the first time. short-chain fatty acids were found to be important in ra mice, where acetate was increased in serum ra mice, while the therapeutic effect of butyrate, which ameliorates the immune-systemic response, was demonstrated [48] . monitoring changes in das28, crp (c reactive protein), and vas (visual analog scale) in ra showed significant improvement after 6 months, while after 3 months, partial improvement in parameters was observed. analyzing the as parameters, crp, vas, and basdai improved after 3 months. these findings confirm that 3m treatment has the same effect as 6m treatment but can retain the treatment effect. the parameters of the disease activity of psa, das28, crp, and vas were not satisfactory in 35% of patients, even after 6m. all these findings reflect the treatment efficacy, showing that fewer treated psa patients are characterized by low disease activity and achieve remission compared to two other diseases, especially as. in the literature, das28 was negatively associated with histidine and was well correlated with its changes [49] . our studies have shown that the increase in statistically important amino acids corresponds to a decrease in all inflammation parameters. however, amino acids, such as proline, isoleucine, tryptophan, valine, arginine, ornithine, kynurenine, 4-hydroxyproline, and leucine, were positively correlated with crp [22] . the negative correlation across all disease entities showed that ethanol, which decreased, clearly reflects the therapeutic efficiency. thus, it seems that ethanol levels decrease during anti-tnf drug administration and are associated with more favorable outcomes of treatment with this biologic agent. notably, a study by azizov et al. suggested that moderate alcohol consumption may be a consistent protective factor for the development of autoimmune diseases in mice [50] . the results described earlier by jansson et al. have shown that this effect of ethanol could be mediated by (i) downregulation of leukocyte migration and (ii) upregulation of testosterone secretion, with the latter leading to decreased nf-κb (nuclear factor kappalight-chain-enhancer of activated b cells) activation [51] . the relationship between anti-tnf treatment efficacy and ethanol levels observed in the present study may also be indirectly affected by the decreased nf-κb expression associated with genetic variability of the gene encoding this transcription factor. indeed, in vitro functional studies [49] have suggested that the presence of the deletion may be associated with diminished expression of the gene. notably, we have previously observed a significantly more efficient response to anti-tnf-α treatment in ra patients carrying the deletion within the nf-κb1 gene [52, 53] . moreover, we have also found some associations between polymorphisms within the tlr4-encoding gene and ra stage, as well as response to anti-tnf-α therapy [53] . thus, our studies indicate a beneficial effect of using a biologic agent to treat ra patients carrying the deletion allele associated with lower nf-κb expression. as suggested by jannson et al., the diminished activity of this transcription factor can mediate the antiinflammatory and anti-destructive properties of ethanol in mice with collagen-induced arthritis. in addition, the previously reported relationships with tlr4 genetic variants (receptors with lps as a ligand) may serve to confirm, as suggested by the results of the present metabolomic study, the involvement of the microbiome in the development of rheumatic diseases [53] . the changes in all perturbed metabolites were subjected to bioinformatics analysis consisting of modules including perturbed compounds, associated enzymes, perturbed pathways, and reactions in which assigned compounds participate (interactive link, supplementary data) [54] . additionally, the meta-analysis was performed, showing the most perturbed biochemical pathways-see figure s6 [55]. altogether, 78 patients with rheumatic diseases were investigated, including patients with ra (n = 26), as (n = 29), and psa (n = 23) hospitalized at the university hospital in wrocław in the department of rheumatology and internal medicine of wroclaw medical university. all patients gave their informed consent for inclusion before they participated in the study. the study was conducted in accordance with the declaration of helsinki, and the protocol was approved by the wroclaw medical university ethics committee (identification code kb-625/2016, 29 december 2016). the exclusion criteria to participate in the study were as follows: clinically significant impairment of hepatic and renal function, coexistence of connective tissue diseases, infections with hepatotropic viruses, or infections resistant to therapy, ongoing history of cancer or uncontrolled diabetes, alcohol abuse, pregnancy, breastfeeding, unwillingness to cooperate, or insufficient clinical records. ra patients met the 2010 european league against rheumatism (eular)/american college of rheumatology (acr) classification criteria. the inclusion criteria were the following: age over 18 years, caucasian origin, high disease activity (das28) ≥ 5.1) before initiating biologic agent therapy, no response to at least two disease-modifying antirheumatic drugs (dmards), and a complete medical history. the disease activity of ra patients was determined using the das28 score based on four components, including the number of swollen and tender joints, c-reactive protein (crp) level, erythrocyte sedimentation rate (esr) and patient's global assessment of general health expressed on a visual analog scale (vas, mm). except for das28 measurement, anti-cyclic citrullinated peptide antibodies (anti-ccp) and rheumatoid factor (rf) levels were determined. the patients were stratified into three subgroups depending on disease activity: high (das28 > 5.1), moderate (3.2 < das28 ≤ 5.1), and low (das28 ≤ 3.2), and their responses to anti-tnf therapy after 3 and 6 months were assessed by the european league against rheumatism (eular) criteria. a response was considered good when reduction of the das28 score value (∆das28) > 1.2 and post-treatment das28 ≤ 3.2, as moderate when ∆das28 > 1.2 and post-treatment das28 > 3.2, or 0.6 < ∆das28 ≤ 1.2 and post-treatment das28 ≤ 5.1. finally, no response was assumed when ∆das28 ≤ 0.6 or 0.6 < ∆das28 ≤ 1.2 and posttreatment das28 > 5.1. as patients were certified according to the modified new york criteria for as and assessment of spondylo arthritis international society (asas) classification criteria for axial and peripheral spondyloarthritis (spa) and the bath ankylosing spondylitis disease activity index (basdai). the following inclusion criteria were applied: age over 18 years, caucasian origin, high disease activity (basdai ≥ 4) before initiation of biologics, resistance to treatment with at least two nonsteroidal anti-inflammatory drugs (nsaids) for at least four weeks at maximum doses (if there were no contraindications) and full medical history. the disease activity of as patients was estimated using the basdai, a number of swollen and tender joints, values of crp and esr, global health evaluation provided by a patient (vas), spinal mobility and assessment of extra-articular manifestations. the disease activity was considered to be high (basdai ≥ 4), moderate (3 ≤ basdai <4), or low (basdai < 3). the asas/eular criteria were employed to assess the clinical outcome after 3 and 6 months of anti-tnf treatment. significant improvement was defined as a reduction in basdai (∆basdai ≥ 2.0), good outcome as ∆basdai ≥ 2.0 and basdai < 3.0 at the endpoint, moderate response as ∆basdai ≥ 2.0 and basdai ≥ 3.0 at the endpoint, and no response as ∆basdai < 2.0. psa patients were diagnosed according to the classification criteria for psoriatic arthritis (caspar criteria). patients included in the study were characterized by subsequent criteria: age over 18 years, caucasian origin, a complete medical history and physical examination, failure with treatment with at least two disease-modifying antirheumatic drugs (dmards) for four months in peripheral form and at least two nonsteroidal anti-inflammatory drugs for at least four weeks at maximum doses (if there were no contraindications) in axial form, and the presence of active disease prior to the initiation of anti-tnf therapy. disease activity in psa patients was calculated using the das28 score, disease activity score (das) or modified criteria for disease activity according to the psoriatic arthritis response criteria (psarc) in peripheral form of psoriatic arthritis and basdai in axial form. high disease activity was defined in axial form as basdai ≥ 4 and in peripheral form as das28 ≥ 5.1 or das > 3.7. the psarc is based on counts of swollen and tender joints, physician global assessment of disease activity (zero-to five-point likert scale), and questionnaires for levels of pain and spinal mobility. tnf-alpha inhibitors were administered to the ra and as patients according to standard protocols: 40 mg of adalimumab (ada) administered subcutaneously every other week; 50 mg of etanercept (eta) administered subcutaneously every week; 400 mg of certolizumab pegol (cer pegol) administered subcutaneously at weeks 0, 2 and 4, then 200 mg every second week thereafter; golimumab (gol) administered subcutaneously 50 mg once a month and on the same day each month. additionally, as patients received infliximab (inf) 3 mg/kg of body weight, given as intravenous infusions at weeks 0, 2, and 6, and every 8 weeks thereafter. psa was treated analogously to ra and as with ada, eta, cer pegol, and gol. patient serum samples were collected at three time points: before treatment (bt) and 3 and 6 months (3m and 6m) after the initialization of anti-tnf drug administration. the patients' characteristics are summarized in table s1 . the serum samples were stored at −80 • c. before preparation, serum samples were thawed in an ice bath, 300 ml was obtained, and it was mixed with 600 ml methanol (merck kgaa, darmstadt, germany). the samples were then mixed for 1 min and placed at −20 • c for 20 min. after that procedure, the samples were centrifuged (30 min, 11,000 rpm, 4 • c), and 700 µl of clarified upper phase was transferred into new tubes. the solvent was evaporated in a vacuum centrifuge (40 • c, 1500 rpm for 4 h). in the next step, 600 µl of pbs buffer (0.5 m, ph = 7.0, tsp (armar ag, döttingen, swisserland) = 0.03 mm, 20% d 2 o (armar ag, döttingen, swisserland) was added to each sample and mixed for 3 min. subsequently, 550 µl of each sample was transferred into 5-mm nmr tubes (5sp, armar chemicals, leipzig, germany). until the measurement was taken, the samples were stored at 4 • c. all nmr spectra were recorded using a bruker 600 mhz avance ii spectrometer and by using the cpmg pulse sequence (cpmgpr1, bruker notation) with following parameters relaxation delay of 3.5 s, acquisition time of 2.72 s, 128 scans, time domain of 65 k, and spectral width of 20 ppm. the line broadening was set at 0.3 hz. all nmr spectra were referenced to the tsp resonance (δ = 0.000 ppm). phased correction and baseline correction were corrected manually. spectra were normalized to the constant sum of the tsp signal. the alignment of resonance signals was performed using the correlation optimized warping (cow) and icoshift functions implemented in matlab environment (v r2019a, mathworks inc., natick, ma, usa) [56, 57] . the relative integral of nmr measured metabolites was obtained as a sum of data points of the nonoverlapping resonances or a cluster of partly overlapping resonances from the data matrix consisting of 60,474 data points for each spectrum in n-dimension. the third quartile value of the noise region was subtracted from the calculated relative integral to decrease the influence on the final values. the metabolite resonances were identified based on chemical shifts and the results of stocsy [58] analysis and according to online databases (biological magnetic resonance data bank [59] and human metabolome data base [60] ) and assignments published in the literature based on 1 h nmr chemical shifts. the calculations for univariate statistics were performed on the relative integral of metabolite values. for normality verification, the shapiro-wilk test was performed. all statistical tests were calculated at a significance level of α = 0.05. all measured metabolites were checked with pearson's rho to verify possible interactions between known small molecular compounds and unidentified compounds. the equality of variances was tested with levene's test. the comparison based on the overtime treatment response was tested dually depending on the number of patients who dropped out. if the number of dropouts was below three, then observations without pairs were removed from the dataset. if the number of dropouts was above three, the testing was based on partially paired data with use of t-test with the pooled fisher's method for p values [61] . the false discovery rate (fdr) based on the benjamini hochberg procedure was applied for the tested metabolites. multiple group comparisons were performed on nonpaired data using anova and tukey hsd or kruskal-wallis and dunn-sidak correction tests depending on the data distribution. the graphical representation and percentage difference were prepared on a general dataset without consideration of the fulfillment of paired samples. the mva analysis was performed on integral relative metabolites. all the relative integral variables were scaled to unit variance (uv). the sample order in the data matrix was randomized. data analysis was performed using two methods: unsupervised principal component analysis (pca) for natural clustering of samples and supervised partial least squares discriminant analysis (pls-da) for identifying variables responsible for biological differences. the mva data visualization marked an ellipse with hotelling's t2 range (95%). partial least squares discriminant analysis with a sevenfold cross-validation procedure was employed to determine variation between studied groups. the reliability of the pls-da models was assessed by cross-validation analysis of variance (cv anova) at a significance level of α = 0.05. the most important variables in discrimination between comparisons, were selected based on the variable importance in projection (vip) values with a cutoff value of 1.00. to obtain kyoto encyclopedia of genes and genomes (kegg) ids from the resulting list of metabolites, the most recent compounds downloaded from the kegg api http://rest. kegg.jp/list/compound (accessed on 19 october 2020) were mapped to the corresponding metabolites. after unification of input, we employed the fella package to build a keggbased hierarchical representation of human biochemistry (pathways, modules, enzymes, reactions, and metabolites). first, we retrieved the tabular kegg data for humans (t01001, release 96.0+/12-13 december 20) to build the knowledge graph. later, we mapped the list of input metabolites to the internal representation, creating an enriched object, and we subsequently ran the propagation algorithm-diffusion method (undirected heat diffusion model) to score graph nodes. additionally, the parametric z-score was computed using normality approximations for statistical normalization [54] . informed consent statement: informed consent was obtained from all subjects involved in the study. the datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. the authors declare no conflict of interest. pathogenesis and treatment of autoimmune rheumatic diseases rheumatoid arthritis metabolomics in the development and progression of rheumatoid arthritis: a systematic review defining response to tnf-inhibitors in rheumatoid arthritis: the negative impact of anti-tnf cycling and the need for a personalized medicine approach to identify primary non-responders ankylosing spondylitis and axial spondyloarthritis ankylosing spondylitis and mesenchymal stromal/stem cell therapy: a new therapeutic approach the assessment of spondyloarthritis international society classification criteria for peripheral spondyloarthritis and for spondyloarthritis in general update of the asas-eular management recommendations for axial spondyloarthritis classification criteria for spondyloarthropathies the pathogenesis of psoriatic arthritis psoriasis, psoriatic arthritis, and rheumatoid arthritis: is all inflammation the same? treating axial spondyloarthritis and peripheral spondyloarthritis, especially psoriatic arthritis, to target: 2017 update of recommendations by an international task force psoriatic arthritis significance of association of hla-c and hla-e with psoriatic arthritis review papers metabolomics and its potential in diagnosis, prognosis and treatment of rheumatic diseases emerging role of metabolomics in rheumatology application of 1 h nmr-based serum metabolomic studies for monitoring female patients with rheumatoid arthritis differences in the serum metabolome and lipidome identify potential biomarkers for seronegative rheumatoid arthritis versus psoriatic arthritis coiled-coil networking shapes cell molecular machinery impact of janus kinase inhibitors on risk of cardiovascular events in patients with rheumatoid arthritis: systematic review and meta-analysis of randomised controlled trials metabolic profiling of human cd4+ cells following treatment with methotrexate and anti-tnf-α infliximab exploring the inflammatory metabolomic profile to predict response to tnf-α inhibitors in rheumatoid arthritis 1h-nmr-based metabolomic study for identifying serum profiles associated with the response to etanercept in patients with rheumatoid arthritis perifolliculitis capitis abscedens et suffodiens treatment with tumor necrosis factor inhibitors: a case report and review of published cases il-17f and il-23r gene polymorphisms in polish patients with rheumatoid arthritis role of amino acids in rheumatoid arthritis studied by metabolomics metabolomics study of fatigue in patients with rheumatoid arthritis naïve to biological treatment global metabolic profiling of human osteoarthritic synovium exploration of the serum metabolite signature in patients with rheumatoid arthritis using gas chromatography-mass spectrometry serum free amino acid levels in rheumatoid arthritis according to therapy and physical disability severe lactic acidosis after an iatrogenic propylene glycol overdose role of glutamine in human carbohydrate metabolism in kidney and other tissues serum metabolic signatures of four types of human arthritis endogenous ethanol production in patients with diabetes mellitus as a medicolegal problem abnormal gut fermentation: the 'auto-brewery' syndrome endogenous ethanol 'auto-brewery syndrome' as a drunk-driving defence challenge autobrewing: fact or fantasy? endogenous ethanol-its metabolic, behavioral and biomedical significance malondialdehyde-acetaldehyde adducts and anti-malondialdehyde-acetaldehyde antibodies in rheumatoid arthritis integrated gc-ms and lc-ms plasma metabonomics analysis of ankylosing spondylitis protein origin of the volatile fatty acids isobutyrate and isovalerate in human stool dissimilatory amino acid metabolism in human colonic bacteria faecal bacterial and short-chain fatty acids signature in hypercholesterolemia can creatine supplementation improve body composition and objective physical function in rheumatoid arthritis patients? a randomized controlled trial glycerol-3-phosphate phosphatase/pgp: role in intermediary metabolism and target for cardiometabolic diseases microbiota-derived metabolites suppress arthritis by amplifying aryl-hydrocarbon receptor activation in regulatory b cells functional annotation of a novel nfkb1 promoter polymorphism that increases risk for ulcerative colitis essential oils: extraction techniques, pharmaceutical and therapeutic potential-a review ethanol prevents development of destructive arthritis significance of polymorphism and expression of mir-146a and nfkb1 genetic variants in patients with rheumatoid arthritis polymorphisms within genes involved in regulation of the nf-κb pathway in patients with rheumatoid arthritis fella: an r package to enrich metabolomics data comprehensive meta-analysis of covid-19 global metabolomics datasets correlation optimized warping and dynamic time warping as preprocessing methods for chromatographic data icoshift: a versatile tool for the rapid alignment of 1d nmr spectra statistical total correlation spectroscopy: an exploratory approach for latent biomarker identification from metabolic1h nmr data sets 0: the human metabolome database for testing equality of means in partially paired data with incompleteness in single response key: cord-0070096-2vaquykw authors: ali, shabana amanda; pastrello, chiara; kaur, navdeep; peffers, mandy j.; ormseth, michelle j.; jurisica, igor title: a network biology approach to understanding the tissue-specific roles of non-coding rnas in arthritis date: 2021-11-03 journal: front endocrinol (lausanne) doi: 10.3389/fendo.2021.744747 sha: 084e5034e2c0ac269c76050c974126accb4d79f0 doc_id: 70096 cord_uid: 2vaquykw discovery of non-coding rnas continues to provide new insights into some of the key molecular drivers of musculoskeletal diseases. among these, micrornas have received widespread attention for their roles in osteoarthritis and rheumatoid arthritis. with evidence to suggest that long non-coding rnas and circular rnas function as competing endogenous rnas to sponge micrornas, the net effect on gene expression in specific disease contexts can be elusive. studies to date have focused on elucidating individual long non-coding-microrna-gene target axes and circular rna-microrna-gene target axes, with a paucity of data integrating experimentally validated effects of non-coding rnas. to address this gap, we curated recent studies reporting non-coding rna axes in chondrocytes from human osteoarthritis and in fibroblast-like synoviocytes from human rheumatoid arthritis. using an integrative computational biology approach, we then combined the findings into celland disease-specific networks for in-depth interpretation. we highlight some challenges to data integration, including non-existent naming conventions and out-of-date databases for non-coding rnas, and some successes exemplified by the international molecular exchange consortium for protein interactions. in this perspective article, we suggest that data integration is a useful in silico approach for creating non-coding rna networks in arthritis and prioritizing interactions for further in vitro and in vivo experimentation in translational research. noncoding rnas are key regulators of gene expression in the musculoskeletal system. these rna molecules are transcribed from dna but not translated into protein (1) . among the many types of non-coding rnas, the most characterized to date are micrornas (mirnas), long non-coding rnas (lncrnas), and circular rnas (circrnas). through seed-sequence binding, mirnas target specific genes and prevent their translation, effectively inhibiting expression. because lncrnas and circrnas are both capable of 'sponging' mirnas, or competitively binding to mirnas, they are considered competing endogenous rnas (cernas) (2) . lncrnas function by binding to mirnas through mirna response elements (mre) present at their 3' ends (3) . therefore, lncrnas act as molecular decoys, sequestering mirnas and preventing their interaction with gene targets. as with circrnas, lncrnas regulate diverse biological processes through their crosstalk with mirnas (4) . this cerna activity is one of many factors contributing to the net effect of mirnas in a specific biological context. there are multiple factors governing mirna expression, from the level of the cell to the organism. mirna biogenesis involves several post-transcriptional processing steps and is under both temporal and spatial control that can introduce variability (5) . once processed, mature mirnas may decay, be degraded, be exported, or be 'sponged' as described above. as a result, the mirnas present in a given cell can vary according to these regulatory factors, but also multiple biological factors, including age, sex, and body mass index (6) . it is therefore not surprising that mirna profiles are dysregulated in several disease contexts and contribute to disease onset and progression. for highly prevalent musculoskeletal diseases like osteoarthritis (oa) and rheumatoid arthritis (ra), multiple tissues in the joint compartment can show pathology that is driven by tissue-specific mirna patterns. this multi-level regulation of mirna expression presents a challenge when attempting to elucidate the function of a particular mirna in disease. mirnas play both protective and destructive roles in the musculoskeletal system (7) . since a mirna can have hundreds of gene targets, its specific effect can be obscure. studies to date have tended to focus on elucidating a single axis involving one or two non-coding rnas, often a lncrna or circrna, and its effects on a mirna and one or two downstream gene targets. while these studies are necessary to demonstrate direct interactions, they may be missing the broader biological context. in fact, the same mirnas are often reported as having important functions through completely different pathways that involve unique upstream regulators and downstream effectors. taking mir-140 as an example, independent studies have investigated its effects on at least 17 different gene targets in various cell types within the musculoskeletal system (8) . though it is now generally accepted that mir-140 plays a beneficial role in oa, promoting cartilage anabolism and inhibiting catabolism, this information was acquired over a decade of research from multiple groups, and remains under investigation due to its tissue-and disease-specific effects. for example in ra, mir-140 has been reported to suppress synovial inflammation (9), again suggesting a beneficial role, but in a different tissue and through a different mechanism than for oa, thereby precluding direct comparison of this mirna across diseases. to improve our understanding of the biological and clinical context of non-coding rna regulation in arthritis, there is an outstanding need for methods to identify the interactions among non-coding rnas and their gene targets in specific cell types. computational biology approaches to predict cerna activity among non-coding rnas, as well as gene targets and pathways, is a useful in silico strategy that can be tailored to cells, diseases, and other factors of interest. to illustrate this, we present two examples that integrate the recent literature on non-coding rnas in oa and ra, focusing on chondrocytes and fibroblast-like synoviocytes (fls), respectively. these examples are proof-of-concept of the relationships (or biological networks) that can be constructed using non-coding rna axes that have been experimentally validated. in building these networks, we encountered key challenges, including inconsistency in annotation of non-coding rnas. these challenges are discussed along with our perspectives on future directions needed to advance the non-coding rna field. to curate relevant literature reporting non-coding rna axes in oa chondrocytes and ra fls, we searched pubmed using combinations of key words including osteoarthritis, rheumatoid arthritis, microrna, long non-coding rna, and circular rna. articles were filtered for those with an experimentally validated lncrna-mirna-mrna or circrna-mirna-mrna axis in chondrocytes from oa subjects or fls from ra subjects. we included 36 studies for oa and 10 studies for ra published between october 2019 and december 2020. the lncrna/circrna, mirna, and mrna targets were extracted from individual papers. common groups of genes were fed into a network prepared in navigator v3.0.14 (10), connecting them using physical protein-protein interactions to visualize potential signaling relationships. the network figure was finalized with legend from an exported svg file in adobe illustrator v25.3.1. network source files are available upon request. physical protein interactions among gene targets were obtained from integrated interactions database (iid) v2021-05, using the entire set of iid interactions (11) . proteins were annotated with gene ontology (go) biological processes using navigator plugin to uniprot. the go biological processes are represented in the figures by node color and are listed in the figure legends. commonly observed in the cartilage, synovium, and bone, but oa research has by far focused on cartilage, including exploration of the role of non-coding rnas (7) . dysregulation of mirnas, lncrnas, and circrnas has been reported in oa, with one molecule having effects on multiple downstream molecules (e.g., mrnas), including other non-coding rnas (2) . these non-coding rna-based regulatory networks represent a molecular mechanism underlying oa that remains poorly understood. the majority of studies we identified explored a single non-coding rna axis in oa chondrocytes, with several studies reporting on the same non-coding rnas, but in different axes, making it difficult to ascertain the net effect of a particular molecule. to gain a better understanding of the networks in which non-coding rnas function in oa, we used a computational biology approach to integrate these individual axes ( figure 1a ; supplementary table 1) . as shown in figure 1a , individual mirnas, lncrnas, and circrnas in oa chondrocytes have multiple and interconnected effects in regulating downstream molecules. for example, a single lncrna can affect multiple mirnas as shown by lncrna malat1 via mir-146a and mir-145 ( figure 1b) . in lipopolysaccharide-treated chondrocytes, malat1/mir-146a modulates extracellular matrix catabolism, inflammation, and apoptosis through pi3k/akt/mtor, impacting oa progression (13) . in addition, in interleukin (il)-1b-treated chondrocytes, malat1/mir-145 increases extracellular matrix degradation by targeting a disintegrin and metalloproteinase with thrombospondin motifs 5 (adamts5) (14) . furthermore, lncrna nkila may compete for mir-145 and thus alter adamts5, while also inhibiting sp1 transcription factor activity and regulating nuclear factor kappa-light-chainenhancer of activated b cells (nf-kb) in chondrocytes. these factors are orchestrated to promote inflammation and apoptosis but inhibit proliferation in a fine-tuned manner (15) . notably, the targets of mir-145 (sp1 and nf-kb) and mir-146a (pi3k/ akt/mtor) interact to form an almost complete clique ( figure 1b) , suggesting a connection between the molecular functions regulated by lncrna malat1 that would not have been identified without integrating the curated data. aside from malat1, lncrnas snhg15 (16, 17) , pvt1 (18, 19) , xist (20, 21) , part1 (22, 23) , and neat1 (24, 25) are each shown to have two mirna targets, which in turn regulate multiple genes with annotated protein-protein interactions ( figure 1a ). this network illustrates non-coding rna function in human oa chondrocytes, the complexities of which are not readily apparent when interpreting studies separately. (26) . like oa, ra can cause cartilage and bone damage, but the major pathological changes in ra are within the synovium. this includes infiltration of immune cells and wildly proliferating invasive fls exhibiting impaired apoptosis and proinflammatory cytokine production (27) . noncoding rnas are known to play important roles in ra fls. early studies found a dysregulation of mir-155 and mir-146a influenced the proliferative, invasive, and proinflammatory phenotype of ra fls (28) (29) (30) (31) . recent studies suggest a more complex picture of non-coding rnas within ra fls (figure 2 and supplementary table 2) . interactions between lncrnas, circrnas, and mirnas contribute to fls abnormalities typical of ra ( figure 2) . for example, lncrna neat1 affects several mirnas as a significant driver of ra joint pathology. neat1 is increased in ra versus control subject synovial tissue (32) and peripheral blood mononuclear cell exosomes (33) . these exosomes can deliver additional neat1 to fls (33) . neat1 sponges mir-410-3p leading to increased yy1 transcription factor which promotes fls proliferation, impaired apoptosis, and increased tumour necrosis factor alpha (tnf-a) and matrix metallopeptidase 9 (mmp-9) expression (32) . neat1 also causes increased fls proliferation and inflammation through sponging mir-23a, leading to an increase in murine double minute-2 (mdm2) (33) . this demonstrates that lncrna neat1 can function through different mirnas to alter expression of unique targets that are further connected by protein-protein interactions as shown for yy1 and mdm2 in figure 2 . other key lncrna axes have been reported in ra. for example, excess lncrna h19 in fls sponges mir-103a, leading to increased expression of multiple direct targets including il-15 and dickkopf wnt signaling pathway inhibitor 1 (dkk1), promoting inflammation and joint destruction in ra (34) . downregulated lncrnas linc-pint, linc01197, and oip5-as1 in ra synovial tissue lead to increased levels of their respective targets mir-155, mir-150, and mir-448 in ra fls, which in turn lead to cell proliferation, invasion, and inflammatory responses (35) (36) (37) . upregulated lncrnas pvt1 and uc.477 lead to sponging of mir-543 (38) and mir-19b (39), respectively, also contributing to ra fls pathology. while fewer circrna axes have been elucidated, one study found excess circ0088036 could sponge mir-140-3p to promote proliferation and migration in fls via sirtuin 1 (sirt1) (40) . as in oa chondrocytes, these studies demonstrate the importance of interconnectivity of non-coding rnas and their gene targets in human ra fls. we hold the perspective that non-coding rnas function in tissuespecific networks that can be elucidated using curation and computational biology to integrate experimentally validated data from individual studies. taking oa chondrocytes and ra fls as two examples, we integrated non-coding rna axes by connecting common molecules and produced networks highlighting connections that may have otherwise been unknown. in particular, mirnas are shown to orchestrate the effects of lncrnas and circrnas on gene expression, which has implications for downstream signaling, including protein-protein interactions and regulation of biological processes. while we did not explore upstream factors regulating the expression of lncrnas and circrnas, nor the direct effects of lncrnas and circrnas on gene expression, these are equally important considerations for understanding the fine-tuning that non-coding rnas drive in disease. since non-coding rnas are highly stable in circulation, they can be used as diagnostic biomarkers for patients with oa, ra, and other musculoskeletal diseases (41) (42) (43) . additionally, we demonstrate that as cernas, lncrnas and circrnas can regulate one or more mirnas, suggesting that mirnas are important coordinating signals and therefore potential therapeutic targets (44) . targeting non-coding rnas with antisense oligonucleotides (asos) and small interfering rnas (sirnas) to selectively modulate their expression in specific tissue types could have therapeutic benefit for oa and ra (45) . to note limitations, our restricted literature search may have missed other reports on the molecules included in the networks, and therefore these are not exhaustive networks. based on the high number of studies reporting non-coding rna axes in oa chondrocytes and ra fls, we focused on these cell types and excluded articles reporting non-coding rna axes in other cells or biofluids for these diseases. similarly, to maximize physiological relevance, we chose to exclude studies that used only animal models or cell lines, instead focusing on studies that used primary human chondrocytes or fls (often in addition to cell lines). by applying these criteria, our intention was to capture axes that were established in comparable biological contexts in order to justify integration across studies. while experimental validation of the networks shown in figures 1 and 2 is required, we have pruned the specific species, cell (chondrocytes and fls, respectively), and disease (oa and ra, respectively) contexts in which these studies should be conducted. this represents a possible workflow in which computational biology methods can inform experimental approaches in non-coding rna research. in constructing these non-coding rna networks, we encountered key challenges pertaining to naming conventions, database maintenance, and target predictions. assigning names to new molecules has historically led to non-standard nomenclature. the most appropriate approach to limit inconsistent naming is to create standard rules, as evidenced by improved gene naming after the hugo gene nomenclature committee (hgnc) released guidelines (https://www. genenames.org/about/guidelines/) (46) . unfortunately, noncoding rnas suffer from naming conventions that are either out-of-date or absent altogether (47, 48) . the mirbase reference database has been used for mirnas since 2004 (49) , and this led to fairly accurate mirna nomenclature in the subsequent 10 years. however, mirbase releases are becoming sparser (the latest being in 2018) and include a small percentage of all the novel mirnas identified in the literature using sequencing techniques, leading to novel mirnas being named inconsistently. while irregular and infrequent database updates create a challenge (50), mirna nomenclature is still better defined than that of other non-coding rnas. hgnc provides naming rules for lncrnas (derived from lncrnadb, which is no longer active), but not for circrnas, though some suggestions are provided (48) . lncipedia (https:// lncipedia.org) provides consistent nomenclature and links to alternative names, a feature that is useful for mapping different publications to the same molecule (51) . unfortunately, the most recent update was in 2018. circpedia (https://www.picb.ac.cn/ rnomics/circpedia/), a reference database for circrnas, had a similar fate, with no up-to-date curations (52) . this lack of nomenclature standards for lncrnas and circrnas leads to considerable variability in naming as shown in our networks, and represents a major roadblock to comprehensive data integration from the literature and across databases (53) . rnacentral (https://rnacentral.org/) is a database that is attempting to overcome such roadblocks, integrating data from multiple reference databases (54) . however, many of the databases lack updates, and the type of multiple mapping provided for the molecules is not conducive to high-throughput workflows or computational analyses. with respect to target predictions, mirna-target predictions have been the focus of several tools and databases over the past 20 years. among the pitfalls, many of these databases are not being updated, some for more than 10 years (55) . this leads to a lack of interactions for more recent mirnas, including both novel mirnas and mirnas in mirbase updates, creating a bias for computational analyses. for lncrnas and circrnas, very few databases curate or predict interactions, and of the few that were created in the past decade, many are already no longer available. the largest lncrna database is lncrna2target (http://123.59.132.21/ lncrna2target/index.jsp), with 2,189 lncrna-target interactions derived from low-throughput experiments and 203,500 from high-throughput experiments, and with a recent update in 2021 (56) . circatlas (http://bio-annotation.cn/lncrna2target/) has annotated 421,501 predicted human circrnas and provides predicted rna-binding proteins and mirna interactions with circrnas (57) . while circatlas is well organized and provides tissue annotation for circrnas, one limitation is that it lacks literature curated data. although data curation is a lengthy process that requires human expertise and dedicated funds, it is necessary to enable trustable and useful computational biology predictions. a preselection of the candidate papers to be curated is necessary to reduce the burden of manual curation. to do this, some databases [e.g., imex and graingenes (58)] prioritize papers from topic-specific journals. many databases allow researchers to submit papers to be curated, or to curate the data themselves; however, that may result in inconsistencies or varying level of detail for included information. multiple efforts using artificial intelligence and text mining have been used over the years to aid the curation process to either prefilter information to be curated, or automate its annotation [e.g., wormbase (59) ]. however, as already highlighted, naming and other inconsistencies create challenges that must be handled manually. since database infrastructure and curation funding are becoming more difficult to obtain, this in part explains the lack of database maintenance and availability, and even data source availability. one approach to promote accurate and upto-date curation is to create consortia among the groups behind the many databases [as proven by the international molecular exchange (imex) consortium for protein interactions, for example (60, 61) ]. the scientific community could contribute to this effort by following naming rules and providing links to databases when describing already curated molecules; in turn, databases should agree on one nomenclature and not create multiple unique ones, and should provide mapping for molecules already described using several different identifiers. again, the protein interaction field provides a successful example of this (62) (63) (64) . as non-coding rna research continues to grow, this strategy is expected to support data integration across studies, and in turn increase the value of biological data for translational research by improving reproducibility and interpretability. while overcoming data integration challenges is not trivial, the networks achieved can provide more complete and more precise insight into the molecular background of a disease of interest. we demonstrate this using an integrative computational biology approach, constructing lncrna/circrna-mirna-mrna networks in oa chondrocytes and ra fls. given the recent surge in studies exploring non-coding rna axes in various diseases, placing findings of individual studies into a broader biological context while maintaining key parameters (e.g., cell types) is expected to advance our understanding of the net effect of manipulating a single non-coding rna. from larger networks, sub-networks may be selected for future investigation based on a specific hypothesis, or simply based on the number or nature of connections. moving forward, concerted efforts to unify nomenclature, maintain databases, and improve context-specific target predictions is expected to support integrative approaches that expedite prioritization of the most promising candidates for experimental exploration in specific biological contexts. data available upon request to the authors as described in methods section. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2021.744747/ full#supplementary-material non-coding rnas in development and disease: background, mechanisms, and therapeutic approaches circular rnas: promising molecular biomarkers of human aging-related diseases via functioning as an mirna sponge crosstalk between long non-coding rnas, micro-rnas and mrnas: deciphering molecular mechanisms of master regulators in cancer long non-coding rna: its evolutionary relics and biological implications in mammals: a review regulation of microrna biogenesis associations of circulating plasma micrornas with age, body mass index and sex in a population-based study the complex landscape of micrornas in articular cartilage: biology, pathology, and therapeutic targets recent progress on the role of mir-140 in cartilage matrix remodelling and its implications for osteoarthritis treatment microrna-140-5p regulates the proliferation, apoptosis and inflammation of ra flss by repressing stat3 network analysis, visualization and graphing toronto update: context-specific physical protein-protein interactions in human, model organisms and domesticated species osteoarthritis in 2020 and beyond: a lancet commission lncrna malat1 mediates proliferation of lps treated-articular chondrocytes by targeting the mir-146a-pi3k/akt/mtor axis lncrna malat1/mir-145 adjusts il-1beta-induced chondrocytes viability and cartilage matrix degradation by regulating adamts5 in human osteoarthritis the reduced lncrna nkila inhibited proliferation and promoted apoptosis of chondrocytes via mir-145/sp1/nf-kappab signaling in human osteoarthritis long non-coding rna snhg15 is a competing endogenous rna of mir-141-3p that prevents osteoarthritis progression by upregulating bcl2l13 expression long non-coding rna (lncrna) small nucleolar rna host gene 15 (snhg15) alleviates osteoarthritis progression by regulation of extracellular matrix homeostasis long non-coding rna pvt1, a molecular sponge of mir-26b, is involved in the progression of hyperglycemia-induced collagen degradation in human chondrocytes by targeting ctgf/tgf-beta signal ways knockdown of pvt1 inhibits il-1beta-induced injury in chondrocytes by regulating long non-coding rna xist contributes to osteoarthritis progression via mir-149-5p/dnmt3a axis xist/mir-376c-5p/opn axis modulates the influence of proinflammatory m1 macrophages on osteoarthritis chondrocyte apoptosis lncrna part-1 targets tgfbr2/smad3 to regulate cell viability and apoptosis of chondrocytes via acting as mir-590-3p sponge in osteoarthritis lncrna part1 modulates chondrocyte proliferation, apoptosis, and extracellular matrix degradation in osteoarthritis via regulating mir-373-3p/sox4 axis neat1/mir-193a-3p/ sox5 axis regulates cartilage matrix degradation in human osteoarthritis microrna-377-3p alleviates il-1beta-caused chondrocyte apoptosis and cartilage degradation in osteoarthritis in part by downregulating itga6 the global prevalence of rheumatoid arthritis: a meta-analysis based on a systematic review rheumatoid arthritis expression of microrna-146 in rheumatoid arthritis synovial tissue altered expression of microrna in synovial fibroblasts and synovial tissue in rheumatoid arthritis upregulated microrna-155 expression in peripheral blood mononuclear cells and fibroblast-like synoviocytes in rheumatoid arthritis deregulation and therapeutic potential of micrornas in arthritic diseases lncrna neat1 targets fibroblast-like synoviocytes in rheumatoid arthritis via the mir-410-3p/yy1 axis delivery of long non-coding rna neat1 by peripheral blood monouclear cells-derived exosomes promotes the occurrence of rheumatoid arthritis via the microrna-23a/mdm2/sirt6 axis blockade of discoidin domain receptor 2 as a strategy for reducing inflammation and joint destruction in rheumatoid arthritis via altered signaling in fibroblast-like synoviocytes lncrna linc-pint increases socs1 expression by sponging mir-155-5p to inhibit the activation of erk signaling pathway in rheumatoid arthritis synovial fibroblasts induced by tnf-alpha inhibiting role of long non-coding rna linc01197 in inflammation in rheumatoid arthritis through the microrna-150/thbs2 axis inhibitory role of long non-coding rna oip5-as1 in rheumatoid arthritis progression through the microrna-448-paraoxonase 1-toll-like receptor 3-nuclear factor kappab axis knockdown of long non-coding rna pvt1 induces apoptosis of fibroblast-like synoviocytes through modulating mir-543-dependent scube2 in rheumatoid arthritis the mechanism of chinese herbal formula hqt in the treatment of rheumatoid arthritis is related to its regulation of lncrna uc.477 and mir-19b hsa_circ_0088036 promotes the proliferation and migration of fibroblast-like synoviocytes by sponging mir-140-3p and upregulating sirt 1 expression in rheumatoid arthritis sequencing identifies a distinct signature of circulating micrornas in early radiographic knee osteoarthritis plasma mirnas improve the prediction of coronary atherosclerosis in patients with rheumatoid arthritis circulating mirnas in bone health and disease antisense oligonucleotide-based therapies for the treatment of osteoarthritis: opportunities and roadblocks oligonucleotide therapies in the treatment of arthritis: a narrative review a uniform system for microrna annotation a guide to naming human non-coding rna genes the microrna registry mirbase tracker: keeping track of microrna annotation changes lncipedia 5: towards a reference set of human long non-coding rnas circpedia v2: an updated database for comprehensive circular rna annotation and expression comparison closing the circle: current state and perspectives of circular rna databases rnacentral 2021: secondary structure integration, improved sequence search and new member databases mirdip 4.1-integrative database of human microrna target predictions lncrna2target v2.0: a comprehensive database for target genes of lncrnas in human and mouse circatlas: an integrated resource of one million highly accurate circular rnas from 1070 vertebrate transcriptomes the art of curation at a biological database: principles and application text mining meets community curation: a newly designed curation platform to improve author experience and participation at wormbase. database (oxford) (2020) 2020:baaa006 towards a unified open access dataset of molecular interactions the imex coronavirus interactome: an evolving map of coronaviridae-host molecular interactions. database (oxford) (2020) 2020: baaa096 capturing variation impact on molecular interactions in the imex consortium mutations data set encompassing new use cases -level 3.0 of the hupo-psi format for molecular interactions protein interaction data curation: the international molecular exchange (imex) consortium key: cord-0035223-78r0rsio authors: jani, meghna; dixon, william g.; matteson, eric l. title: management of the rheumatoid arthritis patient with interstitial lung disease date: 2017-11-29 journal: lung disease in rheumatoid arthritis doi: 10.1007/978-3-319-68888-6_9 sha: f6550d68e2993e371163dc4d6abfad0a47f16a65 doc_id: 35223 cord_uid: 78r0rsio the treatment of rheumatoid arthritis (ra) has undergone considerable changes over the last 15–20 years. with an expansion in the armamentarium of therapies available for ra comes a wider choice in selecting the best treatment in terms of comparative safety in the presence of comorbidities. clinicians frequently encounter patients with ra-associated interstitial lung disease with uncontrolled joint disease and have to make decisions about the safest treatments in this context with the eventual goal of joint remission. in this chapter, available evidence is reviewed on the comparative pulmonary safety of non-biologic disease-modifying antirheumatic drugs (nbdmards), biologic dmards, biosimilars and targeted synthetic dmards in ra-ild. in addition, the potential role for additional immunosuppression in ra-ild is reviewed as well as overarching recommendations proposed for patient assessment to guide treatment decisions and management. the management of rheumatoid arthritis (ra) has changed dramatically over the past 15-20 years. new classification criteria for ra have been introduced [1] that allow the study of patients earlier in their disease course, and recommendations have been developed to treat patients with ra using a strategic approach by targeting an optimal outcome, with the primary goal of joint disease remission [2, 3] . the armamentarium of treatments available for the treatment of ra has also expanded. however, with the introduction of novel therapies comes a wider choice in selecting the best treatment for the individual patient not only in terms of comparative efficacy but also safety in the presence of comorbidities. interstitial lung disease (ild) is one of the most common respiratory manifestations in patients with ra and is a major cause of morbidity and mortality. ra-ild encompasses several histopathologic patterns; the most frequent considered here are usual interstitial pneumonia (uip), nonspecific interstitial pneumonia (nsip), and obstructive pneumonia (op). the development of respiratory complications of treatment is thus particularly problematic in patients with such coexistent lung disease, and there have been many reports of respiratory complications of both non-biologic disease-modifying antirheumatic drugs (nbdmards) and biologic dmard (bdmard) therapy. clinicians are frequently encountered with decisions about balancing risk and benefit of treatments in patients with ra-ild who have active articular disease. serious respiratory adverse events (sraes) in the patient with ra on treatment for their joint disease may be due to induction of 'pneumonitis' or idiosyncratic adverse drug reactions (adrs), acceleration of pre-existing ild or increased predisposition to infection in a susceptible host. several nbdmards and bdmards have been implicated in the development of ild. conversely, treatment of the underlying disease process may be beneficial in halting the progression of the lung disease. this chapter first considers methods for assessing drug safety and then reviews the available evidence for respiratory outcomes of nbdmards and bdmards in ra-ild and the role of additional immunosuppression and overarching recommendations for patient assessment and management of ild. whilst randomised controlled trials (rcts) are considered the gold standard in assessing efficacy of treatments, they have limited utility in terms of assessing safety. although safety has been evaluated as part of a number of rcts, uncommon events such as new-onset ild may not be captured. rcts often have restrictive eligibility criteria to ensure a homogenous population (leading to exclusion of patients with known ra-ild), small numbers of patients and a short follow-up period. this also limits their external validity, as real-life patients may be older and have multiple comorbidities compared to trial patients. open-label extension (ole) studies are often an adjunct to double-blind rcts, and although they do allow surveillance of participants for a longer duration, patients who are intolerant to the drug during the previous study will not be able to participate in the extension period, therefore potentially leading to underreporting of adverse events (aes). ole studies also lack a comparator group and are prone to loss to follow-up, making it difficult to interpret the rate of aes. post-marketing spontaneous pharmacovigilance involves the reporting of suspected adverse drug reactions by healthcare professionals or patients. this is done either in the form of case reports/series to medical journals or to national or international monitoring centres, such as the uk medicines and healthcare products regulatory agency (mhra) 'yellow card scheme', european medicines agency eudravigilance database and the us food and drug administration (fda) medwatch programme. whilst these are useful for drug safety signal detection, there is no available denominator to calculate rates of ae or to determine whether these reported cases represent an increased incidence above the background population rate. observational cohorts have the advantage of being able to examine drug safety in a 'real-world' setting and can follow large numbers of patients for long periods of time. they can therefore study the medium-to long-term side effects of a drug that might otherwise be missed in clinical trials or spontaneous pharmacovigilance. however these come with the challenge of interpreting results in the context of clinical decisions, with their consequent biases and confounding. each of these methods can employ a wide array of terminology to describe various forms of respiratory aes. for example, parenchymal lung disorders are called many things from 'pulmonary fibrosis' to 'allergic pneumonitis'. there are also challenges in differentiating between respiratory conditions with different aetiologies but similar clinical presentations. rcts, for example, can report on culturenegative pneumonias or 'community-acquired pneumonitis', which may indeed be idiosyncratic drug reactions or pneumonias secondary to a resistant microorganism: often patients are administered a combination of glucocorticoids and antibiotics due to initial uncertainty of the diagnosis. the data available for this chapter come from all of the above study designs. having been in use for ra for the longest duration, nbdmards and tumour necrosis factor inhibitors (tnfis) have the most observational evidence, but not in all cases. newer tnfis, nbdmard and emerging novel therapies recently licensed for ra have not had the opportunity for longer follow-up; therefore, experience about pulmonary safety may be restricted to clinical trials and spontaneous pharmacovigilance. methotrexate (mtx) has been described as the anchor drug in ra treatment, as it is often used first line at diagnosis, in combination with other nbdmards and concomitantly with biologics, following failure of traditional nbdmards for controlling articular disease. it inhibits folic acid and purine metabolism along with t-cell activation. mtx-induced pulmonary injury was initially reported in children with leukaemia in the 1960s [4] , followed by a case series in treated patients with ra in 1983 [5] . following a review of 123 cases of methotrexate pneumonitis in the year 2000, 63% of cases arose in patients with ra (dose range 2.5-15 mg/week), 23% occurred during intensification/consolidation treatment for leukaemia (dose range 20-80 mg/week), and 8% were in patients treated for other malignancies (dose range 15-1400 mg/week) [6] . whilst mortality rates have been reported up to 17%, hypersensitivity pneumonitis is reported to be a rare ae in ra patients. in a systematic literature review of 3463 patients with ra on mtx, 84 patients (2%) had some type of lung toxicity, but only 15 patients were felt to be definitive cases of pneumonitis attributable to methotrexate (0.43%) [7] . estimates of reported incidence vary between 0.43 and 1% of treated patients (in up to 3-year follow-up) [8, 9] . the clinical presentation of acute mtx pneumonitis is generally nonspecific, with symptoms (fever, rigors, malaise, nonproductive cough, dyspnoea, chest pain) that can be progressive over several days. criteria proposed by searles and mckendry [10] and carson et al. [11] are generally accepted for defining mtx pneumonitis and can sometimes help in differentiating the disease from ra-ild and respiratory infections, although it is possible to fulfil the criteria with conditions other than pneumonitis, for example, infection or a progression of pre-existing ra-ild (table 9 .1). searles and mckendry criteria have since been adapted by kremer et al. [12] categorising them into major and minor. all rely on a combination of clinical features, radiological, histology and exclusion of infection. studies have explored other factors that might differentiate these clinically similar respiratory diseases. histological findings in mtx pneumonitis such as cellular interstitial infiltrates, diffuse alveolar damage, tissue eosinophils and granuloma formation are nonspecific and have all been seen in ra lung disease [6] . highresolution computer tomography (hrct) studies in mtx pneumonitis typically show ground-glass changes, centrilobular nodules +/− diffuse parenchymal opacification [13] . bronchoalveolar lavage (bal) cell profiles in mtx pneumonitis show a lymphocyte alveolitis with a preferential increase in cd4+ cells compared to normal ra controls [14, 15] , though comparisons have not been made between bal in mtx pneumonitis and ra-ild. a challenge with all these studies is the problem of measuring the accuracy of a test against no accepted gold standard. it has been argued on several counts that mtx hypersensitivity pneumonitis is a unique hypersensitivity reaction rather than the development or progression of ra-ild. firstly, mtx-associated lung injury tends to occur early in the [16] , the close temporal relationship arguing in favour of causality. secondly, acute lung injury has resolved after discontinuation of the drug and treatment with high-dose glucocorticoids [5] . lastly, case reports of ild have been reported in other conditions including psoriatic patients treated with mtx [6, 17] as well as in other conditions not typically associated with ild. however, there are issues with each of these arguments. clinicians are more likely to diagnose and report mtx pneumonitis if there is a close temporal relationship. if cases in question are identified by asking rheumatologists to identify them [16] , it is inevitable that a strong temporal relationship will be found. the second argument of resolution on drug withdrawal would be more convincing if there were no concurrent glucocorticoid treatment. acute exacerbations of ild, which may resemble drug pneumonitis, tend to be responsive to glucocorticoids. that said, there are cases of improvement on mtx withdrawal with no increase in pre-existing glucocorticoid dose [14] . third, dose and duration of mtx treatment does not appear to be associated with pulmonary toxicity in the reported literature [6] . furthermore a recent systematic review and meta-analysis evaluating the risk of pulmonary disease amongst mtx-treated patients with psoriasis, psoriatic arthritis and inflammatory bowel disease in rcts concluded no increased risk [18] . it is not clear whether pre-existing ra-ild increases the risk of mtx pneumonitis with studies both supporting and opposing this view. the largest study was a multicentre case-control study from the usa [12, 19] , which identified 29 cases and 82 controls matched for mtx use. the strongest predictors of lung injury were diabetes, hypoalbuminaemia, previous use of nbdmards, rheumatoid pleuropulmonary involvement (or 7.1 (95% ci 1.1-45.4)) and older age (>60). a history of copd was modestly associated with mtx-induced lung injury. since all the cases were identified following clinical presentation, the observed association between pre-existing lung disease and mtx pneumonitis may represent an increased likelihood of presentation (because of a reduced physiological lung reserve) or a surveillance bias rather than an increased predisposition to pneumonitis. meta-analysis of six studies that suggested an association between pre-existing lung disease (of varying definitions) and an increased risk of mtx pneumonitis found a pooled odds ratio of 7.5 (95% ci 3.6, 15.8) [20] . however, two studies that suggested no such association [11, 21] were not included in the meta-analysis for reasons that are not clear. howes et al. [22] followed 120 patients who received mtx for ra for a median treatment duration of 15 months, all of whom had baseline pfts. three patients developed pulmonary toxicity according the established criteria [5] , all of whom had abnormal baseline pfts. of the patients studied, 3/120 patients had a transfer factor of <70% at baseline, one of whom developed pulmonary toxicity. the authors report a relative risk for pneumonitis of 10, though this estimate is not robust given the small numbers. there is currently no conclusive evidence that lung function is likely to decline faster in patients with ra-ild on mtx who do not develop pneumonitis. in a retrospective cohort analysis, following 6 weeks of high-dose glucocorticoid treatment, one study reported treatment with mtx vs. leflunomide or azathioprine was associated with an improvement in fvc at 6 months in patients with less fibrosis at baseline, although there were no another differences in other major outcomes such as mortality [23] . another study by dawson et al. [7] followed 128 patients with ra for 2 years, 43% of whom were on low-dose mtx (mean 10.7 mg/week), with hrct in all patients at baseline and pulmonary function tests (pfts) at baseline and at 4-month follow-up intervals. there was no significant difference in the change in pfts over the follow-up period either between the mtx and non-mtx patients or between mtx and non-mtx patients with proven ild on hrct at baseline. the authors concluded that they found no association between mtx therapy and progression of chronic pulmonary fibrosis. current guidelines on the use of mtx recommend that all patients should have a baseline chest radiograph with or without pfts. there appears to be little evidence to support this; however, pfts may be useful in patients with ra deemed to be at high risk of ild or known to have ra-ild to assess for progression (discussed further in section "patient management and treatment"). in summary, diagnostic uncertainty and the lack of any gold standard test for mtx pneumonitis leaves ambiguity about the true pattern of disease. however mtx has not been shown to consistently accelerate the progression of underlying ra-ild. whilst few studies have been conducted in pre-existing ra-ild stratified by severity of lung disease, the risk of pneumonitis means that it may not always be the safest first-line nbdmard in patients with ra who have severe pre-existing lung disease. leflunomide is an isoxazole derivative, which inhibits de novo pyrimidine synthesis, resulting in several downstream anti-inflammatory effects such as suppression of tnf-induced cellular responses and inhibition of matrix metalloproteinases and osteoclasts. leflunomide-induced pneumonitis is rare but well reported. a signal of concern was raised in 2004 after an investigation by the japanese ministry of health following post-marketing surveillance in 16 (0.5%) of the first 3000 japanese patients treated with leflunomide, resulting in five fatalities [24, 25] and the committee on the safety of medicines also reporting 17 cases in the uk of which five were fatal. further case reports have subsequently been reported especially in the japanese and korean populations [26] [27] [28] . however leflunomide is often used second line after mtx exposure or in combination with mtx in patients with active articular disease, which make studies evaluating leflunomide-induced pneumonitis difficult to interpret. in a report of 14 such cases from australia and new zealand, 12 were co-prescribed mtx [29] . chikura et al. [30] reported on 32 pneumonitis cases following leflunomide exposure, classified using searles and mckendry criteria (table 9 .1), and found 97% had a history of mtx exposure, whilst 41% were on concomitant mtx at the onset of ild. the majority presented within 20 weeks of initiation. clinical features of those who died were pre-existing ild and diffuse alveolar damage on histology (n = 6/32). pre-existing ild was also shown to be a risk factor for leflunomide-induced pneumonitis in a large case-control study using a canadian claims database [31] . the risk of ild was increased with the use of leflunomide (adjusted rr 1.9 [95% ci 1.1-3.6]); however, in patients without previous mtx exposure or ild history, the risk associated with leflunomide treatment was not elevated (rr 1.2 [95% ci 0.4-3.1]). however, it was acknowledged given the probable association between prior ild and mtx pneumonitis; clinicians may be more likely to prescribe leflunomide than mtx to patients with prevalent ild, leading to channelling bias explaining the increased risk observed. leflunomide-induced pneumonitis appears to occur more frequently in japanese and korean population (reported rate ~1%) [26, 32] , whilst in the western caucasian population, a rate of <0.1% is reported [33] . genetic susceptibility in japanese patients has been described in a study which investigated human leukocyte antigen (hla) class i associations with mtx pneumonitis in japanese patients with ra and found hla-a × 31:01 as a possible predictor. the prevalence of this allele is proportionally higher in the japanese population (8.7%) than in the caucasian population (3.9%) [34] . therefore such genetic differences and undiscovered genetic predictors may explain some of the differences in frequency observed in such populations. several risk factors of leflunomide-induced pneumonitis have been reported in small numbers of patients in case series and retrospective studies including preexisting lung disease [32, 35, 36] , a prescribed loading dose, smoking, low body weight [32] and increased c-reactive protein, hypoalbuminaemia, hypoxia and lymphopaenia [36] . treatment includes cessation of the drug, treatment with glucocorticoids with some benefit reported with activated charcoal and cholestyramine as washout treatments. whilst conclusions of use in ra-ild are limited from studies due to channelling bias, leflunomide should be avoided in patients with previous mtx pneumonitis and should be used with caution in patients with pre-existing ild. sulphasalazine is a 5-aminosalicyclic acid (5-asa) derivative metabolised to sulphapyridine, which is the active moiety in ra. pulmonary hypersensitivity reactions such as eosinophilic pneumonias [37, 38] , fibrosing alveolitis and bronchiolitis obliterans have been well described, with over 50 case reports in the literature [39, 40] . drug reaction with eosinophilia and systemic symptoms (dress) is also reported [41, 42] . typical presentation of sulphasalazineinduced lung disease reported is with new-onset dyspnoea and infiltrates on chest radiograph (with or without peripheral eosinophilia with eosinophilic pneumonitis). cough and fever are the most common symptoms with sputum production, whilst allergy history, rash, chest pain and weight loss were inconsistent findings [40] . histology is often variable; the most frequent appears to eosinophilic pneumonia with interstitial inflammation with or without fibrosis. drug cessation often results in resolution of symptoms in patients who develop eosinophilic pneumonia [43] . the role of systemic glucocorticoids is not wellstudied, as most patients improve with withdrawal of sulphasalazine. there are no studies that have systematically evaluated the safety of sulphasalazine in preexisting ra-ild; however, it is worth observing that the rates of sraes with sulphasalazine appear much lower than those seen with methotrexate and leflunomide in the literature. hydroxychloroquine is an antimalarial drug and is a 4-aminoquinoline derivative, often used in combination with other nbdmards. it is usually well tolerated and serious aes are rare. few cases of drug-induced pneumonitis exist [44] , with some reports in association with drug reaction with eosinophilia and systemic symptoms (dress) syndrome [45, 46] . hydroxychloroquine has been tried in childhood ild (diffuse parenchymal lung diseases) in case reports given the lack of therapeutic options. in a systematic review from 1984 to 2013 [47] , 85 case reports were identified: a favourable response to hydroxychloroquine was reported in 35 cases (41%), with an unclear effect in the rest. whilst idiosyncratic reactions leading to lung toxicity have been reported, no studies have demonstrated a benefit or harm of this drug in ra-ild. in summary, case report evidence for drug-induced pneumonitis exists for all nbdmards commonly used to treat joint disease; therefore, incident ild may be a theoretical risk in most treated patients. with treat-to-target guidelines [2] and the eventual aim of abrogation of joint inflammation as soon as possible, most patients with ra are likely to be treated with a nbdmard early in the course of their disease. whether the risk of progression of ra-ild or sraes increases with nbdmards is not clear and may vary according to the individual patient profile including their comorbidities, concomitant treatments, severity of ild at the outset of treatment and their genotype. evidence to date suggests caution should be exercised whilst treating patients with pre-existing ra-ild with all nbdmards, particularly mtx and leflunomide, with careful monitoring of disease progression and vigilance for (sometimes rare) sraes associated with such treatments (summarised in table 9 .4). five tnfi agents have been approved for treating patients with ra: three monoclonal antibodies (infliximab, adalimumab and golimumab), the recombinant soluble tnf receptor etanercept and pegylated certolizumab. as biologic therapies such as tnfis target key cytokines and cells in the inflammatory cascade with pleiotropic effects on the immune system, there have been particular concerns regarding their long-term safety profile. although these drugs are highly effective in controlling joint disease, and often are used as first-line biologics in combination with mtx, they are reported to be associated with various autoimmune aes [48] . whilst the most common of these appear to be lupus-and vasculitis-like events, tnfiassociated lung injury has been reported [49] . the safety of these drugs in patients with established ra-ild continues to be scrutinised. conversely elevated levels of tnf-α have been detected in the lungs of both experimental animal models [50] and patients with idiopathic pulmonary fibrosis (ipf) [51] . interestingly serum levels of tnf-α have also been found to be elevated in patients with rituximab-induced ild [52] . etanercept has been used in a randomised double-blind placebo-controlled exploratory trial in clinically progressive ipf subjects, however failed to show a difference in primary endpoint of improvement in fvc over 48 weeks. the study was underpowered; however, a nonsignificant reduction in disease progression was seen in several physiologic (including transfer factor), functional and quality-of-life endpoints amongst participants receiving tnfi. importantly there was no decline in lung function or higher incidence of sraes in the etanercept group compared to placebo. therefore whilst legitimate concerns about pulmonary toxicity have been highlighted, tnfi treatments may have plausible bidirectional effects. the next section will review the levels of evidence for both incident ild and safety in pre-existing ra-ild. clinical trials of tnfi therapy have been significantly underpowered to detect even a large increase in the incidence of serious ra-ild. the original rcts leading to eventual approval of tnfi provided no ra-ild safety signals. in the first reported phase iii trial of infliximab and mtx versus placebo and mtx over 30 weeks, there was a death in both the placebo arm due to ild and the infliximab arm due to cardiopulmonary failure as a result of suspected pulmonary embolism or ild [53] . in a two-year extension of this trial, there was a single death attributed to ild in the infliximab arm; however, it was not clear whether this is the same patient reported in the previous paper. a clinical trial of etanercept versus mtx in early ra reported three cases of pneumonitis in the mtx group at 1 year follow-up [54] and four cases at 2 year follow-up [55] with no reported cases in the etanercept arm despite a ratio of etanercept/mtx of 2:1. there was one case of death from respiratory failure in the 5-year ole of this study in the etanercept arm [56] . no cases of ild were reported in the 7-year ole of etanercept [57] . ild was not reported in any of the initial adalimumab clinical trials, in either the adalimumab or placebo arms [58] . however, within an adalimumab ole study [59] , saes coded as 'parenchymal lung disorders' occurred at a rate of 0.2-0.3/100 patient-years, accounting for 20% of all sae's. in this trial, however, there was no placebo-controlled or nbdmardexposed arm, making the interpretation of these figures challenging. similarly no signal of concern for incident ild was observed in the initial golimumab-ra trials [60, 61] in the two-year [62] or five-year [63] extension studies. in the certolizumab rapid 1 and rapid 2 trials [64, 65] , and subsequent oles [66] , no cases of druginduced pneumonitis were reported in any arms. therefore original rcts of tnfi agents did not raise concern over the potential development of ild. these trials typically had short duration, included between 210 and 340 patients in the tnfi arm each and excluded patients with ra-ild at baseline. concerns regarding the respiratory safety of tnfi agents arose initially following three case reports of rapid fatal exacerbations of ra-associated fibrosing alveolitis following commencement of infliximab in 2004 [67] , which was extended in a later publication to five cases [68] . of these cases, three patients were taking concomitant azathioprine, and one taking leflunomide developed rapid progression of ra-ild (known uip preceding treatment). the fifth patient did not have a history of lung disease pretreatment and, following infliximab, developed nonfatal cryptogenic organising pneumonia (cop or bronchiolitis obliterans organising pneumonia [boop]) [68] . in a case series of autoimmune diseases associated with tnfi, 24/226 (~10%) were reported to develop ild (interstitial pneumonitis in 18 patients, sarcoidosis in three, pulmonary haemorrhage in two and boop in one patient) [49] . exacerbation of previous ild was reported in four patients. perez-alvarez et al. described a further series of 122 patient case reports from the literature (89% with ra), with incident ild or exacerbation of pre-existing ild (20 patients, 38%) [69] . the outcome was available in 52 of the described cases: 21 (40%) had complete resolution, improvement or partial resolution in 13 (25%) and no resolution reported in 18 (35%). case reports of incident ild have been reported with newer tnfis such as certolizumab [70] [71] [72] [73] and golimumab [74] , including possible deterioration of pre-existing ild in a patient with ra treated with certolizumab and leflunomide after 3 months of therapy [75] . whilst the temporal association with onset of ild and repeated reporting of such events provides a signal of concern, the interpretation of such data for extrapolation into clinical practice is limited by a likely reporting bias, a possible 'bandwagon' effect (a phenomenon where following the publication of an index case, further cases are consequently more likely to be reported), and lack of an adequate denominator. furthermore, reports of stabilisation of ra-ild in patients exposed to tnfi therapy have also been published [76] [77] [78] , suggesting a further bidirectional influence of tnfi on ild. to accurately evaluate the risk of incident ild or exacerbation in patients with ra-ild on tnfi in patients who are most likely to receive them in the real world, observational studies offer the best design to examine rare outcomes. however all studies evaluating such events have faced methodological issues, namely, with channelling bias and challenges with classification. using the national databank for rheumatic diseases, wolfe et al. used a combination of hospital records, patient descriptive reports and physician and mortality records to classify patients who had severe ra-ild (requiring hospitalisation or who died) [79] . they reported a significant association with previous use of infliximab (hr 2.1, 95% ci 1.1-3.8) and etanercept (hr 1.7, 95% ci 1.0-3.0). however the study was confounded by the fact that physicians were, at the time, prescribing tnfis for treatment of ra-ild, possibly accounting for such an association. the incidence of hospitalisation for ild requiring hospitalisation was reported 260 per 100,000 patient-years with a 27% mortality, likely reflecting the types of patients included in this analysis. whilst the authors concluded that there was no evidence of an association between tnfi and hospitalisation for ild as the observed effect with tnfi was likely to be due to confounding by indication, the specific effect of tnfi on patients with ra-ild at baseline was not assessed [79] . two claims database studies have evaluated ild incidence in patients treated with biologics including tnfis. to assess the risk of incident ild in 8417 patients with autoimmune diseases who were members of kaiser permanente, northern california, herrington and colleagues compared new users of tnfi and nbd-mards [80] . ild cases were identified using icd codes, with a pilot of the first 100 cases verified using ct reports. the study demonstrated no increased risk in the tnfi-exposed group of new ild; however, patients with known ild at baseline were systematically excluded. using data from the commercial claims and benefit (medicare) databases, curtis et al. assessed the ild incidence and exacerbation amongst users of rituximab, abatacept and tocilizumab to tnfi agents [81] . two definitions of ild were used (one more sensitive, the other more specific in the absence of ct results and lung histology). there were no significant differences in the risk of ild and its related complications between patients with ra receiving tnfis and those on non-tnfis. of the patients' studies, 419 patients had a history of ild, of which 232 were put on tnfi agents [81] . however there were clear baseline differences between the groups receiving the different drugs, which were not adjusted in the analysis. therefore the true risk in the tnfi group may be confounded if patients with higher levels of ra joint disease or ild disease severity were channelled to specific therapies, which may have led to an overall underrepresentation of risk in this group. in a recent single-centre retrospective evaluation from japan, nakashita et al. described up to one-year outcomes of 163 ra patients with (n = 58) or without (n = 105) established ild on a biologic drug [82] . in patients with established ra-ild, 14 (24%) had an exacerbation of ild, which the authors concluded was greater in patients exposed to tnfi agents using descriptive statistics. none of the patients on tocilizumab (n = 9) or abatacept (n = 3) were reported to have an exacerbation; however, conclusions were limited due to low numbers. similar to curtis et al.'s study [81] , it did not adjust for severity of ra or any confounders potentially introducing channelling bias. in this case, it is likely that patients on tnfi (approved earlier than the non-tnfi drugs in this study) had more severe disease at the outset, in turn is known to be associated with the outcome. data from the british society for rheumatology biologics register for ra (bsrbr-ra) evaluated the effect of tnfi in patients with ra who have established ild [83] and reported no difference in all-cause mortality between patients on tnfis and nbdmards. however ra-ild as an underlying cause of death was reported in a higher proportion of tnfi-treated patients than nbdmard-treated patients (21 vs. 7%), suggesting a signal for concern. methodological challenges included potential for misclassification, as prevalent ild was identified from clinician questionnaires since attempts to retrospectively verify such cases were unsuccessful. whilst it could be that tnfi truly increased the proportion of deaths attributable to ra-ild, given the well-publicised respiratory concerns with tnfi, it may be that physicians at that time were more inclined to report ra-ild on death certificates of patients exposed to tnfis leading to the observed relationship. pfts, imaging or nonfatal exacerbation of ild details were not available to examine the effect of treatment on such outcomes. challenges in interpreting the data common to all studies is delineating the effect of tnfi on ra-ild in the presence of nbdmards, the majority of which are linked to reports of pneumonitis or exacerbation of ra-ild. furthermore, it can be difficult to ascertain if severity of ra associated with both development of ild and also being the indication for tnfi could explain the association between tnfi and ra-ild exacerbation. however, no studies have completely excluded a clinically meaningful risk, and the multiple case reports, observational evidence and possible increase in ild-specific mortality in patients with known ra-ild indicate that careful monitoring and caution is required when considering use in this setting. with several biologics approaching patent expiration, substantial interest has been in the development of biosimilar products that are not bioidentical but highly similar to already approved reference products listed above [84] . to date, biosimilar infliximab ct-p13 (marketed as remsima and inflectra) and biosimilar etanercept sb4 (marketed as benepali) have been approved for use by the fda and ema. there were no cases of pneumonitis or ild in the biosimilar infliximab ct-p13 trials in ra [85, 86] or ankylosing spondylitis [87] , although there was a case of noninfective dyspnoea reported within the ct-p13 arm of the latter trial felt to be related to the drug by the investigators. open-label extensions of these trials have not suggested a signal for concern [88, 89] . similarly no cases of new ild were reported with biosimilar etanercept sb4, albeit treatment-emergent aes in ≥2% of patients were reported only [90] . one patient in the sb4 arm died due to cardiorespiratory failure (felt to be unrelated to the drug by the investigators), and further details of the event were not provided [90] . whilst this provides some initial reassurance, biosimilars should be considered as having similar risks to the originator reference products and therefore be used with vigilance in the context of established ra-ild. rituximab is a monoclonal chimeric anti-cd20 antibody licensed for the treatment of non-hodgkin lymphoma and ra in tnfi nonresponders. rituximab-induced ild has been a well-described ae in haematology patients often presenting as acute/subacute hypoxaemic organising pneumonia, nsip or hypersensitivity pneumonitis [91] [92] [93] . whilst its pathogenesis is unknown, it may involve induction and release of cytokines, and it has been reported to occur in patients receiving rituximab for several months [94] . in ra, a number of rituximab trials have reported incident ild [95] [96] [97] , including subsequent deaths from acute respiratory distress syndrome (ards) [98] and culture-negative (non-infective) bronchopneumonia [99] . hadjinicolaou et al. identified >120 cases of rituximab-associated ild up to june 2010 in a systematic review of published studies and reports to the fda and ema [100] . in addition, uk spontaneous pharmacovigilance reporting systems (via its yellow card system) have recorded hundreds of rituximab-associated cases of non-infectious respiratory disorders till march 2016 (table 9 .2). of the patients with possible rituximab-associated ild/pneumonitis, 15 deaths have been reported (table 9 .2). further observational evidence has been discussed in section "other agents with limited agents in ra-ild"). abatacept is a selective t-cell costimulation modulator. it is a fully human recombinant protein that comprises of the extracellular domain of ctla4 and the fc portion of an igg1 molecule that has been modified to prevent complement activation. pooled safety data from eight trials of abatacept involving 3173 patients with ra reported incident ild in 11 patients (0.11 cases per 100 person-years), with no events reported in the placebo control groups [101] . patients with pre-existing ra-ild were excluded from all of the initial abatacept trials [101] . therefore limited conclusions can be drawn from existing trial data, which may suggest a small increase in incident ild in patients with ra. despite its fda licence back in 2005, few case reports exist describing abataceptinduced lung injury. one case reported drug-induced respiratory failure, 2 weeks after the second abatacept dose [102] , however was unable to distinguish if infection was the reason for deterioration by the time of death. relatively few respiratory adrs have been reported to the regulatory agencies after treatment with abatacept compared with other biologic agents (table 9. 2). indeed, only one case of pneumonitis and 23 cases of non-infectious respiratory events had been reported through the uk medicines and healthcare products regulatory agency (mhra) 'yellow card' system by march 2016. however it is worth noting at exposure to abatacept may be limited in the uk due to lack of its initial approval by the national institute of health and care excellence (nice) until 2010 as a second-line biologic (following tnfi failure and if rituximab was contraindicated) and first-line biologic approved in 2013. whilst experience of using abatacept in the context of pre-existing ild is limited, a case report of rapid-onset interstitial pneumonia 2 days post initiation of administration, mhra medicines and healthcare products regulatory agency, na data not available, ra rheumatoid arthritis a includes cases reported as idiopathic pulmonary fibrosis, pulmonary fibrosis and pulmonary toxicity treatment has been described in a japanese patient [103] . conversely, there are reports of stabilisation of ra-ild on abatacept [104] and improvement in some patients [105] . in a case series of 16 patients with ra-ild (with varying grades of severity at baseline), all patients completed 52 weeks of abatacept treatment, and no one was reported to have an exacerbation of their pulmonary disease [105] . in the three patients with ra-ild on abatacept, as part of nakashita and colleagues' retrospective study [82] , none had ild-related complications reported by one-year follow-up. however using claims data, in the curtis et al. study [81] , of the 102 patients with ra-ild on abatacept, there was no significant decreased risk of ild events compared to tnfis. in comparison with other biologic agents introduced to the market at around the same time, the spontaneous pharmacovigilance and case report figures are cautiously encouraging. the lack of a clear denominator or the number of patients treated with each drug in these settings makes accurate interpretation challenging. whilst experience in baseline ra-ild is limited, emerging observational data thus far appears tentatively reassuring. additionally patients on abatacept may have a more favourable infection profile in comparison to other biologics in those who have experienced a hospitalised infection previously [106] . this study also forms the basis of its use as a conditional recommendation in the context of prior serious infection in the acr 2015 guidelines for ra [107] . therefore given the limitations of spontaneous pharmacovigilance and inconsistent findings from observational data, more observational data is required before robust conclusions can be formed regarding safety of abatacept in ra-ild. however, there may be a role for abatacept particularly in patients in whom serious infection is a concern. tocilizumab tocilizumab is a humanised monoclonal antibody that inhibits il-6 receptor signalling through its membrane-bound and soluble forms. in the treatment of active joint disease, where mtx is contradicted or not tolerated, tocilizumab monotherapy has been shown to be as effective as combination treatment with mtx [108, 109] . given the potential concerns regarding the respiratory safety profile of mtx, there may be a preference for using this drug over other biologics in ra-ild by some physicians. experimental work has demonstrated profibrotic effects of il-6 on lung fibroblasts, which may have the potential to be antagonised by blocking the il-6/ il-6 receptor pathway, suggesting a potential benefit of tocilizumab in ra-ild [110] . however, clinical data on the use of tocilizumab in pre-existing ra-ild is sparse and inconclusive. unlike other biologics, early rcts of this biologic agent have raised some concerns regarding an association with development of ild. a single case of 'allergic pneumonitis' after tocilizumab monotherapy exposure in 109 patients with ra was reported in 2004 by nishimoto et al. [111] . in 2008, two of 419 patients treated with tocilizumab and mtx in the option study [112] developed ild at weeks 9 and 13, with a further two cases of 'culture-negative pneumonia'. a similar case was also reported in the satori [113] trial, which included patients on tocilizumab monotherapy. in one of the few head-to-head biologics studies, the 2013 adacta trial compared tocilizumab monotherapy with adalimumab monotherapy and reported two deaths in the tocilizumab arm, one of which occurred suddenly in a 56-year-old man with multiple comorbidities, including pre-existing ild [114] . further deaths due to interstitial pneumonitis have been reported in tocilizumabexposed arms in recent trials such as the surprise (tocilizumab + mtx arm) [109] , as well as the summacta trial, the latter reporting one death due to ild and ards each [115] . of note other anti-il-6 drugs being evaluated for ra, such as sarilumab (a fully human anti-il-6rα mab that binds membrane-bound and soluble human il-6rα), have also reported sraes within trials. of the two deaths reported in the saril-ra-mobility trial (sarilumab +mtx), one was due to ards (investigator felt was drug-related) [116] . within the phase iii study published to date, four deaths in the sarilumab arms were described [117] , one due to unspecified respiratory complications post surgery. whilst the consequences of such reports are not clear at present for the patient with pre-existing ild, such observations suggest the need for alertness especially since most trials exclude patients with multimorbidity. case reports have suggested an association between incident ild and tocilizumab exposure in patients on a concurrent nbdmards both in ra [118] [119] [120] and adult-onset still's disease [121] . spontaneous pharmacovigilance figures from the uk have reported five deaths due to sraes in tocilizumab-exposed patients to date, four due to ild and one secondary to ards (table 9 .2). whilst the total number of patients exposed to treatment (denominator) is not available, cautious comparison of these figures with abatacept, which was approved earlier than tocilizumab by fda and ema, is of interest. furthermore, the reaction study, a retrospective study of 229 tocilizumabexposed patients with ra in japan, reported interstitial pneumonia in two of the 229 patients followed up over 6 months and an additional six discontinuations for pneumonia (presumed infective), raising further concerns of pulmonary toxicity in this population. ra-ild was found to have improved in one patient with pre-existing ra-ild within 16 weeks after administration of tocilizumab [122] , whereas another patient with biopsy-proven uip and emphysema developed a fatal exacerbation of ild after treatment with tocilizumab [119] . of the 23 ild events reported in 3881 patients who received tocilizumab every 4 weeks over 28 weeks (1.28 per 100 patient-years) in a post-marketing surveillance programme in japan, the presence of a known ild was a risk factor for acute presentation with ild exacerbation and serious infections [123] . recent small retrospective observational studies demonstrate limited reassurance. in the nine patients with ra-ild selected to receive tocilizumab treatment over 1 year in the nakashita et al. study [82] , no patients were reported to have an ild exacerbation. however patients who did not complete at least 1 year of follow-up lacked imaging data or discontinued treatment because infections were excluded from the study leading to a likely selection bias of reported outcomes. in a retrospective case-control study in ra, patients were stratified according to the presence of baseline ra-ild (n = 78) or without (n = 317) [124] . during observation period of 148.8 patient-years and 629.7 patient-years for the ra-ild and non-ild groups, respectively, six patients developed an acute exacerbation in the ra-ild group (none in the non-ild group). interesting all the patients who developed an exacerbation of ild were on tocilizumab monotherapy, and there was a suggestion that all patients who developed an exacerbation had more uncontrolled articular disease that may in turn be associated with the outcome [124] . the administrative data study by curtis et al. concluded no significant differences between tocilizumab and other biologic drugs in the risk of ild or its exacerbation [81] . however exacerbation of ild can be difficult to define in such databases as recognised by the authors. hence surrogate measures such as hospitalisations for ild, pneumonia or lung transplant were used, which as expected were observed infrequently and capture a heterogeneous overall outcome that includes infection. therefore, whilst current evidence does not allow robust conclusions about the pulmonary safety of tocilizumab in ra-ild, ongoing vigilance is recommended if used in this context in the absence of clear predictors of ild exacerbation. anakinra is a recombinant il-1 antagonist, which binds competitively to the type i il-1 receptor and therefore acts as a competitive antagonist to il-1. initial trials that supported the licensing in ra did not demonstrate a signal of concern in relation to incident ild either as monotherapy [125] [126] [127] or in combination with mtx [128] . whilst such data supported the initial licensing of anakinra in ra, in clinical practice, it is not widely used in ra for active joint disease as it appears less efficacious than tnfi agents and of no additional value in early ra [129] . moreover, combination of anakinra and etanercept provided no added benefit compared to etanercept monotherapy, and indeed increased serious safety concerns were reported including two patients with new ild and pneumonitis in the combination biologic arms [130] . there are sparse reports of non-infective sraes on anakinra from spontaneous pharmacovigilance (table 9 .2), possibly reflecting fewer exposed patients with ra. therefore apart from one case of improvement in tocilizumab-induced pneumonitis following anakinra in a patient with adult-onset still's disease [121] , there is no further literature indicative of non-infective sraes with its use [33] but also minimal evidence to suggest a potential beneficial role in ra-ild. janus kinase (jak) inhibitors, such as tofacitinib and baricitinib, are synthetic orally administered compounds that block jak, a protein that mediates signal transduction of multiple cytokines. although these are not biologic drugs, though they interact with biological pathways, they have been classed as targeted synthetic dmards (tsdmards) for use in moderate to severe ra, who are nonresponders or intolerant to mtx [3, 131] . tofacitinib has been approved in several countries, such as in the usa, latin america and asia although not in the european union or uk. baricitinib has completed phase iii trials, and early results from head-to-head trials even suggest that it may be more efficacious than a tnf inhibitor [132] . rct data thus far with regard to respiratory safety of jak inhibitors are inconclusive. phase iii trials of tofacitinib with concomitant mtx in patients with ra have reported cases of new-onset ild and pulmonary sarcoidosis [133] . other sraes reported in tofacitinib-treated arms in rcts include bronchopneumonia [134] and a death due to ards [135] . a combination of pulmonary fibrosis and chronic obstructive pulmonary disease has been observed in such trials using tofacitinib monotherapy [136] . however data from two long-term extension studies from japan following up 4102 tofacitinib-treated patients over 5963 patient-years reported no additional ild events [137] , nor did subsequent meta-analysis of tofacitinib trials [138] . in addition phase iib studies and phase iii studies of baricitinib also have not reported incident ild cases [139, 140] and is currently under regulatory review. in 2015, the fda released a risk evaluation and mitigation strategy (rems) document highlighting the known concerns with tofacitinib, instructing the pharmaceutical company to send information out to healthcare professionals regarding the risk of serious infections, malignancies, decreases in peripheral lymphocyte counts, neutrophil counts and haemoglobin and derangement in lipid profiles [141] ; lung toxicity was not mentioned. no case reports or regulatory reports of ild have been published to date, although tofacitinib was licensed for the treatment of ra a few years ago in november 2012 in the usa and remains unlicensed in most of europe, limiting the numbers of exposed patients. conclusions drawn regarding the association between jak inhibitors and ild in patients with ra are therefore restricted, given limited data and further observational evidence are required to assess its effect on pre-existing ra-ild. although ra-ild is a fairly heterogeneous extra-articular manifestation of ra, the majority of cases mimic the two idiopathic interstitial pneumonia patterns of nsip and uip. whilst nsip and organising pneumonia appear to be more responsive to glucocorticoid treatment, the presence of the uip pattern on hrct in patients with ra may be associated with a significantly shorter survival compared to other forms of interstitial disease [142] . as a general clinical guidance, lung disease features that may be predictive of treatment benefit include histopathologic patterns other than uip (especially nsip and op, younger age of the patient and worsening of symptoms, pfts or finding on hrct over the preceding 3-6 months [143] [144] [145] . a dlco of less than 54% is associated with progression and poor prognosis and may identify patients who could be considered for treatment [7] . numerous medications have been described for potential therapies for ra-ild, but currently there are no large rcts to help guide physicians specifically in the management of the pulmonary manifestations of ra-ild. in addition to immunosuppressive treatments, general considerations for management of ra-ild include smoking cessation and age-appropriate vaccinations for pneumonia and influenza, as well as prophylaxis for pneumocystis jirovecii pneumonia in patients who are profoundly immunosuppressed [145] . typically glucocorticoids were used as first-line therapies in an attempt to stabilise and improve the disease course of ra-ild based on limited evidence from ipf. because ra-ild is a heterogeneous spectrum of histopathologic patterns beyond uip (ipf), experience extrapolated from management of idiopathic ild suggests that some forms, including nsip and op, may respond to glucocorticoid therapy [145] . given the paucity of evidence in ra-ild, it was felt that the addition of azathioprine maybe beneficial in glucocorticoid-responsive patients and could result in improved survival compared to glucocorticoids alone, again based on historic data from ipf studies [146] . however, the 2012 multicentre prednisone, azathioprine, and (n) acetylcysteine [nac]: a study that evaluates response in ipf (panther-ipf) trial had to be prematurely stopped as it is found that combination of prednisone, azathioprine and nac was associated with greater mortality (eight vs. one death), more hospitalisations (23 vs. 7) and more serious aes (24 vs. 8) compared to placebo in ipf patients with mild to moderate disease [147] . a large proportion of deaths were due to pulmonary infection, highlighting the need for adequate precautions to be taken against respiratory infections in patients with ra-ild who are susceptible to serious infections [148] . indeed in elderly patients with ra, prednisolone has been shown to have a dose-dependent risk of infection, with current/recent glucocorticoid therapy demonstrating the greatest impact on infection risk [149] . therefore use of glucocorticoids specifically for ild in ra is best performed after liaison with respiratory colleagues, with use of the lowest possible dose prescribed for the shortest duration. pirfenidone is an anti-fibrotic drug that inhibits transforming growth factor beta (tgf-β)-stimulated collagen synthesis, decreases the extracellular matrix and blocks fibroblast proliferation. it has demonstrated efficacy in ipf [150] [151] [152] and has been approved by the national institute of health and care excellence in the uk and the fda in the usa for use in patients with mild or moderate ipf. in ctd-ild, specifically secondary to systemic sclerosis, case reports and small retrospective studies have suggested modest benefit [153] [154] [155] [156] , whilst a recent open-label study in systemic sclerosis demonstrated acceptable tolerability of pirfenidone, especially important since 63.5% of patients were on concomitant mycophenolate mofetil [157] . currently there is no evidence for its use in ra-ild. theoretically, however, given its action on tgf-β and fibroblast proliferation, there may be justification of its use in the fibrotic nsip pattern and fibrotic stages of other ra-ild subtypes [158, 159] . currently there is a phase ii trial underway to assess the safety and tolerability of pirfenidone 2403 mg/day for the treatment of ra-ild [160] , with a need for more rcts the effect on respiratory function in this setting. other anti-fibrotic agents such as nintedanib, a tyrosine kinase inhibitor that targets multiple tyrosine kinases, including vascular endothelial growth factor, fibroblast growth factor and platelet-derived growth factor receptors, have been recommended by the international thoracic guidelines for its use in ipf [144] . it was recently found in various murine systemic sclerosis models to effectively inhibit the endogenous as well as cytokine-induced activation of fibroblasts and exert potent anti-fibrotic effects [161] . whilst there is currently no clinical evidence to date in ctd/ra-ild, there may be potential for future clinical trials with this drug. cyclophosphamide has been commonly used to treat ild unresponsive to glucocorticoids. however evidence of its use in ipf is lacking [162] . conflicting data exist regarding its use in scleroderma-related ild [163] , with additional concerns regarding its toxicity profile. rcts using cyclophosphamide suggest moderate benefit in scleroderma-ild patients with early disease [164] [165] [166] , although a previous metaanalysis concluded no improvement in pulmonary function following 12 months of treatment [167] . no rcts have been performed assessing the use of cyclophosphamide in ra-ild. limited evidence suggests that there may be some role in rapidly progressing patients with restricted therapeutic options in the acute or subacute setting [168] or in refractory drug-induced pneumonitis unresponsive to glucocorticoids [169] . the use of this agent is not recommended for mild/moderate stable ra-ild disease. experience of using cyclosporine in the treatment of ra-ild is limited and not recommended currently due to its poor safety profile and absence of proven benefit on pulmonary or joint disease. few publications in ipf have been less than encouraging [170, 171] ; however, it appears to yield some benefits according to anecdotal reports in myositis-related ild particularly anti-synthetase syndrome [172] [173] [174] . mycophenolate mofetil often used in the treatment of scleroderma-ild is an inhibitor of lymphocyte proliferation and additionally targets nonimmune cells such as fibroblasts and smooth muscle cells. the majority of the evidence for use has been derived from small prospective case series and retrospective reviews and has been shown to stabilise scleroderma-ild [175] [176] [177] and ctd-ild [178] . in the latter series, 18 patients with ra-ild were included; mycophenolate was associated with modest improvements in forced vital capacity (fvc), diffusing capacity and reductions in the prednisone dose [178] . a head-to-head rct assessing the use of a twoyear course of mycophenolate compared to oral cyclophosphamide for 12 months in ssc-ild has recently been published, demonstrating a more favourable safety profile with mycophenolate; however, both treatment arms demonstrate similar efficacy on lung function [179] . in the treatment of ra-ild, mycophenolate has been prescribed at doses of 1-2 g per day in patients with ra who have limited pulmonary disease with some benefit [175, 180] . however it is not effective in the treatment of active articular disease on ra, requiring the use of concomitant nbdmards, which may have additional consequences on tolerability. whilst studies supporting the use of mycophenolate in ra-ild represent a relatively small number of patients, further work on tolerability and safety in the context of active ra is required, and if promising well-designed trials may be helpful before advocating its use in this area. of the biologics having potential utility for treatment of ra and other connective tissue disease-related ild, perhaps rituximab has had the most interest and shown some promising results in published case reports and case series, not inclusive of ra [181] [182] [183] [184] . a recent retrospective review of ctd-ild cases treated with rituximab, which included anti-synthetase syndrome (n = 10), dermatomyositis (n = 3), systemic sclerosis (n = 3), systemic lupus erythematosus (n = 2) and unclassifiable ctd-ild (n = 4), suggested stabilisation of lung disease in 11 patients and worsening in nine patients. four patients with myositis had a reported clinically significant improvement with an fvc of >10% post treatment. whilst encouraging in a ctd-ild setting, most published work is in the form of case reports, subject to reporting bias, or retrospective case series. evidence of rituximab use for the treatment of ra-ild has not been encouraging and studies to date have been small or inconclusive. in an open-label pilot study of rituximab in ra-ild [185] of seven patients who completed a 48-week follow-up, one showed improvement in respiratory function, five were stable and one deteriorated. the study initially recruited ten patients, and of the three who did not complete the study, one patient died of ards/possible pneumonia, 6 weeks post treatment (no infective source identified). four small retrospective observational studies assessing the use of rituximab in ra-ild have been inconclusive (all still in abstract form, full papers not published). dass and colleagues in 2011 [186] presented data from 48 patients with baseline ra-ild; three deaths were reported in patients with ra-ild following rituximab treatment, one due to pneumonia and possible acute progression of ild, 4 weeks after the first cycle of rituximab therapy. furthermore, five patients had a decline in transfer factor of >10% over an unspecified time in the presented results of this study. the same group reported their rituximab experience in ra-ild over 10 years (january 2004-july 2014) in 2015 [187] . of the 53 patients with ra-ild on rituximab with a total of 171 patient-years, the authors concluded there was no significant improvement or deterioration in the majority (as measured using fvc/ dlco; actual data not available in abstract). however there were 12 deaths reported, nine of which were attributed to progressive ild (median dlco of 41% [range 35 -64%] pre-rituximab). other reasons for death were lung cancer, colon cancer and infection post surgery (n = 1 each). becerra et al. [188] reported on a single-centre retrospective review of 38 patients with ra and known lung involvement who were undergoing therapy with rituximab, of whom 19 had established ra-ild. progression of ild over 4 years was described in one patient with severe uip at baseline. improvement in lung function was observed in none of the patients, 66% (n = 25) reporting respiratory infections, two of which required hospitalisation. recent work from the bsrbr-ra compared mortality of 353 patients with physician-reported ra-ild, of whom 310 were treated with tnfi and 43 on rituximab [189] . the differences in mortality between the two groups were not statistically significant, and adjustment for baseline confounders made little difference to the estimates (hr adj 0.51, 95% ci 0.25-1.06). however, methodological issues included inherent differences in the two treated groups (the tnfi group being a more historic cohort, therefore likely to have more severe disease), low numbers of deaths and no information on baseline ra-ild severity. the results of all four of these studies are difficult to interpret often due to a lack of well-matched comparator groups and uncertainty about the natural history of ra-ild. therefore, currently, few data exists that suggest that rituximab can markedly improve ra-ild. in comparison to other biologics, there did not seem to be a significantly lower risk of complications related to ild in the curtis et al. study [81] , although channelling bias may exist. furthermore rituximab is known to reduce igg levels, which in turn may be associated with an increased infection risk, which bears consideration whilst making such treatment decisions [190] . in the context of ra overlap with another ctd, however, rtx may have a role in stabilising ild, especially with other extra-pulmonary manifestations of ctd which exist that may benefit from b-cell depletion. a clinical trial is currently underway comparing rituximab versus cyclophosphamide in patients with systemic sclerosis, myositis and mixed connective tissue disease (recital, clinicaltrials.gov identifier: nct01862926). currently, therefore, the evidence does not support preferentially switching to rituximab in all patients with ra-ild who have active joint disease, regardless of the severity of lung disease. in end-stage refractory ra-ild, single lung transplantation may be considered. however there are limited data on long-term outcomes for lung transplant in ra-ild. amongst ten patients with ra-ild who underwent transplantation, survival at 1 year was similar to ipf lung transplant recipients (67 and 69%, respectively), although higher for scleroderma-ild at 82% [191] . eligibility for lung transplantation may be restricted due to age (typically <65 years), contraindicated with certain comorbidities common in ra including osteoporosis, whilst other extra-articular manifestations of ra may in turn complicate transplantation. nonetheless, comparable survival outcomes of ra-ild with ipf are somewhat reassuring. in summary at present, there are no current treatments available that have appeared to consistently stabilise ra-ild as well as adequately treat joint symptoms. the risks and limited benefits of such medications bear consideration, especially since most evidence for efficacy has been demonstrated in ipf and ctd-ild. glucocorticoids may be used in ra-ild, however, with the lowest effective dose for the shortest possible duration to minimise risk of infection amongst others. robust rcts are required to support the use of novel antfibrotic agents or immunosuppressive therapies directed towards treatment of ra-ild. however, at present there is a paucity of evidence to support the routine use of these strategiest. evidence to guide management and treatment of ra-ild is of low quality or absent, hence extrapolated from systemic sclerosis-ild or ipf. however, despite the lack of robust data, clinicians are required to advise patients on the comparative safety of treatment and strategies to minimise complications of ra-ild such as infection and reduce the overall burden of morbidity to prolong survival. in addition ra management has become increasingly complex over the last decade. several effective dmards are available, and treat-to-target strategies support the use of early, rapidly escalating treatment often in combination with each other. as discussed earlier, there are no treatments that conclusively improve both joint and pulmonary disease in all patients; therefore, careful baseline assessment, adequate risk assessment for adverse outcomes and regular monitoring and vigilance whilst using the majority of dmard strategies are essential. to enable guidance of management strategies, a recent approach in idiopathic interstitial pneumonias (iip) has been to classify pulmonary disease as self-limiting, reversible, stable, progressive or irreversible [143, 192, 193] . a thorough assessment of baseline ra-ild severity should consider clinical features, pfts and imaging pattern/extent on hrct, low dlco and a high radiological fibrosis score on hrct being predictors of poor survival [194] [195] [196] . such information in combination with monitoring disease progression over time allows mapping of the disease trajectory and may better inform management decisions in the face of poor evidence. clinical assessment including quantification of exercise tolerance using instruments such as the five-point medical research council (mrc) breathlessness scale [197] can help aid serial assessment of the symptoms of ra-ild (table 9 .3). bibasilar crepitations are an associated feature that may help trigger detection of asymptomatic ild, but have limited value in assessing severity or monitoring progression. the 6-min walk test is a commonly used instrument for assessing ipf and can be performed easily. reduced walk distance and oxygen desaturation below 88% are poor prognostic factors in ipf [144, 198] ; however, practically this may be challenging to perform in ra patients with poor mobility. pfts provide objective measures of lung function and should be performed in all patients with respiratory clinical features, or confirmed ra-ild, prior to decisions about therapy. ilds share a restrictive pattern, with reductions in lung volumes and a reduced dlco [199] . low baseline fvc and dlco (fvc <60% and dlco <40% of predicted values) are independent predictors of early death in patients with ipf [195, 196] . importantly, a 6-12-month decline in fvc of ≥10%, or a decline in dlco of ≥15%, is associated with increased mortality in ipf [200] ; this association might also exist in patients with idiopathic fibrotic nsip. bdmards biologic disease-modifying antirheumatic drugs, dlco diffusing capacity of the lungs for carbon monoxide, fvc forced vital capacity, ild interstitial lung disease, hrct highresolution computed tomography, nbdmards non-biologic disease-modifying antirheumatic drugs, pfts pulmonary function tests, tb tuberculosis, uip usual interstitial pneumonia hrct imaging is indicated in patients with ra-ild clinical features or in asymptomatic patients with a dlco of <70% of predicted [201] . individuals with hrct findings consistent with uip, such as basally dominant honeycomb cysts with little or no ground-glass change, have poorer prognosis than those with hrct-detected features indicative of other types of iip [142, 202] . in ssc-ild, an hrct-based prognostic staging system has been developed, categorising patients as having limited (<20% lung involvement) or extensive (>20% lung involvement) ssc-ild, with extensive disease found to be a strong predictor of mortality [203] . similarly quantifying the extent of fibrosis and disease on hrct has been shown to predict disease progression in ipf [204] . no comparable studies have assessed the use of similar hrct-based scoring in ra-ild, although a small study suggested that the degree of interstitial changes detected using hrct imaging was predictive of prognosis [205] . on the basis of these factors, the authors have previously proposed a framework for assessment of ra-ild, risk assessment before initiation of biologic therapy and post-treatment monitoring [206] (fig. 9.1 ). the proposed approach is designed to help predict short-term progression of ild irrespective of biologic therapy. in patients at higher risk of progression, even in patients who appear to have stable lung disease over a one-or two-year period, treatment decisions should involve a multidisciplinary approach including a respiratory physician as well as a careful discussion with the patient regarding the benefits and risks of treatment options available. in all patients, including those with self-limiting and stable ra-ild on treatment for their joint disease, rheumatologists should prompt questioning of new or evolving respiratory symptoms at follow-up, as patients may not spontaneously disclose such information. all patients who smoke should be educated about the important associated risks. whilst being implicated in the pathogenesis of ra-ild, it may also lead to deterioration of lung disease and is associated with higher joint disease activity and reduction in the effectiveness of medications such as mtx and tnfi drugs [207] . smoking cessation advice and further support such as nicotine replacement therapy should be made available for all patients with ra-ild [159] . following an adequate period of monitored observation, classification of the trajectory of ra-ild may be possible in the individual patient. in patients in whom a 12-24-month period of stability is observed, pft follow-up intervals can be extended to 12 monthly. routine monitoring of pfts is suggested every 3-6 months in individuals at high risk of progression of ra-ild or on any biologic drug [206] . an observed decline following serial pfts could be due to progression of ra-ild, either its natural [206] history or accelerated by therapy or attributable to other causes that may or may not have an association with the dmard prescribed. 'reversible' decline has been categorised into potentially reversible with risk of irreversible disease (e.g. cases of drug-induced lung disease) or reversible disease with risk of progression (e.g. ra-cellular nsip, some ra-fibrotic nsip, ra-organising pneumonia) [143] . management strategy for the former would involve cessation of the putative drug with or without further treatment with glucocorticoids if required, whilst the latter may require more prolonged therapy and monitoring. in practice, the two are difficult to delineate given their similar presentations, and therefore rheumatologists faced with such patients demonstrating worsening respiratory symptoms or lung function should consult with respiratory colleagues early. if the deterioration in clinical picture is found to be due to progression of ra-ild, the decision to continue any dmard, but especially bdmards or tsdmards, necessitates clinical decisions on a case-to-case basis. the choice of whether to continue with effective dmard therapy for joint disease or the risk of potential pulmonary toxicity may be based on the perceived likelihood that the dmard in question is driving deterioration (e.g. temporal relationship of starting the drug with the clinical scenario). currently there is no observational evidence that preferentially switching between biologics, for instance, helps prolong survival or stabilises the disease; however, this may be tried in individuals that require management of their articular disease following careful discussion. in patients deemed to have progressive, irreversible disease, management of patients with ra-ild in a joint pulmonary and rheumatology clinic should be especially considered where possible, with attempts made at stabilisation. in all patients, and especially in those with multimorbidity, conservative and nonpharmacological options that may be worth exploring include education, psychological support, pulmonary rehabilitation and supplemental oxygen if appropriate [143, 159, 208] . sraes following treatments for ra may be due to incidence/exacerbation of ra-ild or due to other reported pulmonary complications summarised in table 9 .4. careful consideration of the underlying aetiology of sraes is essential, as deterioration in chest symptoms may not be due to pneumonitis or progression of ild, which has been the focus of review in this chapter. a thorough investigation of common and opportunistic infections is imperative in patients with ra, especially on high glucocorticoid doses, certain nbdmards, bdmards and tsdmards. a full review of infection risk and biologics is beyond the scope of this chapter, as has been extensively reviewed recently [209, 210] . rare reports of pulmonary manifestations of certain drugs have also been reported in the literature and are summarised in table 9 .4. for instance, lymphoproliferative disease (including non-hodgkin lymphoma) have been described during treatment with mtx and may regress following cessation [211, 212] . tnfis have been associated with paradoxical aes (associated with the 9 management of the rheumatoid arthritis patient with interstitial lung disease treatment and possible induction of the same event) such as sarcoidosis and vasculitis [48] . the authors recommend a low threshold for withholding treatment with such cases, close liaison with respiratory and infectious disease physicians and imaging using hrct, which in turn may help determine the cause of the deterioration. the respiratory safety of ra therapy is an important consideration whilst deciding on the best treatment for patients with ra with active joint disease and coexisting ild. this chapter summarises the current evidence available of the use of dmards in the context of pulmonary toxicity. however several limitations of the existing literature exist. whilst ra-ild is increasingly recognised, its natural history is still poorly understood. with newer treatments, case reports are helpful to identify a signal for concern, although such case reports are insufficient alone to provide a clear picture of drug toxicity. where observational evidence is available, confounding by indication and channelling to certain treatments may limit robust conclusions. the lack of adequate comparator groups in several retrospective studies may no conclusive data on pneumonitis risk bdmards biologic disease-modifying antirheumatic drugs, dress drug reaction with eosinophilia and systemic symptoms, ild interstitial lung disease, nbdmards non-biologic biologic disease-modifying antirheumatic drugs, tsdmards targeted synthetic disease-modifying antirheumatic drugs further limit inferences drawn from the evidence. it appears that legitimate concerns are associated with several therapies; therefore, involving patients and multidisciplinary teams in such decisions is important. careful discussion of the benefits and harms of treatments is encouraged, although this is made more challenging by the uncertainty of the current safety profile. systematic pre-and post-treatment assessment can help identify the trajectory of patients with ra-ild and enable more effective risk stratification, enabling clinicians and patients to make better-informed decisions. unfortunately at present, there are limited therapeutic options for additional treatment for specifically stabilising or reversing established fibrosis; however, emerging treatments such as novel anti-fibrotics may hold promise. given the current evidence base, the decision to start an dmard in patients with ra who have ild should be based on its potential for improving joint disease, individual patient characteristics, patient education about the known risks and benefits of treatment and multidisciplinary team input. rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative treating rheumatoid arthritis to target: 2014 update of the recommendations of an international task force eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update acute lymphocytic leukemia in children: maintenance therapy with methotrexate administered intermittently acute lung disease associated with low-dose pulse methotrexate therapy in patients with rheumatoid arthritis methotrexate pneumonitis: review of the literature and histopathological findings in nine patients investigation of the chronic pulmonary effects of low-dose oral methotrexate in patients with rheumatoid arthritis: a prospective study incorporating hrct scanning and pulmonary function tests long-term safety of methotrexate monotherapy in patients with rheumatoid arthritis: a systematic literature research how common is methotrexate pneumonitis? a large prospective study investigates methotrexate pneumonitis in rheumatoid arthritis: potential risk factors. four case reports and a review of the literature pulmonary disease during the treatment of rheumatoid arthritis with low dose pulse methotrexate clinical, laboratory, radiographic, and histopathologic features of methotrexate-associated lung injury in patients with rheumatoid arthritis: a multicenter study with literature review methotrexate-induced pulmonary injury: serial ct findings spectrum of cd4 to cd8 t-cell ratios in lymphocytic alveolitis associated with methotrexate-induced pneumonitis bronchoalveolar lavage cell profile in methotrexate induced pneumonitis incidence, prevalence and possible risk factors for pneumonitis in patients with rheumatoid arthritis receiving methotrexate pneumonitis complicating methotrexate therapy for pustular psoriasis methotrexate use and risk of lung disease in psoriasis, psoriatic arthritis, and inflammatory bowel disease: systematic literature review and metaanalysis of randomised controlled trials risk factors for methotrexate-induced lung injury in patients with rheumatoid arthritis: a multicenter, case-control study reducing the risk of methotrexate pneumonitis in rheumatoid arthritis pulmonary function in patients receiving long-term low-dose methotrexate can baseline pulmonary function tests predict pulmonary toxicity in patients receiving methotrexate for rheumatoid arthritis? interstitial lung disease related to rheumatoid arthritis: evolution after treatment interstitial lung disease associated with leflunomide japan deaths spark concerns over arthritis drug risk of interstitial lung disease associated with leflunomide treatment in korean patients with rheumatoid arthritis rheumatoid arthritis complicated with acute interstitial pneumonia induced by leflunomide as an adverse reaction leflunomide-related acute interstitial pneumonia in two patients with rheumatoid arthritis: autopsy findings with a mosaic pattern of acute and organizing diffuse alveolar damage pneumonitis associated with leflunomide: a profile of new zealand and australian reports clinical expression of leflunomide-induced pneumonitis leflunomide use and the risk of interstitial lung disease in rheumatoid arthritis leflunomide-induced interstitial lung disease: prevalence and risk factors in japanese patients with rheumatoid arthritis interstitial lung diseases induced or exacerbated by dmards and biologic agents in rheumatoid arthritis: a systematic literature review hla-a*31:01 and methotrexate-induced interstitial lung disease in japanese rheumatoid arthritis patients: a multidrug hypersensitivity marker? risk of interstitial lung disease associated with leflunomide treatment in korean patients with rheumatoid arthritis factors associated with fatal outcome of leflunomideinduced lung injury in japanese patients with rheumatoid arthritis sulfasalazine: a rare cause of acute eosinophilic pneumonia a case of severe pulmonary infiltration with eosinophilia (pie) syndrome induced by sulphasalazine pulmonary infiltration and eosinophilia associated with sulfasalazine therapy for ulcerative colitis: a case report and review of literature sulphasalazine and lung toxicity dress syndrome in a patient on sulfasalazine for rheumatoid arthritis severe drug hypersensitivity syndrome due to sulphasalazine in patient with rheumatoid arthritis review series: aspects of interstitial lung disease: connective tissue disease-associated interstitial lung disease: how does it differ from ipf? how should the clinical approach differ? interstitial lung disease induced by hydroxychloroquine hydroxychloroquine-induced dress syndrome hydroxychloroquine in children with interstitial (diffuse parenchymal) lung diseases biologics-induced autoimmune diseases autoimmune diseases induced by tnftargeted therapies: analysis of 233 cases role of tumor necrosis factor-alpha in the spontaneous development of pulmonary fibrosis in viable motheaten mutant mice expression and localization of tumor necrosis factoralpha and its mrna in idiopathic pulmonary fibrosis a case of interstitial pneumonia induced by rituximab therapy infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase iii trial. attract study group a comparison of etanercept and methotrexate in patients with early rheumatoid arthritis etanercept versus methotrexate in patients with early rheumatoid arthritis: two-year radiographic and clinical outcomes longterm safety, efficacy, and radiographic outcome with etanercept treatment in patients with early rheumatoid arthritis etanercept treatment in adults with established rheumatoid arthritis: 7 years of clinical experience the premier study: a multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previo adalimumab alone and in combination with disease-modifying antirheumatic drugs for the treatment of rheumatoid arthritis in clinical practice: the research in active rheumatoid arthritis (react) trial golimumab, a human anti-tumor necrosis factor alpha monoclonal antibody, injected subcutaneously every four weeks in methotrexatenaive patients with active rheumatoid arthritis: twenty-four-week results of a phase iii, multicenter, randomized, double-bli golimumab, a human antibody to tumour necrosis factor {alpha} given by monthly subcutaneous injections, in active rheumatoid arthritis despite methotrexate therapy: the go-forward study golimumab in patients with active rheumatoid arthritis who have previous experience with tumour necrosis factor inhibitors: results of a long-term extension of the randomised, double-blind, placebo-controlled go-after study through week 160 golimumab in patients with active rheumatoid arthritis after treatment with tumor necrosis factor α inhibitors: findings with up to five years of treatment in the multicenter, randomized, double-blind, placebo-controlled, phase 3 go-after study certolizumab pegol plus methotrexate is significantly more effective than placebo plus methotrexate in active rheumatoid arthritis: findings of a fifty-two-week, phase iii, multicenter, randomized, double-blind, placebo-controlled, parallel-group study efficacy and safety of certolizumab pegol plus methotrexate in active rheumatoid arthritis: the rapid 2 study. a randomised controlled trial long-term safety and efficacy of certolizumab pegol in combination with methotrexate in the treatment of rheumatoid arthritis: 5-year results from the rapid 1 trial and open-label extension fatal exacerbation of rheumatoid arthritis associated fibrosing alveolitis in patients given infliximab pulmonary complications of infliximab therapy in patients with rheumatoid arthritis interstitial lung disease induced or exacerbated by tnf-targeted therapies: analysis of 122 cases interstitial lung disease following certolizumab pegol a case of certolizumab-induced interstitial lung disease in a patient with rheumatoid arthritis severe interstitial lung disease following treatment with certolizumab pegol: a case report acute exacerbation of rheumatoid interstitial lung disease during the maintenance therapy with certolizumab pegol non-infectious pulmonary complications of newer biological agents for rheumatic diseases-a systematic literature review fatal fibrosing alveolitis with certolizumab infliximab treatment in a patient with rheumatoid arthritis and pulmonary fibrosis clinical response of rheumatoid arthritis-associated pulmonary fibrosis to tumor necrosis factor-alpha inhibition infliximab therapy in pulmonary fibrosis associated with collagen vascular disease rheumatoid arthritis treatment and the risk of severe interstitial lung disease association between anti-tnf-alpha therapy and interstitial lung disease incidence and complications of interstitial lung disease in users of tocilizumab, rituximab, abatacept and anti-tumor necrosis factor α agents, a retrospective cohort study potential risk of tnf inhibitors on the progression of interstitial lung disease in patients with rheumatoid arthritis influence of anti-tnf therapy on mortality in patients with rheumatoid arthritis-associated interstitial lung disease: results from the british society for rheumatology biologics register the role of biosimilars in the treatment of rheumatic diseases a randomised, double-blind, parallel-group study to demonstrate equivalence in efficacy and safety of ct-p13 compared with innovator infliximab when coadministered with methotrexate in patients with active rheumatoid arthritis: the planetra study a phase iii randomized study to evaluate the efficacy and safety of ct-p13 compared with reference infliximab in patients with active rheumatoid arthritis: 54-week results from the planetra study a randomised, double-blind, multicentre, parallel-group, prospective study comparing the pharmacokinetics, safety, and efficacy of ct-p13 and innovator infliximab in patients with ankylosing spondylitis: the planetas study efficacy and safety of ct-p13 (biosimilar infliximab) in patients with rheumatoid arthritis: comparison between switching from reference infliximab to ct-p13 and continuing ct-p13 in the planetra extension study efficacy and safety of switching from reference infliximab to ct-p13 compared with maintenance of ct-p13 in ankylosing spondylitis: 102-week data from the planetas extension study a phase iii randomised, double-blind, parallelgroup study comparing sb4 with etanercept reference product in patients with active rheumatoid arthritis despite methotrexate therapy rituximab-induced lung disease: a systematic literature review rituximab-induced interstitial lung disease rituximab-induced interstitial lung disease: five case reports lung involvement and drug-induced lung disease in patients with rheumatoid arthritis safety and effectiveness of rituximab in patients with rheumatoid arthritis following an inadequate response to 1 prior tumor necrosis factor inhibitor: the reset trial the efficacy and safety of rituximab in patients with active rheumatoid arthritis despite methotrexate treatment: results of a phase iib randomized, double-blind, placebo-controlled, dose-ranging trial efficacy and safety of different doses and retreatment of rituximab: a randomised, placebo-controlled trial in patients who are biological naive with active rheumatoid arthritis and an inadequate response to methotrexate efficacy and safety of retreatment in patients with rheumatoid arthritis with previous inadequate response to tumor necrosis factor inhibitors: results from the sunrise trial efficacy of b-cell-targeted therapy with rituximab in patients with rheumatoid arthritis non-infectious pulmonary toxicity of rituximab: a systematic review safety of abatacept administered intravenously in treatment of rheumatoid arthritis: integrated analyses of up to 8 years of treatment from the abatacept clinical trial program a case of acute respiratory failure in a rheumatoid arthritis patient after the administration of abatacept a case of rheumatoid arthritis complicated with deteriorated interstitial pneumonia after the administration of abatacept refractory rheumatoid arthritis with interstitial lung disease: could abatacept be the answer? abatacept can be used safely for ra patients with interstitial lung disease risk of hospitalised infection in rheumatoid arthritis patients receiving biologics following a previous infection while on treatment with anti-tnf therapy american college of rheumatology guideline for the treatment of rheumatoid arthritis adding tocilizumab or switching to tocilizumab monotherapy in methotrexate inadequate responders: 24-week symptomatic and structural results of a 2-year randomised controlled strategy trial comparison of adding tocilizumab to methotrexate with switching to tocilizumab in patients with rheumatoid arthritis with inadequate response to methotrexate: 52-week results from a prospective, randomised, controlled study (surprise study) inverse effects of interleukin-6 on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs treatment of rheumatoid arthritis with humanized anti-interleukin-6 receptor antibody: a multicenter, double-blind, placebo-controlled trial effect of interleukin-6 receptor inhibition with tocilizumab in patients with rheumatoid arthritis (option study): a double-blind, placebocontrolled, randomised trial study of active controlled tocilizumab monotherapy for rheumatoid arthritis patients with an inadequate response to methotrexate (satori): significant reduction in disease activity and serum vascular endothelial growth factor by il-6 receptor inhibition tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind, controlled phase 4 trial efficacy and safety of subcutaneous tocilizumab versus intravenous tocilizumab in combination with traditional dmards in patients with ra at week 97 (summacta) sarilumab, a fully human monoclonal antibody against il-6rα in patients with rheumatoid arthritis and an inadequate response to methotrexate: efficacy and safety results from the randomised saril-ra-mobility part a trial sarilumab plus methotrexate in patients with active rheumatoid arthritis and inadequate response to methotrexate: results of a phase iii study a case of organizing pneumonia induced by tocilizumab a fatal case of acute exacerbation of interstitial lung disease in a patient with rheumatoid arthritis during treatment with tocilizumab exacerbation of combined pulmonary fibrosis and emphysema syndrome during tocilizumab therapy for rheumatoid arthritis acute pneumonitis in a patient with adult-onset disease after toclizumab treatment with good response to anakinra interstitial lung disease in rheumatoid arthritis: response to il-6r blockade postmarketing surveillance of tocilizumab for rheumatoid arthritis in japan: interim analysis of 3881 patients association of disease activity with acute exacerbation of interstitial lung disease during tocilizumab treatment in patients with rheumatoid arthritis: a retrospective, case-control study recombinant human interleukin-1 receptor type i in the treatment of patients with active rheumatoid arthritis dose-range and dose-frequency study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis. the il-1ra arthritis study group treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate: results of a twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial a randomised trial evaluating anakinra in early active rheumatoid arthritis combination therapy with etanercept and anakinra in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully with methotrexate proposal for a new nomenclature of diseasemodifying antirheumatic drugs baricitinib versus placebo or adalimumab in patients with active rheumatoid arthritis (ra) and an inadequate response to background methotrexate therapy: results of a phase 3 study tofacitinib or adalimumab versus placebo in rheumatoid arthritis cp-690,550) in combination with methotrexate in patients with active rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitors: a randomised phase 3 trial cp-690,550) in patients with rheumatoid arthritis receiving methotrexate: twelve-month data from a twenty-four-month phase iii randomized radiographic study placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis safety and efficacy of tofacitinib, an oral janus kinase inhibitor, for the treatment of rheumatoid arthritis in open-label, longterm extension studies efficacy and safety of tofacitinib in the treatment of rheumatoid arthritis: a systematic review and meta-analysis safety and efficacy of baricitinib at 24 weeks in patients with rheumatoid arthritis who have had an inadequate response to methotrexate baricitinib in patients with refractory rheumatoid arthritis risk evaluation and mitigation strategy (rems) nda 203 usual interstitial pneumonia in rheumatoid arthritisassociated interstitial lung disease rheumatoid arthritis and lung disease: from mechanisms to a practical approach an official ats/ers/jrs/alat clinical practice guideline: treatment of idiopathic pulmonary fibrosis: an update of the 2011 clinical practice guideline the lung disease of rheumatoid arthritis azathioprine combined with prednisone in the treatment of idiopathic pulmonary fibrosis: a prospective double-blind, randomized, placebo-controlled clinical trial prednisone, azathioprine, and n-acetylcysteine for pulmonary fibrosis risk of serious infection in patients with rheumatoid arthritis-associated interstitial lung disease immediate and delayed impact of oral glucocorticoid therapy on risk of serious infection in older patients with rheumatoid arthritis: a nested case-control analysis pirfenidone in patients with idiopathic pulmonary fibrosis (capacity): two randomised trials pirfenidone in idiopathic pulmonary fibrosis pirfenidone for idiopathic pulmonary fibrosis: analysis of pooled data from three multinational phase 3 trials open-label compassionate use one year-treatment with pirfenidone to patients with chronic pulmonary fibrosis clinical experience with pirfenidone in five patients with scleroderma-related interstitial lung disease improved pulmonary function following pirfenidone treatment in a patient with progressive interstitial lung disease associated with systemic sclerosis a case report: the efficacy of pirfenidone in a chinese patient with progressive systemic sclerosis-associated interstitial lung disease: a care-compliant article an open-label, phase ii study of the safety and tolerability of pirfenidone in patients with scleroderma-associated interstitial lung disease: the lotuss trial is pirfenidone ready for use in non-idiopathic pulmonary fibrosis interstitial lung diseases? treatment of rheumatoid arthritis-associated interstitial lung disease: a perspective review phase ll study of pirfenidone in patients with ra-ild. clinicaltrials.gov identifier: nct02808871 nintedanib inhibits fibroblast activation and ameliorates fibrosis in preclinical models of systemic sclerosis combined corticosteroid and cyclophosphamide therapy does not alter survival in idiopathic pulmonary fibrosis cyclophosphamide for scleroderma lung disease cyclophosphamide versus placebo in scleroderma lung disease a multicenter, prospective, randomized, doubleblind, placebo-controlled trial of corticosteroids and intravenous cyclophosphamide followed by oral azathioprine for the treatment of pulmonary fibrosis in scleroderma predicting treatment outcomes and responder subsets in scleroderma-related interstitial lung disease effects of cyclophosphamide on pulmonary function in patients with scleroderma and interstitial lung disease: a systematic review and meta-analysis of randomized controlled trials and observational prospective cohort studies efficacy of intensive immunosuppression in exacerbated rheumatoid arthritis-associated interstitial lung disease rheumatoid arthritis associated with methotrexateinduced pneumonitis: improvement with i.v. cyclophosphamide therapy cyclosporin as a treatment for interstitial lung disease of unknown aetiology progression of fibrosis in usual interstitial pneumonia: serial evaluation of the native lung after single lung transplantation cyclosporine in anti-jo1-positive patients with corticosteroid-refractory interstitial lung disease the efficacy of calcineurin inhibitors for the treatment of interstitial lung disease associated with polymyositis/dermatomyositis survival benefit associated with early cyclosporine treatment for dermatomyositis-associated interstitial lung disease experience of mycophenolate mofetil in 10 patients with autoimmune-related interstitial lung disease demonstrates promising effects a prospective observational study of mycophenolate mofetil treatment in progressive diffuse cutaneous systemic sclerosis of recent onset cyclophosphamide versus mycophenolate mofetil in scleroderma interstitial lung disease (ssc-ild) as induction therapy: a singlecentre, retrospective analysis mycophenolate mofetil improves lung function in connective tissue disease-associated interstitial lung disease mycophenolate mofetil versus oral cyclophosphamide in scleroderma-related interstitial lung disease (sls ii): a randomised controlled, double-blind, parallel group trial mycophenolate mofetil is safe, well tolerated, and preserves lung function in patients with connective tissue disease-related interstitial lung disease b-cell depletion salvage therapy in rapidly progressive dermatomyositis related interstitial lung disease successful experience of rituximab therapy for systemic sclerosis-associated interstitial lung disease with concomitant systemic lupus erythematosus severe interstitial lung disease in connective tissue disease: rituximab as rescue therapy long-term efficacy of b cell depletion therapy on lung and skin involvement in diffuse systemic sclerosis open-label, pilot study of the safety and clinical effects of rituximab in patients with rheumatoid arthritis associated interstitial pneumonia safety of rituximab in patients with rheumatoid arthritis and concomitant lung disease efficacy and safety of rituximab in rheumatoid arthritis patients with concomitant interstitial lung disease: 10-year experience at single centre safety and efficacy of rituximab in patients with rheumatoid arthritis and lung involvement mortality in patients with rheumatoid arthritis and interstitial lung disease treated with first line tnfi or rituximab therapies: results from the bsrbr-ra updated consensus statement on the use of rituximab in patients with rheumatoid arthritis survival and quality of life in rheumatoid arthritisassociated interstitial lung disease after lung transplantation pragmatic prognostic approach of rheumatoid arthritis-associated interstitial lung disease an official american thoracic society/european respiratory society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias prevalence and prognosis of unclassifiable interstitial lung disease lung function estimates in idiopathic pulmonary fibrosis: the potential for a simple classification a multidimensional index and staging system for idiopathic pulmonary fibrosis the mrc breathlessness scale an official ats/ers/jrs/alat statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management a clinician's guide to the diagnosis and treatment of interstitial lung diseases fibrotic idiopathic interstitial pneumonia: the prognostic value of longitudinal functional trends rheumatoid arthritis-associated interstitial lung disease: diagnostic dilemma radiological versus histological diagnosis in uip and nsip: survival implications interstitial lung disease in systemic sclerosis: a simple staging system update in diffuse parenchymal lung disease patients with limited rheumatoid arthritis-related interstitial lung disease have a better prognosis than those with extensive disease the safety of biologic therapies in ra-associated interstitial lung disease patients with early rheumatoid arthritis who smoke are less likely to respond to treatment with methotrexate and tumor necrosis factor inhibitors: observations from the epidemiological investigation of rheumatoid arthritis and the swedish rheumatology register cohorts rheumatoid arthritis (ra) associated interstitial lung disease (ild) opportunistic infections and biologic therapies in immune-mediated inflammatory diseases: consensus recommendations for infection reporting during clinical trials and postmarketing surveillance risk of infection with biologic antirheumatic therapies in patients with rheumatoid arthritis lymphoproliferative disorders in rheumatoid arthritis: clinicopathological analysis of 76 cases in relation to methotrexate medication spontaneous remission of 'methotrexate-associated lymphoproliferative disorders' after discontinuation of immunosuppressive treatment for autoimmune disease. review of the literature validated dbs method for filgotinib quantitation in rat dried blood spots and its application to a pharmacokinetic study in rats doi: http://dx.doi.org/10.5599/admet.796 139 admet & dmpk 8(2) (2020) 139-148; doi: http://dx.doi.org/10.5599/admet.796 open access : issn : 1848-7718 http://www.pub.iapchem.org/ojs/index.php/admet/index original scientific paper validated dbs method for filgotinib quantitation in rat dried blood spots and its application to a pharmacokinetic study in rats abhishek dixit, vinay kiran, bhavesh babulal gabani, ramesh mullangi* drug metabolism and pharmacokinetics, jubilant biosys ltd, industrial suburb, yeshwanthpur, bangalore -560 022, india *corresponding author: e-mail: mullangi.ramesh@jubilantinnovation.com; tel.: +91-80-66628339 received: february 24, 2020; revised: april 23, 2020; published online: may 06, 2020 abstract filgotinib is a selective jak1 (janus kinase) inhibitor, showed efficacy in patients suffering from moderateto-severe rheumatoid arthritis. in this paper, we present the data on the development and validation of a sensitive, selective and high-throughput lc-ms/ms (liquid chromatography with tandem mass spectrometry) method for the quantitation of filgotinib from rat dried blood spot (dbs) cards. to the dbs disc cards, 0.2 % formic acid enriched with internal standard (is) was added and sonicated. thereafter the extraction of filgotinib and the is (tofacitinib) was accomplished using ethyl acetate as an extraction solvent. the resolution of filgotinib and the is was achieved on a gemini c18 column with an isocratic mobile phase, which is a mixture of 0.2 % formic acid:acetonitrile (20:80, v/v) at a flow-rate of 0.9 ml/min. the total run time was 2.90 min and the retention time of filgotinib and the is was ~1.31 and 0.89 min, respectively. filgotinib and the is were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 426.3291.3 and m/z 313.2149.2, respectively, for quantitation. the calibration range was 1.37-1937 ng/ml. no matrix effect and carry over were observed. all the validation parameters met the acceptance criteria. the validated method has been applied to a pharmacokinetic study in rats. a good correlation between dbs and plasma concentrations for filgotinib was observed. ©2020 by the authors. this article is an open-access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/). keywords filgotinib; lc-ms/ms; method validation; rat blood; dbs; pharmacokinetics introduction rheumatoid arthritis is a chronic autoimmune inflammatory disease with a prevalence of 1-2 % of the world population. rheumatoid arthritis primarily affects peripheral joints and subsequently damages the synovial tissue and cartilage damage [1]. although disease-modifying anti-rheumatic drugs (dmards: methotrexate, sulfasalazine, leflunomide etc) and biologic dmards (etanercept, adalimumab etc.) are available as first-line drugs due to their low therapeutic benefit and severe side effects they cannot be used for long-treatment [2]. to overcome these drawbacks, janus kinase (jak)/signal transducer and activator of the transcription (stat) signal pathway has been identified as one of the new therapeutic targets to treat rheumatoid arthritis. jak-stat pathway plays a critical role in the downstream signaling of cytokines. the http://dx.doi.org/10.5599/admet.796 http://dx.doi.org/10.5599/admet.796 http://www.pub.iapchem.org/ojs/index.php/admet/index mailto:mullangi.ramesh@jubilantinnovation.com http://creativecommons.org/licenses/by/4.0/ r. mullangi et al. admet & dmpk 8(2) (2020) 139-148 140 inhibition of jaks is an attractive therapeutic target to treat rheumatoid arthritis [3]. tofacitinib and baricitinib are the pan-jak inhibitors (jak1/jak3) approved for the treatment of moderate to severe rheumatoid arthritis but they showed dose-related toxicities [4]. recent findings suggest that selective inhibition of jak1 might reduce the toxicity without a significant detriment to efficacy [2,3]. filgotinib (figure 1; glpg0634), is a selective jak1 inhibitor (ic50: 629 nm) with 30-fold selectivity over jak2 [5]. it has shown dose-dependent reduction of disease progression in a collagen-induced arthritis model post oral administration in rodents [5]. in phase-3 clinical trials, filgotinib was well tolerated, showed efficacy and was found to be safe in rheumatoid arthritis patients, when it was administered as monotherapy at 100 or 200 mg, once daily or with methotrexate [6]. filgotinib is currently being filed in the us and japan to treat the patients suffering from moderate-to-severe rheumatoid arthritis [7,8]. filgotinib tofacitinib (is; internal standard) figure 1. structural representation of filgotinib and tofacitinib (is). to date, two lc-ms/ms (liquid chromatography with tandem mass spectrometry) methods were reported for the quantification of filgotinib. the first method, reported by namuor et al. (2015) employed solid-phase extraction method for the quantitation of filgotinib and its active metabolite from phase-i study plasma samples [100 µl plasma sample was spiked with 20 µl of deuterated filgotinib (125 ng/ml) as an internal standard]. the reported lower limit of quantification (lloq) was 3.00 ng/ml. however, full details on chromatography, mass spectrometer conditions and validation parameters were not reported [9]. very recently, dixit et al. (2020) reported a validated lc-ms/ms method for the quantification of filgotinib. the authors have attained an lloq of 0.78 ng/ml with 50 µl rat plasma. plasma samples were processed using ethyl acetate as an extraction solvent [10]. dried blood spot (dbs) methodology has seen significant progress during recent times in the quantitative analysis of various drugs [11-16]. dbs offers several advantages over traditional sampling (plasma/blood/serum) techniques such as reduction of commercial costs for laboratory equipment, convenience in collection, reduction in collection of blood volume, no requirements for trained phlebotomist, ease of sample handling/storage/shipping, safety in handling, less time in processing and increase in throughput etc. it is anticipated that much clinical development and therapeutic drug monitoring programs in the future may switch to dbs technique to characterize the pharmacokinetic data. to the best of our knowledge, there is no dbs method reported for the quantitation of filgotinib. in this paper, we report the development and validation of an lc-ms/ms method for the quantitation of filgotinib on rat dbs. the applicability of the validated method was shown in a rat pharmacokinetic study. excellent correlation was observed between dbs versus plasma filgotinib concentrations, indicating that the dbs method can be used as an alternative for plasma sampling for pharmacokinetic analysis. admet & dmpk 8(2) (2020) 139-148 dbs method for filgotinib quantitation doi: http://dx.doi.org/10.5599/admet.796 141 materials and methods materials filgotinib (purity: >95 %) was purchased from angene international limited, tsuen wan, hong kong. tofacitinib (is; purity: 98 %) was purchased from sigma-aldrich (st. louis, usa). hplc grade acetonitrile and methanol were purchased from j.t. baker, pa, usa. analytical grade formic acid was purchased from s.d fine chemicals, mumbai, india. all other chemicals and reagents were of analytical grade and used without further purification. the control sprague dawley rat blood was procured from animal house, jubilant biosys, bangalore. liquid chromatography and mass spectrometry conditions we used the similar chromatographic conditions that we previously reported [10]. in brief, a gemini c18 (100 × 4.6 mm, 3 µm) column maintained at ambient room temperature along with an isocratic mobile phase (0.2 % formic acid in milli-q water:acetonitrile, 20:80, v/v) at a flow-rate of 0.9 ml/min was used for separation of filgotinib and the is. the injection volume was 5.0 µl. under these optimized conditions the retention times were 1.31 and 0.89 min, for filgotinib and the is, respectively with a total run time of 2.90 min. the lc was coupled to a sciex 4000 mass spectrometer controlled by sciex analyst 1.6.2 software. the ms was equipped with electro-spray ionization and operated in the multiple reaction monitoring (mrm) mode for the quantitation. ionization was conducted by applying a voltage of 5500 v, and the source temperature was set at 550 °c. the gas settings were as follows curtain gas: 35 psi, gs1: 50 psi, gs2: 55 psi and cad: 8.0 psi. the compound parameters declustering potential, entrance potential, collision energy, and collision cell exit potential were set at 91, 10, 40, and 10 v for filgotinib and 100, 10, 41, and 12 v for the is. the mass transitions m/z (precursor ionproduct ion) 426.3291.3 and 313.2149.2 were monitored for filgotinib and the is, respectively. quadrupole q1 and q3 were set on the unit resolution. dwell time was 150 ms. preparation of stock solutions and standard samples two separate primary stock solutions of filgotinib were prepared at 1670 g/ml in dmso. appropriate secondary and working stocks of filgotinib were prepared from primary stock by successive dilution of primary stock with methanol:water (80:20, v/v) to prepare the calibration curve (cc) and quality controls (qcs). the is primary stock solution was made in dmso at a concentration of 1000 µg/ml, which was diluted with methanol to 500 ng/ml and subsequently used as is working stock solution. the primary stock solutions of filgotinib and the is were stored at -20 °c, which were found to be stable for 50 days. working stock solutions were stored at 4 °c for 25 days. blood spotting freshly drawn rat blood was used to prepare the dbs cards. with the help of a calibrated pipette, 25 μl of the respective spiked cc/qc blood or whole blood collected from the pharmacokinetic study (post administration of filgotinib by intravenous and oral routes) was sampled on dbs card. spiked cards were allowed to dry at ambient room temperature for 3 h and stored appropriately in a sealed bag with desiccant in a desiccator. dbs homogeneity the spot homogeneity was evaluated by punching out the disc from the periphery of the dbs. blood spots at qc low and qc high level were prepared in triplicate. the obtained dbs discs were processed and http://dx.doi.org/10.5599/admet.796 r. mullangi et al. admet & dmpk 8(2) (2020) 139-148 142 analyzed as described in the sample preparation section. dbs sample preparations using a hole puncher (harri-micro-punch®), 3 mm diameter disc was punched from the center of each dbs (fta dmpk-c card) card directly into micro-centrifuge tubes. to each micro-centrifuge tube, 200 µl of 0.2 % formic acid enriched with 100 ng/ml of is was added, thereafter the contents were vortex mixed for 5 min (thermomixer ® , eppendorf) and sonicated (elmasonic s300 h) for 15 min at ambient room temperature. after sonication, to the same micro-centrifuge, 1.0 ml of ethyl acetate was added and the mixtures were vortexed further for 3 min. the samples were centrifuged at 14,000 rpm for 3 min. clear organic layer (800 µl) was pipetted out after centrifugation and dried under gentle stream of nitrogen (turbovap ® , zymark ® ,kopkinton, ma, usa) at 50 °c. the residue was reconstituted with 200 µl of 0.2 % formic acid in acetonitrile and 150 µl clear supernatant was aliquoted into a hplc vial and 5.0 µl was injected onto the column for lc-ms/ms analysis. validation procedure the validation experiments were performed in accordance with the us food and drug administration guideline [17]. various parameters covered under validation are: selectivity, carry over (repetitive punching and auto-injector), recovery, matrix effect, linearity, precision, accuracy, stability (in-injector, 6 h at room temperature and 30-day long-term at -80 ± 10 °c) and incurred sample reanalysis. influence of hematocrit two different hematocrit (hct) blood samples at 30 and 50 % were prepared at lqc and hqc by adding or removing the plasma to the rat blood. these samples (in quadruplicate at each qc) were analysed with calibrators prepared in blood at standard fixed 40 % hct. the relative error of ±15 % and precision of ±15 % was considered acceptable. hct value in rats ranged between 38-46 % (in-house data; measured using mindray bc-5000vet) and it is in line with reported values [18]. pharmacokinetic study in rats all the animal experiments were approved by institutional animal ethical committee (iaec/jdc/2019/189r). male sprague dawley rats (n=8) were procured from vivo biotech, hyderabad, india. the animals were housed in jubilant biosys animal house facility in a temperature (22 ± 2 °c) and humidity (30-70 %) controlled room (15 air changes/hour) with a 12:12 h light:dark cycles, had free access to rodent feed (altromin spezialfutter gmbh & co. kg., im seelenkamp 20, d-32791, lage, germany) and water for one week before using for experimental purpose. following ~12 h fast (during the fasting period animals had free access to water) animals were divided into two groups having four rats in each group. group i animals (205-210) received filgotinib orally as a suspension formulation (prepared using tween-80 and 0.5 % methyl cellulose) at 10 mg/kg (strength: 1.0 mg/ml; dose volume: 10 ml/kg), whereas group ii animals (215-225 g) received filgotinib intravenously (5 % dmso, 5 % solutol:absolute alcohol (1:1, v/v) and 90 % of normal saline; strength: 0.5 mg/ml; dose volume: 4 ml/kg) at 2.0 mg/kg dose. post-dosing serial blood samples (50 µl) were collected through retro-orbital plexus into polypropylene tubes containing k2.edta solution as an anti-coagulant at 0.25, 0.5, 1, 2, 4, 8, 10, 12 and 24 h (for oral study) and 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h (for intravenous study). animals were allowed to access feed 2 h post-dosing. pharmacokinetic analysis blood concentration-time data of filgotinib was analyzed by non-compartmental method and the admet & dmpk 8(2) (2020) 139-148 dbs method for filgotinib quantitation doi: http://dx.doi.org/10.5599/admet.796 143 relevant pharmacokinetic parameters namely auc0- (area under the blood concentration-time curve from time zero to infinite time), c0 (extrapolated blood concentration at time zero), cmax (maximum blood concentration), tmax (time to reach cmax), vd (volume of distribution), cl (clearance) and t½ (terminal halflife) were calculated using phoenix winnonlin software (version 8.1; pharsight corporation, mountain view, ca). absolute oral bioavailability (f) was calculated using this formula [dose (i.v.) × auc(0-)oral / dose (oral) × auc(0-)i.v.] × 100. results and discussion method development the objective of this work was to develop a sensitive and rugged dbs method for the quantification of filgotinib in rat blood and subsequently show the correlation with previously developed method [10] in a pharmacokinetics study so that dbs method can be used as an alternative method to measure the filgotinib concentrations. previously reported lc-ms/ms conditions were utilized for this method [10]. since chromatography and mass spectrometric conditions were optimized, to obtain better sensitivity extraction optimization was done with various extraction solvents namely acetonitrile, methanol, ethyl acetate, acidified methanol/acetonitrile, mixture of water and methanol/acetonitrile, pre-treatment of dbs discs with formic acid followed by extraction with methanol/acetonitrile/ethyl acetate. method validation parameters recovery none of the single organic solvents and pure water gave good recovery of filgotinib from the dbs discs. the recovery with ethyl acetate, water and methanol/acetonitrile was 5, 39 and 43 %, respectively. with methanol/acetonitrile:water 50:50 and 20:80, the recovery was found to be 30 and 43 %, respectively. in order to improve the recovery, the dbs discs were pre-treated with 0.2 % formic acid and extracted with ethyl acetate. this process helped to attain the recovery up to 62 %. the mean ± s.d recovery of filgotinib at lqc and hqc was found to be 62.4 ± 8.39 and 64.2 ± 4.26 %, respectively. the recovery of the is was 95.2 ± 4.85 %. matrix effect the mean absolute matrix effect for filgotinib on rat dbs cards at lqc and hqc was found to be 0.81 ± 0.08 and 0.83 ± 0.08 %, respectively. the matrix effect for the is was 0.97 ± 0.05 % (at 100 ng/ml). these results indicate that the minimal matrix effect did not obscure the quantification of on filgotinib and the is from rat dbs cards. selectivity and carry over figures 2a,b,c show chromatograms for the blank rat dbs cards (free of analyte and the is; figure 2a), blank rat dbs cards spiked with filgotinib at lloq and the is (figure 2b) and an in vivo blood sample obtained at 0.25 h after oral administration of filgotinib along with the is (figure 2c). there was no carryover produced by the highest calibration sample on the following injected blank dbs extract sample. additionally, no dbs-specific device-oriented carry-over was noted. http://dx.doi.org/10.5599/admet.796 r. mullangi et al. admet & dmpk 8(2) (2020) 139-148 144 a) b) c) figure 2. typical mrm chromatograms of filgotinib (left panel) and the is (right panel) in (a) rat blank dbs card (b) rat blank dbs card spiked with filgotinib at lloq (1.37 ng/ml) and the is (c) a 0.25 h in vivo plasma sample showing filgotinib peak obtained following oral administration to rat along with the is. d if fe r e n c e ( % ) 1 10 100 1000 10000 -20 -10 0 10 20 mean val ue (ng/ml )-l og scal e figure 3. bland-altman plot showing the incurred sample re-analysis data for filgotinib on dbs. calibration curve the calibration curve was constructed in the linear range using eight calibrators 1.34, 2.67, 14.7, 26.7, 400, 801, 1603 and 1937 ng/ml. the typical regression equation for calibration curve was y = 0.000849 x + 0.00127. the correlation coefficient (r) average regression (n=4) was found to be >0.9966 for filgotinib. the lowest concentration with the rsd <20 % was taken as lloq and was found to be 1.37 ng/ml. the accuracy observed for the mean of back-calculated concentrations for four calibration curves for filgotinib was within 95.9-110 %; while the precision (cv) values ranged from 3.87-7.94 %. admet & dmpk 8(2) (2020) 139-148 dbs method for filgotinib quantitation doi: http://dx.doi.org/10.5599/admet.796 145 accuracy and precision accuracy and precision data for intraand inter-day dbs samples for filgotinib is presented in table 1. the assay values on both the occasions (intraand inter-day) were found to be within the accepted variable limits. stability the predicted concentrations for filgotinib at 4.01 and 1736 ng/ml samples deviated within ±15 % of the fresh sample concentrations in a battery of stability tests: bench-top (6 h), in-injector (21 h) and freezer stability at -80  10 °c for at least for 30 days (table 2). the results were found to be within the assay variability limits during the entire process. table 1. precision and accuracy determination of filgotinib quality controls in rat plasma lloq qc (1.37 ng/ml) lqc (4.01 ng/ml) mqc (1068 ng/ml) hqc (1736 ng/ml) intra-day (n=6) mean ± s.d 1.34 ± 0.10 4.30 ± 0.45 1136 ± 73.3 1715 ± 124 precision (%rsd) 7.38 10.4 6.46 7.23 accuracy (%re) 1.00 1.07 1.06 0.99 inter-day (n=24) mean ± s.d 1.33 ± 0.15 3.94 ± 0.44 1064 ± 96.8 1663 ± 128 precision (%rsd) 11.0 11.2 9.09 7.71 accuracy (%re) 1.00 0.98 1.00 0.96 rsd: relative standard deviation (sd 100/mean) re: relative error (measured value/actual value) table 2. stability data of filgotinib quality controls in rat plasma rsd: relative standard deviation (sd 100/mean) re: relative error (measured value/actual value) incurred samples reanalysis (isr) as per guidance [17] around 10 % of the study samples should be selected for isr if the total sample size is less than 1000. in this validation a total of 20 samples were chosen for isr from the oral and intravenous rat pharmacokinetic studies. from oral arm samples near cmax (0.5 h) and elimination phase (4 h and 24 h) were selected. however, for intravenous arm representative samples at 0.083, 2 and 8 h time points were selected. figure 3 shows the comparison of isr values versus original values using blandaltman plot suggesting that all the isr samples were within ±20 % of the original values. concentration spiked (ng/ml) bench-top for 6 h (n=6) long-term 30 days at -80 °c (n=6) in-injector for 21 h (n=6) % re %rsd % re %rsd % re %rsd 4.01 0.96 11.1 0.97 15.1 0.91 5.99 1736 0.88 4.09 0.96 6.04 0.88 2.39 http://dx.doi.org/10.5599/admet.796 r. mullangi et al. admet & dmpk 8(2) (2020) 139-148 146 hematocrit effect hematocrit (hct) has a significant effect on the viscosity of the blood, which influences the flux and diffusion properties of the blood through dbs card used for sample collection. it can directly affect the accuracy of the analysis in dbs samples. the reported blood-to-plasma ratio for filgotinib was 1.22 [19] indicating an almost equal plasma and red blood cell distribution. the measured filgotinib concentrations were compared with the results obtained from dbs samples are given in table 3, indicating that hct had no significant influence on the filgotinib concentration. table 3. haematocrit effect analysis concentration spiked (ng/ml) hematocrit (%) concentration found (ng/ml) accuracy (%re) precision (% rsd) 4.01 30 3.95 ± 0.19 0.98 4.81 50 3.90 ± 0.24 0.97 6.29 1736 30 1664 ± 80.4 0.95 4.83 50 1786 ± 88.7 1.02 4.96 0 2 4 6 8 10 12 14 16 18 20 22 24 1 10 100 1000 10000 time (h) m e a n ± s .d (n g /m l ) c o n c e n tr a ti o n o f fi lg o ti n ib iv 2 mg/kg plasma iv 2 mg/kg dbs 0 2 4 6 8 10 12 14 16 18 20 22 24 1 10 100 1000 10000 time (h) m e a n ± s .d (n g /m l ) c o n c e n tr a ti o n o f fi lg o ti n ib po 10 mg/kg plasma po 10 mg/kg dbs figure 4. a) mean plasma concentration-time profiles of filgotinib in rat blood and plasma following intravenous administration of filgotinib; b) mean plasma concentration-time profiles of filgotinib in rat blood and plasma following oral administration of filgotinib. pharmacokinetic study the sensitivity of the present dbs method was found to be sufficient for accurately characterizing the pharmacokinetics of filgotinib by oral and intravenous routes in rats. to assure acceptance of study sample analytical runs, at least two-thirds of the qc samples had to be within ±15 % accuracy, with at least half of the qc samples at each concentration meeting these criteria. results indicated that qcs met the acceptance criteria. figures 4a and 4b show the mean blood concentration versus time for filgotinib post administration of intravenous and oral route, respectively in rats. the pharmacokinetic parameters are summarized in table 4 along with previously reported pharmacokinetic parameters by dixit et al. [10]. filgotinib was quantifiable up to 24 h post intravenous and oral administration to rats. following intravenous administration at 2.0 mg/kg, the plasma concentrations decreased mono-exponentially. filgotinib exhibited moderate clearance (cl) of 18.7 ± 1.86 ml/min/kg, which is ~3-fold lower than hepatic blood flow (55 ml/min/kg) and volume of distribution (vd) was 4.17 ± 0.33 l/kg in rats. the terminal halflife was found to be 3.52 ± 0.23 h. following oral administration filgotinib maximum plasma concentrations (cmax: 804 ± 78.4 ng/ml) attained at 0.50 ± 0.00 h (tmax) in all rats suggesting that filgotinib has a rapid a) b) admet & dmpk 8(2) (2020) 139-148 dbs method for filgotinib quantitation doi: http://dx.doi.org/10.5599/admet.796 147 absorption from the gastrointestinal tract. the auc0- (area under the plasma concentration-time curve from time zero to infinity) was found to be 3722 ± 329 and 1801 ± 188 ngh/ml, by oral and intravenous routes, respectively. the terminal half-life (t½) determined after oral administration was 4.87 ± 0.45 h. the absolute oral bioavailability for filgotinib in rats at 10 mg/kg was 42.5 %. table 4. pharmacokinetic parameters for filgotinib in rats following oral and intravenous administration pk parameters oral intravenous dose (mg/kg) 10 2.0 auc(0-) (ng  h/ml) 3722 ± 329 (3475 ± 547) 1801 ± 188 (1912 ± 123) cmax/c0 (ng/ml) 804 ± 78.4 (802 ± 149) 1602 ± 55.5 (1767 ± 128) tmax (h) 0.50 ± 0.00 (0.50 ± 0.00) -- t1/2 (h) 4.87 ± 0.45 (4.72 ± 0.24) 3.52 ± 0.23 (3.56 ± 0.30) cl (ml/min/kg) -- 18.7 ± 1.86 (17.4 ± 1.08) vd (l/kg) -- 4.17 ± 0.33 (5.41 ± 0.77) f (%) 42.5 (36.3) -- the values in parentheses are reported by dixit et al. (2020) [10]. in figures 4a and 4b the blood versus time concentration profiles of filgotinib obtained from intravenous and oral routes in rats were presented along with plasma versus time concentration profiles reported earlier by us [10] showed excellent correlation (r 2 > 0.99). this indicates that dbs can be used as an alternative strategy for determination of filgotinib circulatory concentration in a pharmacokinetic study. to the best of our knowledge, to date there is no comparison between plasma and whole blood for filgotinib. our study demonstrated that dbs is a promising alternative to plasma sampling for filgotinib in rheumatoid arthritis patients especially with elderly patients in remote or resource-limited settings or immobile patients. conclusions in summary, a simple and rapid method using lc-ms/ms for the determination of filgotinib in rat blood using dbs cards was developed and validated as per us fda regulatory guideline. the validated method suitability was shown in a rat pharmacokinetic study. conflict of interest: the authors are scientists at jubilant biosys ltd. references [1] j.k. buer. a history of the term dmard. inflammopharmacology 23 (2015) 163-171. [2] c. chough, s. lee, m. joung, j. lee, j.h. kim, b.m. kim. design, synthesis and evaluation of (r)-3-(7(methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-5-aza-spiro[2.4]heptan-5-yl)-3-oxopropanenitrile as a jak1-selective inhibitor. medchemcomm 9 (2018) 477. [3] j.j. o’shea, m. gadina. (2019). selective janus kinase inhibitors come of age. nature reviews rheumatology 15 (2019) 74-75. [4] k. yamaoka. janus kinase inhibitors for rheumatoid arthritis. current opinion in chemical biology 32 (2016) 29-33. http://dx.doi.org/10.5599/admet.796 r. mullangi et al. admet & dmpk 8(2) (2020) 139-148 148 [5] l. van rompaey, r. galien, e. m. van der aar, p. clement-lacroix, l. nelles, b. smets, l. lepescheux, t. christophe, k. conrath, n. vandeghinste, b. vayssiere, s. de vos, s. fletcher, r. brys, g. van't klooster, j. h. m. feyen and c.menet. preclinical characterization of glpg0634, a selective inhibitor of jak1, for the treatment of inflammatory diseases. journal of immunology 191 (2013) 3568-3577. [6] m. genovese, r. westhovens, l. meuleners, a. van der aa, p. harrison, c. tasset, a. kavanaugh. effect of filgotinib, a selective jak 1 inhibitor, with and without methotrexate in patients with rheumatoid arthritis: patient-reported outcomes. arthritis research & therapy 20 (2018) 57. [7] pharma japan (2019). https://pj.jiho.jp/article/240785. [8] https://www.gilead.com/news-and-press/press-room/press-releases (accessed may 01, 2020.) [9] f. namour, p.m. diderichsen, e. cox, b. vayssie`re, a.v. aa, c. tasset, v.g. klooster. pharmacokinetics and pharmacokinetic/pharmacodynamic modeling of filgotinib (glpg0634), a selective jak1 inhibitor, in support of phase iib dose selection. clinical pharmacokinetics 54 (2015) 859-874. [10] a. dixit, v. kiran, b.b. gabani, z. mohd, r.r. trivedi, r. mullangi. validated lc-ms/ms method for quantitation of a selective jak1 inhibitor, filgotinib in rat plasma and its application to a pharmacokinetics study in rats. biomedical chromatography (2020) accepted. [11] sulochana sp, daram p, srinivas nr, mullangi r. review of dbs methods as a quantitative tool for anticancer drugs. biomedical chromatography 33 (2019) e4445. [12] k.i. foerster, a. huppertz, a.d. meid, o.j. müller, t. rizos, l. tilemann, w.e. haefeli, j. burhenne. dried blood spot technique to monitor direct oral anticoagulants: clinical validation of an uplc/ms/ms based assay. analytical chemistry 90 (2018) 337-341. [13] h.m. kim, j. park, n.p. long, d.d. kim, s.w. kwon. simultaneous determination of cardiovascular drugs in dried blood spot by liquid chromatography-tandem mass spectrometry. journal of food and drug analysis 27 (2019) 906-914. [14] j. gallaya, s. prod’hom, t. mercierc, c. bardinet, d. spaggiari, e. pothina, t. buclinc, b. gentona, l.a. decosterd. lc-ms/ms method for the simultaneous analysis of seven antimalarials and two active metabolites in dried blood spots for applications in field trials: analytical and clinical validation. journal of pharmaceutical and biomedical analysis 154 (2018) 263-277. [15] m. moretti, f. freni, b. valentini, c. vignali, a. groppi, s.d visonà, a.m.m. osculati, l. morini. determination of antidepressants and antipsychotics in dried blood spots (dbss) collected from post-mortem samples and evaluation of the stability over a three-month period. molecules 24 (2019) pii: e3636. [16] s. velghe, s. deprez, c.p. stove. fully automated therapeutic drug monitoring of anti-epileptic drugs making use of dried blood spots. journal of chromatography a 1601 (2019) 95-103. [17] dhhs, fda, cder, & cvm (2018). guidance for industry: bioanalytical method validation. rockville, md: us department of health and human services, food and drug administration, center for drug evaluation and research and center for veterinary medicine. [18] m. o’mara, b. hudson-curtis, k. olson, y. yueh, j. dunn, n. spooner. the effect of hematocrit and punch location on assay bias during quantitative bioanalysis of dried blood spot samples. bioanalysis 20 (2011) 2335-2347. [19] m.e. dowty, t.h. lin, m.i. jesson, m. hegen, d.a. martin, v. katkade, s. menon, j.b. telliez. janus kinase inhibitors for the treatment of rheumatoid arthritis demonstrate similar profiles of in vitro cytokine receptor inhibition. pharmacology research & perspectives (2019) e00537. ©2020 by the authors; licensee iapc, zagreb, croatia. this article is an open-access article distributed under the terms and conditions of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/) https://pj.jiho.jp/article/240785 https://www.gilead.com/news-and-press/press-room/press-releases http://creativecommons.org/licenses/by/3.0/ key: cord-0071358-8v1oemlt authors: shakeel, faiyaz; alam, prawez; ali, abuzer; alqarni, mohammed h.; alshetaili, abdullah; ghoneim, mohammed m.; alshehri, sultan; ali, amena title: investigating antiarthritic potential of nanostructured clove oil (syzygium aromaticum) in fca-induced arthritic rats: pharmaceutical action and delivery strategies date: 2021-12-02 journal: molecules doi: 10.3390/molecules26237327 sha: c57b9af98c39dfbe780d24ac1079c14b15920b44 doc_id: 71358 cord_uid: 8v1oemlt the combined application of clove oil in a lipid nanocarrier opens a promising avenue for bone and joints therapy. in this study, we successfully developed a tunable controlled-release lipid platform for the efficient delivery of clove oil (co) for the treatment of rheumatoid arthritis (ra). the ultra-small nanostructured lipid carriers co-loaded with co (concs) were developed through an aqueous titration method followed by microfluidization. the concs appeared to be spherical (particle size of 120 nm), stable (zeta potential of −27 mv), and entrapped efficiently (84.5%). in toluene:acetone:glacial acetic acid (90:9:1 percent v/v/v) solvent systems, high-performance thin layer chromatography (hptlc) analysis revealed the primary components in co as eugenol (r(f) = 0.58). the concs greatly increased the therapeutic impact of co in both in vitro and in vivo biological tests, which was further supported by excellent antiarthritic action. the conc had an antiarthritic activity that was slightly higher than neat co and slightly lower than standard, according to our data. the improved formulation inhibited serum lysosomal enzymes and proinflammatory cytokines while also improving hind leg function. this study provides a proof of concept to treat ra with a new strategy utilizing essential oils via nanodelivery. arthritis, or joint inflammation, is a chronic condition caused by a disruption in the production of pro-inflammatory cytokines and enzymes [1] . rheumatoid arthritis (ra) is an autoimmune illness that affects several joints bilaterally and is characterized by increasing tenosynovitis (inflammation of a tendon), which leads to cartilage degradation, bone erosion, and eventually disability [2, 3] . in terms of males to females, the prevalence of ra has been found to be 1:3, with prevalence ranging from 0.4 percent to 1.3 percent of the population depending on both sexes worldwide [4] . analgesics, nonsteroidal anti-inflammatory medicines (nsaids), immunosuppressive glucocorticoids (isgs), anticytokines (acs), and disease-modifying antirheumatic medications (dmards) are currently accessible pharmaceuticals [3] . however, exceptional progress has been observed in the long-term development of dmards, which a high-performance thin layer chromatography (hptlc) method was employed for all analyses of the marker compound as it is a simple and economical method [36] . pharmaceutical evaluation, product stability, and biological studies were performed as per previously published reports [2, 32, 37] . according to the findings, designing appropriate nanolipidic carriers could be a successful method to improve co benefits in ra. we adopted the high-performance thin-layer chromatography (hptlc) method (ca-mag, switzerland) as illustrated in the "materials and method" section for the quantification of eu in prepared formulations and as the crude oil (co). a sharp chromatogram (r f : 0.58 ± 0.02) was observed by densitometric analysis in absorbance mode at 281 nm ( figure 1 ). the developed method was robust and accurate, therefore employed for all estimations. however, the antiarthritic potential of nanostructured co has not been studied via a topical drug delivery system. therefore, the purpose of the study was to develop a lipid nanocarrier of co and to investigate its potential for arthritis by imposing the topical route. we adopted an aqueous titration method followed by high shear mixing for formulation development. a high-performance thin layer chromatography (hptlc) method was employed for all analyses of the marker compound as it is a simple and economical method [36] . pharmaceutical evaluation, product stability, and biological studies were performed as per previously published reports [2, 32, 37] . according to the findings, designing appropriate nanolipidic carriers could be a successful method to improve co benefits in ra. we adopted the high-performance thin-layer chromatography (hptlc) method (camag, switzerland) as illustrated in the "materials and method" section for the quantification of eu in prepared formulations and as the crude oil (co). a sharp chromatogram (rf: 0.58 ± 0.02) was observed by densitometric analysis in absorbance mode at 281 nm ( figure 1 ). the developed method was robust and accurate, therefore employed for all estimations. co was selected as an oil phase containing eu as a functional constituent for antiarthritic activity. standard techniques were used to assess the solubility of co in solid fats/triglycerides, surfactants, and cosurfactants [38] . co was moderately miscible in lipids including stearic acid (sa), cetyl alcohol (ca), and glyceryl oleate (go) at room tem co was selected as an oil phase containing eu as a functional constituent for antiarthritic activity. standard techniques were used to assess the solubility of co in solid fats/triglycerides, surfactants, and cosurfactants [38] . co was moderately miscible in lipids including stearic acid (sa), cetyl alcohol (ca), and glyceryl oleate (go) at room temperature and below 10 • c. co and go in 1:1 ratio demonstrated better solubility performance (table 1) , therefore go was selected as a solid lipid (base) for the study. the surfactant chosen for drug development must be able to (i) lower interfacial tension to a very low value to aid dispersion during nanoemulsion preparation, (ii) provide a flexible film that can easily deform around droplets, and (iii) have the appropriate lipophilic character to provide the correct curvature at the interfacial region for the desired nanoemulsion type (i.e., o/w, w/o, or bicontinuous) [32, [38] [39] [40] . the surface area (mm 2 ) as a function of phase behavior was determined for the co in different surfactants and cosurfactants and the combination thereof to find out the best possibility towards formulation development. we reported the phasic surface area in mm 2 for co in table 1 constructing phase diagrams takes time, especially when the goal is to precisely define a phase boundary. in any case, the pseudo-ternary phase diagrams were created at a constant temperature using the aqueous titration method. visual observation validated the production of mono/biphasic systems; in cases where turbidity developed, the formulation was regarded biphasic; nevertheless, in cases where the clean and transparent mixture was visible after stirring, the formulation was considered monophasic [40] . phase diagrams were constructed separately for each surfactant to co-surfactant (s mix ) combination ( figure 2 ) to identify o/w phases required for the optimization of lipid nanoemulsions. in addition to the water titration approach, the oil and water components were kept constant with varying surfactant/cosurfactant concentrations to complete the nanoemulsion domain. a large nanophasic area (o/w) appeared when a high concentration of s mix was used. tween 80 cannot be used alone, because it precipitates to undesired liquid crystals (lcs). for the above reason, cosurfactant was added to expand the nanoemulsion region and disrupt the lc formation. the phase diagram depicted the nanoemulsion domain obtained by these trials at various ratios of surfactant (tween 80) to cosurfactant (cremophor-el) ( figure 2 ). the s mix (1:0) was not able to break the interfacial tension with co, confirmed by the presence of more lcs in the phase diagram. adding cremophor-el (co-surfactant) to tween 80 (surfactant) in 1:1 ratio (s mix ), made the interfacial film more flexible to accommodate the drug and lcs started disappearing. the phase behavior investigation revealed that the greatest amount of oil that may be added to the nanoemulsion system was when the surfactant to cosurfactant ratio was 1:1. by adding s mix approximately 60.7 ± 5.4 percent v/v, the largest amount of co that solubilized was nearly 21.0 ± 1.2 percent v/v, according to the phase diagram ( figure 2a) . it was discovered that as the cosurfactant concentration in the s mix ratio (1:1, 1:2, and 1:3) increased, the lcs decreased, but the nanophases increased proportionately (figure 2a-c) . inversely, raising the surfactant concentration in the s mix ratio (2:1, 3:1, and 4:1) increased lcs, and phase diagrams revealed a tiny nanoemulsion area ( figure 2d -f). because of the lower interfacial tension and increased fluidity at the interface, the system s entropy increased somewhat, allowing more oil to be integrated into the hydrophobic area of surfactant monomers [40] . when the surfactant concentration in s mix was increased from 2:1 to 3:1, the increase in the nanophase region was minimal ( figure 2d ). actually, this was the lc phase, attributed due to the presence of tween 80 in high concentration which suppressed the effect of the cosurfactant. the above finding revealed that the free energy of nanoemulsion formation is more or less dependent on the extent to which the s mix submissively reduces the interfacial tension of oil and water and dispersion entropy [38, 40] . in such circumstances, the formation of nanoemulsions was spontaneous and produced physically stable dispersions [32, [38] [39] [40] . those formulations were selected from phase diagrams, which can accommodate a high quantity of oil with the low concentration of s mix . the smix (1:0) was not able to break the interfacial tension with co, confirmed by the presence of more lcs in the phase diagram. adding cremophor-el (co-surfactant) to tween 80 (surfactant) in 1:1 ratio (smix), made the interfacial film more flexible to accommodate the drug and lcs started disappearing. the phase behavior investigation revealed that the greatest amount of oil that may be added to the nanoemulsion system was when the surfactant to cosurfactant ratio was 1:1. by adding smix approximately 60.7 ± 5.4 percent v/v, the largest amount of co that solubilized was nearly 21.0 ± 1.2 percent v/v, according to the phase diagram ( figure 2a) . it was discovered that as the cosurfactant concentration in the smix ratio (1:1, 1:2, and 1:3) increased, the lcs decreased, but the nanophases increased proportionately (figure 2a-c) . inversely, raising the surfactant concentration in the smix ratio (2:1, 3:1, and 4:1) increased lcs, and phase diagrams revealed a tiny nanoemulsion area ( figure 2d -f). because of the lower interfacial tension and increased fluidity at the interface, the system′s entropy increased somewhat, allowing more oil to be integrated into the hydrophobic area of surfactant monomers [40] . when the surfactant concentration in smix was increased from 2:1 to 3:1, the increase in the nanophase region was minimal ( figure 2d ). actually, this was the lc phase, attributed due to the presence of tween 80 in high concentration which suppressed the effect of the cosurfactant. the above finding revealed that the free energy of nanoemulsion formation is more or less dependent on the extent to which the smix submissively reduces the interfacial tension of oil and water and dispersion entropy [38, 40] . in such circumstances, the formation of nanoemulsions was spontaneous and produced physically stable dispersions [32, [38] [39] [40] . those formulations were selected from phase diagrams, which can accommodate a high quantity of oil with the low concentration of smix. different formulations from the nanoemulsion area were chosen from each phase diagram and screened further based on their performance during the thermodynamic stability test. the complete spectrum of nanoemulsion was covered by the phase diagrams, different formulations from the nanoemulsion area were chosen from each phase diagram and screened further based on their performance during the thermodynamic stability test. the complete spectrum of nanoemulsion was covered by the phase diagrams, and the formula that used the lowest concentration of s mix for nanoemulsion synthesis was chosen. the composition of the different lipid nanoemulsions is presented in table 2 . the oil (co:go; 1:1) was taken and a measured quantity of s mix (tween 80 and cremophor el) was added in the prescribed ratio. following the slow titration method, the lukewarm water was then added dropwise till a clear and transparent solution was obtained. microfluidization technology is a patented technology used to produce nanoparticles of desired size and features, therefore the obtained nanoemulsion was first solidified in cold water and then microfluidized (high shear homogenization) as illustrated earlier. in vitro thermodynamic stability tests were undertaken to overcome the problem of the metastable form. the thermodynamic stability of the selected formulations, such as the centrifugation (c/t), heating and cooling cycles (h/c), and freeze-pump thaw (f/t) cycles, was tested. table 2 shows the compositions of various formulations. the formulations that passed the thermodynamic stability tests were subjected to additional testing, including size, viscosity, and microscopy. the ee stands for drug encapsulation capacity, which is an evident physicochemical property of lipid nanoparticles. lipid nanocarriers (nlcs) have a better drug loading capacity and entrapment effectiveness than solid lipid nanoparticles (slns) [28, 29] . co was encapsulated in large amounts in the investigated conc, which was attributed to superior lipophilic drug solubilization in the combination of oil and lipids (less ordered recrystallization) than in oil alone (table 3 ). in the present study, the percentage entrapment of the best formulation was 91.34 ± 3.29. the combination of co with go led to high entrapment efficiency, i.e., 63.52% (conc1); 72.30% (conc2); 69.88% (conc3); 82.51% (conc4); 91.35% (conc5), and 79.13% (conc6), perhaps due to their lipophilic character and better loading capacity in stabilized formulations. table 3 . conc formulation physicochemical characterization (mean ± sd, n = 3). diameter ± sd (nm) pdi ± sd zeta potential (mv) ± sd the morphological evaluation was performed by transmission electron microscopy (tem) on a trial nanoformulation using "electron microscope jem 1400-plus (jeol ltd., tokyo, japan)". conc5 particles were found to be spherical in form using tem imaging ( figure 3 ). the particles appeared dark with a bright surrounding in a positive image under tem (table 3) . tem imaging finally concluded the uniform and spherical co nanoparticles with varying sizes, ranging approximately from 120-200 nm. the morphological evaluation was performed by transmission electron microscopy (tem) on a trial nanoformulation using "electron microscope jem 1400-plus (jeol ltd., tokyo, japan)". conc5 particles were found to be spherical in form using tem imaging ( figure 3 ). the particles appeared dark with a bright surrounding in a positive image under tem (table 3) . tem imaging finally concluded the uniform and spherical co nanoparticles with varying sizes, ranging approximately from 120-200 nm. the nano-zs zetasizer was used to analyze the size distribution and surface charge of selected formulations (malvern instruments). the size and charge results of selected nanoformulations are represented in table 3 and figure 3 . the largest droplet size apthe nano-zs zetasizer was used to analyze the size distribution and surface charge of selected formulations (malvern instruments). the size and charge results of selected nanoformulations are represented in table 3 and figure 3 . the largest droplet size appeared in conc2, perhaps due to the presence of a high surfactant concentration which might lead to the formation of a rigid interface. an inadequate quantity of cosurfactant was found to be unable to provide further flexibility to the rigid film for secondary nanosizing. the mean particle size of the optimal formulations (conc1-conc6) ranged from 120 to 190 nm, with pdi values of less than 0.2, indicating that the system has a relatively narrow size distribution. it was observed that particle size increases with an increase in concentration of oil and s mix ratio. conc5 claimed the lowest pdi of 0.148, indicating a narrow distribution of size. a zeta potential of ±30 implies that nanodroplet/nanoparticle production is stable [40, 41] . conc6 formulation showed the highest zp magnitude (−31.12 ± 2.4 mv), which fluctuated greatly, even after three months of storage at a regulated temperature. finally, it was determined that the conc with highly charged surfaces was stable and would resist droplet aggregation in a controlled temperature environment (45 • c/75%). table 3 shows the measured viscosity values of the optimized formulations, which ranged from 110 to 165 cps. viscosity results were correlated with various surfactants and co-surfactant used in the stabilization of nanoformulation compositions. conc5 was found to have a moderate viscosity of 142.73 cps as compared to others, suitable for topical administration. in vitro drug release was performed by using a dialysis membrane fitted over a modified franz diffusion cell. the study was designed to compare the drug release of the optimized formulation and a nonformulated clove oil. the in vitro release demonstrated a prolonged release ( figure 4 ). drug release from conc5 was rapid (81.24% in 24 h), however, the formulations exhibited a comparatively prolonged release pattern [conc5 (81%) > conc6 (79%) > conc4 (75%) > conc3 (70%) > conc2 (64%) > conc1 (59%)] in 24 h. on the other hand, the nonformulated co (neat) exhibited sluggish drug release (38% in 24 h) as compared to the optimized conc. the ex vivo drug transport showed enhanced skin permeation for the optimized formulations as compared to the control (neat co). the maximum transdermal flux value was found to be 82.41 ± 12.39 µg/cm 2 /h (conc5) over the control formulation (28.17 ± 10.29 µg/cm 2 /h) with the enhancement ratio of 6.34 across the rat's skin, possibly due to least droplet size and low viscosity (table 4 ). in fact, the lipid nanoemulsions improve eu penetration via the sc lipid pathway, in which neutral lipids are organized as bilayers with their hydrophobic chains facing each other to produce a lipophilic bimolecular facet [32, 40] . the concs directly penetrate the sc, where they destabilize the bilayer structure due to the involvement of the present surfactant molecule leading to enhanced drug permeability. however, the hydrophilic domain hydrated the sc which led to enhanced percutaneous drug uptake. in terms of pharmaceutical considerations, a high initial flux is always seen as advantageous since a sufficient quantity of bioactives is rapidly released from the lipid nanocarriers to produce a prompt topical effect. the drug's steady state flux (j ss ), permeability coefficient (k p ), and enhancement ratio (e r ) were dramatically raised via the rat skin, possibly due to the synergistic actions of eu and surfactant/cosurfactant combinations. (81%) > conc6 (79%) > conc4 (75%) > conc3 (70%) > conc2 (64%) > conc1 (59%)] in 24 h. on the other hand, the nonformulated co (neat) exhibited sluggish drug release (38% in 24 h) as compared to the optimized conc. the ex vivo drug transport showed enhanced skin permeation for the optimized formulations as compared to the control (neat co). the maximum transdermal flux value was found to be 82.41 ± 12.39 μg/cm 2 /h (conc5) over the control formulation (28.17 ± 10.29 μg/cm 2 /h) with the enhancement ratio of 6.34 across the rat's skin, possibly due to least droplet size and low viscosity (table 4 ). in fact, the lipid nanoemulsions improve eu penetration via the sc lipid pathway, in which neutral lipids are organized as bilayers with their hydrophobic chains facing each other to produce a lipophilic bimolecular facet [32, 40] . the concs directly penetrate the sc, where they destabilize the bilayer structure due to the involvement of the present surfactant molecule leading to enhanced drug permeability. however, the hydrophilic domain hydrated the sc which led to enhanced percutaneous drug uptake. in terms of pharmaceutical considerations, a high initial flux is always seen as advantageous since a sufficient quantity of bioactives is rapidly released from the lipid nanocarriers to produce a prompt topical effect. the drug's steady state flux (jss), permeability coefficient (kp), and enhancement ratio (er) were dramatically raised via the rat skin, possibly due to the synergistic actions of eu and surfactant/cosurfactant combinations. the factual changes in the product stability evaluation have been presented in table 5 . the average particle size of formulation conc5 was not much affected after three months of storage, however other formulations were aggregated/clumped (at a regulated temperature) and showed an increase in particle size. due to the protective ability of tweens in regulated temperature and humidity conditions, the particles remained stable in a normal contour. surfactant ability is one of the solid reasons for product stability. in general, aggregation accelerated with an increase in storage temperatures due to the physical destabilization of conc as a consequence of energy input by the successively higher temperatures. the energy input amplified the system's kinetic energy which led to particle collision, and then, finally, the aggregation of particles. 2.9. pharmacological evaluation 2.9.1. in vitro antiarthritic activity the antiarthritic effects of the produced nanoformulations in vitro were investigated using the protein denaturation method [42] . according to the literature, autoantigen production (in the case of ra) may be caused by protein denaturation. as a result, protein denaturation is one of the most common signs of arthritic inflammation. for this purpose, concs were compared with standard (voltaren; diclofenac gel) products and analyzed. the results revealed that the standard products showed a percentage inhibition of 86.91, however, conc showed 82.73 percent inhibition. the above finding demonstrated the antiarthritic effect of our developed nanoformulations. the volume of the ipsilateral (injected) and contralateral (noninjected) paws increased gradually after subplantar fca injection in the left hind paws of rats [43] . the rats paw volume was measured on the 0th, 7th, 14th, and 21st days after the study began. the paw volume of all groups increased from day 0 to day 7, according to the findings. the results of the conc5 formulation were equivalent to those of the standard (voltaren gel), indicating that the new formulation is therapeutically effective (p < 0.01). the toxic group, on the other hand, experienced a constant increase in paw volume as a result of being the nontreatment group. as shown in table 6 , both the standards and the conc5 formulation resulted in a significant reduction in paw volume throughout the second-third week. the treatment with conc5 demonstrated enhanced cartilage regenerative activity compared with untreated or co-free nc treatment groups. in fca-induced arthritic rats, the examined formulation was shown to limit the immune response more significantly (p < 0.01) than neat co, possibly because of its capacity to reduce acute inflammation by decreasing the inflammatory mediator's cascade and then reducing vascular permeability. table 6 . effect of conc on lysosomal enzymes, c-reactive protein, rheumatoid factor, and serum cytokines in fca-induced arthritic rats. values are mean ± sem for 6 animals. ** p < 0.01 vs. control group, # p < 0.01 when compared to normal control. using one-way anova followed by dunnett test. abbreviations, ast: aspartate aminotransferase; alt: alanine transaminase; alp: alkaline phosphatase; rf: rheumatoid factor, crp: c-reactive protein, tnf-α: tnf: tumor necrosis factor-α; il-6: interleukin-6. table 6 displays the fca induced biochemical changes in "aminotransferase (ast), alanine transaminase (alt), and alkaline phosphatase (alp)" of treated and nontreated rats. in inflammatory progressions, the enzymes ast, alt, and alp play important roles in the production of physiologically active chemical mediators, such as bradykinins. the improved therapeutic efficacy and penetration of the optimized conc formulation may be responsible for the good recovery in the provided state. the level of ast, alt, and alp enzymes increased in all groups treated with fca. the increased level of serum enzymes in arthritic rats was dramatically lowered after treatment with conc5 and voltaren gel. however, when fca-induced arthritic rats were treated with conc formulation, the increased enzyme levels were significantly reduced (p < 0.01), and the impact was superior to plain co. because proinflammatory cytokines (tnf-α and il-6) have been shown to play a significant role in the development of ra, the blood levels of tnf-α and il-6 cytokines in treated arthritic rats were determined and reported in table 6 . tnf-α and il-6 levels were found to be considerably higher in fca-induced arthritic rats (p < 0.01), whereas treatment with the conc formulation effectively reduced these elevated levels. tnf-α (pleiotropic cytokine) is a pleiotropic cytokine produced by activated monocytes and macrophages that regulates immune cells and plays a key role in inflammation (both chronic and acute). tnf-α stimulates inflammatory responses, which can lead to autoimmune diseases such as ankylosing spondylitis and ra. il-6, on the other hand, is said to harm synovial cells by increasing prostaglandin synthesis and fibroblast proliferation in the synovial fluid. as a result, we looked at the levels of tnf-α and il-6 cytokines in the serum of arthritic rats. the levels of tnf-α and il-6 in the fca-induced arthritic rat were significantly raised, while treatment with conc5 decreased the elevated levels of serum tnf-α and il-6. the developed conc formulation was found promising due to its potential to inhibit proinflammatory mediators by arresting tnf-α and il-6 activation. the c-reactive protein (crp) and rheumatoid factor (rf) levels in the blood are indicators of systemic inflammation and antibody formation against the adjuvant administered. the fca control group rats had high levels of serum crp (8.2 mg/l) and serum rf (57.9 iu/l). both crp and rf levels in the serum were dramatically lowered by the conc5 and voltaren gel treatments. because the levels of tnf-α and il-6 were significantly reduced by the topical administration of conc5, it is expected that the levels of other proinflammatory biomarkers such as il-4, il-10, and il-11 would also be reduced by the same formulation. overall, this research suggested that co in a conc formulation significantly decreased the elevated levels of proinflammatory biomarkers, such as tnf-α and il-6 in rats, leading to an anti-inflammatory effect. as a result, the anti-inflammatory potential of co in a conc formulation might be due to the inhibition of tnf-α and il-6 by the topical administration in rats [44] [45] [46] . the working standard of eu (purity: 99.8%), go, brij 98, isopropanol (ipa), and complete fca were obtained from "sigma aldrich (st. louis, mo, usa)". co was purchased from "loba chemie pvt. ltd., (mumbai, india)". capryol-90, transcutol-p, labrasol, and labrafac were obtained from "gattefosse (lyon, france)". cremophore-el, ca, and sa were obtained from basf (darmstadt, germany). tween 80, tween 20, peg-200, and peg-400 were obtained from "e-merck (darmstadt, germany)". hplc grade methanol and ethyl alcohol were purchased from "fluka chemica (darmstadt, germany)". purified water was obtained from "milli-q water purification system (millipore, billerica, ma, usa)". all additional chemicals and reagents used in the experiment were analytical grade and purchased from a reputable vendor. hptlc method was employed for all analyses of marker compound as it is a simple and economical method [36] . the hptlc approach was used to quantify an active ingredient of co, namely eu (camag, muttenz, switzerland). with a camag microliter (µl) syringe, the eu samples were smeared in the shape of bands with a width of 5 mm on a precoated silica-gel aluminum plate 60f254 (20 cm × 10 cm with 0.2 mm thickness) (e-merck, darmstadt, germany) using the camag linomat v sample applicator (camag, muttenz, switzerland). the mobile phase was toluene:acetone:glacial acetic acid (90:9:1 v/v/v), and formed plates were dried and sprayed with vanillin-sulfuric acid reagent to visualize the zones. the camag tlc scanner iii was used to perform densitometric scanning in the absorbance mode at 281 nm. this method was employed for all analytical estimations pertaining to the study. pseudo-ternary phase titration was performed for co at a constant temperature with one of the immiscible components to a turbidimetric end-point by employing conventional water titration method [32] . concisely, the co (oil) and go (fat) were mixed in 1:1 mass ratio (melted on hot water bath) and combination of tween 20 and cremophor-el (surfactant mixture) was dissolved in ipa (co-surfactant) by gentle stirring (table 1) . surfactant/co-surfactant mixtures (s mix ) were obtained in different volume ratios (1:0, 1:1, 1:2, 1:3, 2:1, 3:1, and 4:1) for the titration. thereafter, various combinations of oily phase (co:go; 1:1) and specific s mix were prepared in 9 volume ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:2, and 9:1) to cover the maximum volume ratios, which allowed the boundary between the phases formed during the pseudo-ternary phase construction to be precisely delineated. lukewarm purified water was used as the aqueous phase in the titration. a slow titration was performed for each combination (oil:s mix ) using lukewarm water as an external phase [38, 39] . subsequently, each set was visually examined for transparency and isotropy, as well as for boundaries between homogeneous and heterogeneous mixtures (i.e., single-phase, clear, fluid, and homogeneous nanoformulation). pseudo-ternary diagrams with an aqueous phase on one axis, an oil phase on the other (co:go), and a specific mixture of surfactant/cosurfactant in set volume ratios on the third axis were created. the batches showing significant clarity and transparency were then transferred to cold aqueous medium (1-3 • c) and then homogenized with a microfluidizer (hg-15d daihan scientific co., ltd., chuncheon, korea) operating at 12,000 rpm for 15 min ensuring proper mixing of the system [47] . we adopted the nanoemulsion technique, described by muller et al., to prepare the co nanocarriers (concs) [41] , followed by microfluidization treatment [47] . in brief, from each constructed phase diagram, different formulations were chosen from the nanoemulsion region and selected for the study. furthermore, when distributed in a cold aqueous media (2-3 • c) under light mechanical mixing, these lipid nanoemulsions recrystallized, ensuring that the small size of the particles formed was due to precipitation and not mechanically generated by the stirring process [41, 48] . therefore, microfluidization technique was employed to reduce the particle size of prepared formulation to as small as possible [49] . finally, the produced lipid nanoemulsion (o/w) was stirred for 15 min on a high shear homogenizer at 12,000 rpm, and the resulting formulation was allowed to cool to room temperature. the developed nanoproduct was evaluated for various pharmaceutical properties as per the standard protocol mentioned below [37, 38] . to observe the physical changes, a c/t was used to test the thermodynamic stability of sample formulations at 6000 rpm for 30 min (phase separation, creaming or cracking) [38] . formulations passing the above test were subjected to six consecutive cycles of heating (45 • c) and cooling (0 • c) (h/c) 8 hourly for the next two days. further, the h/c passed formulations were given six cyclic treatments of f/t in between the temperature range of −21 • c to 25 • c for 48 h. out of the test samples, those which survived and passed all the above tests (c/f, h/c, and f/t) were finally selected for biological screening (table 2) . the ee of the developed conc was determined in terms of eu content. the sample was centrifuged (thomas scientific centrifuge, 5418-r, swedesboro, nj, usa) at 12,000 rpm for 30 min to remove noncapsulated co, and the supernatant was gently harvested and diluted with phosphate buffer (ph 7.4). the eu content was then calculated using the hptlc method, as shown in the "analytical procedure section". the percentage entrapment efficiency of the eu was determined by using the following formula: where, 'c t ' is the total concentration of incorporated and nonincorporated eu in conc and 'c r ' is the concentration of free eu in conc. the size and surface morphology of the trial formulation were confirmed using a transmission electron microscope jem 1400-plus (jeol ltd., tokyo, japan) operating at a 200 kv acceleration voltage. the test formulation was diluted in water (1:100) and filtered through a membrane syringe filter with a cut off diameter of 0.2 µm for the tem examination. after complete drying, a drop of the diluted conc was gently placed on a circular copper grid (taab laboratories equipment, berks, uk) and viewed under a microscope. dynamic light scattering (dls) was used to determine particle size and size distribution using a nano-zs zetasizer (zen 3600, malvern instruments, darmstadt, germany) based on the laser light scattering phenomenon, which analyzes variations in light scattering. prior to the size analysis, test samples were adequately diluted at 1:5 (conc/water) and held at room temperature (25 • c) for 5 min. zeta potential (ζ) was determined by nano-zs using the laser doppler velocimetry technique. before being lodged in the instrument for charge measurements, test samples were diluted with kcl 1 mm (ph 7.0). each measurement was carried out three times and the results were recorded individually as mean ± standard deviation (sd). viscidness of lipid nanoemulsion was measured using brookfield viscometer (dvelv ultra, brookfield engineering laboratories, inc., middleboro, ma, usa) at a constant room temperature (25 ± 0.3 • c) at 100 rpm. the calculations were interpreted by using rheocalc software (version #2.6). effect of microfluidization (high shear homogenization) cycles on change of the viscosity value was determined in centipoises (cps). conc formulations were tested for in vitro drug release using a dialysis membrane method with a modified franz diffusion cell (a: 7.16 cm 2 ; v: 37 ml of receiver chamber) [50] . dialysis membrane (2.4 nm, 12,000-14,000 da) previously soaked with dissolution media (7:3; acetate buffer ph 5.4: ethanol) was mounted over franz diffusion cell 12 h before the sample run. the release profile in dissolution media (ph 5.6) was also used to simulate cadaver skin ph. to run the experiment, conc formulations were precisely weighed and placed in a donor compartment with receptor compartment filled with dissolution media. during the experiments, the solution on receptor side was kept at 37 • c ± 0.5 • c with stirring magnetic bead speed of 100 rpm. aliquots (100 µl) were withdrawn from receiver compartment through side tube at a regular interval of time (0, 0.5, 1, 1. 5, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h) and substituted by an equal volume of fresh media each time. the samples were analyzed for the drug content using hptlc method as illustrated earlier. the franz diffusion cell with a diffusion surface area of 7.16 cm 2 and a receiver compartment capacity of 37 ml was used to analyze skin permeation on excised wistar-rat abdomen skin. the full thickness skin was cleaned with purified water and stored in a deep freezer at −20 • c until it was used in the experiment. the skin was warmed to room temperature before being installed atop a diffusion cell in the middle of the donor and receiver compartments, with the stratum corneum (sc) side facing the donor compartment and the dermal side facing the receiver compartment. at 37 ± 1 • c, the receiver chamber was filled with ethanolic acetate buffer ph 5.4 (7:3) and stirred at 100 rpm. in the donor compartment, one ml of conc formulation was inserted. at regular intervals (0. 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 24 , and 48 h), aliquots (1 ml) were taken from the receiver cell, filtered through a 0.45 µm membrane filter, and analyzed using hptlc at 281 nm. for each formulation, the cumulative amount of medication penetrated through the skin (g/cm 2 ) was plotted as a function of time (t). by dividing the slope of the linear component of the graph with the area of the diffusion cell, the j ss was computed. using the equation below, the k p was computed by dividing j ss by the initial concentration of drug in the donor cell (c 0 ) [32] : using the following equation, the e r was computed by dividing the j ss of the test formulation by the j ss of the control formulation [32] : however, the skin retention study was performed to analyze the content of co in the skin after 48 h of diffusion. at the end of experiment, the skin sample was washed with purified water and methanol on both sides respectively and then it was carefully dried. after that methanol (10 ml) was added to the skin and after vortexing for 10 min, the content was kept stirred overnight. the next day, the extracted sample was centrifuged and supernatant was analyzed by hptlc at 281 nm. the product stability of developed conc was examined to ensure the physicochemical stabilities, such as aggregation, precipitation, fusion, and degradation upon storage. in a nutshell, the conc formulations were tested for particle size, zeta potential, and other characteristics over the course of three months (latest time-point evaluated) at 25 • c and 40 • c. male albino wistar rats (180-220 g) of both sexes were used for the pharmacological evaluations of concs. the animals were obtained from "experimental animal care center (eacc) at prince sattam bin abdulaziz university, al-kharj, saudi arabia". the rats were housed under standard animal housing facilities (t = 24 ± 1 • c, %rh = 45-50%, and 12 h light/dark cycle), on standard pellet chow diet and water ad libitum. all experimental protocols and procedures were carried out in compliance with the guidelines of the "eacc, prince sattam bin abdulaziz university, al-kharj, saudi arabia". these studies were approved by the "animal ethics committee of the eacc board (prince sattam bin abdulaziz university, al-kharj, saudi arabia with approval number: berc-016-10-21)". the use of animals and experimental protocols were in accordance with the "european union (eu) directive 2010/63/eu". in vitro antiarthritic potential of prepared concs were compared with topical voltare n-diclofenac sodium gel (standard) by using protein denaturation method [42] . the reaction mixture (0.5 ml) was made up of 0.45 ml bovine serum albumin (5 percent aqueous bsa) and 0.5 g each of the test and control formulations, with the ph adjusted to 6.3 with 1n hcl. both samples were incubated at 37 ± 1 • c for 15 min before being gently heated at 57 • c for 3 min. following cooling, a 2.5 ml aliquot of pbs (ph 6.3) was added to each sample. the spectrophotometer was used to carefully measure turbidity at 660 nm, and the percentage inhibition (pi) of protein denaturation was computed using the formula below: the antiarthritic evaluation in rat model was performed for conc formulation (test) and co-free nc formulation (control) in accordance with our previous research on arthritis [2] . the rats were chosen at random and placed into four experimental groups (each including six rats) for the evaluation of fca-induced arthritis: group i-co-free nc was used as a topically applied control; group ii-no pharmacological therapy was given to the toxic control group, which had been treated with fca.; group iii-treated group, which received 1.2 percent topically administered co loaded nc (i.e., conc); group iv-positive control was 1.16 percent w/v diclofenac gel applied topically, as per normal marketed formulation. the research was scheduled to last three weeks. in the 1st week, all animals in all groups, except group i-normal control, received 0.1 ml of fca subcutaneously into the subplantar area of the left hind paw [43] . fca is made up of mycobacterium tuberculosis that has been heat destroyed, mineral oil, and mannide monooleate. the fca suspension once injected may cause inflammatory reactions within 24 h. on days 1, 4, 7, 10, 12, 14, 17, and 21, the antiarthritic activities of test samples and standard were determined using a "plethysmometer (ugo, basile, italy)" by the hind paw method [43] . the volume of the left paw was measured using the mercury displacement method up to the lateral malleolus shortly before fca injection on the first day, and thereafter using the plethysmometer at various time intervals until the 21st day. the difference between the end and starting paw volumes was used to calculate the changes in paw volume [2, 43] . blood samples were taken from all groups of rats on the 21st day via retro-orbital puncture. harvested blood samples were immediately transferred to the anticoagulation tubes and the required reagents were added precisely. a standard kit (sigma-aldrich assay kit) was used to measure various biochemical parameters such as ast, alt, and alp. the blood samples were maintained undisturbed for around half an hour to estimate proinflammatory biomarkers such as tnf-α and il-6. centrifugation (3000 rpm, 10 min) was used to separate the serum from the blood, which was then stored at −20 • c until further analysis. tnf-α and il-6 levels were measured using ready-to-use elisa reagent kits (biosource inc., camarillo, ca, usa) in accordance with established procedures. routine laboratory techniques, on the other hand, were utilized to estimate serum parameters. commercial kits (aspen labs, england, uk) were used to quantify blood crp and rf levels according to the manufacturer s instructions. statistical analysis was carried out using one-way analysis of variance (anova) with tukey s multiple comparisons test, with a statistical significance level of p < 0.05 (95 percent confidence interval) unless otherwise stated. graph-pad prism software-vi was used to conduct all statistical analyses. the co-loaded nlcs were effectively synthesized using an aqueous titration approach followed by microfluidization, and their antiarthritic potential was studied in vitro and in vivo. the conc was discovered to have a higher transdermal flux value across rat skin in the current investigation. the antiarthritic effects of conc were comparable to those of the standard product, according to in vivo studies. with a fall in the biochemical parameters, the rat paw volume revealed a depleting effect. the findings demonstrated that the optimized formulation has inhibitory effects on enzymes and lowers il-6 and tnf-α levels. the foregoing findings demonstrate that our conc formulation was helpful in the treatment of arthritis. data availability statement: this study did not report any data. early diagnosis and treatment of rheumatoid arthritis antiarthritic potential of calotropis procera leaf fractions in fca-induced arthritic rats: involvement of cellular inflammatory mediators and other biomarkers the immunology of rheumatoid arthritis case series of acute arthritis during covid-19 admission rheumatoid arthritis: current pharmacologic treatment and anesthetic considerations diagnosis and management of rheumatoid arthritis evolving concepts of the pathogenesis of rheumatoid arthritis with focus on the early and late stages functional subsets of cd 4 t cells in rheumatoid synovitis hepatoprotective effect of solanum xanthocarpum fruit extract against ccl 4 induced acute liver toxicity in experimental animals. asian pac anti-inflammatory and antioxidant activity of ficus carica linn. leaves accumulation and clearance of the anaesthetics clove oil and aqui-s™ from the edible tissue of silver perch (bidyanus bidyanus) clove: a champion spice wound healing effects of nanoemulsion containing clove essential oil eugenol: a natural compound with versatile pharmacological actions antimicrobial activity of clove oil dispersed in a concentrated sugar solution pharmacological action of clove oil and its application in oral care products. oral care ind eugenol suppresses cyclooxygenase-2 expression in lipopolysaccharide-stimulated mouse macrophage raw264.7 cells lack of postexposure analgesic efficacy of low concentrations of eugenol in zebrafish study of anti-inflammatory activities of α-d-glucosylated eugenol effect of some penetration enhancers on the permeation of glibenclamide and glipizide through mouse skin essential oils loaded in nanosystems: a developing strategy for a successful therapeutic approach. evid.-based compl characterization and in vitro bioactive studies of lemongrass oil phytonanoemulsion system in the treatment of acne vulgaris changes in aggregation properties of tpgs micelles in the presence of sodium cholate lipid nanoparticles: effect on bioavailability and pharmacokinetic changes lipid-based nanoparticles as carriers for dermal delivery of antioxidants lipid nanoparticles (sln, nlc) in cosmetic and pharmaceutical dermal products lipid-based nanoparticles as drug delivery system for paclitaxel in breast cancer treatment solid lipid nanoparticles (sln) for controlled drug delivery. i. production, characterization and sterilization the production and characteristics of solid lipid nanoparticles (slns) wound healing study of eucalyptus essential oil containing nanoemulsion in rat model wound healing evaluation of self-nanoemulsifying drug delivery system of piper cubeba essential oil nanoemulsification improves the pharmaceutical properties and bioactivities of niaouli essential oil (malaleuca quinquenervia l.) controlled-release antihistamines using supercritical antisolvent process particle size design of acetaminophen using supercritical carbon dioxide to improve drug delivery: experimental and modeling applications of supercritical anti-solvent process in preparation of solid multicomponent systems combining normal/reversed-phase hptlc with univariate calibration for the piperine quantification with traditional and ultrasound-assisted extracts of various food spices of piper nigrum l. under green analytical chemistry viewpoint investigating the feasibility of mefenamic acid nanosuspension for pediatric delivery: preparation, characterization, and role of excipients development and bioavailability assessment of ramipril nanoemulsion formulation development of an alternative, time and cost saving method of creating pseudoternary diagrams using the example of microemulsion critical steps and energetics involved in a successful development of a stable nanoemulsion solid lipid nanoparticles (sln) for controlled drug delivery-a review of the state of the art topical nano-vesicular spanlastics of celecoxib: enhanced anti-inflammatory effect and down-regulation of tnf-α, nf-кb and cox-2 in complete freund's adjuvant-induced arthritis model in rats anti-arthritic activity profile of methanolic extract of ficus bengalensis: comparison with some clinically effective drugs biological investigation of a supersaturated self-nanoemulsifying drug delivery system of piper cubeba essential oil oral and topical anti-inflammatory and antipyretic potentialities of araucaria bidiwilli shoot essential oil and its nanoemulsion in relation to chemical composition antiarthritic activities of herbal isolates: a comprehensive review production of high-oleic palm oil nanoemulsions by high-shear homogenization (microfluidization) preparation and characterization of stearic acid nanostructured lipid carriers by solvent diffusion method in an aqueous system lipid nanoparticles: evaluation of some critical formulation parameters poly propyl ether imine (petim) dendrimer: a novel non-toxic dendrimer for sustained drug delivery authors are thankful to the taif university researchers supporting project (number tursp-2020/124), taif university, taif, saudi arabia for supporting this work. the authors declare no conflict of interest.sample availability: samples of the co compounds are available from the authors. jiims.cdr abstract objective: to determine the effectiveness of intrasheath steroid injection in treating de quervain`s disease. study design: case series observational study. place and duration of study: outpatient department of orthopedics bbs [dhq] teaching hospital abbottabad from april 2006 to sept 2009 including a period of follow up of eighteen months. materials and methods: eighty patients with de quervain`s disease were diagnosed on the basis of positive finkelstein`s test and presence of severe pain for more than four weeks. these patients were given intrasheath injections of 40 mg methyl prednisolone mixed with one ml of lignocaine 2% in the first dorsal compartment of wrist. patients with trauma, infection and rheumatoid arthritis were excluded from study. results: seventy two patients i.e., 90% were cured and out of them 75% required only one injection .only two cases underwent surgical release. conclusion: intrasheath steroid injection is a safe, very useful and cost effective method in treating de quervain`s disease. key words: de quervain's disease, intra sheath injection, steroid injections 87 original article importance of non surgical options is not 4 emphasized. recently there have been a number of claims regarding the efficacy of intrasheath steroid injection. sawaizumi, et. al., reported an efficacy rate of 94% with intrasheath injection of 5 steroids. richie and eriner reviewing seven current reputable papers, concluded that the efficacy rate of injecting the steroid alone 6,7,8,9 was 83%. it is a safe and simple technique which can be carried out in outpatient 10 department. there is very little chance of 11 serious complications as well. hence this study was carried out to further strengthen this claim that injection treatment of de quervain's disease is the best option and it is not associated with any serious complication. this observational study was done in outpatient department of orthopedics of bbs [dhq] teaching hospital abbottabad from april 2006 to sept 2009 including a follow up period of eighteen months. eighty patients, who were diagnosed on the basis of positive finkelstein test, were included in this study. all patients had severe pain materials and methods introduction in a busy outpatient department of orthopedics or rheumatology we come across with a lady having a sore thumb and wrist, almost on daily basis. it was fritz de quervain, a swiss surgeon who in 1895, first described this condition as a tenosynovitis 1 of the first dorsal compartment. most of patients suffering from this problem first try different things either at their own or on the advice of their gp's ranging from natural to physical therapies and nsaid etc. but to their dismay most of these techniques fail and they have to seek the advice of specialist doctor. surgical release of the stenosing sheath is a good option in resistant de quervain's cases. although performed as a day case, it requires about two weeks for complete recovery, besides it is much costly and a s s o c i a t e d w i t h a n u m b e r o f 2,3 complications. surgical treatment is often chosen without careful consideration and ------------------------------------------------correspondence: dr. syed qasim mehmood assistant prof of orthopedics women medical college abbottabad email: sqmehmood@hotmail.com efficacy of intrasheath steroid injection in treating de quervain's disease syed qasim mehmood, johar ali, asif saeed, muhammad nawaz, rifat latif 88 which was interfering their daily activities for more than four weeks. besides other methods of treatment like rest, splints, physiotherapy and nsaids had not given much benefit. all patients with a history of rheumatoid arthritis, trauma or local infection were excluded. we injected 40 mg of methyl prednisolone mixed with one ml of lignocaine 2% in a 3 cc syringe. instead of giving vertical injections we bent the needle at 45 degrees and passed it from distal end proximally parallel to the involved tendon in the first dorsal compartment of wrist. slight ulnar deviation made it easy. intra sheathal status of the needle was ensured by the need of less force to push the syringe and absence of swelling in subcutaneous tissue. all patients were asked to report after two weeks if no complications occurred. success was measured by absence of pain on wrist movements and a negative finkelstein`s test. second injection was given after three weeks in patients who showed less improvement or recurrence. a third injection was given after another three weeks in cases who had still not responded to the treatment. out of 80 patients, 72 were considered to be cured as they remained symptom free after 18 months of follow up. in 75% of these cases only one injection was used (table ii). in others a second injection was given after 03 weeks. out of 80 patients, those who reported with recurrence even after second injection, a third injection has to be given i.e., 08 patients (10%). of these 08 cases, 02 finally underwent surgical release. the fear and doubts about injection were removed, to a large extent by proper counseling. after injection, 90% of patients were satisfied and ready to accept it again if required. slight increase in pain was reported by 70% of patients which however improved over a week in patients who were later declared results cured. depigmentation at the injection site was reported in five patients and atrophy of fat in subcutaneous tissue was seen in one patient only. no tendon rupture, infection or injury to radial nerve was seen. slight increase in pain was reported by 70% of patients which however improved over a week in patients who were letter declared cured depigmentation at the injection site was reported in five patients and atrophy of fat in subcutaneous tissue was seen in one patient only. no tendon rupture, infection or injury to radial nerve was seen. owing to de quervain's tenosynovitis common occurrence, there must be some treatment guidelines and recommendations so as to save time and cost. the brigham and women's hospital guidelines for treatment of de quervain's tenosynovitis, state that corticosteroid injection may be very helpful and that they should be considered if symptoms persist beyond 6 weeks of 1 2 conservative treatment. whereas orthopedic text books recommend corticosteroid injection for de quervain's tenosynovitis after 2 weeks of conservative 1 3 t re a t m e n t h a s f a i l e d . u p t o d a t e recommends steroid injection if pain persists for more than 2 to 6 weeks despite 14 splinting , icing and nsaid therapy. a pooled quantitative literature search concerning the treatment of de quervain's tenosynoritis compared 07 studies (a total of 459 wrists of the 226 cases) treated with steroid injection alone 83% were cured, though 30 of these needed a second injection. sixty one percent of those treated with injection and splint were cured, while 7 14% treated with splint alone reported cure. in another retrospective study comparing injection with splinting and non steroidal anti inflammatory drugs nsaids, authors discussion 89 stratified patients into minimal, mild, or moderate to severe, groups based on their severity of disease. of those cases treated with splinting and nsaids, 15 of 17 in the minimal group had resolution of symptoms, but only 4 of 20 in the mild group and 2 of 8 in the moderate to severe group had symptoms resolved. the injection group showed better results with 100% of cases in the minimal to mild groups resolving and 76% of those in the more severe group resolving completely with an additional 7% 15 reporting improvement. to make steroids more effective, injection needs to be properly placed in the tendon 16 compartment i.e. intra sheath. the efficacy can be enhanced and compilation rate reduced to almost nil by ultrasound guided 17 injections of steroids into tendon sheaths. however an earlier prospective study of 103 patients found suprafibrous injection to be easier to perform than intrasynovial 18 injection and to have the same effects. in our study we gave intra sheath injections and did not find it much difficult, besides it can also avoid potential complications of leakage of steroids in subcutaneous tissue or damage to superficial radial nerve by misplaced needle. we slightly modified the original technique by bending the needle to almost 45 degree and inserting it beneath the 19 tendons along there line proximately. an orthopedics study compared different techniques for injection and found that two point injections vertically in the indurated 5 area is better than injection at one point. but we think if it is properly placed in the sheath or compartment, it does not matter even if there is a septum with in the first dorsal 20, 21 compartment of wrist. this septum would be more effective than surgical as compared to injection as injection liquid would spill over or will he absorbed in the vicinity as well that is why taras js et. al., concluded that exact location of injection into the sheath may not be important in the 22 treatment of trigger digits. surgical release is not a bad option in de quervain's disease but it is not fair to choose an option which is invasive, costly and not without some serious complications, particularly when a simple injection of 23 steroids can cure almost 80-90% of cases. however in chronic cases with much thickening of the sheath and those not responding to repeated local injection can be treated by surgical release with good long 24, 25 term relief. intrasheath steroid injection is a safe, very useful and cost effective method in treating de quervain`s disease. conclusion table i: demographic data of study population (n= 80) table ii: number of injections and their response in the treatment of de quervain's disease (n= 80) 90 references 1. lapidus pw. stenosing tenosynovitis. surg clin north 1953:33:131747. 2. froimson a. tenosynovitis and tennis elbow. in: green dp (ed) operative hand surgery vol 2 3rd edn. churchill livingstone. new york: 1993. pp1989-2006. 3. tak t, eidelman d, thomson jg. patient satisfaction and outcomes of surgery for de quervain's tenosynovitis. j hand surg1999; 24: 1071-7. 4. ahuja nk, chung kc, fritz de quervain. stenosing tendovaginitive at the radial styloid process. hand surg 2004; 29:1164-70. 5. sawaizumi t, nanno m, ito h, de quervain's disease: efficacy of intrasheath triamcinolone injection. int orthop 2007; 31: 265-8. 6. harvey fj, harvey pm. de quervain's disease: surgical or non surgical treatment j hand surg 1990; 15:83-7. 7. richie ca iii, eriner ww jr. corticosteroid injection for treatment of de quervain's tenosynovitis a pooled quantitative literature evaluation. j am board fam pract 2003; 16:102-6. 8. weiss ac, abeiman e, tabatabai m. treatment of de quervain's disease. j hand surg 1994; 19:595-8. 9. zingas c, failla jm, holsbeeb mv. injection accuracy of clinical relief of de quervain's tendinitis. j hand surg 1998; 23: 89-96. 10. tallia. am fam physician 2003; 67:745-50. 11. avei. j hand surg 2002; 27:322-4. 12. brigham and women's hospital upper extremity musculo skeletal disorders: a guide to prevention, diagnosis and treatment. boston, mass: brigham and women's hospital 2003. 13. brinker mr, millor md. the adult wristin: fundamental of orthopeadics. philadelphia, pa: wb saunders; 1999: 179-95. 14. anderson bc, sheon rp. de quervain's tenosynovitis up to date [on line data base] (updated may 7, 2004) waltham, mass: up to date; 2004. 15. lane lb, boretz rs, stuchin sa. treatment of de quervain's disease: role of conservative management. j hand surgery 2001; 26:258-60. 16. ilyas am, astm, schaffer aa. de quervain's tenosynovitis of the wrist. j am acad orthop surg 2007; 15: 757-64. 17. jeyapalen k, chowdhary s. ultrasound guided injection of triamcinolne and bupivicaine in the management of de quervain's disease. skeletal radiol 2009; 38:1099-103. 18. apimonbutr p, budhraje n. suprafibrous injection with corticosteroid in de quervain's disease. j med assoc thai 2003; 86: 232-7. 19. lennard ta. fundamentals of procedural care. in : lannard ta , ed. physiatric procedures. in clinical practice. philadelphia, pa: hanley and belfus; 1995:1-13. 20. jachson wt, viegas sf, coon j mol. anatomical variation in the first extensor compartment of wrist: clinical and anatomical study. j bone joint surg 1986; 68:923-6. 21. leslie bm, ericson wb jr, more head jr. incidence of a septum with in the first dorsal compartment of the wrist. j hand surg 1990; 15:88-91. 22. taras js, rapheal js, pan wt. corticosteroide injections for trigger digits: is intra sheath injection necessary? j hand surg 1998; 23: 8891. 23. anderson bc, manthey r, mathew cb. treatment of de quervain's tenosynovitis with corticosteroids. a prospective study of response to local injection. arthritis rheum 1991; 34:7936. 24. scheller a, schuh r, honle w. long term results of surgical release of de querrain's stenosing tenosynovitis. int orthop 2009; 33: 1301-3. 25. a kt, eidelman d, thomson jg. patient satisfaction and outcomes of surgery for de quervain's tenosynovitis. j hand surg 1999; 24: 1071-7. dr [page 90] [dermatology reports 2011; 3:e40] infliximab-induced intertriginous psoriasis in patient with crohn’s disease federica mola, alberico motolese department of dermatology, circolo hospital and macchi foundation, varese, italy abstract tumor necrosis factor-α (tnfα) inhibition is an effective treatment of moderate-to-severe psoriasis and other diseases (rheumatoid arthritis, ankylosing spondylitis, psoriasis or crohn’s disease). we report a case of a 32years-old patient affected by crohn’s disease since the age of 25 who started infliximab infusion after four years of treatment with prednisone and azathioprine per os without improvement. after the fifth infusion of infliximab, he developed a form of intertriginous psoriasis which was approached with topical steroid cream. the patient never presented psoriasis in the past. new onset of psoriasis in patients without history for skin diseases (as in our case) is a quite uncommon complication of tnfα inhibitor therapy. the increased production of ifnα during tnfα inhibitor therapy is a possible pathophysiologic explanation for this paradoxical effect of the anti-tnfα. introduction tumor necrosis factor-α (tnfα) is a proinflammatory cytokine produced by different cell types (activated t lymphocytes, keratinocytes, langerhans cells, endothelial cells, cardiac myocytes, adipose tissue etc.) and is involved in the pathogenesis of psoriatic skin lesions. tnfα inhibitors have become established agents in the treatment of inflammatory diseases and have shown to be of great benefit in many inflammatory diseases (rheumatoid arthritis, ankylosing spondylitis, psoriasis or crohn’s disease).1 case report we report a case of unexpected induction of psoriasis due to the use of intravenous tnfα inhibitor. a 32-years-old male patient with recalcitrant crohn’s disease of the ileum and descending colon (treated without improvement with prednisone, mesalazine and azathioprine per os) started treatment with infliximab at the dose of 5 mg/kg at the week 0, 2, 6 and afterwards every 14 weeks. after the fifth infusion he developed erythematous patches with peripheral scaling in the axillary folds and inguinal areas, suggesting the diagnosis of flexural psoriasis (figure 1). the face and the neck also presented a form of sebopsoriasis (figure 2). the patient never had psoriasis in the past, and he did not have a familiar history of any skin disease. no signs of infection were shown. the skin biopsy showed psoriasiform hyperplasia, papillary dermal edema with parakeratosis and intracorneal microabscesses of neutrophils (figure 3). the infliximab infusion was continued (seeing the good response of crohn’s disease) and a clinical skin improvement was achieved after 40 days of topical steroid treatment. an expanding literature of experience with anti tnfα associated psoriasis is providing abundant information about this paradoxical effect. many cases are described. the first published report of this association appeared in 2004 and concerned the development of symmetrical psoriasiform plaques in a patient treated with infliximab for crohn’s disease.2 subsequently plaque, guttate, and pustolar psoriasis have all been noted, and palmoplantar pustolar disease appears to be more common than idiopathic psoriasis, accounting for up to the 50% of reported cases. flexural psoriasis and sebopsoriasis are a rare form of presentation. in fact, to our knowledge, ther are only two articles describing cases of flexural psoriasis during infliximab treatment for crohn’s disease.3,4 dermatology reports 2011; volume 3:e40 correspondence: federica mola, department of dermatology, circolo hospital and macchi foundation, viale borri 75, varese, italy. tel. +39.349.2611869. e-mail: federicamola@yahoo.it key words: tumor necrosis factor−α, psoriasis, crohn’s disease. received for publication: 6 june 2011. accepted for publication: 12 september 2011. this work is licensed under a creative commons attribution noncommercial 3.0 license (cc bync 3.0). ©copyright f. mola and a. motolese, 2011 licensee pagepress, italy dermatology reports 2011; 3:e40 doi:10.4081/dr.2011.e40 figure 3. histologic evaluation demonstrates psoriasiform hyperplasia, papillary dermal edema with parakeratosis and intracorneal microabscesses of neutrophils. figure 1. erythematous patches with peripheral scaling in the axillary folds, suggestive for the diagnosis of flexural psoriasis figure 2. typical erithemato-desquamative patches on the face and neck in sebopsoriasis. no nco mm er cia l u se on ly [dermatology reports 2011; 3:e40] [page 91] it is well recognized that blocking tnfα may actually favour specific autoimmune phenomena and may activate autoreactive t cells. in addition, with particular relevance to the skin, it may upregulate interferon (ifn)-α activity.5 immunologically this is not unexpected because tnfα is known to negatively regulate the maturation and function of plasmocytoid dendritic cells, which are the major source of ifn-α. therapeutic inhibition of tnfα signaling would increase ifnα activity and could trigger psoriasis in genetically susceptible individuals.5 on the other hand, some cases can be diagnosed as an adverse drug reaction and may contribute to stop the treatment. in literature two-thirds of patiens who simply continue anti tnfα therapy improve or resolve the skin disease with steroid treatment. the decisions need to be based on individual circumstances as the extent and severity of the disease, the efficacy of the anti tnfα in treating the condition for which it was initiated and the availability of realistic therapeutic alternatives.5 references 1. wollina u, hansel g, koch a, et al. tumor necrosis factor-alpha inhibitor-induced psoriasis or psoriasiform exanthemata: first 120 cases from the literature including a series of six new patients.am j clin dermatol 2008;9:1-14. 2. baeten d, kruithof e, van den bosch f, et al. systematic safety follow up in a cohort of 107 patients with spondyloarthropathy treated with infliximab: a new perspective on the role of host defence in the pathogenesis of the disease? ann rheum dis 2003; 62:829-34 3. peramiquel l, puig l, dalmau jricart e, et al. onset of flexural psoriasis during infliximab treatment for crohn's disease. clin exp dermatol 2005; 30:713-4. 4. avila alvarez a, garcia-alonso l, solar boga a, garcia-silva j. flexural psoriasis induced by infliximab and adalimumab in a patient with crohn's disease. an pediatr (barc) 2009; 70:278-81. 5. shale m, ghosh s. learning the lessons of antitumour necrosis factor therapy-associated psoriasis. can j gastroenterol 2009; 23:674-6. case report no nco mm er cia l u se on ly iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 9 neutrophil / lymphocyte ratio is not correlated with disease activity in rheumatoid arthritis patients ehab m. mikhael *,1 and turathn.ibrahim ** * department of clinical pharmacy college of pharmacy university of baghdad, baghdad ,iraq. ** department of clinical pharmacy college of pharmacy al-mustansria university, baghdad, iraq. abstract rheumatoid arthritis is a chronic systemic inflammatory disease. inflammation leads to joint damage and increases the risk of cardiovascular diseases. neutrophil lymphocyte ratio (nlr) is a measure of inflammation in many diseases. therefore, we aimed to evaluate the usefulness of nlr to detect inflammation in ra, and its correlation to ra disease activity indices and some hematological parameters. a cross-sectional study involving 24 patients with active rheumatoid arthritis (ra) who are using mtx participated in this study. all patients were clinically evaluated using disease activity score of 28 joints (das28) and simplified disease activity index (sdai), whereas functional disability was assessed by health assessment questionnaire disability index (haqdi); moreover, blood specimen of each patient was used for measuring erythrocyte sedimentation rate (esr), c – reactive protein (crp), rheumatoid factor (rf), hemoglobin (hb), white blood cells (wbc) count, platelets and red blood cells (rbcs) count, and nlr ratio.nlr was positively correlated with esr and inversely correlated with hb, but it didn’t show any correlation with other clinical and laboratory parameters. in conclusion nlr is less correlated with inflammation and not suitable to monitor disease activity in ra patients using mtx. keywords: rheumatoid arthritis, inflammation, neutrophil lymphocyte ratio. نسبة كريات الدم البيط العدلة إلى كريات الدم البيط اللوفاوية الترتبط هع فاعلية الورض عند الوصابين بالتهاب الوفاصل الروهاتىيدي ايهاب هضر هيخائيل ،*1 وتراث نبيل ابراهين ** * انؼشاق.تغذاد،فشع انصٛذنح انسشٚشٚح ،كهٛح انصٛذنح ،جايؼح تغذاد ، ** انؼشاق .تغذاد،، انجايؼح انًسرُصشٚحفشع انصٛذنح انسشٚشٚح ، كهٛح انصٛذنح ، الخالصة انرٓاب انًفاصم انشٔياذٕٚذ٘ يشض انرٓاتٙ يزيٍ ْٔزا االنرٓاب ٚؤد٘ إنٗ ذهف انًفصم ٔكًا ٚزٚذ يٍ خطٕسج األيشاض انمهثٛح نك انٕػائٛح. إٌ َسثح كشٚاخ انذو انثٛط انًؼرذنح إنٗ كشٚاخ انذو انثٛط انهًفأٚح ٚؼرثش لٛاسا نُسثح االنرٓاب فٙ كثٛش يٍ األيشاض. ٔنز فائذج ْزِ انُسثح نمٛاس يسرٕٖ االنرٓاب ػُذ يشظٗ انرٓاب انًفاصم انشٔياذٕٚذ٘ ٔػاللح ْزِ انُسثح تفاػهٛح يشض كاٌ ْذفُا ذمٛٛى يصاتا تانرٓاب انًفاصم انشٔياذٕٚذ٘ ٔانزٍٚ ٚسرؼًهٌٕ 42انرٓاب انًفاصم انشٔياذزيٙ ٔتؼط انًؼاٚٛش انذيٕٚح. شًهد انذساسح ٔيهحك فاػهٛح انًشض (das28)يفصم 42شٚشٚا تٕاسطح يؼٛاس فاػهٛح انًشض ل انًٛثٕذشكسٛد. ذى ذمٛٛى جًٛغ انًشظٗ س يهحك انؼجز. إظافح نزنك ػُٛاخ انذو سحثد نمٛاس –, أيا يمذاس اإلػالح فرى ذمًّٛٛ تٕاسطح اسرثٛاٌ ذمٛٛى انصحح (sdai)انًثسط َسثح ذشسة كشٚاخ انذو انحًش, تشٔذٍٛ سٙ انفؼال, يمٛاس انشٔياذٕٚذ, انًٕٓٛغهٕتٍٛ, ػذد كشٚاخ انذو انحًش ٔانثٛط ٔانصفٛحاخ ط انؼذنح إنٗ كشٚاخ انذو انثٛط انذيٕٚح ٔ َسثح كشٚاخ انذو انثٛط انؼذنح إنٗ كشٚاخ انذو انثٛط انهًفأٚح. إٌ َسثح كشٚاخ انذو انثٛ انهًفأٚح كاٌ نٓا ػاللح اٚجاتٛح يغ َسثح ذشسٛة كشٚاخ انذو انحًش ٔػاللح ػكسٛح يغ انًٕٓٛغهٕتٍٛ ٔنكُٓا نى ذظٓش أ٘ ػاللح يغ تالٙ أٚح ذشذثط تؼاللح ظؼٛفح انًؼاٚٛش انسشٚشٚح ٔانًخرثشٚح. ٚسرُرج يٍ رنك إٌ َسثح كشٚاخ انذو انثٛط انؼذنح إنٗ كشٚاخ انذو انثٛط انهًف يغ االنرٓاب ٔغٛش يالئًح نًراتؼح فاػهٛح انرٓاب انًفاصم انشٔياذٕٚذ٘ نهًشظٗ انزٍٚ ٚسرؼًهٌٕ يٛثٕذشكسٛد. . كريات الدم البيط العدلة الى كريات الدم البيط اللوفاويةالكلوات الوفتاحية: التهاب الوفاصل الروهاتىيدي, التهاب, نسبة introduction rheumatoid arthritis (ra) is a chronic systemic inflammatory disease of unknown etiology that characterized by both articular and extra articular features (1) . local inflammation of the joints is correlated to joint damage (2) whereas systemic inflammation increases the risk of atherogenesis and coronary heart disease in ra patients (3) . several studies have explored the relationship between systemic inflammation and cardiovascular mortality (4, 5) .systemic inflammation can be measured by using a variety of biochemical and hematological markers (6) crp is a strong predictor of future 1 corresponding author e-mail:ehab_pharma84@yahoo.com received:3/11/2012 accepted:19/5/2013 iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 10 cardiovascular events in individuals both with and without overt cardiovascular disease (cvd) (7) . additionally, crp correlates directly with the presence of atherosclerosis in patients with ra (8) whereas; esr is significantly associated with the risk of cvd in ra (9). neutrophil lymphocyte ratio (nlr) is an important measure of systemic inflammation as it is readily available and could be calculated easily (10) . many studies found that nlr is a useful measure to detect inflammation and predict long term outcomes in patients with renal failure, cancer and heart diseases (11 -13) . aim of the study to evaluate the usefulness of nlr to detect inflammation in ra, and its correlation to various ra disease activity indices and some hematological parameters. patients and methods a cross-sectional study was conducted in baghdad teaching hospital, rheumatology unit from december 2011 to may 2012. a total of 24 patients (7 males and 17 females) with active ra were involved in this study. patients were diagnosed to have ra by the rheumatologist according to american college of rheumatology (acr) classification criteria for ra (14) . all patients included in the study signed an informed consent form according to the declaration of helsinki. ethical approval was obtained from the ethics committee of baghdad university, college of medicine, department of medicine. patients with diseases other than rheumatoid arthritis were excluded from the study. demographic data of patients were reported regarding their age, duration of the disease, and medication history (table 1). laboratory investigations blood specimens were taken from all patients. hematological investigations that include complete blood count (wbc, rbc and platelet), differential wbc count and hb were measured using hematology auto analyzer (ruby – cell – dyn 08h56 – 02) from abbott company usa. esr was measured by westergren method (15) . crp was measured semi quantitatively according to method of singer et al using serial dilutions of serum; each dilution was mixed with a latex reagent and observed for the presence of agglutination (16) using a ready made kit (agapee, switzerland) whereas rf was measured qualitatively (16) by a ready made kit (spectrum, egypt). clinical evaluation the 28 joints included bilateral knees, shoulders, elbows,wrists, metacarpophalangeal and proximal interphalangeal joints, were palpated to count the number of tender and swelling joints. the patients were asked to mark on the vas of 0 – 100mm according to their global assessment of their general health and pain. the physician marked on the vas of 0-10 cm according to the physician global assessment of the disease activity. disease activity was measured by both das28 and sdai. das28 was calculated from the tjc, sjc and esr according to the following formula [17]: das28 = {(0.56 • √[tjc28]) + (0.28 • √[sjc28]) + (0.70 • in[esr])} • 1.08 whereas sdai was calculated by the following formula [18]: sdai = crp + tjc + sjc + vas (0-10) + ega (0-10)functional status of the patients was measured using health assessment questionnaire disability index [19]. additionally morning stiffness of each patient was calculated according to patient approximate. statistical analysis spss version 12 was used for data input and analysis. shapiro wilk test (web version) used to check if data is normally or abnormally distributed. spearman correlation coefficient was used to assess the correlation between abnormally distributed continuous variables. all p values used were asymptotic and two sided. values with p < 0.05 were considered significant. results table (1) showed general demographic data for the patients that participated in this study. table (2) showed the values (mean ± sd) for the different studied variables, when these valuescorrelated, with neutrophil/lymphocyte ratio there was a significant positive correlation with esr (r = 0.495, p = 0.014) and a significant negative correlation with hb (r = -0.426 , p = 0.034), crp has a weak positive correlation that didn’t achieve statistical significance , whereas other parameters didn’t show a significant correlation with nlr (table 3). iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 11 table (1): general demographic data of the patients parameter patients with moderate disease activity patients with high disease activity all participated patients age, years mean  sd 5112.11 45.2411.08 46.9211.44 female percent 14.85 88.24 66.67% disease duration, years mean  sd 6.714.57 5.885.16 6.134.91 drug used (mtx/hcq) (5/2) 17/0 (22/2) rf positive n (%) 5 (71.4) 9 (52.94) 14 (58.33) smoking percent 42.86 0 12.5% mtx = methotrexate; hcq = hydroxychloroquine; sd = standard deviation; rf = rheumatoid factor. table (2): clinical and laboratory parameters in ra patients participated in table (3): correlation of different parameters with nlr the study jc = tender joint count; sjc = swelling joint count; vas = visual analogue scale; ega = evaluator global assessment; das28 = disease activity score 28 joints; sdai = simplified disease activity score; crp = c – reactive protein; esr = erythrocyte sedimentation rate; rf = rheumatoid factor; hb = hemoglobin; wbc =white blood cells; rbc = red blood cell haqdi = health assessment questionnaire disability index. jc = tender joint count; sjc = swelling joint count; vas = visual analogue scale; ega = evaluator global assessment; das28 = disease activity score 28 joints; sdai = simplified disease activity score; crp = c – reactive protein; esr = erythrocyte sedimentation rate; rf = rheumatoid factor; hb = hemoglobin; wbc =white blood cells; rbc = red blood cell haqdi = health assessment questionnaire disability index. parameter correlation coefficient p value tjc -0.001 0.997 sjc 0.051 0.813 vas 0.179 0.404 ega 0.232 0.276 morning stiffness -0.139 0.516 das28 0.351 0.093 sdai 0.150 0.484 crp (mg/dl) 0.368 0.077 esr (mm/hr) 0.495 0.014 hb ( gm/dl) -0.435 0.034 wbc (cell/ nano liter) 0.306 0.146 rbc (cell/ nano liter) -0.084 0.698 platelet (cell/ nano liter) 0.339 0.105 haqdi 0.146 0.496 parameter value (mean  sd) tjc 10.544.53 sjc 5.133.03 vas 54.1725.70 ega 5.132.35 morning stiffness 26.238.1 das28 5.431.46 sdai 27.9213.62 crp ( mg/dl) 1.652.34 esr ( mm/hr) 3822.14 rf 0.580.50 hb ( g/dl) 12.281.62 wbc (cell/ nano liter) 9.753.27 rbc (cell / nano liter) 4.670.76 platelet (cell/ nano liter) 28887.82 haqdi 1.360.74 iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 12 additionally table (4) showed that nlr not differ significantly between highly active and moderately active ra (p = 0.07), while esr, crp and das28 values varied significantly (p<0.05) between moderately and highly active ra patients. table (5) showed a highly positive correlation between vas and ega. table (4): comparison of some parameters between patients with highly active ra and those with moderately active ra parameter patients with moderate ra disease activity (7) patients with high ra activity ( 17) p value nlr 2.110.46 3.171.68 0.070 das28 3.851.49 6.080.85 0.000 crp 0.430.90 2.152.57 0.047 esr 18.8614.96 45.8819.86 0.004 nlr= neutrophil lymphocyte ratio; das28 = disease activity score of 28 joints; crp = c – reactive protein; esr = erythrocyte sedimentation rate table (5): correlation between visual analogue sale (vas) and evaluator global assessment (ega) disease activity number of cases vas ega spearman correlation coefficient p value moderate 7 31.4320.35 27.1418.90 0.924 0.003 high 17 63.8921.18 60.5616.97 0.795 0.000 moderate and high 24 54.825.35 51.222.97 0.867 0.000 discussion this study showed that nlr as a measure of inflammation in ra patients was positively correlated with esr, similar finding was observed when nlr was evaluated as a measure of inflammation in ulcerative colitis patients (20) . moreover, nlr was inversely correlated with hb, however, there is no any study in this respect and it is the 1 st time to get such result. this finding is acceptable since hb in ra patients is inversely correlated with inflammation and ra disease activity (21) . regarding crp, there is a modest but a non significant correlation with nlr, there is an agreement of this finding in a trial that followed up patients with cancer (22) . according to the above, and since nlr is well correlated with esr and not well correlated with crp, so nlr may be not sufficient laboratory test to predict cvd risk in ra patients and further studies areneeded to evaluate the association of nlr with the severity and extent ofcoronary atherosclerosis. results from the current study also showed that nlr didn’t correlate with ra clinical parameters like tender joint count, swelling joint count and haqdi, this result may be rationale since it has been found that many of clinical parameters that were used to diagnose ra like tjc, sjc, morning stiffness and haqdi were less correlated with inflammation (23-25). this study showed a direct positive correlation between vas and ega and also showed that there is no correlation between vas and ega with nlr. this finding can be explained according to the finding of naredoetal (24) at which vas was not correlated with inflammation ( especially with esr) and since this study showed that nlr was directly correlated with esr, so it can be concluded that there is no correlation between vas or ega with nlr . additionally hematological parameters like wbc, rbc and platelets count were not correlated with nlr. there was very complex hematological parameters in active ra patients that use mtx who participated in this study, since active ra disease is associated with anemia, leucocytosis and thrombocytosis (26,27) , whereas mtx may causes neutropenia and thrombocytopenia (28) . consequently absence of correlation between nlr and hematological parameters may result from the unsuitable patient sample selection and further studies are needed to correlate between nlr and hematological parameters in ra patients using dmards other than mtx. iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 13 finally, results from the current study showed that nlr is not correlated with ra disease activity as measured by either das28 or sdai; similarly fauziaimtiazet al also found that there was a non significant relationship between nlr and rheumatoid arthritis (29). this finding was strengthened by absence of statistical difference in nlr between patients with moderately and those with highly active ra, and only crp and esr showed a significant difference between the 2 groups of patients similar to that of das28; consequently esr and crp is better than nlr to detect ra disease activity. conclusion nlr is less correlated with inflammation and not suitable to monitor disease activity in ra patients using mtx. reference 1. manole cojocaru, inimioara mihaela cojocaru, isabela silosi , et al. extraarticular manifestations in rheumatoid arthritis. maedica (buchar) 2010; 5(4): 286–91. 2. conaghan pg, o'connor p, mcgonagle d, et al. elucidation of the relationship between synovitis and bone damage: a randomized magnetic resonance imaging study of individual joints in patients with early rheumatoid arthritis. arthritis rheum. 2003;48(1):64-71. 3. naveedsattar, david w. mccarey, hilary capell, et al. explaining how “highgrade” systemic inflammation accelerates vascular risk in rheumatoid arthritis. circulation. 2003;108:2957-63. 4. folsom ar, wu kk, rosamond wd, et al. prospective study of hemostatic factors and incidence of coronary heart disease: the atherosclerosis risk in communities (aric) study. circulation. 1997 aug 19;96(4):1102-8. 5. 5 . folsom ar, aleksic n, catellier d, et al. c-reactive protein and incident coronary heart disease in the atherosclerosis risk in communities (aric) study. am heart j 2002;144(2):233-8. 6. pallinti v, ganesan n, anbazhagan m, rajasekhar g.serum biochemical markers in rheumatoid arthritis. indian j biochembiophys. 2009;46(4):342-4. 7. michael b. clearfield. c-reactive protein: a new risk assessment tool for cardiovascular disease. j am osteopathassoc. 2005 ;105(9):409-16. 8. gonzalez-gay ma, gonzalez-juanatey c, piñeiro a, et al.high-grade c-reactive protein elevation correlates with accelerated atherogenesis in patients with rheumatoid arthritis. j rheumatol 2005;32(7):1219–23. 9. myasoedova e, crowson cs, kremers hm, et al. lipid paradox in rheumatoid arthritis: the impact of serum lipid measures and systemic inflammation on the risk of cardiovascular disease. ann rheum dis. 2011;70(3):482-7. 10. zahorec r. ratio of neutrophil to lymphocyte counts rapid and simple parameter of systemic inflammation and stress in critically ill . bratislleklisty 2001; 102 (1): 5–14. 11. turkmen k, guneyi, yerlikaya fh, tonbul hz. the relationship between neutrophilto-lymphocyte ratio and inflammation in end-stage renal disease patients. ren fail. 2012 ; 34(2):155-9. 12. tomita m, shimizu t, ayabe t, onitsuka t.elevated preoperative inflammatory markers based on neutrophil-tolymphocyte ratio and c-reactive protein predict poor survival in resected nonsmall cell lung cancer. anticancer res. 2012;32(8):3535-8. 13. uthamalingam s, patvardhan ea , subramanian s , et al. utility of the neutrophil to lymphocyte ratio in predicting long-term outcomes in acute decompensated heart failure. .am j cardiol. 2011;107(3):433-8. 14. arnett fc, edworthy sm, bloch da, et al. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum 1988; 31(3):31524. 15. jou jm, lewis sm, briggs c, et al . international council for standardization in haematology. review of the measurement of the erythrocyte sedimentation rate. international journal of laboratory hematology 2011; 33(2): 125–32. 16. singer, j.m. c.m., plotz, e. parker & s.k. elster. amer. j. clin. path 1957;28: 611. 17. prevoo ml, van 't hof ma, kuper hh, et al.modified disease activity scores that include twenty eightjoint counts . development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. arthritis rheum 1995; 38(1):44–8. http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bm%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bim%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bim%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20silosi%2bi%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed?term=conaghan%20pg%5bauthor%5d&cauthor=true&cauthor_uid=12528105 http://www.ncbi.nlm.nih.gov/pubmed?term=o'connor%20p%5bauthor%5d&cauthor=true&cauthor_uid=12528105 http://www.ncbi.nlm.nih.gov/pubmed?term=mcgonagle%20d%5bauthor%5d&cauthor=true&cauthor_uid=12528105 http://www.ncbi.nlm.nih.gov/pubmed?term=mcgonagle%20d%5bauthor%5d&cauthor=true&cauthor_uid=12528105 http://www.ncbi.nlm.nih.gov/pubmed/12528105 http://www.ncbi.nlm.nih.gov/pubmed/12528105 http://www.ncbi.nlm.nih.gov/pubmed/9286936 http://www.ncbi.nlm.nih.gov/pubmed?term=pallinti%20v%5bauthor%5d&cauthor=true&cauthor_uid=19788068 http://www.ncbi.nlm.nih.gov/pubmed?term=ganesan%20n%5bauthor%5d&cauthor=true&cauthor_uid=19788068 http://www.ncbi.nlm.nih.gov/pubmed?term=anbazhagan%20m%5bauthor%5d&cauthor=true&cauthor_uid=19788068 http://www.ncbi.nlm.nih.gov/pubmed?term=anbazhagan%20m%5bauthor%5d&cauthor=true&cauthor_uid=19788068 http://www.ncbi.nlm.nih.gov/pubmed?term=rajasekhar%20g%5bauthor%5d&cauthor=true&cauthor_uid=19788068 http://www.ncbi.nlm.nih.gov/pubmed/19788068 http://www.ncbi.nlm.nih.gov/pubmed/19788068 http://www.jaoa.org/search?author1=michael+b.+clearfield&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed/16239491 http://www.ncbi.nlm.nih.gov/pubmed/16239491 http://www.ncbi.nlm.nih.gov/pubmed?term=gonzalez-gay%20ma%5bauthor%5d&cauthor=true&cauthor_uid=15996055 http://www.ncbi.nlm.nih.gov/pubmed?term=gonzalez-juanatey%20c%5bauthor%5d&cauthor=true&cauthor_uid=15996055 http://www.ncbi.nlm.nih.gov/pubmed?term=gonzalez-juanatey%20c%5bauthor%5d&cauthor=true&cauthor_uid=15996055 http://www.ncbi.nlm.nih.gov/pubmed?term=pi%c3%b1eiro%20a%5bauthor%5d&cauthor=true&cauthor_uid=15996055 http://www.ncbi.nlm.nih.gov/pubmed?term=myasoedova%20e%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=crowson%20cs%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=kremers%20hm%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=kremers%20hm%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed/21216812 http://www.ncbi.nlm.nih.gov/pubmed/21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=turkmen%20k%5bauthor%5d&cauthor=true&cauthor_uid=22172001 http://www.ncbi.nlm.nih.gov/pubmed?term=guney%20i%5bauthor%5d&cauthor=true&cauthor_uid=22172001 http://www.ncbi.nlm.nih.gov/pubmed?term=yerlikaya%20fh%5bauthor%5d&cauthor=true&cauthor_uid=22172001 http://www.ncbi.nlm.nih.gov/pubmed?term=tonbul%20hz%5bauthor%5d&cauthor=true&cauthor_uid=22172001 http://www.ncbi.nlm.nih.gov/pubmed?term=tonbul%20hz%5bauthor%5d&cauthor=true&cauthor_uid=22172001 http://www.ncbi.nlm.nih.gov/pubmed/22172001 http://www.ncbi.nlm.nih.gov/pubmed?term=tomita%20m%5bauthor%5d&cauthor=true&cauthor_uid=22843942 http://www.ncbi.nlm.nih.gov/pubmed?term=shimizu%20t%5bauthor%5d&cauthor=true&cauthor_uid=22843942 http://www.ncbi.nlm.nih.gov/pubmed?term=ayabe%20t%5bauthor%5d&cauthor=true&cauthor_uid=22843942 http://www.ncbi.nlm.nih.gov/pubmed?term=onitsuka%20t%5bauthor%5d&cauthor=true&cauthor_uid=22843942 http://www.ncbi.nlm.nih.gov/pubmed?term=onitsuka%20t%5bauthor%5d&cauthor=true&cauthor_uid=22843942 http://www.ncbi.nlm.nih.gov/pubmed/22843942 http://www.ncbi.nlm.nih.gov/pubmed/22843942 http://www.ncbi.nlm.nih.gov/pubmed?term=uthamalingam%20s%5bauthor%5d&cauthor=true&cauthor_uid=21257011 http://www.ncbi.nlm.nih.gov/pubmed?term=patvardhan%20ea%5bauthor%5d&cauthor=true&cauthor_uid=21257011 http://www.ncbi.nlm.nih.gov/pubmed?term=subramanian%20s%5bauthor%5d&cauthor=true&cauthor_uid=21257011 http://www.ncbi.nlm.nih.gov/pubmed?term=subramanian%20s%5bauthor%5d&cauthor=true&cauthor_uid=21257011 http://www.ncbi.nlm.nih.gov/pubmed/21257011 http://www.ncbi.nlm.nih.gov/pubmed?term=arnett%20fc%5bauthor%5d&cauthor=true&cauthor_uid=3358796 http://www.ncbi.nlm.nih.gov/pubmed?term=edworthy%20sm%5bauthor%5d&cauthor=true&cauthor_uid=3358796 http://www.ncbi.nlm.nih.gov/pubmed?term=bloch%20da%5bauthor%5d&cauthor=true&cauthor_uid=3358796 http://www.ncbi.nlm.nih.gov/pubmed?term=jou%20jm%5bauthor%5d&cauthor=true&cauthor_uid=21352508 http://www.ncbi.nlm.nih.gov/pubmed?term=lewis%20sm%5bauthor%5d&cauthor=true&cauthor_uid=21352508 http://www.ncbi.nlm.nih.gov/pubmed?term=briggs%20c%5bauthor%5d&cauthor=true&cauthor_uid=21352508 http://www.ncbi.nlm.nih.gov/pubmed?term=international%20council%20for%20standardization%20in%20haematology%5bcorporate%20author%5d http://www.ncbi.nlm.nih.gov/pubmed?term=international%20council%20for%20standardization%20in%20haematology%5bcorporate%20author%5d http://onlinelibrary.wiley.com/doi/10.1111/ijlh.2011.33.issue-2/issuetoc http://onlinelibrary.wiley.com/doi/10.1111/ijlh.2011.33.issue-2/issuetoc http://www.ncbi.nlm.nih.gov/pubmed?term=prevoo%20ml%5bauthor%5d&cauthor=true&cauthor_uid=7818570 http://www.ncbi.nlm.nih.gov/pubmed?term=van%20't%20hof%20ma%5bauthor%5d&cauthor=true&cauthor_uid=7818570 http://www.ncbi.nlm.nih.gov/pubmed?term=kuper%20hh%5bauthor%5d&cauthor=true&cauthor_uid=7818570 iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 14 18. smolen js, breedveld fc, schiff mh, et al. a simplified disease activity index for rheumatoid arthritis for use in clinical practice. rheumatology (oxford). 2003 ;42(2):244-57. 19. bruce b, fries jf. the stanford health assessment questionnaire: a review of its history, issues, progress, and documentation. j rheumatol 2003; 30:16778. 20. torun s, tunc bd, suvak b, et al. assessment of neutrophil-lymphocyte ratio in ulcerative colitis: a promising marker in predicting disease severity. clin res hepatolgastroenterol. 2012 jul 26. 21. agrawal s, misra r, aggarwal a. anemia in rheumatoid arthritis high prevalence of iron-deficiency anemia in indian patients. rheumatol int. 2006;26(12):1091–5. 22. garcea g, ladwa n, neal cp, et al. preoperative neutrophil-to-lymphocyte ratio (nlr) is associated with reduced disease-free survival following curative resection of pancreatic adenocarcinoma. world j surg. 2011;35(4):868-72. 23. qvistgaard e, røgind h, torp-pedersen s , et al. quantitative ultrasonography in rheumatoid arthritis: evaluation of inflammation by dopplertechnique. ann rheumdis 2001;60(7):690–3. 24. naredo e, bonilla g, gamero f, et al.. assessment of inflammatory activity in rheumatoid arthritis: a comparative study of clinical evaluation with grey scale and power doppler ultrasonography. ann rheum dis 2005;64(3):375–81. 25. udit n verma, ramnathmisra, ram raj singh, s.s. agarwal, sitanaik. serological correlates of inflammation in rheumatoid arthritis: usefulness of acute phase reactants in monitoring disease activity. j indian rheumatolassoc 2002;10: 1 – 4. 26. aisha al-ghamdiand suzan m.(2009) attar. extra-articular manifestations of rheumatoid arthritis: a hospital-based study: ann saudi med., 29(3): 189–93. 27. syed km, pinals rs. leukocytosis in rheumatoid arthritis. j clinrheumatol 1996; 2(4): 197-202. 28. gutierrez-ureña s, molina jf, garcía co, et al. pancytopenia secondary to methotrexate therapy in rheumatoid arthritis. . arthritis rheum. 1996;39(2):272-6. 29. imtiaz f, shafique k, mirza ss, et al. neutrophil lymphocyte ratio as a measure of systemic inflammation in prevalent chronic diseases in asian population. int arch med. 2012; 26;5(1):2. http://www.ncbi.nlm.nih.gov/pubmed?term=smolen%20js%5bauthor%5d&cauthor=true&cauthor_uid=12595618 http://www.ncbi.nlm.nih.gov/pubmed?term=breedveld%20fc%5bauthor%5d&cauthor=true&cauthor_uid=12595618 http://www.ncbi.nlm.nih.gov/pubmed?term=schiff%20mh%5bauthor%5d&cauthor=true&cauthor_uid=12595618 http://www.ncbi.nlm.nih.gov/pubmed/12595618 http://www.ncbi.nlm.nih.gov/pubmed?term=torun%20s%5bauthor%5d&cauthor=true&cauthor_uid=22841412 http://www.ncbi.nlm.nih.gov/pubmed?term=tunc%20bd%5bauthor%5d&cauthor=true&cauthor_uid=22841412 http://www.ncbi.nlm.nih.gov/pubmed?term=suvak%20b%5bauthor%5d&cauthor=true&cauthor_uid=22841412 http://www.ncbi.nlm.nih.gov/pubmed/22841412 http://www.ncbi.nlm.nih.gov/pubmed/22841412 http://www.ncbi.nlm.nih.gov/pubmed?term=garcea%20g%5bauthor%5d&cauthor=true&cauthor_uid=21312035 http://www.ncbi.nlm.nih.gov/pubmed?term=ladwa%20n%5bauthor%5d&cauthor=true&cauthor_uid=21312035 http://www.ncbi.nlm.nih.gov/pubmed?term=neal%20cp%5bauthor%5d&cauthor=true&cauthor_uid=21312035 http://www.ncbi.nlm.nih.gov/pubmed/21312035 http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20al-ghamdi%2ba%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20attar%2bsm%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20attar%2bsm%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed?term=syed%20km%5bauthor%5d&cauthor=true&cauthor_uid=19078065 http://www.ncbi.nlm.nih.gov/pubmed?term=pinals%20rs%5bauthor%5d&cauthor=true&cauthor_uid=19078065 http://www.ncbi.nlm.nih.gov/pubmed/19078065## http://www.ncbi.nlm.nih.gov/pubmed?term=gutierrez-ure%c3%b1a%20s%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=molina%20jf%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=garc%c3%ada%20co%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=garc%c3%ada%20co%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed/8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=imtiaz%20f%5bauthor%5d&cauthor=true&cauthor_uid=22281066 http://www.ncbi.nlm.nih.gov/pubmed?term=shafique%20k%5bauthor%5d&cauthor=true&cauthor_uid=22281066 http://www.ncbi.nlm.nih.gov/pubmed?term=mirza%20ss%5bauthor%5d&cauthor=true&cauthor_uid=22281066 http://www.ncbi.nlm.nih.gov/pubmed/22281066 http://www.ncbi.nlm.nih.gov/pubmed/22281066 editorial rheuminations 1 2 prashant kaushik , aarya a. kaushik i'll be honest…it has been 35+ years since i started medical school [mbbs]. and in the realm of medicine at large, and immunology/rheumatology in specific, we are still struggling to find a 'cure' for chronic systemic inflammatory immune-mediated diseases also known as 'autoimmune rheumatic diseases (ard)'. rheumatology is still in its cradle having gotten the recognition as a subspecialty of medicine only in 1972. i have seen the field evolve in terms of understanding the orchestrated 'play' of the immune cells along with cytokines etc. over the past 3 decades, all of which has led to the concept and birth of 'biologic' disease modifying anti-rheumatic drugs [b-dmards]. the first b-dmard to get approved by the food and drugs administration [fda] was etanercept, a tnf-alpha receptor fusion protein in 1998. infliximab, a chimeric monoclonal antibody against tnf soon followed in 1999. ever since then, there has been a flurry of b-dmards including 3 more in the same family of tnfantagonists, 2 in the interleukin [il]-6 antagonist class, 1 blocker of the second co-stimulatory t-cell signaling: ctla-4ig, 3 il-1 antagonists, b-cell depleting chimeric monoclonal antibody directed against cd-20 etc. also, 3 oral janus-kinase inhibitors have joined the 'gang' and are called targeted synthetic dmards. i still remember the pre-b-dmard era when rip roaring rheumatoid arthritis was still around, and with my ustaad saheb (mentor), all what we had to offer pharmaceutically were the conventional synthetic [cs] dmards including methotrexate, hydroxychloroquine, sulfasalazine, leflunomide and [the younger generation can hold their breath] cyclosporine, d-penicillamine, chlorambucil…gold i n j e c t i o n s a n d e v e n c y c l o p h o s p h a m i d e ! st corticosteroids have been around since 1950! the 1 and “so far” […well, i/you might be next one!] the only nobel prize winning revelation in the realm of rheumatology. the availability of the b-dmards has changed the entire paradigm of pharmaceutical treatment in rheumatology. they are all quite effective yet extremely expensive making them literally unaffordable in the parts of the world, where healthinsurance coverage is suboptimal/non-existent, more so, given the indefinite duration of therapy. although the b-dmards tend to target a specific pathway in the immune system, they are still fraught with some rather challenging adverse drug effects (ade) including infections: bacterial, mycobacterial [especially reactivation of tuberculosis, more so, in the parts of the world where this disease is endemic] fungal and from other pathogens; malignancies, increased incidence of cardiovascular and cerebrovascular events, thromboembolic events etc. all in all, b-dmards are a double-edged sword! they all come with 'a price to pay'! now, let's reflect upon the burden of ard in the community. rheumatoid arthritis, a potentially crippling and life-threatening disease affects at least 1% of the population, with more than 13 million patients in america itself! axial and peripheral spondyloarthritis is now being recognized more and more. various other autoimmune diseases including systemic lupus erythematosus, sjogren's syndrome, scleroderma, autoimmune myopathy including dermatomyositis and polymyositis, vasculitides, and some more novel entities like igg-4 related disease, checkpoint-inhibitor chemotherapy associated rheumatic diseases: the list continues to grow! what can be done to prevent, slow, stop and reverse ard? in fact, the answer is the other side of the question itself! human body has a tremendous capacity of healing itself! only, if we are willing to make healthy choices. truth is self-effulgent! it does correspondence: dr. prashant kaushik, mbbs chief of rheumatology, nhs, tahlequah, ok associate professor of medicine, osu chs cn associate program director, im residency, nhs e-mail: prashantkaushik2508@gmail.com received: november 29,2022; accepted: december 14, 2022 236 1 prashant kaushik mbbs, mnams, facp, facr, rh msus 2 aarya a. kaushik a.b. candidate '24, harvard university english and music secondary in global health and health policy staff writer, the harvard crimson poetry editor, the harvard advocate research associate, joslin diabetes center doi: https://doi.org/10.57234/1621 not require a lab-proof! however, as 'scientists', we are always inclined to see/measure the outcomes. well, it is out there now! systemic inflammatory diseases can be prevented and reversed!!! here are the revelations about some of these ailments including coronary heart disease, diabetes mellitus and even prostate cancer from just a few extremely humbling articles published quite recently. the lifestyle heart trial showed that it is possible to significantly reduce coronary stenoses and risk of cardiac events using aggressive lifestyle changes, and without lipid-lowering medications. a strong doseresponse relationship between self-reported adherence and angiographic changes was found, with excellent adherence during the study. overall, self-reported adherence was highly correlated with 1 percentage of stenosis. it has also been shown that an intense lifestyle treatment can reduce the 2 expenses on cardiac health/procedures drastically. interestingly, adherence to a whole food plant-based nutritional program was found to be more important than the type of diet consumed. more adherent subjects showed greater improvements in weight and cardiac parameters. thus, the intensity of the intervention may be more important than the 3 specific diet for weight loss. by limiting the daily caloric consumption to 600 c a l o r i e s , d e c r e a s e d p a n c r e a t i c a n d l i v e r triacylglycerol stores, improved maximal insulin 4 sensitivity. it's very humbling! , a 12-the dietfits month randomized clinical trial demonstrated that epigenetics must be part of the measures assessed in lifestyle diet intervention studies. diet determines 5 the expression of many genes! the direct study was the first large rigorous trial to show such success in remission of diabetes in a clinical practice setting. diabetes-remission was strongly associated with weight loss in a dose 6,7 response relationship. a fasting-mimicking diet (fmd) demonstrated that the power of dietary interventions may include reprogramming of tissues to restore lost metabolic function, such as beta cells in the pancreas. once confirmed in humans, this could elevate lifestyle medicine intervention as a 'primary' modality for type 2 diabetes mellitus treatment. also, should this treatment lead to beta-cell neogenesis in humans, it 8 could make type 1 diabetes mellitus reversible. from diabetes 'care' to diabetes 'cure': in a seminal article published about 5 years ago, creative ways to implement lifestyle interventions were reiterated. those services would include coaching, information, 9 and communications technology. intensive nutrition and lifestyle changes can even 10 modulate gene expression in the prostate cancer. so, in a nutshell, all chronic lifestyle mediated diseases have systemic inflammation, alteration in the gut microbiome, oxidative stress, allostatic load, and many other features in common! well, then the answer becomes simple! it is possible to prevent, slow, stop the progression and even reverse all these diseases with 'the exact same' 4-pronged approach: eat well, move more, think right, and love all! the s a g e g e n e r a t i o n a l w i s d o m o f d a d i / n a n i (grandmothers) has no ade! it's work, yes, but 'its works'! references 1. ornish d, scherwitz lw, billings jh, et al. intensive lifestyle changes for reversal of coronary heart disease. jama. 1998 dec 16;280(23):2001-7. doi: 10.1001/jama.280.23.2001. erratum in: jama 1999 apr 21;281(15):1380. pmid: 9863851. 2. hambrecht r, walther c, möbius-winkler s, gielen s et al. percutaneous coronary angioplasty compared with exercise training in patients with stable coronary artery disease: a randomized trial. circulation. 2004 mar 23;109(11):1371-8. doi: 10.1161/01.cir.0000121360.31954.1f. epub 2004 mar 8. pmid: 15007010. 3. dansinger ml, gleason ja, griffith jl, selker hp, schaefer ej. comparison of the atkins, ornish, weight watchers, and zone diets for weight loss and heart disease risk reduction: a randomized trial. jama. 2005 jan 5;293(1):43-53. doi: 10.1001/jama.293.1.43. pmid: 15632335. 4. lim el, hollingsworth kg, aribisala bs, chen mj, mathers jc, taylor r. reversal of type 2 diabetes: normalisation of beta cell function in association with decreased pancreas and liver triacylglycerol. diabetologia. 2011 oct;54(10):250614. doi: 10.1007/s00125-011-2204-7. epub 2011 jun 9. pmid: 21656330; pmcid: pmc3168743. 5. gardner cd, trepanowski jf, del gobbo lc, et al. effect of low-fat vs low-carbohydrate diet on 12-month weight loss in overweight adults and the association with genotype pattern or insulin secretion: the dietfits randomized clinical trial. jama. 2018 feb 20;319(7):667679. doi: 10.1001/jama.2018.0245. erratum in: jama. 2018 apr 3;319(13):1386. erratum in: jama. 2018 apr 24;319(16):1728. pmid: 29466592; pmcid: pmc5839290. 6. lean me, leslie ws, barnes ac, et al. primary care-led weight management for remission of type 2 diabetes (direct): an open-label, cluster-randomised trial. lancet. jiimc 2022 vol. 17, no.4 237 doi: https://doi.org/10.57234/1621 2018 feb 10;391(10120):541-551. doi: 10.1016/s01406736(17)33102-1. epub 2017 dec 5. pmid: 29221645. 7. adamson aj, sniehotta ff, mathers jc, et al. primary careled weight management for remission of type 2 diabetes (direct): an open-label, cluster-randomised trial. lancet. 2018 feb 10;391(10120):541-551. doi: 10.1016/s01406736(17)33102-1. epub 2017 dec 5. pmid: 29221645. 8. cheng cw, villani v, buono r, et al. fasting-mimicking diet promotes ngn3-driven β-cell regeneration to reverse diabetes. cell. 2017 feb 23;168(5):775-788.e12. doi: 10.1016/j.cell.2017.01.040. pmid: 28235195; pmcid: pmc5357144. 9. van ommen b, wopereis s, van empelen p, et al. from diabetes care to diabetes cure-the integration of systems biology, ehealth, and behavioral change. front endocrinol ( l a u s a n n e ) . 2 0 1 8 j a n 2 2 ; 8 : 3 8 1 . d o i : 10.3389/fendo.2017.00381. pmid: 29403436; pmcid: pmc5786854. 10. ornish d, magbanua mj, weidner g, et al. changes in prostate gene expression in men undergoing an intensive nutrition and lifestyle intervention. proc natl acad sci u s a. 2 0 0 8 j u n 1 7 ; 1 0 5 ( 2 4 ) : 8 3 6 9 7 4 . d o i : 10.1073/pnas.0803080105. epub 2008 jun 16. pmid: 18559852; pmcid: pmc2430265. jiimc 2022 vol. 17, no.4 238 doi: https://doi.org/10.57234/1621 iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 51 uric acid as a natural scavenger of peroxynitrite in a sample of iraqi patients with rheumatoid arthritis zahraa ma. naji* ,1 , shaimaa m.mohammed * and mays f. muhammad* * department of clinical laboratory science,college of pharmacy,university of baghdad,baghdad, iraq abstract rheumatoid arthritis (ra) is a chronic inflammatory disease associated with decreased antioxidant state .this study aim to investigate the status of oxidant/antioxidant in a sample of iraqi patients with ra and the role of peroxynitrite and its natural scavenger uric acid in them .this case-controlled study was conducted at baghdad teaching hospital /baghdad from december 2010-may 2011 . twenty-five patients with mean age 39 years and 25 apparently healthy subject as controls with mean age 29 years were included in the study .investigations include estimation of serum levels of nitric oxide (no) ,peroxynitrite (pn) , malondialdehyde (mda) , and uric acid (ua) .serum pn levels were significantly elevated in ra patients as compared with control subjects , ua levels were found significantly lowered in ra patients as compared with control subjects. no significant differences were found between no and mda among patients and controls group .in conclusion :this study demonstrated decreased serum uric acid levels in patients with ra accompanied by decreased its effect as a natural scavenger of pn. key word : rheumatoid arthritis ,peroxynitrite ,uric acid. لذي نمىرج من المشضً العشاقُُن للبُشوكسُنتشات ككاسح طبُعٍ دوس حامض الُىسَك المصابُن بالتهاب المفاصل الشثىٌ صهشاء محمذ علٍ ناجٍ ،*1 محمذشُماء منزس و * محمذ و مُس فاضل * * فشع اىعيً٘ اىَخخبشٝت اىسشٝشٝت ، ميٞت اىصٞذىت ، جاٍعت بغذاد ، بغذاد ، اىعشاق . لخالصة ا ٖذف ٕزٓ اىذساست ىخقٌٞٞ ٍشحبػ ٍع اّخفاض ٍعذالث ٍعاداث االمسذة. حاىخٖاب اىَفاصو اىشث٘ٛ ٕ٘ ٍشض اىخٖابٜ ٍضٍِ ٗمزىل دٗس بٖزا اىَشضاىَشظٚ اىعشاقِٞٞ اىَصابِٞ َّ٘رج ٍِ االمسذة ىذٙ اثٍعادٗ االمسذة اىخ٘اصُ بِٞ حاىخٜ حاٍط اىٞ٘سٝل ىذٙ ٕؤالء اىَشظٚ .اجشٝج ٕزٓ اىذساست فٜ ٍسخشفٚ بغذاد اىخعيَٜٞ سح اىطبٞعٜ ىٔ ٕٗ٘ٗ دٗس اىنا اىبٞشٗمسْٞخشاث ٍشٝط ٍصاب باىخٖاب اىَفاصو 02شاسك فٜ ٕزٓ اىذساست . 0200ىغاٝت اٝاس 0202اىعشاق فٜ اىفخشة ٍِ ماُّ٘ االٗه -بغذاد– ٗحٌ حقٌٞٞ ٍعذالث سٞطشةسْت حٌ اخخٞاسٌٕ مَجَ٘عت اى03.3ٌ ٍخ٘سػ اعَاسٕشخص سيٌٞ 02ٗ سْت 93اعَاسٌٕ ٍخ٘سػ اىشث٘ٛ اٗمسٞذ اىْخشاث ,اىبٞشٗمسْٞخشاث ,اىَاىّ٘ذاٝاىذٖٝاٝذ ٗحاٍط اىٞ٘سٝل فٜ َّارج اىذً اىَسح٘بت ٍِ اىَجَ٘عخِٞ .اظٖشث اىْخائج اسحفاعا , بَْٞا ماّج ٍعذالث حاٍط اىٞ٘سٝل شةاىسٞطفٜ ٍعذالث اىبٞشٗمسْٞخشاث ىذٙ ٍجَ٘عت اىَشظٚ باىَقاسّت ٍع ٍجَ٘عت ٍعْ٘ٝا فٜ ٍعذالث اٗمسٞذ اىْخٞشاث ٍعْ٘ٛ, ماُ ْٕاك فشقا غٞش ىسٞطشةىذٙ اىَشظٚ باىَقاسّت ٍع ٍجَ٘عت ا ٍعْ٘ٝاٍْخفعت اّخفاظا اّخفاض اشاسث اىذساست اىٚ اّخفاض ٍعذالث حاٍط اىٞ٘سٝل ىذٙ ٕؤالء اىَشظٚ ٍشحبػ ٍع ٗاىَاىّ٘ذاٝاىذٖٝاٝذ بِٞ اىَجَ٘عخِٞ . دٗسحاٍط اىٞ٘سٝل مناسح غبٞعٜ ىجزس اىبٞشٗمسْٞخشاٝج . . حامض الُىسَك البُشوكسُنتشاَث ،التهاب المفاصل الشثىٌ ، تاحُة :فالكلمات الم introduction rheumatoid arthritis (ra) is a chronic inflammatory and autoimmune disease (1) , characterized by a chronic hypertrophic synovitis leading to destruction of connective tissues and functional damage of cartilage and bony structure (2) . there is evidence indicating that a low antioxidant status is associated with a higher risk of developing ra (3) . the rheumatoid inflammation is associated with an increased generation of oxidants (reactive oxygen and reactive nitrogen species ) , which play an important role in the inflammatory process and contribute to tissue destruction (4) .several mechanisms exist whereby oxygen radicals might be generated within the joint in ra ,these include the release of such species from activated synovial macrophages and polymorphs (5) ,the prostaglandin pathway (6) and xanthine oxidase mediated synovial ischemic reperfusion injury (7) . nitric oxide (no) is a biological messenger mediating many important physiological functions but also pathological process .it play a vital role in host defense and immunity by modulating inflammatory processes (8) . the increased production of no has been linked to both protective and proinflammatory mechanism associated with tissue damage in inflammatory diseases (9) . during acute and chronic inflammation ,superoxide is produced at rates that overwhelm the capacity of the endogenous superoxide dismutase (sod) enzyme defense system to remove it (10) ,superoxide interacts with and destroys the biological activity of 1 corresponding author e-mail: zahraali139@yahoo.com received :25/2/2012 accepted :23/6/2012 iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 52 no (11) ,the rate of interaction between superoxide and no is faster than the rate of removal of superoxide by sod (12) .peroxynitrite is formed by the rapid combination of no and superoxide in a reaction that is limited only bydiffusion rate of the molecule (13) . under normal physiological conditions ,there is a low production of peroxynitrite resulting in a minimal amount of oxidative damage (14) ,while during inflammatory conditions ,large amount of no and superoxide anion are produced ,leading to the formation of a strong oxidant ,peroxynitrite anion (15) . the proposed cytotoxic properties of peroxynitrite include protein nitration, lipid peroxidation, inhibition of cellular metabolic pathways & signal transduction mechanisms and dna strand breakages (15) . important targets for oxidants are the unsaturated fatty acids in cell membranes. malondialdehyde (mda) is a product of lipid peroxidation and thereby functions as a marker of oxidative stress (16) , earlier studies have shown that the level of mda is related to ra disease activity (17) .uric acid is a metabolic product of purine metabolism that may function as an antioxidant (18) .some disease states such as gout have been shown to result when ua levels in the blood are too high ,while other conditions such as neurodegenerative diseases ,may be caused by reduced serum ua levels. the manipulation of serum ua levels has become a popular strategy in the treatment of a variety of disease (19). apart from being able to scavenge peroxynitrite, ua also scavenge singlet oxygen, peroxy and hydroxyl radicals, ozon and hypochlorous acid (20) . by using rat zymosan-induced arthritis; the administration of uric acid, in addition to reducing the inflammatory parameters, also prevents the loss of articular cartilage (21) .in this study we aimed to study the role of nitric oxide and peroxynitrite in ra patients and the role of uric acid as a scavenger of these free radical in those patients and if manipulation of serum ua levels is one of the methods in the treatment of ra. patients and methods this study was conducted at baghdad teaching hospital from december 2010-may 2011. twenty-five patients with ra; their ages ranged from 17-55 years who were attending the rheumatology consultation clinic of baghdad teaching hospital, and twenty-five apparently healthy subjects as control group with age range of 19-50 years were included in the study after inform consent .ra was diagnosed on the basis of the revised criteria of the american college of rheumatology (acr) (22) . exclusion criteria were pregnancy ,the presence of active infection and the presence of cancer ,since all can affect serum no level .ten milliliter blood samples were collected from all patients by vein puncture , 2 ml of each sample were transferred to anticoagulant tube [edta (ethylene diamine tetra acetic acid)] tube for erythrocyte sedimentation rate (esr) determination according to the westergreen method (23) .the rest of 8ml were transferred to 10 ml sterile plane tube, allowed to clot for 30 minutes at room temperature and centrifuged at 3000 rpm for five minute to obtain serum .serum aliquots were divided into four 1 ml eppendroffs tubes for nitric oxide, peroxynitrite ,mda and uric acid estimation .measurement of no level was performed according to the method of miranda et al (2001), absorbance was read at 540nm using elisa reader (biotek elx50 usa) (24). serum peroxynitrite level was determined according to the method described by beckman et al (25) , cited by vanuffelen et al (26) , in which the peroxynitrite mediated nitration of phenol was measured spectrophometrically (ecm lab spectrophotometer ,germany ) at 412 nm. the levels of serum ua was measured spectrophotometrically with kit from biomaghreb company (france) .malondialdehyde level was estimated as described by hunter et al (27) ,the absorbance of the supernatant was determined at 530 nm spectrophotometrically (ecm lab spectrophotometer ,germany ) . statistical analysis statistical analysis was performed using the statistical package for social sciences version 12 (spss inc., chicago, il., usa) .mean and standard error (se) were calculated. student t-test was used to evaluate the significance (p-value) between study variables .a p-value of<0.05 was considered statistically significant. results table -1 show the demographic characteristics of the subjects .there were nonsignificance difference between patients group and control subjects regarding gender, age and body mass index. female represent 40% of total patients ,while male represent 60% . among control subjects , female represent 48% of total group and male represent 42% . the mean age of patients group was (39.19 years) with age range (17-55) years. the mean age of control subjects was (29.9 years) with age range (19-50) years. the mean bmi of patients group was (25.5kg/m 2 ) ,with range of (21.3-35.2) kg/m 2 .the mean bmi of control iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 53 subjects was (24.9kg/m 2 ) ,with range of (1836.7) kg/ m 2 . serum analysis showed significant elevation in esr levels of patients group 88.33 ±4.38 ( mean ±se) mm/hr as compared with control subjects 14±1.29 ( mean ±se) mm/hr ,(p<0.05) . there was non-significant decrease in serum no levels in patients group 163.7±23.6 (mean±se) µmol/l as compared with control subjects 164.3±9.22 (mean ±se) µmol/l,( p>0.05) ,while there was significant increase in serum levels of peroxynitrite in patients group as compared with control subjects 8.71±1.16 (mean±se) µmol/l,(0.53±0.04) (mean±se) µmol/l respectively ,(p <0.05) .there was nonsignificant increase in serum levels of mda in ra patients group 4.76 ±0.31 (mean±se) nmol/ml as compared with control subjects 4.01 ±0.47 (mean±se) nmol/ml, (p >0.05) .the serum levels of ua were significantly decreased in patients group 4.61 ±0.73 (mean±se) mg/dl as compared with control subjects 8.3 ±0.64 (mean±se) mg/dl (p <0.05) table-2 shows the levels of erythrocyte sedimentation rate, nitric oxide, peroxynitrite, malondialdehyde and uric acid in patients with rheumatoid arthritis and healthy control subjects . table1:demographic data of studied groups describe in means ±standard error parameter ra patients control number 25 25 gender f/m 10/15 12/13 age (years) 39±2.5 29.9±2.1 bmi(kg/m 2 ) 25.5±0.62 24.9±0.79 bmi: body mass index . f/m: female /male . table 2: mean±standard error of nitric oxide, peroxynitrite, malondialdehyde, uric acid and erythrocyte sedimentation rates of patients with rheumatoid arthritis and healthy controls group no: nitric oxide onoo :peroxynitrite . mda:malondialdehyde esr:erythrocyle sedimentation rate discussion despite the synovial tissue being highly vascularized , the rheumatoid joint is recognized as a site with typical biochemical features of hypoxia-induced oxidative stress (28) , the accumulation of oxidized dna ,proteins and lipids within the inflamed rheumatoid joint provides evidence for the damaging effects of radicals in this pathology (29) . it was recently reported that no by itself is protective to chondrocyte under oxidative stress in vivo, while reactive oxygen species, including peroxynitrite, promote chondrocyte death (30) . in one study taysi et al (31) found that serum mda correlated positively with disease activity score ,and deaney et al (32) have reported correlation between esr and mda . the result of our study show non-significant increase in serum mda of ra patients compared to controls and correlate with results of kajanachumpol et al study that showed no significant change in mda levels in patients with ra compared to controls (7) . while in another study ; most patients had low to moderate disease activity ,no correlation between urine mda and disease activity variables were found (33) . the serum levels of no of both groups were not different despite the expected elevation in serum no levels in patients group due to the increase of superoxide production which leads to increase in formation of peroxynitrite as it found in this study,the elevation in peroxynitrite level could explained as a result from increasing nitric oxide and super oxide reaction . uric acid is a natural antioxidant, accounting for up to 60% of the free radical scavenging activity in human blood (34) , serum urate concentration in ra correlated inversely with oxidative changes in serum albumin and immunoglobulin g, it is suggested that serum urate might have an antioxidant role under certain conditions by limiting free radical induced oxidative changes to protein during inflammation (35) . although chronic gout and rheumatoid arthritis are common clinical entities, they seldom coexist (36) . in one study the plasma level of uric acid were inversely related to indices of ra disease activity (33) . peroxynitrite ,in particular , is believed to have a significant negative impact on cellular function and survival (14) .ua may assist in the removal of superoxide by preventing against the degradation of superoxide dismutase (14) , removal of superoxide helps to prevent its reaction with nitric oxide , blocking the formation of peroxynitrite (37) .ua is also very effective at preventing peroxynitrite from nitrating the tyrosine residues of proteins , parameter patients group controls group p value no(mol/l) 163.7±23.6 164.3±9.22 0.97 onoo (mol/l) 8.71±1.16 0.53±0.04 0.00 * mda(nmol/l) 4.76±0.31 4.01±0.47 0.183 esr(mm/hr) 88.3±4.38 14±1.29 0.00 * uric acid (mg/dl) 4.61±0.73 8.3±0.64 0.003 * iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 54 thereby preventing the inactivation of cellular enzymes and modification of the cytoskeleton (14) .ua also has the ability to bind iron ,and inhibit iron –dependent ascorbate oxidation ,preventing an increased production of free radicals that can further contribute to oxidative damage (38) ,thus a reduced ua concentration may decrease the ability of the body to prevent peroxynitrite and other free radicals from acting on cellular components and damaging the cell (39) .the decrease in ua levels may be attributed to its oxidation by peroxynitrite and formation of allantoin , described to occur in humans (40) .patients in this study showed a significant decrease in serum levels of uric acid when compared to control subjects , in turn this can result in decrease protective effect of uric acid as a natural scavenger of peroxynitrite which will lead to further damage in those patients. so these patients are unable to prevent free radical toxicity, leading to the development of inflammation and destruction of tissues . on the other hand, the inflammation that occur in ra leads to the consumption of ua to scavenge the excess free radicals produced ,resulting in a lower ua level .we propose for further studies to estimate the relation between ua and oxidation markers other than pn such as glutathione and also study the role of other antioxidant in ra disease process. in conclusion, we can conclude that patients with ra showed lowered serum uric acid levels accompanied by decreased its activity as natural scavenger of peroxynitrite , while the levels mda and no between both groups showed non-significant difference . references 1. phillips d c,dias h k i,kitas g d ,et al .aberrant reactive oxygen and nitrogen spesies generation in rheumatoid arthritis (ra) :causes and consequences for immune function ,cell survival ,and therapeutic intervention .antioxidants & redox signaling . 2010 ;10 (6):743-785. 2. blake dr , merry p ,unsworth j ,et al .hypoxic reperfusion injury in the inflamed human joint .lancet . 1989; 1 :289-93. 3. knekt p ,heliovaara m ,aho k ,et al .serum selenium ,serum alpha-tocopherol ,and risk of rheumatoid arthritis .epidemiology . 2000, 11 :402-405. 4. bauerova k and bezek a .role of reactive oxygen and nitrogen species in etiopathogenesis of rheumatoid arthritis .gen physiol biophys . 1999 ,18:15-20. 5. halliwell b. production of superoxide ,hydrogen peroxide and hydrogen radicals by phagocytes cells : a cause of chronic inflammatory disease .cell biol int rep. 1982; 6 : 529-42. 6. egan rw ,paxton j ,kuehl fa jr .mechanisms for irreversible self deactivation of prostaglandin synthetase .j biol chem .1976 ;251 :7329-35. 7. 7-kajanachumpol s ,vanichapuntu m ,verasertniyom o ,et al . levels of plasma lipid peroxide products and antioxidant status in rheumatoid arthritis .southeast asian j trop med public health .2000 ;31(2) :335-38. 8. farrel aj, blake dr. nitric oxide . ann rheum dis. 1996; 55: 7-20. 9. clancy p.m ,amin a.r ,abramson s.b.the role of nitric oxide in inflammation and immunity .arthritis rheum . 1988; 41 :1141 -1151 . 10. fridovich i. fundamental aspects of reactive oxygen species ,or what's the matter with oxygen ?. ann n y acad sci .1999 :893 ,13-18 . 11. gryglewski r.j, palmer r.m. and moncada s .bioassay of prostacyclin and endothelium-derived relaxing factor (edrf) from porcine aortic endothelial cells . nature .1986; 320 :454-456. 12. becckman j.s., koppenol w.h .nitric oxide ,superoxide and peroxynitrite :the good ,the bad ,and ugly . am j physiol .1996 ;271 :c1424-c1437. 13. beekman js ,ye y z ,anderson pg ,et al .extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry . biol chem hoppeseyler .1994 ;375:81-88. 14. pacher p ,beckman js ,and liaudet l .nitric oxide and peroxynitrite in health and disease .physiol rev. 2007 ; 87 :315-424. 15. beckman js .oxidative damage and tyrosine nitration from peroxynitrite .chem res toxicol .1996 ;9: 836-844. 16. gambhir jk ,lali p and jain ak . correlation between blood antioxidant levels and lipid peroxidation in rheumatoid arthritis .clin biochem .1997 ;30 :351-355. 17. öztürk hs ,cimen my ,cimen ob ,et al.oxidant/antioxidant status of plasma samples from patients with rheumatoid arthritis .rheumatol int. 1999 ;19 :35-37. 18. nieto fj ,iribarren c ,gross md ,comstock gw ,cutler rg .uric acid and serum antioxidant capacity :a reaction to atherosclerosis ? .atherosclerosis. 2000 ;148 :131-9. 19. choi hk, mount db, reginato am .pathogenesis of gout .ann intern med .2005; 143:499-516. 20. whiteman m ,ketsawatsakul u ,halliwell b. a reassessment of the peroxynitrite iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 55 scavenging activity of uric acid .ann n y acad sci .2002 ;962 :242-259. 21. bezerra mm, bran sd, greenacres s, et al. reactive nitrogen species scavenging ,rather than nitric oxide inhibition ,protects from articular cartilage damage in rat zymosan-induced arthritis .br j pharmacol .2004 ;141 (1) :172-182. 22. funovits j,aletaha d,bukert v ,et al. the 2010 american college of rheumatology /european league against rheumatism classification criteria for rheumatoid arthritis: methodological report phase i .ann rheum dis .2010; 69; 1589-95. 23. international council for standardization in haematology (expert panel on blood rheology ).icsh recommendations for measurement of erythrocyte sedimentation rate .j clin pathol.1993 ;46:198-203. 24. mirenda km ,espey mg ,wink da .rapid and simple spectrophotometric method for simultaneous detection of nitrate and nitrite .nitric oxide . 2001 ;5:62-71. 25. beckman js ,ischiropoulos ih,zhu i,van der woerd m,smith c,chen j et al .kinetics of superoxide dismutase and iron catalyzed nitration of phenolics by peroxynitrite .arch biochem biophys .1992 ;298 :438445. 26. van uffelen bg ,van der zee j,dekoster bm ,van stereninck j,elferink jg .intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration .biochem j .1998 ;330(pt2):719-722. 27. hunter mi, niemadim bc, davidson dl. lipid peroxidation products and antioxidant proteins in plasma and cerebrospinal fluid from multiple sclerosis patients .neurochem res .1985 ; 10:1645-52. 28. tak pp ,zvaifler nj ,green df ,and firestein gs .rheumatoid arthritis and p53 :how oxidative stress might alter the course of inflammatory diseases .immunol today. 2000 ; 21 :78-82. 29. eggleton p, haigh r ,and winyard pg .consequences of non-antigenicity of the 'altered self' .rheumatology. 2008 ;47 :567571. 30. del carlo mj ,loeser rf .nitric oxide – mediated chondrocyte cell death requires the generation of additional reactive oxygen species .arthritis rheum . 2002 ;46 :394-403. 31. taysi s,polat f ,gul m ,et al .lipid peroxidation ,some extracellular antioxidants ,and antioxidant enzymes in serum of patients with rheumatoid arthritis .rheumatol int .2002 ;21:200-204 . 32. deaney cl ,feyi k ,forrest cm ,et al .levels of lipid peroxidation products in a chronic inflammatory disorders .res commun mol pathol pharmacol. 2001 ;110 :87-95 . 33. hagfors l ,leanderson p ,sköldstam l,et al .antioxidant intake ,plasma antioxidants and oxidative stress in a randomized ,controlled ,mediterranean dietary intervention study on patients with rheumatoid arthritis .nutrition journal. 2003 ;2:5 34. -ames bn ,cathcart r ,schwiers e,et al . uric acid provides an antioxidant defence in human against oxidant-and radicalcaused aging and cancer ;a hypothesis .proc natl acad sci u s a .1981 ;78 :6858-6862. 35. 35-situnayake r d,thurnham d i ,kootathep s ,et al. chain breaking antioxidant status in rheumatoid arthritis : clinical and laboratory correlates. annals of the rheumatic diseases .1991 ;50:81-86. 36. khosla p, gogia a ,agarwal pk ,et al .concomitant gout and rheumatoid arthritis –a case report .indian j med sci 2004 ;58 :349-352. 37. van der veen rc ,hinton dr ,incardonna f ,and hofman fm.extensive peroxynitrite activity during progressive stages of central nervous system inflammation .j neuroimmunol .1997 ; :77 :1-7 . 38. davies kj ,sevanian a ,muakkassah – kelly sf ,and hochstein p .uric acid-iron ion complexes .a new aspect of antioxidant functions of uric acid .biochem j .1986 ; 235:747-754. 39. meinda k. kutzing and bonnie l .firestein . altered uric acid levels and disease states . jpet . 2008; 324 :1-7 . 40. hellsten y,tullson pc ,richter ea ,bangsbo j. oxidation of urate in human skeletal muscle during exercise .free radical biol med 1997. ;22:169-174. jiimc march 2016.cdr original article abstract objective: to determine the usefulness of anti-cyclic citrullinated peptide and anti-mutated citrullinated vimentin antibody in the diagnosis of seronegative rheumatoid arthritis. study design: descriptive cross-sectional. place and duration of study: this study was conducted over a period of one year from january, 2010 to december, 2010. subjects were recruited from fatima memorial hospital, rheumatology outpatient department, lahore. the research work was conducted at the department of physiology and cell biology of university of health sciences, lahore. materials and methods: a total of 58 known patients of rheumatoid arthritis fulfilling the american college of rheumatology (acr) criteria were included in the study. after selection of subjects, written informed consent was obtained. the venous blood sample was taken and secured in vacutainers. serum was extracted by centrifugation and stored at -20°c till analysis. sera of all study subjects were tested by elisa for presence of rheumatoid factor, anti-mcv and anti-ccp antibodies. the data obtained was analyzed by using spss version 17. the diagnostic significance of anti-ccp and anti-mcv antibody for the diagnosis of sero-negative rheumatoid arthritis patients was determined. results: serum accp antibody was positive in 9 out of 28 rf-ive patients. so the sensitivity of serum accp in rfive group (n=28) was 32.1%. serum amcv antibody was present in 11 out of 28 rf-ive patients. the sensitivity of serum amcv in rf-ive group was 39.2%. conclusion: anti-ccp and anti-mcv had a higher sensitivity for the diagnosis of seronegative ra. keywords: rheumatoid arthritis (ra), rheumatoid factor (rf), anti-mutated citrullinatedvimentin antibody (anti-mcv), anti-cyclic citrullinated peptide antibody (anti-ccp). progressive disease that produces significant morbidity and premature mortality in many 1 patients. many studies have shown that the disease progresses rapidly within first two years of onset and 2 can lead to irreversible erosive joint destruction. the diagnosis of ra depends primarily on history and clinical findings. the gold standard for the classification of ra is the american college of 3 rheumatology criteria (arnett, et al., 1988). this criterion was not designed for diagnosing ra, but rather to harmonize research in population and family studies for epidemiologic purposes. but they are ubiquitously used as a diagnostic aid. patient must satisfy four out of seven criteria to be classified as ra. acr criteria includes: 1)morning stiffness of more than one hour for at least six weeks 2)arthritis and soft tissue swelling of more than 3 of 14 joints/ joint groups,present for at least six weeks. 3) arthritis of hand joints and wrist, present for at least 6 weeks. 4) symmetric arthritis, present for atleast 6 weeks. 5) subcutaneous nodules 6) rheumatoid factor at a introduction rheumatoid arthritis (ra) is a common systemic autoimmune disease of multifactorial etiology characterized by chronic inflammation of synovial joints that often leads to joint destruction. rheumatoid arthritis typically produces symmetrical swelling of peripheral joints of hand and feet, but may affect the large joints as well. rheumatoid arthritis has a worldwide prevalence of 0.5-3%, being 2-3 times more in women than in men, most frequent during fourth and fifth decade of life. once established, rheumatoid arthritis is a lifelong diagnostic usefulness of anti-cyclic citrullinated peptide and anti-mutated citrullinated vimentin antibodies in the diagnosis of seronegative rheumatoid arthritis patients 1 2 3 4 bushra gohar shah , asma saeed , mohammad faisal khan , hamid javaid quershi jiimc 2016 vol. 11, no.1 correspondence: dr. bushra gohar shah assistant professor, physiology avicenna medical college, lahore e-mail: drbushragoharshah@gmail.com 1 2 3 department of physiology /pharmacology / biochemistry avicenna medical college, lahore 4 department of physiology akhtar saeed medical & dental college, lahore funding source: hec ; conflict of interest: nil received: november 10, 2015; accepted: march 06, 2016 accp and amcv antibodies in seronegative ra 3 level above the 95th percentile. 7) radiological changes suggestive of joint erosion/ and or periarticular osteopenia. by the time clinical diagnosis of ra is made, irreversible joint erosions usually have occurred. ongoing research has shown that early therapeutic intervention results in earlier disease control and 2 consequently less joint damage. there is no single test or finding that can diagnose rheumatoid arthritis. rheumatoid factor is the only serological test included in the acr criteria. however, this auto-antibody lacks specificity. it may be found in patients with other autoimmune diseases and infectious disorders. it may also be present in sera of apparently healthy elderly individuals. upto 25% of patients with rheumatoid arthritis have negative rheumatoid factor test 4 (seronegative). therefore, disease-specific auto antibodies are needed for early diagnosis. currently available data suggest that the diagnosis of ra can be made by testing antibodies to citrulline containing peptides such as anti-perinuclear factor(apf), anti-keratin antibody (aka), antifillagrin antibody and anti-cyclic citrullinated peptides (anti-ccp) antibody. these all belong to the family of anti-citrullinated protein/ peptide antibody 5 (acpa). all these antibodies recognize the antigenic 6 epitope containing citrulline, which is generated by post-translational modification of naturally occurring amino acid arginine by the activity of 7 enzyme peptidyl arginine deiminase (pad). citrullinated peptides have been synthesized as 6 antigens for diagnostic immunoassays. several assays for detecting anti-citrullinated peptide antibodies (acpa´s) have been developed employing filaggrin derived peptides (ccp-assay), viral citrullinated peptides (vcp-assay), mutated 8 citrullinatedvimentin (mcv-assay). the anti-mcv assay (elisa) for the detection of antibodies against citrullinatedvimentin uses an antigen with a genetically modified sequence, which is most 8 abundant in patients with rheumatoid arthritis. positivity of these markers in the rheumatoid factor negative ra patients would suggest their additional benefit in the early diagnosis of this subgroup of patients, which are often diagnosed and treated late. it is expected that the results of the present study will help the clinicians in early diagnosis and timely management of this debilitating disease. the aim of this study was to investigate the diagnostic value of antibodies against mutated citrullinatedvimentin (anti-mcv) and antibodies to cyclic citrullinated peptides (anti-ccp) in the diagnosis of seronegative rheumatoid arthritis patients. materials and methods this descriptive cross sectional study was conducted over a period of one year from january, 2010 to december, 2010. fifty-eight subjects were recruited from fatima memorial hospital, rheumatology outpatient department, lahore, by convenient sampling technique. the research work was conducted at the department of physiology and cell biology of university of health sciences, lahore. a total of 58 patients attending the rheumatology outpatient department of fatima memorial hospital, lahore were recruited in the study. all the patients fulfilled the american college of rheumatology criteria for ra and were diagnosed by the rheumatologist. the study was approved by the ethical and review committee of university of health sciences, lahore. informed written consent was taken from each study participant. a purposefully designed proforma was used to record data of the subjects including age, gender, disease duration, clinical characteristics and medication used. the venous blood samples were taken and secured in vacutainers. serum was extracted by centrifugation and stored at -20°c till titer of anticcp and anti-mcv antibodies. rheumatoid factor titers were determined by elisa (highton, et al., 1986) using commercially available immulisa anti-rf antibody igm elisa kit (immco diagnostics, usa), with an automated eia analyzer [coda, bio-rad laboratories, hercules, ca, usa].results were interpreted as follows:rf-igm value of less than 7 iu/ml was considered as negative. rf-igm value of 7-9 iu/ml was considered as borderline. rf-igm value of more than 9 iu/ml was considered as positive. serum anti-mcv antibody levels were determined by 9 elisa using elisa kit (cusabio biotech co., ltd, china), with an automated eia analyzer [coda, biorad laboratories, hercules, ca, usa]. serum anti10 ccp antibody levels were determined by elisa using commercially available elisa kit (immco jiimc 2016 vol. 11, no.1 4 accp and amcv antibodies in seronegative ra diagnostics, usa), with an automated eia analyzer [coda, bio-rad laboratories, hercules, ca, usa]. 25u/ml was taken as cut-off value for anti-ccp antibodies. the data was entered into spss version 17. diagnostic sensitivity of anti-ccp and anti-mcv antibodies for the diagnosis of rheumatoid arthritis in sero-negative patients were calculated by table of 2 x 2. statistical analysis was done to determine the usefulness of the diagnostic sensitivities of anti-ccp and anti-mcv antibodies. p value of < 0.05 was considered to to be statistically significant. results the study population (n=58), comprised of 58 rheumatoid arthritis patients, out of which 38 were females and 20 were males. mean ±sem age of the ra patients was 44±1.2 years. median (iqr) disease duration was 5(4-8) years. median (iqr) anti-ccp antibodies titer (iu/ml) was 10.8(0.00-340.5). median (iqr) anti-mcv antibodies titer (iu/ml) was 19.7(14.2-30.06). (table i) sub-grouping of ra group was done on the basis of presence or absence of rf, accpab, or amcv ab in the sera. subgroups were named rf+ive group (gp), rf-ivegp, accp+ivegp, accp-ivegp, amcv+ivegp, amcv-ivegp. rf testing by elisa technique, was done in a total of 58 ra patients. out of the ra patients (n=58), 30 (52%) were rf+ive and 28 (48%) were rf-negative. in the ra group (n=58), 34 (58%) were accp+ ive and 24(41.4%) were accp –ive. the sensitivity of serum accp antibodies for ra was calculated to be 58.6%. serum accp antibody was positive in 9 out of 28 rfive patients. so the sensitivity of serum accp in rfive group (n=28) was 32.1%. in the ra group (n=58), 20(34.5%) patients were amcv+ and 38(65.5%) were amcv –ive, at cutoff value of 25u/l (table i). the sensitivity of serum amcv antibodies for ra was calculated to be 34.5% at the manufacturer's cutoff value of 25u/l. serum amcv antibody was present in 11 out of 28 rf-ive patients. the sensitivity of serum amcv in rf-ive group was 39.2%. discussion a close relationship exists between autoimmunity and antibodies; despite this, some patients are p e rs i ste n t l y n e ga t i ve fo r d i s e a s e s p e c i f i c autoantibodies. these conditions have been defined as seronegative autoimmune diseases. although the prevalence of seronegative autoimmune diseases is low, they may represent a practical problem because they are often difficult and challenging cases for the 11 clinicians/rheumatologist. about 80% of the patients affected by ra are positive for rf, the rest 20-25% being seronegative. a more disease specific marker for ra may help in diagnosing early disease and seronegative ra patients resulting in reduced joint damage. it is therefore important to differentiate between ra and other forms of arthritis early after the onset of symptoms. therefore, a specific and sensitive serological marker, which is present very early in the disease, is needed so that the rheumatologist are able to target the use of potentially toxic and expensive drugs to those patients, where the benefits clearly outweighs the risk. keeping in view the need of a more sensitive marker, especially in the seronegative cases, the present study aimed to evaluate the sensitivity of anti-ccp and anti-mcv antibodies in local pakistani seronegative ra subjects. in seronegative cases of arthritis, the differential diagnosis is not easily established in the early disease course. especially seronegative patients need the determination of an additional marker for ra besides rheumatoid factor to confirm diagnosis. the high specificity of anti-ccp antibodies has been reported in rf-neative ra patients. in the present study, 9 out of 28 seronegative patients were positive for anticcp antibodies. so, the sensitivity of anti-ccp antibodies in the seronegative sub-group was 32.1%. tablei: serum rf, accp and amcv status in the ra group (n=58) jiimc 2016 vol. 11, no.1 5 accp and amcv antibodies in seronegative ra in a study, conducted by alexiou, et al., sensitivity in seronegative group was reported to be 34.9%, which 12 is almost comparable to our results. similarly, 13 mobini, et al., found sensitivity in seronegative 14 group to be 33.3%, whereas vanichapuntu, et al., found sensitivity value of 20% in the seronegative 15 sub-group whereas serdaroglu, et al., reported sensitivity of 14.3% in seronegative group. thus, anti-ccp antibody serves as a better diagnostic marker in the diagnosis of ra, especially in the seronegative group. the sensitivity of anti-mcv in the sero-negative ra group was 39.2%, as 11 out of the 28 rf-ive patients were anti-mcv positive. this finding is supported by recent results of other authors, that the higher sensitivity of anti-mcv especially in the seronegative patients makes it a more valuable marker in 16 17 the diagnosis of ra. wagner, et al., reported sensitivity of anti-mcv in the sero-negative group of 43% and sensitivity of anti-ccp to be 30.8%. in seronegative ra patients, the sensitivity of anti-mcv 18 was superior over anti-ccp. soos, et al., reported sensitivity of anti-mcv in the sero-negative group to 19 be 29.4%. narvaez, et al. documented sensitivity of 23% in their series of sero-negative ra patients. limitations of the study small sample size was the limitation of this study. further studies with greater number of ra patients are recommended. conclusion both anti-ccp and anti-mcv antibodies can be used for the early diagnosis of sero-negative patients of rheumatoid arthritis. moreover anti-mcv antibody has a significantly higher sensitivity as compared to anti-ccp antibodies for the diagnosis of seronegative ra recommendations clinicians must be aware of the implications of delayed diagnosis in ra. keeping in view the costeffectiveness, this study emphasizes the utility of rf initially for the diagnosis of ra, reserving acpa's for seronegative ra patients where strong clinical suspicion exists. acknowledgement this research was funded by the higher education commission of pakistan. references 1. lee dm, weinblatt me. rheumatoid arthritis. lancet 2001; 358: 903-11. 2. combe b. progression in early rheumatoid arthritis. best pract res clinrheumatol 2009; 23: 59-69. 3. saraux a, berthelot jm, chales g, le henaff c, thorel jb, hoang s, et al. ability of the american college of rheumatology 1987 criteria to predict rheumatoid arthritis in patients with early arthritis and classification of these patients two years later. arthritis rheum 2001; 44: 2485-91. 4. marianna, mn. rheumatoid factors: what do they tell us? j rheumatol. 2002; 29: 2034-40. 5. zendman aj, van venrooij mj, pruijn gj. use and significance of anti-ccp autoantibodies in rheumatoid arthritis. rheumatology. 2006; 45: 20-5. 6. schellekens ga, de jong ba, van den hoogen fh, van de putte lb, van venrooij wj. citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritisspecific autoantibodies. j. clin invest 1998; 101: 273-81. 7. vossenaar er, zendman aj, van venrooij wj, pruijn gj. pad, a growing family of citrullinating enzymes: genes, features and involvement in disease. bioessays 2003; 25: 1106-18. 8. bang h, egerer k, gauliard a, berg w, fredenhagen g, feist e, et al. mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. arthritis & rheumatism. 2007; 56: 2503-11. 9. coenen d, verschueren p, westhovens r, bossuyt k. technical and diagnostic performance of 6 assays for the measurement of citrullinated protein/peptide antibodies in the diagnosis of rheumatoid arthritis. clin chem. 2007; 53: 498-504. 10. bizzaro n, mazzanti g, tonutti e, villalta d, tozzoli, r. diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis. clin chem. 2001; 47: 1089-93. 11. alessandri c, conti f, conigliaro p, mancini r, massaro l,valesini g. seronegative autoimmune diseases. ann ny acad sci. 2009; 1173: 52-9. 12. alexiou i, germenis a, ziogas a, theodoridou k, sakkas li. diagnostic value of anti-cyclic citrullinatedpeptide antibodies in greek patients with rheumatoid arthritis.bmc musculoskeletal disorders. 2007; 8: 37. 13. mobini m, kashi z, mahdavi, mr. the role of rheumatoid factor and anti-cyclic citrullinated peptide antibody in diagnosis of rheumatoid arthritis. ircmj. 2010; 12: 100-3. 14. vanichapuntu m, phuekfon p, suwannalai p, verasertniyom o, nantiruj k, janwityanujit s. are anti-citrulline autoantibodies better serum markers for rheumatoid arthritis than rheumatoid factor in thai population? rheumatol int. 2010; 30: 755-9. 15. serdaroglu m, cakirbay h, deger o, cengiz s, kul s. the association of anti-ccp antibodies with disease activity in rheumatoid arthritis.rheumatol int. 2008; 28: 965-70. 16. ladislav s, walter g, peter s. laboratory biomarkers or imaging in the diagnostics of rheumatoid arthritis?bmc medicine. 2014; 12: 49. 17. wagner e, skoumal m, bayer pm, klaushofer k. antibody against mutated citrullinatedvimentin: a new sensitive jiimc 2016 vol. 11, no.1 6 accp and amcv antibodies in seronegative ra marker in the diagnosis of rheumatoid arthritis. rheumatolint 2009; 29: 1315-21. 18. soos l, szekanecz z, szabo z, fekete a, zeher m, horvath if, e t a l . c l i n i c a l e v a l u a t i o n o f a n t i m u t a t e d citrullinatedvimentin by elisa in rheumatoid arthritis. j rheumatol. 2007; 34: 1658-63. 19. narvaez j, sirvent e, narvaez ja, bas j, gomez-vaquero c, reina d, et al. usefulness of magnetic resonance imaging of the hand versus anticycliccitrullinated peptide antibody testing to confirm the diagnosis of clinically suspected early rheumatoid arthritis in the absence of rheumatoid factor and radiographic erosions.semin arthritis rheum.2007; 38: 101-9. jiimc 2016 vol. 11, no.1 7 accp and amcv antibodies in seronegative ra key: cord-0048681-wp6xmaqe authors: kubota, kazuo; ogawa, mikako; ji, bin; watabe, tadashi; zhang, ming-rong; suzuki, hiromi; sawada, makoto; nishi, kodai; kudo, takashi title: basic science of pet imaging for inflammatory diseases date: 2019-12-21 journal: pet/ct for inflammatory diseases doi: 10.1007/978-981-15-0810-3_1 sha: c840bc6497465963bd4537378a135a3689cab510 doc_id: 48681 cord_uid: wp6xmaqe fdg-pet/ct has recently emerged as a useful tool for the evaluation of inflammatory diseases too, in addition to that of malignant diseases. the imaging is based on active glucose utilization by inflammatory tissue. autoradiography studies have demonstrated high fdg uptake in macrophages, granulocytes, fibroblasts, and granulation tissue. especially, activated macrophages are responsible for the elevated fdg uptake in some types of inflammation. according to one study, after activation by lipopolysaccharide of cultured macrophages, the [(14)c]2dg uptake by the cells doubled, reaching the level seen in glioblastoma cells. in activated macrophages, increase in the expression of total glut1 and redistributions from the intracellular compartments toward the cell surface have been reported. in one rheumatoid arthritis model, following stimulation by hypoxia or tnf-α, the highest elevation of the [(3)h]fdg uptake was observed in the fibroblasts, followed by that in macrophages and neutrophils. as the fundamental mechanism of elevated glucose uptake in both cancer cells and inflammatory cells, activation of glucose metabolism as an adaptive response to a hypoxic environment has been reported, with transcription factor hif-1α playing a key role. inflammatory cells and cancer cells seem to share the same molecular mechanism of elevated glucose metabolism, lending support to the notion of usefulness of fdgpet/ct for the evaluation of inflammatory diseases, besides cancer. fluorine-18-labeled 2-deoxy-2-fluoro-glucose (fdg) is used as a radiopharmaceutical in pet for evaluating glucose metabolism, and accumulates in malignant tissues because of the enhanced glucose utilization by neoplastic cells. because of the increased metabolic demand for glucose, elevated activity of hexokinase and elevated expression of glucose transporter have been shown in tumor tissues [1] . various applications of fdg-pet have been extensively studied in the field of clinical oncology, and the imaging modality, now used worldwide, is recognized as a powerful diagnostic modality for cancer [2, 3] . in 1989, elevated fdg uptake was reported in two patients with abdominal abscesses [4] . this was followed by reports of fdg uptake in brain abscess [5] , tuberculosis, aspergillosis, sarcoidosis, and so on. in addition, early postoperative scarring and early inflammatory reactions after radiotherapy were also reported to show increased fdg uptake. thus, elevation in glucose metabolism is not only specific for cancer but also seen in inflammation. because fdg uptake is seen in benign inflammatory diseases as well, the accuracy of fdg-pet for the diagnosis of cancer is not 100% [6] . although the metabolic fate of fdg is well known, its cellular distribution within tumors or sites of inflammation has not yet been described. to clarify the mechanisms of fdg uptake by inflammatory and tumor tissues, we performed autoradiographic studies. to demonstrate the cellular localization of fdg and [ 3 h]2dg uptake by tumors in vivo, c3h/ he mice with subcutaneously transplanted fm3a tumors were studied 1 h after intravenous injection of fdg or [ 3 h]2dg. newly formed granulation tissue around tumors and macrophages, which had massively infiltrated the marginal areas surrounding the necrotic areas of the tumor, showed a higher fdg uptake than the viable tumor cells. a maximum of 24% of the glucose utilization was derived from the non-neoplastic tissues in these tumors ( fig. 1.1) . the strong accumulation of fdg in these tumors was thought to represent both the high metabolic activity of the viable tumor cells and that of the tumor-associated inflammatory cells, especially activated macrophages. these results indicate that not only glucose uptake by the tumor cells but also that by non-neoplastic cellular elements which appear in association with the growth or necrosis of tumor cells should be considered for a precise analysis of fdg uptake in tumors, especially after radiotherapy ( fig. 1. 2) [7, 8] . fdg uptake by inflammatory tissues was investigated by yamada et al. [9] . a rat model of chemically induced inflammation using turpentine oil was used. a time-course study of the fdg tissue distribution showed that the uptake of fdg in inflammatory tissue increased gradually until 60 min, followed by a steady decrease thereafter. a longitudinal study showed that the uptake increased progressively after the start of inflammation, peaking at 4 days after the inoculation, and then gradually decreased ( fig. 1.3) . these findings suggested that fdg uptake may reach a maximum during the subacute phase of inflammation, and then slightly decreases during the chronic phase of inflammation. an autoradiography study showed a high fdg uptake in the abscess wall, consisting of an inflammatory cell layer and granulation tissue. at the cellular level, the highest radioactivity was found in the marginal zone that contained young fibroblasts, endothelial cells of vessels, and phagocytes consisting of neutrophils and macrophages, followed by that in the neutrophil layer and the granulation tissue layer ( fig. 1.4) . fdg uptake by inflammatory tissue seems to represent the activity of immune cells and fibroblasts mobilized to the lesion. mochizuki et al. [10] compared the fdg uptake by experimental hepatoma and by tissue with experimental infection with staphylococcus aureus. the expressions of glut1 and glut3 were also studied in both the tumor and infected tissue by immunostaining. uptake by the tumor tissue was significantly higher than that by the infected tissue. both the tumor and infected tissue showed strong expression of glut1 and glut 3, although the expression level of glut1 was significantly higher in the tumor than in the infected tissue. glut1 may be responsible for fdg uptake in both tumor tissue and infected tissue. zhao et al. [11] compared the two models of inflammation: bcg-induced granuloma simulating sarcoidosis and turpentine oil-induced inflammation. fdg uptake by the granuloma was significantly higher than that by the tissue with chemical-induced inflammation. their results explained the very high fdg uptake in sarcoidosis, equivalent to the uptake in cancer. however, the main objective of their study was not to compare the models of inflammation, but to compare the uptakes of 14 c-methionine, 18 f-fluorothymidine, and fdg. 14 c-methionine showed the best results for discrimination between tumor and inflammation. deichen et al. [12] studied the uptake of fdg in isolated human monocyte-macrophages (hmms) in vitro. fdg uptake by the hmms increased significantly with the duration of culture, the percent id/100 μg being 7.5% ± 0.9% (% id/100 μg) on day 14. stimulation by lipopolysaccharide further enhanced the fdg uptake in the hmms by a factor of 2. radio-thin layer chromatography of intracellular metabolites revealed that the fdg was trapped by the hmms mainly as fdg-6-phosphate and fdg-1,6-diphosphate. fdg uptake by the hmms was almost equal to that by human glioblastoma and pancreatic carcinoma cells. malide et al. [13] reported the changes in the subcellular localization of the glucose transporter proteins glut-1, glut-3, and glut-5 as the human monocytes differentiated into macrophages in culture, and the effects of the activating agents n-formyl-methionyl-leucylphenylalanine (fmlp) and phorbol myristate acetate (pma). western blot analysis demonstrated progressively increasing glut-1 expression, rapidly decreasing glut-3 expression, and a delayed increase of the glut-5 expression during the differentiation process. confocal microscopy revealed that each isoform exhibited a unique subcellular distribution and cell-activation response. glut-1 was localized primarily at the cell surface, but was also detected in the perinuclear region, in a pattern characteristic of recycling endosomes. activation with fmlp induced similar glut-1 and glut-5 redistributions from the intracellular compartments toward the cell surface. addition of pma elicited a similar translocation of glut-1, but glut-5 was redistributed from the plasma membrane to a distinct intracellular compartment that appeared connected to the cell surface. these results suggest specific subcellular targeting of each transporter isoform and differential regulation of their trafficking pathways in cultured macrophages. not only macrophages but also neutrophil granulocytes play a key role in the pathogenesis of various types of inflammation. jones et al. [14] studied [ 3 h]dg uptake in neutrophils isolated from human peripheral blood. they found elevated uptake of [ 3 h]dg by neutrophils, both after priming with tumor necrotic factor-alpha (tnf-α) and after activation with fmlp. it was not correlated with the respiratory burst or secretory activity, but may reflect the polarization and migrational status of these cells. their study suggested that as far as neutrophils are concerned, priming is the cellular event predominantly responsible for the fdg uptake. ishimori et al. [15] studied immunocompetent balb/c mice and nude mice administered an intravenous injection of 10 mg/kg of concanavalin a (con a). after the injection, the con a-activated lymphocytes actively took up fdg both in vitro and in vivo, and fdg specifically accumulated in the tissues showing con a-mediated acute inflammation in the immunocompetent mice. rheumatoid arthritis (ra) is an autoimmune disorder of unknown etiology and is characterized by systematic, symmetric, and erosive synovitis. ra synovitis is characterized by massive leukocyte infiltration, proliferative synovial membranes, and neovascularization, which give rise to a synovial proliferative fibrovascular tissue known as a pannus. formation of pannus is directly responsible for the cartilage and bone destruction [16] . matsui et al. [17] reported the mechanism of fdg accumulation in ra in vivo using a murine collagen-induced arthritis model, as well as [ 3 h] fdg uptake in vitro using various cell types. they showed that fdg accumulation increased with the progression of joint swelling. fdg uptake began in the area of inflammatory cell infiltration and synovial cell hyperplasia, and the areas showing strong fdg uptake often coincided with the areas where mixed cellular patterns of macrophages and fibroblasts as well as bone destruction by mature osteoclasts were visible. these findings indicated that fdg accumulation reflected the characteristic changes of pathological progression, such as pannus formation and bone destruction. based on the findings of in vitro experiments, matsui et al. suggested that the cell types responsible for fdg uptake were mostly proliferating fibroblasts, with lesser contributions from activated macrophages. fdg uptake by fibroblasts was enhanced in the presence of cytokine stimulation and hypoxia within a joint. hypoxia is a known feature of the microenvironment in inflamed joints [18] . recently, garcia-carbonell et al. directly compared fibroblast-like synoviocytes (fls) from ra patients and osteoarthritis patients [19] . in vitro experiments have shown that the fls from ra patients were dependent on glycolysis rather than on oxidative phosphorylation, and this difference was more pronounced than that for the fls from osteoarthritis patients. the expression of glut-1 messenger rna was correlated with the functions of the fls from the ra patients. these studies showed the importance of fibroblasts in the inflammation in ra. recently, the processes, at the molecular level, occurring in association with hypoxia and glucose metabolism within tumor and inflammatory tissues have been described. cramer et al. reported that activation of hypoxia-inducible factor one-alpha (hif-1α) is essential for myeloid cell infiltration and activation in vivo [20] . they showed that hif-1α is essential for regulation of the glycolytic capacity of myeloid cells; when hif-1α is knocked out, the cellular atp pool decreases drastically. this metabolic defect results in a profound impairment of myeloid cell aggregation, motility, invasiveness, and bacterial killing at sites of inflammation where the tissue environment is hypoxic. thus, hif-1α has a direct regulatory effect on both the survival and functioning of cells in inflammatory microenvironments. furthermore, the enhanced glycolysis in activated macrophages results in elevation of the fdg uptake. regarding the molecular mechanisms responsible for the regulation of glycolysis, hif-1α affects both glucose transporter and hexokinase expressions in tumors and inflamed tissues. the significance of hypoxia in tumor pathophysiology has also been described by denko [21] . usually, proliferation of cancer cells is faster than the growth of capillaries, resulting in inadequate perfusion and hypoxia within the tumor. activation of hif-1α in the hypoxic tumor shifts the energy metabolism from oxidative phosphorylation to anaerobic glycolysis, saves oxygen, and avoids massive cell death. as a result of the elevated glucose metabolism, fdg-pet serves as a useful imaging modality for cancer patients. dominant inflammatory cells activated and responsible for fdg uptake in inflammatory diseases may differ depending upon the disease. all inflammatory cells and cancer cells seem to share the same molecular mechanism of elevated glucose metabolism, lending support to the idea of the usefulness of fdgpet/ct also for the evaluation of inflammatory diseases, besides cancer. although a guideline [22] has been proposed, use of fdgpet/ct for inflammatory diseases is still limited. i hope that this book will help spread knowledge about the application of fdgpet/ct for inflammatory diseases to improve patient management, representing an advance in the field of medicine. atherosclerotic plaques are classified into two types: stable and vulnerable. vulnerable plaques are easy to rupture and may cause stroke and heart attack. therefore, the prompt detection and treatment of vulnerable plaques, prior to the manifestation of symptoms, is of crucial importance. vulnerable plaques are characterized by a lipid-rich atheromatous core, which is infiltrated by macrophages. there is a correlation between the number of infiltrating macrophages and the severity of symptoms in acute myocardial infarction [23, 24] . in contrast, infiltration by macrophages is rarely observed in stable plaques. macrophages are active in energy metabolism. therefore, fdg-pet is a promising tool for the detection of vulnerable plaques, depending on the extent of macrophage infiltration. thus far, numerous clinical and nonclinical studies have investigated the use of fdg for the imaging of vulnerable plaques. in 1994, kubota et al. demonstrated that fdg accumulated more in macrophages than in tumor cells [25] . therefore, it is assumed that vulnerable plaques can be detected depending on the rate of macrophage infiltration in the plaque. in the early 2000s, several clinical studies suggested that it is possible to detect atherosclerosis, according to the level of inflammation [26] [27] [28] . notably, through histological analysis of symptomatic carotid artery plaques, rudd et al. reported the co-localization of [ 3 h]fdg and macrophages [27] . the investigators examined the relationship between the degree of macrophage infiltration and accumulation of fdg in watanabe heritable hyperlipidemic (whhl) rabbits-an arteriosclerotic animal model-, and explored the possibility of detection of vulnerable plaques using fdg-pet [29] . the atherosclerotic plaques were detected by fdg-pet ( fig. 1.5 ). in whhl rabbits, thickening of the intima and infiltration by macrophages was observed in all individuals. we examined the degree of macrophage filtration via histological analysis. the results showed that the fdg uptake and number of macrophages in the atherosclerotic lesions were strongly correlated ( fig. 1.6 ). however, there was no correlation between the degree of macrophage infiltration and that of intimal thickening. these results suggested that macrophages are responsible for the accumulation of fdg in atherosclerotic lesions, and vulnerable plaques may be detected through fdg-pet. studies in humans have shown that the uptake of fdg into vulnerable plaques correlates with the infiltration by macrophages [30, 31] . immunohistochemical staining of macrophage marker cd68 revealed a strong correlation with fdg accumulation. it has also been reported that the incorporation of matrix metalloproteinase-1-an enzyme involved in the destabilization of plaques-and fdg are strongly related [32] . tahara et al. reported correlations between the accumulation of fdg and waist circumference, hypertension, insulin resistance, lowering of high-density lipoprotein cholesterol, etc. [33] . a recent study showed a correlation of fdg accumulation with c-reactive protein [34] . the results of these studies indicate the prospect for the use of fdg-pet in arteriosclerosis-related diseases. foam cell formation is responsible for the vulnerability of plaques. macrophages are recruited from blood monocytes that enter through the endothelium ( fig. 1.7) . scavenger receptors on macrophages mediate the uptake of oxidized low-density lipoprotein (ldl) and cause the accumulation of ldl-derived cholesterol and foam cell formation. foam cells release several proteases and cytokines, which lead to rupture of the plaques. we evaluated the effects of foam cell formation on the uptake of fdg [35] . macrophages were isolated from the peritoneum cavity of mice, and foam cells recent studies have described that polarization of macrophages affects the development of atherosclerosis and rupture of plaques. classically activated m1 macrophages are considered to possess the most proatherogenic phenotype and promote the destabilization of atherosclerotic plaques. in contrast, alternatively activated m2 macrophages possess anti-inflammatory properties and stimulate reparative processes, which lead to the stabilization of atherosclerotic plaques [37, 38] (fig. 1.8) . therefore, the detection of m1 macrophages may assist in predicting cardiovascular events with greater accuracy. in our investigation, m1 macrophages showed a 2.6-fold increased uptake of [ 3 h] fdg versus m2 macrophages [39] . glucose transporter (glut)-1 and glut-3, which are major isoforms of a glucose transporter in macrophages, were significantly upregulated in m1 macrophages versus m2 macrophages. to date, numerous drugs have been developed for the treatment of atherosclerosis. the therapeutic effects of these agents are usually monitored by determining the levels of lipids in the blood. however, lipid-lowering therapy does not always lead to the stabilization of vulnerable plaques. several statins may effectively reduce the levels of cholesterol in the plasma; however, they do not decrease the degree of macrophage infiltration [40, 41] . thus, merely monitoring the levels of lipids in the plasma is not sufficient to determine the therapeutic effect of drugs. macrophage infiltration plays an essential role in the rupture of plaques. therefore, the administration of pharmacological therapy that reduces macrophage infiltration is required to stabilize the vulnerable plaques. considering the individual differences in the stabilization of plaques induced by such drugs, monitoring the therapeutic effect in each individual plaque is important to accurately assess the effect. using probucol as a therapeutic drug in whhl rabbits, we investigated the usefulness of fdg-pet for the monitoring of therapies that target vascular inflammation. in a number of animal studies, the lipid-lowering effect of probucol was moderate, and the drug did not decrease the levels of cholesterol in the plasma [42, 43] . however, probucol can reduce the macrophage-rich plaques even in advanced atherosclerotic lesions [43] . in the present study, the uptake of fdg was significantly decreased in the probucol group versus the pretreatment period ( fig. 1.9 ). however, there was no difference in the levels of cholesterol in the plasma between the probucol and control groups [44] . a large number of macrophages was observed at the initiation of the study. nevertheless, treatment with probucol for 6 months resulted in diminished macrophage infiltration ( fig. 1.10) . notably, the ratio of the intima to the whole cross-sectional area was not affected by treatment with probucol. hence, the observed decrease in the uptake of fdg was attributed to the decrease in the number of infiltrating macrophages. collectively, these studies showed that the therapeutic effect of probucol was successfully monitored by fdg-pet independently of the cholesterol-lowering effect. these results demonstrated the usefulness of fdg-pet for drug development, and numerous clinical studies have been performed [45] [46] [47] [48] [49] . tahara et al. conducted one of the initial clinical trials, showing that the therapeutic effect of simvastatin was successfully monitored by fdg-pet [50] . in 2011, the results of the first clinical multicenter study investigating the efficacy of dalcetrapib against atherosclerotic disease via a novel noninvasive multimodality imaging technique (dal-plaque) were reported. of note, fdg-pet was used to evaluate the level of vascular inflammation [51] . moreover, in the dal-plaque study, the relationship between serum inflammatory biomarkers and plaque inflammation was also assessed using fdg-pet [52] . recently, several pet imaging tracers-apart from fdghave been reported for the imaging of vulnerable plaques. [60] [61] [62] [63] . recently, the β-amyloid imaging agent [ 18 f]flutemetamol showed specific accumulation in human carotid plaques, especially in amyloid betapositive areas [64] . among these tracers, [ 18 f]naf is considered a promising probe for the detection of atherosclerosis, and several comparison studies with fdg have been performed. the results of these studies showed that the area of accumulation differs between fdg and [ 18 f]naf [65] [66] [67] . the accumulation of [ 18 f]naf in atherosclerotic plaques depends on the level of calcification [68] . the [ 18 f]naf-positive area does not completely match with the ct-positive area, and it is thought that [ 18 f]naf accumulates in the early stage of calcification (i.e., microcalcification) [60, 69] . since target molecules are different among these tracers, comparison study should be important to elucidate the specificity of these tracers for the different stages of atherosclerosis progression. this may assist in understanding the mechanism of plaque progression and provide important insight into therapy aimed toward the stabilization of plaques. abstract neuroinflammation is a general event in acute and chronic neurodegenerative disorders. based on the critical role of neuroinflammation characterized by glial activation in neuropathogenesis, in vivo imaging with positron emission tomography (pet) is required in clinical and preclinical studies for the purposes of elucidation of pathogenesis and novel treatment development, because it is commonly available in human and experimental animal models. as a most widely used imaging biomarker for neuroinflammation, 18 kda translocator protein (tspo) imaging has been performed in a large number clinical and preclinical studies. neuropathologyassociated tspo induction has been generally detected in various neurodegenerative animal models with acute and chronic neuroinflammation. however, studies with human subjects showed confusing results likely due to ineffectiveness of the tracers used or impediment of non-microglial tspo expression in human diseased brains. based on the above reasons, recently, alternative molecular targets for microglia imaging instead of tspo have been proposed. colony-stimulating factor 1 receptor (csf1r) is a promising candidate because of its highly specific expression in microglia in the central nervous system (cns). purinergic receptors p2x7r and p2y12r have been proposed as imaging biomarkers of m1 and m2 phenotype microglia, respectively. several pet tracers for these non-tspo biomarkers have been developed, and some of them showed positive results in neuroinflammation is characterized by glial activation, which would lead to an increase in the inflammatory factor level in the cns. microglia are a collection of glial cells that act as the first and main form of active immune defense in the cns. accordingly, its activation is considered to trigger neuroinflammation, and pet tracers bound to active microglia are used for imaging of neuroinflammation. therefore, molecules exclusively expressed in microglia have the potential to be molecular targets for neuroinflammation. because imaging with pet is a commonly available technology for human subjects and experimental animal models, numerous pet tracers have been developed for visualization of microglia in living brains. moreover, increasing evidence has indicated that microglial activation is heterogeneous, and can be categorized into two opposite types: pro-inflammatory (m1) and anti-inflammatory (m2) microglial phenotypes. pet imaging tracers that bind to either general or phenotypespecific microglia are required for studies of neurodegenerative disorders. pet imaging in animal models is an indispensable step for the development and clinical application of pet tracers, as it will provide predictive evidence for tracer utility in human subjects. more importantly, it can easily provide a direct comparison between pet images and histopathology, which is usually difficult in human studies. pet imaging in animal models would supplement the weak points of human studies and provide interpretation for imaging in human subjects. response to neural injury. pet tracers with high affinity for tspo are the most widely used for neuroinflammation imaging. tracers have been developed for tspo imaging for several decades, including typical first-generation tracer 11 c-pk11195 and second-generation tracer 11 c-daa1106 [70] . major parts of these tracers enable the visualization of microglial activation in acute neuroinflammation models triggered by acute events like traumatic brain injury, ischemia, and excitotoxic damages. a longitudinal pet imaging study has shown a continuing increased tspo pet tracer uptake in a mouse model of mesial temporal lobe epilepsy induced by unilaterally intracranial kainic acid injection. the radioactivity uptake reached a peak at 7 days, mostly related to microglial activation. after 14 days, reactive astrocytes provided a major binding site for tspo tracer [71] . the finding of phase-dependent tspo expression in subtypes of glial cells might contribute to the identification of optimal treatment windows in further clinical studies [71] . chronic neuroinflammation is usually observed in many chronic neurodegenerative disorders such as alzheimer's disease (ad), amyotrophic lateral sclerosis (als), and non-ad tauopathies. most animal models of chronic neurodegenerative disorders are genetically modified mouse models. authors have clearly demonstrated glial activation in response to accumulation of amyloid aggregates and treatment with the aid of tspo-pet, indicating the utility of neuroinflammation imaging in monitoring of pathogenesis ( fig. 1.11 ). amyloid accumulation is the earliest pathological change in the brain with ad, followed by tau aggregation and glial activation. glial activation is predominantly concentrated around neuritic plaques, not diffuse plaques [72] . more clinical studies have reported increased tspo tracer accumulation in temporoparietal, entorhinal, and cingulate cortex rather than frontal cortex with rich diffuse plaques and poor neuritic plaques, partially supporting such observation, while a considerable number of studies failed to detect an increase in tspo non-tg representative merged coronal brain images of t2-weighted mr, tspo-pet and merged images showed that tracer binding was greatly increased in transgenic mice (tg) of app and tauopathy models mimicking amyloid (22-month-old) and tau (12-month-old) pathologies, respectively, compared with respective age-matched non-transgenic littermate mice (non-tg). no obvious brain atrophy was observed in hippocampi of app model as well as non-tg mice (white arrows), while tauopathy model showed overtly atrophic hippocampi (white arrowheads). red arrowheads indicated enlarged ventricles due to the hippocampal atrophy. unpublished data expression level [73] . the amyloid plaques accumulated in the amyloid precursor protein (app) model are dense-core plaques, around which there are abundant activated glial cells and tspo expression, indicating a similar glial response between neuritic and dense core plaques. however, immunohistochemical analysis showed that active astrocytes have provided the majority of binding sites for tspo tracer in the app model [74] , while microglia around neuritic plaques expressed abundant tspo [75] . this distinction between species concerning glial characteristics might be attributable to neurodegenerative tau pathologies, which exist in ad but are uncommon in the app model. in vivo tspo imaging also demonstrated increased tspo expression accompanied by tau-related atrophic brain in another model of ad mimicking the tau-related pathologies ( fig. 1.11 ) [72, 76] . considering that predominant microglial tspo expression and age-dependent brain atrophy in these tauopathy models mimicking tau-related pathologies are consistent with the condition in ad patient brains, neuroinflammation imaging in tauopathy models might well reflect the actual situation of pathologies in human subjects. based on the fact that in vivo imaging for amyloid and tau, as well as tspo have been available for clinical and preclinical studies, multiple tracer imaging enables the clarification of the mutual relationship between these ad-related pathologies. for example, longitudinal in vivo imagings with tspo-pet ( 18 f-fedaa1106) and amyloid-pet ( 11 c-pib) have captured the increased level of neuroinflammation triggered by aβ immunotherapy, which greatly reduced amyloid accumulation in an identical app model over the course of treatment ( fig. 1.12 ) [77] . tspo-pet in combination with tau-pet ( 11 c-pbb3) in two tauopathy models showed that neuroinflammation was induced by two different tau aggregates (pbb3-positive and -negative) and thereby accelerated tau-mediated neuron damage via a distinct molecular mechanism [78] . these studies provide experimental evidence for neuroinflammation-targeting therapeutic strategy. similarly, tspo tracer signals were also increased in lesioned brain regions in a mouse model of als, being consistent with microglial immunostaining [79] . in contrast, the results from parkinson's disease (pd) animal models look more complicated. pd animal models are relatively easy to prepare by treating the animals with neurotoxins such as methamphetamine and 6-hydroxydompaine (6-ohda). methamphetamine-induced degeneration of dopaminergic neuron terminals did not trigger tspo-positive gliosis [74] , while 6-ohda induced degeneration of dopaminergic neurons in either striatum or substantia nigra and increased the tspo tracer signal [80] . interestingly, most of the clinical pet studies found no increase in the level of tspo expression in the striatal regions of pd patients [73, 81, 82] despite severe dopaminergic degeneration in these regions, supporting the proposal that tspo imaging is inapplicable to monitoring the degeneration of nonmyelinated nerve [74] . quantitative pet studies require that the input function, representing cumulative availability of authentic radiotracer in arterial plasma, is measured in case of the absence of ideal reference tissue. because of the constitutive expression of tspo in the whole brain, blood collection is needed for calculating the binding potential. however, this usually faces technical difficulties in the actual operation for the small mouse body. so the brain regions without pathological changes, such as cerebellum in rtg4510 mouse [76, 78] or striatum in ps19 mouse [72] , are used as reference tissue when reference-tissue models are used. although it will lead to underestimation of the binding potential due to constitutive low-level tspo expression in reference regions, such analysis can provide relatively stable results compared to the percentage of injection dose, which is greatly affected by body weight, metabolic capacity, or other factors. another issue is a concern for the partial volume effect due to the small volume of the region of interesting (roi) in the case of mouse. this makes it difficult to correctly measure the radioactivity in certain subregions of mouse brain. the use of rat and nonhuman primate models is an alternative, but selectable neurodegenerative models are limited compared to mouse, as most models with pathologies of chronic neurodegenerative disorders, such as ad, als, and non-ad tauopathies [83, 84] , are genetically modified murine models. despite the fact that tspo-pet successfully captured neuroinflammation in various animal models with acute and chronic neuroinflammation, the results in patients with chronic neuroinflammation seemed more complicated [73, 81] . one of the reasons is that a tspo polymorphism (rs6971) greatly influences the affinity of a large majority of tspo tracers in a tracer-dependent manner [85] . another possible reason is the differing cellular distribution of tspo expression. recent studies have shown that tspo expression is not limited to microglia. other brain components, for example, astrocytes and vascular endothelial cells, also express tspo. some studies have demonstrated dominant induction of tspo expression in active astrocytes in a neuropathological type-dependent and phase-dependent manner [71, 74] . vascular expression of tspo is also a factor that disturbs microglial tspo imaging. although no study has quantitatively measured the influence of vascular tspo on tracer binding, inclusion of the additional vascular component in the modified reference-tissue model amplified the binding potential in ad more than in control by decreasing tracer binding to the vasculature in the disease cohort or was better correlated to brain tspo mrna [86, 87] . as few studies have focused on changes in vascular tspo expression in diseased brains, determining which is responsible for the changes in tspo tracer binding in diseased brains is a complicated issue. further studies in combination with postmortem analyses or microglial tspo-specific or vascular tspo-specific knockout animals are desirable. csf1r is a novel imaging biomarker for microglia by its exclusive expression in microglia in the cns [88] . recently, horti et al. reported a newly developed pet tracer, 11 c-cppc, for csf1r imaging. 11 c-cppc showed high initial brain uptake and rapid washout from brain in normal rodent brain, and increased accumulation in inflammatory brains of rodent and nonhuman primate models including lps-injection and ad mouse models. in vitro autoradiogram showed higher binding in postmortem brains with ad compared to healthy control [88] . these results support the concept that csf1r is a suitable molecular target for microglia imaging. however, 11 c-cppc binding is increased in whole brain regions, and is not limited to regions with lesions as exemplified by cerebellum in ad model and intracranial lps injection model, and immunohistochemistry for active microglia and csf1r expression to support imaging results was not provided. another reported csf1r imaging tracer, 11 c-az683, showed high affinity for csf1r (ki = 8 nm) and >250-fold selectivity over other kinases tested, but low brain uptake in rodents and nonhuman primates limited its utility in living brains [89] . further examinations and improvements will be required for their clinical application. it is still unclear which microglia phenotypes predominantly express csf1r because of the lack of data from immunohistochemical and biochemical analyses of the various neurodegenerative models. p2x7r and p2y12r were considered to be predominantly expressed in m1 and m2 microglia phenotypes, respectively [90] . in vitro autoradiographic analysis with 11 c-smw139 showed high specific binding to viral vectormediated human p2x7r expressed in rat brain, but no increase in binding was detected in postmortem brain with ad compared to healthy control [91] . specific binding of 11 c-gsk1482160 is also detectable in p2x7roverexpressing samples under in vitro condition [92] . however, more evidence is required for both of the above two p2x7r tracers to prove their utilities in the living inflammatory brain. in vivo imaging with 18 f-jnj-64413739 with high affinity and selectivity for p2x7r showed significant increase in tracer accumulation in the living lpsinduced neuroinflammatory brain, indicating its leading status among the current p2x7r tracers, while further work is needed to verify its utility in other types of neuroinflammation [93] [94] [95] . villa et al. recently developed a carbon-11-labeled p2y12r-binding compound. in vitro and ex vivo experiments with this radioactive compound showed increased binding in brain sections of mice treated with anti-inflammatory stimuli and decreased binding to brain sections of a murine stroke model and of a stroke patient [96] . immunohistochemistry and immunoblotting also supported the viewpoints that p2y12r is a biomarker for m2 phenotype microglia [90, 96] . abstract neuroinflammation is defined as inflammatory responses in the brain and spinal cord, and microglia and astrocytes are the mainly involved cells. translocator protein (tspo) has been the major target used in positron emission tomography (pet) to detect neuroinflammation with glial activation. in a preclinical study conducted in small animal models of brain ischemia and traumatic brain injury, a second-generation tspo tracer [ 18 f]dpa-714 pet was used for the evaluation of glial activation; the tspo uptake was validated by comparison with the immunohistochemical findings of co-staining for tspo and a microglial/macrophage or astrocyte maker. tspo expression has also been reported in a model of alzheimer's disease, where tspopositive cells with two opposing roles were observed: neuroprotective astrocytes for reversible neuronal injury and detrimental microglia for irreversible neuronal injury. recently, new targets have been identified in microglia, namely, cyclooxygenase-1 (cox-1) and purinergic receptor p2x7, which have been shown to be specifically expressed in the microglia. pet imaging targeting glial cells is a promising modality to characterize and monitor neuroinflammation longitudinally and with quantitative accuracy. the findings of preclinical models need to be confirmed in clinical studies beyond the limitation of animal model and the species differences between rodents and humans. keywords: neuroinflammation, glial activation, translocator protein, microglia, astrocyte neuroinflammation is defined as inflammatory responses in the brain and spinal cord [97] . it is observed in various types of brain diseases, including brain ischemia, traumatic brain injury, neurodegenerative disorders (alzheimer's disease, parkinson's disease, etc.), and neuropsychiatric disorders (schizophrenia, autism spectrum disorder, etc.) [98] [99] [100] [101] . in the central nervous system, two major glial cells are involved in the process of neuroinflammation, namely, microglia and astrocytes [99] . to evaluate neuroinflammation in vivo, translocator protein (tspo) has been the major target used in pet for the detection of neuroinflammation with glial activation [102] . in glial cells, especially microglia, expression of tspo is minimal in the resting state, but upregulated in the activated state [103] . in previous studies, the first-generation tspo-pet tracer, [ 11 c]pk-11195, was mainly used to evaluate glial activation in small animals as well as humans. however, the evaluation using [ 11 c]pk-11195 was found to be highly limited by the high nonspecific binding of the tracer [104, 105] ; therefore, over the last 10 years, second-generation tspo-pet tracers, such as [ 11 c]dpa-713 and [ 18 f]dpa-714, have been used because of their higher specific binding [104, 105] . although the binding affinity in humans has been shown to vary due to polymorphism, tspo pet has been employed as an effective tool to visualize and quantify the degree of neuroinflammation associated with glial activation in preclinical studies conducted using animal models, including rodents [106] . however, attention needs to be paid to the inflammatory cells taking up tspo, as tspo expression is not only observed in activated microglia/macrophages but also in reactive astrocytes [107] . for preclinical studies using small animals, the middle cerebral artery occlusion (maco) model was frequently used owing to its ease of handling for reperfusion and the abundance of accumulated evidence [108, 109] . martin et al. [111] . thus, tspo pet can also be used for monitoring the response to immunomodulatory therapy after brain ischemia. to evaluate traumatic brain injury (tbi) in small animals, a model of controlled cortical impact with a pneumatic impact device has been used for its high reproducibility and low mortality [112] . we reported two peaks of [ 18 f]dpa-714 uptake in this model, namely, on day 7 in the cortical area and on day 21 in the thalamus after induction of the focal brain injury [99] . the enhanced thalamic uptake was sustained until 14 weeks after induction of the tbi (fig. 1.14) . immunohistochemical analysis on day 7 revealed tspo staining mainly co-localized with amoeboid cd11b-positive microglia/macrophages, and weakly with gfap-positive astrocytes in the cortex; however, tspo immunoreactivity was scarcely observed in the thalamus. giemsa staining on day 7 revealed mononuclear cells, such as phagocytic macrophages, in the cortical area, although these cells were not detected in the ipsilateral thalamus. in the macrophage depletion study performed 1 week post-injury, the tspo uptake was decreased in the cortex, but enhanced in the thalamus, suggesting that the cortical uptake was mainly attributable to uptake by phagocytic macrophages recruited from the bloodstream, while the thalamic uptake was most likely attributable to uptake by resident microglia. electron microscopy at 4 weeks post-tbi revealed morphologically activated microglia surrounding the degenerated axons and poorly myelinated neurons in the ipsilateral thalamus, indicating activation of the resident microglia and damage to the remaining neurons. tspo expression was mainly evident in the activated microglia in the thalamus at 6 weeks postinjury. thus, by using [ 18 f]dpa-714 pet, inflammatory migration could be detected from the cortex to the thalamus after focal brain injury. in addition, we also showed that depletion of myeloid-derived suppressor cells (mdscs) was associated with enhanced cortical tspo uptake on day 7 after the induction of focal brain injury, by both immunohistochemistry and cellular phenotype analysis [113] . this finding indicated that mdscs may have strong immunosuppressive characteristics and limit inflammation and facilitate wound healing and recovery. postmortem analyses of the brains of patients with alzheimer's disease (ad) have revealed neuroinflammatory changes, namely, accumulation of activated microglia and astrocytes, around senile plaques and fibrillary tau lesions [114] . in this study, diffuse plaques in the brains of these patients were enveloped by a small number of iba-1positive microglia not expressing tspo, while numerous microglia expressing both iba-1 and tspo were found to be in close contact with neuritic plaques. thus, microglia expressing tspo were recruited to fibrillar tau inclusions, but not to aβ deposits unaccompanied by tau pathology. jin et al. reported that tspo expression was observed, mainly in the astrocytes, in amyloid precursor protein 23 (app23) transgenic (tg) mice which show minimal neuronal loss, while tspo-positive microglia were observed in the tangle-like tau lesions of tau tg mice which show remarkable neuronal loss. they concluded that tspo-positive astrocytes have protective roles against reversible neuronal injury, while tspo-positive microglia have detrimental effects for irreversible neuronal injury [115] . we evaluated tspo expression in the app23 tg mice by immunohistochemistry ( fig. 1.15 ). tspo immunoreactivity was observed around the amyloid beta (aβ) plaques and in reactive astrocytes (gfap-positive shukuri et al. demonstrated that the expression of cyclooxygenase-1 (cox-1) in activated microglia and macrophages during neuroinflammation can be visualized by pet using [ 11 c]ketoprofen methyl ester (ktp-me), with immunohistochemical confirmation [117] . one advantage of [ 11 c]ktp-me pet as compared to tspo pet is that cox-1 is expressed only in activated microglia and not in astrocytes. they also reported that expression of cox-1 could be detected in 16-to 24-month-old app23 tg mice, in accordance with the time of histopathologic appearance of abundant aβ plaques and activated microglia [118] . they also reported the possibility of treating ad by inhibition of cox-1 activity, as a treatment target, with nonsteroidal anti-inflammatory drugs (nsaids). besides tspo, pet imaging of [ 11 c]ktp-me could also be a useful tool for monitoring cox-1 activity in activated microglia in neuroinflammation. imamoto et al. showed that glial activation could be quantitatively imaged in the spinal cord of a rat model of neuropathic pain using [ 11 c]pk11195 pet [119] . it was suggested that high-resolution pet using tspo-specific radioligands is useful for imaging to assess the role of glial activation, including neuroinflammatory processes, in the spinal cord. belolli et al. reported increased enhanced tspo uptake in a mouse model of multiple sclerosis induced by experimental autoimmune encephalomyelitis (eae), with neuropathological confirmation of activated microglia [120] . they concluded that combined use of tspo-pet and mri could provide complementary evidence of the ongoing disease process. evaluation of neuroinflammation by pet in neuropsychiatric disorders, such as schizophrenia and autism spectrum disorder, has been limited to clinical studies, possibly due to the difficulty in establishing appropriate animal models mimicking the pathological condition of the patients. as mentioned in sect. 1.4.4, tspo-positive glial cells have both neurotoxic and neuroprotective effects. two phenotypes of microglia have been recognized, namely, neurotoxic "m1" and neuroprotective "m2," although it cannot be clearly separated as m1 or m2 [121] . tspo-positive microglia that accelerate tau deposition and neuronal deterioration are likely to be m1-polarized. when considering the treatment strategy for neurodegenerative disorders, it is important to detect the detrimental glial cells to suppress their function. expression of the p2x7 receptor (purinergic receptor) has been reported to be observed in m1-polarized microglia, which could be a promising target for the detection of deleterious neuroinflammation [122] . in contrast, expression of the p2y12 receptor has been reported to be observed in the m2-like microglia [123] . recently, a pet radioligand for the p2x7 receptor was synthesized and its efficacy was evaluated in preclinical models. finally, reproducible and dosedependent receptor occupancy studies with a p2x7 receptor antagonist were performed using [ 18 f]jnj-54175446 in rhesus monkeys and humans [124, 125] . the usefulness of pet radioligands for the p2x7 receptor is not only limited to the diagnosis of neuroinflammation. [ 18 f]pttp, which is also a radioligand targeting the p2x7 receptor, has been shown to be potentially useful for the quantification of peripheral inflammation targeting macrophages and to distinguish inflammation from certain solid tumors [126] . other targets in microglia, such as sphingosine-1-phosphate receptor 1 (s1p1) or receptor for advanced glycation end products (rage), are also promising, but only in the preliminary stage of investigation at this moment [127] . findings of pet imaging in animal models of neuroinflammation are summarized in this article. in preclinical studies, the pathology can be confirmed by immunohistochemistry or cellular phenotype analysis, as well as by comparison of the pet results. pet imaging targeting tspo, cox-1, or p2x7 is a promising modality to characterize and monitor neuroinflammation longitudinally and with quantitative accuracy. however, it needs to be borne in mind that preclinical models do not always reflect the pathologies of the heterogeneous characteristics of patients in clinical practice. in addition, we should consider the species differences between rodents and humans. any findings in preclinical models should be confirmed in clinical situations, which is one of the goals of translational research. translocator protein (18 kda; tspo) is the critical component of a multimeric 140-200-kda complex located in the outer mitochondrial membrane and enriched in the outer/inner mitochondrial membrane contact sites [128] . tspo has broad functions related to the regulation of cholesterol transport, steroid hormone synthesis, porphyrin and heme transport, apoptosis, cell proliferation, anion transport, mitochondrial function regulation, immunomodulation, and inflammation [128] . tspo is widely distributed in the peripheral tissues, and the mrna levels of tspo are high in the adrenal glands, kidneys, spleen, skeletal muscle, heart, and lungs, and are low in the liver and brain. tspo expression in blood cells has also been reported, in which phagocytic cells, including monocytes and polymorphonuclear neutrophils, show significantly higher tspo expression than lymphocytes. it has been demonstrated that tspo expression increases in inflammatory cells during the occurrence and progression of inflammation. thus, tspo has become a useful biomarker for monitoring inflammation using pet with a tspospecific radiotracer [128] . since 2002, we have developed more than 10 pet radiotracers for the imaging of tspo in preclinical studies and have translated four new tspo radiotracers to clinical studies in our institute: [ [137] [138] [139] . here, the author reviews their results from pet with [ 18 f]fedac to noninvasively visualize tspo in the peripheral tissues [140] and monitor various inflammatory diseases, such as lung inflammation [141] , nonalcoholic fatty liver disease [142] , liver damage [143] , liver fibrosis [144] , multiple sclerosis [145] , and rheumatic arthritis [146, 147] , in animal models. using small-animal pet with [ 18 f]fedac, we performed tspo imaging to quantify tspo density in rat peripheral tissues, including heart, lungs, and kidneys [140] . pet study showed that the uptake of radioactivity was highly distributed in the lungs, heart, and kidneys, and the tspo-enriched tissues could be clearly visualized after the injection of [ 18 f]fedac ( fig. 1.16) . the kinetics of this radiotracer in the tissues was moderate, which is suitable for determining the in vivo binding parameters and receptor occupancy. the b max values of tspo in the heart, lung, and kidney were 393, 141, and 158 pmol/ml, respectively ( tspo is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lungs. we performed pet imaging of lung inflammation with [ 18 f] fedac and determined cellular sources that enrich tspo in the lung [141] . we prepared an acute lung injury model by using intratracheal administration of lipopolysaccharide (lps) to rats. pet with [ 18 f]fedac demonstrated that, in response to lps treatment, the uptake of radioactivity increased in the lungs as the inflammation progressed ( fig. 1.17 ). pretreatment with a tspo-selective unlabeled ligand pk11195 significantly decreased the lung uptake of [ 18 f]fedac because of competitive binding to tspo. tspo expression was elevated in the inflamed lung section and its level responded to the [ 18 f]fedac uptake and severity of inflammation. the presence of tspo was examined in the lung tissue using western blotting and immunohistochemical assays. the increase of tspo expression was mainly found in the neutrophils and macrophages of inflamed lungs ( fig. 1.18 ). through this study, we demonstrated that pet with [ 18 f] fedac is a useful tool for imaging tspo expression and evaluating the progression of lung inflammation. mitochondrial dysfunction is responsible for liver damage and disease progression in nonalcoholic fatty liver disease (nafld). tspo, as a mitochondrial transmembrane protein, plays important roles in regulating mitochondrial function. we conducted pet with [ 18 f]fedac to explore if tspo could be used as an imaging biomarker of noninvasive diagnosis and staging of nafld [142] . pet with [ 18 f] fedac, ct, autoradiography, histopathology, and gene analysis were performed to evaluate and quantify tspo levels and nafld progression in methionine and cholinedeficient diet-fed mice. the uptake of [ 18 f]fedac increased with disease progression from simple steatosis to nonalcoholic steatohepatitis (nash) (fig. 1.19) . a strong correlation was observed between [ 18 f]fedac uptake ratio and nafld activity score in the liver. the specific binding of [ 18 f]fedac to tspo in the nafld livers was demonstrated by competition experiments with the unlabeled tspo-selective pk11195. autoradiography and histopathology supported these pet imaging results. further, there were greater mrna levels of the functional macromolecular signaling complex composed of tspo, compared to controls (fig. 1.20) . through this study, we demonstrated that tspo expression increased in nafld and was strongly correlated with nafld progression. tspo as a specific molecular imaging biomarker could provide a novel tool for noninvasive, reliable, and quantitative diagnosis and staging of nafld. liver damage induced by drug toxicity is an important problem for both medical doctors and patients. in this study, we noninvasively visualized acute liver damage using pet with [ 18 including tspo (blue) and cd11b (orange). preferential localization of tspo indicated in the liver lesion sites, cd11b/tspo active macrophages and lymphocytes increased in the mcd livers (scale bars = 500 μm). * * p < 0.01 sources enriching tspo expression in the liver [143] . a mild acute liver damage model was prepared by a single intraperitoneal injection of cycloheximide (chx) to rats. treatment with chx induced apoptosis and necrotic changes in hepatocytes with a slight neutrophil infiltration. the uptake of radioactivity in the rats' livers was measured with pet after injection of [ 18 f]fedac. pet with [ 18 f] fedac showed that the uptake of radioactivity increased in livers treated with chx, compared to the controls ( fig. 1.21) . the mrna expression of tspo was increased in the damaged livers compared to the controls, and the tspo level was correlated with the [ 18 f]fedac uptake and severity of damage. tspo expression in the damaged liver sections was mainly found in macrophages (kupffer cells) and neutrophils, but not in hepatocytes ( fig. 1.22) . the increase in expression of tspo mrna was induced by an increase in the numbers of macrophages and neutrophils with tspo in the damaged livers. through this study, we demonstrated that pet imaging with [ 18 f]fedac provided visible evidence that mild liver damage occurs through the enhanced tspo signal in inflammatory cells, and that this method is a useful diagnostic tool in the early stages of acute liver damage. liver fibrosis is the wound healing response to chronic liver injury, which is caused by various factors. in this study, we evaluated the utility of tspo as a molecular imaging biomarker for monitoring the progression of liver fibrosis in response to cirrhosis [144] . to induce cirrhosis, carbon tetrachloride (ccl 4 ) was administered to rats and the subsequent liver fibrosis was assessed. pet with [ 18 f]fedac was used to noninvasively visualize liver fibrosis in vivo. pet scanning, immunohistochemical staining, ex vivo autoradiography, and quantitative reverse-transcription polymerase chain reaction were performed to elucidate the relationships among radioactivity uptake, tspo level, and cellular source enriching tspo expression in damaged livers. pet with [ 18 f]fedac showed that the uptake of radioactivity in livers significantly increased after 2, 4, 6, and 8 weeks of ccl 4 treatment ( fig. 1.23 ). immunohistochemical analysis demonstrated that tspo was primarily expressed in macrophages and liver stellate cells (hscs). tspo-expressing macrophages and hscs increased with the progression of liver fibrosis (fig. 1.24) . the distribution of radioactivity from [ 18 f]fedac was strongly correlated with tspo expression, and tspo mrna levels increased with the severity of liver damage. through this study, we demonstrated that tspo is a useful molecular imaging biomarker for monitoring the progression of liver fibrosis in response to cirrhosis with pet. imaging modalities have the potential to monitor inflammation in order to guide treatment before significant functional impairment or irreversible cellular damage occurs in multiple sclerosis (ms). in this study, [ 11 c]dac, a carbon-11labeled tspo radiotracer derived from [ 18 f]fedac, was used to evaluate neuroinflammation and quantify the therapeutic effects of experimental autoimmune encephalomyelitis (eae), an animal model of ms [145] . pet with [ 11 c] dac was used to visually assess neuroinflammation in eae by measuring tspo expression in the spinal cords; there was the maximal uptake on day 11 and 20 eae rats with significant inflammatory cell infiltration, compared to controls, day 0 and 60 eae rats ( fig. 1.25) . biodistribution studies and in vitro autoradiography confirmed the pet imaging results. doubling immunohistochemical studies demonstrated the infiltration and expansion of cd4+ t cells and cd11b+ microglia; cd68+ macrophages caused the increase in tspo levels visualized by [ 11 c]dac-pet. furthermore, mrna level analysis of the cytokines using quantitative reversetranscription polymerase chain reaction revealed that tspo+/cd4 t cells, tspo+ microglia, and tspo+ macrophages in eae spinal cords were activated and secreted multiple pro-inflammation cytokines, which mediated the inflammation lesions of eae. the eae rats treated with an immunosuppressive agent fty720 exhibited an absence of inflammatory cell infiltrates and displayed a faint radioactive signal, compared to the high accumulation in untreated eae rats ( fig. 1.26) . through this study, we demonstrated that [ 11 c]dac-pet imaging is a useful tool for noninvasively monitoring the neuroinflammation response and evaluating therapeutic interventions in eae. rheumatoid arthritis (ra) is a chronic disease characterized by systemic inflammation that results in the destruction of multiple articular cartilages and bones. activated macrophages have been known to play important roles in the pathogenesis of rheumatoid arthritis (ra). cheon et al. evaluated the feasibility of [ 18 f]fedac-pet in a murine ra model [146] . in the collagen-induced arthritis (cia) mice, joint swelling was apparent on day 26 after the first immunization, and the condition worsened by day 37. in these mice, the mrna and protein expression of the tspo increased in activated macrophages. the uptake of [ 18 f]fedac in activated macrophages was greater compared to the uptake in nonactivated cells, which was inhibited by pk11195. the [ 18 f]fedac uptake by arthritic joints increased early on (day 23), whereas [ 18 f] fdg uptake did not ( fig. 1.27 ). however, [ 18 f]fdg uptake by arthritic joints markedly increased at later stages (day 37) to a higher level than [ 18 f]fedac uptake. the [ 18 f]fedac uptake correlated weakly with the summed severity score, whereas the [ 18 f]fdg uptake correlated strongly with the summed severity score. histologic sections of arthritic joints demonstrated an influx of macrophages compared to that in normal joints ( fig. 1.28) . moreover, these indicated that pet imaging using [ 18 f]fedac may be used as a predictor of the therapeutic effects caused by biological disease-modifying antirheumatic drugs that have anti-inflammatory actions to inhibit activated macrophages [147] . [ 18 f]fedac-pet is a powerful tool for visualizing tspo and monitoring the progress of various inflammatory diseases of the peripheral system. the present studies will contribute to determination of the pathogenic progress and will be of use in evaluating the therapeutic effects of antiinflammatory drugs. in future studies, we will perform or are performing pet imaging with [ 18 f]fedac or other tspo radiotracers to detect various inflammation in humans. hiromi suzuki and makoto sawada abstract at present, common diseases of cns were often chronic and progressive which could affect the patient's quality of life seriously. but the pathogenesis for most of them had not been fully understood and was no effective prevention or treatment. certain pathological injury could cause cellular inflammatory response. microglia, the first activated effector cells, played an important role in the immune defense process. microglia had a two-way effect. on the one hand, activated microglia phagocytosed damaged cell debris and removed antigenic substances, and could release cytotoxic factors which would aggravate the injury of neuron. on the other hand, activated microglia may accumulate around damaged neurons and might induce neurotrophin-dependent protective activity. therefore, to study the mechanism of microglia in the diseases of cns and limit their effects on neuronal injury may help to retard the procession of some neurodegenerative diseases such as alzheimer's disease and parkinson's disease cause neuroinflammation including expression of mhc antigens and production of inflammatory cytokines. however, the condition changes greatly in relation to the onset and the formation of a pathological condition. microglia, macrophage-like cells in the central nervous system (cns), are multifunctional cells; they play important roles not only in the neuro-inflammation but also in the development, differentiation, and maintenance of neural cells via their phagocytic activity and production of enzymes, cytokines, and trophic factors [148] . microglial cells show a rather uniform distribution of cell numbers throughout the brain with only minor population in some brain regions. their in situ morphologies, however, may vary markedly from elongated forms observed in apposition with neuronal fibers to spherical cell bodies with sometimes extremely elaborated branching. human fetal microglia display heterogeneity in phenotype identified by cd68 [149] . this heterogeneity gave rise to the hypothesis that these cells are differentially conditioned by their microenvironment and, therefore, also display specific patterns of differential gene expression. for example, mrnas of tnfalpha, cd4, and fc gamma receptor ii are differentially expressed in microglia isolated from different area of the brain [150] . it has been reported that production of il-12 [151] and histamine [152] are different in the subtypes of microglia. furthermore, the subtypes of microglia show distinct acidification profiles by treatment with pepstatin a [153] and selective clearance of oligomeric beta-amyloid peptide [154] . the distinct phenotypes in activated forms of microglia might result from this heterogeneity. although the origin of microglia is still controversial, resident microglia is thought to be of mesodermal origin, being derived from a bone marrow precursor cell [155] . these cells or a lineage related to monocytes and macrophages may invade the cns at an early embryonic stage to give rise eventually to typical process-bearing resident microglia [155] . like macrophages in other tissues, the growth and activation of microglia is regulated by macrophage colony-stimulating factor (m-csf) [156] . however, unlike macrophages, microglia are observed in macrophagedeficient mice [157] [158] [159] , suggesting the possibility of the presence of m-csf-independent populations. despite the postulate that microglia arise from blood monocytes, microglia appear to display phenotypes differing from macrophages. in vivo, microglia do not normally express mhc class i and ii [160] , cd14 [161] , or scavenger receptor i and ii [162] . their appearance takes a ramified form [160] . in contrast, monocytes and macrophages express these surface antigens absent in microglia and their shape is ameboidal [160] . like their in vivo counterparts, isolated microglia also differ from macrophages. although cultured microglia are ameboidal, they can transform to a ramified form [163] . microglial cell proliferation is induced by conditioned medium from astrocyte-enriched cultures [163] . microglial production of interleukin (il)-5 is stimulated by bioactive phorbol ester but not by interferon-γ, while the opposite is there for macrophages [164] . microglia do not constitutively express il-2 receptors under normal culture conditions, as do monocytes and macrophages [165] . microglia express these receptors only after stimulation such as with lipopolysaccharide (lps) [165] . il-2 increases viability and proliferation of lipopolysaccharide-stimulated microglia [165] , while macrophages respond to il-2 by expressing il-1, il-6, and tumor necrosis factor α mrnas, peroxide production, and microbicidal and tumoricidal activity [166, 167] . lps, cyclosporin a, and fk506 downregulate expression of cd4 in microglia, but not in macrophages [168] . finally, bone marrow chimera experiments have shown that resident microglia form highly stable pool of cns cells displaying very little exchange with the bone marrow compartment [169] [170] [171] . these controversial observations suggest that microglia in the brain may be different from macrophages. recently, we identified two distinct subpopulations of microglia in the normal mouse brain: type i and type ii. both types had similar phagocytic activity, but they showed distinct phenotypes such as cell-surface markers, mrna expression, and growth factor dependency. type i, but not type ii, microglia expressed the hallmarks of microglia such as cd14 and c3b receptor, and production of interleukin 1β. type ii microglia expresse surface markers for immature bone marrow cells, such as antigens against er-mp12 and er-mp20 antibodies, which are not detected in mature monocyte/macrophages. in addition, it also express onetenth amount of mature cell markers compared to monocytes/macrophages. therefore, type ii microglia may arise from progenitor cells similar to immature bone marrow cells which migrate to the cns during the early developmental stages then differentiate in the cns; monocytedifferentiation out of bone marrow like extrathymic differentiation of t-cells [172] . this suggests that the two distinct type of microglia arise from distinct origins. in addition, type i microglia exhibited m-csf-dependent growth, while the type ii were m-csf-independent. a previous report suggested that alveolar macrophages are gm-csf-but not m-csf-dependent [173] . these observations indicate that there are two distinct populations of microglia which are probably of different origins and have different functions in the brain. the presence of oxidative stress and inflammatory activity is one of the significant pathological features of neuroinflammatory diseases [174, 175] . it has been shown that the levels of cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)-1β, and interferon (ifn)-γ are elevated in the substantia nigra of patients with parkinson's disease [176] . since microglia are a principal source of these cytokines, the data support microglial involvement in the pathogenesis of neuroinflammatory diseases. however, the role of activated microglia is controversial. for example, the characteristic pathological features of the parkinson's disease brain are a selective and progressive loss of dopamine neurons of the substantia nigra and their terminals in the caudate-putamen, along with focal accumulation of activated microglia in the substantia nigra and caudate-putamen. the traditional view is that microglia act merely as scavengers and their activation is secondary to the neuronal damage. however, activated microglia have been observed in the limbic system and neocortex, where there are few or no degenerating neurons, in significantly higher numbers in pd brains than in brains from normal controls [177] . activation of microglia has also been identified in the substantia nigra and/or striatum of parkinsonian animal models, such as l-methyl-4-phenyl-1,2,3,6tetrahydropyridine (mptp)-induced parkinsonism [178, 179] . the link between microglia activation and selective neuronal vulnerability has led many researchers to suggest that microglia activity participates in neuronal demise. in this respect, microglial cytotoxicity may contribute to or even promote neuronal damage. activated microglia are capable of releasing numerous cytotoxic agents, including proteolytic enzymes, cytokines, complement proteins, reactive oxygen intermediates, nmda-like toxins, and nitric oxide [148] . in fact, β-amyloid, the senile plaquederived component found in alzheimer's disease, appears to elicit neurotoxic responses through the activation of microglia [180] . however, this suggestion relies solely on in vitro data and as yet no evidence has been presented that indicates that activated microglia destroy neurons under in vivo conditions. recently we showed that highly activated microglia treated with lps may have neurotrophic potential toward dopamine neurons in neonatal mice administered mptp [181] . tyrosine hydroxylase activity and the levels of dopamine and dihydroxyphenylacetic acid (dopac), as well as those of the pro-inflammatory cytokines il-1β and il-6, were elevated in the midbrain of lps-and mptp-treated neonatal mice. the viability of dopamine (a9) neurons was preserved in neonatal mice of the lps-mptp group compared with the mptp group. in contrast, the viability of these neurons in aged mice dropped significantly. these results may suggest that activated microglia show different phenotypes; i.e., microglia activated by lps in the neonatal brain may be neuroprotective for dopaminergic neurons in mptp-treated mice, whereas in aged mice they may be neurotoxic for dopaminergic neurons. the dissociation between injury-induced microglial activation and neuronal degeneration in tnf receptor and colony-stimulating factor 1 (csf-1) knockout mice suggests that microglial activation is not a determinate event in dopaminergic neuronal damage in brain. furthermore, there is a growing body of evidence that microglia may play a beneficial role in ischemia by secreting factors (growth factors or cytokines) that promote survival of neurons. therefore, activated microglia may produce not only neurotoxic effects but also neuroprotective ones, depending upon their environmental situation. we have reported that exogenous microglia enter the brain parenchyma through the blood-brain barrier and migrate to ischemic hippocampal lesions when they are injected into the circulation. by applying this brain-targeted delivery technique, we investigated the effect of exogenous microglia on ischemic pyramidal neurons [182] . to this end, we isolated microglia from neonatal mixed brain cultures, labeled them with a fluorescent dye pkh26, and injected them into the artery of mongolian gerbils subjected to ischemia reperfusion neuronal injury. pkh26-labeled microglia migrated to the ischemic hippocampal lesion and increased the survival of neurons, even when the cells were injected 24 h after the ischemic insult. furthermore, stimulation of isolated microglia with ifn-γ enhanced the neuroprotective effect on the ischemic neurons. microglia also protected ischemia-induced learning disability. as migratory microglia increased the expression of brain-derived neurotrophic factor (bdnf) and glial cell linederived neurotrophic factor (gdnf) in the ischemic hippocampus, they might induce neurotrophin-dependent protective activity in damaged neurons. these results represent the first experimental demonstration of neurotrophic effects of microglia on transient global ischemia in vivo. since peripherally injected microglia exhibit specific affinity for ischemic brain lesions and protect against ischemic neuronal injury in the present model, we suggest that microglia may have the potential to be used as a candidate for cell therapy for cns repair following transitory global ischemia and other neurodegenerative diseases. nef, a multifunctional hiv protein, activates the vav/rac/ p21-activated kinase (pak) signaling pathway. given the potential role of this pathway in the activation of the phagocyte nadph oxidase, we have investigated the effect of the hiv-1 nef protein on microglia superoxide production and toxicity for neurons [183] . microglia were transduced with lentiviral expression vectors to produce a high level of nef protein. expression of nef did not activate the nadph oxidase by itself, but led to a massive enhancement of the responses to a variety of stimuli (ca2+ ionophore, formyl peptide, endotoxin) and induction to produce a large amount of superoxide. these effects were not caused by upregulation of phagocyte nadph oxidase subunits. nef mutants lacking motifs involved in the interaction with plasma membrane and/or other cytosolic proteins failed to reproduce the effects of wild-type nef, suggesting involvement of a certain signaling pathway in microglia for their trophic-toxic control. our results suggest a key role for rac activation in the priming for microglia toxic activation, which is enhanced by nef introduction in the nontoxic form of microglia. rac activation is not sufficient to induce the toxic form of microglia accompanied by stimulation of the phagocyte nadph oxidase; however, it markedly enhances the nadph oxidase response to other stimuli and might be involved in the trophic-toxic control of microglia. cytokines are polypeptidic soluble factors that control the growth and differentiation of cells involved in immune and hematopoietic systems. these factors were initially considered to act on target cells in a cell-type and stage-specific manner. however, it has been shown that their biological actions are pleiotropic, complementary, and counteractive; each of them exerts multiple effects on different cells, and different factors can act on the same cell populations to induce similar or opposite effects. moreover, production of several cytokines is controlled by other cytokines in a stimulatory or inhibitory manner, and, in some cases, acts as a cascade. these complex cytokine actions are therefore referred to as a cytokine network. recent evidence suggests that bidirectional communication occurs between cells of the nervous and immune systems. the basis for this communication is the release of cytokines by immune component cells, as well as hormone products of the neuroendocrine system. cells resident within the cns can synthesize, secrete, and respond to inflammatory cytokines not only contributing to the responses to injury or immunological challenge within the cns but also regulating their own growth and differentiation potential. there are many similarities of cytokine actions between the immune system and the cns. however, there are also several unique modes of cytokine action in the cns (i.e., their target cells, inducing functions on the target cells such as other cytokine production, major histocompatibility complex (mhc) antigen induction, and cell growth control, and inducing agents of their production). therefore, the actions and communication of cytokines in the cns are designated as the cns cytokine network [184] . microglia are one of the most important cells in the cns cytokine network. they produce a variety of cytokines, as well as their proliferation and other functions are regulated by cytokines. astrocytes are another cell population important in the cns cytokine network. there are many similar aspects of cytokine production and responses between microglia and astrocytes; however, the roles in the cns cytokine network may differ between these cells; microglia may have an important function in the removal of dead cells or their remnants by phagocytosis in brain injury, while astrocytes provide structural and environmental support for neurons. there are no clear indications of the difference between the roles of microglia and astrocytes in the cytokine production and response. some of these cytokines control the growth, differentiation, and activation of cells in the cns, and some modulate growth factor production, mhc antigen expression, and morphological changes. their biological functions are mediated by specific receptors, some of which are expressed in the cns, and cellular localization of cytokine receptors in the cns has been demonstrated. neuronal cells and oligodendrocytes as well as microglia and astrocytes expressed several cytokine receptors indicating that certain functions of these cells may also be controlled by some cytokines. however, to determine the involvement of cytokines in controlling the functions and development of cns cells requires elucidating the functional relationships of cytokines and these cells. there are two aspects of roles of the cns cytokine network, one of which is regulation of growth and differentiation of neural cells, and production of cytokines in the cns. another aspect of the cns cytokine network is its interaction with the immune system. since the majority of cytokines are produced by immune cells, it is possible that immune cells may also control the growth and function of cns cells. therefore, the control of microglial growth by cytokines is an important method of regulation of cns cells by peripheral immune cells. in addition to this, cytokines produced in the cns can control immune cell functions. these communications may be important for brain-immune interactions, and some aspects may play crucial roles in certain pathological conditions such as aids-dementia complex and multiple sclerosis as well as several neurological diseases. pet imaging has recently gained wide use not only in clinical stages but also in basic studies. small-animal imaging uses the same technology as clinical imaging. clinical research and problems can thus be introduced to preclinical research directly using small animals. conversely, applying results obtained from preclinical studies to clinical imaging is also possible. imaging has bridged the gap between basic studies and clinical research, and has become widely recognized as a tool for translational research. in traditional animal experiments, relatively high numbers of animals are often required. one reason is that animals are sacrificed to remove organs or tissues for the purposes of observation and measurement. another is the influence of individual differences. in particular, the four elements of pharmacokinetics (absorption, distribution, metabolism, and excretion) are known to be strongly affected by individual differences. the number of samples must therefore be increased for accurate statistical processing. on the other hand, preclinical imaging experiments allow use of the same individual, performing the study and repeatedly acquiring data without killing the individual. this means that applying human imaging techniques to a small animal allows follow-up of changes over time in disease models in the same individual, clarifying changes according to the disease condition more precisely, as in daily practice in the clinical field. in addition, preclinical imaging is an effective technique not only for providing biological information but also for reducing the number of experimental animals. in the area of research into infectious diseases, evaluation of pathogen infection, tracking disease state, and observation over time of individuals are very important, because infectious and inflammatory diseases change with the passage of time. for example, diseases may pass through stages of infection, pathogenesis, ingravescence, remission, and healing. when those stages of infectious disease are studied by sacrificing the animals, the number of animals required to obtain meaningful results will become extremely large due to the large interindi-vidual variance of animals. small animal pet imaging has therefore gained attention as a useful experimental method. to conduct imaging studies using infected model animals, a special laboratory is required. this is because researchers must be protected not only from radiation exposure but also from the risk of infection. biosafety level (bsl) is a classification depending on the degree of pathogen risk. table 1 .2 shows representative pathogens sorted by bsl from the guidelines of the national institute of infectious disease (niid). when dealing with high-risk pathogens, researchers need to use a biocontainment laboratory conforming to the world health organization (who) guidelines. when using imaging systems, measures must also be taken to avoid contamination by biohazards, such as installation in separated safety rooms, prevention of cross-contamination using sealed animal tubes, and so on [185] . in addition, researchers need to wear a tyvek coverall, mask, rubber gloves and face guard ( fig. 1.29 ). given the restrictions as described above, only limited numbers of facilities around the world are capable of performing imaging studies for infectious diseases, and relatively few studies have been conducted. in 2012, a small-animal imaging system was introduced to the bsl-3 section of the radioisotope center at nagasaki university, making this the only facility in which researchers could perform imaging of infectious diseases requiring a high bsl in japan. since then, various infectious diseases imaging studies have been conducted. hayasaka et al. reported an fdg-pet study using mice infected with severe fever with thrombocytopenia syndrome (sfts) virus, which is categorized as bsl-3. sfts is a tickborne infection that causes severe inflammation in the gastrointestinal tract [186] . in that study, sftsv-infected mice were administered about 10 mbq of fdg intravenously via the tail vain. pet acquisition was performed for 30 min from 30 min after fdg injection. fdg uptake was found along the course of the intestine in the sfts-infected mouse ( fig. 1.30 ) [187] . pet studies on sfts have also been performed using 68 ga tracer. fuchigami et al. tried to visualize sfts with 68 ga-citrate [188] . generally, 67 ga-citrate is a radiotracer used for single-photon computed tomography (spect), and is widely used to detect tumors [189, 190] , inflammation, and infectious diseases [190, 191] . since 68 ga-citrate is a pet tracer behaving exactly the same as 67 ga-citrate, targets can be imaged quantitatively and with higher sensitivity. in that study, sftsv-infected mice were administered about 5 mbq of 68 ga-citrate intravenously via the tail vain. pet acquisition was performed for 1 h, starting 2 h after 68 ga-citrate injection. as shown in fig. 1.31 , high accumulation of 68 ga-citrate in the gastrointestinal tract was observed in sftsv-infected mice, and the accumulation pattern of 68 ga-citrate resembled that of fdg. in addition, 68 ga-citrate was used for pet imaging of leishmaniasis [188] . leishmaniasis is a tropical disease caused by leishmania parasites categorized as bsl-2, and leishmania leads to strong localized inflammation [192] . leishmania parasite-infected mice were administered about 4 mbq of 68 ga-citrate intravenously via the tail vein. pet acquisition was performed for 1 h, starting 2 h after 68 ga-citrate injection. as shown in fig. 1.32 , inflammation site-specific accumulation of 68 ga-citrate was observed [188] . the examples given here are only a small selection of many studies underway at nagasaki university. infectious disease studies are more diverse than tumor studies, because infectious diseases have numerous diseases classified as tropical infections and emerging infectious diseases, and many issues have not been elucidated for each disease. fdg and 68 ga-citrate used in the studies introduced this section are widely used general radiotracers that are easily obtained. even without using special or unorthodox radiotracers, new knowledge can be obtained from preclinical pet studies. the pathological condition of the infected animal will change depending on species differences and the infected pathogen. some models only have about 3 days between infection and death. when conducting imaging research into infectious diseases, investigation of the survival rate of model animals as a preliminary experiment and preparation of a survival curve is desirable. also, breeding of model animals requires prevention of infection with other infectious diseases. experiments using infected animals thus require some additional caution, as previously mentioned. in conclusion, small animal imaging using preclinical pet bridges the gap between basic researches and clinical practices and works as an important tool for translational research in infectious disease. from tumor biology to clinical pet: a review of positron emission tomography (pet) in oncology recommendations on the use of 18f-fdg pet in oncology fdg pet/ct: eanm procedure guidelines for tumour imaging: version 2.0 high [18f]fluorodeoxyglucose uptake in abdominal abscess: a pet study ring-like uptake of [18f] fdg in brain abscess: a pet study differential diagnosis of lung tumor with positron emission tomography: a prospective study intratumoral distribution of fluorine-18-fluorodeosxyglucose in vivo: high accumulation in macrophages and granulation tissues studied by microautoradiography fdg accumulation in tumor tissue high accumulation of fluorine-18-fluorodeoxyglucose in turpentine-induced inflammatory tissue fdg uptake and glucose transporter subtype expressions in experimental tumor and inflammation models usefulness of 11c-methionine for differentiating tumors from granulomas in experimental rat models: a comparison with 18f-fdg and 18f-flt uptake of [18f]fluorodeoxyglucose in human monocyte-macrophages in vitro distinct localization of glut-1, -3, and -5 in human monocyte-derived macrophages: effects of cell activation dissociation between respiratory burst activity and deoxyglucose uptake in human neutrophil granulocytes: implications for interpretation of 18f-fdg pet images increased 18f-fdg uptake in a model of inflammation: concanavalin a-mediated lymphocyte activation inflammatory cytokines and hypoxia contribute to 18f-fdg uptake by cells involved in pannus formation in rheumatoid arthritis hypoxia prolongs monocyte/macrophage survival and enhanced glycolysis is associated with their maturation under aerobic conditions critical role of glucose metabolism in rheumatoid arthritis fibroblast-like synoviocytes hif-1α is essential for myeloid cell-mediated inflammation hif1 and glucose metabolism in the solid tumour eanm/snmi guideline for 18f-fdg use in inflammation and infection atherosclerotic plaque caps are locally weakened when macrophages density is increased focal and multi-focal plaque macrophage distributions in patients with acute and stable presentations of coronary artery disease microautoradiographic study for the differentiation of intratumoral macrophages, granulation tissues and cancer cells by the dynamics of fluorine-18-fluorodeoxyglucose uptake fluorodeoxyglucose uptake in the aortic wall at pet/ct: possible finding for active atherosclerosis imaging atherosclerotic plaque inflammation with [18f]-fluorodeoxyglucose positron emission tomography 18f fdg uptake in the large arteries: a correlation study with the atherogenic risk factors 18)f-fdg accumulation in atherosclerotic plaques: immunohistochemical and pet imaging study molecular pathology in vulnerable carotid plaques: correlation with [18]-fluorodeoxyglucose positron emission tomography (fdg-pet) in vivo 18f-fluorodeoxyglucose positron emission tomography imaging provides a noninvasive measure of carotid plaque inflammation in patients characterization of plaques using 18f-fdg pet/ct in patients with carotid atherosclerosis and correlation with matrix metalloproteinase-1 vascular inflammation evaluated by [18f]-fluorodeoxyglucose positron emission tomography is associated with the metabolic syndrome evaluation of carotid plaque inflammation in patients with active rheumatoid arthritis using (18)f-fluorodeoxyglucose pet-ct and mri: a pilot study what can be seen by 18f-fdg pet in atherosclerosis imaging? the effect of foam cell formation on 18f-fdg uptake to macrophages in vitro 18f-fdg pet and intravascular ultrasonography (ivus) images compared with histology of atherosclerotic plaques: 18f-fdg accumulates in foamy macrophages macrophage phenotypes in atherosclerosis macrophage diversity and polarization in atherosclerosis: a question of balance comparison of contrast agents for atherosclerosis imaging using cultured macrophages: fdg versus ultrasmall superparamagnetic iron oxide fibromuscular cap composition is important for the stability of established atherosclerotic plaques in mature whhl rabbits treated with statins statins alter smooth muscle cell accumulation and collagen content in established atheroma of watanabe heritable hyperlipidemic rabbits the effects of probucol on the progression of atherosclerosis in mature watanabe heritable hyperlipidaemic rabbits in vivo inhibition of foam cell development by probucol in watanabe rabbits application of 18f-fdg pet for monitoring the therapeutic effect of antiinflammatory drugs on stabilization of vulnerable atherosclerotic plaques effect of statin therapy on arterial wall inflammation based on 18f-fdg pet/ct: a systematic review and meta-analysis of interventional studies the role of pet in evaluating atherosclerosis: a critical review quantification of atherosclerotic plaque activity and vascular inflammation using [18-f] fluorodeoxyglucose positron emission tomography/computed tomography (fdg-pet/ct) the effects of 3-month atorvastatin therapy on arterial inflammation, calcification, abdominal adipose tissue and circulating biomarkers comparison of atorvastatin 5 and 20 mg/d for reducing f-18 fluorodeoxyglucose uptake in atherosclerotic plaques on positron emission tomography/computed tomography: a randomized, investigator-blinded, open-label, 6-month study in japanese adults scheduled for percutaneous coronary intervention simvastatin attenuates plaque inflammation: evaluation by fluorodeoxyglucose positron emission tomography safety and efficacy of dalcetrapib on atherosclerotic disease using novel non-invasive multimodality imaging (dal-plaque): a randomised clinical trial relationship of serum inflammatory biomarkers with plaque inflammation assessed by fdg pet/ct: the dal-plaque study type 2 diabetes enhances arterial uptake of choline in atherosclerotic mice: an imaging study with positron emission tomography tracer (1)(8) f-fluoromethylcholine evaluation and comparison of 11c-choline uptake and calcification in aortic and common carotid arterial walls with combined pet/ct carotid artery plaque uptake of (11)c-pk11195 inversely correlates with circulating monocytes and classical cd14(++)cd16(−) monocytes expressing hla-dr imaging intraplaque inflammation in carotid atherosclerosis with 11c-pk11195 positron emission tomography/computed tomography measurement of (68)ga-dotatoc uptake in the thoracic aorta and its correlation with cardiovascular risk 64cu-dotatate for noninvasive assessment of atherosclerosis in large arteries and its correlation with risk factors: head-to-head comparison with 68ga-dotatoc in 60 patients noninvasive assessment of hypoxia in rabbit advanced atherosclerosis using (1)(8)f-fluoromisonidazole positron emission tomographic imaging correlation of fluorine 18-labeled sodium fluoride uptake and arterial calcification on whole-body pet/ct in cancer patients predictive value of (18)f-sodium fluoride positron emission tomography in detecting high-risk coronary artery disease in combination with computed tomography in search of the vulnerable patient or the vulnerable plaque: (18)f-sodium fluoride positron emission tomography for cardiovascular risk stratification 18)f-sodium fluoride positron emission tomography for molecular imaging of coronary atherosclerosis based on computed tomography analysis amyloid-targeting pet tracer [(18)f]flutemetamol accumulates in atherosclerotic plaques 18)f-naf and (18)f-fdg as molecular probes in the evaluation of atherosclerosis association between osteogenesis and inflammation during the progression of calcified plaque evaluated by (18)f-fluoride and (18)f-fdg new insight of functional molecular imaging into the atheroma biology: 18f-naf and 18f-fdg in symptomatic and asymptomatic carotid plaques after recent cva. preliminary results quantification of temporal changes in calcium score in active atherosclerotic plaque in major vessels by (18)f-sodium fluoride pet/ct identifying active vascular microcalcification by (18)f-sodium fluoride positron emission tomography recent progress in the development of tspo pet ligands for neuroinflammation imaging in neurological diseases longitudinal imaging of microglia-astrocyte activation in mouse mesial temporal lobe epilepsy with tspo pet to identify the best therapeutic time windows in vivo positron emission tomographic imaging of glial responses to amyloid-beta and tau pathologies in mouse models of alzheimer's disease and related disorders translocator protein-18 kda (tspo) positron emission tomography (pet) imaging and its clinical impact in neurodegenerative diseases imaging of peripheral benzodiazepine receptor expression as biomarkers of detrimental versus beneficial glial responses in mouse models of alzheimer's and other cns pathologies distinct binding of amyloid imaging ligands to unique amyloid-beta deposited in the presubiculum of alzheimer's disease in vivo visualization of tau accumulation, microglial activation, and brain atrophy in a mouse model of tauopathy rtg4510 longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of alzheimer's disease enabled by positron emission tomography comparative in vitro and in vivo quantifications of pathologic tau deposits and their association with neurodegeneration in tauopathy mouse models imaging of brain tspo expression in a mouse model of amyotrophic lateral sclerosis with (18) f-dpa-714 and micro-pet/ct 11c]pbr28 pet imaging is sensitive to neuroinflammation in the aged rat tspo imaging in parkinsonian disorders pet imaging of [(11)c]pbr28 in parkinson's disease patients does not indicate increased binding to tspo despite reduced dopamine transporter binding animal models of amyotrophic lateral sclerosis: a comparison of model validity practical considerations for choosing a mouse model of alzheimer's disease an 18-kda translocator protein (tspo) polymorphism explains differences in binding affinity of the pet radioligand pbr28 kinetic modeling without accounting for the vascular component impairs the quantification of [(11)c]pbr28 brain pet data novel reference region model reveals increased microglial and reduced vascular binding of 11c-(r)-pk11195 in patients with alzheimer's disease pet imaging of microglia by targeting macrophage colony-stimulating factor 1 receptor (csf1r) synthesis and initial in vivo evaluation of [(11) c]az683-a novel pet radiotracer for colony stimulating factor 1 receptor (csf1r) purinergic receptors p2y12r and p2x7r: potential targets for pet imaging of microglia phenotypes in multiple sclerosis identification of the allosteric p2x7 receptor antagonist [(11)c]smw139 as a pet tracer of microglial activation characterization of (11)c-gsk1482160 for targeting the p2x7 receptor as a biomarker for neuroinflammation pet imaging of the p2x7 ion channel with a novel tracer [(18)f]jnj-64413739 in a rat model of neuroinflammation preclinical evaluation and non-human primate receptor occupancy study of (18)f-jnj-64413739, a novel pet radioligand for p2x7 receptors f-jnj-64413739, a novel pet ligand for the p2x7 ion channel: radiation dosimetry, kinetic modeling, testretest variability and occupancy of the p2x7 antagonist jnj-54175446 identification of new molecular targets for pet imaging of the microglial anti-inflammatory activation state neuroinflammation: the devil is in the details inflammatory responses in brain ischemia inflammatory projections after focal brain injury trigger neuronal network disruption: an (18) f-dpa714 pet study in mice role of neuroinflammation in neurodegenerative diseases (review) bridging autism spectrum disorders and schizophrenia through inflammation and biomarkers-pre-clinical and clinical investigations imaging microglial activation with tspo pet: lighting up neurologic diseases? comparison of [ 11 c]-(r)-pk 11195 and [ 11 c]pbr28, two radioligands for translocator protein (18 kda) in human and monkey: implications for positron emission tomographic imaging of this inflammation biomarker 11 c-dpa-713 has much greater specific binding to translocator protein 18 kda (tspo) in human brain than 11 c-( r)-pk11195 as new pet tracers for tspo: a comparison with [ 11 c]-(r)-pk11195 in a rat model of herpes encephalitis an 18-kda translocator protein (tspo) polymorphism explains differences in binding affinity of the pet radioligand pbr28 reactive astrocytes overexpress tspo and are detected by tspo positron emission tomography imaging evaluation of recanalization of occluded middle cerebral artery and restored cerebral blood flow in rats with transient brain ischemia: a combination study of digital subtraction angiography and 15 o-water positron emission tomography experimental studies of ischemic brain edema. a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemia area evaluation of the pbr/ tspo radioligand [(18)f]dpa-714 in a rat model of focal cerebral ischemia reduced pbr/tspo expression after minocycline treatment in a rat model of focal cerebral ischemia: a pet study using temporal changes in cell marker expression and cellular infiltration in a controlled cortical impact model in adult male c57bl/6 mice myeloid-derived suppressor cells infiltrate the brain and suppress neuroinflammation in a mouse model of focal traumatic brain injury in vivo positron emission tomographic imaging of glial responses to amyloid-beta and tau pathologies in mouse models of alzheimer's disease and related disorders imaging of peripheral benzodiazepine receptor expression as biomarkers of detrimental versus beneficial glial responses in mouse models of alzheimer's and other cns pathologies longitudinal pet-mri reveals beta-amyloid deposition and rcbf dynamics and connects vascular amyloidosis to quantitative loss of perfusion in vivo expression of cyclooxygenase-1 in activated microglia and macrophages during neuroinflammation visualized by pet with 11c-ketoprofen methyl ester detection of cyclooxygenase-1 in activated microglia during amyloid plaque progression: pet studies in alzheimer's disease model mice 11c]pk11195 pet imaging of spinal glial activation after nerve injury in rats )f-vc701-pet and mri in the in vivo neuroinflammation assessment of a mouse model of multiple sclerosis in vivo imaging of neuroinflammation in alzheimer's disease p2x7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of alzheimer's disease p2y12 expression and function in alternatively activated human microglia preclinical evaluation and non-human primate receptor occupancy study of (18)f-jnj-64413739, a novel pet radioligand for p2x7 receptors 18)f-jnj-64413739, a novel pet ligand for the p2x7 ion channel: radiation dosimetry, kinetic modeling, test-retest variability and occupancy of the p2x7 antagonist jnj-54175446 p2x7 radioligand (18)f-pttp for the differentiation of lung tumor and inflammation emerging pet radiotracers and targets for imaging of neuroinflammation in neurodegenerative diseases: outlook beyond tspo translocator protein (18 kda): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function c]daa1106: radiosynthesis and in vivo binding to peripheral benzodiazepine receptors in mouse brain novel peripheral benzodiazepine receptor ligand [ 11 c]daa1106 for pet: an imaging tool for glial cells in the brain quantitative analysis for estimating binding potential of the peripheral benzodiazepine receptor with [ 11 c]daa1106 18 f]fmdaa1106 and [ 18 f]fedaa1106: two positron-emitter labeled ligands for peripheral benzodiazepine receptor (pbr) development of a new radioligand, n-(5-fluoro-2-phenoxyphenyl)-n-(2-[ 18 f] fluoroethyl-5-methoxybenzyl)acetamide, for pet imaging of peripheral benzodiazepine receptor in primate brain quantitative analyses of 18 f-fedaa1106 binding to peripheral benzodiazepine receptors in living human brain 11 c-ac-5216: a novel pet ligand for peripheral benzodiazepine receptors in the primate brain quantitative analysis of peripheral benzodiazepine receptor in the human brain using pet with 11 c-ac-5216 18 f]feac and [ 18 f] fedac: two novel positron emission tomography ligands for peripheral-type benzodiazepine receptor in the brain 18 f-feac and 18 f-fedac: pet of the monkey brain and imaging of translocator protein (18 kda) in the infarcted rat brain efficient radiosynthesis and non-clinical safety tests of the tspo radioprobe [ 18 f] fedac: prerequisites for clinical application in vivo imaging and quantitative analysis of tspo in rat peripheral tissues using smallanimal pet with [ 18 f]fedac pet imaging of lung inflammation with [ 18 f]fedac, a radioligand for translocator protein (18 kda) translocator protein (18 kda), a potential molecular imaging biomarker for non-invasively distinguishing non-alcoholic fatty liver disease visualization of acute liver damage induced by cycloheximide in rats using pet with [ 18 f]fedac, a radiotracer for translocator protein (18 kda) utility of translocator protein (18 kda) as a molecular imaging biomarker to monitor the progression of liver fibrosis 11 c]dac-pet for noninvasively monitoring neuroinflammation and immunosuppressive therapy efficacy in rat experimental autoimmune encephalomyelitis model 18 f-fedac as a targeting agent for activated macrophages in dba/1 mice with collagen-induced arthritis: comparison with 18 f-fdg in vivo imaging of activated macrophages by 18 f-fedac, a tspo targeting pet ligand, in the use of biologic disease-modifying anti-rheumatic drugs (bdmards) microglia and neurodegeneration: the role of systemic inflammation microglia in the human fetal spinal cord; patterns of distribution, morphology and phenotype production of interleukin-12 and expression of its receptors by murine microglia histamine production by cultured microglial cells of the mouse pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines interleukin-4-induced selective clearance of oligomeric betaamyloid peptide1-42 by rat primary type-2 microglia the origin and nature of ramified and amoeboid microglia: a historical review and current concepts activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase c identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse the murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene microglia: intrinsic immuneffector cell of the brain cd14: cell surface receptor and differentiation marker macrophage scavenger receptors morphological transformation of microglia in vitro production of interleukin-5 by mouse astrocytes and microglia in culture induction of functional interleukin-2 receptor in mouse microglia il-2 induction of il-1 beta mrna expression in monocytes monocyte interleukin-2-receptor gene expression and interleukin-2 augmentation of microbicidal activity down regulation of cd4 expression in cultured microglia by immunosuppressants and lipopolysaccharide bone marrow derived elements and resident microglia in brain inflammation absence of donor-type major histocompatibility complex class i antigen-bearing microglia in the rat central nervous system of radiation bone marrow chimeras immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to ia-positive cells with dendritic morphology selection of intraepithelial lymphocytes with cd8 alpha/alpha co-receptors by self-antigen in the murine gut granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro nigral dopaminergic cell loss in vitamin e deficient rats cellular and molecular mechanisms of parkinson's disease: neurotoxins, causative genes, and inflammatory cytokines role of cytokines in inflammatory process in parkinson's disease distribution of major histocompatibility complex class il-positive microglia and cytokine profile of parkinson's disease brains blockade of microglial activation is neuroprotective in the 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of parkinson disease nadph oxidase mediates oxidative stress in the 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine model of parkinson disease amyloid-beta peptides induce several chemokine mrna expressions in the primary microglia and ra2 cell line via pi3k/akt and/or erk pathway activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with 1-methyl-4-phenyl 1,2,3,6-tetra-hydropyridine neuroprotective effect of exogenous microglia in global brain ischemia the hiv-1 nef protein and phagocyte nadph oxidase activation cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells validation of an animal isolation imaging chamber for use in animal biosafety level-3 containment severe fever with thrombocytopenia syndrome and its pathogen sftsv 18f-fdg pet imaging for identifying the dynamics of intestinal disease caused by sftsv infection in a mouse model development of a (68) ge/(68)ga generator system using polysaccharide polymers and its application in pet imaging of tropical infectious diseases ga-67 citrate imaging in tumors of the genito-urinary tract: report of cooperative study pediatric solid tumors: evaluation by gallium-67 spect studies mechanism of gallium-67 accumulation in inflammatory lesions cell biology and immunology of leishmania acknowledgements i would like to thank my collaborators: dr. sanae hosomi (department of traumatology and acute critical medicine) for the tbi study and dr. naoyuki sato (department of clinical gene therapy) for the ad study. the author thanks mr. kumata, ms. hatori, dr. xie, dr. yanamoto, and dr. yamasaki in my group for their fruitful experiments and continuous outputs which contributed to this review. the authors are grateful to the american chemical society (acs) for allowing reuse of some images (figs. 1.31 and 1.32) and a direct link to the acs article: https://pubs.acs. org/doi/abs/10.1021/acsomega.7b00147. 33farmeconomia e percorsi terapeutici 2003; 4 (1) © seed tutti i diritti riservati introduzione l’artrite reumatoide (ar) è una malattia infiammatoria cronica caratterizzata da dolore, disabilità funzionale e distruzione delle strutture articolari. a 10 anni dalla diagnosi quasi il 50% dei pazienti risulta inabile al lavoro [1]. la mortalità dei soggetti affetti da ar è significativamente più elevata di quella della popolazione generale [2, 3]. in italia si stima che la prevalenza dell’ar sia lo 0.5% (circa 300.000 malati). le recenti acquisizioni sul processo reumatoide nelle sue diverse componenti di flogosi, proliferazione sinoviale e distruzione articolare hanno permesso di mettere a punto farmaci, ottenuti mediante biotecnologie, selettivamente mirati ai meccanismi patogenetici della malattia. svariati farmaci biologici potenzialmente attivi nell’ar sono attualmente in fase di studio. tra questi, gli agenti in grado di bloccare il tnf-α (etanercept e infliximab) sono quelli maggiormente sperimentati e autorizzati per uso clinico in italia. risulta pertanto interessante valutare il profilo farmacoeconomico degli anti-tnf-α, con particolare riferimento ad etanercept. letteratura economica dell’uso di etanercept esistono diversi lavori pubblicati circa gli aspetti economici della terapia con anti-tnf-α. uno studio farmacoeconomico olandese [4] ha confrontato i costi annuali totali attribuibili a etanercept e infliximab in pazienti adulti affetti da artrite reumatoide. i risultati del lavoro (espressi nel lavoro originale in fiorini olandesi) riportano un minore consumo di risorse economiche in seguito alla somministrazione di etanercept in monoterapia rispetto a trattamento con etanercept dei soggetti con artrite reumatoide in italia: considerazioni economiche lorenzo g. mantovani* * econd, msc, dsc centro di farmacoeconomia università degli studi di milano abstract rheumatoid arthritis is a chronic inflammatory disease affecting the joints and leading to work disability in half of the patients; its prevalence in italy is estimated to be around 0,5%. the understanding of the pathophysiology of the disease has lead to the development of biotechnologically-derived drugs, among which anti-tnf-α agents. at the present moment, two of these drugs are available in italy: etanercept and infliximab. the aim of the present work was to furnish a pharmaco-economical comparison among the two antitnf-α agents in italy. the efficacy of antirheumatic therapies is usually measured in terms of percentage of responders, defined as those patients whose symptoms improve of at least 20, 50 or 70% (acr20, acr50, acr70). a review of the published literature revealed that, according to these accepted parameters, etanercept results superior when infliximab is administered at standard doses, while it is at least as effective as infliximab when the latter is at the highest doses. although drug acquisition costs are comparable for the two drugs at the suggested regimens, economical evaluations performed in several countries indicate that total treatment costs are lower with etanercept as compared to infliximab, mainly because of differences in the route of administration (subcutaneous vs. slow iv infusion), need for patient monitoring and co-administration of methotrexate with infliximab and days at work gained. thus, from this preliminary analysis it appears that etanercept dominates infliximab, i.e. it provides better results at a lower cost, but this result needs to be tested and confirmed by prospective economical evaluations. farmeconomia e percorsi terapeutici 2003; 4 (1): 33-38 analisi economica 34 farmeconomia e percorsi terapeutici 2003; 4 (1)© seed tutti i diritti riservati infliximab (in combinazione con methotrexate). l’evidenza proposta da nujiten e colleghi mostra come a fronte di costi della terapia farmacologica sovrapponibili (14.200-14.300 euro circa), la terapia con infliximab risulta più costosa a motivo del maggior consumo di altre risorse sanitarie (visite, diagnostica, terapie concomitanti, day-hospital etc.) necessarie per la somminstrazione endovenosa di infliximab, la quale richiede l’accesso ospedaliero del paziente. i costi aggiuntivi annui della terapia con infliximab rispetto a etanercept sono quantificabili in circa 5.500 euro, a carico del terzo pagante. tali stime risultano conservative, in quanto nujiten e colleghi non considerano l’escalation nel dosaggio di infliximab. il national institute for clinical excellence (nice) britannico, in “guidance on the use of etanercept and infliximab for the treatment of rheumatoid arthritis” [5], raccomandando l’uso dei nuovi farmaci in europa per la terapia dell’artrite reumatoide nei bambini e negli adulti, pubblica i risultati di una valutazione economica sull’impatto dei farmaci anti-tnf (infliximab, etanercept) nel trattamento dell’artrite reumatoide. l’analisi di costo-utilità incrementale, effettuata mediante un modello preliminare che considerava i costi derivanti dal farmaco, ma non i risparmi in termini di ospedalizzazione, di trasporto, di produttività e di morte, mostrava un costo pari a 63,974£ per qaly (qualityadjusted life-year) guadagnato per etanercept e pari a 99,373£ per infliximab. un’analisi costo-utilità, elaborata con un modello più ampio proposto da wyeth, ha fornito per etanercept un valore di costo-efficacia incrementale di 16,330£ per qaly. un’analisi simile, con un modello proposto da scheringplough, ha fornito perinfliximab un valore costo-efficacia incrementale di 23,936£ per qaly. queste discrepanze hanno portato il nice a rivedere le proprie analisi. è stata pertanto effettuata a cura del nice una rivalutazione dell’analisi costo-utilità fornita dalle case produttrici, considerando valori più conservativi e restrittivi per le variabili. i risultati hanno mostrato un valore di costo-utilità incrementale di 27,000£ per qaly per etanercept e di 35,000£ per qaly per infliximab. questi ultimi valori sono quelli utilizzati dal nice nel prendere la propria decisione (positiva) riguardo alla terapia con anti-tnf-α. diverse valutazioni economiche dell’utilizzo di anti-tnf-α sono state presentate durante il congresso dell’eular nel 2002. i punti che da esse emergono sono i seguenti. innanzitutto la terapia con anti-tnf-α è in assoluto in grado di generare un minor consumo di risorse sanitarie [6], particolarmente nei soggetti sottoposti a terapia con etanercept vs infliximab, a motivo della necessità della somministrazione di quest’ultimo in regime di day-hospital [7]. in secondo luogo, la terapia con anti-tnf-α genera un miglioramento marcato dello status lavorativo dei soggetti che la ricevono in termini sia di giornate lavorate, sia di partecipazione alla forza lavoro [6, 8-9]. tale miglioramento è comunque più marcato per etanercept, vista la possibilità di effettuare la terapia a domicilio, evitando di perdere giornate lavorative a motivo della somministrazione, come invece accade per infliximab. infine la terapia con anti-tnf-α mostra valori di costo efficacia e di costo utilità che rientrano nei parametri giudicati accettabili dalla comunità scientifica, particolarmente quando vengono presi in considerazioni i costi indiretti (status lavorativo). in tale ambito, etanercept mostra un miglior profilo di costo-efficacia e costoutilità sia in assoluto sia in comparazione ad infliximab [10-13]. un lavoro pubblicato sul numero del febbraio 2002 di arthritis & rheumatism, “ guidelines for the management of rheumatoid arthritis: 2002 update” [14], riporta il confronto tra i costi annuali della terapia effettuata con infliximab ed etanercept, con l’esame di diversi dosaggi di infliximab. questi dati non tengono conto del costo aggiuntivo del metotressato. i risultati, in dollari, sono visualizzati nella figura 1. efficacia dell’uso di etanercept i risultati dei trials clinici fino ad ora riportati hanno dimostrato l’efficacia del trattamento con anti-tnf-a. esiste, di fatto, una tendenza ad una efficacia inferiore del dosaggio più basso di infliximab (3 mg/kg ogni 8 settimane) sia rispetto all’alto dosaggio dello stesso farfigura 1 costo annuo della terapia con anti-tnf-a negli stati uniti (us$) trattamento con etanercept dei soggetti con artrite reumatoide in italia: considerazioni economiche 35farmeconomia e percorsi terapeutici 2003; 4 (1) © seed tutti i diritti riservati maco (10 mg/kg) sia, indirettamente, rispetto ad etanercept [15-19]. le tabelle 1 e 2 mostrano l’efficacia di etanercept e infliximab nel generare una risposta secondo i criteri acr 20, 50, 70 a 6 mesi e un anno, in diverse tipologie di pazienti con ar (precoce, attiva) con e senza (per etanercept) la terapia concomitante con metotressato. costo della terapia con anti-tnfααααα infliximab si somministra per infusione lenta (circa tre ore). questo comporta l’ospedalizzazione del paziente (anche in day hospital) con relativi costi diretti di screening (radiografia, tine-test, ecg) e somministrazione. ciò si traduce, nell’ottica dell’ospedale, in un impegno aggiuntivo del personale medico/paramedico specializzato, del farmacista che prepara il farmaco prima della somministrazione, del reumatologo, dell’anestesista. nella prospettiva del servizio sanitario nazionale, la necessità della somministrazione in regime ospedaliero si traduce nel costo di un drg, o di dh o di regime ordinario. al contrario, etanercept si somministra per via sottocutanea anche a domicilio del paziente, in modo più semplice, economico e, prevedilmente, meglio accettato dal paziente. il costo totale della terapia dipende, oltre che dal costo diretto del farmaco, anche da altri costi, quali l’eventuale associazione con altri farmaci, il monitoraggio della tollerabilità, la somministrazione. etanercept è indicato ad una dose fissa di 25 mg due volte alla settimana (riassunto delle caratteristiche del prodotto). il costo di una fiala è 127,56 euro, mentre il costo annuale del farmaco risulta essere di 13.260 euro circa. etanercept può essere usato in monoterapia e non si hanno perciò i costi di una terapia aggiuntiva. inoltre non sono previsti controlli strumentali (radiografia, ecg) o analisi di laboratorio “extra routine” per valutarne la tollerabilità. infine la somministrazione di etanercept, per via sottocutanea, può essere effettuata a domicilio del paziente. non richiede, perciò, il ricovero ospedaliero, con conseguente impegno di personale specializzato. la tabella 3 riassume il costo della terapia con etanercept, facendo riferimento al protocollo dello studio antares. infliximab è stato utilizzato negli studi sull’artrite reumatoide ad una dose variabile dai 3 ai 10 mg per kg di peso (tempo 0, 2, 6 settimane, quindi ogni 4-8 settimane). alcuni studi esteri hanno registrato la necessità di un’escalation nel dosaggio di infliximab nei pazienti non responders. tabella 1 frequenza di responders ad un anno secondo i criteri acr20, acr50, acr70 tabella 2 frequenza di responders a sei mesi secondo i criteri acr20, acr50, acr70 l. g. mantovani oiretirc tpecrenate etnof bamixilfni etnof gm52 repetlov2 anamittes gk/gm3 8ingo enamittes gk/gm3 4ingo enamittes gk/gm01 8ingo enamittes gk/gm01 4ingo enamittes 02rca )onna1( %26 ]51[ %24 %84 %95 %95 ]61[ 05rca )onna1( %54 ]51[ %12 %43 %93 %83 ]61[ 07rca )onna1( %52 ]51[ %01 %71 %52 %91 ]61[ oiretirc tpecrenate etnof bamixilfni etnof 2gm52 repetlov anamittes gk/gm3 8ingo enamittes gk/gm3 4ingo enamittes gk/gm01 8ingo enamittes gk/gm01 4ingo enamittes 02rca )isem6( %95 %17 ]71[ ]81[ %05 %35 %25 %85 ]91[ 05rca )isem6( %04 %93 ]71[ ]81[ %72 %92 %13 %62 ]91[ 07rca )isem6( %51 %51 ]71[ ]81[ %8 %11 %81 %11 ]91[ 36 farmeconomia e percorsi terapeutici 2003; 4 (1)© seed tutti i diritti riservati durez e colleghi hanno aumentato, a partire dalla ventiduesima settimana e in ¼ dei pazienti, la dose in misura pari ad una fiala per paziente per somministrazione, mantenendo la frequenza di somministrazione [20]. nello stesso studio, dei pazienti ancora in trattamento originario un ulteriore 24,2% e 9,2% di pazienti alla quarantaseiesima e sessantaduesima hanno aumentato la dose. dati simili sono stati riportati dallo studio sture: le dosi iniziali di 3 mg/kg sono state aumentate a 5-7 mg/kg in circa il 45% dei pazienti complessivamente [21]. va segnalato che le dosi accresciute vengono mantenute anche durante i periodi successivi, aumentando, prevedibilmente, i costi negli anni successivi al primo. l’esperienza derivante dalla pratica clinica in italia, e riportata in alcuni abstract del 38° congresso sir 2001 [22-24], conferma la necessità dell’aumento del dosaggio pro chilo e/ o della riduzione della frequenza delle somministrazioni e della riduzione dell’effetto clinico nel lungo periodo di terapia con infliximab. nello specifico, fantini e colleghi hanno somministrato infliximab ogni 6 settimane dopo il periodo di induzione, vale a dire a partire dalla dalla 14esima settimana. sulla base di questi dati è stata effettuata una valutazione dei costi annui della terapia con infliximab. il protocollo di fantini e colleghi corrisponde ad un numero di 10 somministrazioni per paziente. in questo protocollo non è stata riportata alcuna escalation nel dosaggio di infliximab. per calcolare il numero di fiale utilizzate si suppone che il 75% dei pazienti abbia un peso corporeo inferiore ai 70 kg richiedendo 2 fiale per somministrazione e che il restante 25% richieda 3 fiale a somministrazione. a tali costi sono stati aggunti i costi dei drg relativi ai day hospital necessari per la somminstrazione di infliximab ed i costi della terapia con metotressato (formulazione orale, ipotesi conservativa). è stata inoltre effettuata un’analisi per verificare quali siano/sarebbero i costi della somministrazione di infliximab come riportato nello studio sture [21], vale a dire ipotizzando un dosaggio iniziale di 3mg/kg con escalation terapeutica fino a 5-7 mg/kg, mantenendo la frequenza ogni 8 settimane durante la fase di mantenimento. anche in questo caso, al fine di calcolare il numero di fiale utilizzate si suppone che il 75% dei pazienti abbia un peso corporeo inferiore ai 70 kg richiedendo 2 fiale per somministrazione e che il restante 25% richieda 3 fiale a somministrazione. la tabella 3 riporta i risultati relativi ad una coorte ipotetica di 100 pazienti per gruppo. conclusioni in virtù delle analisi presentate, non è possibile calcolare un rapporto di costo-efficacia incrementale di etanercept vs infliximab, in quanto la terapia con etanercept risulta contemporaneamente sia meno costosa sia più efficace. essa appare, in altri termini, dominante. la terapia con etanercept appare ugualmente o più efficace rispetto a quella con infliximab tabella 3 costi (in euro) del primo anno di terapia con anti-tnf-α in 100 pazienti italiani § § si suppone che il 75% dei pazienti abbia un peso corporeo inferiore ai 70 kg richiedendo 2 fiale per somministrazione e che il restante 25% richieda 3 fiale a somministrazione * si suppone che la somministrazione avvenga al dosaggio di 3 mg/kg durante tutto il periodo e che a partire dal termine del periodo di induzione (45-esimo giorno) la terapia di mantenimento venga somministrata ogni 45 giorni. # si suppone che infliximab venga somministrato ogni 8 settimane durante tutto il periodo di mantenimento, aumentando il dosaggio nei non responders a 5-7 mg/kg (6 mg/kg in media). @ quantificato utilizzando la tariffa nazionale per il day hospital reumatologico. trattamento con etanercept dei soggetti con artrite reumatoide in italia: considerazioni economiche tpecrenate *]22[bamixilfni #erutsbamixilfni elaiforemun alaifrepotsoc elaizrapotsoc latipsohyad @latipsohyadotsoc elaizrapotsoc otassertotem aiparetotsoc elaizrapotsoc elatototsoc 004.01 5,721 000.623.1 002 142 002.44 ---------000.473.1 002.2 075 000.452.1 000.1 142 000.142 001 45 004.5 066.664.1 402.2 075 082.652.1 008 142 008.291 001 45 004.5 084.454.1 37farmeconomia e percorsi terapeutici 2003; 4 (1) © seed tutti i diritti riservati quando questa è prescritta ai dosaggi minimi, mentre risulta simile ai dosaggi più elevati di infliximab. il costo della terapia con etanercept è superiore al costo di quella con infliximab solo quando questo è somministrato ai dosaggi minimi (3 mg/kg, ogni 8 settimane in mantenimento), caso che appare irrealistico nella pratica per: 1) minor incidenza di responders acr20 a tali dosaggi/frequenza di somministrazione; 2) difficoltà nella divisione delle unità posologiche di infliximab. nelle condizioni reali di utilizzo (3 mg/kg ogni 6 settimane, escalation del dosaggio a 5-7 mg/ kg nei non responders), il costo della terapia con infliximab è superiore a quello di etanercept (dose fissa di 25 mg 2 volte la settimana). in tali condizioni di utilizzo, la terapia con etanercept appare dominante rispetto a quella con infliximab, in quanto meno costosa e maggiormente o ugualmente efficace. questi risultati preliminari andranno comunque verificati e confermati da studi economici prospettici. bibliografia 1. wolfe f et al. the long term outcomes of rheumatoid arthritis. arthritis rheum 1998; 41: 1072-82 2. wolfe f et al. the mortality of rheumatoid arthritis. arthritis rheum 1994; 37: 481-9 3. pincus t. the underestimated long term medical and economic consequences of ra. drugs 1995; 50 (suppl 1): 1-74. 4. nujiten mjc, engelfriet p, duijn k et al. a cost-cost study comparing etanercept with infliximab in rheumatoid arthritis. pharmacoeconomics 2001; 19:1051-64. 5. national institute for clinical excellence (nice). guidance on the use of etanercept and infliximab for the treatment of rheumatoid arthritis, march 2002. disponibile all’indirizzo web: www.nice.org.uk 6. yelin. eh, katz pp, lubeck dp et al. impact of etanercept (enbrel) on health care utilization and employment among persons with early rheumatoid arthritis. disponibile all’indirizzo web: http://www.eular.org/eular2002/ abstract.cfm 7. geborek p, eberhardt k, saxne t. etanercept and infliximab treatment reduces hospital care in arthritis patients. disponibile all’indirizzo web: http://www.eular.org/eular2002/abstract.cfm. 8. geborek p, eberhardt k, larsson b, et al. etanercept and infliximab treatment improves working capacity in arthritis patients. disponibile via url all’indirizzo web: http://www.eular.org/eular2002/abstract.cfm. 9. van vollenhoven r f, harju a, bratt j. treatment with infliximab or etanercept results in significant increases in work force participation: data from the stockholm tnf-alpha antagonist registry (sture). disponibile all’indirizzo web: http://www.eular.org/eular2002/abstract.cfm. 10. bansback n, bansback, brennan a, conway p, reynolds a. the cost-effectiveness of 3rd line etanercept versus other dmards in adults with rheumatoid arthritis in the uk. disponibile all’indirizzo web: http://www.eular.org/ eular2002/abstract.cfm. 11. bansback n, bansback, brennan a, conway p, reynolds a. impact of direct and indirect costs on the costeffectiveness of etanercept in rheumatoid arthritis in the uk. disponibile all’indirizzo web: http://www.eular.org/ eular2002/abstract.cfm. 12. malone dc, ortmeierbg. cost efficacy of etanercept versus infliximab plus methotrexate in rheumatoid arthritis based on radiographic data. disponibile all’indirizzo web: http://www.eular.org/eular2002/abstract.cfm. 13. malone dc, ortmeier bg. cost efficacy of etanercept versus infliximab plus methotrexate in the treatment of dmardresistant rheumatoid arthritis. disponibile all’indirizzo web: http://www.eular.org/eular2002/abstract.cfm. 14. american college of rheumatology. guidelines for the management of rheumatoid arthritis, 2002 update. arthr rheum 2002, 46: 328-346. 15. bathon jm, martin rw, fleischmann rm et al. a comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. nejm 2000; 343: 1586-93. 16. lipsky pe, vdhejide dmfm, st clair ew et al. infliximab and methotrexate in the treatment of rhematoid arthritis. nejm 2000; 343: 1594-602. 17. moreland lw, schiff mh, baumgartner sw et al. etanercept therapy in ra: a randomized controlled study. ann intern med 1999; 130:478-86. l. g. mantovani 38 farmeconomia e percorsi terapeutici 2003; 4 (1)© seed tutti i diritti riservati 18. weinblatt me, kremer jm, bankhurst ad et al. a trial of etanercept, a recombinant tumor necrosis factor receptor:fc fusion protein, in patients with ra receiving methotrexate. nejm 1999; 340:253-59. 19. maini r, st clair ew, breedveld f et al. infliximab vs placebo in ra patients receiving concomitant methotrexate: a randomized phase iii trial. lancet 1999; 354: 1932-1939. 20. durez p, devogelaer jp, van den bosch f et al. infliximab and methotrexate in the treatment of rheumatoid arthritis: clinical results in a belgian observational cohort.eular 2002. disponibile all’indirizzo web: http://www.eular.org/ eular2002/abstract.cfm. 21. van vollenhoven rf, gullstrom e, brannemark s, klareskog l. dose escalation of infliximab in clinical practice: data from the stockholm tnf-alpha registry (sture), eular congress 2002. disponibile all’indirizzo web: http://www.eular.org/eular2002/abstract.cfm. 22. fantini f, tosi s, sinigallia l, et al. risultati ad un anno del trattamento con infliximab in pazienti affetti da artrite reumatoide refrattaria. reumatismo 2001, 53 suppl. 4: 324. 23. botsios c, sfriso p, ostuni pa. applicazione e risultati preliminari del protocollo di studio osservazionale antares in 44 pazienti trattati con infliximab. reumatismo 2001, 53 suppl. 4: 324. 24. covelli m, scioscia c, compagno m et al. studio aperto sulla tollerabilità ed efficacia di un trattamento combinato con methotrexate ed infliximab in poliartriti croniche non responders: dati ad 1 anno e follow-up. reumatismo 2001, 53 suppl. 4: 325. trattamento con etanercept dei soggetti con artrite reumatoide in italia: considerazioni economiche 32 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10671 connective tissue diseases: focus on microcirculatory bed *o.i. zarudna, i.k. venher, a.v. dovbush i. horbachevsky ternopil national medical university, ternopil, ukraine background. a microcirculatory bloodstream is a target, source and reason of the pathological process in patients with systemic connective tissue diseases. objectives. this study is focused on meta-analyses of biopsy material of skin flaps harvested from patients’ fingers to identify specific morphological changes. methods. a retrospective analysis of the medical records of 39 examinees with systemic sclerosis (ssc), 45 with systemic lupus erythematosus (sle), and 45 with rheumatoid arthritis (ra) was performed. the condition of peripheral hemodynamics was examined with longitudinal rheovasography of arms and legs. endothelin-1 (ет1) concentration was evaluated by immunoenzymatic method. we assessed other results of clinical and laboratory tests to compare them with morphological changes of the microcirculatory bed. results. most patients involved suffered from abnormal peripheral hemodynamics. it was revealed that kidneys, lungs or heart were damaged more frequently in the patients with peripheral blood circulation disorders, which were the most significant in the patients with ssc (p<0.05). disorders of peripheral blood flow were exacerbated in case of lengthening of the disease course. concentration of ет1 was relevantly higher in the patients with peripheral blood flow disorders. number of pathologic capillaries was the highest in the ssc patients. conclusions. in terms of integral estimation, extremely significant changes of microcirculatory bloodstream were evidenced in the patients with ssc. however, some morphometric peculiarities were revealed in the patients without peripheral blood flow disorders. thus, normal rheovasography did not exclude any microcirculation disorders. key words: microcirculation; systemic lupus erythematosus (sle); systemic sclerosis (ssc); rheumatoid arthritis (ra); biopsy; rheovasography. *corresponding author: olga zarudna, department of clinical immunology, allergology and general patients’ care, i. horbachevsky ternopil national medical university, 1 maidan voli, ternopil, 46001. e-mail: zarudna@tdmu.edu.ua. introduction manifestations of systemic connective tissue diseases are very diverse. however, one thing they all have in common is vasculitis/ vasculopathy that results in systematicity and development of permanent malfunctioning of body organs and systems. several studies show that patients with systemic lupus erythematosus (sle), systemic sclerosis (ssc), and rheumatoid arthritis (ra) experience such symptoms as raynaud syndrome, livedo reticularis, recurrent thrombophlebitis, digital vasculitis, capillaritis of palms and soles, etc. among the manifestations listed above the most common sign is raynaud syndrome that is characterized by cascade disruption of microcirculation: destruc­ tion of vascular endothelium, capillary basement membrane reduplication, intimal proliferation of smooth muscle cells with collagen hyperproduction and predisposition to vasoconstriction, vascular wall thickening with luminal occlusion, which is manifested by generalized clinical symptoms. meanwhile, endothelium is a target, source and reason of the pathological process. it becomes a receptive field for binding of circulating immunoglobulins, immune complexes, a complement and attacks of sensitized t-lymphocytes. it itself produces vascular endothelial growth factor, endothelin 1, etc. similar findings are presented in the literature [1, 2, 3, 4]. numerous studies have been conducted in order to identify noninvasive methods of studying microcirculatory bloodstream and detect specific pathological characteristics in favour of one or another connective tissue disease to help confirm a diagnosis. consequently, the method of nailfold videocapillaroscopy is relevant [5, 6, 7]. moreover, the results of nailfold videocapillaroscopic examination are included into diagnostic classification by eular criteria of systemic sclerosis [5], indicating their high specificity and sensitivity. for an overall assessment of capillaroscopic pattern, the following indicators are used: capillary length, intercapillary distance, loop diameter, internal diameter, capillary width, international journal of medicine and medical research 2019, volume 5, issue 2, p. 32-39 copyright © 2019, tnmu, all rights reserved o.i. zarudna et al. 33 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 apex width, venous limb, arterial limb [7]. data analysis shows typical changes, for example, ssc: giant capillaries, loss of capillaries with areas of avascularisation, ramified capillaries with pathological neoangiogenesis and severe derangement of capillary structure [5]. nailfold capillaroscopic form changes are less symptomatic in cases of other diseases, for example in sle. according to the literature concerning the above-mentioned pathology, they are present in 30-75% of cases. ssc (in contrast to sle) characteristically reveals these changes that occur in 99% of cases at various stages [5, 6]. the most frequent alterations in the patients with sle include increased tortuosity, elongated loops, and meandering bizarre loops [5]. the patients with ra are reported to have increased capillary tortuosity and prominent, clearly visible subpapillary venous plexus [5]. at the same time, according to the study of auto immune processes, in some pathological con ditions, correlation between the rate of biolo gically active substances and results of capillaroscopy was revealed. for example, in cases of sle a relation between concentration of endothelin­1 (ет­1) and angioscopic characteristics of microcirculatory bloodstream (mcb) was found [8]. meanwhile, increase in anti-endothelial antibody titre in cases of ssc was present in 22-86% of cases and combined primarily with lung damage and peripheral vascular bed [4]. a few researches describe the results of immune histochemical tests that allows establishing that cd20+, b-lymphocytes, macrophagocytes and dendritic cells, ig g, a, m, and a complement in the form of deposits along elastic membrane of a vascular wall prevail among the cells that infiltrate a vessel wall [9]. the revealed morphological abnormalities and immunological disorders in systemic diseases of connective tissue combined with endothelial dysfunction and coagulation system disorders create favourable conditions for development of severe systemic complications. the research is aimed at a retrospective study of morphological condition of a peripheral vascular bed in the patients with sle, ssc, and ra to compare morphological changes with clinical and laboratory data. methods retrospective analysis of the medical re cords of 129 patients with rheumatic diseases was performed: 39 were diagnosed with ssc, 45 – with sle, and 45 – with ra (all were the patients of ternopil university hospital, depart ment of rheumatology, 2001-2004). the statistical analysis revealed a typical situation for this category of diseases – predominance of women (86.05%) of fertile age. the average duration of the disease was 8.14±0.53 years. the archive of biopsy material of skin flaps harvested from patients’ fingers was reviewed that allowed morphometric analysis. the de cision for biopsy was made by a rheumatologist during the treatment at the department of rheumatology. for histological studies, pieces of skin were fixed in 10% neutral formalin solution. subsequent processing of the material followed by embedment in paraffin blocks was carried out by a conventional technique. the sections obtained by a sliding microtome were stained with hematoxylin-eosin. the histological specimens were studied using the optical microscope seo scan and images were made with the vision ccd camera with a histological image display system. the condition of peri pheral hemodynamics was examined by longi tudinal rheovasography of forearm and legs by means of software-hard ware complex of automated analysis usrh­1 (“усрг­01”). in the process of rheovaso­ graphic analysis the following medical parameters were estimated: the regularity of pulse waves, their appearance – upward and downward gradients, the type of a top (apex), extent of incisura, presence of additional waves, their localization on the de scending part of a curve. the following measu rements were analysed: as – percussion systolic wave amplitude of a rheogram, ohm; rі – rheographic index, that measures a magnitude of pulse volume, in other words a systolic flow; that is a correlation of a variable and a constant dimensions of impedance of the researched area, namely, a percussion wave of a rheogram and calibration impulse magnitude, relative units (ru); ai – interval between the isoline and incisures on the descending part of a curve, ohm; di – dicrotic index, which reflects the con­ dition of the tone of arteries and precapillary vessels, %. the main indicator that was used for analysis was a rheographic index that measured the magnitude of pulse volume that was a systolic flow defined by a ratio of a percussion wave of a rheogram and calibration impulse magnitude, relative units (ru). the condition of endothelium was examined in terms of one of the main integral indicators of its functional capacity, i.e. ет1. the contents of ет1 were determined using an immuno­ o.i. zarudna et al. 34 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 enzymatic method, which was based on the principle of competitive immunoenzymatic analysis, involving a reagent kit peninsula laboratories inc. (usa). the severity of inflammation was deter­ mined by erythrocyte sedimentation rate (degree 1 – esr <20 mm/hr, degree 2 – esr 20-40 mm/hr, degree 3 – esr >40 mm/hr). all case histories contained informed consents for the research. the study was conducted in accordance with the declaration of helsinki. the protocol was approved by the committee on bioethics of i. horbachevsky ternopil national medical university. statistical processing of the research results was performed by parametric analysis and nonparametric analysis using the student’s t-test and mann-whitney u-test by means of software package microsoft excel 5.0 and statistica 10 (statsoft). p values lower than 0.05 were considered to be statistically significant. results all cases were divided into two groups according to the state of peripheral hemodynamics, which was examined using longitudinal rheovasography of forearm and legs. the majority of patients, the model participants of the research, had abnormal peripheral hemodynamics (group 2). the ratio of the above-mentioned patients to the patients with normal peripheral hemodynamic parameters (group 1) was 5.1:1.0. clinical assessment of the involved patients is presented in table 1. assessment of peripheral hemodynamics allowed revealing significant changes in indices of longitudinal rheovasography of forearms and legs (table 2). morphometric analysis of skin biopsy was conducted in 26 model patients (table 3, table 4). biopsy results of the patients with different nosology and peripheral blood circulation disorders are presented at figures 1, 2, and 3. notably, microscopic examination revealed that general architectonics of vessel walls was not damaged. visible hyperplasia of endo the liocytes in all categories of patients was also evidenced, which may be treated as an adaptive mechanism in cases of blood circulation dis orders. the results of patients’ blood tests for concentration of ет1 (table 5) pointed to its extre­ mely high content in the patients with peripheral blood circulation disorders in contrast to donors’ indices. table 1. clinical assessment of the patients with/without peripheral blood circulation disorders indicant patients with normal peripheral hemodynamic parameters (1st group) with abnormal peripheral hemodynamics (2nd group) absolute numbers % absolute numbers % age (years): under 20 20-44 45-59 60 and above 3 9 5 4 2.3 6.9 3.9 3.1 4 53 38 13 3.1 41.1 29.5 10.1 males females 6 15 4.7 11.6 12 96 9.3 74.4 sle ssc ra 9 1 11 6.9 0.8 8.5 36 38 34 27.9 29.5 26.4 activity of the 1st degree activity of the 2nd degree activity of the 3rd degree 2 15 4 1.6 11.6 3.1 52 41 15 40.3 31.8 11.6 duration of a disease < 1 yr duration of a disease 1-5 yrs duration of a disease 5-10 yrs duration of a disease >10 yrs 3 6 5 7 2.3 4.7 3.9 5.4 10 38 30 30 7.8 29.5 23.2 23.2 kidney damage no kidney damage 6 15 4.7 11.6 28 80 21.7 62.0 cardiac involvement no cardiac involvement 7 14 5.4 10.9 37 71 28.7 55.0 lung damage no lung damage 5 16 3.9 12.4 23 85 17.8 65.9 o.i. zarudna et al. 35 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 at the same time, concentration of et-1 in the patients without vessel disorders is not substantially different from normal range. discussion among the examinees, the detection rate of peripheral hemodynamic disorders in the table 2. indices of peripheral blood circulation in the patients of group 2 (with peripheral hemodynamic disorders) indices forearms lower legs as, ohm rі, ru ai, ohm di, % as, ohm rі, ru ai, ohm di, % duration of disease < 1 yr, n=10 0.123± 0.002* 1.23± 0.02* 0.090± 0.002* 73.41± 0.08* 0.147± 0.003* 1.47± 0.03* 0.105± 0.002 71.58± 0.08* >1-5 yrs, n=38 0.091± 0.002* 0.91± 0.02* 0.075± 0.002 82.24± 0.06* 0.145± 0.002* 1.45± 0.02* 0.116± 0.002* 80.24± 0.03* >5-10 yrs, n=30 0.087± 0.003* 0.87± 0.03* 0.074± 0.002 84.98± 0.08* 0.127± 0.003* 1.27± 0.03* 0.106± 0.003* 83.49± 0.04* >10 yrs, n=30 0.085± 0.002* 0.85± 0.02* 0.074± 0.001 87.18± 0.05* 0.114± 0.003* 1.14± 0.03* 0.096± 0.002 86.26± 0.04* disease sle, n=37 0.109± 0.002* 1.09± 0.02* 0.083± 0.002* 75.79± 0.05* 0.130± 0.002* 1.30± 0.02* 0.108± 0.008* 83.39± 0.04* ssc, n=37 0.082± 0.002* 0.84± 0.02* 0.072± 0.002 85.73± 0.08* 0.113± 0.004* 1.13± 0.04* 0.095± 0.003 84.41± 0.06* ra, n=34 0.089± 0.002* 0.89± 0.02* 0.070± 0.002 79.06± 0.06* 0.122± 0.003* 1.22± 0.03* 0.099± 0.003 81.11± 0.03* activity of the inflam­ matory process 1st degree, n=52 0.095± 0.002* 0.95± 0.02* 0.080± 0.002* 84.15± 0.05* 0.121± 0.004* 1.21± 0.04* 0.099± 0.002 81.60± 0.09* 2nd degree, n=41 0.095± 0.003* 0.95± 0.03* 0.080± 0.002* 84.17± 0.05* 0.113± 0.002* 1.13 0.02* 0.095± 0.002 84.15± 0.04* 3rd degree, n=15 0.105± 0.005* 1.05± 0.05* 0.080± 0.004 75.52± 0.07* 0.138± 0.002* 1.13± 0.02* 0.111± 0.002* 80.44± 0.06* follow-up control 0.152± 0.008 1.52± 0.08 0.069± 0.005 45.43± 0.04 0.181± 0.012 1.81± 0.12 0.088± 0.008 78.53± 0.07* notes. * – р<0.05 – statistically significant difference between the indices in apparently healthy people of the control group and other studied patients. ra – rheumatoid arthritis; ssc – systemic sclerosis; sle – systemic lupus erythematosus. table 3. average density of skin hemocapillaries of the patients per 1mm² groups no peripheral blood circulation disorders (group1, n=11) peripheral blood circulation disorders (group 2, n=15) normal range 43.0±1.9 – rheumatoid arthritis, n=8 40.0±1.8 p1>0.05 37.0±1.6 p1>0.05 р2>0.05 systemic sclerosis, n=8 38.0±1.7 p1>0.05 21.0±1.0 p1<0.01 p2<0.01 systemic lupus erythematosus, n=10 41.0±1.9 p1>0.05 36.0±1.7 p1<0.05 р2>0.05 notes: p1 – statistically significant difference between the normal range and average density of hemocapillaries per unit area in patients of both groups; р2 – statistically significant difference between the indices in patients of the groups 1 and 2. o.i. zarudna et al. 36 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 patients with ra was 75.6 %, which is slightly higher than the established results of statistical researches. peripheral blood circulation disorders were diagnosed in the patients with sle in 80 % of cases, in the patients with ssc – in 97.4 % of cases, which is consistent with literature [10, 11]. despite nearly equal distribution of patients in every group by age, duration of a disease, activity of the inflammatory process, it has been revealed that a kidney, lung or heart are damaged more frequently in the patients with peripheral blood circulation disorders. such results are probably associated with the development of systematicity, which is caused by peripheral blood flow and microcirculation disorders. table 4. distribution of skin hemocapillaries of the patients over the duct width disease no peripheral blood circulation disorders (group 1, n=11) peripheral blood circulation disorders (group 2, n=15) the number of hemocapillaries and their diameter the number of hemocapillaries and their diameter 8­12 μm 15­25 μm above25 μm 8­12 μm 15­25 μm above 25 μm normal range 100 – – 100 – – rheumatoid arthritis, n=8 80.6±3.6 р1<0.01 12.1±0.6 7.3±0.3 31.3±1.5 р1<0.01 р2<0.01 46.6±2.2 22.1±1.0 systemic sclerosis, n=8 71.7±3.3 р1<0.01 24.5±1.1 3.82±0.17 8.0±0.3 р1<0.01 р2<0.01 58.9±2.8 33.1±1.5 systemic lupus erythematosus, n=10 69.2±3.3 р1<0.01 29.7±1.4 1.13±0.04 7.5±0.3 р1<0.01 р2<0.01 43.2±2.0 49.3±2.4 notes: p1 – statistically significant difference between the normal range and the number of normal-sized capillaries (8-12 μm) in the patients of both groups; p2 – statistically significant difference between the number of capillaries 8-12 μm in diameter in the patients of the groups1 and 2. figure 1. biopsy material of skin flaps harvested from the finger of the patient f. with rheumatoid arthritis, medical record of the in-patient patient no. 01/10064. staining h&e. ×600 figure 2. biopsy material of skin flaps harvested from the finger of the patient sh. with systemic lupus erythematosus, medical record of the in-patient patient no. 01/00920. staining h&e. ×600 figure 3. biopsy material of skin flaps harvested from the finger of the patient kh. with systemic sclerosis, medical record of the in-patient patient no. 01/07587. staining h&e. ×600 o.i. zarudna et al. 37 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 in the rheograms of the patients with peripheral blood circulation disorders the height of percussion wave and ri decrease is related to the increase in duration of the disease. thus, ri in the forearms of the patients, who suffer from the disease for a period of less than 1 year, decreased by 19.07%. at the same time, the systolic blood flow in the forearms of the pa­ tients, who suffer for 10 or more years, decreased by 44.08%. meanwhile, di value points to hypertonia of precapillary vessels in all investigated groups, but this indice is statistically lower (p<0,001) among the patients, who suffer for a period of less than 1 year, and increases if the duration of the disease is longer. in the patients divided by the nosological units, the lowest rates of systolic flow were observed in patients with ssc, meanwhile, ri decreased by 46.05%; the patients with ra and a deficit in ri, representing 41.45 %, were the second; consequently, the smallest changes in ri were found in the patients with sle, whose systolic blood flow in forearms decreased by 28.29 %. the tonus of precapillary vessels, which was significantly higher in the patients with ssc (p<0.001), was changed compare to the abovementioned data. analysis of rheovasography indices in terms of the degrees of inflammatory process acti­ vity (which was determined by the erythrocyte sedimentation rate) revealed no statistically significant difference. however, at the highest degree of the inflammatory process activity di was of the smallest value and significantly different (p<0.001) from the indices at the 1st and 2nd degrees of activity. thus, the most significant peripheral blood circulation disorders were found in the patients with ssc (p<0.05); disorders of peripheral blood flow were exacerbated in case of lengthening of disease course. hypertonia of peripheral vessels and decrease in systolic blood flow were reflected in flattening of a rheographic curve and rheographic index decrease. hypertonia of arterioles and precapillary vessels was evident by high location of incisures in relation to the apex of the rheographic curve and a high di value. the analysis of morphometric data revealed that the average density of capillaries per unit area statistically decreased only in the patients with peripheral blood flow disorders in cases of sle and ssc. along with the decrease in the number of normal-sized capillaries, dilated and mega capillaries were present. meanwhile, their number was small in the patients without peripheral blood flow disorders and statistically significant in the patients with hemodynamic disorders. in terms of relative values, the number of pathologic capillaries was 66.7% in cases of ra, 92.0% in cases of ssc and 91.5% in cases of sle. the concentration of ет­1 was significantly higher in the patients with peripheral blood flow disorders. to examine the changes in concentration of et-1 in detail, all the patients with the signs of vascular bed damage were divided into three groups by the degree of the inflammatory process activity. the revealed changes were reflected in gradual increase in concentration of ет­1 in case of a higher degree of inflammatory process activity, though there was no statistically significant difference between the indices. according to the results of our study it has been established that microcirculatory bed changes in the patient with ssc and sle complied with other literature regarding investigation of non-invasive methods of studying of microcirculatory bloodstream such as nailfold videocapillaroscopy [5, 6, 7]. minimal changes have been revealed in the patients with ra [5]. conclusions it should be noted that peripheral hemodynamic disorders are followed by significant microcirculatory bloodstream disorders such as decrease in average density of hemocapillaries per unit area, decrease in the number of nortable 5. concentration of ет1 in the patients with rheumatic diseases with/without peripheral blood circulation disorders group of patients ет1, pkg/ml donors, n=10 1.8±0.7 group 1, n=5 1.9±0.5 p1>0.05 group 2 sle, n=10 19.0±1.4 p1<0.001 p2<0.001 ssc, n=10 23.1±1.5 p1<0.001 p2<0.001 ra, n=10 18.9±1.4 p1<0.001 p2<0.001 notes: p1 – statistically significant difference between indices in patients and donors; p2 – statistically significant difference between indices in patients of the groups 1 and 2. ra – rheumatoid arthritis; ssc – systemic sclerosis; sle – systemic lupus erythematosus. o.i. zarudna et al. 38 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 mal-sized capillaries, and increase in the number of dilated and megacapillaries. morpho metric changes are accompanied by endothelial dysfunction with a high concentration of ет­1. in terms of integral estimation extremely high level of changes is evidenced in the patients with ssc which complies with current understanding of this disease. however, some morphometric peculiarities were revealed in the patients without peripheral blood flow disorders: in cases of adequate density of hemocapillaries per unit area, there was a decrease in the number of normal-sized capillaries and presence of dilated and megacapillaries. the above-mentioned changes were not followed by endothelial dysfunction in relation to ет1 indicator. thus, normal rheova­ sography did not preclude the presence of microcirculation disorders since biopsy revealed deeper changes in microcirculatory bed. the further research should be focused on the search for non-invasive methods of studying microcirculatory bloodstream in the patients with rheumatic diseases, which is the main cause of the development of systematicity. acknowledgements: the authors are gratefully acknowledged to mariana karanevych (phd in philology, associate professor) with the help in translation of this article. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions zarudna o.i. – conceptualization, data curation, formal analysis, investigation, writing – original draft and review & editing; venher i.k. – conceptualization, data curation, validation, methodology; dovbush a.v. – formal analysis, investigation, visualization, software. системні захворювання сполучної тканини: фокус на мікроциркуляторне русло о.і. зарудна, і.к. венгер, а.в. довбуш тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. прояви системних захворювань є надзвичайно різноманітними. їх об’єднує наявність васкуліту/васкулопатії, що й зумовлює системність і приводить до розвитку стійких порушень функції органів і систем. при цьому мішенню, джерелом та причиною патологічного процесу є ендотелій. мета. провести комплексне вивчення стану мікроциркуляторного русла у хворих на системний червоний вовчак (счв), системну склеродермію (ссд) та ревматоїдний артрит (ра). методи. для досягнення мети проведено ретроспективний аналіз архівних історій хвороби 129 хворих на ревматичні захворювання, з них 39 – з верифікованим діагнозом ссд, 54 – счв та 45 – ра. переглянуто архів біоптатів шкірного клаптя пальця пацієнтів, на основі чого проведено морфометричний аналіз. стан периферичної гемодинаміки вивчали за результатами поздовжньої реовазографії передпліч та гомілок. вміст ет 1 визначено за імуноферментною методикою, яка ґрунтується на принципі конкурентного імуноферментного аналізу. клінічні та лабораторні дані співставлень з результатами морфологічного дослідження біоптатів. результати. у переважної більшості пацієнтів виявлено розлади периферичної гемодинаміки. встановлено, що ураження нирок, легень, серця частіше спостерігається у хворих з розладами периферичного кровоплину, проте найглибші порушення виявлено у хворих на системну склеродермію (p<0,05). порушення периферичної гемодинаміки супроводжуються підвищенням концентрації ендотеліну-1 та поглиблюються за умови зростання тривалості хвороби. кількість патологічних капілярів найвища у хворих на системну склеродермію. висновки. крайній ступінь розладів мікроциркуляторного русла за інтегральною оцінкою клініколабораторних та морфометричних досліджень виявлено у пацієнтів на ссд. однак знайдено деякі морфологічні особливості у пацієнтів без розладів периферичного кровоплину. отже відсутність патологічних відхилень за результатами реовазографії не виключає розладів на мікроциркуляторному рівні. ключові слова: мікроциркуляція; системний червоний вовчак (счв), системна склеродермія (ссд), ревматоїдний артрит (ра); біопсія; реовазографія. o.i. zarudna et al. 39 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 інформація про авторів зарудна ольга ігорівна – канд. мед. наук, доцент кафедри клінічної імунології, алергології та загального догляду за хворими, тернопільський національний медичний університет імені і.я. гор­ бачевського. венгер ігор касянович – доктор медичних наук, професор, завідувач кафедри хірургії №2, тернопільський національний медичний університет імені і.я. горбачевського. довбуш андрій васильович – канд. біол. наук, доцент кафедри гістології та ембріології, тернопільський національний медичний університет імені і.я. горбачевського. information about the authors olga i. zarudna – md, ph.d., associate professor, department of clinical immunology, allergology and general patients′ care, i. horbachevsky ternopil national medical university. orcid 0000­0002­9374­3991, e­mail: zarudna@tdmu.edu.ua ihor k. venher – md, ph.d., dsc, professor, head of the department of surgery no. 2, i. horbachevsky ternopil national medical university. orcid 0000­0003­0170­1995, e­mail: vengerik@tdmu.edu.ua andriy v. dovbush – ph.d., associate professor, department of histology and embryology, i. horbachevsky ternopil national medical university. orcid 0000­0003­2246­6218, e­mail: dovbush@tdmu.edu.ua references 1. bărbulescu al, vreju af, bugă am, sandu re, criveanu c, tudoraşcu dr, gheonea ia, ciurea pl. vascular endothelial growth factor in systemic lupus erythematosus-correlations with disease activity and nailfold capillaroscopy changes. rom j morphol embryol. 2015 jan 1;56(3):1011­6. 2. kuryliszyn-moskal a, klimiuk pa, sierakowski s, ciolkiewicz m. vascular endothelial growth factor in systemic lupus erythematosus: relationship to disease activity, systemic organ manifestation, and nailfold capillaroscopic abnormalities. arch. im munol. ther. exp. 2007;55:179­85. doi: 10.1007/s00005­007­0017­7 3. kuryliszyn-moskal a, klimiuk pa, sierakowski s, ciolkiewicz m. a study on vascular endothelial growth factor and endothelin-1 in patients with extraarticular involvement of rheumatoid arthritis. clinical rheumatology. 2006; 25(3):314­9. doi: 10.1007/s10067­005­0007­2 4. maurizio cutolo m, sulli a, smith v. assessing microvascular changes in systemic sclerosis diagnosis and management. nat rev rheumatol. 2010;6(10):578­87. doi: 10.1038/nrrheum.2010.104 5. mihail c, tervaert jwc. anti-endothelial cell antibodies in systemic sclerosis. bmj journals annals of rheumatic diseases.2010;69(2):319­24. doi: 10.1136/ard.2008.102400 6. chojnowski mm, felis-giemza a, olesinska m. capillaroscopy a role in modern rheumatology. reumatologia. 2016;54(2):67­72. doi: 10.5114/reum.2016.60215 7. ragab o, ashmawy a, abdo m, mokbel a. nailfold capilloroscopy in systemic lupus erythematosus. the egyptian rheumatologist. 2011; 33(1):61­7. doi: 10.1016/j.ejr.2010.12.003 8. etehad tavakol m, fatemi a, karbalaie a, emrani z, erlandsson be. nailfold capillaroscopy in rheumatic diseases: which parameters should be evaluated? biomed research international, 2015. article id 974530. 17 pages. doi: 10.1155/2015/974530 9. ciolkiewicz m, kuryliszyn-moskal a, klimiuk pa. analysis of correlations between selected endothelial cell activation markers, disease activity, and nailfold capillaroscopy microvascular changes in systemic lupus erythematosus patients. clin rheuma tol. 2010;29(2):175­80. doi: 10.1007/s10067­009­1308­7 10. dimitrijevic j, brajuckovic g, cerovic s, popovic z, bogdanovic r, jovanovic d, et al. morphology of autoimmune diseases. arch oncol. 2004;12:27­9. https://pdfs.semanticscholar.org/a10f/9bc9613 9a020f252e49f80d9d65be4b72e24.pdf 11. holovach iyu, egudina ed. systemic scleroderma: modern view on pathogenesis, course, diagnosis and treatment. semeynaya meditsina. 2 0 1 9 ; 3 : 7 ­ 1 6 . h t t p : / / n b u v . g o v . u a / u j r n / s i m ­ med_2019_3_3. (in russian). 12. sinyachenko ov, egudina ed, khaniukov aa, ermolaeva mb, gulmamedova mf, potapov yua. specific course of angiopathy in systemic autoimmune rheumatic diseases. 2017;2:33­9. http://nbuv.gov.ua/ujrn/utj_2017_2_7. received 03 july 2019; revised 03 october 2019; accepted 23 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o.i. zarudna et al. key: cord-0075535-9a9i0pun authors: kriegsmann, mark; kriegsmann, jörg title: synoviale veränderungen bei erkrankungen des rheumatologischen formenkreises und differenzialdiagnosen date: 2022-03-08 journal: arthroskopie doi: 10.1007/s00142-022-00528-4 sha: 8074756dc7fe014ebf9e92495a9742f608ccc614 doc_id: 75535 cord_uid: 9a9i0pun the investigation of alterations to synovial tissue can contribute to the diagnosis of joint and systemic diseases. the domain of histological diagnostics is important to differentiate tumorous and inflammatory conditions. moreover, crystal arthropathies and certain metabolic diseases can be identified. the histological picture of granulomatous synovitis can be caused by a mycobacterial infection, sarcoidosis and foreign body reactions as well as rare genetic diseases. furthermore, subtyping of amyloidosis is possible in the tunica synovialis. molecular methods enable reliable and rapid diagnostics of septic arthritis and a subtyping of microorganisms. additionally, reactive arthritis can also be classified by the identification of dna or rna of microorganisms in joint tissue or fluid. the diagnosis of rheumatoid arthritis is based on the american college of rheumatology (acr) criteria. molecular methods, such as micro-rna technology or proteomics methods can assist in the diagnosis of rheumatoid arthritis. amyloidosen sind durch abnorm gefaltete proteine charakterisiert, die amyloide fibrillen bilden, die sich in verschiedenen geweben und organen ansammeln und mitunter zu organfehlfunktionen, organversagen und tod führen können. heute ist es nicht nur erforderlich, diese krankheitsbilder zu erkennen und eine amyloidose zu diagnostizieren, sondern auch eine subtypisierung vorzunehmen. häufige amyloidosen stellen dabei al-amyloidosen (leichtkettenamyloidosen) bei plasmozytomen und b-zell-lymphomen, aa-amyloidosen bei chronisch-entzündlichen erkrankungen, transthyretin-amyloidose (attr) und dialyseassoziierte amyloidosen dar, die jeweils ein unterschiedliches therapeutisches vorgehen erfordern [1, 2] . bei den entzündlichen veränderungen der tunica synovialis kann morphologisch zunächst zwischen granulomatösen entzündungen, nichtgranulomatösen entzündungen und kristallinduzierten veränderungen (mit sekundärer entzündung) unterschieden werden. daneben sind arthropathien zu erwähnen (amyloidose s. oben, stoffwechselerkankungen). histopathologisch die definitive einordnung erfolgt heute mittels pcr und nachfolgender hybridisierung auf chips, alternativ ist eine sequenzierung möglich. daneben kann auch eine molekularbiologische resistenzbestimmung durch chip-hybridisierung erreicht werden. granulomatöse entzündungen durch pilze können ebenfalls granulomatöse entzündungen der tunica synovialis verursachen [3] und mittels pcr diagnostiziert werden. die korrekte und rasche identifikation von pilzinfektionen ist wichtig, da diese v. a. bei immunkompromittierten patienten auftreten. im rahmen einer sarkoidose, die durch eine granulomatöse entzündung charakterisiert ist, kann ebenfalls eine gelenkbeteiligung beobachtet werden [4] . ein beispiel für eine granulomatöse synovitis ist in . abb. 1 dargestellt. fremdkörperreaktionen treten am häufigsten nach injektion verschiedener medikamente, nach direkter gelenkverletzung durch traumata oder nach gelenkopera-tioneneinschließlichgelenkersatztherapie auf. eine seltene granulomatöse entzün-dung im rahmen einer fremdkörperreaktion stellt die seeigelstachelsynovialitis dar [5] . sehr selten und im kindesalter zu diagnostizieren sind erkrankungen wie die chronische granulomatöse entzündung, die einen vererbten defekt der phagozytenfunktion durch einen defekt der nicotinamidadenindinucleotidphosphat(nadph)-oxidase darstellt [6] . ein zweites beispiel für eine seltene erbliche erkrankung ist das blau-syndrom, welches autosomal-dominant vererbt wird und sich in der frühen kindheit mit dermatitis, uveitis und arthritis manifestiert [7] . kristallinduzierte synovialitiden sind eine arthritis urica oder eine kalziumpyrophosphat-arthropathie. diese können oft schonhistologischunter anwendungpolarisationsoptischer doppelbrechung sicher diagnostiziert werden. die akute septische arthritis bezeichnet eine rasch auftretende entzündungsreaktion im gelenkraum und der ihn begrenzenden synovialmembran. diese kann u. a. durch mikroorganismen (bakterien und pilze) oder deren bestandteile, aber auch durch kristalle verursacht werden. die eitrige arthritis muss schnell erkannt werden und erfordert stets eine interdisziplinäre zusammenarbeit. die verzögerung der diagnose und therapie führt meist zur destruktion und somit funktionseinschränkung des gelenks [8, 9] . eine keimeinordnung septischer arthritiden kann durch pcr der tunica synovialis oder der synovialflüssigkeit erfolgen [10] . eine derartige untersuchung ist auch am formalinfixierten paraffineingebetteten material möglich [11] . bezüglich einer umfassenden diskussion über den begriff der reaktiven arthritis im kontext der sich ändernden krankheitsterminologie, definition und klassifikation wird auf einen review-artikel von zeidler und hudson 2021 verwiesen [12] . dieweltgesundheitsorganisation ursprünglich wurde der terminus rea eingeführt, um eine akute arthritis zu beschreiben, die durch eine bakterielle entzündung getriggert wird, meist gastrointestinal oder urogenital, bei der das verursachende agens nicht aus synovialem gewebe oder synovialflüssigkeit kultiviert werden kann [14] . später wurde vorgeschlagen, die rea in hla-b27-assoziierte und nichtassoziierte formen einzuteilen [15] . in der folgezeit wurde evident, dass patienten mit typischer reaktiver arthritis mikrobielle antigene, bakterielle dna und rna und sogar metabolisch aktive mikroben in der synovialflüssigkeit oder in der tunika synovialis aufweisen [16, 17] . zeidler und hudson (2021) diskutieren, dass eine überlappung zwischen den termini der reaktiven und postinfektiösen arthritis besteht. sie postulieren 2 typen von reaktiver arthritis: "infection reactive arthritis", charakterisiert durch intraartikuläre nichtkultivierbare bakterien wie chlamydien und "infection triggered reactive arthritis", bei der bakterien einer infektion anderer körperregionen in die gelenke gelangen und hier eine immunvermittelte arthritis auslösen [12] . nachfolgend werden folgende kategorien beschrieben: -"well established": primäre ursächliche agentien der reaktiven arthritis; seltene infektiöse agentien, die für die reaktive arthritis ursächlich sind: j urogenitale infektionen, j gastrointestinale infektionen, j respiratorische infektionen, j andere infektionen, einschließlich haut und weichgewebe; neue infektiöse agentien, die an der verursachung einer reaktiven arthritis beteiligt sind. bei den gut etablierten ursächlichen agentien sind gastrointestinale infektionen (yersinien, salmonellen, campylobacter und shigellen) sowie urogenitale infektionen zu nennen, wobei 36 % der rea durch chlamydien und 26 % durch enterische infektionen verursacht werden [18] . reaverursachtdurchchlamydia pneumoniae ist seltener als arthritis verursacht durch c. trachomatis [19] . in einer indischen studie wurde c. trachomatis in urinproben bei 36 % der patienten mit reaktiver arthritis nachgewiesen [20] . bemerkenswert ist, dass in der synovialflüssigkeit die detektionsrate abhängig von der extraktionsmethode ist [21] . wichtig für die praxis ist, dass die detektion von infektionen mit tropheryma whippelei, borrelia burgdorferi, neisseria gonorrhoeaund einigen protozoen spezifische therapeutische interventionen ermöglichen und erfordern. in einer studie mit patienten mit nicht erklärbaren rheumatischen schmerzen (suez canal university hospital) zeigte die untersuchung von stuhlproben, dass folgende parasitosen vorlagen: kryptosporidien (48 %), cyclospora cayetanensis (32 %), giardia lamblia (24 %), blastocystis hominis (20 %) und entamoeba histolytica (8 %; [12] ). medikamentenassoziiert kann eine rea durch bacillus calmette-guérin(bcg)-instillation bei harnblasentumoren auftreten [22] . auch virale infektionen können eine reaktive arthritis triggern. mit auftreten des severe acute respiratory syndrome coronavirus 2 (sars-cov-2) wurden auch akute arthritiden beschrieben [23] . nach der manifestation eines erythrema migrans werden bei unbehandelten pati-arthroskopie 3 enten eine polyarthralgie neben kardialen und/oder neurologischen symptomen beobachtet. später können diese patienten eine lyme-arthritis entwickeln, die sich als mono-oder oligarthritis manifestiert und typischerweise das kniegelenk einbezieht. neben serologischen untersuchungen kann die pcr aus synovialflüssigkeit oder der tunica synovialis die diagnose bestätigen [24] . das histologische und immunhistologische bild der psoriasisarthritis ähnelt mehr dem einer spondylarthropathie als dem einer rheumatoiden arthritis [25] . immer wieder wird das typische gefäßmuster dieser synovialen erkrankung beschrieben [26] . die diagnose einer rheumatoiden arthritis (ra) basiert aktuell auf klinischen kriterien und laborparametern (american college of rheumatology[acr]-kriterien 2010). das histologische muster ist, je nach vorangegangener therapie, sehr heterogen [27] . die diagnose einer ra kann histologisch vermutet werden, wenn eine highgrade-synovitis vorliegt [28] . zur absicherung dieser interpretation kann das mikro-rna-muster herangezogen werden [29] . bei verschiedenen autoimmunerkrankungen wurden mikro-rna (mirna) detektiert, die in regulationsmechanismen eingreifen, die aber auch diagnostische und therapeutische optionen beinhalten. ein muster der mirna mir-146a, mir-155 und mir-223 kann zur abgrenzung der ra von anderen synovialitiden beitragen. eine veränderung der expression der mirna mir-146a wurde bei unterschiedlichen rheumatischen und autoimmunerkrankungen beschrieben. in der histopathologischen, histochemischen und modernen immunhistologischen diagnostik werden proteine detektiert. dies setzt jedoch die kenntnis pathogenetisch oder diagnostisch wichtiger epitope voraus. als neue proteomische methode steht für die gewebediagnostik seit kurzer zeit die massenspektrometrie zur verfügung. neben dem nachweis von masse/ladungsverhältnissen ermög-lichen massenspektrometrische verfahren mittels "matrix-assisted laser desorption ionisation time-of-flight imaging" (maldi-tof-imaging) auch die zuordnung von masse/ladungsverhältnissen und proteinen/peptiden oder auch lipiden zu morphologischen strukturen. in ersten publikationen wurde diese technik an gewebsblöcken unterschiedlicher patienten (tissue-microarray) als hochdurchsatzmethode und zur beschreibung der synovialitis von patienten mit arthrose und arthritis angewendet. proteomische methoden mit 3-d-gel-elektrophorese und proteinidentifizierung durch maldi-tof/tof werden das verständnis der ätiopathogenese rheumatologischer krankheitsbilder in zukunft noch verbessern [30] . eine seltene komplikation rheumatischer erkrankungen stellen bakterielle und mykotische infektionen dar. insbesondere im rahmen immunsuppressiver therapien wird eine steigende anzahl von infektionen beobachtet. da gelenkinfektionen innerhalb kürzester zeit zur gelenkdestruktion und funktionellen einschränkungen führen, ist eine schnelle und zielgerichtete antibiotische therapie unbedingt erforderlich. molekulare testsysteme, die mittels multiplex-pcr die relevanten bakteriellen und mykotischen infektionen erfassen und die auch aussagen bezüglich den wichtigsten resistenzen treffen, sind verfügbar. der vorteil dieser systeme besteht darin, dass auch infektionen bei bereits antibiotisch vorbehandelten patienten detektiert werden können. neben methoden, die auf der detektion von dna-und rna-molekülen beruhen, gewinnen proteomische technologien eine immer größere bedeutung. die proteinexpression im gewebe spiegelt den funktionellen zustand von zellen oder geweben viel besser wider als genetische informationen, da zahlreiche prozesse genetische informationen (posttranslational) modifizieren. epigenetische phänomene sind in den letzten jahren in den fokus des wissenschaftlichen interesses gerückt. the investigation of alterations to synovial tissue can contribute to the diagnosis of joint and systemic diseases. the domain of histological diagnostics is important to differentiate tumorous and inflammatory conditions. moreover, crystal arthropathies and certain metabolic diseases can be identified. the histological picture of granulomatous synovitis can be caused by a mycobacterial infection, sarcoidosis and foreign body reactions as well as rare genetic diseases. furthermore, subtyping of amyloidosis is possible in the tunica synovialis. molecular methods enable reliable and rapid diagnostics of septic arthritis and a subtyping of microorganisms. additionally, reactive arthritis can also be classified by the identification of dna or rna of microorganisms in joint tissue or fluid. the diagnosis of rheumatoid arthritis is based on the american college of rheumatology (acr) criteria. molecular methods, such as micro-rna technology or proteomics methods can assist in the diagnosis of rheumatoid arthritis. tunica synovialis · synovial membrane · rheumatic diseases · molecular pathology · microorganism subtyping with rheumatoid arthritis and osteoarthritis. virchows arch 469 (1) amyloidarthropathy: a review amyloidarthropathy associated with multiple myeloma: polyarthritis without synovial infiltration of cd20+ or cd38+ cells synovitis due to histoplasma capsulatum: a case series and literature review sarcoidosis: a clinical overview from symptoms to diagnosis sea urchin granuloma of the hands: a case report recent advances in chronic granulomatous disease blau syndrome: a rare cause of exuberant granulomatous synovitis of the knee septic hip joint fungal septic knee arthritis caused by aspergillus fumigatus following anterior cruciate ligament reconstruction primary septic arthritis amongchildren6 to48 monthsofage: implications for pcr acquisition and empiric antimicrobial selection the role of molecular diagnostics in implant-associated bone and joint infection reactive arthritis update: spotlight on new and rare infectious agents implicated as pathogens infections and arthritis arthritis associated with yersinia enterocolitica infection twoformsofreactive arthritis? reactive or infectiousarthritis reactive arthritis: clinical aspects and medical management hla-b27-associated reactive arthritis: pathogenetic and clinical considerations chlamydia-induced reactive arthritis: hidden in plain sight? chlamydia trachomatis associated reactive arthritis: a urinary pcr based study detection of chlamydia trachomatis-dna in synovial fluid: evaluation of the sensitivity of different dna extraction methods and amplification systems reactive arthritis induced by intravesical bcg therapy for bladder cancer can sars-cov-2 trigger reactive arthritis? stages of lyme arthritis synovial histopathology of psoriatic arthritis, both oligo-and polyarticular, resembles spondyloarthropathy more than it does rheumatoid arthritis psoriaticsynovitis:singularityandpotentialclinical implications synovial tissue heterogeneity in rheumatoid arthritis and changes with biologic and targeted synthetic therapies to inform stratified therapy fifteen years of the histopathological synovitis score : review and further developments of a diagnostic score expression of mir-146a, mir-155, and mir-223 in formalin-fixed paraffin-embedded synovial tissues of patients key: cord-0042079-2cttcck1 authors: hjelmevik, t. o.; kvalvik, a. g.; knappskog, p. m.; lefsaker, l.; kleiven, a. s.; dyvik, s.; brun, j. g.; østergaard, h.; eiken, h. g. title: infliximab treatment of rheumatoid arthritis patients simultaneously increases tnf‐α protein levels and reduces mrna expression in the blood date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423c.x sha: ab67edff75df91046016461f3b9e6cbfeba8f253 doc_id: 42079 cord_uid: 2cttcck1 objective: tumour necrosis factor‐α (tnf‐α) is an important mediator in the pathogenesis of rheumatoid arthritis (ra). we have investigated long‐term anti‐tnf‐α treatment with infliximab with respect to tnf‐α gene activity and protein levels in the blood of ra patients and disease activity score (das). methods: tnf‐α mrna and plasma protein in ra patients (n = 29) and healthy controls (n = 24) was determined before and during treatment with infliximab (3 mg/kg) using real‐time quantitative reverse transcription‐polymerase chain reaction (rt‐pcr) and high sensitivity enzyme‐linked immunosorbent assay (elisa), respectively. the disease activity of the patients was assessed as das value. results: the tnf‐α mrna levels of ra patients at baseline were higher than that of the control group (p = 0.0135) but were significantly reduced after initiation of treatment (p < 0.001). low mrna levels were sustained throughout the 54 weeks of the study. baseline protein levels of ra patients were similar to the control group. after 2 weeks of treatment, the protein levels were significantly elevated from baseline (p = 0.0353) and increased throughout week 14. clinical improvement for all ra patients was found upon infliximab treatment, as a reduction in das values (p < 0.001). the increase in protein and reduction in das value from week 2–14 was also correlated (p = 0.0374). conclusion: during infliximab treatment of ra patients, there is an accumulation in immune‐reactive tnf‐α protein in blood plasma and simultaneously a reduction in tnf‐α gene expression in pbmc, which may in part explain the beneficial course of ra symptoms. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. 1http://dx.doi.org/10.20396/bjos.v19i0.8656652 volume 19 2020 e206652 original article 1 department of prosthodontics and periodontology, piracicaba dental school, university of campinas (unicamp), piracicaba, são paulo, brazil. corresponding author: renata cunha matheus rodrigues garcia https://orcid.org/0000-0001-8486-3388 department of prosthodontics and periodontology, piracicaba dental school, university of campinas, av. limeira, no 901, bairro areião, piracicaba, sp, brazil, cep: 13414-903, phone number: +55 19 2106-5240 / fax number: +55 19 2106-5211 e-mail: regarcia@fop.unicamp.br received: september 14, 2019 accepted: december 02, 2019 removable prostheses improve oral health-related quality of life and satisfaction of elderly people with rheumatoid arthritis bruna fernandes moreira alfenas1, kelly machado de andrade1, talita malini carletti1, renata cunha matheus rodrigues garcia1,* rheumatoid arthritis (ra) is an autoimmune disease that affects joint tissues and causes severe physical and functional impairments on quality of life due to muscular and articular pain. the involvement of temporomandibular joint in ra interferes with mouth opening and masticatory process. however, no studies addressed the impact of ra on oral health-related quality of life (ohrqol) and satisfaction with prostheses use in elderly people. aim: this study assessed the impact of oral rehabilitation with conventional dentures on the ohrqol and prostheses satisfaction in elderly patients with ra, associated or not with temporomandibular disorder (tmd). methods: forty-five elderly were enrolled and divided into three groups: (1) ra and tmd (n=15, experimental), (2) ra without tmd (n=15, experimental), and (3) without ra and without tmd (n=15, control). the ohrqol and the prostheses satisfaction were evaluated before and after new oral rehabilitation with partial and/or complete dentures. the ohrqol and prosthesis satisfaction were assessed and verified through ohip-14 questionnaire and visual analogue scale, respectively. results: tmd group exhibited the worst mean values (p<0.05) for all ohip-14 domains before insertion of new dentures. group 2 showed worst means (p<0.05) compared to controls for functional limitation and physical pain domains of the ohip-14, but not in the general score. patients showed better outcomes of satisfaction with prostheses use only after the new rehabilitation. conclusion: the use of new and well-fitted dentures improves all domains of ohrqol in patients with ra and tmd and all groups were satisfied with prostheses use after the new rehabilitation with conventional dentures. keywords: arthritis rheumatoid. dental prosthesis. quality of life. oral health. patient satisfaction. https://orcid.org/0000-0001-8486-3388 2 alfenas et al. introduction rheumatoid arthritis (ra) is a chronic auto-inflammatory disease that affects joint tissues and causes severe physical and functional impairments on quality of life (qol) in the disease bearers1-4. the occurrence of ra is over the middle-aged people, between the fourth and sixth decades. it is an autoimmune disease probably caused by environmental factors, such as drugs and diet, in association with regulatory genes responsible to express the immune disease5,6. the american college of rheumatology has updated the guidelines on ra, so that approaches to manage the disease can be applied to prevent pain, loss of function and joint disturbances7. to diagnose ra, in most cases the synovitis is identified through clinical exam in phalangeal joints, in combination with large or other small joints, with swelling and pain episodes1. once temporomandibular joint (tmj) is the unique synovial orofacial joint, it can be frequently affected in ra, including condylar and disc alterations8. however, literature is variable in reporting the frequency of temporomandibular disorder (tmd) due to ra8-10. some authors found the presence of limited mouth opening11, tmj pain10,11, muscular hyperactivity, and masticatory damage12. still, some risk factors have been associated with the involvement of tmj in patients with ra, as being of female gender, presence of mental disorders, insomnia or stroke13. concerning facial joints involvement and the consequences of ra, few reports14,15 verified the impact of oral rehabilitation with conventional dentures in patients with the disease. improvement of masticatory efficiency, bite force and mandibular movements after new prostheses use in individuals with ra has been reported14,15. it is also known that ra is associated with a decreased qol16 due to pain, ageing process17 and, functional limitation18. however, the authors are unaware of studies on ra and oral health-related quality of life (ohrqol) in elderly patients wearing complete or partial conventional dentures. for this reason, considering completely or partially edentulous people wearing old and misadjusted prostheses, the replacement of missing teeth could bring positive results on oral and general health. therefore, the present study aimed to evaluate the impact of oral rehabilitation with conventional dentures on the ohrqol and satisfaction with prostheses use in elderly patients with ra, associated or not with tmd. materials and methods study design this cross-sectional study performed subjective assessments concerning the ohrqol and prosthesis satisfaction among elderly individuals with or without ra and/or tmd, before and after a 2-month prosthetic rehabilitation with new removable dentures. the participants of this clinical research had already participated in previous study14, in which masticatory function and mandibular movements were observed. all participants were first evaluated with their old prosthesis in the mouth, and after receiving new removable dentures. ohrqol was assessed by ohip-14 questionnaire19-21, and prosthesis satisfaction by a visual analogue scale (vas)22. 3 alfenas et al. subjects according to sample size calculation (test power of 80% and α value of 0.05.), a total of 45 participants were considered to be enrolled in the study (figure 1). subjects were selected without restriction of gender and race and were divided into 3 groups: (1) elderly with ra and tmd (n = 15, experimental), (2) elderly with ra without tmd (n = 15, experimental), and (3) elderly without ra and without tmd (n = 15, control). following the inclusion criteria, elderly volunteers must present: 60 years or older; be partially or completely edentulous, and using inadequate removable partial and/ or complete dentures, according to the criteria of vigild23; and present diagnosis of ra. besides these criteria, group 1 participants must present tmd. all volunteers agreed with voluntary participation and signed the informed consent form, approved by the ethics committee of piracicaba dental school, university of campinas (#068/2012). this study was also registered in the brazilian registry of clinical trials (rebec #rbr-6 qkjzy). volunteers with ra were selected from those attended in the medical specialties ambulatory, department of rheumatology (limeira, são paulo, brazil), managed by university of campinas. controls were selected from patients who sought prosthetic treatment at the piracicaba dental school, university of campinas and also met the same inclusion criteria described above, but with absence of both ra and tmd. patients with the presence of severe malocclusions, craniofacial injuries or recent orofacial surgeries, intake of muscle-related medication, or with degenerative diseases, including parkinson’s or alzheimer’s disease, were excluded of this study. the diagnosis of ra was performed by a rheumatologist, according to clinical and laboratory criteria, based on the american college of rheumatology classification: (1) morning joint stiffness (up to 1 hour); (2) arthritis in three or more joints with soft tissue edema or joint effusion; (3) arthritis in the hand joints (wrist, proximal interphalangeal and metacarpophalangeal joints); (4) symmetric arthritis; (5) rheufigure 1. flowchart for selection of volunteers. experimental groups ra medical specialties ambulatory/ unicamp control group without ra piracicaba dental school/ unicamp clinical exam and radiographs rdc/tmd – axis i group 1 (n=15) ra and tmd group 2 (n=15) ra without tmd group 3 (n=15) without both ra and tmd 4 alfenas et al. matoid nodules; (6) high levels of serum rheumatoid factor (destructive antibodies); and (7) radiographic changes (erosions or decalcification located in hands and wrists). in the presence of four of the seven criteria, for at least 6 weeks, (american college of rheumatology subcommittee on rheumatoid arthritis guidelines) it is confirmed the ra diagnosis. meanwhile, for the tmd diagnosis, all volunteers underwent a clinical examination by means of the axis i of research diagnostic criteria (rdc/tmd)24. in the axis i, clinical aspects of tmds are involved and divided into three aspects: (1) muscle disorders, (2) joint disorders and (3) arthralgia, arthrosis, and arthritis. the last one was used to characterize and select volunteers of groups 1 and 2, in the presence of tmd or not, respectively. after clinical examination of muscle insertions, mucosa-bearing area and remaining teeth, vigild´s criteria23 were applied to assess prosthesis conditions with regards to stability, retention, occlusion, vertical dimension and defects. to participate in this study, at least one of these criteria should be unsatisfactory. prosthesis new prosthetic rehabilitation with removable complete and/or partial dentures had been provided to the volunteers. the prosthetic devices were made following conventional techniques25,26. firstly, dental impressions were taken from both arches (hydrogum, zhermack, rovigo, italy) so that stone casts were made for all the volunteers. after that, elderly who received complete dentures had custom trays manufactured with acrylic resin (vipiflash, vipi, são paulo, brazil), while those in need for a removable partial denture had their metallic framework made by a dental technician, following the previous surveying analysis of the casts. for the completely edentulous elderly, functional impressions of both arches were taken. in the partially edentulous arches, the frameworks were proved in the mouth. following these steps, maxillomandibular relationships of all patients were obtained through the occlusal vertical dimension, and dental casts were positioned in a semi-adjustable articulator (a7 plus, bioart, são paulo, brazil), with the help of the facial bow. artificial teeth (biotone, dentsply, ny, usa) were mounted over the wax rims, following the bilateral balanced occlusion. after the prostheses being clinically evaluated by means of aesthetic features, they were finished, polished and delivered to patients. all patients were instructed to keep hygiene and care of the prostheses. prosthetic adjustments on occlusion and acrylic base of the dentures were performed for 2 months to allow patient’s adaptation with no complaints. oral-health related quality of life (ohrqol) to evaluate the ohrqol, the simplified portuguese version of the oral health impact profile (ohip-14) was used20,21. this is a questionnaire comprised of 14 questions, in seven conceptual dimensions of oral health: (1) functional limitation, (2) physical pain, (3) psychological discomfort, (4) physical incapacity, (5) psychological incapacity, (6) social incapacity, and (7) social disadvantages. for each of the domains, volunteers could answer “often”, “sometimes”, “rarely”, “never” and “do not know”; whose weights/ scores were 4, 3, 2, 1 and 0, respectively. the final index could 5 alfenas et al. vary between 0 and 28 points for each volunteer and it was classified as weak (0-9 points), average (10-18 points) and strong (19-28 points). thus, the lower the score, the better the ohrqol. patient satisfaction with the prosthetic treatment volunteer’s satisfaction with their prostheses was measured by a visual analogue scale (vas)22. it consists on a 10 cm ruler, whose extremities indicate the minimum (zero or no satisfaction) and the maximum value (10 or the highest satisfaction), concerning domains of retention, comfort, mastication, speaking, hygiene, aesthetics, and general satisfaction, of both old and new, upper and lower prostheses. the volunteers were instructed to indicate, with a vertical mark over the ruler, the level of satisfaction with prosthesis use before and after the rehabilitation treatment. the greater the score (ranging from 0 to 10), the better the subjective perception with the prosthetic treatment. statistical analysis data were first submitted to the shapiro-wilk test and evaluations of asymmetry coefficient and kurtosis. anova was used when the residues adhered to the gaussian distribution; otherwise, the analysis of variance on ranks (anova-r) was adopted. a generalized linear mixed model with repeated measures was applied to test the effect of prosthetic treatment (before versus after) in each group and for the effect of group (experimental groups versus control) at each time point. tukey-kramer for multiple comparisons was applied as a post-hoc test. all statistical analyses were performed on sas system (release 9.3; sas institute inc., cary, usa) with a significance level of 5%. results sociodemographic data were published in previous studies14. volunteers were aged 60 to 80 years old. a total of 21 subjects received complete dentures in both jaws, while 12 were rehabilitated by upper and lower removable partial dentures. the remaining participants received upper complete dentures and lower removable partial dentures. most of them were married, lived in the urban area, had completed the elementary school and were retired. groups 1 and 2 were diagnosed with ra for, approximately, 10.9 years14. table 1 shows the anova f and p values for main effects of group and time point, and the interaction. the following ohip-14 domains: psychological discomfort, physical incapacity, psychological incapacity, social incapacity, and social disadvantages showed significant group-time point interaction. in contrast, functional limitation and physical pain domains, as well as the general score of the ohip-14 showed significant main effects for both, group and time point factors, while the patient satisfaction with prosthesis revealed significant effect only of time point. 6 alfenas et al. mean comparisons between before and after prosthetic treatment showed that ra and tmd group exhibited the highest means values (p < 0.05) for psychological discomfort, physical incapacity, psychological incapacity, social incapacity, and social disadvantages domains scores on the ohip-14 before insertion of new dentures (table 2). a similar trend was observed for the patients with ra without tmd, however only for psychological discomfort, physical incapacity, and psychological incapacity domains. table 1. f and p values from anova to test ohip-14 domains and prosthesis satisfaction scores. variable group time point group × tp interaction f p f p f p ohip-14 general score 7.87 0.0013 74.93 0.0001 2.07 0.1392 functional limitation 8.64 0.0007 18.78 0.0001 1.52 0.2298 physical pain 12.06 0.0001 31.71 0.0001 2.31 0.1117 psychological discomfort 9.17 0.0005 59.66 0.0001 5.39 0.0082 physical incapacity 3.66 0.0342 51.23 0.0001 4.29 0.0202 psychological incapacity 0.0004 0.0001 0.0172 social incapacity 4.50 0.0170 28.27 0.0001 4.50 0.0170 social disadvantages 0.0043 0.0001 0.0043 prostheses satisfaction 0.3713 0.0001 0.2989 p values < 0.05 indicate significant evidence of the main effects (group and time point) and interaction. table 2. mean (standard deviation) of ohip-14 domains with significant interaction between group and time points. ohip-14 domain groups time point before pt after pt psychological discomfort ra + tmd 1.92 (1.12) a 0.27 (0.69) b ra – tmd 1.49 (1.21) a 0.23 (0.41) b control 0.29 (0.44) b 0.00 (0.00) b physical incapacity ra + tmd 1.74 (1.10) a 0.20 (0.56) c ra – tmd 1.05 (1.10) a 0.07 (0.18) c control 0.50 (0.70) bc 0.10 (0.20) c psychological incapacity ra + tmd 1.71 (0.97) a 0.31 (1.03) bc ra – tmd 0.89 (1.37) b 0.00 (0.00) c control 0.33 (0.64) bc 0.00 (0.00) c social incapacity ra + tmd 1.04 (0.99) a 0.00 (0.00) b ra – tmd 0.28 (0.75) b 0.00 (0.00) b control 0.39 (0.86) ab 0.00 (0.00) b social disadvantages ra + tmd 0.95 (0.90) a 0.00 (0.00) b ra – tmd 0.39 (0.73) b 0.00 (0.00) b control 0.21 (0.58) b 0.00 (0,00) b mean values followed by the same letter do not differ significantly from each other by the tukey-kramer test, with a significance level of 5%. pt, prosthesis treatment. 7 alfenas et al. prior to prosthetic treatment, the control group showed significantly lower mean values (p <0.05) than the ar + tmd group for all the ohip-14 domains, except for social incapacity. besides, controls also presented significantly lower means (p < 0.05) than the ar-tmd group in the psychological discomfort and physical incapacity domains. after the insertion of the new prostheses, no significant differences among the groups were detected (table 2). concerning those ohip-14 domains which showed significant main effects for group and time point (but not for the interaction), their mean values (sd) are shown in table 3. there was a significant reduction in the mean values (p < 0.05) for the general ohip-14 score, as well as for the functional limitation and physical pain domains after the prosthetic treatment for all studied groups, which means that ohrqol was greatly improved after rehabilitation with new dentures. in addition, irrespectively of the prosthetic treatment, patients with ra and tmd presented significant (p < 0.05) higher values than control group. in addition, patients with ra without tmd group showed increased means (p < 0.05) compared to controls for functional limitation and physical pain domains of the ohip-14, but not in the general score. finally, figure 2 illustrates the analysis of patient satisfaction with prostheses use, showing a significant main effect exclusively for the time point factor. thus, regardless of the ra and/or tmd, all patients were greatly satisfied with the new dentures. figure 2. mean values (sd) of patient satisfaction with the prosthetic treatment. p ro st he se s sa tis fa ct io n before time point after 9,40 (0,81) b 4,18 (2,28) a 10,00 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 table 3. mean (standard deviation) of ohip-14 domains with significant main ohip-14 domains time point group before pt after pt ra+tmd ra–tmd control general 6.65 (6.04) a 1.23 (2.23) b 6.34 (6.17) a 3.81 (5.15) ab 1.67 (3.12) b functional limitation 0.85 (0.99) a 0.31 (0.58) b 0.91 (0.91) a 0.71 (0.98) a 0.12 (0.26) b physical pain 1.21 (1.06) a 0.44 (0.65) b 1.34 (1.09) a 0.88 (0.89) a 0.27 (0.45) b effects for group and time points. mean values followed by the same letter do not differ significantly from each other by the tukey-kramer test, with a significance level of 5%. pt, prosthesis treatment 8 alfenas et al. discussion this clinical trial assessed the ohrqol and satisfaction with prosthetic treatment between patients with ra and tmd or not, before and after 2 months of new removable dentures (cd or rpd) insertion. most of ohip-14 domains exhibited significant interactions among groups and time points with increased values in elderly with ra and tmd before new dentures installation, meaning that such elderly had worse ohrqol. the use of old and worn out dentures contributes to the worse perception that elderly may have about their qol27. a recent report showed a correlation between denture wearing and better outcomes of ohrqol4. even though the last authors4 did not mention whether prostheses conditions were satisfactory or not, other reports showed that the use of well-fitted dentures improves masticatory performance, bite force and mandibular movements of patients with ra and tmd14,15. thus, it can be suggested that improvements in such objective variables, could positively influence the subjective measurements, like those of ohip-14 questionnaire. still on groups comparisons before new dentures insertion, the oral implications of tmd in ra11, such as pronounced muscular symptoms, soreness, pain during movement of masticatory muscles, arthralgia of tmj and tinnitus, associated with the absence of teeth and use of misfit dentures may impact the subjective perception of elderly ohrqol. thus, we can suppose that prosthetic problems, resembling the use of misfit dentures, negatively influence ohrqol when tmj are affected by the ra. similarly, patients with ra but without tmd showed worse outcomes of qol before prosthetic treatment, apart from social incapacity and social disadvantages domains, standing for impaired ohrqol in this group. hence, these outcomes possibly indicate that ra does not influence the social life aspects of elderly, but uniquely the presence of tmd and the use of misfit dentures. in this sense, a systematic review exhibited that the use of new and comfortable dentures provided better masticatory efficiency and fitting over the soft tissues, contributing to the higher values of ohip functional and physical domains28. indeed, given the worries concerning the old prostheses conditions, the lack of retention, support, and stability, may bring a psychological discomfort to make social contacts, keep interpersonal interaction, rather than strictly because of an autoimmune disease. preceding prosthetic treatment, controls also presented better values of ohip-14 domains than those with ar and tmd, except for social incapacity. it is reported that personal behavior concerning the social disability present a low impact on ohrqol, once patients with absence of teeth do not avoid social interaction29. thus, our outcome is confirmed by previous data29, which emphasizes that the presence of old dentures does not avoid the social welfare of patients, even in the presence of ra. furthermore, even with the use of old dentures, elderly volunteers without the autoimmune disease (control group) showed better perception of ohrqol in psychological discomfort and physical incapacity domains, compared to patients without tmd. previous studies reported that both domains are of greater impact over elderly patients qol29,30. for this reason, regardless of tmd, ra is a chronic and limiting health condition, due to the severe functional disability on body joints, affecting individuals qol1,2,4. therefore, the psychological concern about teeth, mouth or dentures are mainly related to damage 9 alfenas et al. on masticatory function and changes in diet habits30. a systematic review showed a greater commitment of ra on physical domain than mental health16, since bodily pain and decreased physical functioning is commonly found in ra patients. accordingly, our findings possibly reinforce the idea of a physical-limited life, as well as the psychological processes to identify and deal with daily pain, signs, and symptoms of the disease, which do not occur in a patient without the ra diagnosis. following prosthetic rehabilitation with new conventional dentures, no differences were noticed among the three groups, with respect to the ohip-14 domains. the multidimensional concept of qol involves the individual perception of social, physical, psychological, environmental, spiritual and level of independence31. thereby, since elderly wearing dental prostheses presented prominent expectations regarding the study treatment, our experimental groups reached the same qol values of control group. irrespective of ra and tmd presence, insertion of new dentures improved ohip-14 general, functional limitation and physical pain domains. our outcomes corroborate with those from alves et al.32 (2018), which also showed enhancement of ohip-edent domains after complete dentures rehabilitation. contrastingly, bonnet et al.3 (2016) assessed the qol of patients in need of new prosthetic rehabilitation, however, regardless of prostheses type, whether complete or partial removable dentures, there was not a considerable impact on ohrqol before and after rehabilitation3. despite the fact that implant prostheses were freely offered in the study, these authors adopted gohai questionnaire to assess the qol and, either, did not evaluate elderly with an autoimmune disease3. in addition, irrespective of the prosthetic treatment, controls exhibited greater ohip-14 scores with regards to functional limitation and physical pain. again, this finding confirms the limitations of ra, which causes polyarticular inflammatory alterations, damaging and limiting the physical integrity and the performance of daily activities16-18,33. our results also showed that regardless of groups, patient satisfaction was greater after the installation of the new dentures. these results corroborate with medeiros et al. (2019)28, who also reported the recovery of functional and aesthetical parameters with new rpd and/or cd. in the presence of a damaged health condition, it is reasonable to think that after a deficient dental prostheses use, delivery new prosthetic devices may increase the level of satisfaction of the autoimmune disease bearers’ patients. moreover, all prosthetic devices were made following the conventional technique by experienced professionals, which might have contributed to the positive results. although the literature contains a distinct tool to evaluate the consequences of ra in qol34, the use of the ohip-14 in the present study can figure as a limitation. however, different from a specific instrument to detect the impact of an autoimmune disease on qol, the ohip-14 allowed the perception of questions concerning the mouth, dentures, and teeth deterioration. in addition, a two-month follow-up period is limited and might not reflect the long-term ohrqol or satisfaction with prostheses use of ra patients, thus longer periods can be further assessed. finally, the diagnosis of an autoimmune disease is of great relevance to clinicians and associated professionals. 10 alfenas et al. this way, identifying the patient’s perception about their qol is extremely important to define clinical strategies and organize treatment proposals according to their needs. in conclusion, the use of new and well-fitted dentures improves all domains of ohrqol in patients with ra and tmd. meanwhile, better results of qol after prosthetic rehabilitation were observed for the psychological discomfort, psychological incapacity, and physical incapacity domains in patients with ra without tmd group. all groups were satisfied with prostheses use after the new rehabilitation with conventional dentures. acknowledgments the authors acknowledge the support of the são paulo research foundation (grant number 12/08374-4). references 1. aletaha d, neogi t, silman aj, funovits j, felson dt, bingham co 3rd,et al. 2010 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative. arthritis rheum. 2010 sep;62(9):2569-81. doi: 10.1002/art.27584. 2. hoyuela cp, furtado rn, chiari a, natour j. oro-facial evaluation of women with rheumatoid arthritis. j oral rehabil. 2015 may;42(5):370-7. doi: 10.1111/joor.12255. 3. bonnet g, batisse c, segyo jw, veyrune jl, nicolas e, bessadet m. influence of the renewal of removable dentures on oral health related quality of life. springerplus. 2016 nov 28;5(1):2019. doi: 10.1186/s40064-016-3699-7. 4. chamani g, shakibi mr, zarei mr, rad m, pouyafard a, parhizkar a, et al. assessment of relationship between xerostomia and oral health-related quality of life in patients with rheumatoid arthritis. oral dis. 2017 nov;23(8):1162-1167. doi: 10.1111/odi.12721. 5. scott dl, wolfe f, huizinga tw. rheumatoid arthritis. lancet. 2010 sep 25;376(9746):1094-108. doi: 10.1016/s0140-6736(10)60826-4. 6. nemtsova mv, zaletaev dv, bure iv, mikhaylenko ds, kuznetsova eb, alekseeva ea, et al. epigenetic changes in the pathogenesis of rheumatoid arthritis. front genet. 2019 jun 14;10:570. doi: 10.3389/fgene.2019.00570. 7. american college of rheumatology subcommittee on rheumatoid arthritis guidelines. guidelines for the management of rheumatoid arthritis: 2002 update. arthritis rheum. 2002 feb;46(2):328-46. 8. helenius lm, hallikainen d, helenius i, meurman jh, könönen m, leirisalo-repo m, et al. clinical and radiographic findings of the temporomandibular joint in patients with various rheumatic diseases. a case-control study. oral surg oral med oral pathol oral radiol endod. 2005 apr;99(4):455-63. 9. larheim ta, smith hj, aspestrand f. temporomandibular joint abnormalities associated with rheumatic disease: comparison between mr imaging and arthrotomography. radiology. 1992 apr;183(1):221-6. 10. mortazavi n, babaei m, babaee n, kazemi hh, mortazavi r, mostafazadeh a. evaluation of the prevalence of temporomandibular joint involvement in rheumatoid arthritis using research diagnostic criteria for temporomandibular disorders. j dent (tehran). 2018 nov;15(6):332-8. 11 alfenas et al. 11. crincoli v, anelli mg, quercia e, piancino mg, di comite m. temporomandibular disorders and oral features in early rheumatoid arthritis patients: an observational study. int j med sci. 2019 jan 1;16(2):253-263. doi: 10.7150/ijms.28361. 12. marim gc, machado bcz, trawitzki lvv, de felício cm. tongue strength, masticatory and swallowing dysfunction in patients with chronic temporomandibular disorder. physiol behav. 2019 oct 15;210:112616. doi: 10.1016/j.physbeh.2019.112616. 13. lin cy, chung ch, chu hy, chen lc, tu kh, tsao ch, et al. prevalence of temporomandibular disorders in rheumatoid arthritis and associated risk factors: a nationwide study in taiwan. j oral facial pain headache. 2017 fall;31(4):e29-e36. doi: 10.11607/ofph.1917. 14. andrade km, alfenas bfm, rodrigues garcia rcm. influence of removable prostheses on mastication in elderly subjects with rheumatoid arthritis. j oral rehabil. 2018 apr;45(4):295-300. doi: 10.1111/joor.12592. 15. andrade km, alfenas bf, campos ch, rodrigues garcia rc. mandibular movements in older people with rheumatoid arthritis. oral surg oral med oral pathol oral radiol. 2017 may;123(5):e153-e159. doi: 10.1016/j.oooo.2017.01.014. 16. matcham f, scott ic, rayner l, hotopf m, kingsley gh, norton s, et al. the impact of rheumatoid arthritis on quality-of-life assessed using the sf-36 : a systematic review and meta-analysis. semin arthritis rheum. 2014 oct;44(2):123-30. doi: 10.1016/j.semarthrit.2014.05.001. 17. hillsdon mm, brunner ej, guralnik jm, marmot mg. prospective study of physical activity and physical function in early old age. am j prev med. 2005;28(3):245-50. 18. jakobsson u, hallberg ir. review pain and quality of life among older people with rheumatoid arthritis and/or osteoarthritis: a literature review. j clin nurs. 2002;11(4):430-43. 19. slade gd, spencer aj. development and evaluation of the oral health impact profile. community dent health. 1994 mar;11(1):3-11. 20. oliveira bh, nadanovsky p. psychometric properties of the brazilian version of the oral health impact profile-short form. community dentistry oral epidemiology. 2005 aug;33(4):307-14. 21. pistorius j, horn jg, pistorius a, kraft j. oral health-related quality of life in patients with with removable dentures. schweiz monatsschr zahnmed. 2013;123(11):964-71; 955. 22. awad ma., feine js. measuring patient satisfaction with mandibular prostheses. community dent oral epidemiol. 1998 dec;26(6):400–5. 23. vigild m. denture status and need for prosthodontics treatment among institutionalized elderly in denamark. community dent oral epidemiol. 1987 jun;15(3):128-33. 24. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord.1992 fall;6(4):301-55. 25. telles d. [total prosthesis conventional and on implants]. são paulo: santos; 2009. portuguese. 26. phoenix rd; cagna dr, defreest cf. stewart’s clinical removable partial prosthodontics. 4. ed. quintessence; 2008. 518 p. 27. hadzipasic-nazdrajic a. quality of life with removable dentures. mater sociomed. 2011;23(4):214-20. doi: 10.5455/msm.2011.23.214-220. 28. de medeiros akb, campos mftp, da silva costa rsg, de melo la, barbosa gas, carreiro adfp. improvement in quality of life of elderly edentulous patients with new complete dentures: a systematic review. int j prosthodont. 2019 may/jun;32(3):272-277. doi: 10.11607/ijp.6075. 29. sáez-prado b, haya-fernández mc, sanz-garcía mt. oral health and quality of life in the municipal senior citizen’s social clubs for people over 65 of valencia, spain. med oral patol oral cir bucal. 2016 nov;21(6):e672-8. 12 alfenas et al. 30. echeverria ms, wünsch is, langlois co, cascaes am, ribeiro silva ae. oral health-related quality of life in older adults — longitudinal study. gerodontology. 2019 jun;36(2):118-124. doi: 10.1111/ger.12387. 31. the world health organization quality of life assessment (whoqol): position paper from the world health organization. soc sci med. 1995 nov;41(10):1403-9. 32. alves ac., cavalcanti rva., calderon ps., pernambuco l, alchieri jc. quality of life related to complete denture. acta odontol latinoam. 2018 aug;31(2):91-96. 33. hodkinson b., musenge e., ally m., meyer pw, anderson r, tikly m. functional disability and health-related quality of life in south africans with early rheumatoid arthritis. scand j rheumatol. 2012 oct;41(5):366-74. doi: 10.3109/03009742.2012.676065. 34. de jong z, van der heijde d, mckenna sp, whalley d. the reliability and construct validity of the raqol: a rheumatoid arthritisspecific quality of life instrument. br j rheumatol. 1997 aug;36(8):878-83. drug target insights 2013:7 19–25 doi: 10.4137/dti.s12109 this article is available from http://www.la-press.com. © the author(s), publisher and licensee libertas academica ltd. this is an open access article published under the creative commons cc-by-nc 3.0 license. open access full open access to this and thousands of other papers at http://www.la-press.com. drug target insights r a p i d c o m m u n i c a t i o n drug target insights 2013:7 19 microscopic colitis is associated with several concomitant diseases bodil roth1, jonas manjer2 and bodil ohlsson1 1department of clinical sciences, section of internal medicine. 2department of clinical sciences, plastic surgery, skåne university hospital, lund university, sweden. corresponding author email: bodil.ohlsson@med.lu.se abstract: microscopic colitis (mc) is a disease with intestinal mucosal inflammation causing diarrhea, affecting predominantly middle-aged women. the etiology is unknown, but increased prevalence of autoimmune diseases in these patients has been described, although not compared with controls or adjusted for confounding factors. the aim of this study was to examine the prevalence of common diseases in patients with mc and controls from the general population. hypertension, rheumatoid arthritis, asthma or bronchitis, ischemia, and diabetes mellitus were more prevalent in patients than in controls. the prevalence of gastric ulcer and cancer did not differ between the groups. besides corticosteroids, many patients were also being treated with proton pump inhibitors, antidepressant drugs, angiotensin-converting enzyme inhibitors or angiotensin ii receptor antagonists, statins, thyroid hormones, and beta-blockers. more patients than controls were former or current smokers (72.5% versus 57.7%). thus, mc patients have an increased prevalence of several diseases, not only of autoimmune origin. keywords: concomitant diseases, drug treatments, microscopic colitis, women http://dx.doi.org/10.4137/dti.s12109 http://www.la-press.com http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com mailto:bodil.ohlsson@med.lu.se roth et al 20 drug target insights 2013:7 introduction primary microscopic colitis (mc) is a clinical and histopathological disease of unknown etiology, characterized by chronic gastrointestinal symptoms, and a macroscopically normal or near normal colonic mucosa. the entity includes 2 basic forms: collagenous colitis (cc) and lymphocytic colitis (lc).1 some studies have shown a female predominance in both cc and lc,2 mainly affecting middle-aged women, whereas others have not been able to confirm this in lc.3,4 besides primary mc, a wide array of conditions may lead to secondary lymphocytic inflammation in the intestinal mucosa, which should be distinguished from real mc.1. an increased prevalence of autoimmune diseases and use of anti-inflammatory drugs has been described in retrospective studies of patients with mc. on this basis autoimmunity has been suggested as an etiology.3,5–7 asthma has been associated with lc, but not with cc.8 however, these studies have not compared the prevalence of diseases in patients with mc with controls from the general population, and no adjustment for confounding factors has been performed. furthermore, the coexistence of autoimmune diseases and mc may be due to a high consumption of anti-inflammatory drugs, rather than a common causality.3,5–7 smoking and advanced age are risk factors for developing mc, and individuals over 65 years of age are at least 5 times more likely to be diagnosed as having mc than younger individuals.2,9 in these women of upper middle age, it may be difficult to determine whether the mc is a primary disease, or a secondary effect due to other concomitant diseases and drug treatments influencing the colonic mucosa.1 the aim of this cross-sectional study was to compare the prevalence of concomitant diseases in patients with mc and controls from the same geographic area, after adjustment for confounding factors. materials and methods the ethics committee of lund university approved the study protocol for both patients (dnr 2009/565 and 2011/209) and controls (dnr 51-90). all participants gave their written informed consent to take part in the study. subjects women who had been treated for mc at any outpatient clinic of the departments of gastroenterology, skåne, between 2002 and 2010, were identified by a search for the icd-10 classification for the 2 forms cc and lc (k52.8) in outpatient records, as well as in the local register at the department of pathology, skåne university hospital, malmö. about 1/3 of the total number of identified patients were excluded because they were over 73 years of age, since they had many other concomitant diseases and drug therapies, obscuring the picture as to whether they were suffering from primary or secondary mc.1 of the patients recognized, only the 240 patients (median 63 years, range 22–73 years) who had the diagnoses verified by examination of colonic biopsies by a pathologist specialized in gastrointestinal pathology were invited to participate in the present study. altogether, 159 (median 63 years, range 22–73 years) of the 240 patients invited accepted and were enrolled in the study. one patient was excluded due to another ibd diagnosis a few weeks after inclusion, leaving 158 patients (66%), and of these, 133 also agreed to provide blood samples. these patients represent the majority of female cases of diagnosed mc in the southernmost districts of sweden under the age of 73 years. microscopic colitis is more frequent in women than in men, the small male cohort in our region being unsuitable for statistical calculations. furthermore, as the quality of life and experience of symptoms differ between the genders,2 we chose to include only women in the study. controls the malmö diet and cancer study (mdcs) the mdcs, a population-based prospective cohort study, invited all women in malmö born 1923–1950. recruitment was carried out between 1991 and 1996, and 41% of eligible subjects participated. in all, 17,035 women completed the baseline examination.10 the mdcs baseline examination included a dietary assessment, a self-administered questionnaire about marital status, education, employment, smoking habits, wine consumption, physical activity, medical conditions and medication, anthropometric measurements, and the collection of blood samples.11 menopausal status was defined using information on previous surgery and menstrual status. the classification of pre-, periand postmenopausal women has been described in detail elsewhere.12 http://www.la-press.com mc and concomitant diseases drug target insights 2013:7 21 women selected as controls in a previous study on breast cancer were used in the present study as controls. in all, 737 subjects (median age 56 years, range 45–73 years) were available and the only exclusion criterion was that they should not have had a previous breast cancer at baseline.13 patient recruitment and study design between march and june 2011, invitations including study information and the same self-administered questionnaire as was sent to the controls were sent by mail to all 240 women with mc. in addition, questionnaires about gastrointestinal symptoms, psychological well-being and rome iii criteria were sent. the patients were also invited to visit the outpatient clinics of the departments of gastroenterology, skåne university hospital, malmö or the central hospital in kristianstad, to provide blood samples. a reminding letter was sent a month after the invitation letter to those who had not answered. questionnaires were completed 1–3 weeks before blood samples were collected. medical records were scrutinized, and age, gastrointestinal symptoms, examinations, and treatments were recorded, as well as whether the patients had had a single attack of mc, or had persistent disease. patients were compared with controls from the mdsc study. questionnaire a self-administered questionnaire about marital status, education, employment, smoking habits, wine consumption, medical conditions and medication was completed by both controls and patients. one of the questions was: “have you ever been treated for any of the following diseases, namely, hypertension, rheumatoid arthritis, asthma or chronic bronchitis, gastric ulcer, ischemia including myocardial infarction, intermittent claudication and stroke, cancer, or diabetes mellitus?”. statistical analyses the data were analyzed using the statistical software package spss for windows© (release 20.0; ibm, ny, usa). the patients were significantly older, with a wider age range than controls. therefore, the 12 patients younger and the two patients older than the controls were excluded, as were patients with celiac disease and gastroenteritis (13 patients), leaving 131 of the original 158 patients for statistical analysis. thus, both controls and patients were within the age range 45–73 years. first, the distribution of continuous variables (age, disease duration, days of drinking wine/month) was tested using a onesample kolmogorov-smirnov test. all these distributions differed significantly (p , 0.05) from a normal distribution. therefore, the factors studied were categorized and values were given as median (interquartile range). there was no difference between cc and lc for any characteristics in this mc cohort14 and therefore all calculations were performed independent of the category cc or lc. the number of patients in the study cohort (131 patients) who were under treatment with a drug was given as the percentage of drug users. differences between groups were calculated by the 2-tailed mann–whitney u test. fisher’s exact test was used for categorical variables. p-values , 0.05 were considered statistically significant. age was divided into 5-year intervals. the cohort was divided into quartiles of the number of days wine was taken per month. smoking was divided into 3 categories: subjects who had never smoked, subjects who had stopped smoking, and current smokers, including both regular and occasional smokers. employment was divided into 3 categories: employed, retired, or others, where others included housewives, students, and unemployed. education was divided into patients with or without a university education. some answers concerning days of drinking wine/month and level of education were lacking. these were labeled as separate categories. factors intended to be studied (independent variables) were initially examined using univariate analyses to calculate odds ratios with 95% confidence intervals (or with 95% ci). analyses were then adjusted for age at baseline, smoking, the number of days of drinking wine/month, level of education, and employment, as these characteristics differed by .5 percentage points between controls and patients. results patient characteristics in total, 131 women (median age 63 (59–67) years) with mc were included in the statistical calculations (table 1). collagenous colitis was diagnosed in 82 patients (62.6%) and lc in 49 patients (37.4%). http://www.la-press.com roth et al 22 drug target insights 2013:7 table 1. patient and control characteristics. controls n = 737 microscopic colitis n = 131 age at study (years) 56.16 (50.47–62.36) 63.00 (58.94–67.15) age groups (%) 45–49 17.1 4.6 50–54 22.3 6.9 55–59 22.3 13.7 60–64 19.5 32.1 65–69 11.7 26.0 70–74 7.2 16.8 smoking habits (%) never smoked 42.3 27.5 stopped smoking 29.9 36.6 current smokers 27.8 35.9 days of drinking wine/month (%) missing data 6.8 5.3 0–2 49.5 42.7 3–4 15.2 12.2 5–7 12.3 8.4 .7 16.1 31.3 married women (%) 61.9 58.0 level of education (%) missing 0 2.3 #12 years at school 76.1 67.9 .12 years at school 23.9 29.8 employment (%) employed 65.7 44.3 retired 26.5 49.6 others* 7.9 6.1 notes: *includes housewives, students and unemployed. values are given as median (interquartile range). the duration of the disease was 7 (3–14) years. measurements of hemoglobin (hb) in blood and creactive protein (crp) in plasma were in the majority of patients within reference values, showing that the patients were in an overall inactive phase (data not shown). of the patients, 91.7% were born in sweden compared with 90.2% of the controls. more patients than controls had completed a university degree (p = 0.001). as the patients were older than the controls, more patients were retired (p , 0.001) (table 1). smoking and drinking habits there were more controls than patients who had never smoked, and the prevalence of both current and former smokers was higher among the patients (table 1). more patients than controls drank wine .7 days a month (table 1). concomitant diseases the presence of any concomitant disease was more prevalent in patients with mc (58.8%) than in controls (35.5%) (adjusted or = 1.81, 95% ci = 1.18–2.81). hypertension was present in more than 1/3 of the patients. rheumatoid arthritis was 6 times more common and asthma and bronchitis 3 times as common in patients as in controls (table 2). the type of diabetes mellitus is not known in controls, but 2 of the patients with mc had type 1 diabetes and 7 had type 2 diabetes. there was no difference between those who had had a single attack of mc or a persistent mc in those with concomitant diseases and those without (p = 0.930). there was no difference in duration of mc, or age at inclusion, between those with concomitant diseases and those without in addition to the mc (p = 0.564 and p = 0.146, respectively). the patients were currently being treated with several drugs at the time of inclusion. the most common drug treatments as a percentage of the study cohort were corticosteroids (32.1%), proton pump inhibitors (26.0%), antidepressant drugs, specifically selective serotonin reuptake inhibitors (21.4%), angiotensinconverting enzyme inhibitors or angiotensin ii receptor antagonists (18.3%), statins (17.6%), thyroid hormones (17.6%), and beta-blockers (16.0%). patients on any of these drug treatments were older at inclusion (64.92 (60.00–68.34) years and 62.07 (55.55–64.24) years, respectively, p = 0.012). those who had persistent mc had a higher prevalence of current drug treatment (p = 0.024). 8 of the 31 patients with rheumatoid arthritis used non-steroidal anti inflammatory drugs as well as many other drugs. there was no difference in the prevalence of cc and lc in patients who were on any of these drugs or had any of the concomitant diseases (p = 1.000 and p = 0.931, respectively). discussion in spite of excluding all those over 73 years of age, to get a fairly healthy group with real mc, several concomitant diseases and drugs were still present. all chronic diseases measured were over-represented in patients, in contrast to a history of gastric ulcer or cancer. previous studies have been retrospective, collecting patient cohorts seen at tertiary centers.5–7 in our present study, we used a cross-sectional design, http://www.la-press.com mc and concomitant diseases drug target insights 2013:7 23 collecting patients from the whole region at primary, secondary and tertiary centers. this approach reflects the patient group in a better way, as patients handled at tertiary centers are often selected cases.15 as patients with mc are women of upper middle age with former or current smoking in the anamnesis, it is to be expected that asthma, bronchitis, and cardiovascular diseases will be frequently seen in such a cohort, apart from diseases of autoimmune origin. in the present study, hypertension was the most common concomitant disease, and recent research confirms that smokers have a higher prevalence of hypertension than non-smokers.16 a high prevalence of cardiovascular diseases in patients with mc has been described previously, but this was not compared with a control population.17 the medication of the controls is not reported here because drug recommendations have been table 2. the prevalence of different diseases in microscopic colitis (mc) and controls. controls n = 737 mc n = 131 crude or 95% ci or 95% ci hypertension (%) missing 8.4 1.5 – – no hypertension 77.7 62.6 1.00 1.00 hypertension 13.8 35.9 3.22 (2.12–4.88) 2.73 (1.49–3.78) rheumatoid arthritis (%) missing 8.4 1.5 – – no rheumatoid arthritis 87.9 74.0 1.00 1.00 rheumatoid arthritis 3.7 23.7 7.67 (4.39–13.40) 7.21 (3.81–13.64) asthma and bronchitis (%) missing 8.4 1.5 – – no asthma 85.5 80.9 1.00 1.00 asthma 6.0 16.8 2.97 (1.71–5.16) 3.18 (1.68–6.00) cancer (%) missing 8.4 1.5 – – no cancer 85.9 89.3 1.00 1.00 cancer 5.7 8.4 1.42 (0.71–2.83) 1.14 (0.53–2.47) diabetes mellitus (%) missing 8.4 1.5 – – no diabetes mellitus 90.5 90.8 1.00 1.00 diabetes mellitus 1.1 6.9 6.31 (2.38–16.67) 4.91 (1.62–14.87) gastric ulcer (%) missing 8.4 1.5 – – no gastric ulcer 84.3 80.9 1.00 1.00 gastric ulcer 7.3 16.8 2.39 (1.40–4.08) 1.77 (0.95–3.30) ischemia* (%) missing 8.4 1.5 – – no ischemia 88.7 88.5 1.00 1.00 ischemia 2.8 9.9 3.49 (1.70–7.16) 2.96 (1.30–6.76) notes: *including myocardial infarction, intermittent claudication and stroke. analyses were performed adjusted for age at baseline, smoking, days of drinking wine/month, level of education, and employment, as these characteristics differed by .5 percentage between controls and patients. abbreviations: or, odds ratio; ci, confidence interval. changed since the control cohort was recruited. however, medication in controls should be less than of the patients as they were healthier. in accordance with previous reports,18 the present patients who were taking drugs were older than un-treated ones. it has been suggested in previous studies that the drugs being consumed extensively by the patient group are associated with mc and could explain the persistent character of the disease.6,18–20 the consensus is that drugs suspected to induce mc should be withdrawn prior to diagnosis, and that the introduction of treatment against mc may not be followed in the daily clinic.2 this could contribute to the high prevalence figures of mc in the growing elderly population, with more efficient treatment regimens for cardiovascular diseases.2 prospective studies are needed to determine whether the introduction of a new drug precedes the development of the disease, and whether the disease http://www.la-press.com roth et al 24 drug target insights 2013:7 remains resolved after drug withdrawal, in order to estimate appropriate prevalence figures of mc. ischemic colitis is another frequently diagnosed condition in elderly patients with a history of smoking, cardiovascular diseases and diabetes mellitus, and in those who are on vasoactive drugs.21 these characteristics are similar to those described for the mc population.2,3,5 corticosteroids are used in the treatment of mc, with a better response than antiinflammatory drugs, and corticosteroids can also be useful in the treatment of ischemic, radiatic, and toxic colitis.2,21–23 there are several limitations in this study. one is the use of an external control group, and that recommendations for drug prescription have been changed since our recruitment of controls. it is very difficult to recruit healthy volunteers to clinical studies. the response rate of our control group was 41%, and it is possible that these subjects are healthier than those who did not agree to participate. however, it is a strength of the study to, for the first time, compare patients with mc to such a well-defined control group.13 furthermore, the data concerning smoking, overweight status, and level of education were similar in this control group to a study with 80% participation from the same population.10 we could not find from the medical records whether mc was developed prior to or after the introduction of new drugs, and therefore it is impossible to determine whether the disease is primary or secondary. an additional strength of the study was that the control group is derived from the same geographic area as the patient group, and that calculations are adjusted for age differences, life style factors, and socioeconomic factors. in conclusion, patients with a diagnosis of mc are a selection of middle-aged women, former or current smokers, with several concomitant diseases and cardiovascular ageing, and therefore are under treatment with a wide range of diverse drugs. it is not surprising that these patients exhibit gastrointestinal symptoms and microscopic, intestinal, mucosal inflammation. these changes must be interpreted with caution, before considering them as a separate entity of autoimmune origin, instead of secondary reactions to ischemia and toxic stimulants. efforts must be made to better classify and diagnose patients with real, primary mc, to avoid overprescription of corticosteroids. author contributions conceived and designed the experiments: br, jm, bo. analyzed the data: bo. wrote the first draft of the manuscript: bo. contributed to the writing of the manuscript: br, jm, bo. agree with manuscript results and conclusions: br, jm, bo. jointly developed the structure and arguments for the paper: br, jm, bo. made critical revisions and approved final version: br, jm, bo. all authors reviewed and approved of the final manuscript. funding this study was sponsored by grants from the bengt ihre foundation and the development foundation of region skåne. competing interests authors disclose no potential conflicts of interest. disclosures and ethics as a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with icmje authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. this article was subject to blind, independent, expert peer review. the reviewers reported no competing interests. references 1. carmack sw, lash rh, gulizia jm, genta rm. lymphocytic disorders of the gastrointestinal tract: a review for the practicing pathologist. adv anat pathol. 2009;16(5):290–306. 2. pardi ds, kelly cp. microscopic colitis. gastroenterology. 2011;140(4): 1155–65. 3. olesen m, eriksson s, bohr j, järnerot g, tysk c. lymphocytic colitis: a retrospective clinical study of 199 swedish patients. gut. 2004;53(4):536–41. 4. fernández-bañares f, salas a, forné m, esteve m, espinós j, viver jm. incidence of collagenous and lymphocytic colitis: a 5-year population-based study. am j gastroenterol. 1999;94(2):418–23. 5. bohr j, tysk c, eriksson s, abrahamsson h, järnerot g. collagenous colitis: a retrospective study of clinical presentation and treatment in 163 patients. gut. 1996;39(6):846–51. 6. pardi ds, ramnath vr, loftus ev, tremaine wj, sandborn wj. lymphocytic colitis: clinical features, treatment, and outcomes. am j gastroenterol. 2002;97:2829–33. 7. chande n, driman dk, reynolds rp. collagenous colitis and lymphocytic colitis: patient characteristics and clinical presentation. scand j gastroenterol. 2005;40(3):343–7. http://www.la-press.com mc and concomitant diseases drug target insights 2013:7 25 8. koskela rm, niemelä se, karttunen tj, lehtola jk. clinical characteristics of collagenous and lymphocytic colitis. scand j gastroenterol. 2004;39(9):837–45. 9. yen ef, pokhrel b, du h, et al. current and past cigarette smoking significantly increase risk for microscopic colitis. inflamm bowel dis. 2012;18(10):1835–41. 10. manjer j, carlsson s, elmståhl s, et al. the malmö diet and cancer study: representativity, cancer incidence and mortality in participants and nonparticipants. eur j cancer prev. 2001;10(6):489–99. 11. manjer j, elmståhl s, janzon l, berglund g. invitation to a populationbased cohort study: differences between subjects recruited using various strategies. scand j public health. 2002;30(2):103–12. 12. manjer j, johansson r, berglund g, et al. postmenopausal breast cancer risk in relation to sex steroid hormones, prolactin and shbg (sweden). cancer causes control. 2003;14(7):599–607. 13. almquist m, bondeson ag, bondeson l, malm j, manjer j. serum levels of vitamin d, pth and calcium and breast cancer risk-a prospective nested case-control study. int j cancer. 2010;127(9):2159–68. 14. roth b, gustafsson rj, ohlsson b. auto-antibodies and their association with clinical findings in women diagnosed with microscopic colitis. plos one. 2013;8(6):e66088. 15. zankel e, rogler g, andus t, reng cm, schölmerich j, timmer a. crohn’s disease patient characteristics in a tertiary referral center: comparison with patients from a population-based cohort. eur j gastroenterol hepatol. 2005;17(4):395–401. 16. d’elia l, de palma d, rossi g, et al. not smoking is associated with lower risk of hypertension: results of the olivetti heart study. eur j public health. 2013. 17. bjørnbak c, engel pj, nielsen pl, munck lk. microscopic colitis: clinical findings, topography and persistence of histopathological subgroups. aliment pharmacol ther. 2011;34(10):1225–34. 18. rasmussen ma, munck lk. systematic review: are lymphocytic colitis and collagenous colitis two subtypes of the same disease—microscopic colitis? aliment pharmacol ther. 2012;36(2):79–90. 19. cindoruk m, tuncer c, dursun a, et al. increased colonic intraepithelial lymphocytes in patients with hashimoto’s thyroiditis. j clin gastroenterol. 2002;34(3):237–9. 20. beaugerie l, pardi ds. review article: drug-induced microscopic colitis—proposal for a scoring system and review of the literature. aliment pharmacol ther. 2005;22(4):277–84. 21. o’neill s, yalamarthi s. systematic review of the management of ischaemic colitis. colorectal dis. 2012;14(11):e751–63. 22. kochhar r, patel f, dhar a, et al. radiation-induced proctosigmoiditis. prospective, randomized, double-blind controlled trial of oral sulfasalazine plus rectal steroids versus rectal sucralfate. dig dis sci. 1991;36(1):103–7. 23. onishi h, oosegi t, machida y. efficacy and toxicity of eudragit-coated chitosan-succinyl-prednisolone conjugate microspheres using rats with 2,4,6-trinitrobenzenesulfonic acid-induced colitis. int j pharm. 2008; 358(1–2):296–302. http://www.la-press.com archives of academic emergency medicine. 2020; 8(1): e22 ca s e re p o rt anaphylaxis as a rare side effect of ketorolac; a case report hesam yousefi1, ali sahebi2, mahtab farahani3, mohamad golitaleb4∗ 1. department of pharmaceutical care, sajad hospital, kermanshah, iran. 2. clinical research development unit, shahid mostafa khomeini hospital, ilam university of medical sciences, ilam, iran. 3. student research committee, department of nursing, school of nursing, arak university of medical sciences, arak, iran. 4. department of nursing, school of nursing, arak university of medical sciences, arak, iran. received: january 2020; accepted: february 2020; published online: 17 march 2020 abstract: anaphylaxis is a life-threatening systemic allergic hypersensitivity reaction that may potentially be triggered after the administration of any drug. our case was a 51-year-old man with the history of mild pain in his flanks since the night before he was admitted to our hospital. the patient was diagnosed with urolithiasis and admitted to the emergency department. he developed anaphylaxis after intravenous injection of 30 mg ketorolac. allergic reactions to non-steroidal anti-inflammatory drugs (nsaids) such as ketorolac are rare; nonetheless, they can be life-threatening and should be carefully monitored. keywords: anaphylaxis; drug hypersensitivity syndrome; adverse drug reaction; nsaids; ketorolac cite this article as: yousefi h, sahebi a, farahani m, golitaleb m. anaphylaxis as a rare side effect of ketorolac; a case report. arch acad emerg med. 2020; 8(1): e22. 1. introduction non-steroidal anti-inflammatory drugs (nsaids) constitute a broad spectrum of cyclooxygenase (cox) inhibitors suppressing prostaglandin synthesis. nsaids are used for treating various conditions such as pain, rheumatoid arthritis, osteoarthritis, and musculoskeletal disorders (1). ketorolac is an nsaid, which is used to alleviate renal colic due to its anti-contractile effects on the urethra. considering the pain pathogenesis in renal colic, ketorolac is one of the best painrelieving drugs in these patients (2). in intravenous form, this drug reaches its serum peak level within 1 to 3 minutes. ketorolac is metabolized in the liver and excreted through the kidneys (2). although ketorolac has an excellent safety profile, allergic reactions and anaphylaxis may occur following its administration. even though these reactions, either acute or delayed, are uncommon and rare, they can be fatal (3). a number of studies have reported anaphylactic reactions after ketorolac administration. however, the incidence of these reactions is not predictable (4-6). here, we present a case of ∗corresponding author: mohamad golitaleb; department of nursing, school of nursing, arak university of medical sciences, basij square, arak, iran. email: m.golitaleb@arakmu.ac.ir, phone: +988634173504, fax: +988634173524 anaphylaxis in a male patient admitted to the emergency department of vali-e-asr hospital, arak, iran, following the injection of 30 mg ketorolac. 2. case presentation our patient was a 51-year-old man who had mild pain in his flanks since the night before presenting to the emergency department of vali-e-asr hospital of arak, iran, because the pain had worsened and disseminated to abdominal area. the pain was initially localized in the patient’s right flank propagating to the thighs and testicles afterwards. the pain repeatedly decreased and restarted over a short period of time. the patient was restless and anxious while constantly changing his posture. the patient also suffered from nausea, vomiting, hematuria, polyuria, and dysuria. the patient had a history of surgery due to urolithiasis in his left kidney three years ago. the vital signs upon admission were as follows: heart rate (hr) = 103 beat/min, respiratory rate (rr) = 23 beat/min, o2 saturation = 96% (in room air), and blood pressure (bp) = 153/97 mmhg. after taking medical history from the patient and his companions, the patient was diagnosed with urolithiasis. following insertion of a peripheral iv-line, 30 mg of diluted intravenous ketorolac (produced by: alborz darou pharmaceuticals company, iran) was slowly inthis open-access article distributed under the terms of the creative commons attribution noncommercial 3.0 license (cc by-nc 3.0). downloaded from: http://journals.sbmu.ac.ir/aaem h. yousefi et al. 2 jected. 2 minutes after the initiation of drug injection, the patient developed itching, redness of upper extremities, urticaria, angioedema, hypotension, cyanosis, and dyspnea. the patient’s vital signs were immediately checked and the following values were retrieved: hr = 148 beat/min, rr = 7 beat/min, bp = 75/50 mmhg, and o2 saturation = 72% in room air. the anaphylactic shock was immediately managed by infusion of 30 ml/kg normal saline, 0.5 mg intramuscular epinephrine (1:1000), 200 mg of intravascular hydrocortisone, and 4 mg intravenous chlorpheniramine. oxygen was further administrated through nasal cannula at the rate of 8 liters per minute. fortunately, the patient’s general condition gradually improved and completely recovered after 1 hour. after the reaction, 1 gram of intravenous paracetamol (acetaminophen) diluted in 100 cc in normal saline was used to control the pain. the patient was discharged after 8 hours. 3. discussion anaphylaxis is a life-threatening systemic allergic reaction affecting the cardiovascular and respiratory systems. mast cells and basophils are major contributors to anaphylaxis. drugs are among the most common causes of anaphylaxis. delay in treatment of anaphylaxis can lead to hypoxia, ischemia, encephalopathy, and finally death (7, 8). the side effects of ketorolac include reflux (at prolonged usage), nausea, indigestion, and bronchospasm (in susceptible individuals) (2, 3). acute and delayed systemic allergic reactions can occur following oral or intravenous administration of this drug (9). chung et al. reported a 41-year-old male who had been admitted to the emergency department with the diagnosis of acute gastric ulcer perforation. the patient underwent surgery for correcting perforation and was infused with ketorolac loading dose (i.e. 16.2 mg per hour) after surgery to alleviate pain. the patient developed anaphylactic reaction after 1 hour and forty minutes of the drug administration (6). oliva et al. reported a 53-year old female with severe depression who had committed suicide by consuming 3 vials of 30 mg ketorolac. laboratory investigations (i.e. hematologic tests, as well as histological and toxicological findings) and autopsy results confirmed the cause of death as anaphylactic reaction following ketorolac infusion (4). scala et al. reported a 60-year-old woman developing respiratory problems, decreased consciousness, hypotension, and laryngeal edema after oral consumption of 10 mg ketorolac tablet. the patient and her family had no history of allergic diseases, sinusitis, nasal polyps, or drug hypersensitivity. also, the patient declared that she had not consumed anything before taking ketorolac (5). 4. conclusion allergic reactions following the administration of nsaids such as ketorolac are rare. however, such reactions can be life-threatening if they occur. physicians and nurses working in different hospital wards, especially the emergency department, should be aware of this side effect of ketorolac. they must carefully monitor patients’ condition during and after the injection of this drug to identify this fatal complication in a timely manner. 5. declaration 5.1. ethical consideration the scientific value of presenting this case was fully described to the patient and an informed consent was obtained from him before submission. all procedures performed in the present study were in accordance with the standards of the ethical committee of arak university of medical sciences and the 1964 helsinki declaration. 5.2. authors contribution all authors met the criteria for authorship contribution based on recommendations of international committee of medical journal editors. authors orcids mohamad golitaleb: 0000-0002-9216-9262 hesam yousefi: 0000-0003-3837-8490 ali sahebi: 0000-0003-4662-8998 mahtab farahani: 0000-0001-9841-3716 5.3. conflict of interest authors have no conflict of interest. 5.4. funding none. references 1. cabassi a, tedeschi s, perlini s, verzicco i, volpi r, gonzi g, et al. non-steroidal anti-inflammatory drug effects on renal and cardiovascular function: from physiology to clinical practice. european journal of preventive cardiology. 2019:2047487319848105. 2. mallinson te. a review of ketorolac as a prehospital analgesic. journal of paramedic practice. 2017;9(12):522-6. 3. cerreta aj, masterson ca, lewbart ga, dise dr, papich mg. pharmacokinetics of ketorolac in wild eastern box turtles (terrapene carolina carolina) after single intramuscular administration. journal of veterinary pharmacology and therapeutics. 2019;42(2):154-9. this open-access article distributed under the terms of the creative commons attribution noncommercial 3.0 license (cc by-nc 3.0). downloaded from: http://journals.sbmu.ac.ir/aaem 3 archives of academic emergency medicine. 2020; 8(1): e22 4. oliva a, de fg, arena v, fucci n, pascali vl, navarra p. death due to anaphylactic shock secondary to intravenous self-injection of toradol: a case report and review of the literature. clinical toxicology (philadelphia, pa). 2007;45(6):709-13. 5. scala e, giani m, pirrotta l, guerra ec, locanto m, de pita o, et al. selective severe anaphylactic reaction due to ketorolac tromethamine without nonsteroidal antiinflammatory drug intolerance. journal of allergy and clinical immunology. 2001;107(3):557. 6. chung hs, kim es, you yj, park cs. anaphylactoid reaction after injection of ketorolac in a loading dose for patient-controlled analgesia-a case report. korean journal of anesthesiology. 2010;58(6):565. 7. yousefi h, moayedi s, harorani m, sahebi a, golitaleb m. anaphylaxis as a side effect of pantoprazole. shiraz emedical journal. (in press). 8. faridaalaee g, heris ja. anaphylaxis as a rare side effect of pantoprazole; a case report. emergency. 2018;6(1). 9. castillo-zamora c, castillo-peralta la, nava-ocampo aa. report of an anaphylactoid and an anaphylactic reaction to ketorolac in two pediatric surgical patients. therapeutic drug monitoring. 2006;28(3):458-62. this open-access article distributed under the terms of the creative commons attribution noncommercial 3.0 license (cc by-nc 3.0). downloaded from: http://journals.sbmu.ac.ir/aaem introduction case presentation discussion conclusion declaration references key: cord-0072795-uezqcu8c authors: shin, min jun; park, jun young; lee, dae ho; khang, dongwoo title: stem cell mimicking nanoencapsulation for targeting arthritis date: 2021-12-31 journal: int j nanomedicine doi: 10.2147/ijn.s334298 sha: 29a1a1862a3ebc477b4fb86c263940b4da865ad8 doc_id: 72795 cord_uid: uezqcu8c mesenchymal stem cells (mscs) are considered a promising regenerative therapy due to their ability to migrate toward damaged tissues. the homing ability of mscs is unique compared with that of non-migrating cells and mscs are considered promising therapeutic vectors for targeting major cells in many pathophysiological sites. mscs have many advantages in the treatment of malignant diseases, particularly rheumatoid arthritis (ra). ra is a representative autoimmune disease that primarily affects joints, and secreted chemokines in the joints are well recognized by mscs following their migration to the joints. furthermore, mscs can regulate the inflammatory process and repair damaged cells in the joints. however, the functionality and migration ability of mscs injected in vivo still show insufficient. the targeting ability and migration efficiency of mscs can be enhanced by genetic engineering or modification, eg, overexpressing chemokine receptors or migration-related genes, thus maximizing their therapeutic effect. however, there are concerns about genetic changes due to the increased probability of oncogenesis resulting from genome integration of the viral vector, and thus, clinical application is limited. furthermore, it is suspected that administering mscs can promote tumor growth and metastasis in xenograft and orthotopic models. for this reason, msc mimicking nanoencapsulations are an alternative strategy that does not involve using mscs or bioengineered mscs. msc mimicking nanoencapsulations consist of msc membrane-coated nanoparticles, msc-derived exosomes and artificial ectosomes, and msc membrane-fused liposomes with natural or genetically engineered msc membranes. msc mimicking nanoencapsulations not only retain the targeting ability of mscs but also have many advantages in terms of targeted drug delivery. specifically, msc mimicking nanoencapsulations are capable of encapsulating drugs with various components, including chemotherapeutic agents, nucleic acids, and proteins. furthermore, there are fewer concerns over safety issues on msc mimicking nanoencapsulations associated with mutagenesis even when using genetically engineered mscs, because msc mimicking nanoencapsulations use only the membrane fraction of mscs. genetic engineering is a promising route in clinical settings, where nano-encapsulated technology strategies are combined. in this review, the mechanism underlying msc homing and the advantages of msc mimicking nanoencapsulations are discussed. in addition, genetic engineering of mscs and msc mimicking nanoencapsulation is described as a promising strategy for the treatment of immune-related diseases. given the multi-lineage differentiation abilities of mesenchymal stem cells (mscs) isolated from different tissues and organs, mscs have been widely used in various medical fields, particularly regenerative medicine. [1] [2] [3] the representative sources of mscs are bone marrow, adipose, periodontal, muscle, and umbilical cord blood. [4] [5] [6] [7] [8] [9] [10] interestingly, slight differences have been reported in the characteristics of mscs depending on the different sources, including their population in source tissues, immunosuppressive activities, proliferation, and resistance to cellular aging. 11 bone marrow-derived mscs (bm-mscs) are the most intensively studied and show clinically promising results for cartilage and bone regeneration. 11 however, the isolation procedures for bm-mscs are complicated because bone marrow contains a relatively small fraction of mscs (0.001-0.01% of the cells in bone marrow). 12 furthermore, bone marrow aspiration to harvest mscs in human bones is a painful procedure and the slower proliferation rate of bm-mscs is a clinical limitation. 13 in comparison with bm-mscs, adipose-derived mscs (ad-mscs) are relatively easy to collect and can produce up to 500 times the cell population of bm-mscs. 14 ad-mscs showed a greater ability to regenerate damaged cartilage and bone tissues with increased immunosuppressive ability. 14, 15 umbilical cord blood-derived mscs (uc-mscs) proliferate faster than bm-mscs and are resistant to significant cellular aging. 11 mscs have been investigated and gained worldwide attention as potential therapeutic candidates for incurable diseases such as arthritis, spinal cord injury, and cardiac disease. 3, [16] [17] [18] [19] [20] [21] [22] [23] in particular, the inherent tropism of mscs to inflammatory sites has been thoroughly studied. 24 this inherent tropism, also known as homing ability, originates from the recognition of various chemokine sources in inflamed tissues, where profiled chemokines are continuously secreted and the mscs migrate to the chemokines in a concentration-dependent manner. 24 rheumatoid arthritis (ra) is a representative inflammatory disease that primarily causes inflammation in the joints, and this long-term autoimmune disorder causes worsening pain and stiffness following rest. ra affects approximately 24.5 million people as of 2015, but only symptomatic treatments such as pain medications, steroids, and nonsteroidal antiinflammatory drugs (nsaids), or slow-acting drugs that inhibit the rapid progression of ra, such as diseasemodifying antirheumatic drugs (dmards) are currently available. however, ra drugs have adverse side effects, including hepatitis, osteoporosis, skeletal fracture, steroidinduced arthroplasty, cushing's syndrome, gastrointestinal (gi) intolerance, and bleeding. [25] [26] [27] thus, mscs are rapidly emerging as the next generation of arthritis treatment because they not only recognize and migrate toward chemokines secreted in the inflamed joints but also regulate inflammatory progress and repair damaged cells. 28 however, mscs are associated with many challenges that need to be overcome before they can be used in clinical settings. [29] [30] [31] one of the main challenges is the selective accumulation of systemically administered mscs in the lungs and liver when they are administered intravenously, leading to insufficient concentrations of mscs in the target tissues. 32, 33 in addition, most of the administered mscs are typically initially captured by macrophages in the lungs, liver, and spleen. [32] [33] [34] importantly, the viability and migration ability of mscs injected in vivo differed from results previously reported as favorable therapeutic effects and migration efficiency in vitro. 35 to improve the delivery of mscs, researchers have focused on chemokines, which are responsible for mscs' ability to move. 36 the chemokine receptors are the key proteins on mscs that recognize chemokines, and genetic engineering of mscs to overexpress the chemokine receptor can improve the homing ability, thus enhancing their therapeutic efficacy. 37 genetic engineering is a convenient tool for modifying native or non-native genes, and several technologies for genetic engineering exist, including genome editing, gene knockdown, and replacement with various vectors. 38, 39 however, safety issues that prevent clinical use persist, for example, genome integration, off-target effects, and induction of immune response. 40 in this regard, msc mimicking nanoencapsulations can be an alternative strategy for maintaining the homing ability of mscs and overcoming the current safety issues. [41] [42] [43] nanoencapsulation involves entrapping the core nanoparticles of solids or liquids within nanometer-sized capsules of secondary materials. 44 msc mimicking nanoencapsulation uses the msc membrane fraction as the capsule and targeting molecules, that is chemokine receptors, with several types of nanoparticles, as the core. 45, 46 msc mimicking nanoencapsulation consists of msc membrane-coated nanoparticles, mscderived artificial ectosomes, and msc membrane-fused liposomes. nano drug delivery is an emerging field that has attracted significant interest due to its unique characteristics and paved the way for several unique applications that might solve many problems in medicine. in particular, the nanoscale size of nanoparticles (nps) enhances cellular uptake and can optimize intracellular pathways due to their intrinsic physicochemical properties, and can therefore increase drug delivery to target tissues. 47, 48 however, the inherent targeting ability resulting from the physicochemical properties of nps is not enough to target specific tissues or damaged tissues, and additional studies on additional ligands that can bind to surface receptors on target cells or tissues have been performed to improve the targeting ability of nps. 49 likewise, nanoencapsulation with cell membranes with targeting molecules and encapsulation of the core nps with cell membranes confer the targeting ability of the source cell to the nps. 50, 51 thus, msc mimicking nanoencapsulation can mimic the superior targeting ability of mscs and confer the advantages of each core np. in addition, msc mimicking nanoencapsulations have improved circulation time and camouflaging from phagocytes. 52 this review discusses the mechanism of msc migration to inflammatory sites, addresses the potential strategy for improving the tropism of mscs using genetic engineering, and discusses the promising therapeutic agent, msc mimicking nanoencapsulations. the msc migration mechanism can be exploited for diverse clinical applications. 53 the msc migration mechanism can be divided into five stages: rolling by selectin, activation of mscs by chemokines, stopping cell rolling by integrin, transcellular migration, and migration to the damaged site ( figure 1 ). 54, 55 chemokines are secreted naturally by various cells such as tumor cells, stromal cells, and inflammatory cells, maintaining high chemokine concentrations in target cells at the target tissue and inducing signal cascades. [56] [57] [58] likewise, mscs express a variety of chemokine receptors, allowing them to migrate and be used as new targeting vectors. [59] [60] [61] msc migration accelerates depending on the concentration of chemokines, which are the most important factors in the stem cell homing mechanism. 62, 63 chemokines consist of various cytokine subfamilies that are closely associated with the migration of immune cells. chemokines are divided into four classes based on the locations of the two cysteine (c) residues: cc-chemokines, cxc-chemokine, c-chemokine, and cx3 chemokine. 64, 65 each chemokine binds to various msc receptors and the binding induces a chemokine signaling cascade (table 1) . 56, 66 the mechanisms underlying msc and leukocyte migration are similar in terms of their migratory dynamics. 55 p-selectin glycoprotein ligand-1 (psgl-1) and e-selectin ligand-1 (esl-1) are major proteins involved in leukocyte migration that interact with p-selectin and e-selectin present in vascular endothelial cells. however, these promoters are not present in mscs ( figure 2 ). 53,67 the initial rolling is facilitated by selectins expressed on the surface of endothelial cells. various glycoproteins on the surface of mscs can bind to the selectins and continue the rolling process. 68 however, the mechanism of binding of the glycoprotein on mscs to the selectins is still unclear. 69,70 p-selectins and e-selectins, major cellcell adhesion molecules expressed by endothelial cells, adhere to migrated cells adjacent to endothelial cells and can trigger the rolling process. 71 for leukocyte migration, p-selectin glycoprotein ligand-1 (psgl-1) and e-selectin ligand-1 (esl-1) expressed on the membranes of leukocytes interact with p-selectins and e-selectins on the endothelial cells, initiating the process. 72, 73 as already mentioned, mscs express neither psgl-1 nor esl-1. instead, they express galectin-1 and cd24 on their surfaces, and these bind to e-selectin or p-selectin ( figure 2 ). [74] [75] [76] in the migratory activation step, msc receptors are activated in response to inflammatory cytokines, including cxcl12, cxcl8, cxcl4, ccl2, and ccl7. 77 the corresponding activation of chemokine receptors of mscs in response to inflammatory cytokines results in an accumulation of mscs. 58, 78 for example, inflamed tissues release inflammatory cytokines, 79 and specifically, fibroblasts release cxcl12, which further induces the accumulation of mscs through ligand-receptor interaction after exposure to hypoxia and cytokine-rich environments in the rat model of inflammation. [79] [80] [81] [82] previous studies have reported that overexpressing cxcr4, which is a receptor to recognize cxcl12, in mscs improves the homing ability of mscs toward inflamed sites. 83, 84 in short, cytokines are significantly involved in the homing mechanism of mscs. 53 the rolling arrest stage is facilitated by integrin α4β1 (vla-4) on msc. 85 vla-4 is expressed by mscs which are first activated by cxcl-12 and tnf-α chemokines, and activated vla-4 binds to vcam-1 expressed on endothelial cells to stop the rotational movement ( figure 2 ). 86, 87 cxcl12, cxcl11 karp et al categorized the migration of mscs as either "systemic homing" or "non-systemic homing." systemic homing refers to the process of migration through blood vessels and then across the vascular endothelium near the inflamed site. 67, 88 the process of migration after passing through the vessels or local injection is called nonsystemic homing. in non-systemic migration, stem cells migrate through a chemokine concentration gradient ( figure 3 ). 89 mscs secrete matrix metalloproteinases (mmps) during migration. the mechanism underlying msc migration is currently undefined but msc migration can be advanced by remodeling the matrix through the secretion of various enzymes. [90] [91] [92] [93] the migration of mscs to the damaged area is induced by chemokines released from the injured site, such as il-8, tnf-α, insulin-like growth factor (igf-1), and platelet-derived growth factors (pdgf). [94] [95] [96] mscs migrate toward the damaged area following a chemokine concentration gradient. 87 ra is a chronic inflammatory autoimmune disease characterized by distinct painful stiff joints and movement disorders. 97 ra affects approximately 1% of the world's population. 98 ra is primarily induced by macrophages, which are involved in the innate immune response and are also involved in adaptive immune responses, together with b cells and t cells. 99 inflammatory diseases are caused by high levels of inflammatory cytokines and a hypoxic low-ph environment in the joints. 100, 101 fibroblast-like synoviocytes (flss) and accumulated macrophages and neutrophils in the synovium of inflamed joints also express various chemokines. 102, 103 chemokines from inflammatory reactions can induce migration of white blood cells and stem cells, which are involved in angiogenesis around joints. 101, 104, 105 more than 50 chemokines are present in the rheumatoid synovial membrane ( table 2) . of the chemokines in the synovium, cxcl12, mip1-a, cxcl8, and pdgf are the main ones that attract mscs. 106 in the ra environment, cxcl12, a ligand for cxcr4 on mscs, had 10.71 times higher levels of chemokines than in the normal synovial cell environment. mip-1a, a chemokine that gathers inflammatory cells, is a ligand for ccr1, which is normally expressed on msc. 107,108 cxcl8 is a ligand for cxcr1 and cxcr2 on mscs and induces the migration of neutrophils and macrophages, leading to ros in synovial cells. 59 pdgf is a regulatory peptide that is upregulated in the synovial tissue of ra patients. 109 pdgf induces greater msc migration than cxcl12. 110 importantly, stem cells not only have the homing ability to inflamed joints but also have potential as cell therapy with the anti-apoptotic, anticatabolic, and anti-fibrotic effect of msc. 111 in preclinical trials, msc treatment has been extensively investigated in collagen-induced arthritis (cia), a common autoimmune animal model used to study ra. in the ra model, mscs downregulated inflammatory cytokines such as ifn-γ, tnf-α, il-4, il-12, and il1β, and antibodies against collagen, while anti-inflammatory cytokines, such as tumor necrosis factor-inducible gene 6 protein (tsg-6), prostaglandin e2 (pge2), transforming growth factor-beta (tgf-β), il-10, and il-6, were upregulated. [112] [113] [114] [115] [116] genetically engineered mscs targeting arthritis genetic engineering can improve the therapeutic potential of mscs, including long-term survival, angiogenesis, differentiation into specific lineages, anti-and pro-inflammatory activity, and migratory properties ( figure 4 ). 117, 118 although mscs already have an intrinsic homing ability, the targeting ability of mscs and their derivatives, such as membrane vesicles, which are utilized to produce msc mimicking nanoencapsulation, can be enhanced. 118 the therapeutic potential of mscs can be magnified by reprogramming mscs via upregulation or downregulation of their native genes, resulting in controlled production of the target protein, or by introducing foreign genes that enable mscs to express native or non-native products, for example, nonnative soluble tumor necrosis factor (tnf) receptor 2 can inhibit tnf-alpha signaling in ra therapies. 28 mscs can be genetically engineered using different techniques, including by introducing particular genes into the nucleus of mscs or editing the genome of mscs ( figure 5 ). 119 foreign genes can be transferred into nonbinding chemokine x xcl1, cxcl8, ccl20, cxcl11, cxcl1, cxcl5, cxcl7, cxcl6 abbreviations: cc-family, cistain cistain chemokine structure; cxc-family, cistain one amino acid cistain chemokine structure; cx3-c-family, cistain three amino acid cistain structure. mscs using liposomes (chemical method), electroporation (physical method), or viral delivery (biological method). cationic liposomes, also known as lipoplexes, can stably compact negatively charged nucleic acids, leading to the formation of nanomeric vesicular structure. 120 cationic liposomes are commonly produced with a combination of a cationic lipid such as dotap, dotma, dogs, dospa, and neutral lipids, such as dope and cholesterol. 121 these liposomes are stable enough to protect their bound nucleic acids from degradation and are competent to enter cells via endocytosis. 120 electroporation briefly creates holes in the cell membrane using an electric field of 10-20 kv/cm, and the holes are then rapidly closed by the cell's membrane repair mechanism. 122 even though the electric shock induces irreversible cell damage and non-specific transport into the cytoplasm leads to cell death, electroporation ensures successful gene delivery regardless of the target cell or organism. viral vectors, which are derived from adenovirus, adeno-associated virus (aav), or lentivirus (lv), have been used to introduce specific genes into mscs. recombinant lentiviral vectors are the most widely used systems due to their high tropism to dividing and nondividing cells, transduction efficiency, and stable expression of transgenes in mscs, but the random genome integration of transgenes can be an obstacle in clinical applications. 123 adenovirus and aav systems are appropriate alternative strategies because currently available strains do not have broad genome integration and a strong immune response, unlike lv, thus increasing success and safety in clinical trials. 124 as a representative, the oxford-astrazeneca covid-19 vaccine, which has been authorized in 71 countries as a vaccine for severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which spread globally and led to the current pandemic, transfers the spike protein gene using an adenovirus-based viral vector. 125 furthermore, there are two aav-based gene therapies: luxturna for rare inherited retinal dystrophy and zolgensma for spinal muscular atrophy. 126 clustered regularly interspaced short palindromic repeats (crispr)/cas9 were recently used for genome editing and modification because of their simpler design and higher efficiency for genome editing, however, there are safety issues such as off-target effects that induce mutations at sites other than the intended target site. 127 the foreign gene is then commonly transferred into non-integrating forms such as plasmid dna and messenger rna (mrna). 128 the gene expression machinery can also be manipulated at the cytoplasmic level through rna interference (rnai) technology, inhibition of gene expression, or translation using neutralizing targeted mrna molecules with sequence-specific small rna molecules such as small interfering rna (sirna) or microrna (mirna). 129 these small rnas can form enzyme complexes that degrade mrna molecules and thus decrease their activity by inhibiting translation. moreover, the pretranscriptional silencing mechanism of rnai can induce dna methylation at genomic positions complementary to sirna or mirna with enzyme complexes. cxc chemokine receptor 4 (cxcr4) is one of the most potent chemokine receptors that is genetically engineered to enhance the migratory properties of mscs. 130 cxcr4 is a chemokine receptor specific for stromalderived factor-1 (sdf-1), also known as cxc motif chemokine 12 (cxcl12), which is produced by damaged tissues, such as the area of inflammatory bone destruction. 131 several studies on engineering mscs to increase the expression of the cxcr4 gene have reported a higher density of the cxcr4 receptor on their outer cell membrane and effectively increased the migration of mscs toward sdf-1. 83,132,133 cxc chemokine receptor 7 (cxcr7) also had a high affinity for sdf-1, thus the sdf-1/cxcr7 signaling axis was used to engineer the mscs. 134 cxcr7-overexpressing mscs in a cerebral ischemia-reperfusion rat hippocampus model promoted migration based on an sdf-1 gradient, cooperating with the sdf-1/cxcr4 signaling axis ( figure 6 ). 37 cxc chemokine receptor 1 (cxcr1) enhances msc migratory properties. 59 cxcr1 is a receptor for il-8, which is the primary cytokine involved in the recruitment of neutrophils to the site of damage or infection. 135 in particular, the il-8/cxcr1 axis is a key factor for the migration of mscs toward human glioma cell lines, such as u-87 mg, ln18, u138, and u251, and cxcr1overexpressing mscs showed a superior capacity to migrate toward glioma cells and tumors in mice bearing intracranial human gliomas. 136 the migratory properties of mscs were also controlled via aquaporin-1 (aqp1), which is a water channel molecule that transports water across the cell membrane and regulates endothelial cell migration. 137 aqp1overexpressing mscs showed enhanced migration to fracture gap of a rat fracture model with upregulated focal https://doi.org/10.2147/ijn.s334298 international journal of nanomedicine 2021:16 8492 adhesion kinase (fak) and β-catenin, which are important regulators of cell migration. 138 nur77, also known as nerve growth factor ib or nr4a1, and nuclear receptor-related 1 (nurr1), can play a role in improving the migratory capabilities of mscs. 139, 140 the migrating mscs expressed higher levels of nur77 and nurr1 than the non-migrating mscs, and overexpression of these two nuclear receptors functioning as transcription factors enhanced the migration of mscs toward sdf-1. the migration of cells is closely related to the cell cycle, and normally, cells in the late s or g2/m phase do not migrate. 141 the overexpression of nur77 and nurr1 increased the proportion of mscs in the g0/g1phase similar to the results of migrating mscs had more cells in the g1-phase. msc mimicking nanoencapsulations are nanoparticles combined with msc membrane vesicles and these nps have the greatest advantages as drug delivery systems due to the sustained homing ability of mscs as well as the advantages of nps. particles sized 10-150 nm have great advantages in drug delivery systems because they can pass more freely through the cell membrane by the interaction with biomolecules, such as clathrin and caveolin, to facilitate uptake across the cell membrane compared with micron-sized materials. 142, 143 various materials have been used to formulate nps, including silica, polymers, metals, and lipids. 144, 145 nps have an inherent ability, called "passive targeting," to accumulate at specific sites based on their physicochemical properties such as size, surface charge, surface hydrophilicity, and geometry. [146] [147] [148] however, physicochemical properties are not enough to target specific tissues or damaged tissues, and thus "active targeting" is a clinically approved strategy involving the addition of ligands that can bind to surface receptors on target cells or tissues. 149, 150 msc mimicking nanoencapsulation uses natural or genetically engineered msc membranes to coat synthetic nps, producing artificial ectosomes and fusing them with liposomes to increase their targeting ability figure 7 ). 151 especially, mscs have been studied for targeting inflammation and regenerative drugs, and the mechanism and efficacy of migration toward inflamed tissues have been actively investigated. 152 msc mimicking nanoencapsulation can mimic the well-known migration ability of mscs and can be equally utilized without safety issues from the direct application of using mscs. furthermore, cell membrane encapsulations have a wide range of functions, including prolonged blood circulation time and increased active targeting efficacy from the source cells. 153, 154 msc mimicking encapsulations enter recipient cells using multiple pathways. 155 msc mimicking encapsulations can fuse directly with the plasma membrane and can also be taken up through phagocytosis, micropinocytosis, and endocytosis mediated by caveolin or clathrin. 156 msc mimicking encapsulations can be internalized in a highly cell type-specific manner that depends on the recognition of membrane surface molecules by the cell or tissue. 157 for example, endothelial colony-forming cell (ecfc)-derived exosomes were shown cxcr4/sdf-1α interaction and enhanced delivery toward the ischemic kidney, and tspan8-alpha4 complex on lymph node stroma derived extracellular vesicles induced selective uptake by endothelial cells or pancreatic cells with cd54, serving as a major ligand. 158, 159 therefore, different source cells may contain protein signals that serve as ligands for other cells, and these receptor-ligand interactions maximized targeted delivery of nps. 160 this natural mechanism inspired the application of msc membranes to confer active targeting to nps. cell membrane-coated nps (cmcnps) are biomimetic strategies developed to mimic the properties of cell membranes derived from natural cells such as erythrocytes, white blood cells, cancer cells, stem cells, platelets, or bacterial cells with an np core. 161 core nps made of polymer, silica, and metal have been evaluated in attempts to overcome the limitations of conventional drug delivery systems but there are also issues of toxicity and reduced biocompatibility associated with the surface properties of nps. 162, 163 therefore, only a small number of nps have been approved for medical application by the fda. 164 coating with cell membrane can enhance the biocompatibility of nps by improving immune evasion, enhancing circulation time, reducing res clearance, preventing serum protein adsorption by mimicking cell glycocalyx, which are chemical determinants of "self" at the surfaces of cells. 151, 165 furthermore, the migratory properties of mscs can also be transferred to nps by coating them with the cell membrane. 45 coating nps with msc membranes not only enhances biocompatibility but also maximizes the therapeutic effect of nps by mimicking the targeting ability of mscs. 166 cell membrane-coated nps are prepared in three steps: extraction of cell membrane vesicles from the source cells, synthesis of the core nps, and fusion of the membrane vesicles and core nps to produce cell membrane-coated nps (figure 8 ). 167 cell membrane vesicles, including extracellular vesicles (evs), can be harvested through cell lysis, mechanical disruption, and centrifugation to isolate, purify the cell membrane vesicles, and remove intracellular components. 168 all the processes must be conducted under cold conditions, with protease inhibitors to minimize the denaturation of integral membrane proteins. cell lysis, which is classically performed using mechanical lysis, including homogenization, sonication, or extrusion followed by differential velocity centrifugation, is necessary to remove intracellular components. cytochalasin b (cb), a drug that affects cytoskeleton-membrane interactions, induces secretion of membrane vesicles from source cells and has been used to extract the cell membrane. 169 the membrane functions of the source cells are preserved in cb-induced vesicles, forming biologically active surface receptors and ion pumps. 170 furthermore, cb-induced vesicles can encapsulate drugs and nps successfully, and the vesicles can be harvested by centrifugation without a purification step to remove nuclei and cytoplasm. 171 clinically translatable membrane vesicles require scalable production of high volumes of homogeneous vesicles within a short period. although mechanical methods (eg, shear stress, ultrasonication, or extrusion) are utilized, cbinduced vesicles have shown potential for generating membrane encapsulation for nano-vectors. 168 the advantages of cb-induced vesicles versus other methods are compared in table 3 . after extracting cell membrane vesicles, synthesized core nps are coated with cell membranes, including surface proteins. 172 polymer nps and inorganic nps are adopted as materials for the core nps of cmcnps, and generally, polylactic-co-glycolic acid (plga), polylactic acid (pla), chitosan, and gelatin are used. plga has been approved by fda is the most common polymer of nps. 173 biodegradable polymer nps have gained considerable attention in nanomedicine due to their biocompatibility, nontoxic properties, and the ability to modify their surface as a drug carrier. 174 inorganic nps are composed of gold, iron, copper, and silicon, which have hydrophilic, biocompatible, and highly stable properties compared with organic materials. 175 furthermore, some photosensitive inorganic nps have the potential for use in photothermal therapy (ptt) and photodynamic therapy (pdt). 176 the fusion of cell membrane vesicles and core nps is primarily achieved via extrusion or sonication. 165 cell membrane coating of nps using mechanical extrusion is based on a different-sized porous membrane where core nps and vesicles are forced to generate vesicle-particle fusion. 177 ultrasonic waves are applied to induce the fusion of vesicles and nps. however, ultrasonic frequencies need to be optimized to improve fusion efficiency and minimize drug loss and protein degradation. 178 cmcnps have extensively employed to target and treat cancer using the membranes obtained from red blood cell (rbc), platelet and cancer cell. 165 in addition, membrane from msc also utilized to target tumor and ischemia with various types of core nps, such as msc membrane coated plga nps targeting liver tumors, msc membrane coated gelatin nanogels targeting hela cell, msc membrane coated silica nps targeting hela cell, msc membrane coated plga nps targeting hindlimb ischemia, and msc membrane coated iron oxide nps for targeting the ischemic brain. [179] [180] [181] [182] [183] however, there are few studies on cmcnps using stem cells for the treatment of arthritis. increased targeting ability to arthritis was introduced using msc-derived evs and nps. 184,185 msc membrane-coated nps are proming strategy for clearing raised concerns from direct use of msc (with or without nps) in terms of toxicity, reduced biocompatibility, and poor targeting ability of nps for the treatment of arthritis. exosomes are natural nps that range in size from 40 nm to 120 nm and are derived from the multivesicular body (mvb), which is an endosome defined by intraluminal vesicles (ilvs) that bud inward into the endosomal lumen, fuse with the cell surface, and are then released as exosomes. 186 because of their ability to express receptors on their surfaces, msc-derived exosomes are also considered potential candidates for targeting. 187 exosomes are commonly referred to as intracellular communication molecules that transfer various compounds through physiological mechanisms such as immune response, neural communication, and antigen presentation in diseases such as cancer, cardiovascular disease, diabetes, and inflammation. 188 however, there are several limitations to the application of exosomes as targeted therapeutic carriers. first, the limited reproducibility of exosomes is a major challenge. in this field, the standardized techniques for isolation and purification of exosomes are lacking, and conventional methods containing multi-step ultracentrifugation often lead to contamination of other types of evs. furthermore, exosomes extracted from cell cultures can vary and display inconsistent properties even when the same type of donor cells were used. 189 second, precise characterization studies of exosomes are needed. unknown properties of exosomes can hinder therapeutic efficiencies, for example, when using exosomes as cancer therapeutics, the use of cancer cell-derived exosomes should be avoided because cancer cell-derived exosomes may contain oncogenic factors that may contribute to cancer progression. 190 finally, cost-effective methods for the large-scale production of exosomes are needed for clinical application. the yield of exosomes is much lower than evs. depending on the exosome secretion capacity of donor cells, the yield of exosomes is restricted, and large-scale cell culture technology for the production of exosomes is high difficulty and costly and isolation of exosomes is the time-consuming and low-efficient method. 156 ectosome is an ev generated by outward budding from the plasma membrane followed by pinching off and release to the extracellular parts. recently, artificially produced ectosome utilized as an alternative to exosomes in targeted therapeutics due to stable productivity regardless of cell type compared with conventional exosome. artificial ectosomes, containing modified cargo and targeting molecules have recently been introduced for specific purposes (figure 9 ). 191, 192 artificial ectosomes are typically prepared by breaking bigger cells or cell membrane fractions into smaller ectosomes, similar size to natural exosomes, containing modified cargo such as rna molecules, which control specific genes, and chemical drugs such as anticancer drugs. 193 naturally secreted exosomes in conditioned media from modified source cells can be harvested by differential ultracentrifugation, density gradients, precipitation, filtration, and size exclusion chromatography for exosome separation. 194 even though there are several commercial kits for isolating exosomes simply and easily, challenges in compliant scalable production on a large scale, including purity, homogeneity, and reproducibility, have made it difficult to use naturally secreted exosomes in clinical settings. 195 therefore, artificially produced ectosomes are appropriate for use in clinical applications, with novel production methods that can meet clinical production criteria. production of artificially produced ectosomes begins by breaking the cell membrane fraction of cultured cells and then using them to produce cell membrane vesicles to form ectosomes. as mentioned above, cell membrane vesicles are extracted from source cells in several ways, and cell membrane vesicles are extracted through polycarbonate membrane filters to reduce the mean size to a size similar to that of natural exosomes. 196 furthermore, specific microfluidic devices mounted on microblades (fabricated in silicon nitride) enable direct slicing of living cells as they flow through the hydrophilic microchannels of the device. 197 the sliced cell fraction reassembles and forms ectosomes. there are several strategies for loading exogenous therapeutic cargos such as drugs, dna, rna, lipids, metabolites, and proteins, into exosomes or artificial ectosomes in vitro: electroporation, incubation for passive loading of cargo or active loading with membrane permeabilizer, freeze and thaw cycles, sonication, and extrusion. 198 in addition, protein or rna molecules can be loaded by co-expressing them in source cells via bio-engineering, and proteins designed to interact with the protein inside the cell membrane can be loaded actively into exosomes or artificial ectosomes. 157 targeting molecules at the surface of exosomes or artificial ectosomes can also be engineered in a manner similar to the genetic engineering of mscs. most of the exosomes derived from mscs for drug delivery have employed mirnas or sirnas, inhibiting translation of specific mrna, with anticancer activity, for example, mir-146b, mir-122, and mir-379, which are used for cancer targeting by membrane surface molecules on msc-derived exosomes. [199] [200] [201] drugs such as doxorubicin, paclitaxel, and curcumin were also loaded into msc-derived exosomes to target cancer. [202] [203] [204] however, artificial ectosomes derived from mscs as arthritis therapeutics remains largely unexplored area, while evs, mixtures of natural ectosomes and exosomes, derived from mscs have studied in the treatment of arthritis. 184 artificial ectosomes with intrinsic tropism from mscs plus additional targeting ability with engineering increase the chances of ectosomes reaching target tissues with ligand-receptor interactions before being taken up by macrophages. 205 eventually, this will decrease off-target binding and side effects, leading to lower therapeutic dosages while maintaining therapeutic efficacy. 206, 207 msc membrane-fused liposomes targeting arthritis liposomes are spherical vesicles that are artificially synthesized through the hydration of dry phospholipids. 208 the clinically available liposome is a lipid bilayer surrounding a hollow core with a diameter of 50-150 nm. therapeutic molecules, such as anticancer drugs (doxorubicin and daunorubicin citrate) or nucleic acids, can be loaded into this hollow core for delivery. 209 due to their amphipathic nature, liposomes can load both hydrophilic (polar) molecules in an aqueous interior and hydrophobic (nonpolar) molecules in the lipid membrane. they are well-established biomedical applications and are the most common nanostructures used in advanced drug delivery. 210 furthermore, liposomes have several advantages, including versatile structure, biocompatibility, low toxicity, non-immunogenicity, biodegradability, and synergy with drugs: targeted drug delivery, reduction of the toxic effect of drugs, protection against drug degradation, and enhanced circulation half-life. 211 moreover, surfaces can be modified by either coating them with a functionalized polymer or peg chains to improve targeted delivery and increase their circulation time in biological systems. 212 liposomes have been investigated for use in a wide variety of therapeutic applications, including cancer diagnostics and therapy, vaccines, brain-targeted drug delivery, and anti-microbial therapy. a new approach was recently proposed for providing targeting features to liposomes by fusing them with cell membrane vesicles, generating molecules called membrane-fused liposomes ( figure 10 ). 213 cell membrane vesicles retain the surface membrane molecules from source cells, which are responsible for efficient tissue targeting and cellular uptake by target cells. 214 however, the immunogenicity of cell membrane vesicles leads to their rapid clearance by macrophages in the body and their low drug loading efficiencies present challenges for their use as drug delivery systems. 156 however, membrane-fused liposomes have advantages of stability, long half-life in circulation, and low immunogenicity due to the liposome, and the targeting feature of cell membrane vesicles is completely transferred to the liposome. 215 furthermore, the encapsulation efficiencies of doxorubicin were similar when liposomes and membrane-fused liposomes were used, indicating that the relatively high drug encapsulation capacity of liposomes was maintained during the fusion process. 216 combining 8498 membrane-fused liposomes with macrophage-derived membrane vesicles showed differential targeting and cytotoxicity against normal and cancerous cells. 217 although only a few studies have been conducted, these results corroborate that membrane-fused liposomes are a potentially promising future drug delivery system with increased targeting ability. mscs show intrinsic tropism toward arthritis, and further engineering and modification to enhance their targeting ability make them attractive candidates for the development of drug delivery systems. fusing msc exosomes with liposomes, taking advantage of both membrane vesicles and liposomes, is a promising technique for future drug delivery systems. mscs have great potential as targeted therapies due to their greater ability to home to targeted pathophysiological sites. the intrinsic ability to home to wounds or to the tumor microenvironment secreting inflammatory mediators make mscs and their derivatives targeting strategies for cancer and inflammatory disease. 218, 219 contrary to the well-known homing mechanisms of various blood cells, it is still not clear how homing occurs in mscs. so far, the mechanism of msc tethering, which connects long, thin cell membrane cylinders called tethers to the adherent area for migration, has not been clarified. recent studies have shown that galectin-1, vcam-1, and icam are associated with msc tethering, 53,220 but more research is needed to accurately elucidate the tethering mechanism of mscs. msc chemotaxis is well defined and there is strong evidence relating it to the homing ability of mscs. 53 chemotaxis involves recognizing chemokines through chemokine receptors on mscs and migrating to chemokines in a gradient-dependent manner. 221 ra, a representative inflammatory disease, is associated with well-profiled chemokines such as cxcr1, cxcr4, and cxcr7, which are recognized by chemokine receptors on mscs. in addition, damaged joints in ra continuously secrete cytokines until they are treated, giving mscs an advantage as future therapeutic agents for ra. 222 however, there are several obstacles to utilizing mscs as ra therapeutics. in clinical settings, the functional capability of mscs is significantly affected by the health status of the donor patient. 223 msc yield is significantly reduced in patients undergoing steroid-based treatment and the quality of mscs is dependent on the donor's age and environment. 35 in addition, when mscs are used clinically, cryopreservation and defrosting are necessary, but these procedures shorten the life span of mscs. 224 therefore, nps mimicking mscs are an alternative strategy for overcoming the limitations of mscs. additionally, further engineering and modification of mscs can enhance the therapeutic effect by changing the targeting molecules and loaded drugs. in particular, upregulation of receptors associated with chemotaxis through genetic engineering can confer the additional ability of mscs to home to specific sites, while the increase in engraftment maximizes the therapeutic effect of mscs. 36, 225 furthermore, there are several methods that can be used to exploit the targeting ability of mscs as drug delivery systems. mscs mimicking nanoencapsulation, which consists of msc membrane-coated nps, mscderived artificial ectosomes, and msc membrane-fused liposomes, can mimic the targeting ability of mscs while retaining the advantages of nps. msc-membranecoated nps are synthesized using inorganic or polymer nps and membranes from mscs to coat inner nanosized structures. because they mimic the biological characteristics of msc membranes, msc-membrane-coated nps can not only escape from immune surveillance but also effectively improve targeting ability, with combined functions of the unique properties of core nps and msc membranes. 226 exosomes are also an appropriate candidate for use in msc membranes, utilizing these targeting abilities. however, natural exosomes lack reproducibility and stable productivity, thus artificial ectosomes with targeting ability produced via synthetic routes can increase the local concentration of ectosomes at the targeted site, thereby reducing toxicity and side effects and maximizing therapeutic efficacy. 156 msc membrane-fused liposomes, a novel system, can also transfer the targeting molecules on the surface of mscs to liposomes; thus, the advantages of liposomes are retained, but with targeting ability. with advancements in nanotechnology of drug delivery systems, the research in cell-mimicking nanoencapsulation will be very useful. efficient drug delivery systems fundamentally improve the quality of life of patients with a low dose of medication, low side effects, and subsequent treatment of diseases. 227 however, research on cellmimicking nanoencapsulation is at an early stage, and several problems need to be addressed. to predict the nanotoxicity of artificially synthesized msc mimicking nanoencapsulations, interactions between lipids and drugs, drug release mechanisms near the targeted site, in vivo compatibility, and immunological physiological studies must be conducted before clinical application. mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome engineering mesenchymal stem cells for regenerative medicine and drug delivery regenerative medicine in rheumatic disease-progress in tissue engineering analysis of precursor cells for osteogenic and hematopoietic tissues human adipose tissue is a source of multipotent stem cells a perivascular origin for mesenchymal stem cells in multiple human organs postnatal human dental pulp stem cells (dpscs) in vitro and in vivo human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow mesenchymal stem cells in the wharton's jelly of the human umbilical cord comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue bone marrow-derived from the human femoral shaft as a new source of mesenchymal stem/ stromal cells: an alternative cell material for banking and clinical transplantation factors affecting mesenchymal stromal cells yield from bone marrow aspiration adipose tissue-derived multipotent stromal cells have a higher immunomodulatory capacity than their bone marrow-derived counterparts mesenchymal stem cells of different origin: comparative evaluation of proliferative capacity, telomere length and pluripotency marker expression synovial fibroblasts spread rheumatoid arthritis to unaffected joints japan's approval of stem-cell treatment for spinalcord injury concerns scientists mesenchymal stem cells for spinal cord injury: current options, limitations, and future of cell therapy treatment of spinal cord injury with mesenchymal stem cells mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease use of mesenchymal stem cells for therapy of cardiac disease stem cell therapy for cardiac disease the therapeutic potential of mesenchymal stem cells for cardiovascular diseases inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the global burden of disease study adverse effects of biologics: a network meta-analysis and cochrane overview rheumatoid arthritis: diagnosis and management etanercept-synthesising mesenchymal stem cells efficiently ameliorate collagen-induced arthritis risk factors in the development of stem cell therapy mesenchymal stem cells current clinical applications: a systematic review trends in mesenchymal stem cell clinical trials 2004-2018: is efficacy optimal in a narrow dose range? biodistribution, migration and homing of systemically applied mesenchymal stem/stromal cells quantitative magnetic particle imaging monitors the transplantation, biodistribution, and clearance of stem cells in vivo in vivo tracking of 111in-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis mesenchymal stem cell perspective: cell biology to clinical progress enhancing the migration ability of mesenchymal stromal cells by targeting the sdf-1/cxcr4 axis chemokine receptor 7 overexpression promotes mesenchymal stem cell migration and proliferation via secreting chemokine ligand 12 gene-editing technologies paired with viral vectors for translational research into neurodegenerative diseases cas9-aav6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice the delivery challenge: fulfilling the promise of therapeutic genome editing mesenchymal stem cell-derived extracellular vesicles: challenges in clinical applications mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle micrornas polymer encapsulation of ruthenium complexes for biological and medicinal applications current advances and challenges of mesenchymal stem cells-based drug delivery system and their improvements potential therapeutic usage of nanomedicine for glaucoma treatment therapeutic potential of targeted nanoparticles and perspective on nanotherapies light-triggered switching of liposome surface charge directs delivery of membrane impermeable payloads in vivo focus on fundamentals: achieving effective nanoparticle targeting extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking targeting antigen to the surface of evs improves the in vivo immunogenicity of human and non-human adenoviral vaccines in mice cell membrane camouflaged nanoparticles: a new biomimetic platform for cancer photothermal therapy mesenchymal stromal cell homing: mechanisms and strategies for improvement. iscience concise review: msc adhesion cascade-insights into homing and transendothelial migration the lymphocyte homing receptors: gatekeepers of the multistep paradigm chemokines in the cancer microenvironment and their relevance in cancer immunotherapy the chemokine system in innate immunity secretion of immunoregulatory cytokines by mesenchymal stem cells towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors cxcr1, cxcr2 and ccr2, and migrate upon stimulation with cxcl8 but not ccl2 bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets mesenchymal stem cell therapy assisted by nanotechnology: a possible combinational treatment for brain tumor and central nerve regeneration the role of chemokines in mesenchymal stem cell homing to wounds journey of mesenchymal stem cells for homing: strategies to enhance efficacy and safety of stem cell therapy chemokines from a structural perspective new insights into the structure and function of chemokine receptor: chemokine complexes from an experimental perspective the dual roles of inflammatory cytokines and chemokines in the regulation of autoimmune diseases and their clinical implications homing and migration of mesenchymal stromal cells: how to improve the efficacy of cell therapy? the selectins: vascular adhesion molecules cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells (mscs) and cardiosphere-derived cells (cdcs): pilot validation in porcine ischemia-reperfusion model expression of e-selectin ligands on circulating tumor cells: cross-regulation with cancer stem cell regulatory pathways? front oncol direct observation of catch bonds involving cell-adhesion molecules targeting selectins and selectin ligands in inflammation and cancer chromatographically isolated cd63+cd81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after tbi the use of surface immobilization of p-selectin glycoprotein ligand-1 on mesenchymal stem cells to facilitate selectin mediated cell tethering and rolling mesenchymal stem cells engineered to express selectin ligands and il-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis interaction between mesenchymal stromal cell-derived extracellular vesicles and immune cells by distinct protein content chemokines and chemokine receptors: orchestrating tumor metastasization immunobiology of mesenchymal stem cells tissue production of pro-inflammatory cytokines (il-1beta, tnfalpha and il-6) correlates with the intensity of the systemic inflammatory response and with corticosteroid requirements in giant-cell arteritis the hypoxic tumour microenvironment. oncogenesis cancer-associated fibroblasts: an emerging target of anti-cancer immunotherapy bone marrow-derived cxcr4-overexpressing mscs display increased homing to intestine and ameliorate colitis-associated tumorigenesis in mice overexpression of the mesenchymal stem cell cxcr4 gene in irradiated mice increases the homing capacity of these cells cxcr4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats the integrin vla-4 supports tethering and rolling in flow on vcam-1 a cxcl1 paracrine network links cancer chemoresistance and metastasis tnfalpha-activated mesenchymal stromal cells promote breast cancer metastasis by recruiting cxcr2(+) neutrophils mesenchymal stem cell homing: the devil is in the details the life and fate of mesenchymal stem cells effects of matrix metalloproteinases on the fate of mesenchymal stem cells human mesenchymal stem cells generate a distinct pericellular zone of mmp activities via binding of mmps and secretion of high levels of timps matrix metalloproteinase 1 is necessary for the migration of human bone marrow-derived mesenchymal stem cells toward human glioma matrix metalloproteinases in stem cell mobilization mesenchymal stem cell migration and tissue repair mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating cxcr2-src-pkl/vav2-rac1 a novel strategy to enhance mesenchymal stem cell migration capacity and promote tissue repair in an injury specific fashion successes and failures of chemokine-pathway targeting in rheumatoid arthritis prevalence of rheumatoid arthritis in low-and middle-income countries: a systematic review and analysis mesenchymal stem cells from patients with rheumatoid arthritis display impaired function in inhibiting th17 cells chemokines and chemokine receptors in arthritis role of chemokines and chemokine receptors in rheumatoid arthritis cytokines in the pathogenesis of rheumatoid arthritis role of subnano-, nano-and submicron-surface features on osteoblast differentiation of bone marrow mesenchymal stem cells survival and biodistribution of xenogenic adipose mesenchymal stem cells is not affected by the degree of inflammation in arthritis targeting cellular adhesion molecules, chemokines and chemokine receptors in rheumatoid arthritis chemokines in physiological and pathological bone remodeling stimulation of matrix metalloprotease 3 release from human chondrocytes by the interaction of stromal cell-derived factor 1 and cxc chemokine receptor 4 platelet-derived growth factor and transforming growth factor beta synergistically potentiate inflammatory mediator synthesis by fibroblast-like synoviocytes the in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities human mesenchymal stem cellscurrent trends and future prospective bone marrow mesenchymal stem cells improve bone erosion in collagen-induced arthritis by inhibiting osteoclasia-related factors and differentiating into chondrocytes paradoxical effects of human adipose tissue-derived mesenchymal stem cells on progression of experimental arthritis in skg mice administering human adipose-derived mesenchymal stem cells to prevent and treat experimental arthritis immunosuppressive effects of mesenchymal stem cells in collagen-induced mouse arthritis therapeutic effect of human amniotic membrane-derived cells on experimental arthritis and other inflammatory disorders genetic engineering of mesenchymal stem cells and its application in human disease therapy genetic engineering of mesenchymal stem cells to induce their migration and survival dos santos cc. genetically modified mesenchymal stromal/stem cells: application in critical illness cationic lipids, lipoplexes and intracellular delivery of genes trends and developments in liposome drug delivery systems theory and in vivo application of electroporative gene delivery lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts viral vector platforms within the gene therapy landscape safety and efficacy of the chadox1 ncov-19 vaccine (azd1222) against sars-cov-2: an interim analysis of four randomised controlled trials in brazil, south africa, and the uk recombinant adeno-associated virus gene therapy in light of luxturna (and zolgensma and glybera): where are we, and how did we get here? analysis of off-target effects of crispr/cas-derived rna-guided endonucleases and nickases mammalian cell transfection: the present and the future rna interference: from gene silencing to gene-specific therapeutics stromal cell-derived factor-1 (sdf-1): homing factor for engineered regenerative medicine mesenchymal stem cells markedly suppress inflammatory bone destruction in rats with adjuvant-induced arthritis cell surface engineering to enhance mesenchymal stem cell migration toward an sdf-1 gradient cxcr4 transfection of cord blood mesenchymal stromal cells with the use of cationic liposome enhances their migration toward stromal cell-derived factor-1 cxcr-7 receptor promotes sdf-1alpha-induced migration of bone marrow mesenchymal stem cells in the transient cerebral ischemia/reperfusion rat hippocampus overexpression of cxc chemokine receptors is required for the superior glioma-tracking property of umbilical cord blood-derived mesenchymal stem cells cxc chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule aqp1 enhances migration of bone marrow mesenchymal stem cells through regulation of fak and beta-catenin nuclear receptors nur77 and nurr1 modulate mesenchymal stromal cell migration molecular signature of migratory human mesenchymal stromal cells; influence of the cell cycle cell cycle and cell migration: new pieces to the puzzle interactions of nanoparticles and biosystems: microenvironment of nanoparticles and biomolecules in nanomedicine destroying deep lung tumor tissue through lung-selective accumulation and by activation of caveolin uptake channels using a specific width of carbon nanodrug use of a lipid-coated mesoporous silica nanoparticle platform for synergistic gemcitabine and paclitaxel delivery to human pancreatic cancer in mice lipid-nanodiscs formed by paramagnetic metal chelated polymer for fast nmr data acquisition physical properties of nanoparticles that result in improved cancer targeting selective organ targeting (sort) nanoparticles for tissue-specific mrna delivery and crispr-cas gene editing physical and chemical profiles of nanoparticles for lymphatic targeting nanoparticle design optimization for enhanced targeting: monte carlo simulations targeted nanoparticles for image-guided treatment of triple-negative breast cancer: clinical significance and technological advances cell membrane coated nanoparticles: next-generation therapeutics human mesenchymal stem cells (mscs) for treatment towards immune-and inflammation-mediated diseases: review of current clinical trials cell membrane protein functionalization of nanoparticles as a new tumor-targeting strategy recent advances in mesenchymal stem cell membrane-coated nanoparticles for enhanced drug delivery sizeand coating-dependent uptake of polymer-coated gold nanoparticles in primary human dermal microvascular endothelial cells exosome engineering: current progress in cargo loading and targeted delivery engineering exosomes for targeted drug delivery receptor-ligand interaction mediates targeting of endothelial colony forming cell-derived exosomes to the kidney after ischemic injury toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection cell-based nanoparticles delivery systems for targeted cancer therapy: lessons from anti-angiogenesis treatments cell-mediated and cell membrane-coated nanoparticles for drug delivery and cancer therapy common pitfalls in nanotechnology: lessons learned from nci's nanotechnology characterization laboratory close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems therapeutic nanoparticles and their targeted delivery applications cell membrane coating technology: a promising strategy for biomedical applications doxorubicin delivered using nanoparticles camouflaged with mesenchymal stem cell membranes to treat colon cancer cancer cell membrane-coated nanoparticles for cancer management extracellular blebs: artificially-induced extracellular vesicles for facile production and clinical translation cytochalasin b disrupts the association of filamentous web and plasma membrane in hepatocytes evaluation of cytochalasin b-induced membrane vesicles fusion specificity with target cells cell membrane capsules for encapsulation of chemotherapeutic and cancer cell targeting in vivo preparation and application of cell membrane-camouflaged nanoparticles for cancer therapy poly lactic-co-glycolic acid (plga) as biodegradable controlled drug delivery carrier biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines nanoparticles: properties, applications and toxicities nanoparticle-based photothermal and photodynamic immunotherapy for tumor treatment cell membrane-camouflaged nanoparticles: a promising biomimetic strategy for cancer theragnostics recent advancements in non-invasive formulations for protein drug delivery surface functionalization of polymeric nanoparticles with umbilical cord-derived mesenchymal stem cell membrane for tumor-targeted therapy stem cell membrane-coated nanogels for highly efficient in vivo tumor targeted drug delivery stem-cell-membrane camouflaging on near-infrared photoactivated upconversion nanoarchitectures for in vivo remote-controlled photodynamic therapy bioengineered stem cell membrane functionalized nanocarriers for therapeutic targeting of severe hindlimb ischemia mesenchymal stem cell-derived magnetic extracellular nanovesicles for targeting and treatment of ischemic stroke enhancing extracellular vesicles for therapeutic treatment of arthritic joints nanomaterials for the diagnosis and treatment of inflammatory arthritis biogenesis and function of t cell-derived exosomes mesenchymal stem cell-derived extracellular vesicles as therapeutics and as a drug delivery platform the biology, function, and biomedical applications of exosomes challenges and opportunities in exosome research-perspectives from biology, engineering, and cancer therapy exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in danio rerio therapeutic biomaterials based on extracellular vesicles: classification of bio-engineering and mimetic preparation routes exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools exosomes: biogenesis, biologic function and clinical potential naturally occurring exosome vesicles as potential delivery vehicle for bioactive compounds fully artificial exosomes: towards new theranostic biomaterials exosomes and exosome-inspired vesicles for targeted drug delivery generation of nanovesicles with sliced cellular membrane fragments for exogenous material delivery exosome: a significant nano-scale drug delivery carrier exosomes from marrow stromal cells expressing mir-146b inhibit glioma growth exosomes derived from mir-122-modified adipose tissue-derived mscs increase chemosensitivity of hepatocellular carcinoma employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (ev)-encapsulated microrna-379 paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery targeted doxorubicin-loaded mesenchymal stem cells-derived exosomes as a versatile platform for fighting against colorectal cancer curcumin-primed and curcumin-loaded exosomes: potential neural therapy exosome-based cell-cell communication in the tumor microenvironment hucmsc-exosome mediated-wnt4 signaling is required for cutaneous wound healing systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats liposome: classification, preparation, and applications controlled drug delivery vehicles for cancer treatment and their performance nano based drug delivery systems: recent developments and future prospects engineering smart targeting nanovesicles and their combination with hydrogels for controlled drug delivery pegylation as a strategy for improving nanoparticle-based drug and gene delivery engineering hybrid exosomes by membrane fusion with liposomes extracellular vesicles as an efficient and versatile system for drug delivery stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential exoplexs: chimeric drug delivery platform from the fusion of cell-derived nanovesicles and liposomes macrophagederived exosome-mimetic hybrid vesicles for tumor targeted drug delivery mesenchymal stem cells engineered for cancer therapy mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression chemokines and their receptors: predictors of the therapeutic potential of mesenchymal stromal cells mesenchymal stem cell-based immunomodulation: properties and clinical application shattering barriers toward clinically meaningful msc therapies the current status of mesenchymal stromal cells: controversies, unresolved issues and some promising solutions to improve their therapeutic efficacy improved therapeutics of modified mesenchymal stem cells: an update coating nanoparticles with cell membranes for targeted drug delivery the evolution of commercial drug delivery technologies microfluidic fabrication of cell-derived nanovesicles as endogenous rna carriers preparation of engineered extracellular vesicles derived from human umbilical cord mesenchymal stem cells with ultrasonication for skin rejuvenation large-scale generation of cell-derived nanovesicles msc-derived exosome promotes m2 polarization and enhances cutaneous wound healing exosomes/microvesicles from induced pluripotent stem cells deliver cardioprotective mirnas and prevent cardiomyocyte apoptosis in the ischemic myocardium therapeutic application of extracellular vesicles in kidney disease: promises and challenges exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension trail delivery by msc-derived extracellular vesicles is an effective anticancer therapy this work was supported by the basic science research program through the national research foundation of korea (nrf-2019m3a9h1103690), by the gachon university gil medical center (frd2021-03), and by the gachon university research fund of 2020 (ggu-202008430004). the authors report no conflicts of interest in this work. the international journal of nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. this journal is indexed on pubmed central, medline, cas, scisearch ® , current contents ® /clinical medicine, journal citation reports/science edition, embase, scopus and the elsevier bibliographic databases. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 7 evaluation of rosuvastatin effect as adjuvant therapy to methotrexate on lipid profile and the possibility of its cardioprotective effect in iraqi patients with active rheumatoid arthritis ehab m. mikhael *,1 , ibrahim a. majeed * , faiq i. gorial ** , mohammad h. al-ossamy ** , ali h. falih ** and dhikra abdulhameed *** * department of clinical pharmacy , college of pharmacy, university of baghdad, baghdad, iraq. ** rheumatology unit, college of medicine, university of baghdad, baghdad, iraq. *** teaching laboratories, baghdad teaching hospital. abstract rheumatoid arthritis (ra) is a common inflammatory disease that associated with increased morbidity and mortality due to accelerated atherosclerosis. rosuvastatin is a unique hydroxy methyl glutaryl co a (hmgcoa) reductase inhibitor that has anti inflammatory effects. the aim of this study was to evaluate the effect of rosuvastatin as adjuvant therapy to methotrexate (mtx) on lipid profile and its possible cardioprotective effect in ra patients. a double blinded placebo controlled clinical trial with 8 weeks follow up periods at which 40 patients with active ra using mtx were randomized into 2 groups to receive either rosuvastatin 10mg or placebo as adjuvant therapy to mtx. in addition to twenty healthy subjects as control group. lipid profile and erythrocyte sedimentation rate (esr) was assessed at the start and at the end of the study. at the start of the study total cholesterol (tc), low density lipoprotein cholesterol (ldlc) and high density lipoprotein cholesterol (hdlc) values were not significantly different between ra patients and control group. at the end of the study rosuvastatin significantly reduced esr, tc and ldlc after 8 weeks of treatment. it can be concluded that mtx has the ability to normalize lipid profile in ra patients. rosuvastatin effectively reduce esr, tc and ldlc; moreover, rosuvastatin might have a possible cardioprotective effect in ra patients. keywords: active rheumatoid arthritis, methotrexate, rosuvastatin. عهً انذهىن وجأثُره انقهبٍ انىقائٍ كعالج مضاف نهمُثىجركسُث جأثُر انروسىفاسحاجُهجقُُم انفعال صابُه بانحهاب انمفاصم انرثىٌانمححمم نهمرضً انعراقُُه انم اَهاب مضر مُخائُم *،1 فائق اَشى كىلاير ، ** ابراهُم ادهم مجُذ ، * انعصامٍ محمذ هادٌ ، ** ، عهٍ حسُه فانح ** ركري عبذ انحمُذ و *** * . انؼشاق ،تغذاد ،خايؼح تغذاد،كهٛح انصٛذنح ،فشع انصٛذنح انسشٚشٚح ** . انؼشاق ،تغذاد ،خايؼح تغذاد ،كهٛح انطة ،انشثٕٚح انًفاصم ٔحذج *** . يسرشفٗ تغذاد انرؼهًٛٙ ،انًخرثشاخ انرؼهًٛٛح الخالصة انرٓاب انًفاصم انشثٕ٘ ْٕ يشض انرٓاتٙ شائغ ٔٚرًٛض تضٚادج َسثح انًشاظح ٔانٕفٛاخ تسثة صٚادج سشػح ذصهة األٔػٛح كًا إٌ نّ خاصٛح يعادج (hmgcoa)انذيٕٚح . ٚؼرثش انشٔسٕفاسراذٍٛ يثثطا فشٚذا إلَضٚى ْاٚذسٔكسٙ يثٛم كهٕذاسٚم كٕ ا٘ نالنرٓاب. اٌ انٓذف يٍ ْزِ انذساسح ْٕ نرقٛٛى فائذج انشٔسٕفاسراذٍٛ كؼالج إظافٙ نهًٛثٕذشكسٛد ػهٗ انذٌْٕ ٔذأثٛشِ انًحرًم نٕقاٚح انقهة ػُذ صم انشثٕ٘ انفؼال ٔانزٍٚ يشظٗ انرٓاب انًفاصم انشثٕ٘ انفؼال.انثحث سشٚش٘ يضدٔج اإلػًاء شًم أستؼٌٕ يشٚعا تانرٓاب انًفا يهغشاو أٔ انؼالج 01ٚسرخذيٌٕ نهًٛثٕذشكسٛد ذًد يشاقثرٓى نًذج أساتٛغ تؼذ ذقسًٛٓى ػشٕائٛا نًدًٕػرٍٛ السرؼًال سٔسٕفاسراذٍٛ انٕاْى. تاإلظافح إنٗ ػششٍٚ شخصا كًدًٕػح يقاسَح. الَرٓاء يٍ ْزِ انذساسح. ػُذ تذء ْزِ انذساسح ذثٍٛ إٌ َسثح ذى قٛاط َسثح انذٌْٕ َٔسثح ذشسٛة كشٚاخ انذو انحًش ػُذ انثذء ٔػُذ ا انكٕنسرشٔل ٔانكٕنسرشٔل يُخفط ٔيشذفغ انكثافح الذخرهف تٍٛ انًشظٗ انًصاتٍٛ تانرٓاب انًفاصم انشٔياذٕٚذ٘ ػٍ َظشائٓى ة كشٚاخ انذو انحًش ٔانكٕنسرشٔل, تانًدًٕػح انًقاسَح ٔػُذ اَرٓاء انذساسح قهم انشٔسٕفاسراذٍٛ ٔتشكم يؼُٕ٘ يهحٕظ يٍ َسثح ذشس ٔانثشٔذٍٛ انًشذثط تانكٕنسرشٔل يُخفط انكثافح. َسرُرح يٍ رنك إٌ نهًٛثٕذشكسٛد قاتهٛح ذؼذٚم يسرٕٖ انذٌْٕ ػُذ يشظٗ انرٓاب انًفاصم انشثٕ٘ انفؼال ٔانشٔسٕفاسراذٍٛ نّ انقاتهٛح ذٍٛ انًشذثط تانكٕنسرشٔل يُخفط انكثافح إظافح إنٗ ذأثٛشِ انٕقائٙ ػهٗ ذقهٛم يسرٕٖ ذشسة كشٚاخ انذو انحًش, انكٕنسرشٔل, ٔانثشٔ انًحرًم نهقهة ػُذ يشظٗ انرٓاب انًفاصم انشثٕ٘ انفؼال. انكهمات انمفحاحُة: انحهاب انمفاصم انرثىٌ انفعال, مُثىجركسُث, روسىفاسحاجُه. 1 corresponding author e-mail: ehab_pharma84@yahoo.com received:3 /11/2012 accepted:21 /9/2013 iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 8 introduction rheumatoid arthritis (ra) is a chronic systemic inflammatory disease of unknown etiology characterized by articular and extra articular features (1) . the atherogenic lipid profile and subclinical atherosclerosis are features of early ra (2). rheumatoid arthritis is associated with increased mortality, which is predominantly due to accelerated coronary artery atherosclerosis (3) . cardiovascular (cv) morbidity and mortality are increased twofold in ra patients compared to those of the general population (4, 5) . this increased cv risk may be due to systemic inflammation and its interplay with traditional cv risk factors like smoking, personal and family history of ischemic heart disease, hypertension, hyperlipidemia, higher body mass index and diabetes mellitus (dm) (6) . the association between lipids and cv risk in ra appears to be more complex than in the general population, with systemic inflammation being a notable contributor to the lipid profile changes (7) . inflammation leads to pro-atherogenic changes of the lipoprotein metabolism and an increased disease activity is associated with lower total cholesterol (tc) levels and even more depressed high density lipoprotein – cholesterol (hdlc) levels and lowered apolipoprotein-a1 (apo-a1) levels (4). beside that active inflammation increases oxidized fatty acids in circulating lipoproteins, promoting low density lipoprotein (ldl) oxidation and hdl dysfunction, thereby increasing atherosclerotic risk (8) . rosuvastatin is a unique hydroxy methyl glutaryl coa (hmgcoa) reductase inhibitor that used to treat dyslipidemia (9) . it also exerts important anti-inflammatory effects in addition to its lipid-lowering actions (10) . aim of the study evaluating the effect of rosuvastatin as adjuvant therapy to mtx on lipid profile and its possible cardioprotective effect in iraqi patients with active ra. methods study design this was an 8-week randomized double blind placebo-controlled single center study conducted at rheumatology unit, baghdad teaching hospital, baghdad, iraq from august 2011 till may 2012. patients were randomly allocated to receive each day either rosuvastatin 10mg tablet or capsule prefilled with glucose as placebo (pbo). rosuvastatin was bought from unipharma company/ syria whereas glucose was bought from sdi/ iraq. methotrexate ampoules from ebewe company/ austria. patients were evaluated at baseline and at week 8. sample selection eligible patients had confirmed to have active ra according to the 1987 american college of rheumatology (acr) criteria and had esr values greater than 20 mm/hr for men and greater than 30 for females(11). additionally all patients included were selected to be users of methotrexate (mtx) regularly for at least 3 previous consecutive months. the exclusion criteria included patients who were taking lipid-lowering therapy, had hypersensitivity to statin, pregnancy, breast feeding, renal and liver impairment, patients younger than 18 years old and those using steroids. additionally 20 healthy individuals with age and sex matched were considered as a control group. informed consent was obtained from all participants and this study was approved by the ethical committee of baghdad university, college of medicine medical department. blood sample collection and laboratory evaluation blood specimen collection: 5 ml of venous blood was obtained from forearm for doing laboratory analysis (at baseline and after 8 weeks). 3 ml was transferred to plane test tube and left to be coagulated then centrifuged at 3000 rpm for 10 minutes and then serum obtained for biochemical measurements of total cholesterol, ldlc, hdlc, and triglyceride (tg) and measuring rheumatoid factor. lipid profile tests were done by specialized laboratory workers who did not participate in this study using specialized kits from randox ® company. total cholesterol was measured by cholesterol oxidase peroxidase aminophenazone (chod-pap) enzymatic colorimetric method (12) . tg estimation was done by glycerol-3 phosphate oxidase – peroxidase (gpo-pap) enzymatic colorimetric method under the principle of bucolo and david 1973 (13) .hdlc was measured by polyethylene glycol (peg) / chod – pap method , according to the principle of lopes (14) . whereas ldlc level was estimated according to the friedewald formula (15) . esr was measured by westergren method and also was done by blinded non interested laboratory worker (16) .rheumatoid factor was measured qualitatively just at start of study by serological agglutination test through antigen antibody reaction using specialized kit from spectrum company/ egypt (17) . iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 9 statistical analysis statistical package for social sciences (spss) version 12, was used for data input and analysis. continuous variables were presented as mean ± standard deviation (sd) and discrete variables were presented as numbers and frequencies. chi square test for independence was used to test the significance of association between discrete variables. continuous variables were tested by a web version of shapiro wilk test to determine if they were normally or abnormally distributed. analysis of variance (anova) test was used to test the significance of difference in the mean of 3 independent samples in normally distributed continuous variables. paired t test was used to test the significance of difference in means of pre and post treatment in normally distributed continuous variables, whereas wilcoxon test was used in case of abnormally distributed continuous variables. unpaired t test was used to test the significance of difference in the mean of two independent samples in normally distributed continuous variables and mann whitney test for abnormally distributed data. all p values used were asymptotic and two sided. findings with p value less than 0.05 were considered significant whereas p values less than 0.01 considered highly significant. statistical power was not calculated since it is a pilot study. results of the 74 patients who were randomized in this double-blind study, only 40 patients completed the 8weeks of treatment (20 from the rosuvastatin group and 20 from the pbo). the two groups did not differ significantly in baseline characteristics. another 20 healthy controls aged and sex matched were also participated (figure1, table 1). baseline lipid profile (table 2) showed that there was a non significant difference at baseline level of tc, ldlc and hdlc between ra patients and control subjects and only tg level was significantly higher in ra patients of pbo group than that in the control group. but unfortunately there was a significant difference at a baseline level of tc, ldlc, and tg between rosuvastatin and pbo group (table 3). additionally baseline esr was higher in ra patients, but with no any significant difference between rosuvastatin and placebo group (table 2, 3). after 8 weeks of starting adjuvant treatment with either rosuvastatin or placebo, tc, ldlc and esr decreased significantly by rosuvastatin (p<0.05) while other parameters showed no any difference between the effect of rosuvastatin and placebo (p>0.05, table 4). figure 1: schematic presentation for patient participating in the study iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 10 table ( 1) demographic data and baseline characteristics of both rheumatoid patients and control subjects parameter rosuvastatin pbo control p value age ( years) 43.35 ± 9.96 44.4 ±13.53 42.95±10.39 0.917 female: male ratio 14:6 (70%) 16:4 (80%) 15:5(75%) 0.766 dose of mtx 13.88± 4.40 13.25 ± 3.54 0.624 smoking 3 2 5 0.431 family hx of cvd n (%) 3 (15%) 2 (10%) 3 (15%) 0.865 hypertension n (%) 5 (25%) 5 (25%) 1 dm n (%) 5 (25%) 3 (15%) 0.429 positive rf n (%) 13 (65%) 12 (60%) 0.743 table (2) comparison in baseline laboratory data of rheumatoid arthritis patients with control subjects parm rosuva control p pbo control p tc (mg/dl) 168.9±38.22 180.05±54.29 0.457 202.85±28.68 180.05±54.29 0.105 ldlc (mg/dl) 99.71±33.24 114.78±52.46 0.285 123.21±19.57 114.78±52.46 0.505 hdlc (mg/dl) 42.25±9.42 43.45±6.06 0.635 43.6±10.34 43.45±6.06 0.956 tg (mg/dl) 134.7±63.91 109.1±43.80 0.148 180.2±51.16 109.1±43.80 0.000 esr (mm/hr) 48.95± 31.1 11.7±3.85 0.000 38.25±19.00 11.7±3.85 0.000 tc = total cholesterol, normal range < 200mg/dl; ldlc = low density lipoprotein cholesterol, normal range < 100mg/dl; hdlc = high density lipoprotein cholesterol, normal range 40 – 60mg/dl; tg = triglyceride , normal range < 150mg/dl; esr = erythrocyte sedimentation rate, normal range < 20 for men and < 30 for females. table (3) difference in baseline laboratory parameters between ra patients in rosuvastatin and placebo group parameter rosuva pbo p tc 168.9±38.22 202.85±28.68 0.003 ldlc 99.71±33.24 123.21±19.57 0.01 hdlc 42.25±9.42 43.6±10.34 0.669 tg 134.7±63.91 180.2±51.16 0.017 esr 48.95± 31.1 38.25±19.00 0.197 table (4) change produced in lipid profile and esr after 8 weeks of treatment with either rosuvastatin or placebo parameter rosuvastatin (%) pbo (%) p value tc -39.3 ± 29.21 (-23.27%) -6.25 ± 17.99 (-3.08%) 0.000 ldlc -36.28 ± 27.10 (-36.39%) -8.49 ± 21.61 (-6.89%) 0.001 hdlc 0.7 ± 8.71 (1.66%) 0.4 ± 4.28 (0.92%) 0.956 tg -18.6 ± 33.11 (-13.81%) 9.2 ± 53.97 (5.11%) 0.096 esr -16.85 ± 28.66 (34.42%) -0.55 ± 17.72 (-1.44) 0.012 iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 11 discussion the results of this study showed a non significant difference in tc, ldl-c and hdlc level between ra patients using mtx and control subjects; the results of other studies were controversial: in one study it was found that both tc and ldl-c level were elevated in ra patients whereas hdl-c level was decreased in patients with early ra, and this atherogenic lipid profile can be improved by initiation of therapy (18) . whereas other studies found that systemic inflammation was a notable contributor to the lipid profile changes. growing evidences suggested that patients with active untreated ra have reduced tc, ldl-c and hdl-c levels (7,19) ; and these abnormal lipid profiles were improved through suppression of inflammation by many disease modifying anti rheumatic drugs (dmards) (20) . any how what ever the baseline level of lipid profile in iraqi patients with ra, it seemed that the use of mtx could improve it. the results of this study showed that there was a significant difference in the baseline level of both tc and ldlc between rosuvastatin and placebo group, which may be resulted by chance in such a randomized controlled trial of small sample size. results of this study showed that rosuvastatin produced a significant reduction in both tc and ldlc level; similar finding was reported in many other studies, comparing the effect of different statins, rosuvastatin (21) , atorvastatin (22) and simvastatin (23) to placebo in patients with ra. rosuvastatin reduced tc by more than 23% and ldl-c by more than 36% which was close to that found in uk survey for use of rosuvastatin in general practice (–28% for cholesterol and – 40% for ldl-c ) (24) . such result can be explained according to the fact that rosuvastatin is one of the hmg coa reductase inhibitors which lower plasma cholesterol due to the inhibition of endogenous cholesterol synthesis in the liver, and subsequently increased expression of ldl receptors, resulting in an up-regulated catabolic rate for plasma ldl (25). moreover, the results showed that rosuvastatin was unable to significantly increase hdl-c despite its greater effect when compared to placebo; similar finding was observed in tara study, at which 40mg atorvastatin failed to improve hdl-c (22) ; however, rosuvastatin 10mg was sufficient to increase hdl-c significantly in ra patients after 1 year of therapy (21) , this mean that short duration of follow up period in this study may be a limiting factor in achieving a real result regarding the effect on hdl-c. the results of the current study showed that tg level was higher in active ra patients than those in the control group, and only those in placebo group were significantly higher than control group; similar finding was observed in patients with early active ra by georgiadis et al (18) . limitation in the current study may be related to the significant difference in baseline level of tg between patients in placebo and rosuvastatin group which may be caused by the small sample size; any how only rosuvastatin significantly reduced tg from baseline level, this effect was not statistically significant when compared to that of placebo; similarly the use of low dose rosuvastatin in patients with mildly active ra failed to achieve significant reduction in tg level (21) . the absence of clinically significant effect on tg level may be attributed to the low dose of rosuvastatin that used in the current study, since ooi et al. found that rosuvastatin effect to reduce tg level was dose dependent (26) . regarding esr which is sensitive for most types of inflammation, but cannot distinguish if the underlying cause is infectious, inflammatory, or paraneoplastic (27) , however it provides a reliable means for discrimination between drugs that provide symptomatic relief only and those with a more profound effect in ra (28). the results of this study showed that esr level in ra patients who participated in this study was significantly higher than that in healthy control subjects, which was similar to the finding in the study of yildirim k et al. (29) since we included only patients with high esr level. more importantly, rosuvastatin (but not placebo) significantly reduced esr level; similarly, in two other studies 40mg atorvastatin have the ability to reduce esr significantly (22,30) . however, a study regarding the effect of 10mg rosuvastatin showed no any benefit, which may be explained in that patients who participated in that study had low initial esr level since they have just mildly active ra disease (21) , whereas this study excluded any patient with mild or inactive ra disease who had low esr values ( less than 20 mm/hr ). myasoedova et al found that inflammatory measures (particularly esr) were significantly associated with the risk of cvd in ra (31) . in addition, it was found that controlling both ra disease activity and dyslipidemia is mandatory for minimizing the cardiac risk in ra patients (32). iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 12 this study showed that rosuvastatin significantly reduced both traditional (tc and ldlc) and non traditional (esr) risk factors for cvd in ra patients which agreed with the recent eular recommendation for using statins for cardiovascular risk management in ra patients (33). conclusions methotrexate has the ability to normalize lipid profile in ra patients. rosuvastatin effectively reduce esr, tc and ldlc with little effect on tg and hdlc in ra patients; moreover, rosuvastatin might have a possible cardioprotective effect in ra patients. reference 1. manole cojocaru, inimioara mihaela cojocaru, isabela silosi, camelia doina vrabie, and r tanasescu . extra-articular manifestations in rheumatoid arthritis: maedica (buchar) 2010; 5(4): 286–91. 2. georgiadis an, voulgari pv, argyropoulou mi, et al. early treatment reduces the cardiovascular risk factors in newly diagnosed rheumatoid arthritis patients. semin arthritis rheum 2008; 38(1):13-9. 3. kaplan mj. cardiovascular disease in rheumatoid arthritis. curr opin rheumatol 2006; 18:289-97. 4. nurmohamed mt. atherogenic lipid profiles and its management in patients with rheumatoid arthritis. vasc health risk manag 2007; 3:845-52. 5. nicola pj, maradit-kremers h, roger vl, et al. the risk of congestive heart failure in rheumatoid arthritis: a population-based study over 46 years. arthritis rheum 2005; 52:412-20. 6. kitas gd, gabriel se. cardiovascular disease in rheumatoid arthritis: state of the art and future perspectives: ann rheum dis. 2011 jan;70(1):8-14. 7. choy e, sattar n. interpreting lipid levels in the context of high-grade inflammatory states with a focus on rheumatoid arthritis: a challenge to conventional: cardiovascular risk actions. ann rheum dis 2009; 68:4609. 8. christina cs, david m, yuen yl. et al. oxidation products of arachidonic acid and linoleic acid are increased in high density lipoprotein and low density lipoprotein from patients with active rheumatoid arthritis. arthritis rheum 2011; 63(suppl. 10):763. 9. white cm. a review of the pharmacologic and pharmacokinetic aspects of rosuvastatin. j clin pharmacol 2002; 42:963-70. 10. timothy j stalker, allan m lefer, and rosario scalia. a new hmg-coa reductase inhibitor, rosuvastatin, exerts anti-inflammatory effects on the microvascular endothelium: the role of mevalonic acid: br j pharmacol. 2001 june; 133(3): 406–12. 11. joseph t d, chisholm-burns ma, et al. pharmacotherapy: principle and practice, 1 st edn., mcgraw-hill; 2008:867-79. 12. allain cc, poon is, chan c h g, et al. enzymatic determination of serum total cholesterol. clinchem. 1974; 20: 470-1. 13. bucolo, g. and h. david. quantitative determination of serum triglycerides by the use of enzymes. clinchem. 1973;19: 47682. 14. lopes vmf, stone p, ellis s, colwell ja. cholesterol determination in high density lipoprotein separated by three different methods. clinchem 1977; 23:882-4. 15. friedwall vt, levy ri, fredrickson ds. estimation of low density lipoprotein cholesterol in plasma, without use of preprative centrifuge. clin chem. 1972; 18: 499. 16. j. m. jou, s. m. lewis, et al. review of the measurement of the erythrocyte sedimentation rate: international journal of laboratory hematology 2011; 33(2): 125– 32. 17. singer, j.m. c.m., plotz, e. parker & s.k. elster. amer. j. clin. path 1957;28: 611. 18. georgiadis an, papavasiliou ec, lourida es, et al. atherogenic lipid profile is a feature characteristic of patients with early rheumatoid arthritis: effect of early treatment – a prospective, controlled study: arthritis res ther. 2006;8(3):r82. epub 2006 apr 28. 19. lazarevic mb, vitic j, mladenovic v, et al. dyslipoproteinemia in the course of active rheumatoid arthritis. semin arthritis rheum. 1992;22(3):172-8. 20. steiner g, urowitz mb. lipid profiles in patients with rheumatoid arthritis: mechanisms and the impact of treatment. semin arthritis rheum. 2009 apr;38(5):372-81. 21. tam ls, li ek, shang q, et al. effects of rosuvastatin on subclinical atherosclerosis and arterial stiffness in rheumatoid arthritis: a randomized controlled pilot trial. scand j rheumatol 2011; 40(6):411-21. 22. mccarey dw, mcinnes ib, madhok r, et al. trial of atorvastatin in rheumatoid arthritis (tara): double-blind, randomized placebo-controlled trial. lancet 2004; 363(9426):2015-21. http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bm%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bim%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bim%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20silosi%2bi%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20vrabie%2bcd%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20vrabie%2bcd%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20tanasescu%2br%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed?term=georgiadis%20an%5bauthor%5d&cauthor=true&cauthor_uid=18191989 http://www.ncbi.nlm.nih.gov/pubmed?term=voulgari%20pv%5bauthor%5d&cauthor=true&cauthor_uid=18191989 http://www.ncbi.nlm.nih.gov/pubmed?term=argyropoulou%20mi%5bauthor%5d&cauthor=true&cauthor_uid=18191989 http://www.ncbi.nlm.nih.gov/pubmed?term=athanasios%20n%20g%2c%20paraskevi%20v%20v.%20early%20treatment%20reduces%20the%20cardiovascular%20risk%20factors%20in%20newly%20diagnosed%20rheumatoid%20arthritis%20patients http://www.ncbi.nlm.nih.gov/pubmed?term=kitas%20gd%5bauthor%5d&cauthor=true&cauthor_uid=21109513 http://www.ncbi.nlm.nih.gov/pubmed?term=gabriel%20se%5bauthor%5d&cauthor=true&cauthor_uid=21109513 http://www.ncbi.nlm.nih.gov/pubmed/21109513## http://www.ncbi.nlm.nih.gov/pubmed/21109513## http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20stalker%2btj%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20lefer%2bam%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20scalia%2br%5bauth%5d http://onlinelibrary.wiley.com/doi/10.1111/ijlh.2011.33.issue-2/issuetoc http://www.ncbi.nlm.nih.gov/pubmed?term=georgiadis%20an%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed?term=papavasiliou%20ec%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed?term=lourida%20es%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed?term=lourida%20es%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed/16646989## http://www.ncbi.nlm.nih.gov/pubmed/1295090## http://www.ncbi.nlm.nih.gov/pubmed/1295090## http://www.ncbi.nlm.nih.gov/pubmed/18395771## http://www.ncbi.nlm.nih.gov/pubmed?term=%22tam%20ls%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22li%20ek%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22shang%20q%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=effects%20of%20rosuvastatin%20on%20subclinical%20atherosclerosis%20and%20arterial%20stiffness%20in%20rheumatoid%20arthritis%3a%20a%20randomized%20controlled%20pilot%20trial%20%3a%20scand%20j%20rheumatol%202011 http://www.ncbi.nlm.nih.gov/pubmed?term=effects%20of%20rosuvastatin%20on%20subclinical%20atherosclerosis%20and%20arterial%20stiffness%20in%20rheumatoid%20arthritis%3a%20a%20randomized%20controlled%20pilot%20trial%20%3a%20scand%20j%20rheumatol%202011 iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 13 23. kanda h, yokota k, kohno c, et al. effects of low-dosage simvastatin on rheumatoid arthritis through reduction of th1/th2 and cd4/cd8 ratios. mod rheumatol 2007; 17(5):364-8. 24. george k, john r, cathy e, et al. a uk survey of rosuvastatin in general practice: reaching cholesterol targets: br j cardiol 2008;15(2):95-100. 25. igel m, sudhop t, von bergmann k .pharmacology of 3-hydroxy-3methylglutaryl-coenzyme a reductase inhibitors (statins), including rosuvastatin and pitavastatin. j clin pharmacol 2002; 42(8): 835-45. 26. ooi em, watt gf, nestel pj, et al. dosedependent regulation of high-density lipoprotein metabolism with rosuvastatin in the metabolic syndrome . j clin endocrinol metab. 2008;93(2):430-7. 27. bridgen m. the erythrocyte sedimentation rate. still a helpful test when used judiciously. postgrad med. 1998;103(5):257-62. 28. amos rs, constable tj, crockson ra, et al. rheumatoid arthritis: relation of serum c-reactive protein and erythrocyte sedimentation rates to radiographic changes. br med j. 1977; 1(6055): 195–7. 29. yildirim k, karatay s, melikoglu ma, et al. associations between acute phase reactant levels and disease activity score (das28) in patients with rheumatoid arthritis. ann clin lab sci. 2004; 34(4):423-6. 30. ljung, lotta, leirisalo-repo, et al. improvement of cardiovascular risk markers with atorvastatin treatment in rheumatoid arthritis. arthritis rheum 2009; 60 suppl 10: 430. 31. myasoedova e, crowson cs, kremers hm, et al. lipid paradox in rheumatoid arthritis: the impact of serum lipid measures and systemic inflammation on the risk of cardiovascular disease: ann rheum dis 2011; 70(3):482–7. 32. khaled amer, ahmed m. ibrahim, hosni a. younis and mohamed m. ahmed. evaluation of cardiac changes in hyperlipidaemic rheumatoid arthritis patients. journal of american science 2012; 8(3):517-22. 33. peters mj, symmons dp, mccarey d, et al. eular evidence-based recommendations for cardiovascular risk management in patients with rheumatoid arthritis and other forms of inflammatory arthritis. ann rheum dis. 2010;69(2):32531. http://www.ncbi.nlm.nih.gov/pubmed?term=%22kanda%20h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22yokota%20k%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22kohno%20c%22%5bauthor%5d http://jcp.sagepub.com/search?author1=m+igel&sortspec=date&submit=submit http://jcp.sagepub.com/search?author1=t+sudhop&sortspec=date&submit=submit http://jcp.sagepub.com/search?author1=k+von+bergmann&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed/18029469## http://www.ncbi.nlm.nih.gov/pubmed/18029469## http://www.ncbi.nlm.nih.gov/pubmed/9590999## http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20amos%2brs%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20constable%2btj%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20crockson%2bra%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed?term=yildirim%20k%5bauthor%5d&cauthor=true&cauthor_uid=15648784 http://www.ncbi.nlm.nih.gov/pubmed?term=karatay%20s%5bauthor%5d&cauthor=true&cauthor_uid=15648784 http://www.ncbi.nlm.nih.gov/pubmed?term=melikoglu%20ma%5bauthor%5d&cauthor=true&cauthor_uid=15648784 http://www.ncbi.nlm.nih.gov/pubmed/15648784## http://www.ncbi.nlm.nih.gov/pubmed?term=myasoedova%20e%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=crowson%20cs%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=kremers%20hm%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=peters%20mj%5bauthor%5d&cauthor=true&cauthor_uid=19773290 http://www.ncbi.nlm.nih.gov/pubmed?term=symmons%20dp%5bauthor%5d&cauthor=true&cauthor_uid=19773290 http://www.ncbi.nlm.nih.gov/pubmed?term=mccarey%20d%5bauthor%5d&cauthor=true&cauthor_uid=19773290 http://www.ncbi.nlm.nih.gov/pubmed/19773290## microsoft word 1.doc correlation between some immunological drs nahida , eman , samira and ali parameters and clinical presentation in ra patients j fac med baghdad vol. 50, no.2, 2008 235 correlation between some immunological parameters and clinical presentation in ra patients nahida r. abbas* msc eman sh. alobeidy**m.sc samira n. alnaim***msc ali e. kadim*"*bsc summary: background: complement (c) & crp in patients with rheumatoid arthritis (ra)'could be trigger disease activity. aim: to study the correlation between c, crp, igm,, igg, iga & some clinical presentation in ra patients. methods: latex agglutination test(ag) & single radial immunoassay(sria) were used to asses crp, igmjggjga,, c, in 74 patients with ra. results: iga & igg were significantly increased, while the mean ofc3, c4 were slightly elevated in ra patients . conclusion: there is correlation between igg and igm with joint deformity and joint swelling respectively, while c3 was showed statistically significant p<0.01 with joint stiffness, joint swelling and rheumatoid nodule, where as crp was statistically significant p<0.01withjoint swelling.. key words : ra.c.crp, igm, igg, iga, sri a, ag. introduction: rheumatoid arthritis (ra) is one of the mysterious autoimmune diseases which is still unresolved characterized by inflammation of synovial membrane, principally affecting peripheral joints in a symmetric fashion, commonly leads to cartilage destruction, bone erosion and joint deformities; extra-articular manifestations such as vasculitis and subcutaneous nodules can also occur, hence it's course is quite variable (1 -3). *msc \ immunology,institute of medical technology/al-mansor. ** msc \ immunology, medical city, ***msc \ microbiology,institute of medical technology/al-mansor. ****bsc \ microbiology,institute of medical technology/al-mansor the pathogenesis of ra is incompletely understood ,although there is evidence that cytokines such as tumor necrosis factor a & interleukin-1 are involved (4). furthermore, there is substantial evidence that the complement system(c) is also involved in the pathogenesis of ra(5-9),increased level of c products in the serum & synovial fluid of ra patients (7,8) and correlate with disease activity(9).there are many factors that trigger complement activation in ra patients ,one of these may be c-reactive protein(crp), since this acute phase protein can activate c. original article fac med baghdad 2008; vol.50, no.2 received july 2006 accepted jun.2007 correlation between some immunological drs nahida , eman , samira and ali parameters and clinical presentation in ra patients j fac med baghdad vol. 50, no.2, 2008 236 patients & methods patients: seventy four patients (19 male, 55 female) with ra who met the american college of rheumatology (acr)1987revised criteria (10) attending the rheumatology consultation clinic or admitted to baghdad teaching hospital in a period between november 2001 and february 2002 . fifty patients clinically diagnosed with sle according to acr criteria 1997 for classification of sle (11), thirty healthy individuals chosen from blood bank donors, who have no history or clinical evidence of ra or any chronic disease as a control groups .they were age and sex matched. laboratory investigation: serum c3&c4 complement components & igm, igg, iga were measured using the single radial immunoassay method, and results were expressed in mg / dl ,while crp was detected by latex agglutination test and results were expressed in mg / l . results from 74 ra patients studied , the ratio male to female was 1:2.9 with mean age 42.1±11.syr (range 18-67 )as shown in table 1&2. table 1: age distribution of studied groups. ra sle healthy control age in years n % n % n % <20 1 1.4 4 8.0 2 6.7" 20-29 9 12.2 18 36.0 6 20.0 30-39 23 31.0 17 34.0 13 43.3 40-49 19 25.7 10 20.0 7 23.3 50-59 16 21.6 1 2.0 2 6.7 60+ 6 8.1 total 74 100.0 50 100.0 30 100.0 range 18-67 9-59 18-56 mean 42.1 30.6 33.8 . sd 11.3 10.1 9.4 p (anova) < 0.001 correlation between some immunological drs nahida , eman , samira and ali parameters and clinical presentation in ra patients j fac med baghdad vol. 50, no.2, 2008 237 . table 2: distribution of the studied groups by gender. ra sle healthy control n % n % n % gender female 55 74.3 44 88 23 76.7 male 19 25.7 6 12 7 23.3 total 74 100 50 100 30 100 total igs concentration level-s of iga & igg were significantly increased compared to healthy control (po.ool ,0.006 ) respectively while there was no difference in comparison to sle patients, where as igm level was normal in all studied groups as shown in table 3. table 3 the difference in mean serum immunoglobulin concentration (mg/d!) between studied groups. serum igs (mg/dl) ra (n=74) sle (n=50) healthy control (n=30) p(anova) serum iga <0.001 range 62.3-633.3 . 48-633.3 90-540 mean 350.9 358.3 208.8 sd 129.3 174.7 105.9 serum igg 0.006 range 643.7-2965.9 295.9-3042.3 700-1614.6 mean 1462.8 1481 1114.5 sd 515.9 728.2 282.9 serum igm .0.43wsj range 48.1-277.3 40.8-277.3 93.2-205.2 mean 151.6 140.5 146.1 * * * sd 48.9 49.9 33.1 * normal range of igs iga: 90-540 mg/dl igg: 700-1620 mg/dl igm: 50-250 mg/dl correlation between some immunological drs nahida , eman , samira and ali parameters and clinical presentation in ra patients j fac med baghdad vol. 50, no.2, 2008 238 the mean of c3, c4 were slightly elevated in ra patients as shown in table 4 in comparison with healthy control, it was significantly difference higher po.ool in comparison to sle patients group. . table 4: serum complement component concentration (mg/dl) levels in studied groups. serum complement cone, (mg/dl) ra (n=74) sle (n=50) healthy control (n=30) p (anova) serum c3 <0.001 range 35-260 ' 22.5-220 94.8-250 mean 144.5 96.6 146.6 sd 43.8 47.4 44.8 serum c4 <0.001 range 8.3-84.9 2.5-70 20-72 mean 32.6 19.5 33.1 * it sd 15.4 14.4 12.1 • normal range c3: 84-250 mg/dl c4: 20-72 mg/dl table 5 was showed that the mean of crp levels was significantly higher than in control groups po.ool. table 5: the difference in the mean of parameters between ra patients & control group. parameters ra (n=74) sle (n=50) healthy control (n=30) p (anova) c reactive protein (mg/l) <0.001 range 5-96 • 5-12 5-5 mean 17.6 ±18.4 6.4±2.5 5.0 * normal range crp> 6 mg/l comparing the presence or absence of certain clinical criteria with positivity rate of different parameters as clearly seen in table 6 was showed statistically significant p<0.05 of igg and igm with joint deformity and joint swelling respectively, while c3 was showed statistically significant po.01 with joint stiffness, joint swelling and rheumatoid nodule, where as crp was statistically significant po.01 with joint swelling while others such as iga and c4 was showed with no statistically significant with any one of these clinical criteria correlation between some immunological drs nahida , eman , samira and ali parameters and clinical presentation in ra patients j fac med baghdad vol. 50, no.2, 2008 239 discussion: in this study 19 patients were male while 55were female with ratio 1:2.9 which is comparable to other iraqi study 1:2.7 reportedby ubaid ( 12 ) and constantine 1:3.4 abroad ( 13 ) this is generally accepted to be related to sex hormones eg. estrogen . our data show that patients with ra have elevated levels of iga&jgg were similar to other study (14), possible explanation of the above data propose that high level of igg related to denaturation of igg during initiation phase, while iga concentration is proportionally associated with it's consumption in the synovium may be due to alternative pathway complement activation which confirmed by increased level of c3 in the patients sera. this explain the correlation between c3 complement component level and certain clinical features such as joint stiffness, joint swelling, and rheumatoid nodules which might be related to c. activation and increased opsonization of immune complexes by phagocytosis which is due to cellular infiltration in the synovium by the action of anaphylatoxins complement component resulting in fluid accumulation and hence swelling and stiffness, on the other hand cellular infiltration in extra-articular areas may be lead to nodules formation, these finding supported by abbink,( 15 ) which showed that there was a good contribution between c. component action and damage in arthritis. hence it is very logic to see in this study as well as abroad studies ( 16, 17 ), that increased level of serum complement components c3 & c4 were evidently noticed. assessment of serum concentration of crp has been advocated as a objective measure of disease activity in ra patients , so elevated level were found to be correlated with joint swelling, this may be due to byproduct c. activation which is again a possible reason for swelling of the joint, this is quite accord with abroad studies (17, 18). in conclusion, we found evidence of complement activation c3 in ra patients & also crp this acute phase protein should be considered an inflammation mediation in this disease. table 6: correlation between different parameters with certain clinical criteria.. parameters clinical criteria iga jgg igm c3 c4 crp joint stiffness io.ll3 0.05 0.142 0.298 ** 3.158 0.102 joint swelling 0.183 0.09 0.245 * 0.313 ** 0.077 0.385** joint deformity 0.101 0.242 * 0.017 0.162 0.019 0.15 • rheumatoid nodule 0.121 0.164 0.183 0.302 ** 0.14 0.128 joint effusion 0.199 0.105 0.101 0.086 0.063 0.079 • * correlation significant at the 0.05 level ** correlation significant at the 0.01 level correlation between some immunological drs nahida , eman , samira and ali parameters and clinical presentation in ra patients j fac med baghdad vol. 50, no.2, 2008 240 references . 1-henry jb. clinical diagnosis • and management by laboratory methods. wb saunders company. 19th ed. philadephia. 1996: pp 1019-20. 2-peakman m, vergani d. rheumatic diseases. in: basic and clinical immunology. 1997: 170-175. 3-anderson rj. rheumatoid arthritis, clinical and laboratory features. in: klippel j h, crofford l j, stone j h, ei'al. primer on the rheumatic diseases. 12th edit. atlanta georgia-arthritis foundation. 2001;218-225. t 4-fox da.cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition of tumor necrosis factor . arch intern med 2000; 160:437-44. 5brodeur jp, ruddy s, schwartz lb, moxley g. synovial fluid levels of • complement sc5 b-9 and fragment bb are elevated in patients with rheumatoid arthritis. arthritis rheum. 1991; 34:1531-7. , 6-abbink jj, kamp am, nuijens jh, eerenberg ajm, swaak ajg, hack ce. relative contribution of contact and complement activation to inflammatory reactions in arthritic joints. ann rheum dis. 1992;51:1122-8. 7-swaak ajg, van rooijen a, planten o. han h, hattink o, hack e. an analysis of the levels of complement components in the synovial fluid in rheumatic diseases. clin rheumatol. 1987; 22:350-7 8-shingu m, nagai y, isayama t, naono t, nobunaga m, nagai y. the effects of cytokines on metalloproteinase inhibitors (timp) and collagenase production by human chondrocytes and timp production by synovial cells and endothelial cells. clin explmmunol. 1993; 94:145.9. 9-makinde va, senaldi g, jaward as. berry h, vergani d. reflection of disease activity in rheumatoid arthritis by indices of activation of the classical complement pathway. ann rheum dis. 1989; 48:302-6. 10:arnett fc, edworthy sm, bloch da, mc.shane dj, fries jf, cooper ns. et al. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum 1988;31:315-24. ' 11-hochberg mc. updating the american college of rheumatology revised criteria for the classification of systemic lupus erthymatosus. arthritis and rheum. 1997; 40: (1725-1734). 12-ubaid ah: formal education level as a marker of clinical status in ra among iraqis. a thesis submitted to the college of medicine, university of baghdad for the partial fulfillment of the master degree in community medicine (1996). 13constantin a, cances vl, navaux f, et al. stromelysin 1 (mafrix metalloproteinase 3) and hla-drb1 gene polymorphismsj. arth. & rheum. 2002; 46(7): 1754-62. 14-jorgensen c, legouffe mc, bolongna c, brochier j. sany j. iga isotypes rheumatoid factor in rheumatoid arthritis: clinical implications. clin. exp. rheumatol. 1996; 14(3):301-4. i 15-abbink jj, kamp am, nuijens jh, eerenberg ajm, swaak ajg, hack ce. relative contribution of contact and complement activation to inflammatory reactions in arthritic joints. ann rheum dis. 1992;51:1122-8. . 16-makinde va, senaldi g, jaward as. berry h, vergani d. reflection of disease activity in rheumatoid arthritis by indices of activation of the classical complement pathway. ann rheum dis. 1989; 48:302-6. 17-molenaar et, voskuyl ae, familian a, vanmierlo gj, dijkmans ba, hack ce. complement activation in patients with rheumatoid arthritis mediated in part by creactive protein. arthritis rheum. 2001; 44(5):997-02. 18-wolbink gj, brouwer mc, buysman s.ten berger ijm, hack ce. crpmediated activation of complement invivo: assessment by measuring circulating complementcreactive protein complexes. j. immunol. 1996; 157:473-9. choudhari ok, et al., journal of ideas in health 2020;3(special 1):196-197 © the author(s). 2020 this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article unless otherwise stated. e issn: 2645-9248 journal homepage: www.jidhealth.com open access cotrimoxazole as adjuvant therapy in critical ill covid-19 patients omkar kalidasrao choudhari1*, sonam spalgis2, umesh chandra ojha3 dear editor, the ongoing pandemic of covid-19 has forced us to consider using available drugs in the shortfall of vaccines and established treatment. cotrimoxazole is one of the oldest drugs presently used in the prevention and treatment of opportunistic infections in the human immune deficiency virus (hiv) etc. it is a combination of two drugs trimethoprim and sulfamethoxazole. cotrimoxazole is a potent broad-spectrum antibiotic with antifungal, antiprotozoal, activity. the rationale behind the use of cotrimoxazole is its anti-inflammatory and immunomodulatory action. the mortality among the covid19 patients is mainly due to the acute respiratory distress syndrome or pulmonary embolism and respiratory failure mediated by cytokine storm due to unopposed multiplication of cascade of inflammatory mediators [1]. the immunomodulatory and anti-inflammatory activity of the cotrimoxazole is seen in many studies [2.3]. the arrow trial showed lower concentrations of plasma pro-inflammatory markers like c reactive protein (crp), interleukin 6 in continuous cotrimoxazole prophylaxis, suggesting its role as antiinflammatory and immunomodulation [3]. the role of interleukin 6 (il6) and tumour necrosis factor-alpha (tnf α) in the pathogenesis of covid-19 mortality is well documented [4]. the role of cotrimoxazole in the suppression of tnf α is also well documented [5]. lymphopenia is associated with adverse outcomes in covid-19. cotrimoxazole has shown an increase in lymphocyte count in a short and long therapy duration, but these study findings are not consistent with few other studies; however, no significant impact of cotrimoxazole was seen on immune activation of cd8 t cells [6-8]. hence it should be reserved only for critically ill patients. oxidative stress has an important aspect of the cytokine storm, which is also reduced by cotrimoxazole [9]. various side effects are mentioned in the literature. this cost-effective old drug is well tolerated among the population with the concomitant use of folic acid; moreover, it also looks after the secondary infections [7]. to conclude, cotrimoxazole can be used as critically ill covid-19 patients. abbreviations covid-19: coronavirus disease-19; hiv: human immune deficiency virus; crp: c reactive protein; tnf: tumour necrosis factor; tnf α: tumour necrosis factor-alpha; il6: interleukin 6 declarations acknowledgment none funding the author received no financial support for the research, authorship, and/or publication of this article. availability of data and materials data will be available by emailing omkarchoudhari@yahoo.com authors’ contributions omkar kalidasrao choudhari (okc) is the principal investigator of this manuscript (letter). okc, ss and uco are equally participated in the the study concept, design, writing, reviewing, editing, and approving the manuscript in its final form. all authors read and approved the final manuscript. ethics approval and consent to participate i conducted the research following the declaration of helsinki; however, letter article needs no ethics committee approval. consent for publication not applicable competing interest the author declare that he has no competing interests. open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article unless otherwise stated. author details 1post graduate resident, department of clinical biochemistry vardhman mahavir medical college (vmmc) & safdarjung hospital new delhi. 2department of respiratory medicine, vallabhbhai patel ___________________________________________________ omkarchoudhari@yahoo.com 1post graduate resident, department of clinical biochemistry vardhman mahavir medical college (vmmc) & safdarjung hospital new delhi keywords: cotrimoxazole, pandemic, covid 19 http://www.jidhealth.com/ choudhari ok, et al., journal of ideas in health 2020;3(special1):196-197 197 chest institute, new delhi. 3institute of occupational health and environmental research, basaidarapur, new delhi & department of respiratory medicine, esic pgimsr, new delhi. article info received: 13 august 2020 accepted: 27 august 2020 published: 25 september 2020 references 1. ruan q, yang k, wang w, jiang l, song j. clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intensive care med. 2020;46(5): 846–48. https://doi.org/10.1007/s00134-020-05991-x 2. rozin a, schapira d, braun-moscovici y, nahir am. cotrimoxazole treatment for rheumatoid arthritis. semin arthritis rheum. 2001;31(2):133-41. 3. bourke cd, gough ek, pimundu g, shonhai a, berejena c, terry l, et al. cotrimoxazole reduces systemic inflammation in hiv infection by altering the gut microbiome and immune activation. sci transl med. 2019;11(486): eaav0537. https://doi.org/10.1126/scitranslmed. aav0537 4. liu b, li m, zhou z, guan x, xiang y. can we use interleukin-6 (il-6) blockade for coronavirus disease 2019 (covid-19)induced cytokine release syndrome (crs)? j autoimmun. 2020; 111:102452. https://doi.org/10.1016/j.jaut.2020.102452 5. vickers ie, smikle mf. the immunomodulatory effect of antibiotics on the secretion of tumour necrosis factor alpha by peripheral blood mononuclear cells in response to stenotrophomonas maltophilia stimulation. west indian med j. 2006;55(3):138-41. https://doi.org/10.1590/s004331442006000300002 6. onyebuagu pc, kiridi k, pughikumo dt. effects of septrin administration on blood cells parameters in humans. int. j. basic appl. innov. res, 2014;3(1): 14 -8. https://www.ajol.info/index.php/ijbair/article/view/104688 7. ho jmw, juurlink dn. considerations when prescribing trimethoprim– sulfamethoxazole. cmaj. 2011; 183(16):1851-8. https://doi.org/10.1503/cmaj.111152 8. mahan cs, walusimbi m, johnson df, lancioni c, charlebois e, baseke j, et al. tuberculosis treatment in hiv infected ugandans with cd4 counts .350 cells/mm3 reduces immune activation with no effect on hiv load or cd4 count. plos one.2010;5(2): e9138. https://doi.org/10.1371/journal.pone.0009138 9. varney va, smith b, quirke g, parnell h, ratnatheepan s, bansal as, et al. p49 the effects of oral cotrimoxazole upon neutrophil and monocyte activation in patients with pulmonary fibrosis and healthy controls; does this relate to its action in idiopathic pulmonary fibrosis. thorax. 2017;72: a109. http://dx.doi.org/10.1136/thoraxjnl-2017-210983.191 key: cord-0048700-cgg4u9lv authors: kirpotina, liliya n.; schepetkin, igor a.; hammaker, deepa; kuhs, amanda; khlebnikov, andrei i.; quinn, mark t. title: therapeutic effects of tryptanthrin and tryptanthrin-6-oxime in models of rheumatoid arthritis date: 2020-07-24 journal: front pharmacol doi: 10.3389/fphar.2020.01145 sha: abcbdf17d14b2d18119791c1fccedb9d671b6cfe doc_id: 48700 cord_uid: cgg4u9lv rheumatoid arthritis (ra) is a chronic autoimmune disease involving joint and bone damage that is mediated in part by proteases and cytokines produced by synovial macrophages and fibroblast-like synoviocytes (fls). although current biological therapeutic strategies for ra have been effective in many cases, new classes of therapeutics are needed. we investigated anti-inflammatory properties of the natural alkaloid tryptanthrin (tryp) and its synthetic derivative tryptanthrin-6-oxime (tryp-ox). both tryp and tryp-ox inhibited matrix metalloproteinase (mmp)-3 gene expression in interleukin (il)-1β-stimulated primary human fls, as well as il-1β–induced secretion of mmp-1/3 by fls and synovial sw982 cells and il-6 by fls, sw982 cells, human umbilical vein endothelial cells (huvecs), and monocytic thp-1 cells, although tryp-ox was generally more effective and had no cytotoxicity in vitro. evaluation of the therapeutic potential of tryp and tryp-ox in vivo in murine arthritis models showed that both compounds significantly attenuated the development of collagen-induced arthritis (cia) and collagen-antibody–induced arthritis (caia), with comparable efficacy. collagen ii (cii)-specific antibody levels were similarly reduced in trypand tryp-ox-treated cia mice. tryp and tryp-ox also suppressed proinflammatory cytokine production by lymph node cells from cia mice, with tryp-ox being more effective in inhibiting il-17a, granulocyte-macrophage colony-stimulating factor (gm-csf), and receptor activator of nuclear factor-κb ligand (rankl). thus, even though tryp-ox generally had a better in vitro profile, possibly due to its ability to inhibit c-jun n-terminal kinase (jnk), both tryp and tryp-ox were equally effective in inhibiting the clinical symptoms and damage associated with ra. overall, tryp and/or tryp-ox may represent potential new directions for the pursuit of novel treatments for ra. rheumatoid arthritis (ra) is an autoimmune disease that involves inflammation and progressive damage to distal joints, as well as inflammation and injury to other organs of the body (firestein, 2003; sharif et al., 2018) . thus, optimal therapeutic approaches should be developed to prevent inflammation, immune system dysregulation, and bone destruction associated with this disease, while still exhibiting enhanced safety and efficacy. natural compounds have been considered as potential alternative or complementary treatments, as these compounds have been shown to possess a broader diversity in chemical space and, as a result, have significantly impacted drug development for many diseases (hong, 2011) . indeed, many natural products have been shown to exhibit potential for treatment of inflammatory diseases (lu et al., 2015) and have been evaluated in pre-clinical and clinical trials. for example, triptolide and its derivatives have been evaluated for their therapeutic effects in ra tang and zuo, 2012) . likewise, the plant-derived drug paclitaxel has been shown to inhibit collagen-induced arthritis (cia) in mice (xu et al., 2019) . tryptanthrin (tryp) (indolo[2,1-b]quinazolin-6,12-dione) is a well-known alkaloid and antibiotic that can be isolated from candida lypolica (brufani et al., 1971) , higher plants (bergman et al., 1985) , and several species of marine micro-and macroorganisms [for review (agafonova and moskovkina, 2018) ]. this compound has various pharmacological properties, including anti-inflammatory (recio et al., 2006; iwaki et al., 2011; pathania et al., 2014) , antimicrobial (honda et al., 1979) , antiviral (tsai et al., 2020) , and anti-tumor activities (kimoto et al., 2001; liao and leung, 2013) . for example, tryp has been reported to reduce leukotriene-formation in human neutrophils and rat pleural exudates (pergola et al., 2012) . likewise, tryp was found to be effective in protecting mice against experimentally-induced colitis via regulation of the tumor necrosis factor (tnf)/nuclear factor (nf)-kb and interleukin (il)-6/signal transducer and activator of transcription 3 (stat3) signaling pathways (wang et al., 2018) . although there are no reported studies regarding the effects of tryp on ra, the signaling pathways impacted by tryp clearly play roles in ra pathogenesis [e.g., see (lubberts, 2015; mitchell and carmody, 2018) ]. thus, we hypothesized that tryp or its structural analogs might be effective treatments for ra. structural modification of natural compounds can increase compound potency and selectivity, enhance their pharmacological properties, and significantly diminish their detrimental effects (guo, 2017) . several tryp derivatives with various tetracyclic scaffold modifications have been developed, including compounds with anti-plasmodium and antitoxoplasma properties (krivogorsky et al., 2008; onambele et al., 2015) , indoleamine 2,3-dioxygenase inhibitors (yang et al., 2013) , and dna triplex stabilizing agents (chen et al., 2007) . recently, we found that tryptanthrin-6-oxime (tryp-ox) had high affinity for jnk1-3 and also blocked activation of nf-kb/ap-1 and the production of il-6 by lipopolysaccharide-treated monocytic cells (schepetkin et al., 2019) . since jnk inhibition has potential for reducing inflammation associated with ra, it is reasonable that jnk inhibitors could be developed as ra therapeutics (han et al., 2001; bogoyevitch et al., 2010; koch et al., 2015) . indeed, we found that 11h-indeno [1,2-b] quinoxalin-11-one oxime salt (iq-1s) was an effective jnk inhibitor that blocked proinflammatory cytokine production and that iq-1s treatment had a beneficial therapeutic effect in a mouse model of ra (schepetkin et al., 2012; schepetkin et al., 2015) . moreover, we also found that the indolo[2,1-b]quinazolin-6,12-dione (tryp) structure was analogous to the 11hindeno[1,2-b]quinoxalin-11-one scaffold (schepetkin et al., 2019) . in the present study, we expanded our search for potential biological targets of tryp and tryp-ox using a reverse pharmacophore mapping approach and again found that jnk1 and jnk3 were potential therapeutic targets of tryp-ox but not for tryp. furthermore, we evaluated anti-inflammatory effects of tryp and tryp-ox in vitro and found that tryp-ox effectively inhibited il-1b-induced il-6 secretion by fls, sw-982 synovial cells, and thp-1 monocytic cells, whereas tryp was generally less effective. we also investigated the effect of these compounds in vivo using collagen-induced arthritis (cia) and collagen-antibody-induced arthritis (caia) models of ra and found that tryp-ox significantly reduced the clinical symptoms and cartilage damage in cia and caia. surprisingly, tryp was also effective in treating cia and caia, although inhibition of cartilage destruction was more effective with tryp-ox. the therapeutic effects of tryp and tryp-ox in cia were associated with reduced levels of ciispecific antibodies and inhibition of proinflammatory cytokine production by lymph node (ln) cells. overall, tryp-ox has a relatively greater therapeutic potential for treatment of ra compared to tryp and represents a potential new direction for pursuit of novel treatments for ra. tryp was purchased from combi-blocks (san diego, ca, usa). tryp-ox and iq-1s were synthesized, as described previously (schepetkin et al., 2019) . the jnk inhibitor sp600125 was from tocris bioscience (ellisville, mo, usa). for in vitro studies, the compounds were dissolved in dimethyl sulfoxide (dmso) and diluted into the desired buffer or culture media. for in vivo treatments, the compounds were suspended in sterile phosphate buffer saline (pbs). to induce cia in dba1/j mice, immunization-grade bovine type ii collagen (cii) (chondrex, redmond, wa, usa) was solubilized in 0.05 m acetic acid (2 mg/ml), and 100 mg of cii emulsified in complete freund's adjuvant containing 4 mg/ml mycobacterium tuberculosis (chondrex) were injected subcutaneously (s.c.) in the tail (kochetkova et al., 2010) . using this method, nearly 100% of mice consistently showed clinical symptoms by day 25. suspensions of tryp and tryp-ox or sterile saline were injected intraperitoneally (i.p.) at 30 mg/kg daily beginning on day 8 after the cii challenge, as indicated, and continued until day 42 post-cii challenge. mice were scored using a scale of 0 to 3 for each limb, with a maximal total score of 12, as previously described (schepetkin et al., 2015) : 0, no signs of inflammation; 1, mild redness or swelling of single digits; 2, significant swelling of ankle or wrist with erythema; and 3, severe swelling and erythema of multiple joints. caia was induced in 7-to 8-week-old balb/c mice from charles river laboratories (wilmington, ma, usa) by i.p. injection of a cocktail of monoclonal anti-cii antibodies (chondrex; 1.5 mg/mouse) on day 0, followed by i.p. lipopolysaccharide (lps) injection (30 mg lps from escherichia coli strain 0111:b4 in pbs) on day 3. control animals received an equal volume of pbs. suspensions of tryp, tryp-ox, and iq-1s (all compounds in dose 30 mg/kg) or sterile saline were injected i.p. daily beginning at day 1 after injection of anti-cii abs and continued until day 9. arthritis development was evaluated daily for 10 days postinjection of the monoclonal antibody cocktail. arthritis symptoms were scored on scale of 0 to 4 in accordance with the chondrex mouse arthritis scoring system in a blinded manner, as follows: 0, no signs of inflammation; 1, mild redness or swelling of single joint (one of the interphalangeal joints, metacarpophalangeal joints, carpal joint for the front paw, and tarsal joint for the hind); 2, two joints have redness and swelling; 3, all three joint types have redness and swelling; 4, maximal redness and swelling of entire paw leads to the disappearance of anatomical definition. at 44 days after cii injection (cia) or 14 days after anti-cii antibody injection (caia), the mice were euthanized, and their hind limbs were collected and fixed in 10% neutral buffered formalin. the limbs were decalcified in 5% formic acid for 3 to 6 days, and the joints were embedded in paraffin. eight 8-mm sections were cut using a sagittal projection with 40 µm spacing of the cuts. hematoxylin and eosin (h&e) and toluidine blue (tb) staining were performed for each sample. h&e histopathological scores for the joints were determined by using a graded scale (schramm et al., 2004) , separately for cartilage destruction, pannus formation, and synovial cells changes. for cartilage destruction, the scale was: 0-normal, superficial zone is smooth; 1-superficial fibrillations, some loss of surface lamina; 2-vertical clefts/erosion to the calcified cartilage up to 25% of the surface; 3-vertical clefts/erosion to the calcified cartilage more than 25% of the surface, severe chondrocytes, and cartilage matrix loss with new bone tissue substitution, bone destruction. for pannus formation, the scale was: 0-normal; 1-exudate in the joint; 2-mild infiltration of the inflammatory cells; 3-heavy inflammatory infiltration and debris in the joint). for synovial changes, the scale was: 0normal; 1-initial hyperplasia of the cells, with occasional infiltration of inflammatory cells; 2-focal infiltration of inflammatory cells into the synovial layers; 3-diffused infiltration of inflammatory cells into synovium. all sections of the hind paws and knee joints were examined, the highest score was recorded, with a total maximum score of 36 possible per mouse. the loss of the proteoglycans and cartilage degeneration was also scored in tb-stained sections on a graded scale of 0-3, as previously described (bernotiene et al., 2004) : 0-no cartilage loss; 1minimal chondrocytes and proteoglycan loss in superficial zone; 2-moderate chondrocytes and proteoglycan loss into middle zone but above tidemark; and 3-severe cartilage degeneration through tidemark, with the maximum score of 12 possible per mouse. in caia, animals were sacrificed on day 14, and their forward and hind limbs were processed as described above. three sections in the sagittal projection of joints, 40 µm apart, were examined by the same system with the grades ranging 0 to 4. total h&e histopathological score for three sections included cartilage destruction, pannus formation, and synovial cells changes. for cartilage destruction, the scale was: 0-normal, superficial zone is smooth; 1-superficial fibrillations, some loss of surface lamina; 2-vertical clefts/erosion to the calcified cartilage up to 25% of the surface; 3-vertical clefts/erosion to the calcified cartilage 25% to 50% of the surface, 4-vertical clefts/erosion to the calcified cartilage more than 50% of the surface, severe chondrocytes, and cartilage matrix loss with new bone tissue substitution. for pannus formation the scale was: 0normal; 1-exudate in the joint; 2-single inflammatory cells in the exudate; 3-mild infiltration of the inflammatory cells; 4heavy inflammatory infiltration and debris in the joint. for synovial membrane changes the scale was: 0normal, 1 to 2 layer of synovial lining cells; 1-initial hyperplasia of the cells and increased number of lining cells layers; 2-increased number of lining cell layers, more than 3 to 4 layers and/or proliferation of sub-synovial tissue; 3-more than 4 layers of lining cells, focal infiltration of inflammatory cells, proliferation of subsynovial tissue; 4-more than four layers of lining cells, diffused infiltration of inflammatory cells, proliferation of subsynovial tissue. anti-cii antibody enzyme-linked immunosorbent assay (elisa) serum samples from mice with cia were collected on day 44 after cii injection. samples of the diluted sera were added to microtiter plates (greiner bio-one, monroe, nc, usa) coated with 2 mg/ml elisa grade mouse cii (chondrex). horseradish peroxidase-conjugated goat anti-bovine total igg, igg1, igg2a, igg2b, and igg3 (southern biotechnology associates, birmingham, al, usa) were used for detection. the samples were developed with 2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid (moss inc., pasadena, md, usa), and absorbance was monitored at 415 nm using a spectramax plus microplate reader. endpoint titer represents reciprocal log 2 for the serum dilution with absorbance ≥ 0.1 above negative control. at the end of the study (day 44), we isolated the axillary, inguinal, and popliteal ln and purified mononuclear cells from the ln. these cells were cultured in 6-well plates (5 × 10 6 cells/ml) without or with 50 mg/ml cii for 3 days in rpmi-1640 medium supplemented with 10% of fetal bovine serum (fbs) (invitrogen, carlsbad, ca, usa), 2 mm l-glutamine, 100 u/ml penicillin, 100 mg/ml streptomycin, 1 mm sodium pyruvate, and 0.1 mm nonessential amino acids (kochetkova et al., 2008) . the levels of cytokines, including tumor necrosis factor (tnf), granulocyte-macrophage colony-stimulating factor (gm-csf), il-1b, il-5, il-6, il-10, il-17a, and receptor activator of nuclear factor-kb ligand (rankl) were measured in ln culture supernatants using elisa kits (bd biosciences, san jose, ca, usa) for mouse cytokines. the sw982 human synovial cell line was obtained from the america type culture collection (atcc, rockville, md, usa). sw982 cells were maintained in atcc-formulated leibovitz's l-15 medium supplemented with 10% fbs, 100 u/ml penicillin, and 100 µg/ml streptomycin. human monocytic thp-1 cells (atcc) were cultured in rpmi-1640 medium (mediatech inc., herndon, va, usa) supplemented with 10% (v/v) fbs, 100 u/ ml penicillin, and 100 µg/ml streptomycin. human umbilical vein endothelial cells (huvecs) (atcc, rockville, md, usa) were cultured in vascular cell basal medium (atcc) supplemented with components from the endothelial cell growth kit-vegf (atcc): 2% fbs, 10 mm l-glutamine, 5 ng/ ml recombinant human epidermal growth factor (rh egf), 5 ng/ ml recombinant human vascular endothelial growth factor (rh vegf), 15 ng/ml recombinant human insulin-like growth factor (rh igf-1), 1 mg/ml hydrocortisone hemisuccinate, 0.75 u/ml heparin sulfate, 50 mg/ml ascorbic acid, 100 u/ml penicillin, and 100 mg/ml streptomycin according to the manufacturer's specifications. all cell cultures were maintained in polystyrene tissue culture flasks at 37°c and 5% co 2 . this study was approved by the institutional review board of university of california, san diego school of medicine (la jolla, ca, usa). informed consent was obtained from all participants. anonymous synovial tissue samples were obtained from six patients with ra at the time of total joint replacement (5 females, 1 male; age range, 44-72 years), as previously described (alvaro-gracia et al., 1990) . the diagnosis of ra conformed to american college of rheumatology 1987 revised criteria (arnett et al., 1988) . the synovium was minced and incubated with collagenase type viii (0.5 mg/ml) (sigma-aldrich) in serum-free rpmi-1640 (life technologies, grand island, ny, usa) for 2 h at 37°c, filtered, extensively washed, and cultured in dmem (life technologies) supplemented with 10% fbs (gemini bio products, calabasas, ca, usa), penicillin, streptomycin, gentamicin, and glutamine in a humidified 5% co 2 atmosphere. cells were allowed to adhere overnight, nonadherent cells were removed, and adherent fibroblast-like synoviocytes (fls) were split at 1:3 when 70%-80% confluent. fls were used from passages 3 through 9, during which time they are a homogeneous population of cells (<1% cd11b positive, <1% phagocytic, and <1% fcgrii and fcgriii receptor positive). for mmp mrna analysis, fls were plated in six-well plates, cultured until 80% confluence, and subsequently serum starved (1% fbs/dmem) for 24 h. the cells were treated with tryp or tryp-ox (4, 10, and 25 mm) or vehicle (dmso) for 1 h before il-1b stimulation (2 ng/ml) for 6 h. the mrna was isolated and reverse transcribed to obtain cdna. quantitative real-time polymerase chain reaction was performed using primer probe sets for mmp3 and glyceraldehyde 3-phosphate dehydrogenase (gapdh) (life technologies). threshold cycle values were obtained and normalized to gapdh expression. cultured fls (passage 4) were plated at 2 × 10 5 cells in six-well plates overnight in dmem containing 10% fbs and synchronized for 24 h in dmem/0.1% fbs. the cells were pretreated for 1 h with different concentrations of tryp, tryp-ox, or dmso (vehicle control) and then stimulated with 2 ng/ml il-1b or medium for 15 min at 37°c. the cells were then washed once with ice-cold pbs and lysed with modified radioimmunoprecipitation assay buffer [50 mm hepes, ph 7.4, 150 mm nacl, 1% triton x-100, 10% glycerol, 2.5 mm mgcl 2 , 1.0 mm edta, 20 mm bglycerophosphate, 10 mm naf, 1 mm na 3 vo 4 , and protease inhibitor cocktail (roche, indianapolis, in)]. protein concentration of the lysates was measured using a microbca assay kit (pierce, rockford, il), and 40 mg lysate was subjected to 10% sds-page and western blot analysis. anti-phospho-c-jun (ser63) was purchased from cell signaling technology (danvers, ma), and anti-mouse gapdh was from santa cruz biotechnology (santa cruz, ca). fls, huvec, sw982, and thp-1 cells were plated in 96 well plates (10 4 to 10 5 cells/well) for 48 h before treatment. at 2 days post-confluency, the cells were serum-starved and washed twice (with the exception of thp-1 cell, which were not washed) with fbs-free medium prior to the addition of test compounds. the cells were incubated in the presence or absence of test compounds or dmso (vehicle control) for 30 min at 37°c in a 5% co 2 atmosphere, il-1b (5 ng/ml) was added, and the cells were incubated for an additional 24 h. culture supernatants were collected and stored at -80°c. the levels of mmp-1 and mmp-3 were determined in culture supernatants using commercially available elisa kits according to the manufacturer's instructions (r&d system, minneapolis, mn, usa). il-6 levels were also determined in the supernatants using a human il-6 elisa kit (bd biosciences, san jose, ca, usa). the inhibitory effect of test compounds on ache activity was performed using an ache inhibitor screening kit from sigma-aldrich. the kit is based on an improved ellman method, whereby thiocholine produced from ache activity forms a yellow color with 5,5'-dithiobis(2-nitrobenzoic acid), and the intensity the color (412 nm) is proportional to enzyme activity. the concentration of compound required to cause 30% inhibition (ic 30 ) was used. ic 30 was obtained by graphing the % inhibition of enzyme activity versus the logarithm of concentration of test compound using 5-7 tested concentrations. cytotoxicity was analyzed using a celltiter-glo luminescent cell viability assay kit (promega), according to the manufacturer's protocol. briefly, cells were cultured at a density of 10 4 to 10 5 cells/well with different concentrations of test compounds (final concentration of dmso was 1%) for the indicated periods of time (2, 12, or 24 h) at 37°c and 5% co 2 . following treatment, substrate was added to the cells, and the samples were analyzed with a fluoroscan ascent fl microplate reader. the protein targets of tryp and tryp-ox were analyzed using pharmmapper (liu et al., 2010) . pharmmapper is a tool that identifies potential targets for a given small molecule using an "invert" pharmacophore mapping procedure. pharmmapper utilizes reference databases of protein drug targets encoded by sets of pharmacophore points for faster mapping. initial structures of the test compounds were prepared using chemdraw 16.0 software and saved in tripos mol2 format. the mol2 files of tryp and z-and e-isomers of tryp-ox were uploaded into the pharmmapper server with automatic generation of up to 300 conformers for each compound switched on. we selected the "human protein targets only" database containing 2241 targets for pharmacophore mapping. the top 250 potential targets were retrieved and sorted by normalized fit score value. the physicochemical properties of selected compounds were computed using swissadme (http://www.swissadme.ch) (daina et al., 2017) . a nonparametric mann-whitney u test was used for statistical analysis of cia and caia clinical scores, histology scores, and cartilage destruction. all other data were analyzed by anova with no correction, and differences were considered statistically significant if p<0.05. previously we used kinase profiling to demonstrate that tryp-ox had high binding affinity for jnk1-3, whereas tryp had very low affinity for jnk1 and had no binding affinity for jnk2 and jnk3 (schepetkin et al., 2019) . to expand and enhance our understanding of the potential biological targets of tryp/tryp-ox, we performed reverse pharmacophore mapping on tryp and the z-and e-isomers of tryp-ox using pharmmapper. this program compared a database of pharmacophore patterns with our test compounds and generated target information, including normalized fitness scores and pharmacophoric characteristics. as shown in table 1 , pharmmapper analysis indicated that the top three ranked potential targets for the zand e-isomers of tryp-ox were jnk1, jnk3, and complement factor b (cfb), confirming the high binding affinity for jnk1 and jnk3 that we found using kinase scanning and verifying that these are indeed relevant therapeutic targets for tryp-ox. in contrast, pharmmapper analysis of the parent alkaloid tryp showed relatively low fitness scores for jnk1 and jnk3, with the top three ranked targets for tryp being acetylcholinesterase (ache), carboxylesterase 1 (ces-1), and transthyretin (ttr) ( table 1) . the low fitness for tryp binding to jnk is consistent with our previous kinase scanning studies (schepetkin et al., 2019) . since ache was found to be the top target for tryp and was also on the target list for tryp-ox, although not close to the top targets, we evaluated the direct ache inhibitory effects of both tryp and tryp-ox using an enzymatic test-system but found that both compounds had a relatively low anti-ache activity, with tryp being somewhat more active (table 2) . thus, pharmmapper analysis supported the role of jnk1 and jnk3 as targets for tryp-ox, which is relevant to therapeutic intervention in ra, whereas no obvious targets related to ra intervention were found for tryp. the adme properties determining either the access of a potential drug to the target or its elimination by the organism are necessary during initial stages of drug discovery (doogue and polasek, 2013) . tryp and tryp-ox were analyzed for the most important physicochemical properties in comparison with indenoquinoxaline analogs iq-1 (an oxime formed after spontaneous hydrolysis of the jnk inhibitor iq-1s) and iq-18 (the inactive ketone precursor of iq-1) using the swissadme tool (daina et al., 2017) . we found that these compounds were similar regarding many adme properties (table 3) . nevertheless, they differed markedly in the number of h-bond acceptors and donors, which we showed previously affected jnk binding affinities and locations within the jnk binding site for (schepetkin et al., 2012; schepetkin et al., 2015) . we also created bioavailability radar plots that display an assessment of the druglikeness of each compound. six physicochemical properties are considered: lipophilicity, size, polarity, solubility, flexibility, and saturation. the physicochemical range on each axis is depicted as a pink area in which the radar plot of the molecule has to fall entirely to be considered drug-like (daina et al., 2017) . we found that all four compounds had similar bioavailability radar plots ( figure 1) . moreover, the charge distributions for iq-1 and tryp-ox were very similar, which is not surprising based on their similar structures (schepetkin et al., 2019) . tryp and tryp-ox inhibit mmp3 gene expression and mmp1/3 and il-6 secretion fls obtained by arthroplasty from tissue of patients with ra are an excellent in vitro model for screening novel compounds with antiarthritic potential (bartok and firestein, 2010) . we evaluated whether tryp and tryp-ox altered mmp3 expression by il-1b-stimulated human fls and found that cells pretreated with either of these compounds exhibited a dose-dependent reduction in mmp3 mrna levels compared with the vehicle-treated group, but that tryp-ox was significantly more active than tryp ( figure 2) . numerous studies suggest that human sw982 synovial sarcoma cells share similar physiological and immunological properties with primary fls and that il-1b stimulation of sw982 cells can mimic the inflammatory status of synovial cells typically seen in ra patients (tsuji et al., 1999; kim et al., 2008; chang et al., 2014) . thus, sw982 cells can be useful in screening of potential antiarthritic compounds, as fls are in limited supply. indeed, we found that tryp and tryp-ox inhibited il-1b-induced il-6 secretion by fls and sw982 cells, as well as by huvecs and thp-1 monocytic cells, in a dosedependent manner, with the most potent being tryp-ox (table 4a) . these compounds also inhibited il-1b-induced mmp-1/3 secretion by fls, sw982 cells, and huvecs, with the most potent being tryp-ox in fls and sw982 cells ( table 4a) . as examples, the dose-dependent effects of tryp and tryp-ox on il-1b-induced secretion of il-6 by fls, sw982 cells, and huvecs are shown in figure 3 . note that tryp-ox was significantly more effective than tryp in inhibiting il-6 production in all three of these cell types. to ensure that the effect of our compounds on functional activity in vitro were not affected by possible toxicity, we evaluated cytotoxicity of tryp and tryp-ox at various concentrations (up to 50 µm) in all cultured cells (table 4b) . while tryp-ox exhibited no cytotoxicity in all of the cells tested, tryp exhibited some cytotoxic effects, albeit at higher concentrations that those required to inhibit il-6 and mmp-1/3 production. thus, tryp cytotoxicity may have some contribution to the inhibition of pro-inflammatory cytokine/ enzyme production by some cell lines. we also evaluated the effects of sp600125, a commonly used jnk inhibitor , on il-6 secretion in sw982 synovial cells. since sp600125 increased cytotoxicity of tryp during a 24 h incubation, we used a shorter incubation period (12 h) that did not affect cell viability. we found that sp600125 dose-dependently inhibited il-1b-induced il-6 secretion by sw982 cells (ic 50 = 1.3 ± 0.5 µm). in addition, combined treatment of sp600125 plus tryp was evaluated. in the presence of 5 µm tryp, the ic 50 value for sp600125 was the similar to that observed when tryp was absent (1.2 ± 0.2 µm), although tryp acted synergistically with sp00125 to significantly increase the overall inhibition compared to sp00125 alone (figure 4) . thus, tryp and sp600125 may inhibit il-6 production through different pathways. previously, we showed that the jnk inhibitor iq-1s, which is a close chemical analog of tryp-ox (see table 3 ), inhibited c-jun phosphorylation in fls (schepetkin et al., 2015) . analysis of the effects of tryp and tryp-ox on c-jun phosphorylation in il-1b-stimulated fls showed that tryp-ox also inhibited c-jun phosphorylation, whereas tryp had no effect ( figure 5) , confirming our previous conclusion that tryp-ox, but not tryp, is a jnk inhibitor (schepetkin et al., 2019) . note that neither tryp nor tryp-ox affected fls viability during the 90min period. previously, we evaluated a range of iq-1s doses (5, 20, 30, and 50 mg/kg i.p.) and found that 5 mg/kg was not very effective, whereas 30 and 50 mg/kg were equally effective in reducing arthritis in the cia model (schepetkin et al., 2015) . preclinical pharmacokinetic studies in rats demonstrated that after i.p. administration of iq-1s, a rapid rise in the serum concentration was observed, peaking at 5 min, and the half-life in circulation was 12 h (plotnikov et al., 2020) . when mice were dosed with 30 mg/kg iq-1s i.p., the serum exposure of the compound was also good, with auc 0-12 h values of 7.4 µm/h (schepetkin et al., 2012) . thus, based on the similarity in physicochemical properties of tryp-ox and iq-1s, their high jnk binding affinity, the effectiveness of 30 mg/kg iq-1s in previous cia experiments, and the known preclinical pharmacokinetic studies for iq-1s, we selected a 30 mg/kg dose for tryp-ox and tryp treatment in our in vivo studies. cia was induced in arthritis-susceptible dba1/j mice by immunization with bovine cii emulsified in complete freund's adjuvant. to test the therapeutic potential of tryp and tryp-ox on cia, treatment was initiated at day 8 after cii challenge. although disease symptoms appear usually at day 21 postinduction, immune pathologic processes begin soon after cii injection (brand et al., 2003) . thus, we consider administration of compounds between days 0 and 21 following cia initiation to be a combined prevention/treatment model. we found that mice treated i.p. daily with 30 mg/kg of tryp or tryp-ox displayed significant reductions in cia symptoms compared with vehicletreated mice ( figure 6 ). both tryp and tryp-ox seem to be equally effective treatments for reducing clinical scores in the cia model. histopathological analysis was performed on ankle joints collected from cia mice treated with tryp or tryp-ox and compared with control, saline-treated mice. representative images of stained joint tissue from mice treated with 30 mg/kg compounds are shown in figure 7a . in contrast to the severe cartilage erosion, synovial hyperplasia, and infiltration of inflammatory cells observed in the joints of saline-treated cia mice, there was little cartilage erosion, synovial hyperplasia, or cellular infiltration in the joints of tryp and tryp-ox-treated cia mice. furthermore, histological scoring showed significant differences between treated and control groups ( figures 7b, c) , whereas the differences between tryp-and tryp-ox-treated mice were not significant. thus, consistent with the reductions in cia clinical score, tryp and tryp-ox treatment clearly protected these mice from cartilage destruction. injected cii-specific abs can induce transient arthritis, indicating that ab responses are important in cia pathogenesis (myers et al., 1997; nandakumar et al., 2003) , and it has been shown that the anti-cii igg antibody titer correlates with arthritis severity in cia mice (williams et al., 1998) . we measured the levels of cii-specific abs in tryp-, tryp-ox-, and vehicle-treated cia mice and found that the titers of the igg, igg1, igg2a, igg2b, and igg3 were significantly lower in the compound-treated groups compared to those in the saline-treated group (figure 8 ). proinflammatory cytokines produced by myeloid cells can facilitate inflammation, cartilage damage, and joint destruction in ra (schurgers et al., 2011; zhou et al., 2011; azizi et al., 2013) . to test whether tryp and tryp-ox affected the production of inflammatory cytokines by cii-specific t cells, we evaluated ciiinduced cytokine production by ln cells isolated from control and treated mice. we cultured the same numbers of cells from compound-treated and saline-treated cia mice for 72 h in the presence of cii and found that the generation of proinflammatory cytokines (il-1b, il-5, il-6, il-17a, tnf, gm-csf, and rankl) was significantly reduced in compound-treated mice (30 mg/kg) compared to saline-treated cia mice. in contrast, the production of the anti-inflammatory cytokine il-10 was higher in compoundtreated mice (figure 9 ). note that the levels of il-17a, gm-csf, and rankl production by ln cells isolated from tryp-oxtreated mice were significantly lower compared to mice treated with tryp. the caia mouse model is another widely used animal model to screen for potential anti-ra compounds [for example (chang et al., 2011) ]. to evaluate the therapeutic potential of tryp and tryp-ox in caia, treatment was initiated at day 1 after anti-cii antibody injection. we found that mice treated i.p. daily with 30 mg/kg tryp or tryp-ox displayed significant reductions in the severity of caia compared with vehicle-treated mice, as assessed by the mean arthritis score (figure 10) . although the differences in therapeutic effects of tryp and tryp-ox were not significant, the therapeutic effects of tryp and tryp-ox were significantly greater at days 6 and 7 compared to mice treated with iq-1s, a jnk inhibitor that we previously showed to inhibit arthritis in the cia model (schepetkin et al., 2015) . histopathological analysis was also performed on ankle joints collected from caia mice treated with 30 mg/kg tryp or tryp-ox and compared to that of control mice (figure 11) . severe cartilage erosion, synovial hyperplasia, and infiltration of inflammatory cells was seen in the joints of saline-treated caia mice. in contrast, there was little cartilage erosion, synovial hyperplasia, or cellular infiltration in tryp and tryp-oxtreated caia mice. furthermore, histological scoring showed significant differences between treated and control groups ( figures 11b, c) . thus, consistent with the reduced caia clinical score and the results using the cia model, tryp and tryp-ox treatment clearly protected caia mice from cartilage damage, as well as from severe joint inflammation. ra is a chronic destructive autoimmune disorder that commonly leads to significant joint destruction that is mediated in part by proteases and cytokines produced by macrophages and fls. although current biological therapeutic strategies for ra have been effective in many cases, there are still issues of cost, loss of efficacy over time, toxicity, and protein degradation. thus, new classes of therapeutics are needed that can be used alone or in combination with the current biologic ra treatments. in the present study, we identify tryp-ox and possibly tryp as potentially novel ra therapeutics that could be pursued in drug development. previously, we demonstrated that tryp-ox had high affinity for jnks and also inhibited pro-inflammatory cytokine production in vitro (schepetkin et al., 2019) , suggesting that it might be have potential in the development of treatments for inflammatory diseases, such as ra. to further evaluate potential targets of tryp and tryp-ox, we performed a broad pharmacophore mapping screen using pharmmapper, which screens a large database of human protein targets. screening of tryp-ox showed that jnk1, jnk3, and cfb were the top potential protein targets. these mapping results are supported by our previous kinase screening, which demonstrated that tryp-ox had affinity for all three jnks, with the highest affinity for jnk1 (150 nm) and jnk3 (275 nm) (schepetkin et al., 2019) . indeed, a number of studies have shown that jnks play an important role in ra (han et al., 2001; figure 4 | effects of jnk inhibitor sp600125 and tryp on il-1b-induced il-6 secretion by sw982 cells. cells were treated with 5 µm tryp or dmso combined with the indicated concentrations of sp600125 for 30 min, followed by stimulation with 5 ng/ml il-1b for 10 h. il-6 levels were measured by elisa. values are expressed as mean ± s.d. of one experiment that is representative of 2 independent experiments. *p < 0.05 compared with sp600125 treatment alone. the cells were treated with compounds for 30 min and then activated with 5 ng/ml il-1b. after 24 h incubation, mmp-1/3 and il-6 were evaluated in the supernatants by elisa. ic 50 values are presented as the mean ± s.d. of three to four independent experiments. # p < 0.05 compared with tryp. 2010; koch et al., 2015) , as well as many other inflammatory diseases (thalhamer et al., 2008; nikoloudaki et al., 2020) . cfb may also represent a therapeutic target in ra, as cfb deficient mice (cfb -/-) are highly resistant to cia and caia and have a decreased cii-specific igg antibody response in cia (hietala et al., 2002; banda et al., 2006) . furthermore, local complement production, including cfb, may play a role in ra pathogenesis. thus, the reduction local complement generation may represent a reasonable therapeutic approach for ra (neumann et al., 2002) . in contrast to tryp-ox, the three top ranked targets for tryp were ache, ces-1, and ttr, which do not seem to be obvious targets associated with ra. thus, it is interesting that tryp was still fairly effective in vivo in treating cia/caia. figure 5 | tryp-ox inhibits c-jun phosphorylation induced by il-1b in fls from ra patients. fls were pretreated for 1 h with the indicated concentrations of tryp-ox, tryp, or dmso and then stimulated with 2 ng/ ml il-1b or medium for 15 min at 37°c. equal concentrations of cell lysates were separated by sds-page, followed by western blotting for phospho-c-jun (p-c-jun) and gapdh, as described. the blot shown is from one experiment that is representative of 2 independent experiments. figure 6 | therapeutic effects of tryp and tryp-ox in cia mice. starting at day 8 after cii challenge, mice were treated i.p. daily with 30 mg/kg tryp ( ) or 30 mg/kg ( ) tryp-ox until day 42, and clinical scores were determined, as described. control mice were treated i.p. daily with saline ( ). results are shown as the mean of 10 mice per group ± s.d. *p < 0.05 compared with saline control. although ttr has been reported to be significantly upregulated in the plasma of ra patients , this protein has not yet been established as a pharmacological target for ra treatment. ces-1 may play important role in pharmacokinetics and pharmacodynamics of some anti-inflammatory drugs (mishima et al., 1991) , but ces-1 also has not been reported as a therapeutic target for ra. recently rivastigmine, a known ache inhibitor, was reported to have therapeutic effects in a complete freund's adjuvant-induced model of arthritis (shafiey et al., 2018) . furthermore, it was found that ache activity was increased with age and may increase ra risk and severity in elderly people (sato et al., 2015) . therefore, ache could be an figure 9 | effect of tryp and tryp-ox on cytokine production by ln cells. starting at day 8 after cii challenge, mice were treated i.p. daily with saline (0), 30 mg/ kg tryp, or 30 mg/kg tryp-ox until day 42. mice were euthanized, and their ln mononuclear cells were purified and cultured for 3 days with cii. cytokine levels were measured in supernatants by elisa. the results show the mean of one experiment (three to four replicates) ± s.d., which is representative of three independent experiments. a p < 0.05 compared with the dmso controls; b p < 0.05 compared with tryp. additional co-target for agents with anti-arthritic activity. however, we found here that both tryp and tryp-ox have relatively low anti-ache effects, suggesting that ache is probably not a therapeutic target for these compounds in cia or caia. note that tryp has been reported to act on multiple signaling cascades associated with inflammation, including tnf/ nf-kb and il-6/stat3 (wang et al., 2018) , leukotriene secretion (pergola et al., 2012) , and several cytokines required for th17 polarization (cheng et al., 2020) . tryp has also been reported to inhibit macrophage nitric oxide and prostaglandin e production upon exposure to oxidative stress (ishihara et al., 2000) , as well as inhibit cyclooxygenase-2 and indoleamine 2,3dioxygenase activities (danz et al., 2001; pergola et al., 2012; yang et al., 2013) . overall, the putative tryp anti-inflammatory target associated with ra is still not clear and will require further investigation. we demonstrate here for the first time that tryp and tryp-ox may be promising therapeutics for treating ra, as shown by their ability to ameliorate clinical outcomes in vivo in cia and caia mice, as well as reduce disease-associated anti-cii antibody production in cia mice. cia progression requires both adaptive and innate immune responses through the production of anti-cii-specific antibodies and the development of cii-specific t cells (brand et al., 2003) . we found that both tryp and tryp-ox inhibited the production of anti-cii antibodies by b cells, especially igg1 and igg3, which represent t cell-dependent and t cell-independent responses, respectively (stevens et al., 1988) . thus, therapeutic effects of tryp and tryp-ox in cia may be associated with direct inhibition of b cell activity or indirect effects on innate immune cells. indeed, in both cia and caia models, anti-cii antibodies have been reported to form immune complexes that figure 10 | therapeutic effects of tryp and tryp-ox in caia mice. starting at day 1 after the anti-cii antibody injection, mice were treated i.p. daily with control saline ( ), tryp ( ), tryp-ox ( ), or iq-1s ( ) (30 mg/ kg of each compound) until day 9, and clinical scores were determined. results are shown as the mean of 5 mice per group ± s.d. a p < 0.05; b p < 0.01; c p < 0.001 compared with saline control, and d p < 0.05 compared with the iq-1s-treated group. activate myloid cell fcgr, leading to infiltration of monocyte/ macrophages, neutrophils, and mast cells into the joints, where they release inflammatory mediators that initiate synovitis and bone destruction (delire, 1994; firestein and corr, 2005) . macrophages and fls invade the synovium in ra and secrete proinflammatory cytokines and mmps that enhance and sustain the inflammatory response (kinne et al., 2000; davignon et al., 2013) . the primary cytokines required for inducing arthritis in autoimmune models are il-1b, il-6, il-17a, tnf, gm-csf, and rankl (nakae et al., 2003; guma et al., 2010; li et al., 2012; tanaka, 2019) . these cytokines play a key role in ra pathogenesis through their ability to recruit and stimulate macrophages, neutrophils, and mast cells (drexler et al., 2008; guma et al., 2010; tanaka, 2019) . here, we found that tryp and tryp-ox down-regulated il-1b, il-5, il-6, il17a, tnf, gm-csf, and rankl production by ln cells, while il-10 production was increased. levels of il-17a, gm-csf, and rankl production by ln cells isolated from tryp-oxtreated mice were much lower compared to mice treated with tryp. this finding may suggest jnk-dependent regulation of these cytokines. indeed, rankl and gm-csf expression and (or) secretion in other cells, such as macrophages, osteocytes, and fls, can be blocked by sp600125, another jnk inhibitor (xu et al., 2015; liu et al., 2016; takeno et al., 2018) . among the different t cell subsets, a population of il17-producing t helper cells (th17) has been shown to be involved in model systems of autoimmunity (weaver et al., 2007) . recently cheng et al. (cheng et al., 2020) reported that tryp exhibits anti-th17 activity through its ability to repress the expression of several cytokines required for th17 polarization. il-1b plays important roles in inflammation and destruction of synovial tissue, cartilage, and bone in patients with ra (tak and bresnihan, 2000) . il-1b induces synoviolin, an e3 ubiquitin ligase, in synovial fibroblasts, which is involved in the overgrowth of synovial cells during ra (gao et al., 2006) . therapeutic effects of some kinase inhibitors is associated with decreased il-1b levels in the joints of cia mice (mcintyre et al., 2003; nishikawa et al., 2003) . here, we show that tryp and tryp-ox significantly inhibited il-1b-induced il-6 secretion by sw982 synovial sarcoma cells and thp-1 monocytic cells, with tryp-ox being far more effective than tryp. the joint tissue damage associated with ra is due primarily to the aggressiveness of fls in the synovial intima, and it is known that rheumatoid fls produce mmps that participate in extracellular matrix destruction in articular cartilage and cytokines that prolong inflammation (burrage et al., 2006; bartok and firestein, 2010) . furthermore, jnk plays a key role in fls mmp gene expression (han et al., 1999; liacini et al., 2002; inoue et al., 2005; kanai et al., 2020) . in our studies, tryp and tryp-ox inhibited il-1b-induced mmp-1/3 secretion by huvecs and sw982 cells and mmp3 gene expression in fls, with the most potent being tryp-ox in fls and sw982 cells. thus, part of the therapeutic effects of tryp and tryp-ox may be due to their ability to target fls and potentially reduce their aggressiveness in ra [e.g., (mcintyre et al., 2003; nishikawa et al., 2003; hegen et al., 2008) ]. these results are consistent with our observations in cia mouse model, where tryp and tryp-ox decreased joint edema, cell migration, and cartilage erosion. jnks play important roles in many pathological processes, including autoimmune inflammatory disorders, such as ra (bogoyevitch et al., 2004; bogoyevitch et al., 2010; mehan et al., 2011) . a number of jnk inhibitors with antiinflammatory properties have been developed (bhagwat, 2007 ), yet few have been evaluated for treatment of arthritis. previous studies demonstrated that sp600125 and iq-1s, which are both jnk inhibitors, could reduce the development and pathogenesis of arthritis when evaluated in animal models (han et al., 2001; schepetkin et al., 2015) . interestingly, the iq-1s structure is related to tryp-ox, and, similarly to iq-1s, tryp-ox was specific for jnk as compared to a variety of other kinases screened (schepetkin et al., 2019) . moreover, tryp-ox is likely a prodrug for tryp, because microsomal metabolism of aryloximes leads to the formation of ketone derivatives (andronik-lion et al., 1992) . although iq-18, the ketone precursor of iq-1s, did not have any anti-inflammatory activity or therapeutic effects in cia (schepetkin et al., 2012; schepetkin et al., 2015) , we show here that tryp, the ketone precursor of tryp-ox, has therapeutic effects in both cia and caia, although with lower efficacy. thus, the therapeutic effects observed for tryp-ox in vivo are likely due to the combined effects of both tryp-ox and its metabolite tryp. in conclusion, we demonstrated that tryp and tryp-ox have anti-inflammatory properties and potentially represent a novel class of quinazoline-based therapeutic agents that could be used in the development of treatment for ra. tryp-ox has additive beneficial therapeutic properties as a jnk inhibitor. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by institutional review board, university of california, san diego school of medicine, la jolla, ca. the patients/ participants provided their written informed consent to participate in this study. the animal study was reviewed and approved by institutional animal care and use committee, montana state university, bozeman, mt 59717. low-dose action of tryptanthrin and its derivatives against developing embryos of the sea urchin strongylocentrotus intermedius cytokines in chronic inflammatory arthritis. v. mutual antagonism between interferon-g and tumor necrosis factor-alpha on hla-dr expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes formation of nitric oxide by cytochrome p450-catalyzed oxidation of aromatic amidoximes the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis th17 cells in immunopathogenesis and treatment of rheumatoid arthritis alternative complement pathway activation is essential for inflammation and joint destruction in the passive transfer model of collagen-induced arthritis fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis sp600125, an anthrapyrazolone inhibitor of jun n-terminal kinase the structure and properties of some indolic constituents in couroupita guianensis aubl delayed resolution of acute inflammation during zymosan-induced arthritis in leptin-deficient mice map kinase inhibitors in inflammation and autoimmune disorders targeting the jnk mapk cascade for inhibition: basic science and therapeutic potential c-jun n-terminal kinase (jnk) signaling: recent advances and challenges immunopathogenesis of collagen arthritis structure of tryptanthrin matrix metalloproteinases: role in arthritis the bruton tyrosine kinase inhibitor pci-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells validity of sw982 synovial cell line for studying the drugs against rheumatoid arthritis in fluvastatin-induced apoptosis signaling model specific stabilization of dna triple helices by indolo[2,1-b]quinazolin-6,12-dione derivatives the anti-th17 polarization effect of indigo naturalis and tryptanthrin by differentially inhibiting cytokine expression swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules identification and isolation of the cyclooxygenase-2 inhibitory principle in isatis tinctoria targeting monocytes/macrophages in the treatment of rheumatoid arthritis different modes of action of high-dose immunoglobulins in rheumatoid arthritis the abcd of clinical pharmacokinetics cell signalling in macrophages, the principal innate immune effector cells of rheumatoid arthritis common mechanisms in immune-mediated inflammatory disease evolving concepts of rheumatoid arthritis the proinflammatory cytokines il-1b and tnf-a induce the expression of synoviolin, an e3 ubiquitin ligase, in mouse synovial fibroblasts via the erk1/2-ets1 pathway jnk1 controls mast cell degranulation and il-1b production in inflammatory arthritis the modification of natural products for medical use jun n-terminal kinase in rheumatoid arthritis c-jun n-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis different roles of peripheral mitogen-activated protein kinases in carrageenan-induced arthritic pain and arthritis in rats triptolide in the treatment of psoriasis and other immune-mediated inflammatory diseases utility of animal models for identification of potential therapeutics for rheumatoid arthritis complement deficiency ameliorates collagen-induced arthritis in mice the antimicrobial specificity of tryptanthrin role of natural product diversity in chemical biology regulation of p38 mapk by mapk kinases 3 and 6 in fibroblast-like synoviocytes tryptanthrin inhibits nitric oxide and prostaglandin e(2) synthesis by murine macrophages tryptanthrin inhibits th2 development, and ige-mediated degranulation and il-4 production by rat basophilic leukemia rbl-2h3 cells the jnk pathway represents a novel target in the treatment of rheumatoid arthritis through the suppression of mmp-3 effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and inos expression in sw982 cells cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (u-937 and hl-60) by tryptanthrin, an active ingredient of polygonum tinctorium lour macrophages in rheumatoid arthritis inhibitors of c-jun n-terminal kinases: an update vaccination without autoantigen protects against collagen ii-induced arthritis via immune deviation and regulatory t cells il-35 stimulation of cd39 + regulatory t cells confers protection against collagen ii-induced arthritis via the production of il-10 inhibition of toxoplasma gondii by indirubin and tryptanthrin analogs managing macrophages in rheumatoid arthritis by reform or removal inhibition of interleukin-1-stimulated map kinases, activating protein-1 (ap-1) and nuclear factor kb (nf-kb) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma la-n-1 cells pharmmapper server: a web server for potential drug target identification using pharmacophore mapping approach lentinan mitigates therarubicin-induced myelosuppression by activating bone marrow-derived macrophages in an mapk/nf-kb-dependent manner the treatment of rheumatoid arthritis using chinese medicinal plants: from pharmacology to potential molecular mechanisms role of t lymphocytes in the development of rheumatoid arthritis. implications for treatment a highly selective inhibitor of i kb kinase, bms-345541, blocks both joint inflammation and destruction in collagen-induced arthritis in mice jnk: a stress-activated protein kinase therapeutic strategies and involvement in alzheimer's and various neurodegenerative abnormalities enzymatic hydrolysis of indometacin farnesil, a prodrug of indomethacin, by carboxylesterase in cultured synovial cells nf-kb and the transcriptional control of inflammation collageninduced arthritis, an animal model of autoimmunity suppression of immune induction of collagen-induced arthritis in il-17-deficient mice induction of arthritis by single monoclonal igg anti-collagen type ii antibodies and enhancement of arthritis in mice lacking inhibitory fcgriib local production of complement proteins in rheumatoid arthritis synovium jnk signaling as a key modulator of soft connective tissue physiology, pathology, and healing prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogen-activated protein kinase inhibitor fr167653 synthesis and evaluation of the antiplasmodial activity of tryptanthrin derivatives the synthetic tryptanthrin analogue suppresses stat3 signaling and induces caspase dependent apoptosis via erk up regulation in human leukemia hl-60 cells on the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo antihypertensive activity of a new c-jun n-terminal kinase inhibitor in spontaneously hypertensive rats anti-inflammatory and antiallergic activity in vivo of lipophilic isatis tinctoria extracts and tryptanthrin donepezil prevents rank-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase identification and characterization of a novel class of c-jun n-terminal kinase inhibitors anti-inflammatory effects and joint protection in collagen-induced arthritis after treatment with iq-1s, a selective c-jun nterminal kinase inhibitor synthesis, biological evaluation, and molecular modeling of 11h-indeno[1,2-b]quinoxalin-11-one derivatives and tryptanthrin-6-oxime as c-jun n-terminal kinase inhibitors susceptibility to collagen-induced arthritis is modulated by tgfb responsiveness of t cells collagen-induced arthritis as an animal model for rheumatoid arthritis: focus on interferon-g paroxetine and rivastigmine mitigates adjuvant-induced rheumatoid arthritis in rats: impact on oxidative stress, apoptosis and rankl/opg signals rheumatoid arthritis in review: clinical, anatomical, cellular and molecular points of view identification of autoantibodies against transthyretin for the screening and diagnosis of rheumatoid arthritis regulation of antibody isotype secretion by subsets of antigen-specific helper t-cells the pathogenesis and prevention of joint damage in rheumatoid arthritis: advances from synovial biopsy and tissue analysis glucose uptake inhibition decreases expressions of receptor activator of nuclear factor-kb ligand (rankl) and osteocalcin in osteocytic mlo-y4-a2 cells rankl is a therapeutic target of bone destruction in rheumatoid arthritis immunosuppressant discovery from tripterygium wilfordii hook f: the novel triptolide analog (5r)-5-hydroxytriptolide (lldt-8) mapks and their relevance to arthritis and inflammation antiviral action of tryptanthrin isolated from strobilanthes cusia leaf against human coronavirus nl63 effects of mitogenactivated protein kinase inhibitors or phosphodiesterase inhibitors on interleukin-1-induced cytokines production in synovium-derived cells tryptanthrin protects mice against dextran sulfate sodium-induced colitis through inhibition of tnf-a/nf-kb and il-6/stat3 pathways il-17 family cytokines and the expanding diversity of effector t cell lineages correlation between igg anti-type ii collagen levels and arthritic severity in murine arthritis interleukin-29 induces receptor activator of nf-kb ligand expression in fibroblast-like synoviocytes via mapk signaling pathways taxol alleviates collagen-induced arthritis in mice by inhibiting the formation of microvessels discovery of tryptanthrin derivatives as potent inhibitors of indoleamine 2,3-dioxygenase with therapeutic activity in lewis lung cancer (llc) tumor-bearing mice the latest developments in synthetic peptides with immunoregulatory activities type 2 dm is known to be a disease characterized by an inadequate beta-cell response to the progressive insulin resistance that typically accompanies advancing age, inactivity, and weight gain [9] iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 105 the correlation between hyperglycemia and rheumatoid factor in type 2 diabetic patients in alrisafa area, baghdad raya k. mohammed salih* ,1 , ali m. al-gharawi** and khalid i. al-lehibi*** * ministry of health, kimadia /dgmi relation section, baghdad, iraq. ** ministry of higher education , college of health and medical technology, baghdad, iraq . ***ministry of health, specialized center for endocrinology and diabetes , baghdad, iraq . abstract diabetes mellitus type 2 (t2dm) formerly called non-insulin dependent diabetes mellitus (niddm) or adult-onset diabetes is a common disease. rheumatoid factor is a well-established test used in the diagnosis and follows the prognosis of rheumatoid arthritis (ra). rheumatoid factor is sometimes found in serum of patients with other diseases including diabetes mellitus (dm), due to the presence of pro-inflammatory cytokines such as tnfα which play an important role in chronic inflammatory and autoimmune diseases like rheumatoid arthritis (ra). the aim of the study is to investigate the associations between type 2 diabetes mellitus (t2dm) and rheumatoid arthritis (ra) in scope of rheumatoid factor (rf), hyperglycemia and body mass index (bmi), in patients with t2dm lived in al-risafa area -baghdad. one hundred twenty five (125) type 2 diabetes mellitus (t2dm) patients were selected from the out patients department of the specialized center for endocrinology and diabetes, baghdad; in addition to (70) apparently healthy non diabetic, non arthritic subjects as control, during the period from sep. dec./2010. the ages of both patients and control subjects were within (35-75) years. this study focus to search for the correlation between t2dm and rf "qualitative and quantitative" in relation to body mass index (bmi) and gender. out of 125 dm-patients (73 female and 52 male), 44 (35.2 %) showed positive rf when compared with healthy controls (n=3, 4.3%). [p value =0.01 is significant] with female dominance (n=28, 63.6%) in compared to males (n=16, 36.4 %), when these diabetics with rf positive were titered for rf (8, 16, 32 and 64 iu/ml), the following results were obtained. the highest percentage of titer observed with 34.1% in those with rf titer 64 iu/ ml [p value = 0.01] when compared with healthy control. 18.2 % had rf titer of 8 iu/ ml, 20.4 % had rf titer of 16 iu/ ml, 27.3 % had rf titer of 32 iu/ ml and 34.1 % had rf titer of 64 iu/ ml. the highest percentage among the overweight, dm patients (38.9 %) have a mean titer 64 iu/ml, a percentage decrease respectively as below: 38.9 % had rf titer of 64 iu/ ml, 27.8% had rf titer of 32 iu/ ml, 16.6 % had rf titer 16 iu / ml and 16.6% had rf titer 8 iu/ ml. the highest number and percentage of dm with rf positive (n=17, 38.6 %) were located among higher age (50-59), (60-69) & (70 -79) year groups (n=17, 38.6%), (n=13, 29.5%) & (n=8, 18.2%) respectively, [pvalue < 0.01] when compared to the corresponding controls. the effect of fasting plasma glucose level of type 2 dm in patients who have rf positive titer, is found that > 7.2 mmol/l glucose in plasma contribute the highest titer (n=28, 63.6 %), in comparison with group of plasma glucose levels < 7.2 mmol/l patients (n=16, 36.4%). with a highly significant difference, p-value = 0.006.smokers diabetic patients with rf positive (n=27, 61.4%) dominate over nonsmokers with rf positive (n=17, 38.6%). the results of this study indicate that there is a reasonable increased frequency of positive rheumatoid factor (rf) in type 2 diabetic patients. poor glycemic control is associated with higher rf titer in positive cases. the titer of t2dm smoker patients is associated with positive rf values that exceed the titer of the nonsmoker rf positive patients. thus, smoking might not be correlated significantly to dm, but may contribute to its complications. key words: t2dm, rf, bmi, smoking. منطقة نعامم انرثىانً فًاننىع انثانً وظهىر ا -انعالقة بٍن ارتفاع انسكر عند مرضى انسكري بغداد انرصافه ٌا كمال محمد صانح* ر ،1 ٍبً*** ابراهٍمانغروي** و خاند محمد ، عًه انهه ٚا ٔصاسج انظحح ، * ًاد ، تغذاد ، انؼشاق . كٛ ح، تغذاد ، انؼشاق . ** ٛاخ انطٛث ح انرُم ٙ ،كٛه ى انؼان ٔصاسج انرؼٛه شكض صاسج انظحح ، اًن ٘ ، تغذاد ،انؼشاق. ***ٔ ٔانسكش ًرخظض نهغذد ان الخالصة ع انثاَٙيُٚؼذ ٍ كٌا انز٘ type 2 diabetes mellitus (t2dm) شع انسكش٘ انُٕ ٕٛن ذ ػٗه االَس ٚسًٗ ساتما داء انسكش٘ انغٛش يؼًر (niddm) ٘انؼايم انشٕثٌٔأ فحض يٍ االيشاع انشائؼح انثانغٍٛ ٘ٔأ سكش rf ًفاطمهرحمك يٍ نٚسرخذو ٕاَٙ ذشخٛض ذطٕس يشع ان ) انشث ra) rheumatoid arthritis م ع يٍ انرحٛه زا انُٕ ْ ح جاٛت ٓاب rfٚالحع ٚا ٕد يسثثاخ االنر ج رنك ٕن ٔ ٓا انسكش٘ ًُ ؼ ٚ ش يٍ االيشاع نكٛث ٍ كٛ ٕٚر ّ كًش ( α tnf-)يثم cytokineانسا ّ انزاٛذ ُاػ ٔايشاع اًن ٍ ضي ٓاب اًن ٙ االنر ًا ف ٙ ذهؼة دٔسا يٓ ٕاَٙ انر ٓاب انشث شذثط raع االنر ٔكزنكٚ كٍٛ ٕٚر ٕنٍٛ cytokine ْزا انسا يح االَس يمٔا ٔ ًُّ َا تانس ػششٌٔ ).أخرش خًسحٔ ٔ 521ٌياّئ ؼإَ ىٚ ٙ -يٍ يشع انسكش٘ ( يشٚؼا أثثد أَٓ ع انثَا انُٕ type 2 diabetes mellitus (t2dm) يُطمح ان ٙ ٘ ف ٔانسكش ٙ نهغذد انظى شكض انرخظظ ٙ اًن ٍ يشاجؼ ٗ ) -شطاّفي ًاً )07تثغذاد ، إَػافح إن سٛه ٙ َٕا ٌ انؼايم انشث ًٕه ح الٚ ٔ٘ ٍ تانسكش ى غٛش يظاٛت ك أَٓ ش يغ انرذٛل كًجًٕػح سٛطشج. rheumatoid factor (rf)تانظْا 1 corresponding author email : rayakamal38@yahoo.com received : 17/1/2012 accepted : 24/4/2012 iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 106 ؤالء ٕاَٙ ، أُدخمْ ًفاطم انشث ٓاب ان ٔانر ع انثاَٙ ٍ يشع انسكش٘ انُٕ ِز انذساسح نرحش٘ انؼال ّل ٛت ْ rheumatoid arthritis (ra)األشخاص ٙف ٕاَٙ ًال انؼايم انشث ًَّاً( rheumatoid factor (rf)تاسرؼ ك ػأً َٕ(qualitative & quantitative) ؼايم كرهح انجسى ًا ًت ػاللٓر ٔ)body mass index (bmi) ل أشٓش ) أستؼح. أُجش٘ انثحث خالل ٕٚه ٌ األٔل -أ د 2252كإَ كَا ًاس(ٔ كزنك يجًٕػّ أػ شػٗٔ ٍ انسٛطشجاًن ح ٛت ذرشٔا ى ػاو. )51 -51) ػذْد ٕاَٙ يٕجثاً % (51.2)٘أيشٚغ 44ٌأ ذثٍٛ 521يٍـ يجًٕع يشػٗ انسكش٘ٔ ى لذ أظٓش انؼايم انشث ًماسَحيُٓ يغ تان ى انسٛطشجح ػيجًٕ ٛاَسثح يهحٕظح يغ .%(4.5٘أ ) 5 ٔػذْد ٕاَٙ أكثش يٍ إطاتح انشجال [p= 0.01] . .إحظائ ساء تانؼايم انشث د إطاتح اُن 22 ٔكَا ًِٛـ53.4ّ٘أ )يشٚغ 53إٗن %(35.3٘أ )يشٚؼّ ٚمح انك ٕاَٙ تانطش ح انؼايم انشث جاٛت ٔا ٚا ؤالء انزٍٚ أظٓش ْ ُذ فحض ٙ. ػ ٕاـن ّ%( ػهٗـ انر quantitative ٚٙه نذسجح حع يا ٕاَٙ ، ٕن ش54.5ِ : انؼايم انشث ؼٚا ٓى أظٓش دسجح اًن ى أظٓش iu/ ml ٔ 25.5% 64 % يُ شِ دسجح يُٓ ؼٚا 52 اًن iu/ ml ٔ4ٔ22 ِش ؼٚا ى أظٓش دسجح اًن شِ iu/ml ٔ52.2 16% يُٓ ؼٚا ى أظٓش دسجح اًن ٗ .iu/ ml 8 % يُٓ شػ ؤالء اًن سّث يٍْ َ ٌأ أػٗه ٕا ػً ٕاَٙ ٘ر دسجح انكى كَا يٍ ثى ذمم ػٗه انرؼالة. iu/ ml 64 ٍ يجًٕػح انؼايم انشث ٚادج يهحٕظح [،ٔ ٍ .] .p= 0.01ص ٍُٛ ي أظٓش أغهة انثٚذ ى ػًٍ يجًٕػح ٕاَٙ overweight bmi (52.3%)يشػٗ انسكشْ٘ ٛاً يٍ انؼايم انشث شِ ػان ٌ (iu/ml 64) يؼٚا ٔا ػٗه انرؼالة. ٔانثإل ظٓش شِ% 52.3 ٓى أظٓش دسجح يؼٚا ِ iu/ ml ٔ 25.2 64 يُ ش ٓى أظٓش دسجح يؼٚا شِ iu/ ml ٔ 5363 52 % يُ ى أظٓش دسجح يؼٚا /iu 16% يُٓ ml ٔ 53.3ِش ى أظٓش دسجح يؼٚا ٕا ػًٍ يجًٕػح . iu/ ml 8 % يُٓ ٕاَٙ كَا ظٓش٘ انؼايم انشث ٔاًن َٔسّث يٍ يشػٗ انسكش٘ ٌإ أػٗه ػذد ح ًاس انؼاٛن )13-12)األػ ٔ)32-33( ى )52-53(ٔ َٔسثٓر ) 52.3يشٚغ، 55( ػاو ٗ 52.2، ػٗيش 2% ( ٔ ) 23.1، يشٚغ%55 (ٔ % ( ػه سّث يشػٗ انسكش٘ انرؼالة. َ كٌٕ فّٛ سكش انذو ٌأ ٔانز٘ٚ ٕاٙذ )يٙه يٕل نكم نرش 5.2≤ تذٌٔ سٛطشج ى انؼًم انشث %( أكثش 3563 ، 22ٔانزٍٚ نذٓٚ شع انسك سثح اًن َ سٛطش ػهّٛ > يٍ ٕاَٙ يٙه يٕل نكم نرش 5.2ش٘ اًن ى انؼًم انشث ٘ . (5364 ، 53) ٔانزٍٚ نذٓٚ ظاتٍٛ تانسكش ٍُٛ اًن ذخ ٌإ اًن ٕاَٙ ) ظٓش٘ انؼايم انشث ٕاَٙ )35.4، يشٚغ 25ٔاًن ظٓش٘ انؼايم انشث ٔاًن ظاتٍٛ تانسكش٘ ٍُٛ اًن ذخ ٕا اكثش يٍ غٛش اًن ، يشٚغ55%( كَا ّٛ أخشٖ ٌأ52.3 َاح يٍ ٕاَٙ ) %(ٔ. ٓى انؼايم انشث س نٚذ ٔٛن ٘ ظاتٍٛ تانسكش ٍُٛ اًن ًذخ ٓى 55.2، يشٚغ 35ػذد غٛش ان س نٚذ ٔٛن ٍُٛ ًذخ %( أكثش يٍ ان ٕاَٙ ) ٚادج يهحٕظح ػانّٛ 22.2يشٚغ ، 52انؼايم انشث ح ٙف ظٕٓس (.p.v= 0.000001 %(.)ص جاٛت ّٕن ٚا ٚاِد يؼم ُانك ص ْ ِز انذساسّ ذثثد ٌأ َرائجْ ٕاَٙ ن ٙ. -ٖذ يشػٗ انسكش٘ انؼايم انشث ع انثَا سثح انسكش ٙف انذو يغ اسذفاع انُٕ َ ٛاس انرشدد انشئٚشذثط ػؼف انسٛطشج ػٗه ٕاَٙؼ ٙف انحاالخ ث ح. ح انؼايم االٚجاٛت جاٛت ٛاس ٚا شذثٌا يؼ زا فٌا انرذخٍٛ لذ الٚ ظاتٍٛ تانسكش٘ ٓن ٔاًن ٍُٛ خ ظاتٍٛ تانسكش٘ أكثش يٍ غٛش اًن ٔاًن ٍُٛ ذخ ٕاَٙ ٙف اًن ط انشث ًا. ّ ٓت ًا تانراكٛذ راخ طه َٔا ٘ كًشع ًفاطم انشٕث ٔايشاع ان ٘ ِ تانسكش يثاشش انتدخٍن ،,rf, bmi انكهمات انمفتاحٍة : مرضى انسكري اننىع انثاًن introduction type 2 diabetes mellitus (t2dm) which was formerly called non-insulin dependent diabetes mellitus (niddm) or adult-onset diabetes, is a metabolic disorder that is characterized by high blood glucose level (1) .in t2dm, insulin concentrations may be normal or even high, there is insensitivity of the tissues to the effects of insulin(an effect termed insulin resistance) (2) .type 2 dm (t2dm) occurs as a result of chronic insulin resistance and subsequent beta-cell dysfunction that appears to be reversible, particularly in the early stages of the disease (3) . type 2 diabetes, is characterized by progressive insulin resistance that typically accompanies advancing age, inactivity, and weight gain (4) . rheumatoid factor (rf) rheumatoid factor is an antiimunglobulin with a course against fragment fc of igg human molecule. rheumatoid factor is present in (70-80 %) of patients with rheumatoid arthritis (ra), where the disease is defined as a seropositive athropathy (5) .the rfs frequently occur in a variety of other diseases such as, systemic lupus erythromatus (sle) (15-35%), systemic sclerosis (20-30%), juvenile rheumatoid (7-30%), poliomyelitis (5-10%) and infection (0-5%) (6) . rheumatoid factor is a well-established test used in the diagnosis and prognosis of rheumatoid arthritis (ra) (7) . in addition, rheumatoid factor precedes the appearance of rheumatoid arthritis. rheumatoid factor is sometimes found in serum of patients with other diseases including diabetes mellitus (dm), due to the presence of pro-inflammatory cytokines such as tnfα which play an important role in chronic inflammatory and autoimmune diseases like rheumatoid arthritis (ra). the tnfα has also been closely linked to obesity and insulin resistance (8) .substantial studies have been conducted in several iraqi regions, however; this study was planned to investigate the possible association between t2dm and rf in relation to bmi, age and smoking in alrisafa baghdad area. material and methods one hundred twenty five (125) patients with t2dm were attending the specialized center for endocrinology and diabetes, at al-risafa, baghdad, during the period from september to december/ 2010.age ranging between (35–75) years. in addition to seventy (70) age matched apparently healthy persons as controls, were selected from neighbours, friends and staff members of the college and who attended al-kindy general hospital for checking. their fasting plasma glucose (fpg) was within the normal range. all plasma specimens were submitted to fasting plasma glucose by enzymatic colorimetric method and rf – latex by slide agglutination test. assay methods 1. blood glucose determination (enzymatic). enzymatic, colorimetric method, based on glucose oxidase with reference for serum or plasma in fasting state (9) reference value of : 4.2-6.4 mmol/l 75-115 mg/dl 2. qualitative determination of rheumatoid factor [latex slide agglutination] principle of method: the rflatex is a slide agglutination test for the qualitative and semiqualitative detection of rf titer in human serum. latex particles coated with human e-globulin are agglutinated when mixed with sample http://en.wikipedia.org/wiki/metabolic_disorder http://en.wikipedia.org/wiki/blood_glucose iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 107 containing rf. the test occurs by using a kit (omega diagnostics avitex rf).uk. calibration the rf-latex sensitivity is a calibration against the word health organization (who) 1/64 rheumatoid arthritis serum. sample fresh serum should be used to detect measurable titer of anti-igg (rheumatoid factor). reading and interpretation the presence or absence of visible agglutination was observed by necked eye immediately after removing the slide from the rotator. semi-quantitative determination the semi-quantitative test was performed in the same way as the qualitative test using dilution of the serum with phosphate buffered saline as follows statistical analysis the parameters were treated and computerized by using spss version 15. p value < 0.05 is considered significant, while p value > 0.05 is considered non significant. dilutions 1/2 1/4 1/8 saline 50 µl 50 µl 50 µl serum 50 µl ─ ─ dilution serum 1/2 ─ 50 µl ─ dilution serum 1/4 ─ ─ 50 µl 8 x no of dilution 8x2 8x4 8x8 iu/ml 16 32 64 results out of diabetic patients, 44 (35.2%) are positive for rf, 16(36.4%) males and 28(63.6%) females, respectively. while the number and percentage of rf positive subjects out of healthy control are only 3 (4.3%), 1(33.33%) male and 2(66.66%) females, respectively. the differences are significant (p= 0.01). table 1: distribution of studied groups according to rf agglutination test diabetes mellitus patient healthy control comparison of significant gender rf male female total percent male female total percent p= 0.01 positive 16 28 44 35.2% 1 2 3 4.3% negative 36 45 81 64.8% 29 38 67 95.7% total 52 73 125 100% 30 40 70 100% table (2) shows the comparison of t2 dm patients & healthy control with rf positive in addition to rf negative according to their bmi(kg/m 2 ).the largest number and percentage of t2dm patients are the overweight patients 47 (37.6%). and when added to obese groups the total will be 108(86.4%).while the largest number and percentage for the control lie in the normal group of bmi 50 (71.4%), with a highly significant difference, p value < 0.01 between bmi and rf in dm as well as in control table 2: comparison of diabetic patients with healthy control who have rf positive and negative according to body mass index bmi (kg/m 2 ) diabetes mellitus patient healthy control comparison of significant rf bmi positive (n=44) negative (n=81) total (n=125) percent positive (n=3) negative (n=67) total (n=70) percent p < 0.01 under weight 16.5-18.4 0 3 3 2.4 % 0 0 0 0% normal 18.5-24.9 0 14 14 11.2 % 0 50 50 71.4 % overweight 25.0-29 18 29 47 37.6 % 1 14 15 21.4 % obese class i 30.034.9 12 16 28 22.4% 2 3 5 7.1 % obese class ii 35 40 10 12 22 17.6 % 0 0 0 0 % obese class iii over 40 4 7 11 8.8 % 0 0 0 0 % total 44 81 125 100% 3 67 70 100 % iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 108 table 3: distribution of diabetic patients who have rf positive and negative within age groups (years) compared with healthy control. diabetes mellitus patient healthy control comparison of significant rf age positive (n=44) negative (n=81) total (n=125) percent positive (n=3) negative (n=67) total (n=70) percent p =0.00l8 30-39 0 6 6 4.8% 0 7 7 10.0% 40-49 6 22 28 22.4% 0 21 21 30% 50-59 17 26 43 34.4% 1 25 26 37.1% 60-69 13 18 31 24.8% 2 9 11 15.7% 70-79 8 9 17 13.6% 0 5 5 7.1% total 44 81 125 100% 3 67 70 100% table (3) shows that the highest number and percent of t2dm with rf positive values are located within the age group (50-59) years. at the same age, healthy controls show also the highest number and percent. (pvalue =0.00l8) table 4:distribution of diabetic patients who have rf positive according to their glycemic control and rf titer. rf titers glycemic control level 8 16 32 64 total percent comparison of significant fpg > 7.2 mmol/l > (130 mg/dl) 2 5 9 12 28 63.6% p = 0.006 fpg < 7.2 mmol/l < (130 mg/dl) 6 4 3 3 16 36.4% total 8 9 12 15 44 100% table (4) summarizes the effect of fasting plasma glucose level of type 2 dm in relation to rf titer. it is found that > 7.2 mmol/l glucose in plasma contribute the highest titer (n=28, 63.6 %), in comparison with group of plasma glucose levels < 7.2 mmol/l patients (n=16, 36.4%). with a highly significant difference, p-value = 0.006. table 5: distribution of diabetic patients with rf positive according to their titer, in relationship to body mass index groups bmi (kg/m 2 ) rf titers iu/ml bmi 8 16 32 64 total percent under weight 16.5-18.4 0 0 0 0 0 0% normal 18.5-24.9 0 0 0 0 0 0% overweight 25.0-29 3 3 5 7 18 40.9% obese class i 30.034.9 4 3 3 4 14 31.8% obese class ii 35 40 1 2 3 2 8 18.2% obese class iii over 40 0 1 1 2 4 9.1% total 8 9 12 15 44 100% as indicated in table (5) it could be conclude the following two points. most of type 2 dm patients show rf positive (34.1%) high titer in 64 iu/ml: titer ( iu/ml) number percent 64 15 34.1 % 32 12 27.3 % 16 9 20.4 % 8 8 18.2 % most of overweight patients show high titer 64 iu/ml: (38.9 %) titer (iu/ml) number percent 64 7 38.9 % 32 5 27.8% 16 3 16.6 % 8 3 16.6% iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 109 table 6: distribution of smokers among diabetic patients with either rf positive or rf negative results rf smoke positive negative total percent comparison of significant positive 27 (61.4%) 18 (22.2%) 45 36.0% p= 0.000001 hs negative 17 (38.6%) 63 (77.8%) 80 64.0% total 44 (100%) 81 100%) 125 100 % a shown in table (6) smokers with rf positive t2 dm patients (n=27, 61.4%), dominate over the nonsmoker patients. the non smoker's diabetic patients who have rf negative (n=63, 77.8%) are nearly four times as many as smokers (n=18, 22.2%). table 7: distribution of rf positive diabetic patients according to smoking habit and rf titer rf titer iu/ml smoke 8 16 32 64 total percent comparison of significant positive 4 5 8 10 27 61.4% p= 0.000001 hs negative 4 4 4 5 17 38.6% total 8 9 12 15 44 100% table (7) explains the effect of smoking on t2dm/rf positive patients where the smokers are doubling as many as the nonsmokers, in titers 32 & 64 iu/ml, and the smoker's numbers are more than the non-smokers (4:4, 5:4, 8:4, and 10:5; total 27:17). the number of patients increases as the titer increases. discussion this work shows that about (35.2%) of the iraqi population sample (125 subjects) diabetic patients in al-risafa region have rf positive in their blood when compared with apparently normal subjects (4.3%) as referred in table (1). these percentages coincide with the results obtained by moustschen (10) in 1992.also coincide with searches done by al-gharawi at 2009, in medical city, baghdad, who observed that 62.5% out of dm patients showed rf positive (11) ; 49% have been recorded in al-umara city by khalawi (12) and 15.5% have been reported in al-adhamiya, baghdad city by al-hammami (13) .the differences are possibly due to geographical reason and because the attendants of the medical city are collection from different districts and most severely diseased. umara & a'dhamyah are confined areas. also they have different lifestyles, types of food & behavior. the result of this study (35.2%) rf positive dm patients seem to be affected by the same reasons stated above, and lie in between the results of the other authors. table (1) shows that 28 females and 16 males had rf positive (approximately 2:1 ratio) which coincides with the work that showed the women presented with ra more often than men, with a ratio of 3:1 (14) , indicating that hormone levels are of importance (15) . epidemiological and immunological evidence share suggested that female sex hormones could play a role in the etiology and course of chronic inflammatory diseases because of the menstrual cycle, pregnancy, and menopausal status which are important influencing factors (16) . table (2) shows the prevalence of t2dm with rf positive according to their bmi. a significantly higher level concentrated at overweight and obese groups. the t2dm is associated strongly with overweight, independent of age, gender and family history of dm. this relationship has been found consistently in other populations. (17-21) diabetes mellitus and ra are associated with an adverse cardiovascular risk profile, particularly dyslipidaemia (22, 23) and obesity is a state of lowgrade chronic inflammation, as indicated by the increased concentrations of creactive protein, il-6, and other inflammatory markers identified in the plasma of obese individuals (24,25) . indeed, pro-inflammatory cytokines (tnf-α, il-18, il-6) were found to be increased in patients with t2dm. (26, 27) the tnf-α, a pivotal proinflammatory cytokine in ra, arises from adipose tissue during chronic hyperglycemia in t2dm and has harmful effects on the pathway of insulin signaling. (28) the coincidence of bmi level and severity of t2dm resembles the work which concluded that obesity is well recognized as important risk factor for t2dm and impaired glucose tolerance (29, 30) . parallel to this idea, in same table (2) which shows that the positive rf is concentrated in the overweight and obese; while all of the normal bmi(patients & control) had negative rf.table (3) shows that increased number of t2dm patients who have rf positive are aged 50 and older, which resembles that of the american diabetes association (ada), showing that approximately 18.3% (8.6 million) of the americans aged 60 and older have diabetes (31) and t2dm patients are at ages (40-70) years, which coincide with other workers (32,33) .diabetes mellitus in these tables show that the prevalence increases ../اطروحه/4%20encyclopedia/diabetes_mellitus_2.htm#cite_note-dlife-34#cite_note-dlife-34 iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 110 with age. in a previous review, female's dominant over males explain by the role of the menopause on pro-inflammatory cytokine activity (34) .this review focused on the increase of pro-inflammatory cytokines with the menopause (the fall of estrogens and other gonadal steroids), another review on gonadal steroids and t and b cell immunity was presented 10 years ago, but since then, a lot of new information has been generated (35) . this is particularly true with respect to chronic diseases that formerly have not been allocated to “inflammatory diseases” such as bone resorption. this is important because rate of incidence over age for osteoporosis almost matches incidence rates of inflammatory markers, as rheumatoid arthritis (ra). table (4) shows that most of diabetic patients who have rf positive (n=28 , 63.6%) lie within fpg ≥ 7.2 mmol/l category in comparison with those with lower fpg (n=16 , 36.4%) which lie in fpg < 7.2 mmol/l. these results agree with other workers who showed that 68% of dm patients had fpg > 11.1 mmol/l and 32% of them had fpg < 11.1 mmol/l (13) .glucose intolerance table (5), presents in ra and diabetes, is another parallel, and indicator for direct correlation between the degree of impaired glucose handling and inflammation (36) . the diabetic patients who have rf positive titer, which mean the severity of disease go parallel with the increase of rf titers. comparatively, the rf in blood of the control, 4.3%, is too near to the international ratio, 3 %. in this study the results are in agreement with several studies have discussed the association between chronic inflammatory disease states and disorders in intermediary metabolism (37-41) , in particular, peripheral insulin resistance (ir).the overweight groups also gain the highest score (38.9%) among the highest rf positive titer, the 44 diabetic with rf positive value agrees with other studies (12, 13) .tables (6 and 7) show the prevalence of t2dm smokers over t2dm non-smokers to have rf positive. issues of smoking and diabetes are correlated effectively in the ada technical review (42) , it is concluded that smoking might not be the causative agent for t2dm, but definitely is related to it. these tables also show the prevalence of rf positive in smokers over non-smokers, accordingly this model explains that ra results from a complex gene–environment interaction, in which ra only develops after the immune systems has been triggered by several environmental factors, a process which may take years (43) . one of the environmental factors that have been clearly shown to trigger ra is smoking (44) . references 1. giuffrida fm, reis af. genetic and clinical characteristics of maturity-onset diabetes of the young. diabetes obesmetab 2005; 7(4): 318– 326. 2. henry m. kronenbery. kenneth s. polonsky and p. reed. williams textbook of endocrinology, 11th ed. saunders eisevier 2008;(30): p: 13301389. 3. wajchenberg bl. β-cell failure in diabetes and preservation by clinical treatment.endocr rev. 2007; 28: 187–218. 4. stumvoll m, goldstein bj, van haeften tw. type 2 diabetes: pathogenesis and treatment. lancet 2008; 371:2153-6. 5. rosmus k., sandow d., paulke b.r., et al. detection of igm rheumatoid factors using elisa and agglutination tests with new latex. z. gesamte. hyg.1990; 36(6): 223-225. 6. helen.ch. mansel, h., siraj m., neil s., essential of clinical immunolology. 7 th ed. 1984 chap 15 p: 270. 7. halldorsdottir hd, jonsson t, thorsteinsson j, valdimarsson h. a prospective study on the incidence of rheumatoid arthritis among people with persistent increase of rheumatoid factor. ann rheum dis 2000; 59: 149–151. 8. borst se. the role of tnf-alpha in insulin resistance. endocrine 2004; 23: 177–182. 9. tietz, n.w. clinical guide to laboratory tests. 2 nd edition. philadelphia, pa:wb saunders co.;1990; p. 246-250. 10. moustschen, m.d., a., j., scheen, p., j., and lefebvre (1992) impaired immune response in d.m. analysis of the factor and mechanism involved relevance to the increase susceptibility of diabetic patient to specific infection. diabetes and metabolism 18 th ed, 1992; p: 187 – 201. 11. al-gharawi, ali. m. the relationship between hyperglycemia and the rheumatoid factor in the serum of diabetic patients. umsalama science journal, 2009; 6(1): 321 12. khalawi, m.m. the correlation between hyperglycemia and rheumatoid factor in diabetic patients type 2 with anthropometric measurement, h. diploma thesis, college of health & medical technology, 2009;p:76 13. hammami, a.t. the correlation between hyperglycemia and rheumatoid factor in diabetic patients type 2 with anthropometric measurement, h. diploma thesis, college of health & medical technology, 2010;p: 71 14. oliver je, silman aj. why are women predisposed to autoimmune rheumatic diseases? arthritis res ther. 2009; 11(5): 252. 15. jochems c, islander u, kallkopf a, lagerquist m, ohlsson c, carlsten h. role of raloxifene as a potent inhibitor of experimental postmenopausal polyarthritis and osteoporosis. arthritis rheum. 2007 ;56(10): 3261-70. 16. cutolo m, wilder rl different roles for androgens and estrogens in the susceptibility to autoimmune rheumatic diseases. rheum dis clin north am ,2000 ;26: 825–839. 17. al-nuaim ar: prevalence of glucose intolerance in urban and rural communities in saudi arabia. diabet med 1997;14: 595-602. ../اطروحه/4%20encyclopedia/diabetes_mellitus_2.htm#cite_note-dlife-34#cite_note-dlife-34 ../اطروحه/4%20encyclopedia/diabetes_mellitus_2.htm#cite_note-dlife-34#cite_note-dlife-34 iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 111 18. baltazar jc, ancheta ca, aban ib, fernando re, baquilod mm: prevalence and correlates of diabetes mellitus and impaired glucose tolerance among adults in luzon, philippines. diabetes res clin pract 2004;64: 107-115. 19. tamayo-marco b, faure-nogueras e, rocheasensio mj, rubio-calvo e, sanchez-oriz e, salvador-olivan ja: prevalence of diabetes and impaired glucose tolerance in aragon, spain. diabetes care 1997; 20: 534-536. 20. satman i, yilmaz t, sengul a, salman s, salman f, uygur s, bastar i, tutuncu y, sargin m, dinccag n, karsidag k, kalaca s, ozcan c, and king h: population-based study of diabetes and risk characteristics in turkey: results of the turkish diabetes epidemiology study (turdep). diabetes care 2002; 25: 15511556. 21. sullivan pw, morrato eh, ghushchyan v, wyatt hr, hill jo: obesity, inactivity, and the prevalence of diabetes and diabetesrelated cardiovascular comorbidities in the u.s., 20002002. diabetes care 2005;28: 1599-1603. 22. steiner g, urowitz mb. lipid profiles in patients with rheumatoid arthritis: mechanisms and the impact of treatment. semin arthritis rheum 2009; 38: 372–381. 23. chahil tj, ginsberg hn. diabetic dyslipidemia. endocrinol metab clin north american 2006; 35:491–viii. 24. gimeno, r. e., and l. d. klaman.. adipose tissue as an active endocrine organ: recent advances. curr. opin. pharmacol. 2005; 5: 122– 128. 25. yudkin, j. s., c. d. stehouwer, j. j. emeis, and s. w. coppack. c-reactive protein in healthy subjects: associations with obesity, insulin resistance, and endothelial dysfunction. a potential role for cytokines originating from adipose tissue? arterioscler. thromb. vasc. biol. 1999;19: 972–978. 26. crook, m. a., p. tutt, and j. c. pickup.. elevated serum sialic acid concentration in niddm and its relationship to blood pressure and retinopathy. diabetes care. 1993;16: 57–60. 27. pickup, j. c., m. b. mattock, g. d. chusney, and d. burt.. niddm as a disease of the innate immune system: association of acute-phase reactants and interleukin-6 with metabolic syndrome x. diabetologia.1997;40:1286–1292. 28. 28fukuzawa m, satoh j, qiang x, miyaguchi s, sakata y, nakazawa t, et al.inhibition of tumor necrosis factor-alpha with anti-diabetic agents. diabetes res clin pract 1999; 43: 147– 154. 29. hasletl, c., edwin, p.c., john, a., a. principle and practice of medicine, 18 th ed., edinburgh, london, 1999; p: 524 – 525. 30. naomi, s., levilt, j., deborah, m.n. and francois, prevalence and identification of risk factors for niddm in urban african in cape town, south africa. diabetes care, 1993; 16, 601 – 607. 31. seniors and diabetes, elderly and diabetes diabetes and seniors. life med media (2006). retrieved on 2007; p. 5-14. 32. harrison, e., b.;"principle of internal medicine". part four, nutritional hormonal and metabolism dieses, 2001;p. 2109 – 2001. 33. halett, c, chilvers e., hunter j.a, boon n.a. "davidson principle and practice of medicine, edinburgh, churchill livingstone", 1996; p. 254 – 247, p. 578 – 584. 34. pfeilschifter j, koditz r, pfohl m, schatz h changes in pro-inflammatory cytokine activity after menopause. endocr rev 2002; 23: 90– 119. 35. olsen nj, kovacswjgonadal steroids and immunity. endocr rev 1996;17: 369–384. 36. dessein ph, joffe bi, stanwix ae, christian bf, veller m. the acute phase response does not fully predict the presence of insulin resistance and dyslipidemia in inflammatory arthritis. j rheumatol 2002; 29: 462-466. 37. aggarwal bb. signaling pathways of the tnf super family: a double-edged sword. nat rev immunol 2003; 3(9): 745-756. 38. firestein gs. the t cell cometh: interplay between adaptive immunity and cytokine networks in rheumatoid arthritis. j clin invest 2004; 114(4): 471-4. 39. migita k, maeda y, miyashita t, kimura h, nakamura m, ishibashi h, et al. the serum levels of resistin in rheumatoid arthritis patients. clin exp rheumatol 2006; 24(6): 698-701. 40. popa c, netea mg, van riel pl, van der meer jw, stalenhoef af. the role of tnf17 alpha in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. j lipid res 2007; 48(4): 751-762. 41. tam ls, tomlinson b, chu tt, li tk, li ek. impact of tnf inhibition on insulin resistance and lipids levels in patients with rheumatoid arthritis. clin rheumatol 2007; 26(9): 14951498. 42. haire-joshu d, glasgow re, tibbs tl. smoking and diabetes. diabetes care 1999; 22: 1887–1898. 43. klareskog l, padyukov l, ronnelid j, et al. genes, environment and immunity in the development of rheumatoid arthritis. curr opin immunol 2006; 18:650–655. 44. lundstrom e, kallberg h, alfredsson l, et al. geneenvironment interaction between the drb1 shared epitope and smoking in the risk of anti-citrullinated protein antibody-positive rheumatoid arthritis: all alleles are important. arthritis rheum 2009; 60: 1597–1603. ../../../../wiki/index.php key: cord-0070899-gke00q25 authors: gholami, ahmad; azizpoor, jassem; aflaki, elham; rezaee, mehdi; keshavarz, khosro title: cost-effectiveness analysis of biopharmaceuticals for treating rheumatoid arthritis: infliximab, adalimumab, and etanercept date: 2021-11-28 journal: biomed res int doi: 10.1155/2021/4450162 sha: 2a9494775130ba7fc8b596912f2acd91fb9d5e44 doc_id: 70899 cord_uid: gke00q25 introduction: rheumatoid arthritis (ra) is a chronic progressive inflammatory disease that causes joint destruction. the condition imposes a significant economic burden on patients and societies. the present study is aimed at evaluating the cost-effectiveness of infliximab, adalimumab, and etanercept in treating rheumatoid arthritis in iran. methods: this is a cost-effectiveness study of economic evaluation in which the markov model was used. the study was carried out on 154 patients with rheumatoid arthritis in fars province taking infliximab, adalimumab, and etanercept. the patients were selected through sampling. in this study, the cost data were collected from a community perspective, and the outcomes were the mean reductions in das-28 and qaly. the cost data collection form and the eq-5d questionnaire were also used to collect the required data. the results were presented in the form of an incremental cost-effectiveness ratio, and the sensitivity analysis was used to measure the robustness of the study results. the treeage pro and excel softwares were used to analyze the collected data. results: the results showed that the mean costs and the qaly rates in the infliximab, adalimumab, and etanercept arms were $ 79,518.33 and 12.34, $ 91,695.59 and 13.25, and $ 87,440.92 and 11.79, respectively. the one-way sensitivity analysis confirmed the robustness of the results. in addition, the results of the probabilistic sensitivity analysis (psa) indicated that on the cost-effectiveness acceptability curve, infliximab was in the acceptance area and below the threshold in 77% of simulations. the scatter plot was in the mentioned area in 81% and 91% of simulations compared with adalimumab and etanercept, respectively, implying lower costs and higher effectiveness than the other two alternatives. therefore, the strategy was more cost-effective. conclusion: according to the results of this study, infliximab was more cost-effective than the other two medications. therefore, it is recommended that physicians use this medication as the priority in treating rheumatoid arthritis. it is also suggested that health policymakers consider the present study results in preparing treatment guidelines for ra. rheumatoid arthritis (ra) is a progressive inflammatory disease characterized by inflammation of the synovial mem-brane and may eventually lead to joint destruction [1, 2] . due to its long-term chronic and safety course, it is immediately required to treat with immunomodulatory medications [3] . this debilitating condition is supposed to affect 0.3-1.2% of the world's population [4] . uncontrolled ra leads to progressive joint destruction and performance reduction [5] . these conditions impose a significant underlying economic burden, reduce the quality of life (qol), and lead to productivity loss [6] . disease-modifying antirheumatic medications (dmards) such as methotrexate, sulfasalazine, and hydroxychloroquine may delay the disease progression [7] . however, many patients do not achieve an appropriate response, and some do not maintain a reaction due to ineffectiveness or toxicity [8] . nowadays, physicians are trying to achieve less disease activity or, preferably, recovery, rather than simply slowing the progression of the disease and controlling the symptoms [9] . biopharmaceuticals are drugs that are obtained from biological sources by biotechnological methods [10] [11] [12] . the more important these drugs become in medicine, the more attention is paid to concerns such as biosimilars, cost-effectiveness, and price control. the therapeutic value of biopharmaceuticals for the healthcare system is not yet well understood, and this only happens when policymakers understand the effects of these biological products on the economic system of healthcare facilities [13, 14] . the discovery of biopharmaceuticals leads to a dramatic change in the therapeutic approach to ra and results in better qol [15] . however, success requires the purchase of these medications at high prices [4, 5] , which may ultimately increase the financial burden that ra imposes on the community. such a scenario represents the need for pharmacoeconomics evaluations to inform policymakers and decision-makers about the cost-effectiveness of biological dmards [5, 16] . tnf inhibitors are a class of biopharmaceuticals applicable for treating crohn's disease, ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, and/or juvenile idiopathic arthritis. according to the fda, this category of medicines includes infliximab, etanercept, adalimumab, certolizumab pegol, and golimumab. although the side effects of these medicines are not yet fully understood, several side effects are still under investigation. some of these well-known adverse effects are bacterial, fungal, viral, or atypical infections, the risk of malignancies, especially lymphomas, congestive heart failure nyha class iii or iv, drug-induced lupus demyelinating disorders, including optic neuritis, multiple sclerosis, and local injection site reaction/ erythema. infliximab is a chimeric monoclonal antibody composed of fixed human and variable mouse regions [17] . this medication can only be used intravenously and should be used in combination with methotrexate if possible. the starting dose of the medicine is 3 mg per kg of body weight and can be increased up to 10 mg/kg with an interval of 4-8 weeks. in mid-2001, the fda/ema approved infliximab combined with methotrexate to treat ra [18] . infliximab inhibits tnf-α binding to its target receptors and prevents the production of other proinflammatory cytokines, including interleukin and gcsf [19] . common side effects of infliximab therapy include acute injection reactions, infections, and delayed hypersensitivity reactions. the medication is contraindicated in people with moderate to severe heart failure and tuberculosis or other severe or opportunistic infections [20] . adalimumab is a recombinant human igg1 monoclonal antibody with no mouse ingredient produced by phage display technology. the fda/ema approved it in 2002 to treat moderate to severe ra to be used alone or in combination with other dmards. adalimumab is injected subcutaneously every two weeks [21] . the common side effects of the medication include injection reactions and site infection. adalimumab is contraindicated in people with moderate to severe heart failure and active tb or people with other severe or opportunistic infections. before starting the treatment, physicians should examine the patients for active and inactive (latent) tuberculosis infection [20] . etanercept is also a recombinant human tnf receptor fusion protein that attenuates the effects of endogenous tnf by competitively inhibiting its interaction with cell surface receptors. etanercept has been proved to be effective in patients with rheumatoid arthritis and is injected subcutaneously at 25 or 50 mg once or twice a week [22] . considering different medical costs, medication of various financial and economic consequences of these biopharmaceuticals is not clear on the health system, and there is limited knowledge about their cost-effectiveness. since the researchers could not find any studies that have compared these medications' cost-effectiveness, the present study was conducted to determine and compare the cost-effectiveness of infliximab, adalimumab, and etanercept for patients with ra. this is a cross-sectional study for the economic evaluation of cost-effectiveness in patients with ra in fars province in 2019. the study population included all the patients with ra referred to the rheumatology department of hafez hospital and the rheumatologists' offices in 2019 and who were treated with one of the following three medications: adalimumab, infliximab, and etanercept. the sample sizes of the patients treated with adalimumab, infliximab, and etanercept based on previous studies, 80% power, and 5% error using the ncss statistical software were 48, 53, and 53, respectively. 2.1. description of the model. in this study, the markov model was used to evaluate the cost-effectiveness of infliximab, adalimumab, and etanercept for treating patients with ra and describing the progression of the disease. as in previous studies, three-month markov cycles and the time horizon until the end of life were considered. the disease activity score-28 (das-28 due to the evaluation of 28 joints) was used to show the clinical course of the disease. das-28 is a standard measure of ra activity, and the score it provides indicates whether the current treatment has worked for the patient. the doctor or nurse calculates the das-28 with a special calculator based on several tests, including joint examinations, blood tests, and a selfassessment of how the condition is felt during the investigations. as a rule, the lower the das-28 score, the better the patient's condition has been controlled. more severe joint damage is often associated with a higher das-28 score [23, 24] . figure 1 shows a schematic diagram of the markov model for ra. biomed research international the costs and outcomes used in the model were discounted based on the discount rates of 5.8% [25] and 3% [26] , respectively. furthermore, microsoft excel and tree-age pro softwares were used to analyze the collected data. probabilities. all transition probabilities are reported in table 1 , based on the previously published studies. 2.3. cost data. in this study, the societal perspective was used to extract the costs. the related costs from a societal perspective included direct medical costs (dmc), direct nonmedical costs (dnmc), and indirect costs (ic). dmc related to each of the three medications were retrospectively collected from january 1, 2019, to december 31, 2019, using a researcher-made checklist by referring to the rheumatology department of hafez hospital and the personal offices of rheumatologists. dnmc, as well as ic, were also collected using the cost data collection form and the patients' selfreport. the human capital approach was applied to calculate the indirect costs. furthermore, for international comparisons, the costs were converted into dollars (ppp) using international dollars using a purchasing power parity (ppp) $ exchange rate of 22075 rials per 1 $ rial in 2019 [29] . utility values were also extracted using the eq-5d questionnaire, and the health outcomes were evaluated based on quality-adjusted life years (qaly) [30] . to measure the utility scores, we carried out face-to-face interviews or telephone calls with 154 ra patients in 2019. the interviews were conducted with the outpatients referring to the hospitals and clinics affiliated to shiraz university of medical sciences. it should be noted that an eq-5d questionnaire is a standard tool for measuring health outcomes, introduced by the euroqol group in 1990 (https://euroqol.org/). it includes five questions on five aspects of mobility, self-care, routine activities, pain/discomfort, and anxiety/depression. the respondents' scores range from 0 to 1, and higher scores mean better utility. the patients with ra who were willing to participate in this study were interviewed accordingly. once the eq-5d questionnaire was completed, the values of iran, determined in a separate survey of goudarzi et al. [31] using time tradeoff (tto), were considered, and the 5-digit codes of the questionnaire were changed to numerical utility. after obtaining the costs and utilities through the previous steps, the incremental cost-effectiveness ratio (icer) was calculated using the following formula: 2.6. uncertainty analysis. finally, the one-way sensitivity analysis and probabilistic sensitivity analysis (psa) were used to investigate the effects of parameter uncertainty on the results. to do the one-way sensitivity analysis, some critical parameters of the study, such as cost and utility, were changed by 20% for each medication strategy. then, the results were presented in the form of a tornado diagram. also, the psas were conducted since the utility and cost variables in the present study were measurable and probabilistic, and they were considered distributions so that beta distribution (β) was used to determine the distribution of utility values (0 to 1). the gamma distribution was also used to determine the cost distribution, based on which secondorder monte carlo simulation was performed using 5000 trials. the psa results are presented using the costeffectiveness acceptability curve and the incremental costeffectiveness scattered plot. the cost-effectiveness acceptability curve is one of the best curves for planning and policy-making. it can help the policymakers and planners of the health system measure the cost-effectiveness probability of each intervention in return for willingness to pay for the expenses. on the other hand, the scatter plot provides more detailed information in individual comparisons. it indicates the percentage of the points in the acceptance area, i.e., below the threshold [32] . an explicit threshold for willingness to pay (wtp) is not available in iran. therefore, according to who suggestion for developing countries, the willingness to pay was determined as one to three times the gross domestic product (gdp) per capita qaly [33] . gdp was about $ 12547 in iran in 2019, used as the threshold for willingness to pay [34] . according to the present study results, a majority of the patients were females (73.37%) and housewives (62.33%), and all the patients had insurance coverage. besides, 94.34%, 87.5%, and 88.68% of those treated with infliximab, adalimumab, and etanercept were 18-65 years old, respectively. given that in economic studies, the ages 18 to 65 are considered the productivity ages, they are economically significant. in general, according to table 2 , the total treatment costs for infliximab, adalimumab, and etanercept were $ 11,675.21, $ 12337.62, and $ 11406.79, respectively. thus, the cost of treatment with etanercept was the lowest. as shown in table 2 , the number of people whose das-28 (biologic medication threshold) dropped from 5.1 to <2.6 was 27 (51%), 33 (68.75%), and 29 (54.72%) in the case of infliximab, adalimumab, and etanercept, respectively. according to qaly, the highest utility scores of the patients with ra obtained from the eq5d questionnaire were those of the patients using etanercept who had das-28 < 2:6 (0.891). as shown in figure 2 and table 3 , the results of utility cost analysis using the markov model showed that the mean costs and qaly in infliximab, adalimumab, and etanercept arms were $ 79,518.33 and 12.34, $ 91,695.59 and 13.25, and $ 87,440.92 and 11.79, respectively. these results indicate that treatment with infliximab or adalimumab was predominant over treatment with etanercept and was more costeffective. however, the cost-effectiveness ratio calculated for adalimumab treatment compared to infliximab was $ 13,420.09, suggesting that $ 13,420.09 had to be spent for each additional qaly in the patients treated with adalimumab. in this case, icer had to be compared with the threshold to decide. the method provided by the who was used to calculate the threshold; thus, if the icer were lower than one times gdp per capita, the program would be much cost-effective, and if it were lower than three times gdp per capita, the program would be cost-effective [33] . the gdp per capita was $ 12547 in 2019 [34] . besides, considering that the icer was $ 13,420.09, more than one times gdp per capita, adalimumab treatment was not more costeffective than infliximab treatment due to the icer of over one times threshold. 3.1.1. one-way sensitivity analysis. figure 3 shows the percentage of change in the incremental cost-effectiveness ratio in treating infliximab vs. adalimumab. the total cost-effectiveness ratio is also presented with $ 13,420.12. according to the tornado diagram results, icer had the highest sensitivity to the reduction of utility in the treatment with adalimumab in remission mode and the minor sensitivity to the decrease in other costs of adalimumab in the weak state of the disease. therefore, if utility in the treatment with adalimumab changes in remission mode, considering that the icer value will still become a positive number, it cannot be decided with certainty that infliximab has superiority over adalimumab. figure 4 shows the percentage of change in the incremental cost-effectiveness ratio of infliximab treatment compared to the treatment with etanercept. the number $ -14,348.30 indicates the incremental cost-effectiveness ratio. the tornado diagram results show that icer was the most sensitive to reducing utility in treatment with infliximab in an intermediate state and had the least sensitivity to the reduction of other utilities of the infliximab in the low and remission states. furthermore, given that in this case, the icer value was again negative, it could be decided with certainty that infliximab was superior to etanercept. 3.2. probabilistic sensitivity analysis (psa). the psa results were uncertainly presented using the cost-effectiveness acceptability curve and the incremental cost-effectiveness scattered plot. the acceptability curve result based on qaly shows that infliximab was below the cost-effectiveness threshold of $ 12547 ppp (one times gdp) in 77% of the simulations and, therefore, was the most cost-effective medication therapy strategy ( figure 5 ). in addition, the results of the scatter plots based on qaly (figures 6(a) and 6(b) ) showed that compared to adalimumab and etanercept, infliximab was in the acceptance area and below the threshold in 81% and 91% of the 5 biomed research international simulations, respectively. this result indicates lower costand higher effectiveness than the other two alternatives and, therefore, is a more cost-effective strategy. for the first time, this study was conducted to evaluate the cost-effectiveness of infliximab, adalimumab, and etanercept in patients with ra in iran. this study is aimed at comparing three medicines that act against tnf-alpha, which was widely used to treat ra. all three medicines are considered equally effective in terms of clinical value for physicians, and the main difference is in their price. therefore, the subject of the present study was which ones are more cost-effective used against tnf-alpha? all three medicines studied in this research are used subcutaneously through an autoinjector pen and are no different in this regard. according to the present study findings, treatment with infliximab, adalimumab, and etanercept had a mean cost of $ 11,675.21, $ 12,337.62, and $ 11,406.79 ppp, respectively, for each one-year treatment course. thus, the mean treatment cost per patient taking etanercept was lower than treatment with the two other medications. in this regard, the results are consistent with those of the studies by tang et al., carter et al., and ramírez-herráiz et al. [35] [36] [37] . the dmc, dnmc, and ic of the patients using infliximab were $ 9004.00 (77.12% of the total costs), $ 2484.67 (21.28% of the total costs), and $ 186. 53 [38] [39] [40] [41] [42] . the results of this study showed that the number of the patients whose das-28 dropped from 5.1 (biologic medication biomed research international threshold) to <2.6 in the infliximab, adalimumab, and etanercept groups was 27 (51%), 33 (68.75%), and 29 (54.72%), respectively. this result indicates that adalimumab was the most effective medication. a study carried out by cárdenas et al. examined the costeffectiveness of infliximab, adalimumab, and etanercept over two years showing that adalimumab was more effective than the other two medications [43] . furthermore, the results of the study by wiens et al. that entitled the analysis of effectiveness and safety of adalimumab, etanercept, and infliximab for the treatment of ra indicated that shortterm therapy with etanercept and adalimumab was most effective, while long-term treatment with adalimumab was the most effective [44] . in a study entitled direct comparison of therapeutic responses, disease control, and medication adherence in patients with ra treated with adalimumab, etanercept, and infliximab, hetland et al. (2009) concluded that infliximab had the lowest therapeutic response, the lowest rate of recovery, and the lowest rate of medication adherence. however, adalimumab had the highest therapeutic response and remission rate, while etanercept had the highest medication adherence [45] . in this respect, the results are consistent with the findings of the present study. the study results by santos-moreno et al. conducted as a cohort in colombia to directly compare the effectiveness of adalimumab, etanercept, and infliximab showed that in the beginning, the das-28 was 4.1 but it changed to 2.39 after 36 months. the most common complication was dermatitis. it was finally concluded that all three medications reduced the severity of the disease, and etanercept had a lower incidence of side effects than the other two medications. it is in line with the present study regarding the effectiveness of all three medications in reducing the symptoms and controlling the disease [46] . according to the present study results, the highest utility of each medication was found in the patients with das-28 < 2:6, and as the disease activity score-28 (das-28) increased, the life desirability decreased. as the disease activity score-28 (das-28) increased, more joints got involved in the disease, and the effect of the medications was usually reduced. therefore, the patients entered the severe phase of the disease, and it could be natural that their life desirability decreased [47] . the cost-utility analysis results using the markov model showed that the mean costs and qaly amount in the infliximab, adalimumab, and etanercept arms were $ 79,518.33 and 12.34, $ 91,695.59 and 13.25, and $ 87,440.92 and 11.79, respectively. thus, treatment with infliximab or adalimumab was predominant over etanercept and was also more effective. besides, the comparison of the threshold introduced by the who (one times gdp-per capita) and zrubka et al. conducted a systematic study and evaluated the long-term efficacy and cost-effectiveness of infliximab as a first-line treatment for ra. the results showed that the recovery of the ra patients treated with infliximab was significant within six months compared to the control group. over a year, the improvement was remarkable in those who used infliximab than the control strategies [47] . in this respect, the results are consistent with those of the present study. in taiwan, chen et al. examined the cost-effectiveness of tofacitinib vs. adalimumab and concluded that the qaly obtained in treatment with tofacitinib was 0.09 more than adalimumab (5.13 vs. 5.04). besides, the incremental cost-effectiveness was 143122 qaly/$ nt. the one-way sensitivity analysis confirmed that the results were robust [49] . these results are in line with those of the present study. fatemi et al. conducted a study in iran and examined the cost-effectiveness of tofacitinib vs. adalimumab and etanercept. they concluded that tofacitinib was more costeffective than the two others, and although tofacitinib had fewer qalys than etanercept (6.664 vs. 6.876), it cost less on the lifetime horizon ($ 42,565.04 vs. $ 58,696.29). tofacitinib also cost less than adalimumab ($ 50,299.91 vs. $ 51,550.29) and had more qalys (6,900 vs. 6,687). the sensitivity analysis also showed that the results were sensitive to the cost of the medications [50] . these findings are in line with those of the present study. in a study in brazil entitled the cost-effectiveness analysis of ra medications, dos-santos et al. suggested that golimumab was the most effective medication. it was also the dominant option compared to etanercept. on the other hand, the adalimumab icur was $ 95,095.37. the sensitivity analysis indicated that the results were robust [51] . the results of a study by chastek et al. on the comparative efficacy of tnf blockers in ra patients treated with adalimumab, etanercept, and infliximab from january 1, 2006 january 1, , to 2008 showed that etanercept had the lowest dose and the patients showed the best response to infliximab [52] . this study is in line with the results of the present study. in their study entitled "biological medications for ra in medicare: cost-effectiveness analysis", wailoo biomed infliximab was more costly [53] . this conclusion might be due to the higher price of this medication. curtis et al. conducted a study on the cost-effectiveness of biological medications in ra patients with commercial insurance, in which the subjects were 18 to 63 years old. they finally indicated that etanercept was the most costeffective option [54] . their study results are inconsistent with the present research, which could be the lower price of etanercept compared to other medications. also, the one-way sensitivity analysis results on infliximab and etanercept confirmed the robustness of the study results and indicated that infliximab could be a superior medication compared to etanercept. the probabilistic sensitivity analysis results showed that on the cost-effectiveness acceptability curve, infliximab was in the acceptance area and below the threshold in 77% of the simulations. the medication was also in the acceptance area of cost-effectiveness scattered plot, e.g., below the threshold in 81% and 91% of the simulations compared to adalimumab and etanercept. this finding indicates its lower costs and higher effectiveness than the other two alternatives, and therefore, the strategy was more cost-effective. the present study had some limitations as the limited data required, especially for the disease transition probabilities. hence, fixed rates were used in this study. in addition, intangible costs were not calculated in this study due to the impossibility of measuring them accurately. regarding the generalizability of the results, it can be said that since the medications are used in all provinces and medical centers of iran to treat ra patients and their 9 biomed research international prices are the same throughout the country, the results of this study can be generalized to other provinces and the whole country. however, it is necessary to consider the following items to generalize the results to other countries: epidemiology of the disease and demographic structure, existence of resources, prices, evaluation of outcomes by individuals, threshold, and the use of various effectiveness indicators in different studies that may affect the results of the present study. therefore, caution is needed when generalizing the results to other countries. according to the results of this study, infliximab was more cost-effective than the other two medications. therefore, based on the sensitivity analysis results, as long as the study parameters do not change significantly, it is suggested that infliximab should be used as the priority for treating patients with ra. also, health policymakers and managers should try to increase insurance coverage and reduce outof-pocket payments. all data used to support the findings of this study are included within the article. the study protocol was approved by the ethics committee of shiraz university of medical sciences under the ethical code ir.sums.rec.1399.100. all participants were informed both verbally and through written information of their right to withdraw from the study at any time. all participants gave their written consent to participate in the study. the authors declare that they have no competing interests. agh participated in the study's design, supervised the whole study, and revised the paper critically for important intellectual content. ja is assigned to the study concept and design and participated in literature bibliography, acquisition of data, analysis, and interpretation of data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. ea drafted the manuscript and revised the paper. mr participated in the analysis of data, drafting of the manuscript, and editing the article. khk participated in the design of the study, drafting of the manuscript, and final revision. all authors read and approved the final manuscript. evidence-based review of biologic markers as indicators of disease progression and remission in rheumatoid arthritis biological agents in rheumatoid arthritis safety of rituximab in rheumatoid arthritis modelling cost-effectiveness of biologic treatments based on disease activity scores for the management of rheumatoid arthritis in spain health economics: implications for novel antirheumatic therapies expanding role of biologic agents in rheumatoid arthritis reduction in long-term disability in patients with rheumatoid arthritis by disease-modifying antirheumatic drug-based treatment strategies second-line drug therapy for rheumatoid arthritis biologic agents for rheumatoid arthritis-negotiating the nice technology appraisals recent biotechnological approaches for treatment of novel covid-19: from bench to clinical trial functionalized graphene oxide with chitosan for protein nanocarriers to protect against enzymatic cleavage and retain collagenase activity cloning, characterization and bioinformatics analysis of novel cytosine deaminase from escherichia coli agh09 a comprehensive insight towards pharmaceutical aspects of graphene nanosheets computational design of a chimeric epitope-based vaccine biomed research international to protect against staphylococcus aureus infections cost effectiveness of tnf-inhibitors in rheumatoid arthritis cost utility of tumour necrosis factor-α inhibitors for rheumatoid arthritis infliximab in the treatment of rheumatoid arthritis treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor α anti-tnf alpha therapy is useful in rheumatoid arthritis and crohn's disease: analysis of the mechanism of action predicts utility in other diseases adalimumab, etanercept, infliximab, rituximab and abatacept for the treatment of rheumatoid arthritis after the failure of a tumour necrosis factor inhibitor: a systematic review and economic evaluation monoclonal antibodies in rheumatoid arthritis etanercept as monotherapy in patients with psoriasis validation of the 28-joint disease activity score (das28) and european league against rheumatism response criteria based on c-reactive protein against disease progression in patients with rheumatoid arthritis, and comparison with the das28 based on erythrocyte sedimentation rate interpretation of das28 and its components in the assessment of inflammatory and noninflammatory aspects of rheumatoid arthritis estimation social discount rate for iran estimation of private and social time preferences for health in northern tanzania an evaluation of the public health responsibility deal: informants' experiences and views of the development, implementation and achievements of a pledge-based, publicprivate partnership to improve population health in england the mortality of rheumatoid arthritis ppp conversion factor, gdp (lcu per international $) -iran, islamic rep quality of life assessment using euroqol eq-5d questionnaire in patients with deep infiltrating endometriosis: the relation with symptoms and locations valuation of quality weights for euroqol 5-dimensional health states with the time tradeoff method in the capital of iran a costutility and cost-effectiveness analysis of different oral antiviral medications in patients with hbeag-negative chronic hepatitis b in iran: an economic microsimulation decision model thresholds for the cost-effectiveness of interventions: alternative approaches iran-gdp per capita treatment persistence with adalimumab, etanercept, or infliximab in combination with methotrexate and the effects on health care costs in patients with rheumatoid arthritis efficiency of adalimumab, etanercept and infliximab in rheumatoid arthritis patients: dosing patterns and effectiveness in daily clinical practice adalimumab, etanercept, and infliximab utilization patterns and drug costs among rheumatoid arthritis patients the effect of dose escalation on the cost-effectiveness of etanercept and adalimumab with methotrexate among patients with moderate to severe rheumatoid arthritis cost-effectiveness of adalimumab, etanercept, and tocilizumab as first-line treatments for moderate-tosevere rheumatoid arthritis cost per treated patient for etanercept, adalimumab, and infliximab across adult indications: a claims analysis cost-effectiveness of real-world infliximab use in patients with rheumatoid arthritis in sweden cost-effectiveness modelling of biological treatment sequences in moderate to severe rheumatoid arthritis in france real-world costeffectiveness of infliximab, etanercept and adalimumab in rheumatoid arthritis patients: results of the create registry meta-analysis of the efficacy and safety of adalimumab, etanercept, and infliximab for the treatment of rheumatoid arthritis direct comparison of treatment responses, remission rates, and drug adherence in patients with rheumatoid arthritis treated with adalimumab, etanercept, or infliximab: results from eight years of surveillance of clinical practice in the nationwide danish danbio registry direct comparative effectiveness among 3 anti-tumor necrosis factor biologics in a real-life cohort of patients with rheumatoid arthritis long-term efficacy and cost-effectiveness of infliximab as first-line treatment in rheumatoid arthritis: systematic review and meta-analysis cost-utility analysis of second-line therapy with rituximab compared to tumour necrosis factor inhibitors in rheumatoid arthritis tofacitinib in the treatment of moderate-tosevere rheumatoid arthritis: a cost-effectiveness analysis compared with adalimumab in taiwan cost-effectiveness analysis of tofacitinib compared with adalimumab and etanercept in the treatment of severe active rheumatoid arthritis; iranian experience cost-utility analysis of the anti-tnf therapy for rheumatoid arthritis in a real-world based model comparative effectiveness of tnf blockers in rheumatoid arthritis patients biologic drugs for rheumatoid arthritis in the medicare program: a cost-effectiveness analysis cost and effectiveness of biologics for rheumatoid arthritis in a commercially insured population this manuscript is retrieved from the pharmacy thesis supported and approved by shiraz university of medical sci key: cord-0030844-qv8oznba authors: song, jihyeon; kim, jun yeong; you, gayeon; kang, yoon young; yang, jiwon; mok, hyejung title: formulation of glycyrrhizic acid-based nanocomplexes for enhanced anti-cancer and anti-inflammatory effects of curcumin date: 2022-05-02 journal: biotechnol bioprocess eng doi: 10.1007/s12257-021-0198-7 sha: 77bbccc4fd30b4c897c6a3ba6a2fea365ec328a6 doc_id: 30844 cord_uid: qv8oznba in this study, nanocomplexes composed of glycyrrhizic acid (ga) derived from the root of the licorice plant (glycyrrhiza glabra) were formulated for the delivery of curcumin (cur). sonication of amphiphilic ga solution with hydrophobic cur resulted in the production of nanosized complexes with a size of 164.8 ± 51.7 nm, which greatly enhanced the solubility of cur in aqueous solution. a majority of the curs were released from these ga/ cur nanocomplexes within 12 h. ga/cur nanocomplexes exhibited excellent intracellular uptake in human breast cancer cells (michigan cancer foundation-7/multi-drug resistant cells), indicating enhanced anti-cancer effects compared to that of free cur. in addition, ga/cur nanocomplexes demonstrated high intracellular uptake into macrophages (raw264.7 cells), consequently reducing the release of the pro-inflammatory cytokine tumor necrosis factor-α. furthermore, ga/cur nanocomplexes successfully reduced the levels of serum pro-inflammatory cytokines and splenomegaly in a rheumatoid arthritis model. taking into consideration the wide range of phytochemicals that have been used for combination therapies in herbal medicine, it is crucial to elucidate the mechanisms of herb-herb interactions and herb-drug interactions [1, 2] . licorice is a popular guide drug used to harmonize different ingredients in traditional herbal medicines [3] . glycyrrhizic acid (ga), in particular, is a major component of licorice [4] . ga, composed of glycyrrhetinic acid in combination with two glucoses, is well known for its anti-inflammatory, antivirus, anti-oxidant, and anti-cancer effects [5, 6] . in addition, ga has been widely used and studied as a natural sweetener and solubilizer in the cosmetic as well as pharmaceutical industries [7] . more recently, ga has been under study as a carrier material for hydrophobic drugs, such as paclitaxel and camptothecin, owing to its amphiphilic characteristics [8] . curcumin (cur), derived from the rhizomes of the curcumin longa l. plant, is popularly proposed as a pleiotropic molecule due to its promising and diverse biological effects, including various anti-cancer, anti-malarial, anti-oxidant, and anti-inflammatory properties [9, 10] . despite the superior bioactivity and therapeutic potential of cur, its poor aqueous solubility and absorption in vivo limit its practical application as a drug [11, 12] . to overcome these limitations, nanomaterials have been developed to enhance the stability and solubility of cur; for example, liposomes, polymer nanoparticles, exosomes, and conjugates [13, 14] . however, simple formulation processes for cur that eliminate laborious formulation steps are yet to be established. in this study, cur was formulated in conjunction with ga to enhance its solubility and bioavailability in vitro as well as in vivo. the solubility of cur in aqueous solution was examined by transmission (%) and particle sizes at various ga/cur weight ratios using a uv spectrophotometer and via dynamic light scattering (dls). similarly, the antioxidant effect of ga/cur nanocomplexes was assessed via a hydrogen peroxide (h 2 o 2 ) scavenging assay. moreover, the anti-cancer and anti-inflammatory effects of ga/cur nanocomplexes were evaluated in michigan cancer foundation-7/multi-drug resistant cells (mcf-7/mdr cells) and raw264.7 macrophages, respectively. the levels of cytokines tumor necrosis factor-α (tnf-α) and interleukin-1β (il-1β) in serum were quantitatively measured in a rheumatoid arthritis (ra) mouse model, following the treatment with ga/cur nanocomplexes in vivo. preparation and characterization of ga/cur nanocomplexes ga (10 mg) dissolved in etoh and cur (10 mg) in dmso were mixed at diverse ga/cur weight ratios (ga/ cur weight ratio = 0, 0.5, 1, 1.5, and 4), followed by the dropwise addition of samples into distilled water (dw) under sonication (branson digital sonifier 450; branson ultrasonics, danbury, ct, usa) in an ice bath for 10 min using 30% amplitude, pulse on 8 sec, pulse off 2 sec [15, 16] . the transmission (%) of the ga/cur nanocomplexes in 8% etoh solution was measured at a wavelength of 600 nm using a uv-vis spectrophotometer (beckman coulter du730; beckman coulter, inc., brea, ca, usa) [17] . after various types of ga/cur nanocomplexes were diluted with dw to a final cur concentration of 200 µg/ml, the hydrodynamic size of each particle was measured via dls (malvern instruments ltd., malvern, uk). subsequently, the different ga/cur nanocomplexes were incubated for 5 min in pbs solution containing 5% tx-100, followed by dls at room temperature in order to evaluate the particle sizes. the release profile of cur from ga/cur nanocomplexes was analyzed in vitro using the dialysis diffusion method, as outlined in a previous study [18] . briefly, 2 ml of freshly prepared ga/cur nanocomplexes (ga/cur weight ratio = 4) and cur in dw were transferred to dialysis membranes (mwco = 3.5 kda) and dialyzed in 100 ml of pbs solution (ph 7.4). after different incubation times (0, 1, 3, 6, 12, 24, and 36 h) under stirring at 60 rpm, the solution in the dialysis membrane was diluted with pbs solution containing 1% tx-100. the quantity of cur in the solution was assessed by measuring the absorbance at a wavelength of 425 nm using a uv-vis spectrophotometer. in order to evaluate the antioxidant activity of the ga/cur nanocomplexes, an h 2 o 2 scavenging assay was performed as described in our previous study [19] . cur and ga/ cur nanocomplexes (ga/cur weight ratio = 4) were prepared at various cur concentrations (0, 4, 8, 12, 16, and 20 µg/ml) in dw. ga dissolved in etoh was further diluted with dw at the corresponding concentrations of the ga/cur nanocomplexes (0, 16, 32, 48, 64, and 80 µg/ml). each solution was subsequently mixed with h 2 o 2 (20 mm) in pbs solution at a sample:h 2 o 2 solution volume ratio of 1:2. following incubation for 10 min at room temperature, the absorbance of each solution was measured at a wavelength of 230 nm using a uv-vis spectrophotometer to quantify the remaining hydrogen peroxide. since the absorbance of ga was detected at a wavelength of 230 nm, the correction value was obtained by subtracting the absorbance of the nanoparticle solution excluding the h 2 o 2 . 2.5. anti-cancer effect of ga/cur nanocomplexes in mcf-7/mdr cells mcf-7/mdr cells were maintained in rpmi-1640 supplemented with 10% fbs, 100 u/ml penicillin, and 100 µg/ml streptomycin at 37°c in a humidified atmosphere of 5% co 2 . to observe the differential uptake of ga/cur nanocomplexes at the specific ga/cur weight ratios of 1 and 4, mcf-7/mdr cells were seeded in 4-well plate chambers (falcon culture slides; falcon, franklin lakes, nj, usa) at a density of 2.5 × 10 5 cells per well 24 h prior to the treatment. subsequently, cur and ga/cur nanocomplexes were prepared as described above and used to treat the cells at a cur concentration of 25 µm (9.2 µg/ml) in serum-free medium, followed by incubation for 2 h at 37°c in a humidified atmosphere of 5% co 2 . after washing thrice with pbs containing 5% fbs, the transfected cells were fixed with 3.7% formaldehyde in pbs solution for 10 min at room temperature. the fixed cells were subsequently washed with pbs solution for 1 min and then stained with mounting medium and covered with a cover slip. the fixed cells were visualized using an inverted fluorescence microscope (axio200; carl zeiss, land baden-württemberg, germany) at an excitation wavelength of 525 nm and emission wavelength of 550 nm. mcf-7/mdr cells were seeded in 6-well plates at a density of 5 × 10 5 cells per well, 24 h prior to treatment. cur and ga/cur nanocomplexes at a ga/cur weight ratio of 4 were used to treat cells for 4 h at a cur concentration of 25 µm (9.2 µg/ml), in the presence of 10% fbs. following this treatment, cells were washed with pbs and detached via centrifugation at 5,000 rpm at 4°c for 10 min. the resulting cell pellet was washed thrice with pbs solution containing 5% fbs and lysed in 1% tx-100 in pbs solution for 10 min. after centrifugation of the cell lysate at 13,000 rpm at 4°c for 10 min, the fluorescence intensity (fi) of the supernatant was analyzed using a fluorescence microplate reader at excitation and emission wavelengths of 430 nm and 520 nm, respectively. to quantify the anti-cancer effect of cur, mcf-7/mdr cells were seeded in 24-well plates, at a density of 6 × 10 4 cells per well 24 h prior to the treatment. cur and ga/ cur nanocomplexes at a ga/cur weight ratio of 4 were added to the cells at a final cur concentration of 10 µm. after treatment for 24 or 48 h, the viable cells were stained with 0.4% trypan blue solution in pbs solution and counted using a bright-line™ hematocytometer. 2.6. cellular uptake of ga/cur nanocomplexes in raw264.7 cells raw264.7 cells (belonging to a murine macrophage cell line) were maintained in dmem supplemented with 10% fbs, 100 u/ml penicillin, and 100 µg/ml streptomycin at 37°c in a humidified atmosphere of 5% co 2 , as described previously [20] . the cells were seeded in 6-well plates at a density of 6 × 10 5 cells per well, 24 h prior to the treatment. cur and ga/cur nanocomplexes were used to treat the cells for 2 h at a cur concentration of 25 µm (9.2 µg/ml) in serum-containing medium. after the cells were collected using a scraper, the cells were spun down and the pellet was lysed in 1% tx-100 in pbs solution. this was followed by centrifugation of the cell lysate at 13,000 rpm at 4°c for 10 min. subsequently, the fi in the supernatant of each cell lysate was measured using a fluorescence microplate reader. the concentration of tnf-α in the conditioned medium of lps-stimulated raw264.7 cells induced via lps was evaluated [21] . raw264.7 cells were initially plated in 6well plates at a density of 6 × 10 5 cells per well, 24 h prior to the treatment. subsequently, the cells were pre-treated with cur and ga/cur nanocomplexes (ga/cur weight ratio of 1.5 and 4) at a cur concentration of 10 µm (3.7 μg/ml) for 6 h. ga (18 µm, 15 μg/ml) in complete media was used as a control. cells were further stimulated with lps (100 ng/ml) in fresh complete media in the presence of cur, ga/cur nanocomplexes, and ga. after co-treatment for 18 h, the concentration of tnf-α released in each sample was quantified using a tnf-α elisa kit, according to the manufacturer's protocol. all animal care and experimental procedures were approved by the animal care committee of konkuk university. the ra models were prepared according to a previous study [22] . briefly, bovine type ii collagen (4 mg/ml) dissolved in 0.1 m acetic acid was homogenized on ice to block protein denaturation, followed by the dropwise addition of complete freud's adjuvants to form a mixture with a 1:1 volume ratio. the resulting emulsion (100 μl) was intradermally injected into the tail of dba-1/j mice (female, 6week-old). following the first immunization, a secondary injection of emulsion was performed on the 21st day after the initial immunization for boosting. after ra modeling, the dba-1/j mice were randomly divided into five groups: non-treated group (normal group, nor); ra mice treated with pbs solution (control group, con); ra mice treated with ga bare complexes (80 mg/kg) in pbs solution (ga); ra mice treated with cur (20 mg/kg) in pbs solution (cur); and ra mice treated with ga/cur nanocomplexes (ga 80 mg/kg and cur 20 mg/kg; ga/cur in pbs solution), according to previous studies [23] . all samples (500 μl) were freshly prepared and injected intraperitoneally every alternate day, from the 21st days post first immunization till the 45th day. upon completion of 45 days following the initial immunization, whole mouse blood was isolated from the heart via cardiac puncture and the mice were then euthanized. blood sera were subsequently isolated by centrifuging whole blood at 2,000 g for 15 min [24] . the levels of tnf-α and il-1β in the serum were quantitatively measured using mouse tnf-α and il-1β elisa, respectively. additionally, all isolated spleens and thymuses were weighed. in this study, to enhance the aqueous solubility of cur, ga was complexed with cur via tip sonication [25] . during sonication, cur was homogeneously dispersed with amphiphilic ga, resulting in the formation of ga/cur nanocomplexes in aqueous solution through hydrophobic interactions (fig. 1) . after formulation, ga/cur nanocomplexes were used to treat two types of cells (breast cancer cells and macrophages) to assess their anti-cancer and anti-inflammatory activities as compared to free cur in vitro. moreover, following the preparation of the ra models, ga/cur nanocomplexes were administered intraperitoneally to examine the anti-inflammatory effects in vivo. as the weight ratio of ga/cur increased from 0 to 4, the solubility of cur in dw increased proportionately, as investigated by measuring the transmission (%) of the solution. fig. 2a depicts the transmission (%) of cur solution at a wavelength of 600 nm, after mixing cur with ga at different ga/cur weight ratios. while transmissions of the ga/cur solution were below 5% at a ga/cur weight ratio of 0.5, those at ga/cur weight ratios of 1, 1.5, and 4 were observed to be 71.3 ± 10.9, 89.6 ± 11.1, and 94.4 ± 11.8%, respectively. the inset in fig. 2a presents photographic images of the ga/cur solution at various ga/cur weight ratios. by the formation of ga/ cur nanocomplexes, cur was homogeneously dispersed up to 200 μg/ml in our study. considering that the solubility of free cur is approximately below 11 ng/ml in aqueous solution and ~183.4 ng/ml in oil, ga/cur nanocomplexes could successfully increase the solubility of cur [12, 26] . the particle sizes of the ga/cur nanocomplexes were examined at various ga/cur weight ratios of ga/cur by dls, as represented in fig. 2b . as the ga/cur weight ratios increased, the hydrodynamic sizes of the ga/cur nanocomplexes decreased correspondingly. in the absence of ga, cur exists as larger aggregates in an aqueous solution, with sizes over 500 nm. however, the size of ga/ cur nanocomplexes at a ga/cur weight ratio of 4 was 164.8 ± 51.7 nm, with a polydispersity index of 0.3 ± 0.1. in addition, ga/cur nanocomplexes at a ga/cur weight ratio of 4 showed homogeneous size distribution (inset of fig. 2b ). when ga/cur nanocomplexes were prepared at a ga/cur weight ratio of 4, the ga concentration was 1 mm, which is the critical micelle concentration of ga [15, 27] . after incubation of ga/cur in the presence of a surfactant (5% tx-100 in pbs solution), no noticeable nanoparticles were observed. this result indicates that ga/ cur nanocomplexes were formed via reversible noncovalent interactions; for example, hydrophobic interactions. to further examine whether free cur could be released from ga/cur nanocomplexes, ga/cur nanocomplexes contained within dialysis bags (3.5 kda) were incubated in pbs solution for predetermined time intervals, and the released cur was measured using a uv-vis spectrophotometer. as depicted in fig. 2c , cur was almost completely released (approximately 95.6%) in a sustained manner over 12 h. cur is a well-known antioxidant molecule that is crucial for its biological effects. to examine whether the antioxidant effect of ga/cur nanocomplexes was reduced, an h 2 o 2 scavenging assay was performed (fig. 2d) . ga/cur nanocomplexes at a ga/cur weight ratio of 4 exhibited excellent h 2 o 2 scavenging activity (95.3 ± 1.4%), at a cur concentration of 20 μg/ml. this result indicates that complexation with ga has negligible harmful effects on the antioxidant activity of cur bioactivity. according to our previous study, cur nano-formulation using nanosized exosomes also produced enhanced antioxidant effects compared to free cur [19] . it is likely that the homogeneous dispersion of cur via nano-complexation might provide excellent antioxidant effects in aqueous solutions. to examine the intracellular delivery of ga/cur nanocomplexes, mcf-7/mdr cells were treated with cur and ga/cur nanocomplexes for 2 h. in our previous study, the expression levels of p-glycoprotein in mcf-7/mdr cells were higher than that in mcf-7 cells [28] . because of the high expression of p-glycoprotein on cellular membranes, free cur demonstrated negligible intracellular accumulation, as depicted in fig. 3a . however, ga/cur nanocomplexes with a ga/cur weight ratio of 4 exhibited much higher intracellular localization than free cur. the amount of intracellular cur was also quantitatively measured via the fluorescence intensity of cur within cells, after incubation of the mcf-7 and mdr cells with cur and ga/cur nanocomplexes, respectively (fig. 3b) . intracellular fluorescence intensities of cur and ga/cur were observed to be 14.3 ± 1.8 and 21.2 ± 2.7, respectively. fig. 3a and 3b indicate that ga/cur nanocomplexes allowed increased intracellular localization of cur, which might be crucial for bioactivity. to determine the anti-cancer effect of ga/cur nanocomplexes, cancer cell viability was assessed via a cell counting after treatment of the samples for 24 h. as presented in fig. 3c , the viability of cells with cur and ga/cur nanocomplexes was 57.1 ± 4.4 and 21.3 ± 4.4%, respectively, when compared to that of the control group (100% viability). this result indicates that the anti-cancer activity demonstrated by ga/cur nanocomplexes was approximately 2.6-folds higher than that of cur, which might be attributed to the increased intracellular localization of cur. however, ga exerted negligible effects on the viability of mcf-7/mdr cells at various concentrations (fig. 3d) . although several studies have reported the anti-cancer effects of ga in breast cancer cells, noticeable effects of ga were not observed in our results, possibly due to the multidrug-resistance of breast cancer cells [29, 30] . according to previous studies, cur exhibits antiinflammatory properties via the suppression of prostaglandin synthesis, which is closely related to cyclooxygenase-2 and inducible nitric oxide synthase signaling for pro-inflammatory reactions [31, 32] . to assess the anti-inflammatory activity of macrophages, intracellular uptake of ga/cur nanocomplexes was examined in raw264.7 cells, as depicted in fig. 4a . while cells treated with ga/cur nanocomplexes demonstrated a fluorescence intensity of 54.8 ± 6.2, those that underwent treatment with cur had a fluorescence intensity of 29.5 ± 8.8. this result clearly indicates that the ga/cur nanocomplexes could be efficiently delivered into the macrophages. furthermore, in order to investigate the anti-inflammatory effects of ga/cur nanocomplexes, raw264.7 cells were treated with lps at a concentration of 100 ng/ml for 18 h in vitro to activate inflammatory responses for macrophages, as described in a previous study [21] . after pre-treatment of the ga/cur nanocomplexes and cur only for a duration of 6 h, the same were treated with lps at a concentration of 100 ng/ml for 18 h. the level of inflammatory responses was quantitatively assessed by measuring the amount of released tnf-α, a representative pro-inflammatory cytokine, using elisa (fig. 4b) . the concentration of released tnf-α from cells treated with cur and ga/cur nanocomplexes was observed to be 44.3 ± 11.1 and 30.1 ± 6.8 ng/ml, respectively, at a cur concentration of 10 μm, while the cytokine level in cells treated with lps only was 70.8 ± 6.4 ng/ml. in previous studies, cur and ga exhibited anti-rheumatoid effects in vivo that were correlated to their anti-inflammatory activity in vivo [33] . to determine the anti-inflammatory effects of ga/cur nanocomplexes, a collagen-induced ra mouse model was prepared by intradermal administration of bovine type ii collagen and complete freund's adjuvants (fig. 5a) . following the 13 times injection of ga/cur nanocomplexes at concentration of 20 mg/kg of cur every alternate day, sera of all mice were collected on the 45th day to determine the amount of pro-inflammatory cytokines, tnf-α and il-1β [34] . the concentration of tnf-α in blood sera was observed as follows: 3.4 ± 3.0, 18.1 ± 6.4, 9.7 ± 4.9, 8.3 ± 5.4, and 6.3 ± 4.2 pg/ml after treatment with nor, con, ga, cur, and ga/cur nanocomplexes, respectively (fig. 5b ). in addition, the levels of il-1β in blood sera were as follows: 1.1 ± 0.8, 2.4 ± 0.5, 0.9 ± 1.0, 3.1 ± 0.8, and 1.2 ± 0.8 pg/ml after treatment with nor, con, ga, cur, and ga/cur nanocomplexes, respectively (fig. 5c) . these results clearly indicate that ga/cur nanocomplexes greatly reduced tnf-α as well as il-1β levels in the ra model, which suggests that ga/cur nanocomplexes produce enhanced anti-inflammatory effects in the ra model, as compared to cur alone. furthermore, splenomegaly and thymus enlargement observed in the ra model are sometimes used as indicators of rheumatoid arthritis [35] . the weights of spleen were 76.5 ± 10.5, 130.8 ± 25.0, 97.6 ± 20.0, 138.6 ± 29.4, and 95.3 ± 15.0 mg in the mice treated with nor, con, ga, cur, and ga/cur, respectively (fig. 5d ). while con and cur groups possessed enlarged spleens, the spleen sizes in the ga and ga/cur groups were similar to that of the nor group. however, there were no noticeable differences in the weight of the thymus in any of the groups (fig. 5e ). this result suggests that ga/cur nanocomplexes can reduce inflammation in vivo without producing any side effects, such as splenomegaly [36] . this study demonstrated the formulation and application of a ga-based nano-complex as a simple and facile carrier for cur, both in vitro and in vivo. ga/cur nanocomplexes with a size of 164.8 ± 51.7 nm exhibited promising antioxidant activity and anti-cancer activity in mcf-7/mdr cells, along with anti-inflammatory activity in raw264.7 cells in vitro. the same could potentially be attributed to increased aqueous solubility. in the ra mouse model, ga/cur nanocomplexes revealed remarkably reduced release of inflammatory cytokines (tnf-α and il-1β) after intraperitoneal administration, without any splenomegaly. taken together, ga/cur nanocomplexes could serve as a promising delivery system for cur with improved in vivo activity and safety. synergistic effects of chinese herbal medicine: a comprehensive review of methodology and current research complementary medicine use during cancer treatment and potential herb-drug interactions from a cross-sectional study in an academic centre liquorice, a unique "guide drug" of traditional chinese medicine: a review of its role in drug interactions ga/cur) nanocomplexes in the rheumatoid arthritis (ra) mouse model. (b, c) the extent of released (b) tumor necrosis factor-α (tnf-α) and (c) interleukin-1β (il-1β) levels in serum (*p < 0.05). (d, e) the weight of (d) spleen and (e) thymus after administration of nanocomplexes biosynthesis-based quantitative analysis of 151 secondary metabolites of licorice to differentiate medicinal glycyrrhiza species and their hybrids glycyrrhizic-acid-based carbon dots with high antiviral activity by multisite inhibition mechanisms glycyrrhizic acid nanoparticles as antiviral and anti-inflammatory agents for covid-19 treatment therapeutic effects of glycyrrhizic acid glycyrrhizic acid as a multifunctional drug carrier -from physicochemical properties to biomedical applications: a modern insight on the ancient drug therapeutic effects of curcumin in inflammatory and immune-mediated diseases: a nature-made jack-of-all-trades? health perspectives of a bioactive compound curcumin: a review recent advances in colloidal delivery systems for nutraceuticals: a case study -delivery by design of curcumin highly bioavailable forms of curcumin and promising avenues for curcumin-based research and application: a review curcumin nanoformulations: a review of pharmaceutical properties and preclinical studies and clinical data related to cancer treatment commensal flora triggered target anti-inflammation of alginate-curcumin micelle for ulcerative colitis treatment formulation and evaluation of novel glycyrrhizic acid micelles for transdermal delivery of podophyllotoxin bioavailability enhancement of paclitaxel via a novel oral drug delivery system: paclitaxelloaded glycyrrhizic acid micelles physical and chemical stability of curcumin in aqueous solutions and emulsions: impact of ph, temperature, and molecular environment stable curcumin-loaded polymeric micellar formulation for enhancing cellular uptake and cytotoxicity to flt3 overexpressing eol-1 leukemic cells evaluation of the enhanced antioxidant activity of curcumin within exosomes by fluorescence monitoring exosomemodified plga microspheres for improved internalization into dendritic cells and macrophages nardochinoid b inhibited the activation of raw264.7 macrophages stimulated by lipopolysaccharide through activating the nrf2/ho-1 pathway collagen-induced arthritis curcumin loaded solid lipid nanoparticles ameliorate adjuvant-induced arthritis in rats byakangelicin as a modulator for improved distribution and bioactivity of natural compounds and synthetic drugs in the brain biobased polymeric surfactant: natural glycyrrhizic acid-appended homopolymer with multiple ph-responsiveness an insight into curcumin-based photosensitization as a promising and green food preservation technology polyakov (2021) ph-sensitive glycyrrhizin based vesicles for nifedipine delivery enhanced intracellular uptake and stability of umbelliferone in compound mixtures from angelica gigas in vitro glycyrrhizic acid-induced differentiation repressed stemness in hepatocellular carcinoma by targeting c-jun n-terminal kinase 1 formulation of injectable glycyrrhizic acidhydroxycamptothecin micelles as new generation of dna topoisomerase i inhibitor for enhanced antitumor activity glycyrrhizic acid and 18β-glycyrrhetinic acid modulate lipopolysaccharideinduced inflammatory response by suppression of nf-κb through pi3k p110δ and p110γ inhibitions glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing tlr4 recruitment into lipid rafts in raw264.7 cells curcumin attenuates collagen-induced rat arthritis via anti-inflammatory and apoptotic effects evaluation of tnf-alpha and il-1 blockade in collagen-induced arthritis and comparison with combined anti-tnf-alpha/anti-cd4 therapy angiotensin-(1-7) attenuates collagen-induced arthritis via inhibiting oxidative stress in rats combined use of etanercept and mtx restores cd4 in spleen and thymus in collagen-induced arthritis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations this study was supported by a grant (nrf-2020r1a2b5b 01001677) from the national research foundation funded by the ministry of education, science, and technology, korea. the authors declare no conflict of interest. neither ethical approval nor informed consent was required for this study. 163farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati abstract bisphosphonates (bps) are osteoclast-mediated bone resorption inhibitors and the nature of the groups attached to the central carbon atom determines the drug potency. neridronate is an injectable aminobisphosphonate, structurally similar to alendronate and pamidronate, authorized for the treatment of osteogenesis imperfecta (oi). this drug has often been used to treat other pathologies, as an off-label option, to increase tolerability and ameliorate compliance, partly because the management of orally administered bisphosphonates, with their gastrointestinal side effects, results complicated. in this paper pharmacokinetic, pharmacodinamic and main placebo-controlled clinical trials on oi patients are reviewed. the available scientific evidence demonstrates the neridronate efficacy to improve spine and hip bone mineral density, to lower markers of skeletal turnover and to decrease fracture incidence, compared with controls. we also report clinical trials results and data about the effect of intravenous infusions of neridronate in patients with postmenopausal osteoporosis, paget’s disease and rheumatoid arthritis. finally, we consider the economical impact of chronic and incapacitating pathologies, like osteogenesis imperfecta, on family’s total income and the influence of the disease on quality of life of pediatric and adult patients. farmeconomia e percorsi terapeutici 2004; 5 (3): 163-178 neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano orietta zaniolo*, mario eandi* introduzione i bifosfonati sono analoghi del pirofosfato in cui il ponte p–o–p è stato sostituito con un ponte p–c–p, non idrolizzabile. grazie alla loro particolare attività nel mantenere l’omeostasi minerale ossea, sono attualmente i farmaci più utilizzati nel trattamento dell’osteolisi tumorale con ipercalcemia e del morbo di paget, oltre che nella prevenzione primaria e secondaria dell’osteoporosi. il neridronato, un amino-bifosfonato di recente introduzione, è stato utilizzato con successo in alcuni trial clinici per il trattamento dell’osteoporosi post-menopausale, del morbo di paget e soprattutto dell’osteogenesi imperfetta (oi), malattia rara e gravissima contro la quale questo farmaco ha mostrato risultati molto incoraggianti. l’oi è una patologia ereditaria del tessuto connettivo, caratterizzata da fragilità ossea e fratture ricorrenti che, in molti casi, conducono a deformità scheletriche. la maggior parte dei pazienti con osteogenesi imperfetta presenta una diminuzione della densità ossea anche se a volte, sia negli adulti sia nei bambini, l’aspetto radiologico appare normale. la densitometria ossea a doppio raggio-x conferma la diminuzione della densità dell’osso sia trabecolare (vertebre, coste) sia corticale (omero, femore), soprattutto in caso di gravidanza [1], dopo la menopausa e, negli uomini, dopo i 50 anni [2]. pur trattandosi di una patologia eterogenea in termini sia di ereditarietà (dominante, recessiva, sporadica/nuova mutazione) sia di espressività fenotipica, gli individui affetti da *farmacologia clinica, università di torino review 164 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati oi comunemente presentano bassa statura, sclere blu, dentinogenesi imperfetta, perdita di udito nell’età adulta, scoliosi e lassità legamentosa. generalmente, sulla base delle caratteristiche cliniche e genetiche, si distinguono quattro tipi di oi (tabella 1). i soggetti affetti dalle forme più gravi presentano fratture multiple sin dalla nascita e sviluppano precocemente deformazioni scheletriche: il decesso nella grande maggioranza dei casi avviene nelle prime ore o nei primi giorni di vita; la sopravvivenza oltre l’anno di vita è rara. nelle forme severe (tipo iii e iv) c’è sempre il rischio di complicazioni cardio-polmonari, dovute a deformità spinali e toraciche, che possono portare all’exitus durante l’infanzia [3]. il numero e l’età di inizio delle fratture e delle deformità hanno un ruolo nel determinare il raggiungimento della capacità di camminare e quindi la futura autonomia del bambino [4]. la causa principale di questa patologia è da ricercare nella mutazione dei geni del collagene di tipo i [6]; non sono state rilevate, al momento, particolari correlazioni tra i fenotipi clinici e specifiche mutazioni, anche se sembra che quelle localizzate nella parte n-terminale dell’elica del collagene siano associate a fenotipi meno gravi, mentre quelle della parte c-terminale sarebbero associate a forme di malattia più gravi. nei casi più lievi le deformità scheletriche non sono costanti, a dispetto delle fratture multiple, e l’altezza dei pazienti può essere quasi normale [5]. in questo caso, la capacità motoria non è ridotta in maniera grave, per cui spesso è mantenuta la possibilità di deambulare e di una vita autonoma. invece quasi tutti i pazienti con oi tipo iii e una buona parte dei pazienti con tipo iv sono costretti sulla sedia a rotelle, malgrado gli ausili e una riabilitazione intensiva. profilo farmacologico clinico del neridronato farmacodinamica il neridronato appartiene alla classe dei bifosfonati, composti che agiscono sul metabolismo osseo impedendo il riassorbimento, con conseguente diminuzione del turnover osseo. la struttura chimica p–c–p possiede grande affinità per il ca++ (base della selettività per l’osso di questi composti) ed è molto resistente all’idrolisi in ambiente acido e all’azione delle pirofosfatasi: caratteristiche fondamentali per l’azione farmacologica. gli effetti scheletrici dei bifosfonati sono mediati da un’azione inibitoria diretta sul reclutamento, la differenziazione, l’attività e il ciclo vitale degli osteoclasti, nonché da un’azione indiretta sugli osteoblasti, in cui induce una diminuzione nel rilascio dei mediatori che in situazioni normali attivano gli osteoclasti. ciò determina una rapida inibizione dell’attività osteoclastica e un effetto, più ritardato nel tempo, sulla deposizione osteoblastica [7-9]. nella maggior parte dei casi, si ottiene un sostanziale incremento della densità minerale ossea [10]. il meccanismo con cui questa classe di farmaci esplica i propri effetti non è completamente chiaro, ma pare che essi rallentino la formazione e la dissoluzione dei cristalli di idrossiapatite mimando l’azione del loro analogo naturale, il pirofosfato. in particolare, l’elevata affinità in vivo della struttura p–c–p per l’idrossiapatite induce la deposizione dei bifosfonati nella matrice ossea [11-13]. si ipotizza che una volta incorporati nella matrice ossea, i bifosfonati siano inglobati durante i processi di riassorbimento osseo dagli osteoclasti maturi mediante endocitosi, inibendo la loro attività mediante il blocco della produzione di enzimi litici, delle secrezioni acide e tabella 1 classificazione dell�osteogenesi imperfetta (generalmente, sulla base delle caratteristiche cliniche e genetiche, si distinguono quattro tipi di oi) neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano enoizacifissalc iopit erelcs,enegallocled)%05acric(ovitatitnauqticifednuadatazzirettarac,evargonemeetneuqerfùipamrof atuicsonocsimosseps.ataznavaàte'lleneaiznafni'lleneruttarfidaznedicniatatnemua,ulb iiopit elatanirepetromnocevargùipamrofalè iiiopit iedatividàtitnauqeàtilauqonanoizidnocehcehcirtelehcsàtimrofedivarg,arutatsassabnocarevesamrof attefrepmiisenegonitnedeulberelcs,itneizap viopit ehcirtelehcsàtimrofedetsedomeassabotsottuiparutatsnociiieiopitliartaidemretniamrof 165farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati delle prostaglandine. inoltre essi indurrebbero negli osteoclasti alterazioni morfologiche apoptotiche (retrazione, condensazione nucleare, frammentazione cellulare) e biochimiche (frammentazione del dna e attivazione delle caspasi), riducendo la durata media della vita di queste cellule e i correlati processi di riassorbimento. un’altra possibilità è che il bifosfonato accumulato all’interno dell’osso venga nel tempo rilasciato in piccole quantità alle circostanti aree di elevato turnover; ciò spiegherebbe il fatto che, nonostante i bassi livelli di farmaco attivo circolante, una singola somministrazione spesso sia sufficiente a mantenere una sostanziale inibizione del riassorbimento osseo anche per alcuni anni. l’azione indiretta svolta dai bifosfonati si esplica a livello degli osteoblasti, i quali, in condizioni fisiologiche, attivano, mediante un legante presente sulla loro membrana (rankl), un recettore espresso sulla membrana del precursore emopoietico dell’osteoclasta (rank) inducendo la sua differenziazione e maturazione a osteoclasta attivo. il farmaco probabilmente interferisce con questo meccanismo, inducendo la produzione di osteoprotegerina (opg) da parte degli osteoblasti. l’opg è una proteina naturale che, legandosi essa stessa al rankl, impedisce l’azione del ligando sul rank e la conseguente osteoclastogenesi [14,15]. analizzando le curve dose-risposta relative al neridronato e agli altri bifosfonati è possibile osservare che anche basse concentrazioni di farmaco sono in grado di indurre gli effetti clinici desiderati; per esempio osteoblasti esposti per soli 5 minuti a una modestissima dose di bifosfonato sono stati in grado di inibire la maturazione osteoclastica [16,17]. tali considerazioni suggeriscono l’esistenza di un recettore o di un sito di legame cellulare specifico per questa classe di farmaci, anche se a oggi esso non è stato ancora identificato. studi in vitro e in vivo hanno dimostrato che il trattamento con neridronato comporta riduzioni significative dei parametri di riassorbimento e della fosfatasi alcalina indice di turnover osseo, senza però modificare il processo di mineralizzazione. i marker biochimici del riassorbimento subiscono decrementi molto rapidi (pochi giorni nel caso di somministrazione parenterale) anticipando di alcune settimane la riduzione della fosfatasi alcalina, che mostra un andamento più lento e graduale. la rapida inibizione del riassorbimento osseo provoca un transitorio disaccoppiamento tra i processi di neoformazione e quelli di riassorbimento; questo stato perdura sino a quando non venga raggiunto un nuovo equilibrio, con l’adeguamento della neosintesi ossea ad un livello di turnover più basso. la prevalenza dei processi di osteosintesi durante la fase di disaccoppiamento determina la riduzione dell’afflusso di calcio verso l’ambiente extracellulare e un temporaneo aumento della secrezione di paratormone. l’insorgenza di un iperparatiroidismo secondario permette di contrastare l’ipocalcemia incrementando la sintesi di calcitriolo e stimolando l’assorbimento intestinale di calcio. inoltre l’aumento del paratormone diminuisce il riassorbimento tubulare dei fosfati, diminuendone in tal modo la concentrazione sierica [18]. farmacocinetica i bifosfonati hanno una bassissima biodisponibilità orale, peraltro condizionata dalla contemporanea assunzione di cibo o farmaci: dopo somministrazione per via orale solo una minima quantità di bifosfonato viene assorbita (biodisponibilità orale inferiore all’1% per i bifosfonati azotati). questo è parzialmente spiegato dalla loro bassa lipofilia, dalla elevata carica negativa e dalla forma insolubile con cui si presentano a livello intestinale a causa del legame con il calcio. il contatto di questi farmaci con la mucosa dell’apparato digerente può provocare fenomeni irritativi [19,20]: risulta quindi importante assumere i bifosfonati con abbondante acqua demineralizzata calda, a completo digiuno (sia prima che dopo) e stando in piedi per almeno trenta minuti [21]. al contrario, le formulazioni parenterali, come quelle con cui viene somministrato il neridronato, sono quelle che garantiscono una piena biodisponibilità del principio attivo nel caso di somministrazione endovenosa e, spesso, anche intramuscolare. le caratteristiche farmacocinetiche del sodio neridronato sono state valutate nel plasma e nelle urine di ratto dopo somministrazione endovenosa singola di 10mg/kg. i risultati dell’analisi hanno dimostrato una concentrazione massima (0,06 mg/ml) raggiunta subito dopo la somministrazione, seguita da una fase rapida di riduzione delle concentrazioni plasmatiche, dovuta ai fenomeni di distribuzione tissutale e di eliminazione; nello studio il 31,7% del farmaco veniva escreto nelle urine durante le 24 ore successive alla somministrazione. nell’uomo l’infusione endovenosa lenta di 25, 50 e 100 mg ha mostrato una chiara linearità e proporzionalità fra le dosi; con la somministrazione intramuscolare, i profili di escreo. zaniolo, m. eandi 166 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati zione, a parità di dose, sono confrontabili con quelli osservati dopo infusione endovenosa [18]. nel plasma i bifosfonati sono legati alle proteine, ma la rilevanza di questo legame varia a seconda del ph e della concentrazione di calcio: esso viene favorito dalla presenza di calcio e di ph elevati. questa classe di farmaci tende a scomparire rapidamente dal circolo sanguigno: una percentuale compresa tra il 20 e il 50% della dose è captata dall’osso entro 12-24 ore; essi presentano un tropismo particolare per il tessuto trabecolare, caratterizzato da un alto turnover [22]. in passato si è sempre pensato che questi farmaci si depositassero nelle zone ossee di neoformazione; recentemente però sono stati scoperti alcuni depositi di farmaco in siti di riassorbimento [9]. pare che la distribuzione in una zona piuttosto che un’altra dipenda fondamentalmente dalla quantità di farmaco somministrata: modeste quantità favoriscono l’accumulo nelle aree di riassorbimento, mentre elevate quantità si distribuiscono abbastanza equamente nei due diversi tipi di siti. solitamente i bifosfonati non si accumulano nei tessuti molli, tuttavia si può verificare un deposito a livello di stomaco, fegato e milza in particolari condizioni in cui, a causa di una somministrazione endovenosa troppo elevata o troppo rapida, si possono formare grossi complessi con il ferro e con il calcio, fagocitati poi dai macrofagi del sistema reticoloendoteliale. sempre a causa della formazione di questi aggregati insolubili, infusioni troppo rapide e dosi troppo alte possono risultare dannose anche a livello renale [22]. l’emivita di eliminazione è di circa 7 ore e, circa la metà della dose somministrata per infusione endovenosa o per via intramuscolare, viene escreta nelle urine dopo ogni somministrazione. il farmaco in vivo non è metabolizzato, probabilmente a causa dell’elevata stabilità del legame p–c–p nei confronti degli enzimi idrolitici: esso viene escreto pressoché inalterato. l’eliminazione di questi farmaci è molto lenta, infatti essi permangono a lungo a livello scheletrico; è stata calcolata un’emivita nello scheletro di diversi anni [23]. non sono disponibili dati che rilevino il comportamento farmacocinetico del farmaco in soggetti con insufficienza renale o epatica. efficacia terapeutica sono oggi disponibili i dati relativi al trattamento dell’osteogenesi imperfetta con neridronato sia nei pazienti in età pediatrica che in quelli adulti [24,25]. 81 pazienti di ambo i sessi, 52 sopra i 20 anni (range 21-71) e 29 di età inferiore (range 517), in gran parte affetti dalla malattia nella forma più lieve, ma con qualche caso di media (tipo iv=20) ed elevata (tipo iii=10) gravità [26] sono stati trattati con un protocollo terapeutico che prevedeva la somministrazione trimestrale di una dose di neridronato compresa fra un minimo di 2 mg/kg e un massimo di 100 mg/kg. nel gruppo pediatrico, in cui la dose somministrata è stata di 2 mg/kg di peso corporeo, si sono osservati i maggiori incrementi di massa ossea, accompagnati da significativi miglioramenti a livello delle vertebre e di tutti i distretti studiati. gli effetti, evidenti già dopo 6 mesi di terapia, sono stati osservati per tutta la durata del trattamento (figura 1). figura 1 variazioni densitometriche in corso di terapia con neridronato in soggetti di età inferiore a 20 anni [26] figura 2 confronto tra il numero di eventi fratturativi verificatisi nei due anni precedenti il trattamento e durante i due anni di terapia con neridronato nel gruppo pediatrico [26] neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano 167farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati sempre nel gruppo pediatrico, tra i due anni precedenti l’inizio del trattamento e i due anni di terapia si è verificato un calo di circa il 50% dell’incidenza di frattura (figura 2). oltre al miglioramento di tali parametri oggettivi, i pazienti del gruppo pediatrico hanno riportato anche una notevole attenuazione dei dolori ossei, caratteristici della patologia, e un incremento della mobilità generale: aspetti da non trascurare in vista del considerevole impatto che hanno sulla qualità della vita dei pazienti. il protocollo clinico che prevede l’utilizzo del neridronato nel trattamento dei bambini affetti da oi, anche a partire da pochi mesi di vita, è stato valutato dall’équipe di finocchiaro della clinica pediatrica dell’università la sapienza di roma. in questo studio sono stati analizzati gli aminoacidi urinari, indici di metabolismo osseo come l’idrossiprolina e tutta una serie di altre molecole urinarie come il calcio, il fosfato, la fosfatasi alcalina, l’osteocalcina, la taurina, la prolina e il c-telopeptide/creatinina. dopo due cicli di somministrazione è stato osservato un aumento del calcio sierico e una diminuzione del fosfato sierico, della fosfatasi alcalina e dell’escrezione del calcio urinario [27]. per quanto riguarda il trattamento di soggetti adulti sono disponibili i dati relativi ad uno studio controllato e randomizzato svolto da adami e colleghi (università di verona), in collaborazione con l’associazione italiana osteogenesi imperfetta. i pazienti sono stati randomizzati a ricevere trimestralmente 100 mg di neridronato ev (in 250 ml di fisiologica infusi in 30 minuti) o placebo, con un rapporto di 2:1. l’apporto dietetico di calcio è stato valutato regolarmente e mantenuto al di sopra di 1.000 mg giornalieri in tutti i pazienti. supplementi di vitamina d 2 sono stati somministrati se i livelli ematici di 25oh-vitamina d scendevano al di sotto di 20 ng/ml: 5 pazienti hanno ricevuto supplementi di calcio o di vitamina d 2 . dopo i primi 12 mesi di follow up, anche i pazienti del gruppo di controllo hanno iniziato la terapia con neridronato. dei 78 partecipanti al trial, 46 hanno terminato lo studio (dopo due anni di follow up); di questi 31 erano stati randomizzati a ricevere neridronato, mentre i rimanenti 15 pazienti facevano parte del gruppo di controllo [24]. nel gruppo trattato con neridronato, dopo i primi 12 mesi di trattamento si è determinato un aumento del 3,0 ± 4,6% nei valori di densità ossea (bmd) della colonna e del 4,3 ± 3,9% in quelli dell’anca; valori che, nel corso del secondo anno, sono ulteriormente aumentati rispettivamente al 3,9% e all’1,5%. l’analisi densitometrica dei pazienti del gruppo controllo, che durante il primo anno non ha mostrato variazioni significative, nel corso del secondo anno, e cioè dopo il passaggio alla terapia, ha mostrato valori molto simili a quelli ottenuti il primo anno dal gruppo randomizzato a neridronato (figura 3). i valori dei marker di turnover osseo durante il trattamento con neridronato si sono ridotti significativamente; le riduzioni medie sono riportate nei grafici di figura 4. tutti i pazienti mostravano al baseline alcune deformità delle vertebre toraciche e lombari, e in 21 di essi vennero trovate evidenti fratture (schiacciamento delle vertebre). nei quattro anni precedenti l’inizio dello studio, 16 figura 3 variazioni della densità minerale ossea durante il trattamento con neridronato ev (linea continua) e durante il periodo di controllo (linea tratteggiata) [24] o. zaniolo, m. eandi 168 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati figura 4 variazioni della fosfatasi alcalina ossea (bap), del livello sierico di telopeptide c-terminale (sctx) e del rapporto deossipiridinolina libera urinaria/creatinina (ufdpd) durante il trattamento con neridronato ev (linea continua) e durante il periodo di controllo (linea tratteggiata)[24] pazienti avevano riportato complessivamente 18 fratture clinicamente evidenti di cui la più neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano recente si era verificata cinque mesi prima dell’arruolamento. nel gruppo di controllo, una frattura vertebrale e una frattura a un arto si sono verificate durante il primo anno, mentre nessuna durante il secondo anno di follow up. all’interno del gruppo randomizzato a neridronato si è verificata una sola frattura nel corso del trial, una riduzione statisticamente significativa dell’incidenza di fratture rispetto agli anni precedenti lo studio. come si evince dai grafici riportati in figura 3, nei pazienti che non hanno seguito alcun trattamento non si sono verificate variazioni significative della densità ossea; al contrario, nei pazienti trattati, la massa è aumentata sia rispetto al valore iniziale che al gruppo di controllo, con una crescita confermata anche nel secondo anno di studio. questi risultati sommati all’aumento della mobilità, all’attenuazione del dolore osseo e, soprattutto, alla diminuzione del rischio di fratture, costituiscono un grosso beneficio per i pazienti affetti dalla patologia. l’efficacia e la tollerabilità del neridronato sono state valutate anche nella terapia dell’osteoporosi nell’adulto, patologia estremamente diffusa fra le donne nel periodo postmenopausale, ma in progressiva diffusione anche fra i soggetti più giovani, sia donne che uomini. il possibile utilizzo di un trattamento farmacologico endovenoso intermittente in questa patologia potrebbe aumentare l’adesione alla terapia, rappresentando un considerevole vantaggio per il paziente. i bifosfonati orali, pur essendo utilizzati con successo nella terapia dell’osteoporosi, sono poco assorbiti dal tratto gastrointestinale e la presenza di cibo nello stomaco abbassa ulteriormente la percentuale di assorbimento; anche la tollerabilità gastrointestinale è molto scarsa, infatti per ridurre gli effetti collaterali il farmaco deve essere ingerito con notevoli quantità d’acqua e mantenendo rigorosamente la posizione eretta per almeno mezz’ora: il che li rende totalmente inaccessibili a coloro che sono costretti a letto, anche solo temporaneamente [28]. per valutare l’eventuale possibilità di adottare una strategia terapeutica alternativa, basata su neridronato somministrato in modo intermittente per via parenterale, è stato condotto uno studio su 78 donne in menopausa da almeno cinque anni, con un’età non superiore a 80 anni e riduzione della densità ossea del rachide pari ad almeno -2,5 [29]. il razionale dell’utilizzo dei bifosfonati in maniera intermittente è basato sul fatto che anche una breve inibizione dell’attività osteoclastica lascerà spazio a un periodo di attività osteo169farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati blastica incontrastata, con conseguente miglioramento del bilancio osseo. le pazienti hanno ricevuto per due anni 50 mg ev di neridronato o placebo, in 50 ml di fisiologica ogni 2 mesi; inoltre, entrambi i gruppi hanno ricevuto un supplemento giornaliero di calcio e vitamina d per tutta la durata del trial. tutte le pazienti del gruppo neridronato hanno completato lo studio, mentre quattro pazienti del gruppo controllo lo hanno abbandonato per cominciare un trattamento attivo. nel gruppo neridronato i valori di densità ossea sono cresciuti progressivamente nei due anni di trattamento e queste variazioni si sono mantenute costanti anche durante un ulteriore anno di follow up in cui è stato somministrato solo il supplemento giornaliero di calcio e vitamina d (figura 5). nel gruppo neridronato, inoltre, la fosfatasi alcalina ossea si è ridotta significativamente (23±17%) nei confronti dei valori basali e del gruppo di controllo entro quattro mesi dall’inizio dello studio, restando soppressa per tutta la durata del trattamento. i valori sierici di ctx si sono ridotti in modo significativo già due mesi dopo la prima infusione. dati clinici comparabili, in termini di incremento della bmd e di riduzione dei marker biochimici, sono stati ottenuti con cicli di somministrazione intramuscolare di neridronato: 25 mg ogni due settimane o 25 mg/die per sei giorni consecutivi ogni tre mesi [30]. in questo studio è stato rilevato un rapido incremento della densità ossea durante i primi sei mesi di terapia, con una tendenza all’incremento ancora evidente alla fine dei due anni di durata dello studio. gli studi, di fatto, confermano la sostanziale equivalenza in termini di incremento di massa ossea fra la somministrazione endovenosa e quella intramuscolare. nonostante ciò, sembrerebbe che il trattamento sia più efficace nel caso in cui venga adottato uno schema posologico più regolare: infatti i risultati in termini di bmd a lungo termine (dopo 24 mesi) sono decisamente migliori [15] nel gruppo randomizzato a ricevere neridronato bisettimanalmente. questi studi hanno rilevato che le variazioni del bmd indotte da neridronato 50 mg/bimestre sono dello stesso ordine di grandezza di quelle ottenute mediante il trattamento orale con alendronato 10-20 mg/die [31]; queste variazioni appaiono, inoltre, superiori a quelle ottenute con altri aminobifosfonati somministrati con terapia intermittente endovenosa: 4 mg/anno di zoledronato o 8 mg/anno di ibandronato aumentano, nei 12 mesi, la bmd del rachide e dell’anca rispettivamente del 5% e del 1-3% [32-34], mentre con l’utilizzo del clodronato le variazioni di bmd registrate durante i trial clinici risultano essere ancora più basse [35-38]. tali risultati collocano il neridronato tra le terapie più valide potenzialmente a disposizione del sistema sanitario. inoltre, dal momento che l’osteoporosi è una patologia cronica che va trattata per tempi molto lunghi, la collaborazione del paziente riveste un ruolo fondamentale nel raggiungimento degli effetti sperati: alcune tipologie di pazienti (ad esempio allettati o con problemi gastrointestinali) trarrebbero considerevoli benefici da una terapia endovenosa con ampi intervalli fra una somministrazione e l’altra, e ciò ne aumentefigura 5 variazioni % della bmd del rachide lombare e del femore a 6, 12, 18 e 24 mesi; la linea tratteggiata indica il periodo di follow up [29] o. zaniolo, m. eandi 170 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati rebbe notevolmente la compliance. il neridronato è risultato essere efficace anche nei confronti di altri disturbi a carico dell’apparato figura 6 variazioni medie percentuali rispetto al baseline della fosfatasi alcalina totale (ap totale), ossea (ap ossea) e dell�escrezione di n-telopeptide (ntx/cr) [41] neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano scheletrico, come ad esempio il morbo di paget. questa patologia, ad eziologia sconosciuta, è caratterizzata da un aumentato riassorbimento scheletrico con successiva formazione di tessuto osseo patologico a causa del netto incremento della componente vascolare e fibrosa; secondo alcuni studi radiografici su pazienti con più di 45 anni la sua incidenza è compresa fra il 3 e il 3,7%. le manifestazioni cliniche del morbo di paget dipendono dall’estensione e dalla localizzazione della malattia; la colonna vertebrale è una delle localizzazioni più tipiche di questa patologia (soprattutto il segmento lombare), così come il femore, la tibia e le ossa pelviche. la sintomatologia dolorosa può essere causata da fratture locali, dall’aumento di dimensioni e di vascolarizzazione dell’osso o da compressione delle strutture nervose adiacenti al segmento interessato. la calcemia, la calciuria e la fosforemia sono in genere nella norma, ad indicare un aumento simile di distruzione e di sintesi del tessuto osseo, mentre si nota un’aumentata concentrazione plasmatica delle idrossipiridinoline, delle lisilpiridinoline e di n-telopeptide. particolare valore è da attribuire alla fosfatasi alcalina, i cui livelli sierici si correlano sia con l’estensione che con l’attività della malattia [39,40]. uno studio condotto da adami e colleghi aveva come obiettivo quello di valutare l’efficacia di una terapia endovenosa di breve durata, costituita da 2 infusioni consecutive di neridronato ev in pazienti affetti da morbo di paget in fase attiva. 83 pazienti sono stati randomizzati a ricevere 12.5, 25, 50 o 100 mg al giorno per due giorni consecutivi con complessive dosi totali rispettivamente di 25, 50, 100 e 200 mg e un follow up di 180 giorni. la variazione del livello sierico di fosfatasi alcalina, che durante il baseline era superiore alla norma almeno del 10%, costituiva l’end point primario dello studio, unitamente a quella del suo isoenzima osseo e all’escrezione urinaria di n-telopeptide. tutte le dosi di neridronato hanno diminuito significativamente i marker biochimici di attività di malattia; in particolare, il nadir (la media dei tre valori più bassi registrati consecutivamente) dei livelli di fosfatasi alcalina totale era compreso tra –16% e –57,5% rispetto ai valori iniziali, con una significativa correlazione alla dose somministrata; correlazione riscontrabile anche per quanto riguarda la percentuale di pazienti che dopo sei mesi presentava ancora una risposta, sebbene parziale (figura 6). 171farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati la variazione della fosfatasi alcalina ossea e dell’escrezione di n-telopeptide, inoltre, è risultata correlata alla diminuzione del dolore osseo collegato alla patologia, significativamente diminuito nei responders biochimici [41]. un altro studio, condotto per un periodo di 12 mesi su 32 pazienti affetti da morbo di paget, ha valutato l’efficacia di 200 mg di neridronato ev somministrato in singola dose, o in due dosi elargite in giorni consecutivi a pazienti che non erano mai stati trattati con agenti antiriassorbitivi (n = 15) e a pazienti che non avevano avuto risultati soddisfacenti tramite terapia con clodronato (n = 17). all’inizio della terapia non sono state rilevate differenze statisticamente significative nelle concentrazioni dei marker biologici assunti come indicatori del riassorbimento osseo (isoenzima osseo della fosfatasi alcalina, deossipiridinolina, peptide n-terminale e c-terminale del collageno di tipo 1) fra i pazienti precedentemente in terapia con clodronato e quelli mai trattati. dopo la prima somministrazione il neridronato ha indotto una diminuzione media percentuale dell’eccesso (definito come differenza tra i valori misurati e il punto medio del range di normalità) di isoenzima osseo della fosfatasi alcalina (bap) circa del 68% e dell’eccesso di deossipiridinolina, di peptide n-terminale e di peptide c-terminale rispettivamente di 68,1%, 60,6% e 86,7%. l’analisi ha rilevato che i marker di riassorbimento osseo diminuivano più lentamente nei pazienti che avevano assunto precedentemente clodronato, anche se l’entità della variazione appariva sostanzialmente uguale. in 15 dei 21 pazienti che facevano uso di analgesici per controllare il dolore correlato alla patologia, si è verificato un miglioramento sintomatologico con un controllo completo del dolore in otto pazienti; 21 pazienti hanno manifestato remissione della malattia (rientro della bap in un range di normalità), senza sostanziali differenze fra la somministrazione unica e quella in due fasi, e fra i pazienti precedentemente trattati e quelli non [42]. questi dati confermano l’efficacia del neridronato nel trattare il morbo di paget sia come bifosfonato di prima scelta, sia come terapia alternativa in caso di recidiva con clodronato. oltre ad essere potenti inibitori del riassorbimento osseo, alcuni bifosfonati hanno dimostrato di avere proprietà antinfiammatorie: in vitro, essi inibiscono la proliferazione linfocitaria, la sintesi delle prostaglandine [43-45] e l’enzima collagenasi-3 [46]. ad esempio, in un trial controllato, la somministrazione di alendronato per tre mesi in pazienti affetti da artrite reumatoide (ra) ha diminuito considerevolmente i livelli sierici di il-1, il-6 e tnf-α [47]; in contrasto con questi dati, i risultati relativi ad uno studio svolto su 18 pazienti ra a cui e stato somministrato per via endovenosa pamidronato, un aminobifosfonato, non hanno rilevato nessun effetto antinfiammatorio. in considerazione di questi dati contrastanti e del fatto che recentemente alcune analisi in vitro e in vivo [48,49] hanno addirittura rilevato una possibile azione proflogistica da parte degli aminobifosfonati (probabilmente mediante stimolazione della sintesi di istamina e della maturazione di macrofagi e granulociti), è stato svolto uno studio al fine di valutare se il neridronato potesse avere effetti sul sistema immunitario e, se sì, di quale entità. 45 pazienti con artrite reumatoide attiva sono stati randomizzati in doppio cieco a ricevere una singola infusione di 25 mg (n = 15) o di 50 mg (n = 15) del farmaco in esame contro placebo (n = 15). dopo 7 giorni dall’infusione, nel gruppo randomizzato a 25 mg, si è assistito ad un calo significativo della proteina c reattiva e della ves, mentre in nessuno dei tre gruppi si è verificata una variazione nell’indice articolare di ritchie (i tre parametri assunti come indicatori dell’attività della malattia) [50]. tali risultati suggeriscono che il neridronato possa modulare la flogosi in modo diverso a seconda della posologia con cui viene assunto; questo potrebbe rappresentare un fattore sfavorevole al suo utilizzo, anche se i risultati positivi ottenuti a livello di concentrazione di proteina c reattiva e velocità di eritrosedimentazione possono essere delle ottime basi per progettare studi più accurati volti a testare l’azione a lungo termine di questo farmaco. recentemente è stato segnalato che il neridronato, e gli aminobifosfonati in generale, potrebbero inibire la sintesi dello squalene e del colesterolo, al contrario dei bifosfonati non contenenti un gruppo amminico, che non interferiscono nei processi di sintesi degli steroli. per stimare la rilevanza di tale effetto, durante un trial volto a determinare gli effetti del neridronato sul riassorbimento osseo, sono stati monitorati i profili lipidici delle 87 pazienti incluse nello studio, randomizzate a ricevere 50 mg ev del farmaco ogni due mesi (n = 44) o placebo (n = 43). il trattamento con neridronato ha determinato in queste pazienti una riduzione significativa nelle concentrazioni sieriche di colesterolo ldl (-4% al secondo mese) e apo b (-6%), e aumenti rilevanti nei valori di apo a-1 e di colesterolo hdl (+4% al secono. zaniolo, m. eandi 172 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati do mese e +17% al 12º mese) [51]. questi effetti potrebbero portare a lungo termine un rilevante miglioramento del quadro lipidico del paziente, anche se nel confronto con i valori calcolati al baseline o con quelli riferiti al gruppo placebo, non sempre si raggiunge la significatività (figura 7). sicurezza e tollerabilità l’acido neridronico è un aminobifosfonato con proprietà simili a quelle degli altri bifosfonati. questi farmaci possono causare disturbi gastrointestinali quali dolore addo-minale, nausea e vomito, diarrea o stitichezza, ma nel caso di somministrazione parenterale, come per il neridronato, la reazione avversa principale è una sindrome simil-influenzale caratterizzata da febbre, malessere, brividi e dolori ossei o muscolari. nella maggior parte dei casi, comunque, non è necessario alcuno specifico trattamento e i sintomi regrediscono nel giro di poche ore o giorni. nel trial condotto da adami e colleghi per il trattamento dell’osteo-genesi imperfetta, 13 pazienti sui 31 randomizzati a neridronato hanno segnalato questo genere di reazione avversa 24-36 ore dopo la prima infusione ev, la cui durata non è stata comunque superiore alle 36 ore; di questi, quattro pazienti hanno riportato una reazione attenuata anche dopo la seconda infusione [24]. l’assunzione di neridronato ha causato una reazione di fase acuta (con dolore muscolare e febbre fino a 37,7 ºc) anche nel 13% delle pazienti affette da osteoporosi post-menopausale esaminate da braga e colleghi, percentuale che supera il 19% fra i pazienti pagetici sottoposti a terapia endovenosa a breve termine [41]. durante lo studio condotto da mazzantini e colleghi al fine di valutare i possibili effetti del farmaco sulla risposta infiammatoria, due pazienti (n = 15) dopo aver ricevuto 50 mg di neridronato ev hanno riportato un innalza-mento della temperatura, mentre nessuno dei pazienti randomizzati a ricevere 25 mg del farmaco ha riportato reazioni di questo genere dimostrando un’apparente correlazione fra effetti indesiderati e dose somministrata [50]. saltuariamente, durante il trattamento con questo farmaco, possono verificarsi anche disturbi elettrolitici, più frequentemente ipocalcemia e ipofosfatemia; per tale ragione, durante il trattamento con neridronato devono essere monitorati la funzionalità renale, nonché il calcio e il fosfato sierici, anche se nessuno dei pazienti partecipanti ai trial sopracitati ha riportato questo genere di effetto indesiderato. più raramente il farmaco può portare a vertigini, cefalea, orticaria o ad altri rari effetti indesiderati fra cui disturbi ematici, quali leucopenia, e alterazioni degli enzimi epatici. la somministrazione per via intramuscolare è spesso accompagnata da dolore al sito di iniezione, che comunque si attenua dopo pochi minuti: disturbo riportato dal 45% dei pazienti partecipanti al trial condotto da filipponi e colleghi [30]. in seguito alla somministrazione di bifosfonati recentemente è sorto un problema relativo alla comparsa di disturbi visivi, quali congiuntiviti, uveiti, scleriti, fotofobia, ecc. un elevato numero di segnalazioni di questo tipo è stato riportato sul new england journal of medicine a carico di pamidronato, alendronato, etidronato, risedronato e clodronato, con una numerosità che riflette la loro diffusione di utilizzo. nel database del gif (gruppo interregionale di farmacovigilanza) sono presenti 10 segnalazioni di sospette reazioni avverse oculari non gravi da acido alendronico (6 nel 2003) con disturbi dell’accomodazione, visione offigura 7 variazioni % dei valori sierici delle lipoproteine nel gruppo neridronato (linea grigia) rispetto al baseline. i simboli (*p < 0,05; #p < 0,01; +p < 0,001) sopra la linea grigia indicano significatività statistica del gruppo neridronato rispetto ai valori basali, mentre quelli sopra la linea nera (gruppo controllo) indicano la differenza tra gruppi neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano 173farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati fuscata, dolore oculare, tre segnalazioni da acido clodronico e due da acido risedronico (una irite nel 2003). anche se nessun caso di reazioni simili è stato riportato a carico del neridronato, questo tipo di evento collaterale va sempre tenuto sotto controllo [52]. la reazione infiammatoria non pare in relazione alla dose, alla via di somministrazione o alla gravità della malattia che viene trattata. considerazioni farmacoeconomiche in vista dei buoni risultati ottenuti in termini di efficacia nel trattamento dell’osteogenesi imperfetta e dietro sollecitazione di alcune associazioni di malati, il ministero della salute italiano ha concesso l’autorizzazione al commercio del neridronato mediante le procedure preferenziali disposte per i farmaci orfani. il farmaco è stato dunque collocato in fascia a, senza note cuf, per la cura dell’osteogenesi imperfetta, prima che l’azienda produttrice terminasse la sperimentazione anche per l’osteoporosi. questa situazione ha generato un difetto di comunicazione tra asl, medici e pazienti, in quanto alcuni specialisti, essendo a conoscenza dell’efficacia del farmaco su pazienti affetti da oi, lo prescrivono off label anche per il trattamento dell’osteoporosi. secondo il decreto-legge n. 23 del 17 febbraio 1998 (convertito in legge l’8 aprile 1998), in singoli casi il medico può, sotto la sua diretta responsabilità e previa informazione del paziente e acquisizione del consenso dello stesso, impiegare un medicinale prodotto industrialmente per un’indicazione o una via di somministrazione diversa da quella autorizzata. secondo questo decreto, è lecito effettuare tale scelta qualora il medico ritenga, in base a dati documentabili, che il paziente non possa essere utilmente trattato con medicinali per i quali sia già approvata quella indicazione terapeutica (o quella via o modalità di somministrazione), e purché tale impiego sia noto e conforme a lavori apparsi su pubblicazioni scientifiche accreditate in campo internazionale. in nessun caso il ricorso del medico alla prescrizione off label può costituire riconoscimento del diritto del paziente alla erogazione dei medicinali a carico del servizio sanitario nazionale, a meno che il farmaco non presenti caratteristiche particolari disciplinate dall’articolo 1, comma 4, del decreto-legge 21 ottobre 1996, n. 536. per il neridronato è quindi ammessa la rimborsabilità del farmaco soltanto per il trattamento dell’osteogenesi imperfetta; per tali ragioni focalizzeremo la nostra attenzione sulle conseguenze farmacoeconomiche derivanti dall’utilizzo del farmaco esclusivamente nel trattamento di tale patologia. questo genere di analisi ha il fine di fornire al medico e agli altri decisori della spesa sanitaria gli strumenti adatti per scegliere razionalmente tra due o più strategie terapeutiche o tra una terapia e il “non trattamento”; ciò vale in particolar modo per quelle patologie come l’osteogenesi imperfetta che, pur non avendo un’incidenza elevata, sono malattie gravi, croniche e, di conseguenza, con un grande impatto sulla qualità della vita del paziente e dei suoi familiari. la maggiore conoscenza delle sue basi genetiche e l’aumento delle possibilità di diagnosi, anche in casi precedentemente insospettabili, hanno indotto il passaggio dell’osteogenesi imperfetta dallo stato di malattia genetica “rara” a quello di uno dei disturbi genetici rilevati con maggior frequenza nella popolazione; anche se le forme più gravi sono clinicamente evidenti sin dalla nascita, forme più lievi di oi possono non essere sospettate sino a età avanzata [53]. sulla base di queste considerazioni, si stima che attualmente negli stati uniti vi siano circa 50.000 individui affetti da oi, di cui il 60% con forme lievi; la forma letale più grave si verifica in circa 3-4 casi ogni 100.000 nascite. le stime basate sulla presenza di fratture alla nascita variano da 1,6/100.000 a singapore [54], 3,3/100.000 in francia [55], e 15/100.000 nel regno unito [56], dati sottostimati a causa dei possibili aborti nei casi molto gravi, in cui pertanto la diagnosi alla nascita non è possibile; nell’insieme si stima l’esistenza di circa 0,5 milioni di persone con oi nel mondo (0.008%). in italia ci sono circa 3.800 individui affetti da osteogenesi imperfetta in qualsiasi forma: la prevalenza è di circa 5 casi ogni 100.000 bambini nati [57]. al momento sono disponibili tre tipi di trattamento: management non operativo (terapia fisica, riabilitazione, uso di busti ortopedici e apparecchi gessati), approccio chirurgico e approccio farmacologico. l’approccio chirurgico, previsto soprattutto nelle forme di oi più gravi, prevede principalmente il posizionamento intramidollare di chiodi e ha lo scopo di prevenire il rischio di fratture e garantire una migliore funzionalità degli arti inferiori. questo tipo di intervento, sebbene produca un notevole miglioramento della capacità motoria e del conseguente livello di autonomia del paziente, comporta un elevato tasso di complicanze. innanzitutto finché il paziente ha un’età in cui permane una significativa crescita ossea, risulta necessario infibulare l’osso con chiodi telescopici che, seguendo la creo. zaniolo, m. eandi 174 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati scita, possono essere tenuti in sede per un periodo più lungo rispetto ai chiodi non allungabili. questo tipo di chiodi però richiedono interventi più complessi e sembrano aumentare il rischio di osteoporosi. le complicazioni più frequenti sono la migrazione del chiodo, spesso associata alla perforazione dell’articolazione, dell’osso e del tessuto molle periarticolare o la frattura stessa dell’osso infibulato; il rischio che si verifichino questi eventi è maggiore nei pazienti di età inferiore ai 5 anni e nelle tibie rispetto ai femori. dopo l’intervento è importante limitare l’immobilizzazione, per riprendere la riabilitazione il prima possibile; l’intervento, infatti, non è un fatto isolato ma è solo una tappa del cammino riabilitativo [58] nell’oi severa e moderatamente severa, i farmaci che agiscono favorendo la produzione di collagene tipo i, non rappresentano, in linea di principio, una terapia efficace. al momento una terapia farmacologica eziopatogenetica non esiste, mentre sono disponibili alcuni trattamenti sintomatici; in passato a tale scopo sono stati utilizzati gli steroidi anabolizzanti, la vitamina d, la vitamina c, il fluoruro di sodio, l’ossido di magnesio, i flavonoidi e la calcitonina, ma nessuno di questi ha mai dato buoni risultati nel lungo periodo. negli ultimi anni due sono i tipi di farmaci maggiormente usati nel trattamento dell’oi: i bifosfonati, di cui il neridronato è l’unico indicato per questa patologia, e l’ormone della crescita (gh). in letteratura si trovano pochi riferimenti relativi all’esperienza con gh soprattutto per quanto riguarda i trial controllati; dai dati analizzati si può concludere che l’ormone della crescita è una terapia utile nei pazienti con forme moderate di oi, sui quali ha dimostrato un’azione positiva sul turn over osseo, sulla densità minerale e sulla velocità di crescita; tuttavia i pazienti con scoliosi pre esistente o deformità ossee vanno trattati con particolare cautela visto il potenziale rischio di peggioramento di questi problemi [59]. i pazienti con oi grave non sembrano avere alcun vantaggio sostanziale da questa terapia [60-62]; inoltre, non sono noti i suoi effetti a lungo termine: il gh non ha ancora un’indicazione approvata per l’oi e i protocolli che lo utilizzano in associazione con altri farmaci nel trattamento di questa patologia sono al momento solo alla fase iniziale di sperimentazione. risultati più incoraggianti, come detto in precedenza, sono stati ottenuti con l’utilizzo dei bifosfonati e in particolar modo i derivati con un gruppo amino terminale: il neridronato ha dato ottimi risultati sia nei pazienti adulti che nel trattamento sintomatico dei bambini affetti da gravi forme di oi. per capire l’importanza di questi dati occorre soffermarsi sul notevole abbassamento della qualità di vita di un bambino affetto da oi e della sua famiglia. nelle forme più gravi il bambino, ammesso che riesca a sopravvivere al parto, frequentemente nasce già con fratture ed è costretto a convivere costantemente con il rischio di andare incontro a deformità dovute all’incurvamento delle ossa, a corrosione dei denti e sordità e, ovviamente, a nuove fratture. ciò comporta tutta una serie di precauzioni che devono essere prese per evitare di esporre il paziente a ulteriori rischi, proteggendolo da situazioni che per le altre persone sono del tutto normali: il bambino affetto da oi difficilmente potrà frequentare la scuola, fare sport, giochi di gruppo o attività esterne, e spesso non sarà neanche in grado di deambulare senza il supporto di una sedia a rotelle, con tutti i problemi di discriminazione, di insicurezza e di sfiducia che ne derivano [63]. utilizzare un farmaco in grado di ridurre, anche solo parzialmente, questi gravi disagi significa migliorare notevolmente la qualità di vita del bambino e dei suoi familiari. per le famiglie che hanno un bambino affetto da oi la situazione descritta porta a conseguenze drastiche anche sul piano economico. circa il 67% delle donne con un figlio disabile non ha la possibilità di trovare un lavoro o di mantenere quello che avevano prima della nascita del bambino [64], al contrario delle altre madri che, in seguito alla nascita del figlio, riescono a ritornare al lavoro nel 65% circa dei casi [65]. in media, questo tipo di introito rappresenta circa il 13% del reddito totale della famiglia e, in inghilterra, esso risulta determinante affinché circa un milione di nuclei familiari non scendano sotto la soglia di povertà. il motivo fondamentale per cui le madri di figli disabili sono impossibilitate a tornare al lavoro è certamente riscontrabile nel maggior bisogno di cure di cui essi necessitano [64]. uno studio condotto in gran bretagna ha analizzato questa complicata situazione facendo un confronto fra le esperienze di famiglie con bambini disabili e quelle di famiglie con bambini non disabili. l’analisi è stata svolta mediante l’utilizzo di un “event diary” nel quale è stato chiesto alle famiglie di annotare tutte le cure dedicate al figlio, intendendo per cure tutte quelle azioni svolte al fine di preservare neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano 175farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati la sua salute, il suo benessere e la sua educazione. inoltre alle famiglie è stato richiesto di compilare un questionario dettagliato sulla loro situazione finanziaria, sulla composizione del nucleo familiare, sugli aiuti che ricevevano da parenti, amici e istituzioni, sulle visite ospedaliere e sul tipo di diagnosi che era stata fatta al bambino (epilessia, autismo, osteogenesi imperfetta, ecc.); tale questionario è stato compilato con l’ausilio di un intervistatore incaricato della raccolta dei dati. dai risultati di questo studio appare chiaro che i bambini invalidi necessitano di un numero di cure per unità di tempo (waking hour) molto più elevato degli altri e soprattutto che, mentre per i bambini non disabili, questo numero decresce con l’età, per i bambini affetti da patologie gravi e croniche, il bisogno di sorveglianza e di protezione rimane costante. esso infatti dipende solo ed esclusivamente dalla gravità della malattia nel periodo considerato. delle 16 madri con figli disabili intervistate, 12 hanno dichiarato di non aver potuto riprendere il lavoro; questo, come già accennato, è una grave perdita a livello di budget familiare, ma il problema risulta ancora più serio se si considera che la maggior parte delle famiglie con disabili, per fronteggiare i costi delle cure, ha riportato un esborso di denaro molto maggiore rispetto a quelle con figli non disabili. l’ammontare di questa differenza di spesa è risultato in media di £ 97,450, delle quali £ 31,050 finanziate tramite sovvenzioni governative, £ 26,000 elargite da organizzazioni di beneficenza e £ 40,400 versate dalla famiglia stessa: come si può osservare quasi il 50% dell’ammontare resta a carico dei nuclei familiari, mettendo in grave difficoltà economica la maggior parte degli interessati [66]. in italia l’osteogenesi imperfetta rientra nell’elenco delle malattie rare per le quali è riconosciuto il diritto all’esenzione dalla partecipazione al costo per le correlate prestazioni; l’assistito riconosciuto esente ha dunque diritto alle prestazioni, efficaci ed appropriate, incluse nei livelli essenziali di assistenza per il trattamento e il monitoraggio della malattia dalla quale è affetto e per la prevenzione degli ulteriori aggravamenti [67]. la qualità di vita per il paziente e per la sua famiglia, così come la disponibilità economica, restano tuttavia decisamente ridotte rispetto alla normalità: considerazione che sottolinea marcatamente la necessità di avere a disposizione una terapia in grado di migliorare il quadro clinico e sintomatico del paziente. una confezione di neridronato costa 16,82 euro e contiene una fiala iniettabile da 25 mg. la posologia orientativa è di 2 mg/kg di peso corporeo ogni tre mesi. il costo per anno di terapia, assumendo che venga somministrata in tutti i casi la dose massima di 100 mg, risulta di 269,12 euro. nel trial condotto da adami et al [25], l’incidente di fratture registrate nei pazienti affetti da oi durante i due anni di terapia aveva dimostrato un calo significativo del 45% rispetto al numero di fratture verificatesi nei due anni precedenti l’inizio del trattamento. come si evince da questi dati, il costo del trattamento con neridronato risulta molto contenuto rispetto ai benefici derivanti dal suo utilizzo, soprattutto considerando che proprio la frattura rappresenta il maggior ostacolo che deve superare il paziente per potersi permettere una vita “normale”. conclusioni il neridronato è un aminobifosfonato di ultima generazione indicato nel trattamento dell’osteogenesi imperfetta, malattia rara e gravissima, per la cui indicazione è stato approvato con la procedura semplificata degli “orphan drugs”. la sua particolare formulazione lo rende adatto ad una somministrazione trimestrale per via endovenosa: ciò migliora notevolmente la compliance del paziente visto che i bifosfonati per via orale richiedono particolare attenzione nell’assunzione (sono scarsamente assorbiti) e comportano svariati effetti collaterali, soprattutto a livello gastrointestinale. dai trial effettuati, neridronato è risultato ben tollerato e molto efficace nell’aumentare la densità minerale ossea e nel diminuire il rischio di fratture nei pazienti affetti da osteogenesi imperfetta; inoltre la sua efficacia è stata testata con buoni risultati anche su pazienti affetti da osteoporosi post-menopausale e morbo di paget. dal punto di vista economico, il prezzo di acquisto del neridronato è tra i più bassi della sua classe; se paragonato ai benefici prodotti in termini di innalzamento della qualità di vita questo dato rivela l’efficienza farmacoeconomica del neridronato nella terapia dell’osteogenesi imperfetta. infatti situazioni o movimenti che per una persona “normale” non rappresentano assolutamente un pericolo, per gli individui affetti dalla patologia possono diventare motivo di grave rischio: nelle forme più gravi la frequenza con cui avviene una frattura è elevatissima. si può facilmente dedurre quanto questo condizioni le normali attività del paziente, soprattutto perché una frattura rappresenta un evento traumatico sia dal punto di vista fisico o. zaniolo, m. eandi 176 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati che psicologico; inoltre spesso questo evento richiede ospedalizzazione, con conseguente aumento drastico delle risorse consumate e dei mancati guadagni da parte della famiglia o del paziente stesso, se adulto. un farmaco che è in grado di diminuire sostanzialmente l’incibibliografia 1. cole wg et al. collagen genes: mutations affecting collagen structure and expression. prog nucleic acid res mol biol. 1994;47:29-80. 2. paterson cr, mcallion s, stellman jl. osteogenesis imperfecta after the menopause. n engl j med. 1984 jun 28;310(26):1694-6. 3. mcallion sj, paterson cr. causes of death in osteogenesis imperfecta. j clin path 1996; 49: 627-30. 4. shapiro f. consequences of an osteogenesis imperfecta diagnosis for survival and ambulation. j pediatr orthop 1985; 5: 456-62. 5. sillence do, senn a, danks dm. genetic heterogeneity in osteogenesis imperfecta. j med genet. 1979 apr;16(2):10116. 6. byers ph. brittle bones-fragile molecules: disorders of collagen gene structure and expression. trends genet. 1990 sep;6(9):293-300. 7. hughes de et al. bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. j bone miner res. 1995 oct;10(10):1478-87. 8. hughes de et al. inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. j clin invest. 1989 jun;83(6):1930-5. 9. sato m et al. bisphosphonate action. alendronate localization in rat bone and effects on osteoclast ultrastructure. j clin invest. 1991 dec;88(6):2095-105. 10. heaney rp. the bone-remodeling transient: implications for the interpretation of clinical studies of bone mass change. j bone miner res. 1994 oct;9(10):1515-23. 11. compston je. prevention and management of osteoporosis. current trends and future prospects. drugs. 1997 may;53(5):727-35. 12. tobias jh. how do bisphosphonates prevent fractures? ann rheum dis. 1997 sep;56(9):510-1. 13. fleisch h. bisphosphonates: mechanisms of action and clinical use in osteoporosis an update. horm metab res. 1997 mar;29(3):145-50. 14. gallini g, cangini f. ruolo dei mediatori dell’infiammazione nei meccanismi di regolazione e nelle interazioni tra cellule osseeseconda parte. il dentista moderno (aggiornamento monografico). 2003 gen. 15. sartori l et al. injectable bisphosphonates in the treatment of postmenopausal osteoporosis. aging clin exp res. 2003 aug;15(4):271-83. 16. gotcher je, jee ws. the progress of the periodontal syndrome in the rice cat ii. the effects of a diphosphonate on the periodontium. j periodontal res. 1981 jul;16(4):441-55. 17. li m et al. parathyroid hormone monotherapy and cotherapy with antiresorptive agents restore vertebral bone mass and strength in aged ovariectomized rats. bone. 1995 jun;16(6):629-35. 18. nerixia® riassunto delle caratteristiche del prodotto. 19. maconi g, porro gb. multiple ulcerative esophagitis caused by alendronate. am j gastroenterol. 1995 oct;90(10):188990. denza di fratture e di preservare la massa ossea per un’età più avanzata può migliorare notevolmente la qualità di vita dei pazienti affetti da osteogenesi imperfetta, riducendone l’impatto clinico, sociale ed economico a carico delle famiglie e dell’intera società. neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano 177farmeconomia e percorsi terapeutici 2004; 5 (3) © seed tutti i diritti riservati 20. ettinger b, pressman a, schein j. clinic visits and hospital admissions for care of acid-related upper gastrointestinal disorders in women using alendronate for osteoporosis. am j manag care. 1998 oct;4(10):1377-82. 21. adami s, zamberlan n. adverse effects of bisphosphonates. a comparative review. drug saf. 1996 mar;14(3):158-70. 22. fleisch h. bisphosphonates: mechanisms of action. endocr rev. 1998 feb;19(1):80-100. 23. kasting gb, francis md. retention of etidronate in human, dog, and rat. j bone miner res. 1992 may;7(5):513-22. 24. adami et al. intravenous neridronate in adults with osteogenesis imperfecta. j bone miner res. 2003 jan;18(1):12630. 25. adami s et al. intravenous neridronate in the treatment of patients with osteogenesis imperfecta. sixth workshop on bisphosphonates from laboratory to the patient, davos 2002. 26. gatti d, colapietro f, adami s. bisfosfonati e osteogenesi imperfetta. gibis – rivista bisfosfonati 2002 giu; 3 (2):3-8. 27. xix convegno nazionale as.it.o.i. caserta 16-18 maggio 2003. sito ufficiale dell’associazione italiana osteogenesi imperfetta www.asitoi.it. 28. aki s et al. gastrointestinal side effect profile due to the use of alendronate in the treatment of osteoporosis. yonsei med j. 2003 dec 30;44(6):961-7. 29. braga v et al. intravenous intermittent neridronate in the treatment of postmenopausal osteoporosis. bone. 2003 sep;33(3):342-5. 30. filipponi p et al. two years neridronate increases bone mineral density in postmenopausal women affectes by osteoporosis. bone 2002; 30 (suppl 3): s48. 31. wimalawqansa sj. intermittent intravenous pamidronate therapy: highly effective treatment for postmenopausal osteoporosis. j bone miner res 2000; 16 (suppl 1): s405. 32. thiebaud d et al. three monthly intravenous injections of ibandronate in the treatment of postmenopausal osteoporosis. am j med. 1997 oct;103(4):298-307. 33. adami s et al. three-monthly 2 mg intravenous ibandronate injections restore bone turnover to premenopausal levels. j bone miner res 2002; 17 (suppl 1): s472. 34. reid ir et al. intravenous zoledronic acid in postmenopausal women with low bone mineral density. n engl j med. 2002 feb 28;346(9):653-61. 35. filipponi p et al. cyclical clodronate is effective in preventing postmenopausal bone loss: a comparative study with transcutaneous hormone replacement therapy. j bone miner res. 1995 may;10(5):697-703. 36. heikkinen je et al. short-term intravenous bisphosphonates in prevention of postmenopausal bone loss. j bone miner res. 1997 jan;12(1):103-10. 37. rossini m et al. intramuscular clodronate therapy in postmenopausal osteoporosis. bone. 1999 feb;24(2):125-9. 38. filipponi p et al. intermittent versus continuous clodronate administration in postmenopausal women with low bone mass. bone. 2000 mar;26(3):269-74. 39. singer fr, krane sm. paget’s disease of bone. in metabolic bone diseas. accademic press, 1998: 545-582. 40. delmas pd, meunier pj. the management of paget’s disease of bone. n engl j med. 1997 feb 20;336(8):558-66. 41. adami s et al. short-term intravenous therapy with neridronate in paget’s disease. clin exp rheumatol. 2002 janfeb;20(1):55-8. 42. filipponi p et al. paget’s disease of bone: benefits of neridonate as a first treatment and in cases of relapse after clodronate. bone. 1998 dec;23(6):543-8. 43. de vries e et al. in vitro effect of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonic acid (apd) on the function of mononuclear phagocytes in lymphocyte proliferation. immunology. 1982 sep;47(1):157-63. 44. felix r, bettex jd, fleisch h. effect of diphosphonates on the synthesis of prostaglandins in cultured calvaria cells. calcif tissue int. 1981;33(5):549-52. 45. ohya k et al. effect of bisphosphonates on prostaglandin synthesis by rat bone cells and mouse calvaria in culture. clin sci (lond). 1985 oct;69(4):403-11. 46. konttinen yt et al. collagenase-3 (mmp-13) and its activators in rheumatoid arthritis: localization in the pannus-hard tissue junction and inhibition by alendronate. matrix biol. 1999 aug;18(4):401-12. o. zaniolo, m. eandi 178 farmeconomia e percorsi terapeutici 2004; 5 (3)© seed tutti i diritti riservati 47. cantatore fp, acquista ca, pipitone v. evaluation of bone turnover and osteoclastic cytokines in early rheumatoid arthritis treated with alendronate. j rheumatol. 1999 nov;26(11):2318-23. 48. richards pj et al. pro-inflammatory effects of the aminobisphosphonate ibandronate in vitro and in vivo. rheumatology (oxford). 1999 oct;38(10):984-91. 49. nakamura m et al. contrast between effects of aminobisphosphonates and non-aminobisphosphonates on collageninduced arthritis in mice. br j pharmacol. 1996 sep;119(2):205-12. 50. mazzantini m et al. single infusion of neridronate (6-amino-1-hydroxyhexylidene-1,1-bisphosphonate) in patients with active rheumatoid arthritis: effects on disease activity and bone resorption markers. aging clin exp res. 2002 jun;14(3):197-201. 51. adami s et al. chronic intravenous aminobisphosphonate therapy increases high-density lipoprotein cholesterol and decreases low-density lipoprotein cholesterol. j bone miner res. 2000 mar;15(3):599-604. 52. fraunfelder fw. ocular side effects associated with bisphosphonates. drugs today (barc). 2003 nov;39(11):829-35. 53. shapiro jr. osteogenesi imperfetta e altri difetti dello sviluppo osseo come cause occasionali di osteoporosi dell’adulto. estratto da osteoporosis. second edition. volume 2, capitolo 50. 54. tan kl, tock ep. osteogenesis imperfecta congenita. aust paediatr j. 1971 mar;7(1):49-53. 55. robert jm et al. hereditary bone fragility. lyon med. 1968 mar 17;219(11):881-982. 56. navani sv, sarzin b. intrauterine osteogenesis imperfecta. review of the literature and a report of the radiological and necropsy findings in two cases. br j radiol. 1967 jun;40(474):449-52. 57. sito ufficiale di uildm unione italiana lotta alla distrofia muscolare. www.uildm.org. 58. osteogenesi imperfetta: linee guida di trattamento. comitato scientifico dell’as.it.o.i www.asitoi.it. 59. antoniazzi f. therapeutic approach with growth hormone. 7th international conference on osteogenesis imperfecta. montreal, canada, 29 august 1 september 1999. 60. marini jc et al. the growth hormone and somatomedin axis in short children with osteogenesis imperfecta. j clin endocrinol metab 1993; 76: 251-6. 61. marini jc et al. positive linear growth and bone responses to growth hormone treatment in children with types iii and iv osteogenesis imperfecta: high predictive value of the carboxyterminal propeptide of type i procollagen. j bone miner res 2003;18: 237-43. 62. sillence d et al. factors which influence the efficacy of growth hormone in 15 children with osteogenesis imperfecta types i and iv. sixth international conference on osteogenesis imperfecta. utrecht, the netherlands, 19-21 september 1996. 63. rauch f, glorieux fh. osteogenesis imperfecta. lancet. 2004 apr 24;363(9418):1377-85. 64. dobson b, middleton s. paying to care: the cost of childhood disability. york, uk: joseph rowntree foundation. 1998. 65. lawton d. the family fund database. york, uk: university of york. 1996. 66. curran al et al. time costs of caring for children with severe disabilities compared with caring for children without disabilities. dev med child neurol. 2001 aug;43(8):529-33. 67. sito del ministero della salute www.ministerosalute.it. neridronato nel trattamento dell’osteogenesi imperfetta: prestazioni cliniche ed economiche di un farmaco orfano 77farmeconomia e percorsi terapeutici 2003; 4 (2) © seed tutti i diritti riservati valutazione economica di infliximab (remicade®) vs etanercept (enbrel®) nel trattamento dell’artrite reumatoide colombo g. l.*, muzio a.*, longhi a.* * s.a.v.e. studi analisi valutazioni economiche, milano introduzione l’artrite reumatoide (ar) è una delle più severe malattie croniche, con sintomatologia dolorosa, che comporta una progressiva perdita della mobilità e la diminuzione della qualità della vita a causa della progressiva distruzione delle strutture articolari [1]. l’esordio avviene tra i 25/50 anni, ma non sono rari i casi di artrite reumatoide infantile [2]. è una malattia invalidante che colpisce l’organismo nel suo insieme; le articolazioni colpite più frequentemente sono mani, ginocchia, anche e piedi, la cui struttura, nei casi più gravi viene alterata causando deformità [3]. la prevalenza dell’ar in italia è stimata pari allo 0,5%, circa 300.000 persone, in prevalenza donne [2]. a causa dei gravi effetti invalidanti presenta costi elevati per il sistema sanitario nazionale e per la società anche quando è trattata con le migliori terapie tradizionali [4]. nel lavoro di leardini [5] sul costo sociale dell’artrite reumatoide in italia, si è stimato un costo diretto annuo che varia tra 1.643 euro e 5.697 euro per persona, a seconda delle 4 classi di gravità della malattia, e dei costi indiretti annui (le perdite di produzione sul sistema economico) che variano da 2.705 euro a 17.249 euro secondo la gravità della malattia. il trattamento con farmaci antinfiammatori (fans e corticosteroidi) e con i cosiddetti dmard, disease modifying antirheumatic drugs (ad esempio methotrexate), purtroppo cura solo i sintomi, ma non il progredire della malattia e nemmeno i danni irreversibili alla capacità motoria, che rendono impossibili le normali attività quotidiane [6]. studi anglosassoni indicano che a 10 anni dalla diagnosi il 44% dei pazienti risulta inabile al lavoro e dopo abstract rheumatoid arthritis is one of the most severe chronic pathologies, affecting the whole organism, with invalidating outcomes that affect the quality of life of the patients. its prevalence is estimated to be about 0,5% in italy, with elevated costs for the national health system (nhs) and the society, in spite of the best treatment with traditional therapies that include anti-inflammatory and disease modifying antirheumatic drugs (dmards). the introduction of new drugs with biological activity, mainly acting through an antagonism of tumor necrosis factor (anti-tnf), is a great advance in the management of the disease, as their use has been shown to be effective in slowing the progression of the joint damage, and sometimes in reversing it,. the present article present a cost-minimization study conducted by comparing the two anti-tnfs available in italy, etanercept and infliximab, assuming equal efficacy and approached from the perspectives of the italian nhs and society. only differential costs were considered, i.e. drug acquisition, drug administration and patient monitoring costs, and the analysis comprised two treatment years, in order two account for the cost differences between the first treatment year and the following. the analysis showed that infliximab represents the more convenient alternative from both the nhs and the society points of view, mainly due to lower drug acquisition costs, which offset the higher drug administration costs: the total two year treatment costs per patient with etanercept resulted of 20.173 euro and 22.164 euro from the societal and nhs perspectives, respectively, while the same costs resulted of 13.715 euro and 14.795 euro with infliximab. these results were tested with thorough sensitive analyses, conducted by varying the principal cost and time consumption estimates, that showed that etanercept results less convenient in all the hypothesis tested. farmeconomia e percorsi terapeutici 2003; 4 (2): 77-86 analisi economica 78 farmeconomia e percorsi terapeutici 2003; 4 (2)© seed tutti i diritti riservati 20 anni il 25% dei pazienti subisce un intervento chirurgico di artroprotesi [2]. le nuove terapie a base di farmaci biologici, gli anti-tnf (antagonism of tumour necrosis factor) sono in grado di inibire la progressione del danno strutturale agendo direttamente sul tnf (antagonism of tumour necrosis factor), il principale mediatore dell’immunoflogosi reumatoide, inoltre “nel 40-55% dei pazienti trattati con infliximab arruolati nello studio attract, l’evidenza radiologica del danno articolare si è ridotta, indicando la riparazione di parte del danno stesso.” [7]. queste terapie sono state testate con risultati positivi su pazienti che non avevano risposto positivamente alle terapie classiche (interrotte perché inefficaci o a causa di eventi avversi intollerabili); in questa popolazione è stato dimostrato un rallentamento della progressione del danno articolare [8]. i farmaci in questione sono tre: infliximab (remicade®), etanercept (enbrel®) e kineret (anakinra®); nella presente analisi ci si è concentrati sui farmaci attualmente in commercio: infliximab (remicade®) vs. etanercept (enbrel®), entrambi prescrivibili e distribuibili solo in centri specializzati [2]. obiettivo del presente lavoro è quello di presentare una valutazione economica di infliximab vs. etanercept nel trattamento dell’artrite reumatoide; a tal proposito, si svilupperà un’analisi di minimizzazione dei costi che indichi quale terapia, a parità di efficacia, consenta un maggior risparmio per la società e per il ssn, al fine di garantire un miglior impiego delle risorse disponibili. materiali e metodi è stata costruita un’analisi di cost minimization al fine di stimare i costi totali annuali derivanti dall’impiego di infliximab vs. etanercept, ipotizzando che i due trattamenti presentino pari efficacia e probabilità di eventi avversi, anche se dal confronto dei dati pubblicati risulta che solo infliximab è in grado di arrestare la riduzione della rima articolare (erosione cartilaginea), oltre a fermare ed in alcuni casi riparare l’erosione ossea [9]. l’orizzonte temporale prescelto è stato di 2 anni, dal momento che il costo affrontato il primo anno è maggiore di quello affrontato per gli anni successivi. nell’analisi si è adottato il punto di vista della società, comprendendo anche i costi indiretti per la perdita di produttività. per l’individuazione degli elementi di costo si è fatto riferimento principalmente ad un precedente studio olandese [10] e allo studio osservazionale antares [2], un protocollo di monitoraggio per il trattamento dei pazienti affetti da artrite reumatoide con farmaci “biologici” anti-tnf (alfa). consumo di risorse sono stati considerati solo i costi differenziali delle due terapie, ignorando i costi comuni (ad esempio visite di controllo ed esami di laboratorio), che sono uguali e presentano la stessa cadenza temporale per i due trattamenti, come indicato nello studio osservazionale antares [2]. durante le visite avviene una valutazione delle articolazioni tumefatte e dolenti e dell’attività della malattia, gli esami di laboratorio consistono prevalentemente nel controllo dei dati bioumorali e in uno screening per la tubercolosi. le differenze tra le due terapie sono invece riconducibili più che altro alle modalità di somministrazione. etanercept viene assunto per via sottocutanea due volte la settimana, mentre infliximab mediante infusione endovenosa ogni 8 settimane, dopo un periodo iniziale in cui la somministrazione avviene alle settimane 0, 2, 6; dunque in 2 anni (104 settimane) il farmaco verrà somministrato 15 volte: 3 volte per la terapia di induzione e 12 per la prosecuzione (98 settimane/8). pertanto i costi ospedalieri influiscono prevalentemente su infliximab, mentre per il primo si mantiene valida l’ipotesi dello studio di nujten [10], secondo cui il 10% dei pazienti necessita di un infermiere a domicilio per l’iniezione. la valorizzazione delle terapie farmacologiche è stata effettuata mediante l’applicazione del prezzo al pubblico del principio attivo (prezzo relativo alla specialità in commercio a minor prezzo) [11], scontato del 50% nel caso il consumo del farmaco avvenisse in regime di ricovero ospedaliero (cfr. legge 386/ 75). da tale valore, nell’analisi dal punto di vista della società, è stata dedotta l’iva (10%), dividendolo per 1,1. le visite di controllo avvengono con la stessa cadenza e contestualmente avviene l’infusione per i pazienti in trattamento con infliximab e la consegna di etanercept per gli altri. methotrexate, che viene assunto in terapia combinata con infliximab, può essere acquistato in farmacia e la ricetta viene fatta durante la visita di controllo. per questa ragione non abbiamo considerato i costi di trasporto dei pazienti verso l’ospedale e i costi di stoccaggio, dal momento che entrambi i farmaci devono essere conservati in frigorifero. per i dosaggi dei farmaci si è fatto riferimento alle rispettive schede tecniche e al protocollo antares (3 mg/kg di infliximab per ogni valutazione economica di infliximab (remicade®) vs etanercept (enbrel®) nel trattamento dell’artrite reumatoide 79farmeconomia e percorsi terapeutici 2003; 4 (2) © seed tutti i diritti riservati infusione, 25 mg di etanercept per iniezione e 12,5 mg di methotrexate alla settimana). i tempi impiegati da infermieri e reumatologi sono stati stimati rispettivamente di 15 e 5 minuti. considerando che l’infusione dura circa 2 ore e che è previsto un periodo di osservazione successivo di 1 o 2 ore, si è ipotizzato che i pazienti con una attività lavorativa si debbano assentare per almeno mezza giornata. per i pazienti che non presentano un’attività lavorativa, invece, non si è tenuto conto della perdita di tempo libero. per calcolare il peso medio si sono utilizzati i dati di una indagine doxa combinati con quelli dell’istat [12, 13]; il risultato, 67 kg, richiede la somministrazione di 2 fiale di infliximab per infusione, mentre per un peso compreso tra i 67 kg e i 100 kg ne sono richieste 3, non essendo possibile conservare il contenuto della fiala dopo l’apertura. la quantificazione del costo del personale infermieristico e medico durante l’infusione è stata fatta sulla base del costo/minuto della stessa, desunto dalle strutture ospedaliere lombarde: il costo medio annuo di un infermiere professionale per la struttura è di circa 39.000 euro; tale cifra è stata rapportata a 1.512 ore (36h per 42 settimane), per ottenere il costo orario, valore poi rapportato al minuto lavorato. il costo/minuto così elaborato è risultato essere di 0,43 euro. il costo medio annuo di un medico specialista è di circa 83.000 euro; tale costo è stato rapportato a 1.428 ore lavorate/ anno (34 ore per 42 settimane, tenendo conto che 4 ore possono essere dedicate all’aggiornamento), con un costo al minuto di 0,97 euro. il costo del materiale di somministrazione dell’infusione, comprensivo di fisiologica, cotone idrofilo, alcool, soluzione di cloruro di sodio, sacca per infusioni, set infusioni, pompa lifecare, guanti di plastica sterili, siringa ed ago a farfalla, è stato stimato pari a 14,94 euro (iva compresa). altra voce di costo differenziale è risultata lo smaltimento dei rifiuti ospedalieri trattati (rot), stimata in circa 0,96 euro/kg (iva compresa). ai costi ospedalieri è stata infine aggiunta una quota di costi comuni del 20%, sulla base di una rilevazione campionaria in alcuni ospedali della regione lombardia. per la stima delle perdite di produzione, si è fatto riferimento al reddito procapite annuo [14], tramite l’impiego del valore della retribuzione lorda per unità di lavoro dipendente, da cui si è ottenuta, dividendo per il numero di giorni lavorativi (220), la retribuzione giornaliera, pari a 95 euro. per calcolare il numero degli occupati sulla popolazione con ar, si è considerato il numero di occupati nel 2002 [14], moltiplicato per la prevalenza dell’artrite reumatoide in italia; il peso rispetto al numero di pazienti è pari al 49,2%. considerando che la popolazione colpita da ar è piuttosto anziana e composta prevalentemente da donne e che la malattia è molto invalidante, questa cifra appare attendibile; comunque nell’analisi di sensibilità è stata testata anche l’ipotesi che tutti avessero un’occupazione per l’esposizione dei risultati ci si è concentrati, in una prima fase, sui costi differenziali del singolo paziente nell’anno 1 e 2 (essendo il numero di somministrazioni nel primo anno maggiori rispetto ai successivi), per poi calcolare il costo totale dell’intero periodo. per considerare la diversa distribuzione dei costi, i costi del secondo anno sono stati attualizzati con un tasso compreso tra 0%, 3%, 5%. per testare i risultati ottenuti sono state fatte analisi di sensibilità sulle variabili principali: sulle principali tipologie di costo, sul dosaggio di infliximab, sui tempi di somministrazione, sulla percentuale di occupati, sulla percentuale di necessità di un infermiere a domicilio e sulla quantità di lavoro perso. risultati si sono costruite due serie di tabelle, una secondo il punto di vista della società (tab. 1-4), al netto dell’iva (quella applicata ai farmaci è del 10%) e considerando i costi indiretti generati dalla perdita di lavoro, e l’altra dal punto di vista del ssn (tab. 5-8), considerando solo i costi sanitari. nelle tabb. 1, 2 (punto di vista della società) e 5, 6 (punto di vista del ssn) vengono mostrate le diverse voci di costo relative a infliximab, che si suddividono in costi sanitari (costi di farmaci, personale, materiale per la somministrazione, smaltimento rifiuti a cui sono aggiunti i costi comuni) e costi indiretti relativi alla perdita di lavoro. le tabelle 1 e 5 contengono i dati relativi al primo anno ed i totali parziali, che ammontano rispettivamente a 7.311 euro e 7.839 euro; le tabelle 2 e 6 si riferiscono ai valori relativi al secondo anno, 6.403 euro per la società e 6.865 euro per il ssn. nelle tabb. 3 e 7 sono invece contenuti i costi relativi ad etanercept che sono stati scomposti in costi per il farmaco e costi per l’intervento dell’infermiere a domicilio; i totali annuali sono pari a 10.087 euro per la società e a 11.082 per il ssn. per ciascuna voce di costo sono espressi l’unità di misura, la dose per trattamento, la quantità totale e il costo, sia unitario sia totale. g.l. colombo, a. muzio, a. longhi 80 farmeconomia e percorsi terapeutici 2003; 4 (2)© seed tutti i diritti riservati ta b e lla 1 in fl ix im a b , ri s o rs e im p ie g a te ( a n n o 1 ) d a l p u n to d i v is ta d e lla s o c ie tà ( v a lo ri in e u r o ) valutazione economica di infliximab (remicade®) vs etanercept (enbrel®) nel trattamento dell’artrite reumatoide aigolopi t airogeta c enoizircse d arusi mid àtin u otunetno c .fnoc enoizartsini m mos otso c oiratinu .tart rep àtin u otso c otne mattart azneuqerf itne mattart àtitnau q .tart elaunna otso c elaunna inoizatonn a itso c iratinas ica mraf ba mixilfni etaxertohte m )rpc( )g m001( alaif .rpc 52 enoizefnoc g m001 )alaif 1( g m5,26 )rpc52( gk/g m3 .ttes alla g m5,21 21,824 45,4 2 2,0 42,658 19,0 e 6/2/0.ttes alla .ttes 8ingoiop .ttes 1 8 25 29,9486 22,74 osep g k76=oide m .rpc elanosrep irei mrefni ilanoisseforp igolota mue r ituni m ituni m 34,0 79,0 00,51 00,5 54,6 58,4 e 6/2/0.ttes alla .ttes 8ingoiop e 6/2/0.ttes alla .ttes 8ingoiop 8 8 06,15 08,83 ilaireta m enoizartsini m mos .tart 24,21 e 6/2/0.ttes alla .ttes 8ingoiop 8 63,99 itsocirtl a t o r )otne mitla ms( .g k 1 50,0 08,0 50,0 40,0 e 6/2/0.ttes alla .ttes 8ingoiop 8 23,0 .rg 05idisetopi ituifirid )ica mraf otsoc osulcse( elaizrap elatot 80,091 ) %02(inu mocitsoc atou q 20,83 ica mrafi osulcse 1 onnairatinasitsoc elatot 32,521.7 aigolopi t airogeta c enoizircse d itapucco % otidder etipacorp elaunna etipacorp otidder oreilanroig rep àtin u .tart .tart otso c .tart azneuqerf àtitnau q .ttart elaunna otso c rep elaunna otapucco otso c rep elaunna itapucco % inoizatonn a itso c itteridni atidrep enoizudorp inroig atidrep ivitaroval atanroig azze m otne mattart rep 91802 36,49 azze m atanroig 23,74 e 6/2/0.ttes alla .ttes 8ingoiop 8 35,873 42,681 ied %2,94li olos atlusiritala m otapucco 1 onna otsoc elatot 74,113.7 81farmeconomia e percorsi terapeutici 2003; 4 (2) © seed tutti i diritti riservati ta b e lla 2 in fl ix im a b , ri s o rs e im p ie g a te ( a n n o 2 ) d a l p u n to d i v is ta d e lla s o c ie tà ( v a lo ri in e u r o ) g.l. colombo, a. muzio, a. longhi aigolopi t airogeta c enoizircse d arusi mid àtin u otunetno c enoizefnoc enoizartsini m mos otso c oiratinu .tart rep àtin u otso c otne mattart azneuqerf itne mattart àtitnau q .tart elaunna otso c elaunna inoizatonn a itso c iratinas ica mraf ba mixilfni etaxertohte m )rpc( )g m001( alaif .rpc 52 enoizefnoc g m001 )alaif 1( g m5,26 )rpc52( gk/g m3 .ttes alla g m5,21 21,824 45,4 2 2,0 42,658 19,0 ena mittes 8ingo .ttes 1 7 25 86,3995 22,74 osep g k76=oide m .rpc elanosrep irei mrefni ilanoisseforp igolota mue r ituni m ituni m 34,0 79,0 00,51 00,5 54,6 58,4 ena mittes 8ingo ena mittes 8ingo 7 7 51,54 59,33 ilaireta m enoizartsini m mos .tart 24,21 ena mittes 8ingo 7 49,68 itsocirtl a t o r )otne mitla ms( .g k 1 50,0 08,0 50,0 40,0 ena mittes 8ingo 7 82,0 id.rg 05idisetopi ituifir )ica mraf otsoc osulcse( elaizrap elatot 23,661 ) %02(inu mocitsoc atou q 62,33 ica mraf osulcse 2 onn airatinasitsoc elatot 84,0426 aigolopi t airogeta c enoizircse d itapucco % otidde r etipacorp elaunna otidde r etipacorp oreilanroig rep àtin u .tart otso c otne mattart azneuqerf otne mattart àtitnau q .tart elaunna otso c rep elaunna otapucco otso c rep elaunna itapucco % inoizatonn a itso c itteridni atidrep àtivittudorp inroig atidrep ivitaroval atanroig azze m otne mattart rep 91802 36,49 azze m atanroig 23,74 ena mittes 8ingo 7 12,133 69,261 ied %2,94li olos atlusiritala m otapucco 2 onna otsoc elatot 44,304.6 iratinasitsoc elatot 17,563.31 .dorp atidrepitsoc elatot 91,943 )onna 2+1(ilatotitso c 09,417.31 82 farmeconomia e percorsi terapeutici 2003; 4 (2)© seed tutti i diritti riservati tabella 3 etanercept, risorse annue impiegate dal punto di vista della società (valori in euro) le differenze nelle tipologie di costo rilevate nei due trattamenti derivano essenzialmente dalla diversa modalità di somministrazione: per l’infusione di infliximab si sono dovuti stimare i relativi costi ospedalieri, mentre per l’iniezione sottocutanea a domicilio richiesta da etanercept i costi riguardano solo l’infermiere a domicilio, stimato necessario solo dal 10% dei pazienti. i costi più rilevanti sono quelli per l’acquisto del farmaco, che incidono per il 93,7% sul costo totale per paziente trattato con infliximab e per il 98,7 % per etanercept. i costi ospedalieri e indiretti, per infliximab, hanno una incidenza minima sul totale, ammontano solo a 473 euro, e quindi non influenzano il segno dell’analisi. dall’analisi di minimizzazione dei costi è emerso che infliximab è la terapia più conveniente, sia dal punto di vista della società (tab. 4: costi per paziente dal punto di vista della società) sia da quello del ssn (tab. 8: costi per paziente dal punto di vista del ssn), con un risparmio rispettivamente di 6.458 euro e di 7.459 euro per paziente trattato. analisi di sensibilità per verificare la validità dei risultati ottenuti e la consistenza del risparmio che può determinare l’adozione di infliximab vs. etanercept è stata condotta un’analisi di sensibilità sui principali fattori di costo considerati. in particolare, si sono fatti variare i valori dei prezzi dei farmaci (±20%), del tempo di infusione e del costo di medici (0-10 minuti) ed infermieri (5-60 minuti), del costo per il materiale di infusione (± 30%) e dei costi e quantità di rifiuti da smaltire (± 20% e 0-0,15 kg). si è fatto poi variare il costo del lavoro (+ 45% e + 35%) e si è ipotizzato che per ogni infusione si perdesse una giornata intera, invece di mezza; si è quindi aumentato il numero degli occupati, ipotizzando che tutti i pazienti con ar lavorassero, ed infine si è supposto che nessuno avesse bisogno di un infermiere a domicilio per la somministrazione di etanercept. in nessuna circostanza si è invertito il segno dell’analisi e infliximab è risultato in ogni caso la terapia più economica (tabella 9: analisi di sensibilità). data la scarsa incidenza dei costi ospedalieri e indiretti, le loro variazioni incidono poco sul livello di risparmio, mentre i prezzi dei farmaci presentano la maggiore incidenza facendo variare notevolmente il livello di risparmio. è particolarmente significativa la variazione del risparmio in caso di impiego di 3 fiale di infliximab invece di due, ipotesi che si verifica quando il paziente pesa più di 67 kg: in questo caso il risparmio va dai 37 euro (senza attualizzazione) ai 4 euro per paziente (attualizzazione al tasso del 5%). attualizzando il valore della terapia a tassi crescenti (del 3% e del 5%) il valore del risparmio si assottiglia: il motivo risiede nel fatto che il costo della terapia a base di infliximab è maggiore nel primo anno. è stata svolta, infine, un’analisi di soglia sui costi ospedalieri, che comprendono il costo per materiale, personale, rot e costi comuni; affinché il costo del trattamento con etanercept risulti più conveniente, i costi ospedalieri devono essere pari a 6.886 euro vs. i 427,67 euro stimati durante l’analisi. la stessa analisi è stata ripetuta per le ore del personale e da tale analisi è risultato che sia infermiere sia reumatologo dovrebbero dedicare al paziente 4,75 ore per ciascuna infusione, ovvero più della durata dell’infusione stessa, affinché le due terapie si equivalgano. iratinasitsoc .dorpatidrepitsoc ilatotitsoc bamixilfni 663.31 943 517.31 tpecrenate 371.02 371.02 .ffid 708.6943 854.6tabella 4 risultati: punto di vista della società, costo per paziente (anno 1+2) valutazione economica di infliximab (remicade®) vs etanercept (enbrel®) nel trattamento dell’artrite reumatoide aigolopit airogetac enoizircsed idàtinu arusim otunetnoc enoizefnoc enoizartsinimmos otsoc oiratinu repàtinu otnemattart otsoc otnemattart azneuqerf itnemattart àtitnauq elaunna .ttart otsoc elaunna inoizatonna itsoc iratinas ocamraf tpecrenate alaif ).gm52( elaif4 gm52 97,283 52,0 07,59 .ttesalla2 401 45,259.9 elanosrep aereimrefni oilicimod itunim 03 34,0 03 9,21 .ttesalla2 401 61,431 è.tototsocli àig otacilpitlom %01lirep ounnaotsocelatot 07,680.01 )onna2+1(ilatotitsoc 04,371.02 8 3 f a rm e c o n o m ia e p e rc o rs i te ra p e u tic i 2 0 0 3 ; 4 (2 ) © s e e d t u tti i d iritti ris e rv a ti tabella 5 infliximab, risorse impiegate (anno 1) dal punto di vista del ssn (valori in euro) tabella 6 infliximab, risorse impiegate (anno 2) dal punto di vista del ssn (valori in euro) g .l . c o lo m b o , a . m u zio , a . l o n g h i aigolopit airogetac enoizircsed arusimidàtinu otunetnoc .fnoc enoizartsinimmos otsoc oiratinu .tartrepàtinu .tartotsoc .tartazneuqerf .tartonnaq otsoc elaunna inoizatonna itsoc iratinas icamraf bamixilfni etaxertohtem )rpc( )gm001(alaif .rpc52enoizefnoc gm001 )alaif1( gm5,26 )rpc52( gk/gm3 .ttesallagm5,21 39,074 99,4 2 2,0 58,149 00,1 e6/2/0.ttesalla .ttes8ingoiop .ttes1 8 25 08,435.7 09,15 osep gk76=oidem .rpc elanosrep ireimrefni ilanoisseforp igolotamuer itunim itunim 34,0 79,0 00,51 00,5 54,6 58,4 e6/2/0.ttesalla .ttes8ingoiop e6/2/0.ttesalla .ttes8ingoiop 8 8 06,15 08,83 ilairetam enoizartsinimmos .tart 49,41 e6/2/0.ttesalla .ttes8ingoiop 8 94,911 itsocirtla tor )otnemitlams( .gk 1 50,0 69,0 50,0 50,0 e6/2/0.ttesalla .ttes8ingoiop 8 83,0 id.rg05idisetopi ituifir )icamrafotsocosulcse(elaizrapelatot 82,012 )%02(inumocitsocatouq 60,24 icamrafisulcse 1onnairatinasitsocelatot 30,938.7 itsoc iratinas icamraf bamixilfni etaxertohtem )rpc( )gm001(alaif .rpc52enoizefnoc gm001 )alaif1( gm5,26 )rpc52( gk/gm3 .ttesallagm5,21 39,074 99,4 2 2,0 58,149 00,1 enamittes8ingo .ttes1 7 25 59,2956 09,15 osep gk76=oidem .rpc elanosrep ireimrefni ilanoisseforp igolotamuer itunim itunim 34,0 79,0 00,51 00,5 54,6 58,4 enamittes8ingo enamittes8ingo 7 7 51,54 59,33 ilairetam enoizartsinimmos .tart 49,41 e6/2/0.ttesalla .ttes8ingoiop 7 65,401 itsocirtla tor )otnemitlams( .gk 1 50,0 69,0 50,0 50,0 enamittes8ingo 7 43,0 id.rg05idisetopi ituifir )icamrafotsocosulcse(elaizrapelatot 99,381 )%02(inumocitsocatouq 08,63 icamrafisulcse 2onnairatinasitsocelatot 46,5686 )onna2+1(iratinasitsocelatot 66,407.41 84 farmeconomia e percorsi terapeutici 2003; 4 (2)© seed tutti i diritti riservati discussione il presente studio ha mostrato le risorse consumate da due terapie alternative (infliximab vs. etanercept) per pazienti affetti da artrite reumatoide mediante un’analisi di cost minimization. nell’analisi proposta si è fatto riferimento alle sole voci di costo che costituivano un differenziale tra i due trattamenti. nel lavoro si è ipotizzata la stessa efficacia e gli stessi eventi avversi, simili per entrambe le terapie. nel complesso si è stimato in 6.458,5 euro il risparmio per la società per paziente trattato con infliximab vs. etanercept; tale importo diventerebbe oltre 1.933 mila euro se rapportato a tutti i pazienti affetti da artrite reumatoide in italia ed eligibili al trattamento con queste due molecole. i risultati emersi mostrano un considerevole risparmio, garantito soprattutto dal minor costo di acquisto di infliximab (5.994 euro vs. 9.953 euro). occorre tuttavia ricordare in sede di discussione alcuni problemi che si incontrano nel calcolo di tempi e costi ospedalieri. innanzitutto, è opportuno aver presente che si tratta di tempi e costi indicativi, seppure ottenuti dall’osservazione dell’attività ospedaliera, lo stesso vale per i costi di somministrazione. è pertanto chiaro che questi valori non sono il risultato di tabella 7 etanercept, risorse annue impiegate dal punto di vista del ssn (valori in euro) ilatotitsoc bamixilfni 507.41 tpecrenate 461.22 aznereffid 954.7tabella 8 risultati: punto di vista ssn, costo per paziente (anno 1+2) estese sperimentazioni sul campo, anche se l’analisi di sensibilità, sviluppata sui tempi e costi delle figure professionali sanitarie coinvolte nella somministrazione, ha determinato un’equivalenza di costo delle due alternative solo nel caso in cui al paziente, durante l’infusione, vengano dedicate almeno 4,75 ore (più della durata dell’infusione stessa) sia dal reumatologo sia dall’infermiere. è stata svolta anche un’analisi di soglia sui costi ospedalieri e ne è risultato che le due terapie avrebbero lo stesso costo qualora i costi ospedalieri fossero pari a 6.886 euro vs. i 427,67 euro, che si sono stimati durante questa analisi. dato il piccolo peso di tutte queste variabili rispetto al costo totale della terapia, le questioni inerenti la loro stima non influenzano il risultato dell’analisi, come si è potuto ampiamente testare nell’analisi di sensibilità, dove, in particolare, si sono impiegati i tempi del personale dello studio olandese e dai costi è stata sottratta l’aliquota media dei contributi. occorre infine ricordare che effettuare il calcolo con le modalità ora descritte significa assumere che tutto il tempo di lavoro del personale sia completamente saturato da operazioni “produttive”, nel senso di operazioni direttamente rivolte alla cura dei pazienti, come lo è la somministrazione dei farmaci. in realtà le cose non stanno così. le 1.512 ore annue convenzionali utilizzate come base per il calcolo dei costi unitari sono “lorde” e comprendono, accanto ai tempi dedicati a operazioni produttive nel senso sopra indicato, anche i tempi dedicati ad altre operazioni. comprendono infatti anche i tempi di lavoro dedicati ad attività generali (come l’aggiornamento professionale e le attività di coordinamento) e preparatorie, i tempi morti (spesi nel passaggio tra un tipo e un altro di operazione), le pause fisiologiche di riposo e spesso quelle (meno fisiologiche) dovute a un’organizzazione poco efficiente del lavoro e così via. queste premesse metodologiche sono necessarie, ma non impediscono di valutare il costo del personale secondo la tecnica illustrata, in quanto fornisce valutazione economica di infliximab (remicade®) vs etanercept (enbrel®) nel trattamento dell’artrite reumatoide aigolopit airogetac enoizircsed idàtinu arusim otunetnoc enoizefnoc enoizartsinimmos otsoc oiratinu repàtinu otnemattart otsoc otnemattart azneuqerf itnemattart àtitnauq elaunna .ttart otsoc elaunna inoizatonna itsoc iratinas ocamraf tpecrenate ).gm52(alaif gm001 )elaif4( gm52 560,124 52,0 72,501 .ttesalla2 401 96,749.01 elanosrep aereimrefni oilicimod itunim 03 34,0 03 9,21 .ttesalla2 401 61,431 è.tototsocli otacilpitlomàig %01lirep ounnaotsocelatot 58,180.11 )onna2+1(ilatotitsoc 07,361.22 85farmeconomia e percorsi terapeutici 2003; 4 (2) © seed tutti i diritti riservati comunque utili informazioni, soprattutto ai fini di un confronto fra diverse alternative. riguardo ai dati sulla popolazione trattata si è ipotizzato che tutti i malati di artrite reumatoide venissero trattati con i farmaci biologici, ed i valori impiegati per il calcolo degli occupati e il reddito pro-capite sono stimati sulla base di medie nazionali; questi valori potabella 9 analisi di sensibilità dal punto di vista della società (valori in euro) trebbero non cogliere con precisione le caratteristiche della popolazione affetta da artrite reumatoide. ma anche in questo caso, l’analisi di sensibilità proposta mostra il ruolo dominate di infliximab in termini di risparmio di risorse. questo studio è stato reso possibile da un finanziamento messo a disposizione da schering-plough spa bibliografia 1. wong, singh, kavanaugh, estimating the cost effectiveness of 54 weeks of infliximab for rheumatoid arthritis, the american journal of medicine, 2002, vol 113 2. studio osservazionale antares, http://www.reumatologia.it/ 3. erkan, yazici, harrison, paget, physician treatment preferences in rheumatoid arthritis of differing disease severity and activity: the impact of cost on first-line therapy, arthritis and rheumatism, 2002, vol.47 (3) 4. lubeck, a review of the direct cost of rheumatoid arthritis, managed care versus fee-for service settings, pharmaeconomics, 2001, vol.19 (8) 5. leardini, salaffi, montanelli, et al. a multicenter cost-of-illness study on rheumatoid arthritis in italy, clinical and experimental rheumatology, 2002, 20: 505-515. 6. maetzel, strand, tugwell, wells, bombardier, economic comparison of leflunomide and methotrexate in patients with rheumatoid arthritis, pharmacoeconomics, 2002, 20 (1), pp.61-70 g.l. colombo, a. muzio, a. longhi airogetac elibairav ollavretni etneizaprep)tpecrenate-bamixilfni(eiparetelledotsoclenaznereffid ollavretni'llederoirefnietimilla ollavretni'llederoirepusetimilla %0.tta %3.tta %5.tta %0.tta %3.tta %5.tta ocamraf bamixilfniozzerp etaxertohtemozzerp ozzerp tpecrenate bamixilfnioiggasod %02+;%02%02+;%02%02+;%02elaif3;2 720.9774.6774.2854.6588.8073.6824.2153.6597.8203.6793.2382.6098.3044.6934.0173718.3333.6472.0171177.3562.6961.014elanosrep ereimrefniopmet enoisufnirep ogolotamueropmet enoisufnirep aereimrefniopmet oilicimod idogeipmion aereimrefni oilicimod ereimrefniotsoc ogolotamuerotsoc itunim06;5 itunim01;0 itunim06;01 %01;%0 %0;%53%0;%54635.6645.6082.6091.6504.6894.6824.6734.6571.6780.6992.6093.6953.6863.6801.6120.6132.6123.6011.6173.6727.6854.6854.6854.6800.6562.6616.6153.6153.6153.6349.5891.6545.6382.6382.6382.6elairetam otsoc enoizartsinimmos torotsoc toràtitnauq %03+;%03%02+;%02.gk51,0;.gk0 625.6954.6954.6714.6153.6253.6943.6382.6482.6193.6854.6754.6582.6153.6053.6812.6382.6282.6otteridni idatidrepotsoc oroval idatidrepàtitnauq oroval ilgedoremun itapucco %02+;%021;onroig2/1 onroig %001;%05 itneizapied 825.6854.6854.6024.6153.6153.6153.6382.6382.6983.6901.6890.6282.6700.6699.5512.6249.5139.586 farmeconomia e percorsi terapeutici 2003; 4 (2)© seed tutti i diritti riservati 7. lipsky, van der hejde, st.clair et al., infliximab and methotrexate in the treatment of rheumatoid arthritis, the new england journal of medicine, 2000, vol.30 8. kobelt, jonsson, young, eberhardt, the cost-effectiveness of infliximab (infliximab) in the treatment of rheumatoid arthritis in sweden and the united kingdom based on the attract study, rheumatology, 2002, 42:326-335 9. strand, sharp, radiographic data from recent randomized controlled trials in rheumatoid arthritis, arthritis & rheumatism 2003, vol. 48, pp. 21-34. 10. nujiten, engelfriet, duijn, et al., a cost-cost study comparing etanercept with infliximab in rheumatoid arthritis pharmaeconomics, 2001, 19 (10): 1051-1064 11. informatore farmaceutico, 2000 12. doxa, http://web.tiscali.it/no-redirect-tiscali/exob1/obes/ 13. istat, statistiche demografiche, 2000. 14. istat, (2000), relazione generale sulla situazione economica del paese, aprile, 2000 15. drummond m.f., o’ brien b., stoddard g.l., torrance g.w., (1997), methods for the economic evaluation of health care programmes, oxford university press, second edition. 16. jefferson t., demicheli v., mugford m., (1998), la valutazione economica degli interventi sanitari, il pensiero scientifico editore, roma. 17. jobanputra, barton, bryan, burls, the effectiveness of infliximab and etanercept for the treatment of rheumatoid arthritis: a systematic review and economic evaluation, health technology assessment, 2002, vol.6, n°21 valutazione economica di infliximab (remicade®) vs etanercept (enbrel®) nel trattamento dell’artrite reumatoide simpo pdf merge and split unregistered version http://www.simpopdf.comsimpo pdf merge and split unregistered version http://www.simpopdf.com simpo pdf merge and split unregistered version http://www.simpopdf.comsimpo pdf merge and split unregistered version http://www.simpopdf.com simpo pdf merge and split unregistered version http://www.simpopdf.comsimpo pdf merge and split unregistered version http://www.simpopdf.com simpo pdf merge and split unregistered version http://www.simpopdf.comsimpo pdf merge and split unregistered version http://www.simpopdf.com simpo pdf merge and split unregistered version http://www.simpopdf.comsimpo pdf merge and split unregistered version http://www.simpopdf.com key: cord-0071025-bkalx73u authors: valori, miko; jansson, lilja; tienari, pentti j. title: cd8+ cell somatic mutations in multiple sclerosis patients and controls—enrichment of mutations in stat3 and other genes implicated in hematological malignancies date: 2021-12-07 journal: plos one doi: 10.1371/journal.pone.0261002 sha: 2262a4cd9165afc24899e25b8899aa63ad8e3532 doc_id: 71025 cord_uid: bkalx73u somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. previously we and others have demonstrated that especially cd8+ t cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (ms) and rheumatoid arthritis. here we concentrated on cd8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between ms patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? we separated peripheral blood cd8+ cells from newly diagnosed relapsing ms patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the cd8+ cells’ dna, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. we discovered nonsynonymous somatic mutations in all ms patients’ and controls’ cd8+ cell dna samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2–8.6%). the mutations showed statistically significant clustering especially to the stat3 gene, and also enrichment to the smarca2, dnmt3a, socs1 and ppp3ca genes. known activating stat3 mutations were found both in ms patients and controls and overall 1/5 of the mutations were previously described cancer mutations. the detected clustering suggests a selection advantage of the mutated cd8+ clones and calls for further research on possible phenotypic effects. somatic mutations can occur early or late in life and lead to mosaicism in different cell lineages. the mutation rates differ between tissues, ranging from 3.5 x 10 −9 (small intestine) to 1.6 x10 -7 (skin) mutations per base-pair [1] . blood cells accumulate mutations during lifetime and mutations that provide a survival advantage or promote proliferation will enrich by time in a process called clonal hematopoiesis. somatic mutations have been shown to increase in number during aging. in population cohorts not selected for cancer or hematologic phenotypes somatic mutations in whole blood dna with allelic fraction of 10-20% have been found in approximately 1% of subjects under age 50, and in 10% of subjects older than 70 years [2, 3] . somatic mutations have an established role in cancer; their role in non-malignant diseases is also gaining attention. single cell sequence analyses have raised the hypothesis that somatic mutations may modulate brain aging and neurodegeneration [4] . activating kras mutations have been observed in arteriovenous malformations in the brain [5] . there are also examples of rare autoimmune diseases in which somatic mutations play a role. in children with an autoimmune lymphoproliferative syndrome, somatic mutations in fas have been discovered, and these mutations induced a defect in t-cell apoptosis [6] . similar childhood autoimmune disease has also been described with activating somatic mutations in the kras gene, which also impair t-cell apoptosis [7] . recently, somatic mutations in x-chromosomal upa1 gene were reported in men with an adult-onset inflammatory syndrome called vexas (vacuoles, e1 enzyme, x-linked, autoinflammatory, somatic) [8] . the role of somatic mutations in more common autoimmune disorders is not clear. activating jak1 and stat3 mutations have been discovered in the gut intraepithelial lymphocytes that underwent malignant transformation in refractory celiac disease [9] . another example in the border-zone of cancer and autoimmunity is the discovery of activating stat3 mutations in cd8+ cells of patients with large granular lymphocyte (lgl) leukemia and rheumatoid arthritis [10, 11] . in a subsequent analysis of 25 newly-diagnosed rheumatoid arthritis patients and 20 controls stat3 mutations were not found, but other somatic mutations were discovered in the expanded cd8+ effector-memory subset in 20% of patients vs. 5% of controls [12] . multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system and among the most common causes of neurological disability in young adults. in relapsing ms, which constitute about 90% of cases, there is evidence based on genetics, environmental risk factors and treatment paradigms that peripheral leukocyte dysfunction plays a major role in the disease [13] [14] [15] . inflammation in ms occurs in plaques, and analyses of rare cases of acute plaques have identified clonal expansion of cd8+ t cells [16] . recent analyses of ms patient's t-cell receptor vβ repertoire suggest that the cd8+ clones present in ms plaques can be detected in the cerebrospinal fluid and blood [17] . indirect evidence of somatic mutations in ms patients' cultured t-lymphocytes was reported in 1990s by using the hypoxanthine guanine phosphoribosyltransferase (hprt) assay. this assay measures 6-thioguanine resistance of cultured cells, caused by inactivating somatic mutations of the x-chromosomal hprt gene or other mechanisms. ms patients' hprt-deficient t-lymphocyte clones, but not wild-type clones, were potentially autoreactive, i.e. proliferated in response to myelin autoantigen [18] and a higher hprt-deficient t lymphocyte frequency was reported in ms patients as compared to controls [19] . a pilot study on amplified dna of cd4+ cells, derived from two ms patients' csf reported thousands of mutations in an exome-wide analysis of 21 individual cells. these mutations were considered pcr amplification artifacts and this study points to important technical limitations, when small amounts of dna are amplified [20] . we and others have subsequently demonstrated that nonsynonymous somatic mutations are detectable in ms patients blood cells in about 60% of cases and that the mutant clones persist over time [21] [22] [23] . mutations were predominantly (85%) found in the cd8+ fraction [21] . here we extend these findings by using an improved methodology for mutation discovery and analyze a cohort of 21 newly-diagnosed ms patients and 21 matched controls. the comparison between groups allows us to form an initial picture of whether the previously reported findings in ms are disease specific or not. another major goal enabled by the sample size (n = 42 participants total) is an assessment of non-random accumulation of somatic mutations in certain genes, which we carry out in addition to the case-control comparison. because the detection of somatic variants in small cell clones is very dependent on sequencing depth, we opted to limit our search to a subset of the exome containing key immunological and cancerrelated genes, which allowed us to reach high sequencing depths of over 2000x and obtain a more detailed view over small allelic fraction events. patients (n = 21) were recruited at the helsinki university hospital department of neurology outpatient clinic during their diagnostic examinations and were selected from a larger collection of patients to fulfill the inclusion criteria and age-and sex-matching. inclusion criteria for ms patients were: 1. mcdonald 2010 criteria for relapsing ms, 2. csf oligoclonal bands and 3. barkhof's magnetic resonance imaging criteria (20 patients fulfilled 4/4 criteria one patient fulfilled ¾) for anatomical dissemination of demyelination [24] . the location of presenting symptom was spinal in 9, brainstem in 4, optic neuritis/hemispheral in 5 and multifocal in 3 patients. their subsequent treatments were the following: injectables in 5 (beta-interferons and glatiramer acetate), oral in 13 (teriflunomide, dimethyl fumarate, fingolimod) and infusions in 2 (natalizumab, rituximab). one patient did not want to start any treatment. none of the patients had started disease-modifying treatment when the blood sample was taken. ageand sex-matched controls (n = 21), free of autoimmune disease or cancer, were selected from other neurological diseases visiting the outpatient clinic and from staff of the campus. the demographic features of the patients and controls are shown in table 1 and more detailed data in s1 table (ms patients) and s2 table (controls) . this study has been approved by the regional ethics committee (dno 83/13/03/01/2013). all participants gave informed written consent. peripheral blood mononuclear cells (pbmcs) were extracted from 120-140 ml venous edta blood using ficoll-paque plus (ge healthcare). first, 13 ml of ficoll-paque was added to a centrifuge tube. then, 9 ml of blood diluted with 28 ml of pbs was layered on top of it. the tube was centrifuged at 800 x g for 30 minutes after which the pbmc layer was transferred to a new tube with a pipette. the cells were washed twice, using pbs and centrifugation at 500 x g for 15 minutes and at 500 x g for 10 minutes. from the pbmcs, positive separation with macs cd8 antibody microbeads (catalogue number130-045-201, miltenyi biotec, bergisch gladbach, germany) was performed using an octomacs magnetic separator (miltenyi biotec) following the manufacturers protocol. we simultaneously separated also other cells, which will be analyzed later. from the separated cell populations, dna and rna were extracted a gene panel that consists of 2524 genes related to immunity and cancer (immunopanel-2524) was designed for mutation screening [25] . the gene list is given in s3 a "reference dna" pool was collected from 8 whole blood dnas of healthy donors, to act as technical control material. these dna samples were sequenced in an identical manner to a combined median depth of 4343x. the samples for the reference dna pool were obtained via meilahti biobank from individuals aged 20-35 years without hematological or autoimmune conditions. the sequencing reads were trimmed for adapters and base quality using trimmomatic [26] , after which they were mapped to the grch37 reference genome with bwa mem [27] . pcr duplicates were removed using samblaster [28] . somatic variant calling was performed with tnscope [29] , using a pool of 8 whole blood samples as a technical reference to discard common sequencing, library preparation and mapping artefacts. the separated cd8+ cell fraction of a participant was used as the "tumor" bam file for tnscope, and the whole blood pool reference as the "normal" bam file. tnscope was set to only consider bases of quality score 30 or higher. variant annotation was performed using annovar [30] . the somatic variant calls were filtered using several methods. tnscope produced vcf filter statistics were checked, and suspect artefact or possible germline calls were removed from each sample's data. germline variant calls were removed within-sample based on a call's allelic fraction, and germline variants detected in any of the other samples were also removed to avoid possible cross-contamination. furthermore, somatic variants were only called at high depth (>100x) locations in order to facilitate accurate calculation of the allelic fraction and to avoid germline mix-up. strand bias filtering was used to discard systematic sequencing errors that preferably show up in one sequencing direction only. known segmental duplication areas were excluded from the analysis to avoid errors from mismapping of pseudogenes at these loci. errors from mismapping of reads were mitigated by discarding highly clustered variants and by requiring a similarly high mapping quality from variant reads as from the reference allele containing reads. variants with a frequency of >10 −5 in the exac or gnomad germline population databases were also filtered to remove remaining possible germline contamination and to exclude common mapping errors and other artefacts. to remove common erroneous variant calls, the exac and gnomad database variant lists that were used were purposefully of the unfiltered type i.e., they included even the population variant calls that don't pass the quality control filters of exac or gnomad, and likely represent repeating artefacts. in order to automatically calibrate the variant calling sensitivity level against several classes of library preparation related base substitution errors, an empirical trinucleotide-context aware noise frequency was calculated for each possible base substitution, separately for each sample. the noise frequencies were calculated from the data by counting the number of base mismatches against the reference genome, for all sequenced coordinates, excluding germline variant sites, tabulated separately for each possible 192 trinucleotide context and base substitution combinations. using the empirical noise frequencies calculated this way, a binomial test that takes into account the number of variant observations, the sequencing depth, and the noise frequency was used to discard likely library preparation related artefacts, by requiring more evidence at noisier locations. we paid especial attention to base changes of type c>t (g>a) because previous studies have indicated a risk of erroneous c>t (g>a) substitutions arising during library preparation [31] . these are caused by deamination of cytosine residues, typically at cpg dinucleotides, which are often in the methylated state [32] . artifact substitutions of this class can even be generated by thermal cycling without any dna synthesis [31] . in our data-derived trinucleotide context aware noise estimates, abundant c>t (g>a) reference genome mismatches were present an order of magnitude more often than most other substitutions, especially when encountered in a cpg sequence context (s4 table) . to limit the effect of c>t (g>a) and other false positives in our results, we chose to set our sensitivity cutoff favoring specificity so that the base distribution of the somatic variant calls remained similar to the germline variant calling performed on the same data. increasing sensitivity beyond this point to pick up events at even smaller allelic fractions was found to greatly increase the rate of c>t (g>a) somatic variant calls in comparison to the baseline germline rate. these we considered likely false positives because we assume that the somatic mutations present at higher variant allele fractions should follow a similar base distribution to those at modestly lower allelic fractions rates, instead of an increasing c>t (g>a) bias for the lower allelic fraction calls that are supported by less evidence (number of sequencing reads). after filtering for quality, all synonymous variants were excluded (n = 160) from detailed analysis. the variant list was also filtered by excluding genes that are not expressed in cd8 + pbmcs according to rna-seq data that we had previously generated [21] , which resulted in removal of 76 mutations in ms patients and 88 in controls. see s5 table for the full list of variant filtering procedures applied. deleteriousness of the somatic variants was assessed using the cadd [33] and polyphen [34] algorithms, with a cadd score of > 20 considered possibly deleterious. enrichment of somatic mutations on certain genes was assessed using a poisson test, where the per-gene expected mutation rate parameter was obtained from the size of each gene, the size of the full gene panel target area, and the total number of mutations found in all participants. synonymous somatic variants were evaluated using the trap [35] and surf [36] algorithms. as a quality control measure, targeted pcr amplicons from cd8+ cell dna were designed to verify some of the discovered variants. the amplicons were sequenced using illumina miseq with 2x300bp reads. variant loci were inspected using samtools mpileup and base counts were compared to a control dna amplified in the same manner. we were able to perform unusually high depth sequencing of over 2000x median coverage for 42 cd8+ cell dna samples. all of the sequenced cd8+ samples were found to contain highconfidence somatic variants. after quality filtering, and including synonymous variants, the number of detected somatic mutations was 652 (median and mean numbers of mutations per subject were 13.5 and 15.5). the total number of mutations was 307 in ms patients (median 13, mean 14.6, range 3-41 per subject) and 345 in controls (median 14, mean 16.4, range 4-35 per subject). after removing synonymous variants and variants in genes not expressed in cd8 + cells a total of 225 nonsynonymous somatic mutations were detected in genes expressed in cd8+ cells. the number of nonsynonymous mutations in genes expressed in cd8+ cells was 104 in ms patients and 121 in controls (mann-whitney u-test, p = 0.60). a comparison of mutations between cases and controls is shown in table 2 . the majority of the mutations were predicted to affect the function of the gene product in both groups and all participants had at least one such mutation. mutation type was classified as (i) predictably deleterious single nucleotide variation (both cadd and polyphen2 algorithms), (ii) stop-codon, (iii) frameshift insertion/deletion, or (iv) splice-site mutation in 62 (60%) of the mutations found in cases and in 69 (57%) of the mutations in controls ( table 2 ). the distribution of the mutations by allelic fraction is illustrated in fig 1. as in our previous study (20) , most of the mutations (77%) were found in allelic fractions of <1%. the 225 nonsynonymous somatic mutations in genes expressed in cd8+ cells were found in 186 different genes and the mutations mostly did not recur between samples. three mutations were found in two participants (igfr2 � g712dl, tab3 � p182del, trpv2 � t96g), in one ms patient and in one control. otherwise, the mutations were singleton observations. since aging is associated with the number of somatic mutations in most tissues, we tested whether there would be an association between age and the number of mutations (all mutations included). there was an association between participant age and the number of somatic mutations, when normalized by the sequencing depth (p = 0.01, s2 fig) . age also associated with the allelic fraction of the mutations (surrogate of clone size), the mean allelic fractions in subjects 23-29 yrs vs. 41-57 yrs were 0.71% vs. 1.24% (mann-whitney u-test, p<0.001). the number of detected somatic mutations was spread similarly between males and females, with no statistical difference detected between these groups (mann-whitney u-test p = 0.36). as expected, the median sequencing depth had a modest association to the number of somatic mutations detected in that sample (kendall's tau p = 0.02). we analyzed enrichment of nonsynonymous mutations to specific genes. because a large coding region size of a gene increases the likelihood of mutations, we calculated poisson p-values that take into account the size of each gene, the number of mutations in that gene, the total gene panel area, and the total number of mutations. there were 17 enriched genes that were mutated in two or more participants at p<0.05 (table 3) . interestingly, the stat3 gene stood out as the most enriched locus (p = 9 x 10 −8 ) with a total of 6 nonsynonymous somatic mutations (2 in cases and 4 in controls, table 3 ). stat3 is a gene known to be mutated in various hematological malignancies, and some of the mutations that we found were previously known activating mutations (d661y and s614r each in one ms patient, y640f and h410r each in one control, table 4 ). the top 5 mutated genes (p<0.01) and their mutations in cases and controls are shown in table 4 in more detail. in addition to stat3, these included smarca2, a tumor suppressor involved in various cancers, socs1, a stat-inhibitor, dnmt3a, with known somatic mutations in the context of clonal hematopoiesis and ppp3ca encoding calcineurin-a, involved in t-cell receptor signaling. all 225 nonsynonymous somatic mutations in genes expressed in cd8+ cells, their allelic fractions, base counts, presence in databases, discovery p-values and predicted deleteriousness are shown in s6 table. to test our somatic variant calling sensitivity and specificity settings (see 2.4 data analysis), we prepared confirmation pcr amplicons for the two high-confidence stat3 somatic mutations detected in our ms patients (d661y and s614r variants). for comparison we chose a low-confidence but recurring c>t variant itpr3 � t2584m, which was filtered out in our pipeline. a more relaxed sensitivity level would have called this variant in three ms patients and one control. this variant call represented a potential mutational hotspot, and it has been previously reported in cancer tissue (cosmic database id cosm1643037). hence, we chose to test this variant call with amplicon sequencing. by comparing the variant allele fractions in the original panel data and amplicon sequencing data (s7 table) , we found that the allele fractions were in agreement for the stat3 variant (fold change only 0.76-0.85 in amplicon sequencing) but not for the itpr3 variant (fold change 0,06-0,075 in amplicon sequencing). moreover, the itpr3 variant was found in amplicon sequencing data of the reference-dna and in a cell-line control dna close to the same allele fraction as in the tested cd8+ cell dnas. these results indicate that the stat3 variants are true, while the itpr3 c>t variant is a likely artifact, and support the use of stringent filtering to avoid false positives. we carried out an additional analysis of synonymous somatic variants in order to include possible effects on rna structure and other effects not examined by our primary analysis of nonsynonymous mutations. it should be noted that we targeted the coding region of each gene, untranslated regions were not included. after applying the same filters to synonymous variants as we used for the nonsynonymous variants, a total of 72 synonymous somatic mutations were detected in genes expressed in cd8+ cells. out of these, 43 were found in cases and 29 in controls with no statistical difference in number between the groups (mann-whitney u-test p = 0.25). a full list of the detected synonymous mutations is shown in s8 table. a clustering of somatic synonymous mutations was detected in the map3k12 gene (poisson test p = 0.00011), with a total of 4 synonymous mutations, 2 in cases and 2 in controls. all four had the same recurring mutation, nm_001193511:c.279a>g, which was novel. the general purpose cadd algorithm did not predict this mutation to be deleterious (cadd phred score 9.024), nor did the algorithm trap, specialized in synonymous variants, classify it as damaging (trap score 0.038). however, we also utilized the surf rna structural predictivity index that combines different secondary structure effects into one summary metric, and this algorithm did predict the mutation to be deleterious. the somatic map3k12 synonymous mutation had a surf phred score of 21.46, which is inside the 99 th percentile and is higher than the scores obtained by the surf authors for nine known pathogenic mutations affecting rna structure [36] . this suggests that the synonymous mutation may have a deleterious effect on the stability of map3k12 rna, which is a gene associated with jnk signaling [37] . although a few singleton mutations had high surf scores s8 table) , no other genes showed significant (p<0.01) clustering of synonymous mutations. we had previously shown that it is possible to detect somatic mutations in about 60% of ms patient blood samples, and that the mutations were mostly (85%) discovered in cd8+ cells in comparison to other, cd4+ or cd19+ cells. in the present work, we thus chose to concentrate on the cd8+ cell fraction and included age-and sex-matched controls to assess whether the finding is ms specific, or generalizes to the population at large. moreover, we were able to perform especially deep sequencing, reaching coverages greater than 2000x, which enabled a sensitive screen of low allelic fraction events. we found 225 nonsynonymous somatic mutations in genes expressed in cd8+ cells and these were distributed evenly between ms patients and healthy controls, with each sample carrying at least one (median 4 mutations in both groups). including also synonymous and non cd8+ expressed gene variants in the target area there were a total of 652 high-confidence somatic mutations (median and mean numbers of mutations per subject were 13.5 and 15.5). in order to understand what would be the expected (theoretical) number of detectable mutations in our experiment, we need to consider some technical issues regarding the number of cells and detectable clones. we used 1000 ng of dna, which corresponds to 167,000 individual cells (6 pg of dna per cell), of which �90% (150,000) were cd8+ cells after the immunomagnetic purification scheme. our median sequencing depth 2349x, due to the excess of cells, produces essentially haploid sequence from ca. 2100 cd8+ cells (90% of total cells). usually about half of the blood cd8+ cells are naïve cells, with small clone sizes [38] , not detectable with our methodology. the other half (1050 cells) constitutes memory cd8+ cells, which have larger, detectable clones. the mean number of reads in the detected variants was 19.3. using the mean number of reads as a surrogate for the mean clone size, the estimated mean number of cd8+ memory clones would be 54 (1050/19.3) with large variation in size. given the non-gaussian distribution of t cell clones that fits with power law [38, 39] the estimated 54 clones is likely the maximum number of detectable clones in our experiment. somatic mutations start to occur and accumulate since the first cell division of the fertilized egg. a recent analysis utilizing individual dna molecule sequencing of hematopoietic stem cell/progenitor cell colonies and circulating granulocytes from different ages suggest that mutational burden increases linearly with age [40] based on these data (figs 2b and 3p in [40] ) the expected number of substitutions and small indels in the diploid genome (6200 mb) at the age of 35 would be �880 (35 yrs was the mean age of our donors). the expected number of mutations per cell in our haploid target region (5 mb) would be 0.71 (880 x 5 mb/6200 mb). assuming that the estimated maximum of 54 clones in our experiment would represent extremely independent phylogenies we would arrive to the estimate of 38 detectable mutations per subject (0.71 x 54). the observed numbers in our study, median 13.5 and mean 15.5 mutations per subject are not far from this figure. this estimation gives an idea of the magnitude of the expected number of mutations. there are several factors that make accurate estimation difficult, but these do not necessarily change the order of magnitude of the expected number of mutations. shared phylogenies of the clones is very likely, which would result in lower number of independent clones and mutations. the mutation rate of cd8+ cells is currently not known and it may be higher than the rates observed so far in hematopoietic stem cells and granulocytes [40] . additionally, it should be noted that our variant calling scheme was conservative with a bias towards false negatives rather than false positives. most of the discovered mutations were predictably deleterious ( table 2 ) and many of the mutations confer proliferation and/or survival advantage. of notable interest were the somatic mutations detected in the stat3 gene, most of which (4 out of 6 mutations) have been shown to cause an activating phenotype which affects growth and apoptosis properties of t lymphocytes [10, 41] . in our results, stat3 was the gene containing more nonsynonymous somatic mutations than any other gene, showing a statistically significant enrichment. it is likely that the clones that carry stat3 mutations have a growth advantage over other clones, increasing their size in the sequenced sample, and thus bring the somatic mutation closer to detection threshold more often than mutations in many other genes. it is of note that the stat3 � d661y mutation found in one ms patient was also previously discovered in our pilot study in another patient [21] . we are collecting a larger dataset for analyzing the frequencies of somatic stat3 variants in ms patients and controls. the other genes that stood out in the number of enriched nonsynonymous mutations were smarca2, socs1, dnmt3a and ppp3ca. the smarca2 (also known as brahma, brm) gene is involved in chromatin remodeling processes and is considered a tumor suppressor gene that is very often downregulated in various cancers [42] . on the other hand, in pancreatic cancer cells smarca2 activates epigenetically stat3 signaling and may promote metastasis [43] . somatic mutations in smarca2 have been reported in chronic lymphocytic leukemia [44] and, most typically, in adenoid cystic carcinoma [45] . socs1 is a gene with known regulatory functions in autoimmunity and a tumor suppressor in cancer [46] . stat3 activation is inhibited by socs1 via its binding to receptor-associated tyrosine kinases jak2 and tyk2 [47] . dnmt3a codes a methyltransferase protein that is essential for establishing normal epigenetic dna methylation patterns. somatic mutations in dnmt3a have been implicated in age-related clonal hematopoiesis, wherein a hematopoietic stem cell has acquired a growth advantage mutation resulting in an increased clonal size of its peripheral blood cell progeny [48] . on the other hand, dnmt3a loss-of-function somatic mutations are also known for phenotypic effects more specifically tied to t cell behavior. in cd8+ t cells specifically, defects in dnmt3a will cause an accelerated shift towards a long-living memory phenotype [49] . ppp3ca encodes calcineurin-a, which mediates one of the three major signaling pathways of t cell receptor signaling [50] . ca2+-calcineurin signaling activates t-cells by mediating nuclear translocation of nuclear factor of activated t cells (nfat) and plays a role in the maintenance of tumor cells in t-cell leukemia [51] . one potentially functional and recurring synonymous somatic mutation was found in map3k12 encoding mitogen-activated protein 3-kinase 12, also known as dual leucine zipper kinase (dlk). it acts upstream of c-jun n-terminal kinase (jnk) and p38 map kinases in the jnk signalling pathway. there are multiple functional outcomes of this pathway in different cell types, one of which is the regulation of stat3 [52] . amplification of the map3k12 locus has been reported in non-hodgkin's lymphomas [53] . the most commonly mutated genes we found in blood cd8+ cells differ as compared to the top5 mutated genes reported in sun-exposed normal skin (notch1, notch2, fat1, tp53, notch3) and normal oesophagus (notch1, tp53, notch2, fat1, notch3) [54, 55] . this is consistent with the view that the cell type and its' environment shape the mutations that provide selection advantage. it is of note that the same mutations found in non-neoplastic cells also occur commonly in cancers of the same cell types. the mutations common in normal skin and oesephagus are common in respective cancers [54, 55] and the commonest mutated genes we found in cd8+ cells have all been implicated in hematological malignancies. it is interesting to consider whether some of the detected somatic mutations might have a role in disease. they can theoretically add some level of cancer risk, as 1/5 of the mutations have been found in cancer cells, even if they likely drive only a limited growth potential in most people, when normal t-cell apoptosis occurs. the mutations were detected in cd8+ cells, and because cd8+ t cells are a potent effector of the immune system, a link to autoimmune disorders is also possible. in our data the somatic mutations were equally distributed between ms patients and healthy controls. however, the antigen specificities and general phenotypic features of the mutated cd8+ clones may be differ in ms patients as compared to controls. an autoreactive clone with an activating somatic mutation would offer a potential mechanistic explanation for a chronic disease like ms. previously, using the hprt reporter assay it was shown that ms-patients' mutated t-lymphocyte clones proliferated in response to myelin antigen [18] . future work is needed to address this hypothesis. the sequencing depths of over 2000x that we attained in this research call for special care in handling potential false positive errors that may arise in the sample preparation and sequencing processes. misclassification of low frequency technical artefacts as reportable variants becomes more likely at low allelic fractions. the sequencing artefacts can arise from several different biochemical processes affecting dna while it is being processed in the laboratory, such as heating induced spontaneous deamination, base oxidization caused by energy from ultrasonic shearing, or damage from free oxygen radicals [56] . to combat these, we applied several strict filters in our variant calling procedure, considering different false positive classes, and aimed to emphasize specificity over sensitivity. one of the more impactful filters employed in this work was based on calculating the data-derived empirical noise frequency at different possible trinucleotide contexts. as diverse biochemical processes can cause different types of error signatures, this allowed us to require higher levels of evidence at locations where a false positive risk was more present, without a priori knowledge of the error generating processes our library preparation procedure would most induce. we found c>t/g>a substitutions to be highly noisy at low allelic fractions in our sequencing data, especially when encountered in a cpg context. our pipeline automatically corrected for this by being extra strict at such loci, at the possible cost of missing some true positives. thermocycling (such as in pcr) induced c>t/g>a errors have been reported previously in the literature, in fact forming 97% of errors in one study [31] , which is in line with our data. furthermore, we used amplicon sequencing to confirm that a repeating false positive of the c>t/g>a was correctly classified, and that the stat3 somatic mutations in our ms patients were actual true positives. other experiments may witness a different base substitution error profile depending on the specific library preparation conditions they employ [56] . as we sequenced a panel of genes chosen because of their association to immunity and cancer, we were not able to run generic enrichment analyses of biological functions using our result variant list, and this is a limitation of the study-the somatic mutations we detected are found in immunity and cancer related genes by design. however, inside the immunity context, stat3-signaling emerged as one clear pathway possibly affected. moreover, the repeated hits in genes known to give cd8+ t cells a growth and survival advantage suggest that these mutations may have functional consequences as algorithms predict. as there was an association in our data between the number of mutations detected and the sequencing depth reached by a sample, it is reasonable to expect that even deeper sequencing would increase the number of discovered deleterious somatic mutations and potentially dysregulated clones, even in normal healthy adults such as the control subjects in this study. in conclusion, our results demonstrate that somatic mutations are present both in ms patients' and controls blood cd8+ t cells and that they cluster on certain genes such as stat3. somatic mutations can be important modifiers of the characteristics of cd8+ t cell clones and may result in dysregulated immune responses. the role of somatic mutations in driving autoimmune responses is presently unclear but given the abundance of mutated clones deserves further attention. variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates clonal hematopoiesis and blood-cancer risk inferred from blood dna sequence age-related clonal hematopoiesis associated with adverse outcomes aging and neurodegeneration are associated with increased mutations in single human neurons somatic activating kras mutations in arteriovenous malformations of the brain autoimmune lymphoproliferative syndrome with somatic fas mutations somatic kras mutations associated with a human nonmalignant syndrome of autoimmunity and abnormal leukocyte homeostasis somatic mutations in uba1 and severe adult-onset autoinflammatory disease interleukin-15-dependent t-cell-like innate intraepithelial lymphocytes develop in the intestine and transform into lymphomas in celiac disease somatic stat3 mutations in large granular lymphocytic leukemia somatic stat3 mutations in felty syndrome: an implication for a common pathogenesis with large granular lymphocyte leukemia somatic mutations in clonally expanded cytotoxic t lymphocytes in patients with newly diagnosed rheumatoid arthritis the international multiple sclerosis genetics consortium & the wellcome trust case control consortium 2. genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis complete epstein-barr virus seropositivity in a large cohort of patients with early multiple sclerosis multiple sclerosis: mechanisms and immunotherapy clonal expansions of cd8+ t cells dominate the t cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction expanded cd 8 t-cell sharing between periphery and cns in multiple sclerosis t cells responsive to myelin basic protein in patients with multiple sclerosis longitudinal study of frequency of hprt mutant t cells in patients with multiple sclerosis exome sequencing in single cells from the cerebrospinal fluid in multiple sclerosis a novel class of somatic mutations in blood detected preferentially in cd8 + cells somatic variants: new kids on the block in human immunogenetics a robust pipeline with high replication rate for detection of somatic variants in the adaptive immune system as a source of common genetic variation in autoimmune disease comparison of mri criteria at first presentation to predict conversion to clinically definite multiple sclerosis somatic mutations and t-cell clonality in patients with immunodeficiency trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform samblaster: fast duplicate marking and structural variant read extraction accurate detection of somatic mutations with haplotype-based variant candidate detection and machine learning filtering annovar: functional annotation of genetic variants from high-throughput sequencing data examining sources of error in pcr by single-molecule sequencing mutagenic deamination of cytosine residues in dna cadd: predicting the deleteriousness of variants throughout the human genome predicting functional effect of human missense mutations using polyphen-2 annotating pathogenic non-coding variants in genic regions synonymous variants that disrupt messenger rna structure are significantly constrained in the human population mbip (map3k12 binding inhibitory protein) drives nsclc metastasis by jnk-dependent activation of mmps quantitative characterization of the t cell receptor repertoire of naïve and memory subsets using an integrated experimental and computational pipeline which is robust, economical, and versatile assessing t cell clonal size distribution: a non-parametric approach somatic mutation landscapes at single-molecule resolution activating somatic mutations outside the sh2-domain of stat3 in lgl leukemia high expression of smarca4 or smarca2 is frequently associated with an opposite prognosis in cancer brm transcriptionally regulates mir-302a-3p to target socs5/stat3 signaling axis to potentiate pancreatic cancer metastasis new mutations in chronic lymphocytic leukemia identified by target enrichment and deep sequencing the mutational landscape of adenoid cystic carcinoma emerging concepts targeting immune checkpoints in cancer and autoimmunity intrinsic virulence factors, and treatment of covid-19 clonal hematopoiesis in human aging and disease effector cd8 t cells dedifferentiate into long-lived memory cells regulatory mechanisms in t cell receptor signalling targeting calcineurin activation as a therapeutic strategy for t-cell acute lymphoblastic leukemia the dlk signalling pathway-a double-edged sword in neural development and regeneration integrative genomic profiling reveals conserved genetic mechanisms for tumorigenesis in common entities of non-hodgkin's lymphoma high burden and pervasive positive selection of somatic mutations in normal human skin somatic mutant clones colonize the human esophagus with age enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations the authors wish to thank all participants for the blood samples, csc-it center for science, finland, for computational resources, dr. nina peitsaro and noora aarnio in the biomedicum helsinki flow cytometry core facility for facs services and drs. satu mustjoki, mikko keränen and sofie lundgren at the university of helsinki for establishing the immunopanel-2524. pekka ellonen from finnish institute of molecular medicine is thanked for his excellent work in the next-generation sequencing. writing -original draft: miko valori, pentti j. tienari. miko valori, lilja jansson, pentti j. tienari. microsoft word document1 33 cultural adaptation and optimization of the compliance questionnairerheumatology (cqr) through statistical methods for patients with rheumatic diseases mihaela-simona subtirelu faculty of pharmacy university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 mihaela.subtirelu@yahoo.com adina turcu-stiolica faculty of pharmacy university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 adina.turcu@gmail.com florentin-ananu vreju faculty of medicine, department of rheumatology university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 florin_vreju@yahoo.com paulina lucia ciurea faculty of medicine, department of rheumatology university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 stefan cristian dinescu faculty of medicine, department of rheumatology university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 andreea lili barbulescu faculty of medicine, department of pharmacology university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 johny neamtu faculty of pharmacy university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 jneamtu@yahoo.com raluca christiana dănciulescu faculty of medicine university of medicine and pharmacy of craiova str. petru rareș 2, craiova 200349, romania phone: 0351 443 500 ralucadanciulescu@yahoo.com brain – broad research in artificial intelligence and neuroscience volume 10, issue 3 (september, 2019), issn 2067-3957 34 abstract there is a growing interest in the management of long-term conditions and increasing awareness of the need for compliance/adherence's assessment. an inexpensive and simple way to measure patients’ treatment compliance is pharmacoeconomic measurement tools. the aim of the study is to increase the quality of assessment of patients’ compliance through statistical methods in order to obtain a more accurate estimation and a shorter, easy-to-respond selfadministered questionnaire. we used the compliance questionnaire-rheumatology (cqr) translated into romanian and we verified the reliability and the internal consistency of this instrument. the research included 140 consecutive patients diagnosed with rheumatoid arthritis according to currently used criteria, with different treatment strategies and from different parts of romania. two methods for cqr's optimization were used. the first method identified associations between cqr score and patient compliance. an exploratory factor analysis was performed, as an alternative method, to reduce the number of items in cqr. the factor analysis identified a two-dimensional 9 item model that explains 66.78% of the variance in adherence and has a good internal consistency and fits the data. a shortened and optimized version of the cqr increases the clinical utility by reducing the patient’s burden while maintaining a good level of reliability. keywords: compliance; adherence; reliability; compliance questionnaire-rheumatology (cqr); pharmacoeconomics. 1. introduction more and more, pharmacoeconomic instruments have been used to predict the quality of life of patients, but also to ease efforts to identify associated pathologies. as defined by the us food and drug administration (fda), pro (patient-reported outcomes) is “a measurement based on a report that comes directly from the patient about the status of a patient’s condition without amendment or interpretation of the patient’s response by a clinician or anyone else” (u.s. food and drug administration, 2009). pro (patient-reported outcomes) can be easily implemented and in the same time can be associated with lower costs because the interval to complete these forms is shorter, and most often no specific training is required in order to complete. the management of rheumatic diseases is particularly important because they have increased levels of influence on patient’s quality of life, mortality, and morbidity, thus the drug therapy plays a major role in the disease outcome. in order to obtain optimal management, there is a need for an accurate understanding of the pathology but, it is clear that the effectiveness of any subsequent therapy depends on each patient’s decision to take the treatment correctly or not. thus, we have several international studies that showed patients suffering from rheumatic diseases are often not compliant with the treatment prescribed by the specialist. rheumatic disorders are a group of inflammatory conditions that have similar immunopathological mechanisms. these disorders are characterized by a complex pathology involving multiple organs and systems, thus increasing the rate of morbidity and mortality. as with other chronic rheumatic conditions, besides an accurate periodic clinical and paraclinical evaluation, using modern imaging techniques (vreju et al., 2016; filippou et al., 2018), patient adherence to treatment is an important part of therapy success. since rheumatic diseases involve lifelong treatment, the impact of low adherence to treatment can also influence its effectiveness. a lack of adherence to treatment can be misinterpreted as low efficacy or lack of response to treatment (anghel, farcaş & oprean, 2018). there are studies (kardas, 2007; haddad, brain & scott, 2014) which demonstrated that good patient compliance with treatment leads directly to stable clinical effects. non-compliance causes a number of serious problems in properly managing the medical condition involving very high costs and treatment-related changes. for this reason, measuring and predicting the compliance of a particular patient is a piece of useful information for the optimal management of the disease. m.-s. subtirelu, a. turcu-stiolica, f.-a. vreju, p. l. ciurea, s. c. dinescu, a. l. barbulescu, j. neamtu, r. c. dănciulescu cultural adaptation and optimization of the compliance questionnaire-rheumatology (cqr) through statistical methods for patients with rheumatic diseases 35 pednekar (2019) assessed the multiple methods for measuring medication compliance/adherence and self-reported questionnaire was the most used from these methods. the number of pharmacoeconomic instruments that measure directly the level of adherence or compliance of the patient to drug treatment for rheumatic diseases is quite low (van den bemt et al., 2009). a single questionnaire was specifically developed to assess patients’ compliance to treatment for various rheumatic diseases and is called the compliance questionnaire for rheumatology (subtirelu, turcu-stiolica, vreju & neamtu, 2017; de klerk, van der heijde, van der tempel & van der linden, 1999). the compliance questionnaire-rheumatology (subtirelu, turcustiolica, vreju & neamtu, 2017, table 1) is a standardized, self-administrated compliance instrument that can be used by patients diagnosed with rheumatic diseases or specialist in the field of rheumatology. this pharmacoeconomic tool contains 19 questions with four response options (also called the 4-point likert scale). table1. the full compliance questionnaire for rheumatology (cqr19) questions q1 if the rheumatologist tells me to take the medicines, i do so. q2* i take my anti-rheumatic medicines because i then have fewer problems. q3 i definitely don’t dare to miss my anti-rheumatic medications. q4 if i can help myself with alternative therapies, i prefer that to what my rheumatologist prescribes. q5* my medicines are always stored in the same place and that’s why i don’t forget them. q6* i take my medicines because i have complete confidence in my rheumatologist. q7 the most important reason to take my anti-rheumatic medicines is that i can still do what i want to do. q8 i don’t like to take medicine. if i can do without them, i will. q9 when i am on vacation, it sometimes happens that i don’t take my medicines. q10 i take my anti-rheumatic drugs, for otherwise what’s the point of consulting a rheumatologist? q11 i don’t expect miracles from my anti-rheumatic medicines. q12 if you can’t stand the medicines you might say: “throw it away, no matter what”. q13* if i don’t take my anti-rheumatic medicines regularly, the inflammation returns. q14* if i don’t take my anti-rheumatic medicines, my body warns me. q15* my health goes above everything else and if i have to take medicines to keep well, i will. q16* i use a dose organizer for my medications. q17* what the doctor tells me, i hang on to. q18* if i don’t take my anti-rheumatic medicines, i have more complaints. q19 it happens every now and then, i go out for the weekend and then i don't take my medicines. note: items denoted with * have been retained in the final 9 items cqr9 questionnaire. using an already validated questionnaire has many advantages over the other commonly used methods; it is considered a procedure that is not invasive, it has minimal costs, but can also provide relatively accurate measures of compliance with the actual information provided by the patient. the questionnaire is based on a four-point likert scale, ranked from "strongly agree" to "strongly disagree". a lower score indicates lower levels of adherence. if the adherence score is higher than 80, the patient is adherent to treatment (subtirelu, turcu-stiolica, vreju & neamtu, 2018). the time spent by the patient with completing the questionnaire is short, up to 10 minutes. regarding the measurement of adherence for rheumatic diseases, there are few pharmacoeconomic instruments in english-language, but in romanian, there is no validated questionnaire that measures the level of adherence for patients. usually, people seek treatment at the final stage of the disease, when the various conditions tend to become more serious and lead to functional disabilities, pain, tiredness, even restrictions on social participation. this requires a better understanding of the systematic monitoring of these results, in addition to traditional clinical outcome measurements. we translated cqr19 (subtirelu, turcu-stiolica, vreju & neamtu, 2017) according to international translation guidelines (beaton, bombardier, guillemin & ferraz, 2000). the translation of the cqr19 was made by two of the authors, native in romanian language and fluent brain – broad research in artificial intelligence and neuroscience volume 10, issue 3 (september, 2019), issn 2067-3957 36 in english. both authors were connected in the health/pharmacoeconomic field. the two translations were carried out independently and for the final version of the questionnaire, both checked out for possible misunderstandings, choosing a final questionnaire. each item translated must meet certain criteria related to clarity, good understanding in the romanian language because patients coming from different social environments will respond to the questionnaires. our aim was to optimize the cqr19, investigating if it is possible to reduce the number of items in the cqr19 while maintaining internal reliability. a shorter questionnaire would be quicker and easier to administer during a routine outpatient clinic appointment. in the same time, the reduction version allows for the cqr to be incorporated into a battery of questionnaires for research purposes (turcu-stiolica, subtirelu, vreju & neamtu, 2018; subtirelu, turcu-stiolica, vreju, neamtu, 2017). 2. methods 2.1. patients a total of one hundred forty romanian patients diagnosed with rheumatoid arthritis (ra) according to currently used criteria, with different treatment strategies (n1=103 patients treated with conventional and n2=37 treated with biologic drugs) were enrolled in this study. the patients were enrolled from the rheumatology clinic of emergency county hospital craiova, city hospital filiasi and city hospital dragasani. the questionnaire was completed under the guidance of a pharmacist and a physician, senior specialist in the field of rheumatology. the local ethics committee approved this study and patients’ informed consent was obtained, during which they were informed of their rights and obligations and other study details. the diagnosis date for rheumatic conditions is different for each of them, ranging between 2007 and 2018. at the 103 patients we found diseases like: gonarthrosis, spondylosis, acute lumbago, coxarthrosis and most patients claim to have chronic degenerative rheumatism (arthrosis). the cqr was translated into romanian (subtirelu, turcu-stiolica, vreju & neamtu, 2017) and the final version was optimized after the obtained value of adherence of the ra patients treated with biologics and conventional treatment. in addition to the biological treatment, the patients also had conventional medicines such as: nsaids, ipp (pantoprazole) and some otcs used to treat rheumatic pain. in romania, the national health insurance house (nhih) governs health insurance for ra patients (kawalec et al., 2017). a doctor diagnoses the ra patient and issues prescriptions for symptomatic therapy and synthetic or biologic disease modifying antirheumatic drugs (dmard) that will be dispensed by a pharmacist in a community pharmacy. the patients could pay a difference for symptomatic drugs, but in the case of dmards, the drugs are totally reimbursed by nhih. more, ra patients treated with biologic dmards have better results than the ra patients treated with conventional dmards. so, the patients treated with biologics should be more adherent than patients treated with conventional drugs. the romanian cqr-19 score was first calculated as the sum of all scored item (every item has the score 1, 2, 3 or 4) minus 19 and divided by 0.57: 𝐶𝑄𝑅𝑠𝑐𝑜𝑟𝑒 = ∑ . (equation 1) a patient is adherent if the score is bigger than 80%. the influence of demographic factors (age, gender, living place and level of education) was assessed. 2.2. optimization of cqr19 the use of tests with multiple items is inconvenient for many reasons. one of these is the time consumed with their application; the other may be the uselessness of keeping items whose contribution to the overall score is small or going in a different direction. identifying and removing m.-s. subtirelu, a. turcu-stiolica, f.-a. vreju, p. l. ciurea, s. c. dinescu, a. l. barbulescu, j. neamtu, r. c. dănciulescu cultural adaptation and optimization of the compliance questionnaire-rheumatology (cqr) through statistical methods for patients with rheumatic diseases 37 or optimizing these items is one of the objectives of this study. the basic criteria for this operation are the value of the cronbach alpha index, which has a variation range between 0 and 1. in our study, we used two methods for optimization of cqr19. method i to adapt and optimize the questionnaire, the cqr score was analyzed using pearson correlation coefficients. the validity of cqr was assessed by identifying associations between cqr scores and patient compliance. some of the items in the cqr received a negative score if the patient indicated less compliance. the items of some questions were recoded (score 1→4, 2→3, 3→2, 4→1) to obtain a useful total score, where high scores are related to higher compliance. test-retest reliability was measured by calculating the intraclass correlation coefficient (icc), a measure of concordance that corrects for systemic errors (fleiss, 1986). the number of items was reduced for predicting an acceptable amount of medication adherence. the negative issue regarding the recoding of the elements is that although this may significantly increase the adherence score, the use of inverse questions may reduce the patients’ response tendency. if questionnaires are used with inverse questions, they must be carefully formulated, ensuring that they are properly interpreted. multiple regression analyses were used to obtain the weights that increased the cqr score. method ii the self-reporting questionnaire cqr19 was interpreted with a multivariate statistical procedure, exploratory factor analysis, for reducing the number of variables and optimizing it (williams, brown & onsman, 2012). we aimed to reduce the number of items by removing those not adding to the explained variance of the factor, that is adherence, in this study. a factor can be described in terms of the variables measured and their relative importance for the factor. in our questionnaire, variables were measured with the initial 19 items. therefore, having discovered which factors exist, we analyzed correlation coefficients between responses and factors. according to tabachnick and fidell (2007), the correlation matrix must be inspected for correlation coefficients over 0.30. factorability of 0.3 indicates that the factors account for approximately 30% relationship within the data, or in this study, it would indicate that a third of the variables share too much variance, and hence becomes impractical to determine if the variables are correlated with each other, or if they are influenced by the dependent variable (multicollinearity). factor analysis produces a matrix of coefficients (or factor loadings) that describes the interrelationships between the measures under study and the underlying factors (like the correlation coefficients) (kleinbaum & kupper, 1978). not all factors were retained because we want to assess only treatment adherence of ra patients. this is the reason why we used another approach: catell's screen test that involves plotting each of the eigenvalues of the factors. the process of deciding how many factors to keep is called extraction. eigenvalues associated with a variate indicate the substantive importance of that factor (field, 2013), so we retained only factors with large eigenvalues. the eigenvalues represent the amount of variation explained by a factor and that an eigenvalue of 1 represents a substantial amount of variation. catell recommended plotting each eigenvalue (y-axis) against the factor with which it is associated (x-axis), retaining all factors above the elbow, or break in the plot, as these factors contribute the most to the explanation of the variance in the data set (pallant, 2007). prior to the extraction of the factors, kaiser-meyer-olkin (kmo) measure of sampling adequacy and bartlett’s test of sphericity. a kmo index of 0.5 and significant bartlett’s test of sphericity (p<0.01) are considered suitable for factor analysis (hair, 1995). once the final model was determined, the internal consistency was tested using cronbach’s α with a threshold of >0.8 being considered as having high internal consistency (>0.7 is a good consistency, <0.7 is considerably questionable). statistical analysis was carried out using spss version 20.0. the quantitative variables were characterized by the mean and standard deviation (normal distribution), and qualitative variables brain – broad research in artificial intelligence and neuroscience volume 10, issue 3 (september, 2019), issn 2067-3957 38 with absolute frequency. for qualitative data, the comparison between groups was realized with the chi-square test (χ2). for comparison between means of two groups of parametric data of independent samples, student t-test was used. pearson correlation was used for analyzing two quantitative variables. a p-value <0.05 was considered statistically significant. in spss, patients’ data were stored in rows and variables were stored in columns. we had 26 variables (age, gender, environment, education, employed, q1-q19, treatment, adherence score). 3. results the demographic characteristics of the patients in the cultural adaptation and optimization analysis are summarized in table 2. the mean (± standard deviation) age of the patients was 54.31 (±14.48) years. the majorities of the participants were aged over 40 years (76%) and most were females (66%). the most part of the patients live in the urban environment (73%) and one third have a university background (38%). table 2. demographic characteristics of the patients (n=140) patients characteristics total (n=140) n (%) patients treated with biologic drugs (n=37) n (%) patients treated with conventional drugs (n=103) n (%) χ2 (p) age, yr (mean± sd) min max 54.31 (±14.48) 20 83 51.97 (±12.78) 21 83 55.16 (±15.01) 20 82 age category 18-29 30-39 40-49 50-59 60-69 70+ 12 (9%) 12 (9%) 22 (16%) 37 (26%) 38 (27%) 19 (14%) 4 (11%) 2 (5%) 7 (19%) 13 (35%) 9 (24%) 3 (8%) 8 (8%) 10 (10%) 15 (15%) 24 (23%) 29 (28%) 17 (17%) t=1.148 (p=0.253) gender male female 47 (34%) 93 (66%) 7 (19%) 30 (81%) 40 (39%) 63 (61%) 4.842 (0.028) living place urban rural 102 (73%) 38 (27%) 30 (81%) 7 (19%) 72 (70%) 31 (30%) 1.720 (p=0.281) level of education 8 classes highschool post-high school university 40 (29%) 27 (19%) 19 (14%) 54 (38%) 4 (11%) 10 (27%) 5 (13%) 18 (49%) 36 (35%) 17 (17%) 14 (13%) 35 (35%) 8.439 (p=0.038) employment situation employee pensioner unemployed 74 (53%) 48 (34%) 18 (13%) 23 (63%) 12 (32%) 2 (5%) 51 (50%) 36 (35%) 16 (15%) 3.046 (p=0.218) m.-s. subtirelu, a. turcu-stiolica, f.-a. vreju, p. l. ciurea, s. c. dinescu, a. l. barbulescu, j. neamtu, r. c. dănciulescu cultural adaptation and optimization of the compliance questionnaire-rheumatology (cqr) through statistical methods for patients with rheumatic diseases 39 3.1 method i compliance, as measured using different formulas, is summarized in table 3. in order to determine the questionnaire questions to be recoded, ibm spss statistics 20.0 is used as a working tool. table 3. compliance value: cqr scores compliance’s patients treated with cd mean (sd) compliance’s patients treated with bd mean (sd) low adherence (% of patients cd with less than 80% compliance) low adherence (% of patients bd with less than 80% compliance) initial formula (equation 1) 57.13 (7.71) 65.86 (7.15) 100% 100% recoded formula (5-q4, 5-q12, 5-q19) 56.16 (8.56) 73.49 (10.41) 97% 89% weight formula (q4, q8, q9, 5q12, 5-q19) 63.13 (10.90) 77.98 (12.98) 92% 51% cd=conventional drugs, bd=biologic drugs compliance is done in the following particular cases:  in the initial version when all 19 questions are considered, coded according to the likert scale;  in the recoded version, when certain questions are selected (q4, q12, q19) and recoded;  in the weight formula where using spss, the program considers or not the value of the answers given. the results obtained with weight formula are better because of the increased values of the cqr score in the case of biologic drugs. the results obtained in the first case are presented on line 1 of the table. in the current study, for the 103 patients treated with conventional drugs, an compliance of 57.13% with a standard deviation of 7.71 results; this means that the 103 patients are 100% non-adherent to treatment. also for the 37 patients treated with biological drugs an compliance of 65.86% and a standard deviation of 7.15 was obtained. all 37 patients are non-adherent (non-compliant). these conclusions are erroneous leading to the need to perform the optimization of the questionnaire. line 2 of the table presents the results obtained in the case of recoding of questions no. 4, 12, and 19. in this case, the 103 patients treated with conventional drugs showed an adhesion of 56.16% with a standard deviation of 8.56; so 97% of patients are non-adherent to treatment. at the same time, the 37 patients treated with biological drugs have an adhesion of 73.49% with a standard deviation of 10.41. the percentage of non-adherent patients decreased from 100% to 89%. an improvement in the adhesion score is observed for both categories of patients. 3.2. method ii the measure of sampling adequacy, the kaiser-meyer-olkin (kmo) is considered high at a value of 0.87, so there isn’t any problem with the sample size. the kmo can be calculated for individual and multiple variables and represents the ratio of the squared correlation between variables to the squared partial correlation between variables. the kmo statistic varies between 0 and 1. a value of 0 indicates that the sum of partial correlations is large relative to the sum of correlations, indicating diffusion in the pattern of correlations (hence, factor analysis is likely to be brain – broad research in artificial intelligence and neuroscience volume 10, issue 3 (september, 2019), issn 2067-3957 40 inappropriate). a value close to 1 indicates that patterns of correlations are relatively compact and so factor analysis should yield distinct and reliable factors. the test of sphericity of bartlett is significant (p<0.001), so we have at least one significant correlation between two of our items and we can run factory analysis. we don’t have any value of less than 0.3 in the communalities table and no items’ problems. there were 5 eigenvalues of >1 which explained 64.03% of the total variance. according to the parallel analysis, we should retain the same five new factors. we analyzed the component correlation matrix to determine if the new factors are going to be orthogonal or oblique. the values were not greater than 0.5, the new factors presented a weak or moderate correlation. table 4. cronbach’s alpha factor component from rotated component matrix initial reliability cronbach’s alpha cronbach’s alpha if item deleted 1 q1, q5, q6, q15, q16, q17, q19 0.683 0.861 (if q19 item was deleted) 2 q2, q3, q4, q13, q14, q18 0.615 0.834 (if q4 item was deleted) 3 q6, q7, q11, q12 0.251 0.722 (if q12 item was deleted) 4 q1, q8, q9, q19 0.019 0.499 (if q1 item was deleted) 5 q3, q10, q11 0.391 0.471 (if q11 item was deleted) after successive exploratory factor analysis, the msa and factor loadings were recalculated. table 4 shows that checking the reliability on each of our factors, we obtained better cronbach’s alpha if we would delete q4, q12, and q19. table 5 shows the outcomes we obtained for every new type of cqr questionnaire if we remove some items. table 5 exploratory factor analysis of the full and reduced versions of the compliance questionnaire for rheumatology factor analysis no. of items kmo items removed msa of removed items highest factor loading of removed item number of items total variance explained 1-cqr19 0.870 na na na 5 64.028 2-cqr16 0.864 4 12 19 0.303 0.474 0.520 0.452 0.806 0.517 4 63.761 3-cqr15 0.862 7 0.376 0.551 4 65.123 4-cqr12 0.859 1 3 10 0.376 0.309 0.309 0.483 0.606 0.856 4 65.781 5-cqr9 0.855 8 9 11 0. 192 0. 192 0.479 0.818 0.596 0.849 3 62.892 6-cqr9 0.865 2 66.778 cqr19=19 item compliance questionnaire for rheumatology, cqr15=15 item compliance questionnaire for rheumatology, kmo=kaiser-meyer-olkin, msa=measure of sampling adequacy m.-s. subtirelu, a. turcu-stiolica, f.-a. vreju, p. l. ciurea, s. c. dinescu, a. l. barbulescu, j. neamtu, r. c. dănciulescu cultural adaptation and optimization of the compliance questionnaire-rheumatology (cqr) through statistical methods for patients with rheumatic diseases 41 inspecting figure 1 of the initial cqr19 we can see a point at which the shape of the curve changes direction and becomes horizontal. we observe in figure 2 that the final cqr9 has two components with eigenvalue bigger than 1, that reflecting the outcome of compliance. in fig.1 is presented a capture of the results obtained with the spss program in the case of the analysis of the main components (a component is a group of variables or questions that have a certain characteristic). analyzing the diagram (method 2, graphics) only those components (factors) those have their own values greater than 1 are retained because these factors contribute most to explaining the variance in the data set. practically a straight line is drawn over the points that have the lowest eigen values; there are 5 components (factors, question groups) that are important for the analysis of the study. brain – broad research in artificial intelligence and neuroscience volume 10, issue 3 (september, 2019), issn 2067-3957 42 according to the parallel analysis, we should keep the same five new factors. the component matrix was analyzed to determine whether the new factors would be orthogonal or oblique. values were no more than 0.5 and the new factors were weak or moderately correlated. once we have obtained the final model, it is tested the internal consistency using the cronbach index; if the value of this index is greater than 0.8 then we can assume that we have a high internal consistency of the questionnaire; if the value of the index is between 0.7 and 0.8 then we have a good consistency and if the index value is less than 0.7 then we have a weak consistency. it can be seen from the figure that the last cqr-9 (9 questions questionnaire) has two components with an eigen value greater than 1 which reflects the adhesion result. the final value of cronbach alpha of 0.865 (see table 5) suggests a very good internal reliability of the scale coherence of this questionnaire. the final cronbach’s alpha value (0.865) suggested very good internal consistency reliability for the scale with this questionnaire. the two remaining components show us two different ways to calculate adherence:  component 1: q 5 (“my medicines are always stored in the same place and that’s why i don’t forget them.”), q6 (“i take my medicines because i have complete confidence in my rheumatologist.”), q15 (“my health goes above everything else and if i have to take medicines to keep well, i will.”), q16 (“i use a dose organizer for my medications.”), q17 (“what the doctor tells me, i hang on to.”)  component 2: q2 (“i take my anti-rheumatic medicines because i then have fewer problems.”), q13 (“if i don’t take my anti-rheumatic medicines regularly, the inflammation returns.”), q14 (“if i don’t take my anti-rheumatic medicines, my body warns me.”), q18 (“if i don’t take my anti-rheumatic medicines, i have more complaints.”) both two final components, like two subscales of compliance, had very good internal reliability: 0.849 and 0.853, respectively. component 1 emphasizes the belief in rheumatologist and medications. component 2 emphasizes the fear that not taking the prescribed drugs could lead to distinct worsening of ra symptoms. cqr9 (q2, q5, q6, q13, q14, q15, q16, q17, q18) will assess the compliance using the formula: 𝐶𝑄𝑅𝑠𝑐𝑜𝑟𝑒 = ∑ . (equation 2) m.-s. subtirelu, a. turcu-stiolica, f.-a. vreju, p. l. ciurea, s. c. dinescu, a. l. barbulescu, j. neamtu, r. c. dănciulescu cultural adaptation and optimization of the compliance questionnaire-rheumatology (cqr) through statistical methods for patients with rheumatic diseases 43 using the above formula, patients treated with biological drugs had an adherence average of 81.08% (standard deviation of 14.10) versus 65.86% (standard deviation of 7.15) than they had in the initial case (questionnaire with 19 questions). patients treated with conventional drugs had an average adherence of 62.75% (standard deviation of 13.20) versus 57.13% (standard deviation of 7.71) as compared to baseline. 4. discussion in this study, we developed and optimized a romanian version of the cqr for the patients with ra. the original cqr measured compliance in patients with ra, polymyalgia rheumatica and gout (de klerk, van der heijde, landewé, van der tempel & van der linden, 2003) and we could forward the next research to patients with other diseases. noncompliance in ra can lead to brief aggravation of arthralgia, while there is no such immediate effect in gout. some authors (grymonpre, didur, montgomery & sitar, 1998) have suggested that selfreporting is a useful measurement technique if the patient is interviewed at home. however, selfreporting questionnaires still have the inevitable limitation that it is purely subjective. refill compliance based on pharmacy refill data is a method that best reflects real-life compliance. in romania, the two health insurance houses (cnas=national house of health insurance and opsnaj=house of health insurance of defence, public order, national security, and judicial authority) could enable us to measure pharmacy refill data accurately. thereby, in the case of biologic drugs, we could measure compliance patients with almost 100% accuracy. factor analysis allows us to condense a large set of scale items (19) down to a smaller, more manageable number of items (9). the compliance’s size obtained with cqr9 formula was closer to reality. the two subscales of cqr9 highlights the fact that psychosocial factors, particularly perceptions, are strong predictors of compliance to anti-rheumatic medication. this study benefitted from having a substantial sample size and patients that had a large treatment experience as well as different socio and geographic demographics and ages. although the cqr9 performed well compared to the cqr19, the next step would be to validate the reduced version against a measure of adherence to determine the sensitivity of assessment of suboptimal adherence in a more objective manner. 5. conclusions in conclusion, we optimized the romanian version of a compliance questionnaire for rheumatoid arthritis. it showed adequate reliability for clinical application. thus, the new optimization questionnaire responds to the needs of the target population in romania with adequate reliability for the following clinical applications. references anghel, l., farcaş, a., & oprean, r. (2018). medication adherence and persistence in patients with autoimmune rheumatic diseases: a narrative review. patient preference and adherence, volume 12, 1151-1166. beaton, d., bombardier, c., guillemin, f., & ferraz, m. (2000). guidelines for the process of cross-cultural adaptation of self-report measures. spine, 25(24), 3186-3191. de klerk e., van der heijde d., van der tempel h., van der linden s. (1999). development of a questionnaire to investigate patient compliance with antirheumatic drug therapy. de klerk e., van der heijde d., landewé r., van der tempel h., van der linden s. (2003). the compliance-questionnaire-rheumatology compared with electronic medication event monitoring: a validation study. field a. (2013). discovering statistics using ibm spss statistics. 4th edition. sage publications ltd. brain – broad research in artificial intelligence and neuroscience volume 10, issue 3 (september, 2019), issn 2067-3957 44 filippou, g., scirè, c., adinolfi, a., damjanov, n., carrara, g., & bruyn, g. et al. (2018). identification of calcium pyrophosphate deposition disease (cppd) by ultrasound: reliability of the omeract definitions in an extended set of joints—an international multiobserver study by the omeract calcium pyrophosphate deposition disease ultrasound subtask force. annals of the rheumatic diseases, 77(8), 1194-1199. fleiss j.l. (1986). the design and analysis of clinical experiments. new york: john wiley and sons. grymonpre, r., didur, c., montgomery, p., & sitar, d. (1998). pill count, self-report, and pharmacy claims data to measure medication adherence in the elderly. annals of pharmacotherapy, 32(7-8), 749-754. haddad, p., brain, c., & scott, j. (2014). nonadherence with antipsychotic medication in schizophrenia: challenges and management strategies. patient related outcome measures, 5, 43-62. hair j., anderson r.e., tatham r.l., black w.c. (1995). multivariate data analysis. 4th ed. new jersey: prentice-hall inc. kardas p. (2007). compliance, clinical outcome, and quality of life of patients with stable angina pectoris receiving once-daily betaxolol versus twice daily metoprolol: a randomized controlled trial. vascular health and risk management, 3(2), 235-242. kawalec, p., tesar, t., vostalova, l., draganic, p., manova, m., & savova, a. et al. (2017). pharmaceutical regulation in central and eastern european countries: a current review. frontiers in pharmacology, 8. kleinbaum d.g., kupper l.l. (1978). applied regression analysis and other multivariate methods. duxbury, ma, duxbury press, pallant j. (2007). a step-by-step guide to data analysis using spss. open university press pednekar, p., ágh, t., malmenäs, m., raval, a., bennett, b., & borah, b. et al. (2019). methods for measuring multiple medication adherence: a systematic review–report of the ispor medication adherence and persistence special interest group. value in health, 22(2), 139156. subtirelu m., turcu-stiolica a., vreju f.a., neamtu j. (2018). the study on the medical adherence of patients to rheumatoid arthritis treatment. management and marketing journal. subtirelu m., turcu-stiolica a., vreju f., neamtu j. (2017). the post-traumatic stress disorder and medication adherence encountered at patients with rheumatoid arthritis. value in health, 20(9). subtirelu m., turcu-stiolica a., vreju f., neamtu j. (2017). translation and validation of the cqr19 for romanian patients with rheumatic diseases. value in health, 20(5). tabachnick b.g., fidell l.s. (2007). using multivariate statistics. boston: pearson education inc. turcu-stiolica a., subtirelu m., vreju f., neamtu j. (2018). the correlation between mental disorder parameters and medical adherence in patients with rheumatoid arthritis. value in health, 21(3). van den bemt, b., van den hoogen, f., benraad, b., hekster, y., van riel, p., & van lankveld, w. (2009). adherence rates and associations with nonadherence in patients with rheumatoid arthritis using disease modifying antirheumatic drugs. the journal of rheumatology, 36(10), 2164-2170. vreju, f., ciurea, m., popa, d., popa, f., parvanescu, c., & chisalau, b. et al. (2016). ultrasonography in the diagnosis and management of non inflammatory conditions of the hand and wrist. medical ultrasonography, 18(1), 90. williams b., brown t., onsman a. (2012). exploratory factor analysis. a five-step guide for novices. australian journal of paramedicine, 8(3). patient-reported outcome measures: use in medical product development. (2009). retrieved 10 february 2019, from https://www.fda.gov/regulatory-information/search-fda-guidancem.-s. subtirelu, a. turcu-stiolica, f.-a. vreju, p. l. ciurea, s. c. dinescu, a. l. barbulescu, j. neamtu, r. c. dănciulescu cultural adaptation and optimization of the compliance questionnaire-rheumatology (cqr) through statistical methods for patients with rheumatic diseases 45 documents/patient-reported-outcome-measures-use-medical-product-development-supportlabeling-claims www.cnas.ro, www.aopsnaj.ro key: cord-0065630-6aztp98i authors: boutet, marie-astrid; nerviani, alessandra; lliso-ribera, gloria; leone, roberto; sironi, marina; hands, rebecca; rivellese, felice; del prete, annalisa; goldmann, katriona; lewis, myles j.; mantovani, alberto; bottazzi, barbara; pitzalis, costantino title: circulating and synovial pentraxin-3 (ptx3) expression levels correlate with rheumatoid arthritis severity and tissue infiltration independently of conventional treatments response date: 2021-06-25 journal: front immunol doi: 10.3389/fimmu.2021.686795 sha: 310c27b484abfbe0e1c058cabcae97b5860e1605 doc_id: 65630 cord_uid: 6aztp98i aims: to determine the relationship between ptx3 systemic and synovial levels and the clinical features of rheumatoid arthritis (ra) in a cohort of early, treatment naïve patients and to explore the relevance of ptx3 expression in predicting response to conventional-synthetic (cs) disease-modifying-anti-rheumatic-drugs (dmards) treatment. methods: ptx3 expression was analyzed in 119 baseline serum samples from early naïve ra patients, 95 paired samples obtained 6-months following the initiation of cs-dmards treatment and 43 healthy donors. rna-sequencing analysis and immunohistochemistry for ptx3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess ptx3 gene and protein expression. immunofluorescence staining was performed to characterize ptx3 expressing cells within the synovium. results: circulating levels of ptx3 were significantly higher in early ra compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. six-months after commencing cs-dmards, a high level of ptx3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. rna-seq analysis confirmed that synovial transcript levels of ptx3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. ptx3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. accordingly, ptx3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. the percentage of ptx3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-dmards responders and non-responders. conclusion: this study demonstrates that, early in the disease and prior to treatment modification, the level of circulating ptx3 is a reliable marker of ra activity and predicts a high degree of structural damages at 12-months. in the joint, ptx3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. high synovial and peripheral blood levels of ptx3 are associated with chronic inflammation characteristic of ra. additional studies to determine the mechanistic link are required. rheumatoid arthritis (ra) is the most common chronic inflammatory autoimmune joint disease. it is characterized by synovitis and progressive cartilage destruction and bone erosions, and can lead to persistent joint pain and functional disability. defective control of the systemic inflammatory response and the development of autoimmunity, often emerging long before the clinical onset of the disease, result in the synovial infiltration and activation of immune and stromal cells that lead to the production of high levels of inflammatory mediators such as pro-inflammatory interleukins (il-1, tumor necrosis factor-tnf-a, il-6) or matrix metalloproteinases (mmp) (1) . these molecules contribute to the maintenance of synovitis, which displays histopathological variability that has been categorized into three partially overlapping pathotypes (2) . these include the lympho-myeloid pathotype characterized by the presence of b/t cells forming organized ectopic lymphoid structures (els), plasma cells and macrophages; the diffusemyeloid pathotype with a predominant infiltration of macrophages without organized els, and the pauci-immune fibroid pathotype defined by a scant immune cell infiltrate. these pathotypes have been shown to stratify disease activity and severity, as well as response to conventional synthetic (cs) disease modifying anti-rheumatic drug (dmards) (3) . furthermore, soluble proteins such as the classical short pentraxin c-reactive protein (crp), in addition to acting as acute phase reactants and indicator of systemic inflammation, also reflect joint disease activity and tissue pathology (3, 4) . the long pentraxin 3 (ptx3) is a soluble pattern recognition molecule highly conserved in evolution (5) . ptx3 is generally poorly expressed in homeostatic conditions, but is rapidly upregulated in response to inflammatory stimuli. for instance, the proinflammatory molecules il-1 and tnf are the most important inducers of ptx3 production by myeloid and endothelial cells. when appropriately stimulated, fibroblasts and other stromal cells also release ptx3. ptx3 is a multifunctional protein exerting non-redundant roles in the innate resistance to pathogens, the regulation of inflammation and tissue repair. it has been shown that ptx3 can limit tissue damage in murine models of acute lung injury and ischemia/ reperfusion-induced kidney injury by regulating neutrophil recruitment at inflamed sites (6, 7) . in experimental models of chemically-induced sterile liver and lung injury, skin wound healing and in arterial thrombosis, ptx3-deficiency is associated with altered remodeling of the fibrin-rich inflammatory matrix and impairment of normal tissue repair (8) . similarly to crp, ptx3 circulating levels are increased in several inflammatory or infectious pathological conditions and are associated with disease severity and mortality risk (9) (10) (11) . in the context of ra, ptx3 has been shown to be constitutively highly expressed by synovial fibroblasts (12); however, as described in other tissues, various resident or infiltrating synovial cells like macrophages or endothelial cells might also represent a source of ptx3 upon inflammatory activation. several groups found that ptx3 is upregulated in the serum of ra patients compared to healthy donors (13) (14) (15) or patients with osteoarthritis (16) . however, conflicting results have been published regarding the relationship between the level of ptx3 in the serum and clinical features of the disease. on the one hand, studies on different cohorts of 111 (17) , 29 (18) , 60 (13) , 83 (19) , 58 (14) or 41 (15) ra patients reported no relationship between ptx3 and the disease activity score (das)-28. conversely, other groups found an association between ptx3 serum levels and either crp (16) or the progression of joint damage assessed by increase in the total van der heijde modified sharp score (shss) and joint space narrowing over 3 years (20) . in addition, weitoft and colleagues reported that the level of ptx3 in synovial fluid from ra patients is higher in seropositive patients (anti-cyclic citrullinated protein -ccp-antibody or rheumatoid factor -rf-) when compared to seronegative patients, and correlates with disease activity and local expression of markers of inflammation such as il-6 or vascular endothelial growth factor (vegf) (21) . these contradictory results likely depend on the heterogeneity of the cohorts analyzed including, for example, diverse disease stages, treatment exposure (cs-or biologic-dmards, known to influence various biologic processes), and disease phases (acute flare/latent chronic). for example, during acute flares, a dysregulated production of synovial fluid with a predominant infiltration of neutrophils will significantly influence ptx3 level. here, to clarify the relationship between ptx3 systemic and synovial levels and clinical parameters while minimizing population heterogeneity and drug-exposure, we investigated a large cohort of treatment-naïve early ra patients. we performed a thorough analysis of ptx3 circulating and transcript/protein synovial expression at baseline and 6-months after cs-dmards exposure in matched individuals. here, we provide important insights into the relevance of ptx3 in ra disease tissue pathology, disease severity and whether it is a useful biomarker predictive of treatment response in ra. blood samples and synovial tissues were collected from early (<12 months of symptoms) treatment-naïve ra patients enrolled into the pathobiology of early arthritis cohort (peac) at bart's health nhs trust (http://www.peac-mrc.mds.qmul.ac.uk/). all ra patients fulfilled the 2010 american college of rheumatology/european league against rheumatism (acr/eular) criteria (22) . the study was approved by the national research ethics service committee london dulwich (rec 05/q0703/198). a signed informed consent was obtained by all patients enrolled in the study. at baseline, patients underwent ultrasound-guided needle synovial biopsy of an actively inflamed joint, as previously described (23) , before commencing cs-dmards treatment. synovial tissues were embedded in paraffin for histological characterization or preserved in rna later (ambion, invitrogen, carlsbad, ca, usa) for gene expression analysis. sera were obtained by centrifugation of blood at room temperature for 10 minutes, aliquoted and stored at -80°c for further use. six-months after starting treatment, patients were consented to undergo a repeated synovial biopsy of the same joint as baseline and a post-treatment serum sample was also collected. at both baseline and 6-months, number of swollen and tender joints, patient visual analogue score (vas) for pain, fatigue, global health, and physician assessment of global health were recorded. erythrocyte sedimentation rate (esr) and crp were also measured. rheumatoid factor (rf) and anti-cyclic citrullinated peptide (ccp) antibodies in the serum were assessed and recorded at baseline, as per the ra 2010 classification criteria (22) . the clinical response to cs-dmards was assessed according to eular criteria based on the achieved das28 and the difference between baseline/post-treatment das28. accordingly, patients were classified as non-, moderate-or good-responders (24) . structural damage was assessed at baseline and at 12-month follow-up by scoring hand and feet radiographs according to the shss performed by a single reader blinded to all clinical/ histological data. control serum samples were collected from 43 age-and gendermatched healthy volunteers at humanitas research hospital. total rna was extracted from ra synovial tissues using a trizol/chloroform method. bulk rna-seq was performed on an illumina hiseq2500 platform (illumina inc., san diego, ca, usa). raw data quality control, normalization and analysis of regularized log expression read counts were performed as previously described (4) . rna-seq data were uploaded to arrayexpress and are accessible via accession e-mtab-6141. gene set enrichment analysis (gsea) was performed using the r interface to the enrichr database (https://cran.r-project.org/ package=enrichr). the ptx3 gene module was composed of the 30 genes encoding for the protein integrated in the string network for ptx3 (25) . ptx3 levels were measured in serum samples from healthy donors (n=43) and ra patients (119 baseline and 95 follow-up samples) as previously described (26) , using a sandwich elisa assay developed in-house (detection limit 0.1 ng/ml, inter-assay variability from 8% to 10%), by staff blinded to patients' characteristics. all samples were run in technical duplicates. histology, immunohistochemistry (ihc) and immunofluorescence (if) sections (3 mm thick) of synovial tissue were stained by ihc using antibodies specific for b cells (cd20, dako, agilent technologies, santa clara, ca, usa), t cells (cd3, dako), plasma cells (cd138, dako) or macrophages (cd68, dako) to determine their degree of cellular infiltration. synovial samples were categorized into three pathotypes (pauci-immune, diffuse myeloid or lympho-myeloid) following semi-quantitative scoring by two independent observers (2, 3) . briefly, the lympho-myeloid pathotype is characterized by the presence of b/t cells frequently forming highly-organized ectopic lymphoid structures (els), plasma cells and abundant macrophages in the sublining; the diffuse-myeloid pathotype is marked by a predominant infiltration of cd68+ macrophages without distinctly organized follicular structures; and the pauciimmune pathotype is defined by a scant immune cells infiltrate and a fibroblast-rich stroma. samples were classified as ungraded if there was no recognizable synovial tissue or if the tissue was of insufficient quality. synovial tissues were also stained for ptx3 [affinity purified rabbit igg anti-human ptx3, generated in-house (27) ]. matching isotype controls were used to confirm the specificity of the primary antibodies. slides were counterstained with hematoxylin and mounted with distyrene plasticizer xylene (dpx) mounting medium (sigma-aldrich, saint-louis, mo, usa). all sections were digitally scanned using nanozoomer s210 (hamamatsu photonics, japan). quantitative digital image analyses were performed to determine the percentage of ptx3positive cells within the synovial tissue using qupath software (28) . double fluorescent labeling was performed on synovial sections by multiplex immunofluorescence staining using a tyramide signal amplification protocol (invitrogen, thermo fisher scientific, waltham, ma, usa). ptx3 expression was evaluated in combination with the expression of the following markers: cd68, cd138, cd55 (thermo fisher scientific), te-7 (merck, darmstadt, germany), lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1, abcam, cambridge, uk), von willebrand factor (vwf, dako) and neutrophil elastase (ne, novus biologicals, centennial, co, usa). slides were counterstained with 40,6-diamidino-2-phenylindole (dapi) (invitrogen, thermo fisher scientific) and mounted with prolong antifade mountant (thermo fisher scientific). sections were digitally scanned using nanozoomer s60 (hamamatsu photonics). differences in continuous variables were evaluated by the mann whitney u test (unpaired samples, two groups, <30) or wilcoxon test (paired samples, two groups, <30), unpaired or paired student's t test (two groups, >30) or kruskal wallis with dunn's post-test (multiple groups). chi-squared/fisher's exact test were used to evaluate associations of categorical variables. correlations were evaluated by spearman's bivariate analysis. statistical analyses were performed using graphpad prism-v9 software (graphpad, san diego, ca, usa). p-values <0.05 were considered significant. baseline demographics and clinical features of the 119 ra patients included in this study are summarized in table 1 . as expected for a cohort of ra patients, the female-to-male ratio was approximately 3:1 (68.9% female), the median age was 51.0 and 67.2% of the patients were positive for either rf or anti-ccp autoantibodies. as per the inclusion criteria, patients had early ra with a median disease duration of 5.3 months and all patients had active joint disease (tender joint count/28 11/28; swollen joint count/28 6/28; das28 5.9). at baseline, 38.7% of the synovial tissues were classified as lympho-myeloid, 28.6% as diffuse-myeloid, 21% as pauci-immune and 11.8% were ungraded. as previously reported by our group (3), patients presenting a lympho-myeloid pathotype had significantly more active disease, with higher esr and crp levels and das28 scores. in addition, patients with a lympho-myeloid pathotype were more likely to be seropositive for rf or anti-ccp, though the result did not reach statistical significance. following the baseline clinical assessment, 94.1% of the patients received cs-dmards and 47.9% received adjunctive corticosteroid treatment. ninety-five patients (79.8% of the baseline population) successfully completed their 6-month follow-up visit and, at this timepoint, 77.9% of patients were classified as responders to treatment according to eular response criteria, with a similar response rate in the three pathotype groups. disease severity in early untreated ra patients, but not with clinical response to cs-dmards treatment ptx3 was significantly higher in the serum of ra patients (n=119) in comparison with age-and gender-matched healthy donors (n=43), as expected in association with the inflammatory status of these patients (healthy donors median 1.25, iqr 0.76-1.64; ra median 3.23, iqr 2.28-4.99; figure 1a ). the overall ptx3 systemic levels were similar at baseline and at 6-months after treatment with cs-dmards (n= 94 paired samples) ( figure 1b) . however, paired analysis revealed that patients with a high baseline ptx3 serum level (values higher than the median, i.e. >3.23 ng/ml) also had significantly higher circulating ptx3 at 6-months after starting treatment compared to the low-ptx3 (≤3.23 ng/ml) baseline patients group ( figure 1c , left panel); consistently, we detected a significant correlation between ptx3 levels at baseline and at 6-months ( figure 1c , right panel). we observed that serum ptx3 positively correlated with clinical parameters such as crp, esr, das28, tender and swollen joints, vas physician assessment of global health and haq at baseline. however, for most of these variables, except for the crp, the correlation was lost at 6-months after starting treatment ( figure 1d ). importantly, the baseline serum level of ptx3 also positively correlated with the degree of bone erosions assessed by the shss at 12-months ( figure 1e ). however, a low/high clinical activity or erosion status at baseline did not influence the evolution of ptx3 levels between baseline and 6-months (supplementary figure 1a) . although all clinical variables assessed at 6-months were significantly reduced in patients who responded to treatment according to eular response criteria ( figure 1f ), ptx3 expression was not affected by cs-dmards, and both ptx3 concentrations at each timepoint and the delta-ptx3 between baseline and 6-months were similar in all patients, regardless of their response status (none, moderate or good responders) ( figure 1g and supplementary figure 1b) . moreover, there was no difference between patients treated with different cs-dmards or who received steroids (data not shown). next, we analyzed the expression and distribution of ptx3 in the synovial tissue of a subset of patients both at the gene (n=79) and protein (n=58) level by rna-seq and ihc, respectively. as shown in supplementary table 1 , these subgroups of patients displayed similar demographic and clinical characteristics as the total population (n=119) above in which we analyzed systemic serum levels. here, we assessed the relationship between synovial ptx3 expression, clinical disease activity and local inflammation. at baseline, rna-seq analysis revealed that transcript levels of ptx3 in the synovial tissue, similarly to what was observed in the peripheral blood ( figure 1d ), strongly correlated with disease activity, assessed by esr and circulating crp concentrations and das28 (figure 2a ). in addition, synovial ptx3 mrna also correlated with the gene expression of other acute phase reactants, such as crp or serum amyloid a (saa), selected chemokines, cytokines and molecules involved in their signaling pathways, as well as mediators involved in promoting tissue remodeling and bone resorption, such as matrix metalloproteinases (mmp) or receptor activator of nf-kb (rank, gene tnfsfr11a) ( figure 2b ). conversely, we observed a negative correlation between ptx3 transcript expression and tnfsfr11b, the gene encoding for osteoprotegerin (opg), decoy receptor of rank ligand (rankl) and negative regulator of osteoclastogenesis. of note, although the correlation between ptx3 and tnf expression was not significant, several mediators involved in the tnf signaling cascade and cell death, such as bcl2 antagonist/killer 1 (bak1), bh3 interacting domain death agonist (bid), lymphotoxin beta (ltb) and ltb receptor (ltbr) were positively and significantly associated with ptx3 expression ( figure 2b ). lympho-myeloid n=22 diffuse-myeloid n=25 pauci-immune we then applied blood transcript modules to the ra synovium rna-seq dataset, as described in (4) (and see https://peac.hpc.qmul.ac.uk/). ptx3 was present in four different modules, "pro-inflammatory cytokines and chemokines" (m29), "enriched in monocytes" (m118.0), "immune activation" (m37.0) and "antiviral ifn signature" (m75) (29) ( figure 2c) . importantly, 3 out of 4 of these modules were significantly upregulated in synovial tissue presenting lympho-myeloid and diffuse-myeloid pathotypes. in addition, a similar pattern of expression was observed using synovium modules, defined by weighted gene correlation network analysis (wgcna) (4), in which ptx3 was present in the "m1 macrophages, chemokines and cytokines" module (sc43) (supplementary figure 2a , and see https://peac.hpc. qmul.ac.uk/). accordingly, a direct comparison of synovial ptx3 transcript expression in the three pathotypes highlighted a higher expression of ptx3 in the lympho-myeloid compared to the pauci-immune pathotype while the correlation was not obvious with the krenn synovitis score (supplementary figures 2b, c) . string network analysis identified 30 relevant known and predicted ptx3 p hysical a nd functional partners (supplementary figure 2d) . we used these 30 molecules to define a ptx3 gene module. using gsea, we observed that this module was particularly highly associated with the development of inflammation, arthritis, and sepsis ( figure 2d ) and with relevant ontology pathways, such as neutrophil-mediated immunity, cytokine signaling pathways or regulation of leukocytes migration (supplementary figure 2e) . the enrichment of this ptx3 module associated with the presence of a lympho-myeloid pathotype ( figure 2e) , as observed at the single ptx3 gene expression level, which prompted us to further investigate how ptx3 protein expression related to synovial histopathology and to characterize synovial ptx3producing cells. to understand the relationship between ptx3 synovial expression and specific histopathological features of the tissue, we performed ptx3 ihc staining and positive cell quantification. representative pictures of the staining in ra synovial tissue, classified as lympho-myeloid, diffuse myeloid or pauci-immune, are shown in figure 3a . as observed at the transcript level, the percentage of ptx3-positive cells was significantly higher in synovial tissue presenting a lymphomyeloid pathotype, characterized by the presence of els, compared to diffuse-myeloid and pauci-immune pathotypes ( figure 3b ). next, we investigated whether, in line with its higher expression in the lympho-myeloid tissue, ptx3 protein expression was linked to the presence of ra auto-antibodies, which have been reported to associate with els-positive tissues (3). we showed a higher expression of synovial ptx3-positive cells in rf/ccp seropositive compared to seronegative patients ( figure 3c ). in addition, in keeping with the higher expression of ptx3 in the lympho-myeloid pathotype, the percentage of ptx3 positive cells strongly correlated with synovial infiltration of cd3+ t cells, cd20+ b cells, cd138+ plasma cells and lining and sublining cd68+ macrophages ( figure 3d ) and the total krenn score (supplementary figure 3a) . double immunofluorescence staining confirmed that ptx3 was expressed by a large variety of synovial cells, mainly by cd55+ and te7+ fibroblasts, cd138+ plasma cells, lyve-1+ endothelial lymphatic cells, vwf+ endothelial cells, lining and sublining cd68+ macrophages, and ne+ neutrophils ( figure 3e and supplementary figure 3b) . these results are in accordance with previously rna-seq published data that highlighted the expression of ptx3 in both fibroblast and monocyte populations and a higher expression between monocytes sorted from leukocyte-rich and leukocyte-poor synovial tissue (30) (supplementary figure 3c , and see https://immunogenomics. io/ampra/). importantly, although we noticed a non-linear correlation between synovial ptx3 transcripts and protein expression (supplementary figure 4) , the strong association between histopathology, and in particular the presence of a lymphomyeloid pathotype, and a high ptx3 expression was observed at both transcript and protein levels. to further characterize the effects of cs-dmard treatments on joint inflammation, a total of 24 synovial tissue biopsies collected at 6-months post-cs-dmard treatment were stained for ptx3. conversely to what was observed in serum, where ptx3 levels remained high at 6-months ( figure 1b) , as a result of global decreased synovial cellularity, paired analysis revealed that the percentage of synovial positive ptx3 cells was significantly decreased following csdmard treatment ( figure 4a) . indeed, in line with the association between ptx3 expression and the immune synovial infiltrate observed at baseline, decreased ptx3 levels were significantly associated with the reduction of semiquantitative scores for inflammatory cells markers, in particular macrophage and b/plasma cell infiltration ( figure 4b) . as observed at the peripheral level ( figure 1g ), baseline and 6month ptx3 synovial expression levels did not distinguish patients who responded from those who were resistant to cs-dmards ( figure 4c) . importantly, both at peripheral and local levels, the baseline level of ptx3 was not predictive of specific response status (figures 1g and 4c ). here, for the first time, we analyzed ptx3 expression profiles in a cohort of early and treatment-naïve ra patients at baseline, and 6-months after cs-dmard treatment. as previously described, we confirmed that ra patients presented a higher concentration of circulating ptx3 compared to healthy donors (13) (14) (15) and that ptx3 levels positively correlated with crp (16) . in addition, we showed that, at the onset of the disease prior to any treatment modification, circulating ptx3 levels also correlated with esr, das28 and the count of swollen and tender joints, therefore being a strong marker of disease activity. however, serum ptx3 level did not differentiate patients presenting distinct synovial pathotypes, while, for example higher systemic levels of crp were found in patients with a lympho-myeloid pathotype compared to other pathotypes, in line with a previous report from our group (3). we also found that ptx3 serum levels were not reduced after treatment in paired samples, with 6-months values being proportional to baseline values and independent of a low/high clinical activity or erosion status at baseline. these results are in line with previously published evidence demonstrating that ptx3 serum levels are not significantly affected by various treatments for inflammatory arthritis, either cs-dmards (methotrexate) or anti-tnf or a combination of both, at different timepoints (6 weeks or 6 months) in a cohort including patients with ra, psoriatic arthritis and spondylarthritis (31) . as previously reported by our group, steroid treatment might have opposite effects on the release of ptx3 by different cell types involved in the same inflammatory microenvironment, ptx3 expression being for example inhibited in myeloid cells and upregulated in fibroblasts and endothelial cells (32) , illustrating the complexity of ptx3 production balance in response to therapies. also, as reported by satomura and colleagues, ptx3 expression is enhanced by other acute-phase reactants, such as saa produced by hepatocytes, independently of treatment influence (33) . accordingly, we observed a positive correlation between saa and ptx3 synovial transcripts abundance in our cohort. the maintenance of high peripheral levels of ptx3, despite cs-dmard treatment, might therefore participate in the perpetuation of systemic inflammation in ra and promote the occurrence of disease flares. importantly, as synovitis and overall local joint inflammation is considered a hallmark of ra, we provided, for the first time, complementary analysis in matched blood and synovial samples from patients with ra at the same timepoint before and after treatments. we demonstrated that, as a result of the global decreased synovial cellularity, local expression of ptx3 significantly diminished following cs-dmard treatment. cs-dmard are indeed known to influence the phenotypes and functions of several synovial immune cells, including for example macrophages (34) or neutrophils (35) , which both represents major sources of synovial ptx3. the decreased ptx3 expression following cs-dmard treatments was, however, independent of the response status, and deciphering the exact mechanisms by which cs-dmard influence ptx3 production in the complex ra local and peripheral environments still requires further investigations. here, we confirmed that ptx3 cannot be considered a predictive marker of response to cs-dmards either in the circulation or at the local disease tissue level. post-transcriptional modifications of ptx3 protein have been shown to finely regulate ptx3 functions in native immunity and inflammation (36) . ptx3 is characterized by a n-glycosylation site in its c-terminal domain and ptx3 glycosylation pattern changes depending on cell types and inducing stimuli, which contribute to modulate ptx3 interactions with its ligands (e.g., complement components, extracellular matrix proteins) to tune related functions, including the recruitment of inflammatory cells to the site of inflammation (6, 37) . although we observed that synovial transcript and protein levels did not significantly correlate, suggesting that local post-transcriptional regulations may affect ptx3 expression in the joint, a strong association between ptx3 and the presence of specific pathotypes was maintained at both levels. notably, ptx3 protein was significantly more expressed in synovial tissues displaying a lympho-myeloid pathotype, characterized by the presence of els, compared to diffusemyeloid or pauci-immune pathotypes. accordingly, ptx3 synovial expression positively correlated with the levels of cxcl-and ccl-chemokines involved in els formation and maintenance, such as cxcl13 or ccl19 (38) . in keeping with the high levels of ptx3 in highly infiltrated synovial tissues, we observed a significant correlation between ptx3 synovial expression and either the total krenn score or semiquantitative assessment of lining and sublining cd68+ macrophages, cd3+ t cells, cd20+ b cells and cd138+ plasma cells infiltration. ptx3 was expressed in numerous infiltrating and resident cell types activated during synovial inflammation, such as vascular and lymphatic endothelial cells, fibroblasts, plasma cells, macrophages and neutrophils. importantly, we demonstrated for the first time that a high ptx3 synovial tissue expression was associated with seropositivity for rf/anti-ccp autoantibodies, as highlighted by weitoft and colleagues in the synovial fluid of ra patients (21) . thus, synovial ptx3 might act as a bridge between innate and adaptive immunity, as previously reported in other tissues. for example, in the spleen ptx3 produced by activated neutrophils can bind b cells in the marginal zone, promote homeostatic production of igm and class-switched igg antibodies to microbial capsular polysaccharides and enhance the immunogenic response to blood-borne bacterial infection (39) . similarly, ptx3 has been shown to bind microbial moieties from neisseria meningitidis, act as an endogenous adjuvant of response and amplify the induction of an effective adaptive antibody response (40) . recently, the presence of anti-ptx3 autoantibodies has been described in ra and shown to positively correlate with ptx3 serum levels, disease activity and circulating levels of pro-inflammatory cytokines, such as il-6 or il-1b (41), further confirming ptx3 immunogenicity in the ra context. ptx3 roles within the arthritic tissue is still being debated. our rna-seq analysis confirmed a strong association between ptx3 expression in the synovial tissue, disease activity and the presence of cytokines and chemokines driving local inflammation, tissue remodeling and bone erosions. although these correlations are not proof of a causal link between ptx3 expression and disease features, it provides important insights into the relationship between ptx3 synovial levels and the inflammatory environment associated with synovial histopathology. importantly, these results are consistent with protein data. in addition, the expression of the 30 genes included in our ptx3 module are highly associated with the development of inflammation, arthritis, and sepsis. of note, ptx3 has been described by our group and others as a functional player and relevant biomarker in sepsis (42) . recently, wu and colleagues (43) also demonstrated an important role of il-6 in driving ptx3/c1q-mediated pyroptosis and inflammasome activation in ra. in addition, garcia and colleagues demonstrated that mmp8 deficiency leads to an increased disease severity, accompanied by an increase expression of il1b and ptx3 in the joints (44) . while the participation of ptx3 in driving joint inflammation is now well documented, its contribution to bone remodeling is more controversial and still requires further investigations (45) . for example, a recent report highlighted a protective role of ptx3 through the inhibition of fibroblast growth factor 2 (fgf-2) associated osteoclastogenesis in the collagen-induced arthritis model (46) . in this model, the injection of recombinant ptx3 reduces inflammatory scores and bone erosions. in addition, the binding of ptx3 to fgf-2 could reverse the inhibitory effect of this growth factor on osteoblastogenesis, therefore confirming its influence on physiological and pathological bone remodeling (47) . in contrast, and aligned with recent studies (20, 48) demonstrating an association between ptx3 circulating levels and the progression of joint damage in ra, osteoporosis and osteoarthritis bone phenotype, we confirmed that ptx3 serum levels measured at the onset of ra and prior to treatment modification correlates with radiographic damage observed at 12 months follow-up. in addition, in line with our previous report that patients presenting with a lympho-myeloid pathotype were more likely to develop joint damage progression, compared with the diffuse-myeloid and pauci-immune-fibroid pathotypes (3), here we observed that ptx3 expression was significantly elevated in the lympho-myeloid pathotype. this work therefore provides a better characterization of the relationship of ptx3 in the inflamed ra synovial tissue, and further improves our understanding of key pathways associated with the development of pathotypes in individual patients and joint erosions. hence, the measurement of ptx3 circulatory levels provides relevant information about disease activity and evolution of radiographic damage and its expression within the synovium also significantly associates with deleterious tissue infiltration. noteworthy, ptx3 expression has also been associated with the development of ra comorbidities such as vasculitis (49, 50) or coronary artery disease (51, 52) . therefore, serum and synovial levels of ptx3 in ra provide additional insights into the disease pathogenesis and help anticipating increased risks of complications. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found below: arrayexpress [accession: e-mtab-6141]. the studies involving human participants were reviewed and approved by the national research ethics service committee london dulwich (rec 05/q0703/198). the patients/ participants provided their written informed consent to participate in this study. all authors have significantly contributed to enable the delivery of the present manuscript, either by performing the experiments, analyzing the results, recruiting patients, writing or critically revising the manuscript. all authors have read and approved it for publication. all authors contributed to the article and approved the submitted version. rheumatoid arthritis new learnings on the pathophysiology of ra from synovial biopsies synovial cellular and molecular signatures stratify clinical response to csdmard therapy and predict radiographic progression in early rheumatoid arthritis patients molecular portraits of early rheumatoid arthritis identify clinical and treatment response phenotypes ptx3, a humoral pattern recognition molecule, in innate immunity, tissue repair, and cancer regulation of leukocyte recruitment by the long pentraxin ptx3 endogenous and exogenous pentraxin-3 limits postischemic acute and chronic kidney injury an acidic microenvironment sets the humoral pattern recognition molecule ptx3 in a tissue repair mode macrophage expression and prognostic significance of the long pentraxin ptx3 in covid-19 pentraxin 3 in patients with severe sepsis or shock: the albios trial prognostic significance of the long pentraxin ptx3 in acute myocardial infarction expression and production of the long pentraxin ptx3 in rheumatoid arthritis (ra) levels of serum pentraxin 3, il-6, fetuin a and insulin in patients with rheumatoid arthritis the relationship between serum pentraxine 3 levels and hematological markers in patients with rheumatoid arthritis is pentraxin 3 level an effective biomarker in disease activity in patients with rheumatoid arthritis acute phase reactant, pentraxin 3, as a novel marker for the diagnosis of rheumatoid arthritis blood monocyte chemotactic protein-1 (mcp-1) and adapted disease activity score28-mcp-1: favorable indicators for rheumatoid arthritis activity differential associations of inflammatory and endothelial biomarkers with disease activity in rheumatoid arthritis of short duration messias-reason ijt. serum pentraxin 3 levels are negatively associated with carotid intima media thickness in non-obese rheumatoid arthritis patients plasma pentraxin 3 is associated with progression of radiographic joint damage, but not carotid atherosclerosis, in female rheumatoid arthritis patients: 3-year prospective study pentraxin 3 in serum and synovial fluid of patients with rheumatoid arthritis with and without autoantibodies rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative ultrasound-guided synovial biopsy: a safe, well-tolerated and reliable technique for obtaining high-quality synovial tissue from both large and small joints in early arthritis patients the disease activity score and the eular response criteria string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets pentraxin-3 as a marker of advanced atherosclerosis results from the bruneck, army and arfy studies the long pentraxin ptx3 as a correlate of cancer-related inflammation and prognosis of malignancy in gliomas open source software for digital pathology image analysis. sci rep molecular signatures of antibody responses derived from a systems biology study of five human vaccines defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry anti-rheumatic treatment is not associated with reduction of pentraxin 3 in rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis cell-specific regulation of ptx3 by glucocorticoid hormones in hematopoietic and nonhematopoietic cells saa) induces pentraxin 3 (ptx3) production in rheumatoid synoviocytes novel insights into macrophage diversity in rheumatoid arthritis synovium neutrophil function in inflammation and inflammatory diseases side of a long pentraxin: how glycosylation affects ptx3 functions in innate immunity and inflammation structure and function of the long pentraxin ptx3 glycosidic moiety: fine-tuning of the interaction with c1q and complement activation role of chemokines in ectopic lymphoid structures formation in autoimmunity and cancer the soluble pattern recognition receptor ptx3 links humoral innate and adaptive immune responses by helping marginal zone b cells recognition of neisseria meningitidis by the long pentraxin ptx3 and its role as an endogenous adjuvant novel autoantibodies identified in acpa-negative rheumatoid arthritis the long pentraxin ptx3 as a humoral innate immunity functional player and biomarker of infections and sepsis complement c1q synergizes with ptx3 in promoting nlrp3 inflammasome over-activation and pyroptosis in rheumatoid arthritis matrix metalloproteinase-8 deficiency increases joint inflammation and bone erosion in the k/bxn serum-transfer arthritis model the long pentraxin ptx3 in bone homeostasis and pathology pentraxin 3 inhibits fibroblast growth factor 2 induced osteoclastogenesis in rheumatoid arthritis the long pentraxin 3 plays a role in bone turnover and repair the long pentraxin ptx3: a novel serum marker to improve the prediction of osteoporosis and osteoarthritis bone-related phenotypes ptx3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation ptx3 intercepts vascular inflammation in systemic immune-mediated diseases patients with inflammatory rheumatic disease are associated with subclinical atherosclerosis in recent-onset rheumatoid arthritis the authors would like to thank all ra patients and healthy donors who participated in this study and laboratory staff who helped with the processing of the synovium and blood samples. 177 the potential application of stem cell in dentistry ketut suardita department of conservative dentistry faculty of dentistry airlangga university surabaya indonesia abstract stem cells are generally defined as cells that have the capacity to self-renewal and differentiate to specialize cell. there are two kinds of stem cell, embryonic stem cell and adult stem cells. stem cell therapy has been used to treat diseases including parkinson’s and alzheimer’s diseases, spinal cord injury, stroke, burns, heart diseases, diabetes, osteoarthritis, and rheumatoid arthritis. stem cells were found in dental pulp, periodontal ligament, and alveolar bone marrow. because of their potential in medical therapy, stem cells were used to regenerate lost or damage teeth and periodontal structures. this article discusses the potential application of stem cells for dental field. key words: embryonic stem cell, adult stem cells, dentistry correspondence: ketut suardita, c/o: bagian konservasi gigi, fakultas kedokteran gigi universitas airlangga. jln. mayjend. prof. dr. moestopo 47 surabaya 60132, indonesia. introduction the inability of most tissues and organs to repair and regenerate after damage is a problem in medical and dentistry that should be solve. to repair or regenerate damage tissues and organs, many materials and devices were used, but the results are not good. many complications including infection, inflammation, impaired function, and loosening were happen. these conditions make opportunities to scientist improved therapies. in medical therapy, stem cells have been used for engineering many tissues and organs. stem cell researches are interesting knowledge about how to regenerate healthy cells, tissues, and organs from a single cell. stem cell is a multipotent cell, which can proliferate and differentiate to specific cell. these cells have the capacity to form many different tissue types. stem cell therapy has been used to treat diseases including parkinson’s and alzheimer’s diseases, spinal cord injury, stroke, burns, heart diseases, diabetes, osteoarthritis, and rheumatoid arthritis.1 regeneration of damage periodontal tissue, bone, pulp, and dentin are problems that the dentist should solve. at present, some traditional approaches are used to repair damage dental tissues. direct pulp capping using calcium hydroxide is a conventional technique to repair the damage of tooth pulp. demineralized bone graft was used in order to repair fractured bone. furthermore, guided tissue regeneration (gtr) and growth factor, for example bone morphogenic proteins (bmps) were used to regenerate new periodontal tissues. in fact, it is difficult to predict the result of treatments stated above.2 since, stem cells were used to regenerate damage tissue in medical therapy successfully; it is possible that the dentist use stem cell to regenerate lost or damage dental and periodontal structures. the purpose of this article is to describe and discuss stem cell potential application in dentistry. in future, stem cell therapy will enable new dental treatments for caries, endodontic, periodontal and oral-maxillo facial surgery, alveolar ridge augmentation, and cartilage in the temporomandibular joint. what is stem cell? stem cell can be described as an immature or undifferentiated cell that is capable of producing an identical daughter cell. stem cell has two important characteristics that distinguish them from other types of cells. first, they are unspecialized cells that renew themselves for long periods through cell division. the second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions. stem cell self-renewal may be perpetuated over many generations, resulting in considerable amplification of stem cell numbers. a stem cell is able to produce at least one type of highly differentiated cell. in traditional thinking, stem cells have been generally recognized as undifferentiated cells with varying degrees of potency. there are three basic measures of stem cell potency i.e. totipotent, pluripotent, and multipotent.3 stem cell has been identified in two kinds of tissues that are in adult tissues, so-called adult stem cell and in embryo, called embryonic stem cell. in a blastocyst of a developing embryo, stem cells differentiate into all of the specialized embryonic tissues. in adult organisms, stem cells act as a repair system for the body; replenishing specialized cells.4 generally adult stem cells present a more limited range of differentiated lineages. compared to embryonic stem cells, adult stem cell are preferable for therapeutic purposes since they are considered safer for implantation, with lesser 178 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 177–180 proliferation capacity and tumorogenecity. adult stem cells are also easier to differentiate to specific lineages.5 source of stem cell embryonic stem cells are isolated from the inner cell mass of the preimplantation blastocyst and have been derived from mice, non-human primates, and human. they are pluripotent cells, retaining the capacity to generate any and all fetal and adult cell types in vivo and in vitro. by manipulating the culture conditions under which embryonic stem cell differentiate, it has been possible to control and restrict the differentiation pathways. embryonic stem cell, especially mouse embryonic stem cell have been used to generate a range of distinct phenotypes including haematopoietic precursors, neural cells, adipocytes, muscle cells, myocytes, chondrocytes, pancreatic islet, and osteoblasts in vitro. because of their capability to differentiate into many different cell types, embryonic stem cell have been recognized as a valuable model system for studying the mechanisms underlying lineage specification during the early stages of mammalian development.6,7 stem cell can be identified in many adult mammalian tissues. in some tissues, such as epithelia, blood, and germ line, stem cells contribute to replenishment of cells lost through normal cellular senescence or injury. stem cells may also be present in other adult organs, such as the brain and pancreas, which normally undergo very limited cellular regeneration or turnover. adult stem cells are found in specific niches or tissue compartment including skin, liver, intestine, brain, skeletal muscle, myocardium, fatty tissue, and bone marrow.8 bone marrow contains hematopoetic stem cells, which differentiate into every type of mature blood cell; endothelial cell progenitors; and marrow stromal cells, also called mesenchymal stem cells (msc). msc can fabricate a spectrum of specializes mesenchymal tissues including bone, cartilage, muscle, marrow stroma, tendon, ligament, fat, and variety other connective tissues.9,10,11 in oral environment, stem cells were isolated from adult dental pulp tissues, periodontal ligament and alveolar bone marrow.12,13,14,15 there was an evidence that remnant dental pulp derived from exfoliated deciduous teeth contains a multipotent stem-cell population.16 application of stem cell for medical and dental therapies because of their abilities of unlimited expansion and pluripotency, embryonic stem cells are a potential source for regenerative medicine and tissue replacement after injury or disease. to date, no approved medical treatments have been derived from embryonic stem cell research. controversies surrounding the legal and moral status of human embryos and the use of embryonic stem cells encompass fundamental issues such as contraception, abortion, the definition of human life, and the rights and legal status of an embryo. the use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does require the destruction of an embryo. adult stem cells have been proven clinically useful because they can be isolated, transplanted, and effectively reconstitute the damage tissues. using autologous mscs dispersed in a collagen-type i gel, wakitani et al.17 succeeded in repairing full-thickness defects on the weight-bearing surface of medial femoral condyles. furthermore, treatment of msc with synthetic glucocorticoid dexamethasone stimulates msc proliferation and support osteogenic lineage differentiated.18 adult stem cells have been used to repairing or regenerating tissues because the limitation of adult cell for tissue regeneration. grande et al.19 reported that autologous chondrocyte cultures could be utilized to repair articular cartilage defects in the rabbit knee. subsequently, this technique has been applied to the clinical treatment of articular cartilage defects. although repairing the defect with chondrocytes is attractive, there are limitations related to the harvesting of chondrocytes and expanded these cells. we need large amount of biopsy to get enough cells for transplantations. in addition to the well-established bone and cartilage lineages, the induction of msc differentiation into other connective tissues, such as muscle, tendons, and ligaments is also being investigated. for a tissue-engineering approach, marrow-derived mscs have been used for achilles tendon repair. mscs seeded onto a collagen-type i construct incorporated into healing tendons. these mscs-loaded scaffolds had better alignment of cells and collagen fibers and were more similar to the native tendon than unloaded controls.20 recently, some groups have been examined the treatment of myocardial infarction by application of autologous mscs in the pig model, and these studies show engraftment, differentiation, and improved function in animals treated with autologous marrow mscs.21 in periodontal treatment, bone marrow-derived mesenchymal stem cells were transplanted to experimental class iii periodontal defects. the aim of this research was to elucidate the behavior of transplanted mscs in periodontal defects. four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue.22 discussion caries, pulpitis, apical periodontitis and another craniofacial diseases increase health costs and attendant loss of economic productivity. they ultimately result in premature tooth loss and therefore diminishing the quality of life. within the next few decades, changes in the methods and materials used to treat dental disease will take place. tissue engineering is a new concept that might be solves the problem in craniofacial regeneration. tissue engineering is the science of design and manufacture of new tissues to replace damage tissues because of diseases and trauma. the three key elements of tissue engineering are signal for morphogenesis, stem cells for responding to morphogens, and the scaffold of extra cellular matrix. stem cells are generally defined as cells that have the 179suardita: the potential application of stem cell capacity to self-renewal and differentiate to specialize cell. stem cells are present in small numbers in many vertebrate adult and fetal tissues. they are responsible for tissue renewal and for regeneration of damaged tissues. during wound healing, dental pulp stem cells have the potential to proliferate and to differentiate into odontoblasts to form dentin.23 in the other hand, stem cell derived from periodontal ligament may migrate into periodontal defect, proliferate, and differentiate.24 the ability of high expansion and multipotent of differentiation make stem cells are the cell sources for potential therapeutic use and tissue engineering in dentistry. several studies indicated that stem cells are present in dental pulp, alveolar bone and in the periodontal ligament.12,13,14 in endodontic and conservative dentistry, to restore and regenerate the dentin-pulp complex is a problem that very difficult to solve. direct pulp capping using calcium hydroxide cannot induce new dentin regeneration when there are no odontoblasts remain in dental pulp. stemcell based tissue engineering may be a new technique to regenerate dentin-pulp complex. using methodology developed to isolate and characterize mesenchymal stem cells, clonogenic and highly proliferative dental pulp stem cells (dpscs) have been isolated from adult human teeth. these stem cells maintained their high rate of proliferation even after extensive sub culturing and generated s dentin/ pulp-like complex. furthermore, it is noteworthy that the amount of dentin and pulp-like tissue formed in transplant far exceeds the amount that would be generated in situ during the lifetime of an organism. consequently, there is a great potential for the isolation of a large number of dpscs from a single tooth that could be used for dentinal repair of a number of teeth.12 in addition, iohara et al.25 in their research stated that the autogenous transplantation of bmp2-treated dental pulp stem cell pellet culture onto the amputated pulp stimulated reparative dentin formation. these results prove that stem cell therapy has considerable promise in dentin regeneration. in near future, stem cells in combination with appropriate scaffolds and growth factors are materials, which may use for direct pulp capping. in addition, multipotent stem cells were isolated from the remnant pulp of exfoliated deciduous teeth. it found that these stem cells are distinct from dpscs with respect to their high proliferation rate, increases cell-population doublings, sphere-like cell-cluster formation, osteoinductive capacity in vivo, and failure to reconstitute a dentin-pulp like complex. it is indicate that deciduous teeth may be an ideal resource of stem cells to repair damage tooth structure, induce bone regeneration, and possibly to treat neural injury or degenerative diseases. as we know, periodontal diseases can destroy the periodontal ligament, bone, and cementum. destruction of this tissue is a cause of tooth loss. recently, seo et al.26 discovered stem cells from human periodontal ligament. these stem cells have the potential to generate periodontal ligament and cementum. in another research, mesenchymal stem cells have been used for periodontal defect treatment. hasegawa et al.22 was transplanted bone marrow-derived mesenchymal stem cell to experimental class iii periodontal defects. four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue. cementoblasts, osteoblasts, osteocytes, and fibroblasts of the regenerated periodontal tissues were detected. as a dentist, we frequently encounter defects in alveolar bone caused by trauma or inflammatory processes. due to the high healing capacity of oral tissues, small defects frequently heal without major problems. however, if the affected area is large or complex tissues are involved, regeneration is generally incomplete. to regenerate the deteriorated tissues biologically based technique are required. stem cell based-tissue engineering is a good alternative therapy. several studies have already detailed the ability of mscs transduced with bmp7 to elicit periodontal bone formation.27 stem cell has a clinical potential for bone defect therapy. until now, bone graft technique have been used for bone graft therapy. autogenous bone graft from iliac bone make good healing, but this therapy is too expensive and morbidity. furthermore, 8% of iliac graft make infection, nerve injury, blood loss, short and long-term pain and functional deficit. recently, ueda 28 were able to demonstrate the transplantibility and therapeutic effects of msc in bone defect. he formed mscs transplantation in combination with biodegradable scaffold (beta tcp). the result of this research was the increased of bone regeneration in the defect. stem cells are also found in alveolar bone marrow. isolated stem cells from alveolar bone marrow were cultured and expanded. these stem cells had potent osteogenic potential in vitro and in vivo. based on the results, the researchers hope that transplantation of alveolar bone marrow stem cell can promote regeneration of alveolar bone in patients with periodontal diseases.15 in conclusion, stem cell present in dental pulp, periodontal ligament and alveolar bone marrow, and has a potential to repair and regenerate tooth and periodontal structures. stem cells can be harvested from dental pulp, periodontal ligament, alveolar bone marrow, expanded, embedded in a appropriate scaffold, and transplanted back into defect to regenerate bone and tooth structures. references 1. bonassar lj, vacanti ca. tissue engineering: the first decade and beyond. j cell biochem supp 1998; 30(31): 297–303. 2. nakashima m, reddi h. the application of bone morphogenetic proteins to dental tissue engineering. nature biotechnology 2003; 21:1025–32. 3. ballas cb, zielske sp, gerson s. adult bone marrow stem cell and gene therapies: implications for greater use. j cell biochemistry supp 2002; 38:20–8. 4. caplan ai, bruder sp. mesenchymal stem cells: building blocks for molecular medicine in the 21st century. trends in mol med 2001; 7:259–64. 5. pelled g, turgeman g, aslan h, gazit z, gazit d. mesenchymal stem cells for bone gene therapy and tissue engineering. current pharmaceutical design, 2002; 8:1917–28. 180 dent. j. (maj. ked. gigi), vol. 39. no. 4 october–december 2006: 177–180 6. rathjen pd, lake j, whyatt lm, bettes md, ratjhen j. properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. reprod fertil dev 1998; 10:31–47. 7. odorico js, kaufman ds, thomson ja. multilineage differentiation from human embryonic stem cell lines. stem cell 2001; 19:193–204. 8. kuehnle i, goodell m. the therapeutic potential of stem cells from adults. bmj 2002; 325:372–6. 9. caplan ai. the mesengenic process. clin plastic surg 1994; 21:42935. 10. makino s, fukuda k, miyoshi s, kinoshi f, kodama h, pan j, sano m, takahashi t, hori s, abe h, hata j, umezawa a, ogawa s. cardiomyocytes can be generated from marrow stromal cells in vitro. j clin invest 1999; 103:697–705. 11. bianco p, robey pg. marrow stromal stem cells. j clin invest 2000; 105:1663–8. 12. gronthos s, mankani m, brahimj, robey pg, shi s. postnatal human dental pulp stem cells (dpscs) in vitro and in vivo. proc natl acad sci usa 2000; 97:13625–30. 13. gronthos s, brahim j, li w, fisher lw, cherman n, boyde a, denbesten p, robey pg, shi s. stem cell properties of human dental pulp stem cells. j dent rest 2002; 81:531–5. 14. batouli s, miura m, brahim j, tsutsui tw, fisher lw, gronthos s, robey pg, shi s. comparison of stem-cell-medicated osteogenesis and dentinogenesis. j dent res 2003; 82:976–81. 15. matsubara t, suardita k, ishii m, sugiyama m, igarashi a, oda r, nishimura m, saito m, nakagawa k, yamanaka k, miyazaki k, shimizu m, bhawal u, tsuji k, nakamura k, kato y. alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells. j bone and min. res. 2005; 20: 399–409. 16. miura m, gronthos s, zhao m, lu b, fisher lw, robey pg, shi s. shed: stem cells from human exfoliated deciduous teeth. proccedings of the national academy of science. 2003; 100: 5807–12. 17. wakitani s, gotot, pineda sj, young rg, mansour jm, caplan ai, goldberg vm. mesenchymal cell-based repair of large, fullthicknesss defects of articular cartilage. j bone joint surg am 1994; 76:579–92. 18. liu f, aubin, je, malaval l. expression of leukimia inhibitory factor (lif)/interleukin-6 family cytokines and receptors during in vitro osteogenesis: differential regulation by dexamethasone and lif. bone 2002; 31:212–9. 19. grande, da, pitman mi, peterson j, menche d, klein m. the repair of experimentally produced defect in rabbit articularcartilage by autologous chondrocyte transplantation. j orthop res 1989; 7:208–18. 20. shake jg, gruber pj, baumgartner wa, senechal g, mryers j, redmont jm, pittenger mf, martin bj. mesenchymal stem cell implantation i a swine myocardial infarct model: engraftment and functional effects. ann thorac surg 2002; 73: 1919–26 21. young rg, butler dl, weber w, caplan ai, gordon sl, fink dj. use of mesenchymal stem cells in a collagen matrix for achilles tendon repair. j orthop res 1998; 16: 406–13. 22. hasegawa n, kawaguchi h, hirachi a, takeda k, mizuno n, nishimura m, koike c, tsuji k, iba h, katoy, kurihara h. behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects. j periodontol 2006; 77(6):1003–7. 23. tziafas d, smith aj, lesot h. designing new treatment strategies in vital pulp therapy. j dent 2000; 28: 77–92. 24. cho mi, garant pr. development and general structure of the periodontium. periodontol 2000; 24: 9–27. 25. iohara k, nakashima m, ito m, ishikawa m, nakashima a, akamine a. dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. j dent res. 2004; 83(8): 590–5 26. seo bm, miura m, gronthos s, bartold pm, batouli s, brahim j, young m, robey pg, wang cy, shi s. investigation of multipotent postnatal stem cells from human periodontal ligament. the lancet 2004; 364:149–155. 27. jin qm, anusaksathien o, webb sa, rutherford rb, giannobile wv. gene therapy of bone morphogenic protein for periodontal tissue engineering. j periodontol 2003; 74:202–13. 28. ueda m. maxillofacial bone regeneration using tissue engineering concepts. dentistry in japan 2003. 39:199–205 key: cord-0066794-iw0rjwtd authors: dow, coad thomas title: warm, sweetened milk at the twilight of immunity alzheimer’s disease inflammaging, insulin resistance, m. paratuberculosis and immunosenescence date: 2021-08-05 journal: front immunol doi: 10.3389/fimmu.2021.714179 sha: 85921645c8964500a5db909c73a4eda0a433817f doc_id: 66794 cord_uid: iw0rjwtd this article prosecutes a case against the zoonotic pathogen mycobacterium avium ss. paratuberculosis (map) as a precipitant of alzheimer’s disease (ad). like the other major neurodegenerative diseases ad is, at its core, a proteinopathy. aggregated extracellular amyloid protein plaques and intracellular tau protein tangles are the recognized protein pathologies of ad. autophagy is the cellular housekeeping process that manages protein quality control and recycling, cellular metabolism, and pathogen elimination. impaired autophagy and cerebral insulin resistance are invariant features of ad. with a backdrop of age-related low-grade inflammation (inflammaging) and heightened immune risk (immunosenescence), infection with map subverts glucose metabolism and further exhausts an already exhausted autophagic capacity. increasingly, a variety of agents have been found to favorably impact ad; they are agents that promote autophagy and reduce insulin resistance. the potpourri of these therapeutic agents: mtor inhibitors, sirt1 activators and vaccines are seemingly random until one recognizes that all these agents also suppress intracellular mycobacterial infection. the zoonotic mycobacterial map causes a common fatal enteritis in ruminant animals. humans are exposed to map from contaminated food products and from the environment. the enteritis in animals is called paratuberculosis or johne’s disease; in humans, it is the putative cause of crohn’s disease. beyond crohn’s, map is associated with an increasing number of inflammatory and autoimmune diseases: sarcoidosis, blau syndrome, autoimmune diabetes, autoimmune thyroiditis, multiple sclerosis, and rheumatoid arthritis. moreover, map has been associated with parkinson’s disease. india is one county that has extensively studied the human bio-load of map; 30% of more than 28,000 tested individuals were found to harbor, or to have harbored, map. this article asserts an unfolding realization that map infection of humans 1) is widespread in its presence, 2) is wide-ranging in its zoonosis and 3) provides a plausible link connecting map to ad. common age-related neurodegenerative diseases are alzheimer's disease (ad), parkinson's disease (pd), huntington's disease (hd) and amyotrophic lateral sclerosis (als). while these diseases are distinct clinical entities, at their core, they are all proteinopathies and share a common feature: misfolded and aggregated proteins. for ad, the proteins are amyloid and tau; for pd, synuclein; for hd, htt; and for als, tdp-43 (1) . in disease, these proteins lose their physiological functions, aggregate and acquire neurotoxic potential (2) . in ad, impairment of protein elimination is central to amyloid accumulation; there is stable production, but inadequate amyloid clearance (3) . autophagy is the conserved phylogenetic mechanism critical for intracellular clearance and recycling of aged and/or damaged protein elements occurring in all cell types, including neurons. in the brain, astrocytes and subtypes of microglia play important "janitorial" roles in the phagocytosis and subsequent autophagic elimination of neurotoxic proteins (4) . autophagy is also critical for the regulation of a wide range of immune responses including innate immunity, inflammation, and antibacterial defense (5, 6) . when pathogenic microbes are the target of autophagy the process is called "xenophagy," a form of selective autophagy (7) . consideration of an infectious contribution to ad is not new. the "usual suspects" are herpes simplex virus 1 (hsv-1), cytomegalovirus cmv), borrelia burgdorferi, chlamydia pneumoniae, helicobacter pylori and porphyromonas gingivalis (8, 9) . giving credence to a microbial cause of ad is the recognition that amyloid is an antimicrobial peptide (amp), and the accumulation of this amp may be reflective of an increasing infectious burden (10, 11) . indeed, the risk of ad appears to increase as the number of concurrent infections increases (12) . this article presents an additional potential ad precipitant, the zoonotic pathogen mycobacterium avium ss. paratuberculosis (map). map has been killing livestock, contaminating food products and has been associated with human inflammatory and autoimmune diseases at a steadily increasing global rate for 100 years (13) (14) (15) . moreover, map can contribute to cellular and metabolic invariant features of ad; namely dysfunctional autophagy and insulin resistance ( figure 1) . before an in-depth discussion of map and ad, it is relevant to review systemic states that are at the intersection of age and ad: inflammaging and immunosenescence. inflammaging, coined by franceschi in 2001 (16) refers to a lowgrade inflammatory state manifest by 1) expression of genes linked to inflammation 2) higher levels of cytokines in serum and 3) activation of nuclear factor (nf) signaling, the master regulator of inflammatory responses (17) (18) (19) . inflammaging is associated with a decline in autophagic capacity impairing figure 1 | map can impair autophagy and promote insulin resistance; these respectively, are the cellular and metabolic changes seen in alzheimer's disease. mycobacterium avium subspecies paratuberculosis -map is a notable bacterium responsible for disease of animals, contamination of food and water and associated with an increasingly long list of human diseases. cellular housekeeping which leads to protein aggregation and accumulation of dysfunctional mitochondria (20) . this agerelated phenotype is a risk factor for morbidity and mortality of the elderly and is implicated in the pathogenesis of a number of human maladies including type-2 diabetes, coronary artery disease and alzheimer's (21, 22) . paralleling inflammaging is an age-related reduction in immune competency termed immunosenescence (23) . immunosenescence not only lessens the capacity to respond to infections but also contributes to a number of age-related non-infectious diseases (24, 25) . while aging of the immune system has been often described as a shift from naive lymphocytes to memory lymphocytes, that shift is relative; aging itself is not the sole determinant of an accumulation of memory t cells and b cells. for instance, cytomegalovirus infection is associated with an acceleration of immunosenescence (26) as well as increased risk of mycobacterial infection (27, 28) . there is a bi-directional communication, a crosstalk, between the gut microbiota and cns; the gut-brain-microbiome axis is increasingly understood to play a major role in the pathogenesis of many neurodegenerative disorders including alzheimer's disease (ad) (29) . there is increasing evidence suggesting that the development of the hallmark pathologic features of ad, amyloid plaque, and tau tangles, can be linked to microbes (30) . this is tied to the aforementioned function of amyloid as an antimicrobial peptide (amp). studies found that amyloid exerts antimicrobial activity against eight common, clinically relevant microorganisms. previously unrecognized as an amp, this contrasting model of amyloid-mediated pathology has important implications for ongoing and future ad treatment strategies (31) . though none of the tested microbes were mycobacteria, it is hoped that this manuscript would prompt future testing of mycobacteria, specifically map. while there are known factors associated with ad: cerebral ischemia, hypertension, type 2 diabetes metabolic syndrome, high and low body weight, tobacco use and traumatic brain injury (32) , change to the microbiome is considered a newly added factor (33) (34) (35) . microbial residents of the intestine are able to modulate the activities of distant sites including the brain via bidirectional communication of the gi tract through interactions between the enteric nervous system (ens) and central nervous system (cns) (36, 37) . metabolic products of a healthy microbiome are required for the optimal function of the cns (38) . the largest reservoir of tissue macrophages in the body is contained within the gastrointestinal system. macrophage response to bacterial presence results in phagocytosis. the response to map, however, is blunted due to the fact that map prevents phago-lysomal maturation allowing map to persist within the macrophage (39) . there is broad acceptance of mycobacterial presence in pulmonary disease, certainly for tuberculosis, and increasingly so for non-tuberculous mycobacteria (40) . conversely, it is unlikely that map would be reported in an enteric microbiome report: molecular targets fail to report or under report map as it is in low abundance and very difficult to extract from the cell (41, 42) ; the target genetic sequences are not map specific; assays are performed on the gut contents, not the submucosa where map thrives (43, 44) ; map overcomes its host, not by sheer numbers but by map's own virulence factors (45) . the proposed mechanism by which map promotes ad is the following: map persists by inhibition of phagosome maturation, inhibition of toll-like receptor signaling and inhibition of interferon-gamma (ifn-g) signaling by map (13), altered composition of the enteric microbial community by map "bio-load" causes and increased mucosal inflammation which loosens epithelial tight junctions, "leaky gut" (46, 47) . this permits map and/or its byproducts to enter the circulation that contribute to a permeable blood-brain barrier (bbb) allowing their introduction into the brain wherein they produce inflammation and amyloid accumulation/aggregation promoting ad (30) . although nascent, the study of gut bacteria metabolites upon the brain is an area of active investigation (48) . in addition to regulation of both the intestinal barrier and the bbb, several gut microbiota metabolites are able to cross both the intestinal barrier and the bbb presenting a means of communication between the microbiota and the brain. an animal study looking at dietary polyphenols showed minimum absorption with the remainder extensively metabolized by the gut microbiota. twenty-three polyphenol microbial metabolites were isolated then given intravenously; the brain was found to be a targeted organ for ten of the metabolites (49). short-chain fatty acids (scfas), such as butyrate, are speculated to have a key role in microbiome brain communication; the scfas are produced in the gut by bacterial fermentation of dietary fiber (50). these key products of microbiome fermentation may directly or indirectly mediate these interactions via signaling routes including immune, endocrine neural and humoral pathways (50) . humans lack the pathway to produce essential aromatic amino acids; for instance, humans rely on gut bacteria for biosynthesis of phenylalanine, tyrosine, and tryptophan. multiple studies demonstrate reduced plasma tryptophan levels in probable ad and in clinical ad (51) . map infection results in upregulation of the enzyme that controls tryptophan metabolism; indoleamine 2,3 -dioxygenase (ido) levels are increased in map infected monocytes, infected ileum and in peripheral blood of infected ruminants. ido breaks down tryptophan reflected by decreased plasma tryptophan levels and correlated with onset of clinical paratuberculosis (52) . this offers a conceptual link between map infection and ad. a recent longitudinal study measuring nine cytokines of 298 cognitively normal elderly found that a higher ifn-g level was associated with slower cognitive decline independent of amyloid deposition (53) . ifng plays a central role in immune defense against a variety of intracellular pathogens, including mycobacteria (54) . map, specifically, inhibits ifn-g signaling (13) , presenting an additional plausible mechanism for the association or map and ad. only one country, india, has assessed the "bio-load" of map in a human population study: over 30% of nearly 30,000 tested positive for map: this represents a composite result: 1/3 of serum elisa tests were positive (past or present exposure), 8 .8% of blood samples tested positive by pcr and 22.4 of stool samples tested positive by pcr (55) . there are greater than 140 known mycobacterium species; most of which are considered non-pathogenic or "environmental" (56) . m. tuberculosis maintains such a prominent place within the species that all others are referred to as non-tuberculous mycobacteria (ntm), many of which have clinical significance particularly in immunocompromised individuals (57) . based upon genetic sequencing, the mycobacteria responsible for tuberculosis, leprosy and paratuberculosis, are proposed to have gone through an "evolutionary bottleneck" about 10,000 years ago. it is speculated that this was due, in part, to the domestication of and living closely with animals (58) . two of these mycobacteria are well known and studied: tuberculosis has claimed more lives than any other bacterium and a third of the world population is latently infection with m. tuberculosis (59). m. leprae, responsible for leprosy, is literally biblical in presence and continues today. official world health organization figures report there were more than 202,000 new cases in 2019 (60). the third mycobacterial agent emerging through this evolutionary bottleneck, map, is the long-recognized cause of johne's disease. map infection known also as paratuberculosis, and recognized worldwide, is an enteric inflammatory infectious disease, mostly studied in ruminant animals: cattle, sheep, and goats. it is very difficult to diagnose the map infection in the early, subclinical stage of the disease. map will colonize the intestines of infected animals for years while the animal exhibits no symptoms. however, sub-clinically infected animals continue to shed map bacilli in their milk (61) and feces contaminating pastures, the environment, and the food chain (62) . a majority of the dairy herds in the united states and europe have infected animals within the herd (63) . indeed, according to the usda, the herd-level prevalence of map infection in us dairy herds has markedly increased from 21.6% in 1996 to 91.1% in 2007 (64) . as noted, paratuberculosis is a global disease. extensive testing in india describes an increasing map "bio-load" in cattle (43%), buffalo (36%), goats (23%) and sheep (41%). moreover, in this same geographic area, 30.8% of 28,291 humans (via serum elisa, blood pcr and stool pcr) tested positive for map (48) . similarly, testing of ruminants in saudi arabia found map: 26% of sheep, 27% of goats, 30% of cattle and 15% of camels (65) . milk and related dairy products are considered to be the primary source of map infection in humans (66); products from pasteurized milk constitute a risk as pasteurization only reduces the map load originally present in milk (66, 67) . map is present in yogurt (68), cheese (69), muscle meat (70) and hamburger (71) . though the link of map zoonosis to crohn's disease has been controversial for over one hundred years (13), validation of this association has come from studies showing crohn's disease resolution with anti-mycobacterial therapy targeted against map (72) (73) (74) (75) . moreover, map is now linked to an increasing list of inflammatory and autoimmune diseases (13, 76) . to date, map has been causally associated with granulomatous diseases: crohn's (77), sarcoidosis (78, 79) and blau syndrome (80) . through molecular mimicry from mycobacterial heat shock protein (hsp65) (81), map induces autoantibodies in autoimmune diabetes (t1d) (82), multiple sclerosis (83, 84) , autoimmune thyroiditis (85), lupus (86), rheumatoid arthritis (87, 88) and possibly, sjogren's syndrome (89) . neuronal homeostasis is dependent upon autophagy; the soma of a neuron is the primary site for the degradative pathways while the axon, which extends to synaptic sites as far as a meter away, traffics the lysosome cargo to the soma to complete the degradative process (90) . axonal lysosomes are abundant but are separated from the soma by a selectivity filter that regulates trafficking of the lysosomes to the soma (91) . evidence suggests that amyloid deposits cause a local impairment of retrograde axonal transport of lysosomes leading the further amyloid accumulation. although an enormous effort has been given to develop ad therapies, there has been little success in finding effective treatments. currently fda-approved cholinesterase inhibitors and memantine, while addressing some ad symptoms, lack the ability to slow or stop disease progression (92) . considering the impact of dysfunctional autophagy on both pathogenic mycobacterial infection and ad, perhaps the most compelling argument for mycobacterial involvement in ad can be found in reviewing therapeutic agents that have been found to favorably impact ad and mycobacterial infection. a complex interaction between macrophages and pathogenic mycobacterial agents determines the outcome of an infection with these organisms (93) . for example, map has the ability to retard lysosomal maturation by limiting acidification which improves its virulence and facilitates its survival (45) . the remainder of this section identifies therapeutic agents that boost autophagy and by doing so, have potential benefit in both pathogenic mycobacterial infection and ad. the mammalian target of rapamycin (mtor) signaling pathway is a well described controller of autophagy. rapamycin and related "rapalogs" are protein kinases that inhibit mtor (94) . as neurons are small, polarized and are post-mitotic they are sensitive to the accumulation of aggregated and damaged cellular proteins and as such are dependent upon efficient autophagy for survival (95) . mounting evidence suggests that ad may be related to mtor protein synthesis and impaired autophagy (96, 97) . rapamycin is also an effective inhibitor of map; its benefit may have unknowingly been an anti-map antibiotic (98) . everolimus, as a rapalog, inhibits mtor and boots autophagy. it has been studied and showed benefit in animal models of ad (99, 100) as well as in mycobacterial infection (101, 102) . this includes map infection wherein, like with rapamycin, it may act as an anti-map antibiotic (98) . both rapamycin and everolimus are from the macrolide antibiotic family of medications, amongst the most potent anti m. avium antibiotic families (103) . the silent information regulator 1 (sirt1) function is linked to cellular metabolism and is activated by l-serine (104) . sirt1 induces autophagy (105) and plays a critical role in controlling mycobacterial disease (106) . activation of sirt1 reduces the intracellular growth of both drug-susceptible and drug-resistant strains of m. tuberculosis and induces phagosome maturation fusion and autophagy in a sirt1-dependent manner (107) . serine, a dietary amino acid, is currently being studied for early stage ad: clinicaltrials.gov identifier: nct03062449, (108). vitamin d has had an increasingly recognized roll in an expanding variety of diseases including mycobacterial disease and ad; calcitriol causes a dose-dependent inhibition of map (109) . calcitriol induces the synthesis of the archetypical antimicrobial peptide ll-37, (cathelicidin), which enhances autophagy (110) (111) (112) (113) . specific binding interactions between ll-37 and amyloid complexes may inhibit amyloid aggregation (114) . a 1992 epidemiological study revealed that japanese patients treated with anti-mycobacterial drugs for leprosy had a significantly lower incidence of ad dementia compared with an untreated group (115) . moreover, subsequent histological analyses indicated that non-demented, treated japanese leprosy patients aged over 70 years showed significantly lower levels of senile plaques in the brain than age-matched non-demented non-leprosy subjects (116, 117) . these studies have brought attention to the anti-mycobacterial drugs rifampin and dapsone. dapsone, an anti-leprosy drug, has neuroprotective effects (118) ; there is a single reported case of an individual who recovered from ad to mild cognitive impairment, mci, while on dapsone (119) . rifampin may have the greatest potential as a repurposed drug for ad (120) . rifampicin is a well-known antibiotic used in the treatment of mycobacterial infections including tuberculosis and leprosy. rifampin is available for oral and intravenous use; rifampin induces autophagy (121) . clinical trials of therapies that target amyloid-b in patients with ad have revealed that initiating therapy after the onset of clinical symptoms has little effect on cognitive function (122) (123) (124) suggesting that preventive therapy should start prior to clinical symptoms. treatment efforts with rifampin failed in cohorts of mild and moderate ad individuals (125) . however, when rifampin was used in preclinical and prodromal ad, it showed preventative effects (126) . this amplifies the need for novel plasma biomarkers that identify ad risk which then can be used in clinical trials of individuals with prodromal ad (127) . as attractive as repurposed rifampin may be for ad clinical trials, it is notably hepatotoxic (128) and has multiple adverse drug-drug interactions (129). the nasal route of drug administration has several advantages over oral or intravenous administration, which include noninvasiveness, self-administration, shorter time to onset of effect and higher bioavailability due to avoidance of hepatic first-pass metabolism (130) . intranasal rifampin delivery has direct access to the brain due to the olfactory and trigeminal neural pathways that connect the nasal mucosa with the brain (131) . moreover, rifampin has advantages for the treatment of ad as it can cross the bbb preventing production of amyloid aggregates as well as amyloidassociated cellular toxicity (132, 133) . the permeabilityglycoprotein (p-gp) is considered the most important transporter modulating the entry of drugs into the central nervous system (cns) (134) ; rifampin, as a potent p-gp inducer, facilitates reduced accumulation of amyloid (135) . thus, intranasal rifampin can access the brain directly via ante grade olfactory nerve access, retrograde trigeminal nerve access and via a favored permeability status through the bbb. a further benefit of the anti-mycobacterial agent rifampin, as suggested from animal studies, is that it works favorably in carriers of the apoe4 allele (136) . also, as with ad, rifampin has a neuroprotective role in parkinson's disease (137, 138) . map has also been associated with parkinson's disease (139, 140) . alzheimer's and insulin resistance accumulating evidence indicates that ad is an age-related, metabolic disease. impaired cerebral glucose metabolism is an invariant pathophysiological feature in ad and its occurrence precedes cognitive dysfunction and pathological alterations even for decades (141) . compared with age-matched controls, ad individuals show regional glucose metabolism impairment in parieto-temporal lobe, posterior cingulate cortex, and the frontal areas during disease progression (142) . some investigators refer to ad as "type 3 diabetes mellitus" (143) . insulin has been implicated in clearance of amyloid across the bbb, in tau phosphorylation, and in memory via its effects on synaptic function and long-term potentiation (144) . there is a proposed bi-directional relationship between insulin resistance and mycobacterial infection (145, 146) ; this occurs to the degree that insulin resistance could be considered both a biomarker and risk factor for active mycobacterial infection (147) . insulin resistance is associated with alzheimer's disease reflected in a two to five fold increased probability of a type 2 diabetic developing ad (148) . as with rifampin, enhancing brain insulin function with intranasal delivery may be a viable approach to ameliorating ad symptoms and attenuating ad-related pathophysiologic processes (149) ; this is accomplished without perturbation of the peripheral glucose level as little of the intranasal peptide reaches the peripheral circulation (150) . unlike rifampin, cerebral insulin treatment appears to have less benefit for those carrying the apoe4 allele(s) (151) . the primary use of bcg is for the prevention of tuberculosis (152) . there is increasing evidence that bcg provides protection against ntm infections (153) (154) (155) . this extends to leprosy (156) . this is unsurprising as bcg, a live attenuated vaccine, shares epitopes with mycobacteria other than tuberculosis (157) . a recent population study found an inverse relationship between the incidence of alzheimer's disease and bcg vaccination. the populations studied showed a lower prevalence of ad in countries with high bcg coverage. the authors hypothesized that exposure to bcg decreases the prevalence of ad due to a modulation of the immune system. they proposed testing their hypothesis by evaluating bladder cancer patients who received bcg comparing them to bladder cancer patients for whom bcg was not part of their recommended treatment (158) . they found that bladder cancer patients treated with bcg were significantly less likely to develop ad compared to those not similarly treated. the mean age at diagnosis of bladder cancer was 68 years. ad was diagnosed at a mean age of 84 years. bcg dramatically reduced the risk of developing ad. those treated with bcg had four-fold less risk for developing ad compared to patients not treated with bcg. the authors state that confirmation of their retrospective study would support prospective studies of bcg in ad (159) . a follow up multi-cohort study again showed protective benefit of intravesicular bcg and risk of ad; it also showed protection against parkinson's disease (160) . increasingly appreciated is the protective benefit of not only bcg, but also other live-attenuated vaccines against all-cause infection (161) (162) (163) . bcg vaccination for autoimmune diseases type 1 diabetes (t1d) and multiple sclerosis (ms) have shown benefit in these disparate diseases; both diseases associated with map: t1d (164, 165) and ms (166, 167) the positive response to bcg in t1d (168) and ms (169), may be due to a mitigation by bcg of the consequences of map infection. the role of microbial agents in ad is gaining recognition. the current and projected demographics of ad is dire and mandates broad approaches to mitigate the impact ad, not only for individuals and families, but also for global social and health systems. map is closely related to the greatest pathogen in human history, m. tuberculosis, a microbe that continues to latently infect one third of the world population. map may have a role in ad. this article suggests steps to further investigate this potentially fertile line of inquiry: 1) determine population-based map "bio-load", 2) use optimized blood-based biomarkers to determine ad risk, 3) test for map in those with elevated ad risk vs. healthy controls. concurrently, interventions could be initiated to 1) eliminate map from animals, the environment, and the food chain, 2) initiate clinical trials to test iterations of anti-mycobacterial agents shown to have benefit for ad. parsimoniously, when searching for new directions in the efforts against ad, look at the map. the author confirms being the sole contributor of this work and has approved it for publication. promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities decreased clearance of cns beta-amyloid in alzheimer's disease diversity of astroglial responses across human neurodegenerative disorders and brain aging autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms autophagy and cellular immune responses time to test antibacterial therapy in alzheimer's disease the microbiome and disease: reviewing the links between the oral microbiome, aging, and alzheimer's disease infection of fungi and bacteria in brain tissue from elderly persons and patients with alzheimer's disease role of microbes in the development of alzheimer's disease: state of the art -an international symposium presented at the 2017 iagg congress in san francisco a study on the association between infectious burden and alzheimer's disease systematic assessment of mycobacterium avium subspecies paratuberculosis infections from 1911-2019: a growth analysis of association with human autoimmune diseases. microorganisms (2020) mycobacterium avium ss. paratuberculosis zoonosis -the hundred year war -beyond crohn's disease global warming' to mycobacterium avium subspecies paratuberculosis meta-analysis of age-related gene expression profiles identifies common signatures of aging inflammatory markers in population studies of aging activation of innate immunity system during aging: nf-kb signaling is the molecular culprit of inflamm-aging inflammaging: disturbed interplay between autophagy and inflammasomes tnf and ager polymorphisms and late diabetic complications systemic inflammation and disease progression in alzheimer disease inflammation and alzheimer's disease on the immunological theory of aging the role of immunosenescence in the development of age-related diseases aging and cytomegalovirus infection differentially and jointly affect distinct circulating t cell subsets in humans cytomegalovirus infection modulates the phenotype and functional profile of the t-cell immune response to mycobacterial antigens in older life cytomegalovirus infection is a risk factor for tuberculosis disease in infants healthy gut, healthy brain: the gut microbiome in neurodegenerative disorders microbial involvement in alzheimer disease development and progression the alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers the gut-brain axis, including the microbiome, leaky gut and bacterial translocation: mechanisms and pathophysiological role in alzheimer's disease alzheimer disease: host immune defense, amyloid-b peptide and alzheimer disease role of gut microbiota and nutrients in amyloid formation and pathogenesis of alzheimer disease the gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems gut microbiota and their neuroinflammatory implications in alzheimer's disease host microbiota constantly control maturation and function of microglia in the cns mycobacterium avium subspecies impair dendritic cell maturation profiling mycobacterial communities in pulmonary nontuberculous mycobacterial disease development of a reference standard for the detection and quantification of mycobacterium avium subsp efficient, and safe method to prepare high quality dna from mycobacteria and other challenging cells crohn's disease may be differentiated into 2 distinct biotypes based on the detection of bacterial genomic sequences and virulence genes within submucosal tissues mannosylated lipoarabinomannans from mycobacterium avium subsp. paratuberculosis alters the inflammatory response by bovine macrophages and suppresses killing of mycobacterium avium subsp. avium organisms elevated fecal calprotectin in patients with alzheimer's dementia indicates leaky gut age-associated microbial dysbiosis promotes intestinal permeability, systemic inflammation, and macrophage dysfunction the role of gut bacterial metabolites in brain development fate of microbial metabolites of dietary polyphenols in rats: is the brain their target destination? the role of short-chain fatty acids in microbiota-gut-brain communication geographical distribution and diversity of gut microbial nadh: ubiquinone oxidoreductase sequence associated with alzheimer's disease indoleamine 2,3-dioxygenase, tryptophan catabolism, and mycobacterium avium subsp. paratuberculosis: a model for chronic mycobacterial infections plasma il-12/ifn-g axis predicts cognitive trajectories in cognitively unimpaired older adults mutation in the signal-transducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection mycobacterium avium subspecies paratuberculosis -an important food borne pathogen of high public health significance with special reference to india: an update non-tuberculous mycobacteria: classification, diagnostics, and therapy of tuberculosis and non-tuberculous mycobacterial infections -a comparative analysis of epidemiology, diagnosis and treatment evolutionary bottlenecks in the agents of tuberculosis, leprosy, and paratuberculosis presence, characterization, and genotype profiles of mycobacterium avium subspecies paratuberculosis from unpasteurized individual and pooled milk, commercial pasteurized milk, and milk products in india by culture, pcr, and pcr-rea methods mycobacterium avium ssp. paratuberculosis detection in animals, food, water and other sources or vehicles of human exposure: a scoping review of the existing evidence johne's disease: a hidden threat herd-level prevalence of mycobacterium avium subsp. paratuberculosis infection in united states dairy herds in 2007 serological and molecular characterization of mycobacterium avium subsp. paratuberculosis (map) from sheep, goats, cattle and camels in the eastern province, saudi arabia mycobacterium avium subsp. paratuberculosis in dairy products, meat, and drinking water contamination of food products with mycobacterium avium paratuberculosis: a systematic review survival of mycobacterium avium ssp. paratuberculosis in yoghurt and in commercial fermented milk products containing probiotic cultures detection of mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in tuscany isolation of mycobacterium avium subsp. paratuberculosis from muscle tissue of naturally infected cattle inactivation of mycobacterium avium subsp. paratuberculosis during cooking of hamburger patties anti-map triple therapy supports immunomodulatory therapeutic response in crohn's disease through downregulation of nf-kb activation in the absence of map detection profound remission in crohn's disease requiring no further treatment for 3-23 years: a case series antimicrobial treatment with the fixed-dose antibiotic combination rhb-104 for mycobacterium avium subspecies paratuberculosis in crohn's disease: pharmacological and clinical implications anti-mycobacterial therapy in crohn's disease heals mucosa with longitudinal scars cows get crohn's disease and they're giving us diabetes. microorganisms the consensus from the mycobacterium avium ssp cardiac sarcoidosis resolved with mycobacterium avium paratuberculosis antibiotics (map). sarcoidosis vasc diffuse lung dis serologic reactivity against mycobacterium paratuberculosis antigens in patients with sarcoidosis detection of mycobacterium avium ss. paratuberculosis in blau syndrome tissues paratuberculosis heat shock protein 65 and human diseases: bridging infection and autoimmunity exploring the role of mycobacterium avium subspecies paratuberculosis in the pathogenesis of type 1 diabetes mellitus: a pilot study a sardinian map for multiple sclerosis mycobacterium avium subspecies paratuberculosis and myelin basic protein specific epitopes are highly recognized by sera from patients with neuromyelitis optica spectrum disorder proposing a relationship between mycobacterium avium subspecies paratuberculosis infection and hashimoto's thyroiditis detection of m. paratuberculosis bacteremia in a child with lupus erythematosus and sjogren's syndrome ptpa and pkng proteins secreted by mycobacterium avium subsp. paratuberculosis are recognized by sera from patients with rheumatoid arthritis: a case-control study interferon regulatory factor 5 is a potential target of autoimmune response triggered by epstein-barr virus and mycobacterium avium subsp. paratuberculosis in rheumatoid arthritis: investigating a mechanism of molecular mimicry what is the evidence that mycobacteria are associated with the pathogenesis of sjogren's syndrome? axonal autophagy: mini-review for autophagy in the cns massive accumulation of luminal protease-deficient axonal lysosomes at alzheimer's disease amyloid plaques the use of antimicrobial and antiviral drugs in alzheimer's disease sequential patterns of gene expression by bovine monocyte-derived macrophages associated with ingestion of mycobacterial organisms rapamycin and mtor kinase inhibitors liaisons dangereuses: autophagy, neuronal survival and neurodegeneration fighting neurodegeneration with rapamycin: mechanistic insights mammalian target of rapamycin: a valid therapeutic target through the autophagy pathway for alzheimer's disease? on the action of cyclosporine a early intrathecal infusion of everolimus restores cognitive function and mood in a murine model of alzheimer's disease everolimus, a mammalian target of rapamycin inhibitor, ameliorated streptozotocin-induced learning and memory deficits via neurochemical alterations in male rats investigating the role of everolimus in mtor inhibition and autophagy promotion as a potential host-directed therapeutic target in mycobacterium tuberculosis infection antimycobacterial effects of everolimus in a human granuloma model activities of fluoroquinolone, macrolide, and aminoglycoside drugs combined with inhibitors of glycosylation and fatty acid and peptide biosynthesis against mycobacterium avium activation of sirt1 by l-serine increases fatty acid oxidation and reverses insulin resistance in c2c12 myotubes sirt1 protects against apoptosis by promoting autophagy in the oxygen glucose deprivation/reperfusion-induced injury role of sirt1 in innate immune mechanisms against mycobacterium tuberculosis via the inhibition of tak1 activation host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis a naturally-occurring amino acid with therapeutic potential vitamins a & d inhibit the growth of mycobacteria in radiometric culture mycobacterium tuberculosis, autoimmunity, and vitamin d vitamin d status and risk of dementia and alzheimer's disease: a meta-analysis of dose-response † the influence of vitamin d on neurodegeneration and neurological disorders: a rationale for its physio-pathological actions vitamin d -a host directed autophagy mediated therapy for tuberculosis evidence that the human innate immune peptide ll-37 may be a binding partner of amyloid-b and inhibitor of fibril assembly prevalence of dementia amongst elderly japanese with leprosy: apparent effect of chronic drug therapy neurofibrillary tangles and senile plaques in brain of elderly leprosy patients decreased b-amyloid and increased abnormal tau deposition in the brain of aged patients with leprosy protective effect of dapsone on cognitive impairment induced by propofol involves hippocampal autophagy rifampicin attenuates rotenone-treated microglia inflammation via improving lysosomal function rifampicin is a candidate preventive medicine against amyloid-b and tau oligomers recovery of dementia syndrome following treatment of brain inflammation g-secretase inhibitors and modulators for the treatment of alzheimer's disease: disappointments and hopes testing the right target and right drug at the right stage is there still any hope for amyloid-based immunotherapy for alzheimer's disease? controlled trial of doxycycline and rifampin for patients with alzheimer's disease preventive effect of rifampicin on alzheimer disease needs at least 450 mg daily for 1 year: an fdg-pet follow-up study a blood-based diagnostic test incorporating plasma ab42/40 ratio, apoe proteotype, and age accurately identifies brain amyloid status: findings from a multi cohort validity analysis evaluation of intranasal delivery route of drug administration for brain targeting intranasal delivery: circumventing the iron curtain to treat neurological disorders rifampicin prevents the aggregation and neurotoxicity of amyloid beta protein in vitro inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. its possible function as a hydroxyl radical scavenger drug efflux transporters in the cns up-regulation of pglycoprotein reduces intracellular accumulation of beta amyloid: investigation of p-glycoprotein as a novel therapeutic target for alzheimer's disease apoe genotype-dependent pharmacogenetic responses to rapamycin for preventing alzheimer's disease rifampicin and parkinson's disease rifampicin inhibits alpha-synuclein fibrillation and disaggregates fibrils paratuberculosis and parkinson's disease-is this a trigger is there a role for mycobacterium avium subspecies paratuberculosis in parkinson's disease? decoding alzheimer's disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies metabolic reduction in the posterior cingulate cortex in very early alzheimer's disease alzheimer's disease is type 3 diabetes-evidence reviewed insulin signaling in alzheimer's disease and diabetes: from epidemiology to molecular links bi-directional screening for tuberculosis and diabetes: a systematic review the association between tuberculosis and the development of insulin resistance in adults with pulmonary tuberculosis in the western sub-district of the cape metropole region, south africa: a combined cross-sectional, cohort study insulin resistance: a potential marker and risk factor for active tuberculosis? cognitive decline and dementia in diabetes mellitus: mechanisms and clinical implications effects of regular and long-acting insulin on cognition and alzheimer's disease biomarkers: a pilot clinical trial sniffing neuropeptides: a transnasal approach to the human brain effects of intranasal insulin on cognition in memory-impaired older adults: modulation by apoe genotype optimal models to evaluate the protective efficacy of tuberculosis vaccines bcg vaccination induces m. avium and m. abscessus cross-protective immunity increase in childhood nontuberculous mycobacterial infections after bacille calmette-gueŕin coverage drop: a nationwide, population-based retrospective study six years' experience with the discontinuation of bcg vaccination. 4. protective effect of bcg vaccination against the mycobacterium avium intracellulare complex the role of bcg in prevention of leprosy: a meta-analysis does bcg vaccination protect against nontuberculous mycobacterial infection? a systematic review and meta-analysis can immunization with bacillus calmette-gueŕin (bcg) protect against alzheimer's disease? med hypotheses bacillus calmette-gueŕin (bcg) therapy lowers the incidence of alzheimer's disease in bladder cancer patients bladder cancer immunotherapy by bcg is associated with a significantly reduced risk of alzheimer's disease and parkinson's disease. vaccines (2021) non-specific effects of vaccines: current evidence and potential implications optimize prime/boost vaccine strategies: trained immunity as a new player in the game bcg immunomodulation: from the 'hygiene hypothesis' to covid-19 humoral immune responses of type 1 diabetes patients to mycobacterium avium subsp. paratuberculosis lend support to the infectious trigger hypothesis specific immunoassays confirm association of mycobacterium avium subsp. paratuberculosis with type-1 but not type-2 diabetes mellitus association of mycobacterium avium subsp. paratuberculosis with multiple sclerosis in sardinian patients seroprevalence of igg1 and igg4 class antibodies against mycobacterium avium subsp. paratuberculosis in japanese population bcg therapy for type 1 diabetes: restoration of balanced immunity and metabolism use of bacille calmette-guèrin (bcg) in multiple sclerosis the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.copyright © 2021 dow. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage doi: https://doi.org/10.31351/vol29iss1pp260-267 260 effects of vitamin d3 on methotrexateinduced jejunum damage in rats farah k. abdul-wahab *, 1 and nada n. al-shawi * *department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract both methotrexate and vitamin d3 are used in combination for the treatment of various diseases. the aim of this study is to highlight the effect of vitamin d3 on methotrexate-induced jejunum damage using biochemical and histopathological studies. seven groups of both sexes of rats were selected and treated as follows: (group i and group ii) : control 1,control 2 (i.p normal saline) daily for 14 and 21 days respectively ; (group iii and group iv) :vitamin d3 groups (500 iu/rat/day) orally for 14 and 21 days, respectively;(gro up v): a single dose of methotrexate 20mg/kg, i.p injected for 4 days;(group vi):vitamin d3 (500 iu/rat/day) single for 14 days and methotrexate (20 mg/kg i.p) injected only at day 10;.(group vii) vitamin d3 (500 iu/rat/day) orally for 21 days and methotrexate (20 mg/kg i.p) injected only at day 17; then the jejunum was removed and used for measuring malondialdehyde (mda) content, total antioxidant capacity (taoc) level; in addition histopathological study of jejunum tissue. administration of vitamin d3 for 21 days and a single dose of methotrexate at day 17 resulted in non-significant difference (p>0.05) in mda; while significant reduction (p<0.05) in the taoc level in jejunum tissue; furthermore , sever villi damage ,crypts abscess, epithelial atrophy , mixed inflammatory cells infiltrate and goblet cells depletion were observed in comparison with methotrexate group. so the study demonstrates that vitamin d3 plays a synergistic role with methotrexate therefore the combined use of vitamin d3 and methotrexate may be used as a strategy to overcome dose limitations and side effects when use for the treatment of cancer, rheumatoid arthritis and psoriasis. key words: jejunum damage, methotrexate, oxidative stress, rats, vitamin d3. عه المَثوحرٍكسَج فٌ الجرذان علي حلف الصائم الىاجم 3 دحأثَر فَخامَه فرح قَس عبدالوهاب ،*1 و ودى واجٌ الشاوً * .*فشع االدٌٔح ٔانسًٕو ، كهٍح انصٍذنح، خايعح تغذاد، تغذاد،انعشاق الخالصة. انضٕءعهى ْٕذسهٍظ انذساسح ْزِ يٍ أيشاض يرعذدج.انٓذف نعلج ًٌكٍ اسرخذايًٓا يعا 3د ٔفٍرايٍٍ انًٍثٕذشٌكسٍد يٍ لا ك اٌ دشراٌان يٍ يدايٍع اخرٍاسسثع ذى ٔانُسٍدٍح.، انحٌٍٕح انكًٍٍائٍح انذساساخ تاسرخذاو انًٍثٕذشٌكسٍد عٍ انُاخى انصائى ذهف عهى 3د ذؤثٍشفٍرايٍٍ صسلد داخم انصفاق 2انسٍطشج ٔيدًٕعح 1يدًٕعح انسٍطشج : ( )انًدًٕعح االٔنى ٔانثاٍَح انُحٕانرانً: عهى ٔاعطٍد اندُسٍٍ يٍ كل دٔنٍح /خشر/ٌٕو( ٔحذج 555 ) 3د فٍرايٍٍ )انًدًٕعح انثانثح ٔانشاتعح(: يدايٍع ، انرٕانً عهى ًٌٕيا 21ٔ 14نًذج )يحهٕل انًهح انطثٍعً( ٌٕيًٍا أٌاو؛4كغى ٔصٌ اندسى داخم انصفاق( نًذج يهغى/ 25ٔاحذج ) خشعح )انًدًٕعح انخايسح(:انًٍثٕذشٌكسٍد ؛ انرٕانً ًٌٕياعهى 21ٔ 14نًذج فًٌٕا يهغى/كغى( انزي25) ٔانًٍثٕذشٌكسٍد ًٌٕيا 14نًذج ٌٕيًٍا ٔاحذج يشج] ( ٌٕو خشر/ دٔنٍح / ٔحذج555 3د [ ()انًدًٕعح انسادسح(:فٍرايٍٍ ًٌٕيا 21نًذج انفى طشٌك دٔنٍح / خشر/ ٌٕو( عٍ ٔحذج 555 ) 3د فٍرايٍٍ داخم انصفاق؛)انًدًٕعح انساتعح(: 15انٍٕو فً فمظ صسق ٔانسعح انًانَٕذاي انذٌٓاٌذ، نمٍاط ٔاسرخذايّ انصائى إصانح ذًد ؛ثى داخم انصفاق 11انٍٕو فً فمظ حمٍ يهغى / كغى( انزي 25ٔانًٍثٕذشٌكسٍد ) يٍ ٔاحذج ٔخشعح ًٌٕيا 21( نًذج 3دفٍرايٍٍ ) إعطاء يٍ انصائى. َرح يشضٍح َسٍدٍحألَسدح فضل عٍ دساسح نألكسذج انًضادج انكهٍح نـهسعح انكهٍح انًضادج نألكسذج اَخفاضا يعٌُٕا كاٌ ( تًٍُاp>0.05انًانَٕذاي انذٌٓاٌذ ) َسثح فً غٍش يعُٕي اَخفاًضا 11انٍٕو فً انًٍثٕذشٌكسٍد (p<0.05ٔذهف ) تًدًٕعح يماسَح انكؤط خلٌا َٔضٕب االنرٓاتٍح انًخرهطحانخلٌا ٔذسهم ٔضًٕسانطلئٍح االلثٍح فً ٔخشاج انضغة فً شذٌذ ٔانًٍثٕذشٌكسٍد3د ًٌكٍ اسرخذايٓا يعا فٍرايٍٍ نزنك ، انًٍثٕذشٌكسٍد يع دًٔسايرآصًسا ٌهعة 3د فٍرايٍٍ أٌ انذساسح ذٕضح ، . نزنكانًٍثٕذشٌكسٍد ٔانرٓاب انًفاصم انشٔياذٌٕذي ٔانصذفٍح. اندشعح ٔاَثاس انداَثٍح عُذ علج انسشطاٌ حذٔد عهى نهرغهة كإسرشاذٍدٍح . 3 فَخامَه د,الجرذان ,االكسدة ,المَثوحرٍكسَج ,الكلماث المفخاحَت : حلف الصائم 1 corresponding author e-mail: farah77kais@yahoo.com. received: 20/ 12 /2019 accepted:8 /2 /2020 iraqi journal of pharmaceutical science iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 261 introduction methotrexate (mtx), a folic acid antagonist, inhibits the enzyme dihydrofolate reductase (dhfr) and it is utilized as an antineoplastic and anti-rheumatoid agent (1) . the effects of mtx are not specific in action against tumour cells but also it can damage normal rapidly proliferating cells, namely intestinal epithelial cells (2) .the jejunal damage induced by mtx may be due to several factors including oxidative stress (os), inflammation and apoptosis .therefore, it is imperative to combine it with another drug, which could decrease its toxicity and increase its efficacy. it has been reported that different in vitro and in vivo studies have shown that os and dysregulation of antioxidant enzymes can play an important role in mtx-induced jejunal damage (3, 4) . moreover, researchers reported that there is no specific treatment for mtx-induced jejunum damage once it has occurred (2, 5) . vitamin d, a fat-soluble vitamin obtainable from the diet as well as produced in the skin from sunlight. vitamin d as a precursor of a potent steroid hormone, undergoes two-step metabolism in the liver and kidney to synthesize a biologically active form, calcitriol, which binds to the vitamin d receptor (vdr) (6, 7) that found in most cells, resulting in wide spread actions of vitamin d3 on most physiologic and pathologic processes. the primary sites of action of such vitamin are the intestine, bone, and kidneys; thus, the biologic functions of vitamin d3 are multiple and include its classic role in bone and mineral metabolism, and other non-classic actions, including cell proliferation, immunomodulation and cell differentiation (8) .vitamin d3 implicated in the pathophysiology of immune-mediated diseases including multiple sclerosis (ms) and inflammatory bowel disease (ibd) and it insufficiency has been linked to higher rates of cancers including colorectal, prostate and breast cancers (9,10) . objective: this study aims to study the effect of vitamin d3 at two different administration durations against mtx-induced jejunal damage in rats using biochemical and histopathological studies. materials and methods experimental animals forty nine (49) adult albino rats of both sexes, weighing 150-250gm were randomly allocated into seven groups (7 animals each) were used in this study. rats were obtained from and maintained in the animal house of the college of pharmacy, university of baghdad under conditions of controlled temperature. the animals were fed commercial pellets and tap water ad libitum throughout the experiment period. the study was approved by the scientificand the ethical committees of the college of pharmacy/ university of baghdad. chemicals and drugs methotrexate vial [(50mg/2ml vial) milan, italy]; vitamin d3 [(10,000 drops), diabase, italy]; malondialdehyde (mda) elisa kit (elabscience biotechnology, china); total anti-oxidant capacity kit (elabscience biotechnology, china). experimental protocol experimental rats used in this study were randomly divided into seven groups (7 animals /group) as follows: group i: control 1 [(i.p normal saline (0.9% nacl)] daily for 14, this group served as negative control; group ii: control 2 (i.p 0.9% nacl) daily for 21 days this group served as negative control; group iii: vitamin d3 group (500 iu/rat/day) (11) orally for 14; group iv: vitamin d3 group (500 iu/rat/day) (11) orally for 21 days; group v: methotrexate (mtx) single dose (20mg/kg, i.p) for 4 days, this group served as positive control (12) ; group vi: vitamin d3 orally (500 iu/rat/day) single for 14 days and methotrexate (20 mg/kg i.p), which injected only at day 10; group vii: vitamin d3 (500 iu/rat/day) orally for 21 days and methotrexate (20 mg/kg i.p), which injected only at day 17. at the end of the experiment, all rats were anesthetized by diethyl ether and sacrificed by cervical dislocation. then, the jejunum tissue samples were taken for the determination of mda contents, total antioxidant capacity (taoc), and for histopathological examination. determination of malondialdehyde (mda) contents contents of mda in the jejunum tissue homogenate were quantitatively estimated using mda kit based on elisa method (#e-el-0060; elabscience).the content of mda in jejunum tissue homogenate samples can be calculated by comparing the od of the samples with the standard curve. level of mda is expressed as ng/ml (13) . determination of total antioxidant capacity (taoc) level total antioxidant capacity(taoc)level was measured according to the manufacturers’ protocol using colorimetric method by the utilization of the total antioxidant capacity assay kit (#e-bc-k136; elabscience).many antioxidants in the body can reduce fe +3 to fe +2 , which in turn can form stable complex with phenanthroline substance. the taoc level in jejunum tissue samples can be calculated by measuring the absorbance at 520 nm (14) . histopathological examination the jejunum tissue samples were fixed in 10% formalin, dehydrated in graded ethanol, and https://en.wikipedia.org/wiki/jejunum iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 262 embedded in paraffin. sections of jejunal tissue were cut with a microtome set at a thickness of 5 μm, mounted on clear glass slides, and stained with haematoxylin and eosin (h&e). sections were examined and photographed using light microscopy (micros) and evaluated by the specialist pathologist (15) . an overall score for the severity of the jejunal damage was assessed in stained jejunum tissue sections as follows: avilli damage (shortening and fusion); bcrypt damage; c-epithelial atrophy; dinflammatory cells infiltrate in the lamina propria; and egoblet cell depletion as 0, none; 1, mild; 2, moderate; 3, severe (16) . statistical analysis data were expressed as the mean ± standard error of the mean (sem). the statistical significance of the differences among various groups was determined by one-way analysis of variance (anоva) followed by least significant difference (lsd) test by spss statistics version 25. differences were considered statistically significant for p-value less than 0.05. results figure 1 showed that there were nonsignificant differences (p>0.05) in the content of mda in jejunum tissue homogenate among group i (control 1), group ii (control 2), group iii (vitamin d3 for 14 days) and group iv (vitamin d3 for 21 days ); while the content of mda in the jejunum tissue homogenate in group of rats ip injected with group v (mtx) was significantly elevated(p<0.05) compared to groupi (control 1, group ii (control 2), group iii (vitamin d3 for 14 days) and group iv (vitamin d3 for 21 days ) furthermore, although the content of mda was reduced in group vi (mtx+ vitamin d3 for 14 days) and group vii (mtx+ vitamin d3 for 21 days) but still non-significantly different when each compared to mtx-treated rats (group v) (p>0.05). figure 1. effects of various treatments on malondialdehyde (mda)contents in the jejunum tissue homogenate of rats. each value represents mean ± standard error of means (sem). values expressed in small letters (a, and b) are significantly different (p<0.05). figure 2 showed that there were non-significant differences (p>0.05) in the taoc level in jejunum tissue homogenate among group i (control 1), group ii (control 2), group iii (vitamin d3) and group iv (vitamin d3); furthermore, figure 2 also showed that the level of taoc in jejunum tissue homogenate of rats ip injected with mtx (group v) was significantly reduced (p<0.05) compared to group i (control1), group ii (control 2), group iii (vitamin d3) and group iv (vitamin d3); additionally, there were significant reduction (p<0.05) in the taoc level in jejunum tissue sample in group of rats treated with mtx+vit d3 (group vi) and mtx+vit d3 (group vii) compared to mtx-treated rats (group v) (p<0.05). figure 2. effects of various treatments on total antioxidant capacity (taoc) levels in the jejunum tissue of rats. each value represents mean ± standard error of means (sem). values expressed in small letters (a, b, c, and d) are significantly different (p<0.05). histopathological examination of rats' jejunum tissue for histopathological study in the jejunum tissue of control 1 (group i), and control 2 (group ii) of rats, there were normal appearance of villi and crypts, with no epithelial atrophy,but a mild– moderate degree of mixed inflammatory cells infiltrate were seen within lamina propria; additionally, no goblet cell depletion was observed in figure 3 (a and b). furthermore, in group of rats orally-administered vitamin d3 for 14 days (group iii), there wasa mild-villous and crypts damage, -epithelial atrophy, and –mixed inflammatory cells infiltrate were seen in the lamina propria; furthermore, mild goblet cells depletion was also observed (figure 3-c). while in group of rats orally-administered vitamin d3 for 21 days (group iv), there are focal widening ,and shorting of villi, mild crypt damage (fusion of crypt) and a mild mixed inflammatory cells infiltrate in the lamina propria and mild goblet cells depletion were observed in (figure 3-d). concerning section of jejunum tissue of methotrexate-treated rats (group v), there were moderate villous (v)and crypts (c)damage which was represented by shortening, widening and fusion of villi with v/c ratio 2/1; moreover, moderate epithelial atrophy , moderate inflammation is seen in lamina propria and a moderate degree of goblet cells depletion. (figure 3-e ). iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 263 in group vi of rats-treated with methotrexate+ vitamin d3 (for 14 days), figure 3-f showed a severe damage that represented by shorting, widening and fusion of villi (villous atrophy),and crypt damage (abscess); with villous to crypts ratio 1/1; furthermore, moderate epithelial cells atrophy, a heavy mixed inflammatory cells infilterate , and moderate goblet cells depletion was also observed. moreover, in group vii of rats treated with methotrexate+ vitamin d3 (for 21 days), figure 3g showed that there were severe damages that represented by loss, widening and fusion of villi with v/c ratio 1/1, severe crypt damage (the crypt epithelium is atrophied and few cells are sloughed out in the lumen of the crypt(apoptosis); additionally, severe epithelial cells atrophy , heavy mixed inflammatory cells infiltrate were seen in the lamina propria and sever goblet cells depletion. figure 3. sections of the jejunum tissue of various rats groups (haematoxylin and eosin (h&e) x 10).a and b) group i and group ii: normal appearance of villi (v) and crypts (c), no epithelial atrophy, with a mild to moderate degree of mixed inflammatory cells infiltrate is seen in lamina propria, and no goblet cell depletion. c) group iii: a mild villous, crypts damage, mild epithelial atrophy and goblet cells depletion compared to the area pointed by the large arrow which shows goblet cells. d) group iv: focal widening ( )and shorting of villi, a mild crypt damage, a mild mixed inflammatory cells infiltrate in the lamina propria and the tips of villi showed mild goblet cell depletion compared to the sides of the villi where by goblet cells are still present. e) group v: moderate villous and crypts damage represented by shortening, widening and fusion of villi with v/c ratio 2/1, moderate epithelial atrophy and moderate inflammation were seen in lamina propria and moderate goblet cells depletion. f) group vi: severe damage that represented by shorting, widening and fusion of villi (villous atrophy) with v/c ratio 1/1, crypt abscess, moderate epithelial cells atrophy, a heavy mixed inflammatory cells infiltrate is also seen in lamina propria, and goblet cells depletion. g) group vii: severe damage represented by loss, widening and fusion of villi with v/c ratio 1/1, crypt abscess, severe epithelial atrophy, heavy mixed inflammatory cells infiltrate is seen in the lamina propria and sever goblet cells depletion. yellow arrows indicate villi damage, white arrows indicate crypt abscess, and blue arrow indicates goblet cells and black arrows indicate goblet cells depletion. a b d e f g c iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 264 discussion the intestinal epithelium is the largest surface area of the body in contact with the external environment (17) . concerning mtx, such chemotherapeutic drug belongs to the antimetabolite class of medication; where, it structurally resembles folic acid, and it competitively inhibited dihydrofolate reductase (dhfr) enzyme (18, 19) . researchers described that the therapeutic use of mtx has been limited by its impact on the rapidly-dividing cells of crypts through the inhibition of the epithelial cell proliferation and induced apoptosis in the small intestinal crypts (20) ; moreover, multiple factors may have role in mtx-induced small intestine damage such as the dose and the treatment duration of such drug, type of disease, in addition to apoptotic factors (2) . additionally, several mechanisms have been hypothesized to underline small intestine injury induced by mtx such as oxidativeand nitrosative stress, up-regulation of inflammatory mediators that may promote apoptosis which in turn may augment inflammation and tissue injury of intestinal tissue which play important role in the pathogenesis of small intestine damage induce by mtx in rat (21, 22) . regarding the oxidative stress marker malondialdehyde (mda), which is an end product of lipid peroxidation an events in the cells are recognized as an indicator of oxidative stress (23) . figure 1 showed that there were non-significant differences (p>0.05) in the content of mda in jejunum tissue homogenate among group i (control 1), group ii (control 2), group iii (vitamin d3) and group iv (vitamin d3); while , in group of rats ip injected with mtx 20mg/kg (group v) caused elevation in mda contents accompanied by the reduction of taoc level in rats' jejunum tissue homogenate compared to control1 (group i), control 2 (group ii), vitamin d3-orally administered rats for 14 days (group iii) and vitamin d3-orally administered rats for 21 (group iv), this comes in line with previous animal and clinical studies, which demonstrated that os and lipid peroxidation are hallmarks of mtx-induced jejunum damage (24-26) ; furthermore, authors reported that the cell damage can be initiated by the reaction of free radicals with biological macromolecules producing lipid peroxides with the depletion of first-line antioxidant enzyme systems, including reduced glutathione (gsh) (3, 27) .although there were reduction in mda contents in rats' jejunum homogenate that treated with mtx with vitamin d3 orally-administered for 14 days (group vi) and mtx with vitamin d3 orallyadministered for 21 days (group vii) but still nonsignificantly different when each compared to mtx-treated rats (group v). on the other hand, results of this study showed that the taoc level was significantly reduced in groups vi and vii rats each compared to groups v rats as shown in figure 2. for histopathological study in the jejunum tissue of control 1 (group i), and control 2 (group ii) of rat treated with normal saline for 14 and 21 days, respectively, there were normal appearance of villi and crypts, with no epithelial atrophy, but a mild– moderate degree of mixed inflammatory cells infiltrate were seen within lamina propria; additionally, no goblet cell depletion was observed in figure 3 (a and b). furthermore, in group of rats orally-administered vitamin d3 for 14 days (group iii), there was a mild -villous and crypts damage, -epithelial atrophy, and –mixed inflammatory cells infiltrate were seen in the lamina propria; furthermore, goblet cells depletion was also observed [figure 3-c]. while in group of rats orally-administered vitamin d3 for 21 days (group iv), there are focal widening, and shorting of villi, mild crypt damage (fusion of crypt) and a mild mixed inflammatory cells infiltrate in the lamina propria were observed in (figure 3-d). the suggestion concerning the effect of vitamin d3 may be due to its pro-oxidant effect that caused an increase in ros, which in turn may decrease gsh level; thus os can consequently be resulted in cells. in addition, this study showed that there were histopathological changes in the jejunum tissue of rat treated with mtx for 4 days which characterized by moderate villous damage that represented by shortening, widening and fusion of villi and crypts damage with v/c ratio 2/1, moderate epithelial atrophy and moderate inflammation were also seen in the lamina propria; furthermore, a moderate degree of goblet cells depletion is seen compared to group i, group ii, group iii, and group iv rats; and these changes are consistent with that observed in previous studies (11, 28) .authors mentioned that mtx act as a prooxidant that may cause depletion of the tetrahydrofolate, suppressed dna synthesis, inhibited epithelial proliferation, and induced apoptosis in the small intestinal crypt (4) . in addition, the release of free radicals by moderate infiltrate inflammatory cells in lamina propria may have a role in jejunum pathogenesis and this comes in line with previous study (29) . iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 265 moreover, many authors described that the oxidative damage induced by mtx in jejunum tissue can be prevented by different vitamins and natural plant extracts that may act as cytoprotectors to protect normal cells of the jejunum from mtx damage (2, 5) ; but, results of the current study showed that histopathological jejunum sections of rats administered vitamin d3 for 14 days and mtx at day 10 (group vi) (figure 3-f); where severe damage that represented by loss, widening and fusion of villi, crypt abscess, severe epithelial cells atrophy, a heavy mixed inflammatory cells infiltrate were seen in the lamina propria and sever goblet cells depletion (figure 3-g).flanagan l, et al (2016) reported that vitamin d3 can synergistically act with mtx; where, vitamin d3 can induce apoptosis which may be due to the action of such vitamin as pro-oxidant and caused an induction of reactive oxygen species (ros) by altering redox state of cell (30) . in addition, abu el maaty ma and wölfl s (2017) described that vitamin d3 can enhance the activity of mtx by increasing intracellular availability (31) . additionally, zhao r et al in 2013 demonstrated that the intestinal absorption of mtx and folate is mostly mediated by the proton-coupled folate transporter (pcft), which mainly expressed in the proximal part of the small intestine at the apical brush-border of enterocytes' membrane; and vitamin d3 can increase expression of intestinal pcft and an enhancement of cellular folate uptake (32) . moreover, the potential mechanism for increasing intestinal absorption of mtx can be brought about via simultaneous treatment of mtx with vitamin d3; in other word, vitamin d3 can affect the bioavailability of mtx (33) ; additionally, vitamin d3-mtx interaction was reported to be through transporters; where, such vitamin can alter the pharmacokinetics and pharmacodynamics of mtx that can result in variability of efficacy and involved in the mtx disposition in the body and in the regulation of intracellular metabolism in targets cells (31, 34) . thus, beneficial effects may be brought about when vitamin d3 and mtx used together for the treatment of different diseases such as cancer, inflammatory bowel disease, rheumatoid arthritis (ra), and psoriasis by decreasing the dose of mtx so decrease side effects and increase efficiency; but, adverse effects instead of beneficial effects were observed in this study. conclusion the present study demonstrates that vitamin d3 plays a synergistic role with methotrexate by causing histopathological damage in jejunum tissue of rats; where, vitamin d3 can act as pro-oxidant and/or it can enhance the entrance of mtx inside the cell; and, adverse effects resulted from combination of vitamin d3 and mtx are observed in this study. acknowledgments this article was abstracted from the phd thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors gratefully thank family, friends in the college of pharmacy of baghdad university, dr. ban a. abdul majeed, working at department of molecular pathology, pioneer molecular pathology laboratory, baghdad, iraq and deep thanks to dr. rima hussain, consultant histopathologist for her assistance during study. references 1. raimondi mv, randazzo o, la franca m, barone g, vignoni e, rossi d, collina s. dhfr inhibitors: reading the past for discovering novel anticancer agents. molecules. 2019 jan;24(6):1140. 2. yucel y, tabur s, gozeneli o, kocarslan s, seker a, buyukaslan h, şavik e, aktumen a, ozgonul a, uzunkoy a, aksoy n. the effects of lycopene on intestinal injury due to methotrexate in rats. redox rep. 2016 may 3;21(3):113-8. 3. arslan a, ozcicek f, cimen fk, altuner d, yarali o, kurt n, tumkaya l, ozturk c, suleyman h. protective effect of resveratrol against methotrexate-induced oxidative stress in the small intestinal tissues of rats. int j clin exp med. 2015;8(7):10491.–10500. 4. chang cj, lin jf, chang hh, lee ga, hung cf. lutein protects against methotrexateinduced and reactive oxygen species-mediated apoptotic cell injury of iec-6 cells. plos one 2013; 8(9):e72553. 5. de almeida sb, monteiro mc, de lima av, de menezes db, monteiro sm. protective effect of camellia sinensis on methotrexateinduced small intestinal mucositis in mice. food and nutrition sciences 2014; 5 (5): 443 448. 6. wang z, zhang h, sun x, ren l. the protective role of vitamin d3 in a murine model of asthma via the suppression of tgfβ/smad signaling and activation of the nrf2/ho-1 pathway. mol med rep 2016; 14 (3): 2389-96. 7. jeon sm, shin ea. exploring vitamin d metabolism and function in cancer. exp mol med. 2018; 50(4):1-4. 8. christakos s, dhawan p, verstuyf a, verlinden l, carmeliet g. vitamin d: metabolism, molecular mechanism of action, and pleiotropic effects. physiol rev.2016 jan;96(1):365-408. 9. raman m, milestone an, walters jr, hart al, ghosh s. vitamin d and gastrointestinal diseases: inflammatory bowel disease and colorectal cancer. therap adv gastroenterol. 2011; 4 (1): 49-62. https://en.wikipedia.org/wiki/jejunum https://int.search.myway.com/search/ggmain.jhtml?p2=%5ey6%5exdm269%5ettab02%5eiq&ptb=ba591e76-7ee8-438e-8fcb-e7964d1bd1a9&n=783aa9e7&cn=iq&ln=ar&si=cohe5ty6p9ccfqs77qodrq8oua&trs=wtt&brwsid=7f900d18-cf9e-4b87-b7e8-c0021fc69283&st=tab&tpr=sc&searchfor=synergistically&ots=1575849298139 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4565222/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4565222/ https://www.ncbi.nlm.nih.gov/pubmed/27484042 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5938036/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5938036/ https://www.ncbi.nlm.nih.gov/pubmed/26681795 iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 266 10. narula n, marshall jk. protective effect of camellia sinensis on methotrexate-induced small intestinal mucositis in mice.j crohns colitis 2012 may 1;6(4):397-404. 11. ashour th. effect of vitamin d supplementation with pegylated interferon-α and ribavirin on erythrocyte indices, iron parameters and erythropoietin expression in male wistar rats. clin exp pharmacol. 2014; 4(160): 2161-1459. 12. türkcü g, alabalık u, keleş an, bozkurt m, i̇biloğlu i̇, fırat u, büyükbayram h. protective effects of carvacrol and pomegranate against methotrexate-induced intestinal damage in rats. int j clin exp med. 2015;8(9):15474. – 15481. 13. bahadır a, ceyhan a, gergin öö, yalçın b, ülger m, özyazgan tm, yay a. protective effects of curcumin and beta-carotene on cisplatin-induced cardiotoxicity: an experimental rat model. anatol j cardiol. 2018 mar;19(3):213-221. 14. al-balawi aa, ahmed ym, albukhari a, alghamdi sa, zeyadi ma, maddah mr, huwait eh, ali s, kumosani ta, moselhy ss. modulation the neuro-toxicity induced by aluminum chloride in rats using beetroots and broccli extracts. ijpr. 2018: 25(3):1-18 15. natarajan k, abraham p, kota r. activation of the mitochondrial apoptotic pathway contributes to methotrexate‐induced small intestinal injury in rats. cell biochem funct. 2017; 35 (7): 378-91. 16. abd-allah om, el-din aa. the possible protective effect of ginger against intestinal damage induced by methotrexate in rats. med j cairo univ. 2013; 81 (1):1073-84. 17. vereecke l, beyaert r, van loo g. enterocyte death and intestinal barrier maintenance in homeostasis and disease. trends mol med 2011;17 (10):584-93. 18. al-darraji as, mohamed mh. synthesis and preliminary anticancer evaluation of 6mercaptopurine–methotrexate conjugate as possible mutual prodrug. ijps. 2019 jun 9;28(1):113-23. 19. nadhum sa, mohammed mh. design, synthesis, characterization and preliminary anticancer study for methotrexate silibinin conjugates. ijps.2015;24(1):74-84. 20. li t, ito k, sumi si, fuwa t, horie t. antiapoptosis action of aged garlic extract (age) protects epithelial cells from methotrexate induced injury. gut. 2005;54 (12):1819-20. 21. el-sheikh aa, morsy ma, hamouda ah. protective mechanisms of thymoquinone on methotrexate-induced intestinal toxicity in rats. pharmacogn mag. 2016;12(suppl 1):s76 22. zhou b, xia x, wang p, chen s, yu c, huang r, zhang r, wang y, lu l, yuan f, tian y. induction and amelioration of methotrexateinduced gastrointestinal toxicity are related to immune response and gut microbiota. ebiomedicine. 2018; 33:122-33. 23. jeon sm, shin ea. exploring vitamin d metabolism and function in cancer. exp mol med.. 2018 apr 16;50(4):1-4. 24. zhou b, xia x, wang p, chen s, yu c, huang r, zhang r, wang y, lu l, yuan f, tian y. induction and amelioration of methotrexateinduced gastrointestinal toxicity are related to immune response and gut microbiota. ebiomedicine. 2018 jul 1;33:122-33. 25. gulgun m, erdem o, oztas e, kesik v, balamtekin n, vurucu s, kul m, kismet e, koseoglu v. proanthocyanidin prevents methotrexate-induced intestinal damage and oxidative stress. exp toxicol pathol. 2010 mar 1;62(2):109-15. 26. famurewa ac, folawiyo am, enohnyaket eb, azubuike-osu so, abi i, obaje sg, famurewa oa. beneficial role of virgin coconut oil supplementation against acute methotrexate chemotherapy-induced oxidative toxicity and inflammation in rats. integrative medicine research. 2018 sep 1;7(3):257-63. 27. arslan a, ozcicek a, suleyman b, coban ta, cimen fk, nalkiran hs, kuzucu m, altuner d, cetin n, suleyman h. effects of nimesulide on the small intestine mucositis induced by methotrexate in rats. experimental animals. 2016:15-0122. 28. koppelmann t, pollak y, mogilner j, bejar j, coran ag, sukhotnik i. dietary l-arginine supplementation reduces methotrexateinduced intestinal mucosal injury in rat. bmc gastroenterology. 2012 dec;12(1):41. 29. acipayam c, bayram i, daglioglu k, doran f, yilmaz s, sezgin g, ateş bt, ozkan a, tanyeli a. the protective effect of hesperidin on methotrexate-induced intestinal epithelial damage in rats: an experimental study. med princ pract. 2014;23(1):45-52. 30. flanagan l, meyer m, fay j, curry s, bacon o, duessmann h, john k, boland kc, mcnamara da, kay ew, bantel h. low levels of caspase-3 predict favourable response to 5fu-based chemotherapy in advanced colorectal cancer: caspase-3 inhibition as a therapeutic approach. cell death dis. 2016;7(2):e2087. 31. abu el maaty ma, wölfl s. effects of 1, 25 (oh) 2d3 on cancer cells and potential applications in combination with established and putative anti-cancer agents .nutrients 2017;9 (1):87. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4658926/ https://www.ncbi.nlm.nih.gov/pubmed/29521316 https://www.ncbi.nlm.nih.gov/pubmed/28871597 https://www.ncbi.nlm.nih.gov/pubmed/21741311 https://www.ncbi.nlm.nih.gov/pubmed/27041864 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5938036/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5938036/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4849164/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4849164/ iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 267 32. zhao r, goldman id. the proton-coupled folate transporter: physiological and pharmacological roles. curr opin pharmacol. 2013;13 (6):875-80. 33. eloranta jj, zaïr zm, hiller c, häusler s, stieger b, kullak-ublick ga. vitamin d3 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter. mol pharmacol. 2009;76 (5):106271. 34. inoue k, yuasa h. molecular basis for pharmacokinetics and pharmacodynamics of methotrexate inrheumatoid arthritis therapy . drug metab pharmacokinet. 2014; 29 (1): 1219. creative commons attribution 4.0 is licensed under a bijpsbaghdad iraqi journal pharmaceutical sciences by d.university of baghda -copyrights© 2015 college of pharmacy .international license https://www.ncbi.nlm.nih.gov/pubmed/24383099 https://www.ncbi.nlm.nih.gov/pubmed/19666701 https://www.ncbi.nlm.nih.gov/pubmed/?term=inoue%20k%5bauthor%5d&cauthor=true&cauthor_uid=24284432 https://www.ncbi.nlm.nih.gov/pubmed/?term=yuasa%20h%5bauthor%5d&cauthor=true&cauthor_uid=24284432 https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.ncbi.nlm.nih.gov/pubmed/24284432 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ simpo pdf merge and split unregistered version http://www.simpopdf.com simpo pdf merge and split unregistered version http://www.simpopdf.com simpo pdf merge and split unregistered version http://www.simpopdf.com jiimc march 2016.cdr 1 one of the most difficult task in clinical medicine is to evaluate a patient who presents with joint pain. if you were to open the index of a rheumatology text you would find a list of over hundred different types of arthritides (table i). fortunately the more commonly seen musculoskeletal conditions can be divided into five different groups. if approached logically a working diagnosis can usually be easily obtained. editorial early diagnoses of rheumatoid arthritis is important. it should be clinical not lab dependent asim zulfiqar ------------------------------------------------correspondence: prof. dr. asim zulfiqar professor of medicine islamic international medical college riphah international university islamabad e-mail: asimzulfiqar786@gmail.com received: february 15, 2016; accepted: march 06, 2016 window of therapeutic opportunity (a time span in which the institution of effective therapeutic strategy in the form of dmards and biologicals) is important to modify the course of disease significantly, decelerating the progression of disease and minimizing joint damage and disability. early diagnosis of ra is important as early therapeutic intervention reduces the accrual of joint damage and 3 disability. in the first decade of current century the classification criteria set that was in widespread international use to define ra were the 1987 acr 4 (american college of rheumatology) criteria. those criteria gave emphasis to serological tests, rheumatoid nodules and joint erosions which are actually late features of disease. in fact these late features of disease which are pathognomonic for the diagnosis of ra can be prevented if effective therapy is given in early phase of the disease. keeping in view the problems in diagnosing era the working group developed acr/eular classification criteria for ra in 5 2010. these classification criteria were introduced to select amongst the newly presenting patients with undifferentiated inflammatory synovitis, the subset of patients who are at sufficiently high risk of persistent and/or erosive disease (this being the appropriate current paradigm underlying the disease construct ra). these classification criteria can be applied to any patient or otherwise healthy individual as long as two mandatory requirements are met. first there must be evidence of currently active synovitis in at least one joint. secondly the criteria must be applied to those patients in whom the observed synovitis is not better explained by another diagnosis. four additional criteria (table ii) can then be applied to eligible patients to identify definite ra. application of these criteria provides a score from 0 to 10 with score of 6 or >6 being indicative of ra. a patient with a score<6 cannot be classified as having definitive ra at the moment but might fulfill the criteria at a later time point. to classify a patient as having definite ra or not a history of symptom duration, a thorough joint evaluation of both small and large joints and at least one serological test (rf or acpa) and one acute phase response measure (esr/crp) must be obtained. it is acknowledged that an individual table i: simple classifica�on of arthri�c and rheuma�c 1 disorders one of the most common and devastating disease that has been encountered in clinical medicine practice is rheumatoid arthritis (ra). ra is a chronic inflammatory disease characterized by joint swelling, joint tenderness and destruction of synovial joints leading to severe disability and 2 premature mortality. ra affects between 0.5-1% of the general population, mainly during their working age affecting thus the functional capacity, with great economic burden to the individual and the society. in the last decades there was a clear evolution in knowledge about pathophysiology of the disease resulting in its approach and treatment. the association between symptom duration and ra persistence is not linear suggesting the presence of a confined period in which ra is most susceptible to treatment. early ra (era) is defined as the diagnosis given in the first weeks or months of joint symptoms or signs. the concept of era and existence of a 2 jiimc 2016 vol. 11, no.1 early diagnoses of rheumatoid arthritis is important potentiate your clinical diagnosis of ra but the presence of characteristic pattern of joint involvement of greater than six weeks duration almost makes certain the diagnosis of era. a due consideration of early aggressive treatment should be made in such patients to arrest the progressive disease at an earlier stage. references 1. alderdice c. approach to the patient with polyarthritis. can fam physician 1990; 36: 553-4. 2. pincus t, callahan lf, sale wg, brooks al, payne le, vaughn wk, et al. severe functional decline, work disability and increased mortality in ra patients. arthritis rheum 1984; 27: 864-72. 3. van nies j a b, tsonaka r, gaujoux-viala c, fautrel b, van der helm-van mil a h m. evaluating relationship between symptom duration and persistence of ra. does a window of opportunity exists. ann rheum dis 2015; 74: 806-812 4. arnett fc, edworthy sm, bloch da, mcshane dj, fries jf, cooper ns, et al. the american rheumatism association 1987 criteria for the classification of ra. arthritis rheum 1988; 31: 315-24. 5. neogi t, aletaha d, silman aj, naden rl, felson dt, aggarwal r, et al. the american college of rheumatology/ european league against rheumatism classification criteria for ra. arthritis rheum 2010; 62: 2582-91. patient may meet the definition of ra without requiring lab test or even if the serological tests are negative (seronegative ra) e.g. patients with a sufficient number of joints and longer duration (> 6 weeks) of symptoms will achieve 6 points regardless of their serological or acute phase response status. in conclusion ra is entirely a clinical diagnosis. presence of positive serology (rf/acpa) may microsoft word gjphm-2022 methotrexatedocx.docx 618 global journal of public health medicine 2022, vol 4, issue 1 gggggglo original research adherence to methotrexate in iraqi patients with rheumatoid arthritis : a cross-sectional study ali m. kadhim al-tuma¹ & nizar abdulateef jassim 2 1 department of internal medicine ,college of medicine, karbala university , karbala, iraq 2 department of internal medicine,college of medicine , university of baghdad , baghdad, iraq *corresponding author: doctor88ali@gmail.com abstract introduction: methotrexate (mtx) is the most widely used disease modifying antirheumatic drugs (dmards) in the treatment of rheumatoid arthritis (ra). in ra, medication adherence is variable and sub-optimal. poor adherence affects 20-70% of patients. adherence to mtx is the key to attaining the goal of low disease activity or disease remission. the aim of the study is to determine adherence of ra patients to mtx when used as a monotherapy and when combined with anti-tumor necrosis factor (tnf) and to look for the factors that may positively and negatively affect adherence. methods: an observational cross sectional study was conducted at rheumatology clinic/baghdad teaching hospital over a period from january-june 2020. a total of 100 patients diagnosed with ra according to american college of rheumatology (acr) and european league against rheumatism (eular), 2010 criteria are included. all were on mtx for more than 3 months. a questionnaire was used to collect information from them. results: young age, middle-high educational level and good socio-economic state increase the regular mtx intake and thus improve the adherence. long duration of the disease and treatment, oral and combination therapy decrease the patients’ adherence. conclusion: non-adherence to mtx is noted frequently in ra patients and variable factors tend to affect adherence. multiple factors encourage or discourage the continuity of mtx intake, some are related to patients themselves while others are related to the course of the disease or mtx itself. keywords: adherence, non-adherence, methotrexate , rheumatoid arthritis. 619 global journal of public health medicine 2022, vol 4, issue 1 gggggglo introduction: rheumatoid arthritis (ra) is a chronic, systemic, inflammatory disorder of joints and connective tissues (jeffery, 2014). the primary site of involvement is the synovium of the joints which become inflamed and proliferate (firestein, 2017). extra-articular manifestations might accompany the joint disease such as eye involvement, rheumatoid nodules, cardiovascular and hematological changes (angelotti et al., 2017). the exact cause of ra is still unknown, but it tends to be multi-factorial. genetic and environmental factors (smoking, pollutants, and others) play important roles (lin et al., 2016). the incidence of ra is estimated to be 1%, females are more affected than males with a predisposition for more severe disease manifestations. the peak age of presentation is around 30-45 years old and its incidence increases with age (silman, 2001 ; feist and burmester, 2013). the characteristic, typical presentations of ra are pain and swelling in the small joints of hands, wrists and feet and prolonged morning stiffness, often more than 1 hour (odells, 2014). patients are classified as having ra according to the 2010 american college of rheumatology (acr)/ european league against rheumatism (eular) classification criteria as shown in figure 1. they are scored from 0 to 10, a patient with a score equal to 6 is classified under the name ''definite ra'' (neogi et al., 2010). figure 1: acr/ eular classification criteria of ra,2010 (neogi et al., 2010). *rf: rheumatoid factor, acpa: anti-citrullinated protein antibody, crp: c reactive protein, esr : erythrocyte sedimentation rate. 620 global journal of public health medicine 2022, vol 4, issue 1 gggggglo methotrexate is one of the most frequently used dmards for ra either as a monotherapy or combination therapy. methotrexate when used as monotherapy, it may induce low disease activity in about 30% of patients. (singh et al., 2015). the precise mechanisms of action are not understood completely, it seems to have both anti-inflammatory and immunemodulatory actions (ranganathan and mcleod, 2006). insight to the molecular pathogenesis of ra, the need of using targeted diseases modifying anti-rheumatoid drugs (dmards) has increased significantly to get a state of low disease activity or disease remission(mcinnes and schett, 2007). early intervention with these dmards prevents joint damage and improves long term functional outcomes (escalas et al., 2012). according to acr and eular treatment guidelines for ra, mtx is an anchor drug whether alone or in combination with conventional or biologic dmards (curtis et al., 2016). as in all chronic diseases, compliance to therapy plays a vital role in treatment success. adherence to mtx is the key to attaining the goal of disease remission or low disease activity. non adherence (na) is defined as poor implementation of a generally continued therapy for one reason or another. it has been reported that na to mtx is considered a major challenge in the real-world treatment of ra patients (salt et al., 2010). various social and economic issues predispose to na and adherence could be promoted by physician counselling. however, due to the lack of follow up studies among mtx nonadherent ra patients, it is difficult to precisely assume the possible factors which might affect patients’ adherence (müller et al., 2017). na to treatment may impair the patient’s health by a progressively severe joint damage, functional disability, poor health-related quality of life and higher disease morbidity and mortality (rapoff and pediatr, 2002). in addition, na increases the unnecessary clinic appointments and diagnostic tests with increased usage of additional treatments' modalities with an ultimate result of increased treatment cost (de achaval and suarez-almazor, 2010). so, the study aimed to explore the patient and drug-related aspects of methotrexate adherence and it was one of few studies that deal with the mtx adherence. it tried to investigate or evaluate the mtx adherence and the barrier to the adherence to be considered and discussed clearly with the patients before the start of therapy methods: this study is an observational cross sectional study was conducted at rheumatology clinic in baghdad teaching hospital over a period from january 2020 to june 2020. a total of 100 patients (male =30, female=70) diagnosed with ra according to acr and eular, 2010 criteria were included. the sample size was calculated according to the prevalence of ra in the population. all had been on mtx for more than 3 months. informed consent was taken from the patients and the study was done under the supervision of scientific council of the iraqi board for medical specializations in partial fulfilment of the requirement for the 621 global journal of public health medicine 2022, vol 4, issue 1 gggggglo degree of the fellowship of the iraqi board for medical specialization in rheumatology and medical rehabilitation. inclusion criteria: 1. patients with ra diagnosed according to acr /eular 2010 criteria for more than 1year duration. 2. taking mtx for more than 3 months. exclusion criteria: 1.patients with chronic diseases which necessitate chronic drug intake: ht, dm, asthma and epilepsy. 2.patients with multi-drug usage (combination therapy with dmards other than anti-tnf e.g. hydroxychloroquine, prednisolone and sulfasalazine). 3. patients with mental illness: dementia and memory loss. a questionnaire was used to collect information from the patients and verbal consent was taken from them to be included in the study. adherence questionnaires, the medication adherence report scale (mars-5) and the compliance-questionnaire-rheumatology (cqr) as well as a visual analogue scale (vas) measuring mtx adherence, were administered to these patients as the following (de cuyper et al., 2016). demographic data; age in years, sex, address, educational level (primary, secondary and tertiary), socioeconomic state (according to the monthly income of the family). duration of ra (years), duration of mtx intake (years), the current dose of mtx mg/week), mode of intake: oral or s.c/ i.m. combined with anti-tnf or not (type, dose and duration). disease activity at the time of visit according to clinical disease activity index (cdai) score as shown in figure 2 and table 1 (mild when cdai of 2.8-10, moderate from 10-22 and severe 22 and above) (jeka et al., 2018). 622 global journal of public health medicine 2022, vol 4, issue 1 gggggglo . figure 2: clinical disease activity index (cdai) score (jeka et al., 2018) table 1: calculation scores of cdai (jeka et al., 2018). variable range value tender joint score (0-28) swollen joint score (0-28) patient global score (0-10) provider global score (0-10) add the above values to calculate the cdai score (0-76) to calculate both the patient global assessment disease activity and provider global assessment disease activity, the doctor must consider all the ways that arthritis affects the patient, and ask the patient to rate how well he/she is doing on the following scale ranging from very well 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 very poor. joint left right tender swollen tender swollen shoulder elbow wrist mcp 1 mcp 2 mcp 3 mcp 4 mcp 5 pip 1 pip 2 pip 3 pip 4 pip 5 knee total tender: swollen: 623 global journal of public health medicine 2022, vol 4, issue 1 gggggglo adherence to mtx: when 2 or less of prescribed mtx doses are omitted in the previous 8 weeks. nonadherence to mtx: when 3 or more doses are omitted in the previous 8 weeks. possible reasons which encourage or discourage the patients to take the drug were discussed. data were analyzed statistically by ibm spss statistics for windows version 24.0 (ibm corp., armonk. n. y,. usa). in each group, either mean ± sd for continuous data or number and percentage for categorical data was calculated. an independent sample student t-test was used for comparing parameters between both groups. for categorical values, chisquare was used. only at a p-value ≤ 0.05 the differences between the values were considered as significant statistically. results: below are demographic characteristics of both groups. there was a significant statistical difference between both groups regarding age (the adherent group is younger with a mean of 48.4± 10.4 vs 54.3±10.7 years old), educational level, socio-economic state (in which the non-adherent patient exhibited both low educational and socio-economic levels respectively) at a significant p-value of less than 0.05. table 2: demographic data of the studied groups. parameter adherent=58 non-adherent=42 p-value age m±sd 48.4± 10.4 54.3 ±10.7 0.008* sex male 16 14 0.53 female 42 28 educational level high 6 (10.3%) 2 (4.76%) 0.009* medium 34 (58.6%) 14 (33.3%) low 18 (31%) 26 (61.9%) socio-economic state high 4 (6.89%) 2 (4.76%) 0.001* middle 44 (75.8%) 18 (42.85%) low 10 (17.2%) 22 (52.3%) table 3 illustrates the medical history of the patients in both groups represented by the duration of ra, disease activity (depending upon cdai score), duration of mtx treatment, the dose of mtx, mode of mtx intake (oral vs parenteral) and type, duration of treatment (mono vs combined). there was a significant difference between both groups regarding duration of ra ( the duration of ra is less in the adherent group with a mean 7.6±3.8 vs 12.0±6.3 years non-adherent one), duration of mtx treatment (being less in the adherent group 4.5±3.0 vs 7.3±5.8 years), mode of intake (37.9% of the adherent group were on oral 624 global journal of public health medicine 2022, vol 4, issue 1 gggggglo treatment and 62% on parenteral, while 76.19% of non-adherent group on oral and only 23.8% on parenteral treatment) and type of treatment ( 58.6% of adherent patients used mtx monotherapy and 41.37% used mtx in combination with anti-tnf in contrast to 28.5% and 71.42 respectively in the non-adherent group). while, the activity of the disease, dose of mtx and duration of combined therapy showed no significant difference between both groups with a p-value> 0.05. table 3: medical history of patients in both studied groups. parameter adherent, 58 non-adherent, 42 p-value duration of ra(years) 7.6 ±3.8 12.0± 6.3 0.0001* disease activity mild (2.8-10) 0 (0%) 0 (0%) 0.58 moderate (10-22) 48 (82.75%) 33 (78.57%) sever >22 10 (17.2%) 9 (15.5%) duration of mtx (years) 4.5 ±3.0 7.3 ±5.8 0.02* dose of mtx 15.9± 4.2 15.2± 4.7 0.44 mode of mtx intake oral 22 (37.9%) 32 (76.19%) 0.001* parenteral 36 (62.06%) 10 (23.80%) type of treatment mono 34 (58.62%) 12 (28.57%) 0.003* combined with anttnf 24 (41.37%) 30 (71.42%) duration of combined (years) 3.7± 2.3 3.8± 2.4 0.90 table 4 shows the likely reasons which encourage the patient to take the drug regularly and in turn improve adherence to mtx. improvement of joint pain is at the top of the list 86.2%, followed by ra control 79.3%, improvement of quality of life 65.5%, fear from ra complications 44.8%, fear from ra morbidity & mortality 31% and the lowest reason is fear from disability 24%. 625 global journal of public health medicine 2022, vol 4, issue 1 gggggglo table 4: possible reasons for increased adherence to mtx while table 5 explains the major reasons which decrease the ra patient adherence to mtx. starting from forgetfulness 61.9%, lack of awareness of its importance in disease control 57.1%, lack of availability and lack of awareness regarding its’ long term intake 42.5%, lack of affordability and fear from se 38.0%, peoples’ negative advice 33.33%, lack of family support and intractable se 23.8% and only 14.2% due to difficulty regarding the mode of intake. no one of the patients had a concept of mtx dependence in long term use. table 5: possible reasons for decreased adherence to mtx. parameter total number percentage forgetfulness 26/42 61.9% lack of affordability 16/42 38.09% lack of availability 18/42 42.58% fear from dependence 0/42 0% fear from side effects 16/42 38.09% peoples’ negative advice 14/42 33.33% lack of awareness of its importance in disease control 24/42 57.14% lack of awareness regarding its long term intake 18/42 42.85% lack of family support 10/42 23.80% difficulty regarding the mode of intake: oral or parenteral 6/42 14.28% intractable side effects: nausea, vomiting, stomatitis, oral ulcers, epigastric pain…etc. 10/42 23.80% parameter total number percentage pain improvement 50/58 86.20% improve quality of life 38/58 65.51% disease control 46/58 79.31% fear from ra complications 26/58 44.82% fear from handicap & disability 14/58 24.13% fear from ra morbidity & mortality 18/58 31.03% 626 global journal of public health medicine 2022, vol 4, issue 1 gggggglo discussion the current study showed that about 58% of patients with ra exhibited adherence to mtx which is consistent with the results of many studies; one large american cohort study that included more than 14,000 ra patients and a prevalence study of 2662 ra patients who reported an adherence rate up to 65% (mcinnes and schett, 2007 ; escalas et al., 2012 ). however, a higher adherence rate of 80% was registered by a longitudinal study conducted in denmark which followed 941 patients with ra for 10 years (curtis et al., 2016). the adherent patients in this study were significantly younger, with middle educational and socio-economic levels in comparison to non-adherent who exhibited an older age and low both educational and socio-economic levels. similar results were obtained by arshad et al. in 2016 despite no significant differences among these parameters (arshad et al., 2016). for disease activity, disease duration, dose and duration of the mtx therapy, the study showed a significant difference between both groups. the duration of ra and mtx intake was longer in the non-adherent group while both disease activity and dose of mtx showed no significant difference. this means that with increasing the duration of both disease and treatment, drug adherence is decreased. with the exception of disease activity, this result is in some agreement with what was recognized by some studies in which the adherent patients had the more active disease (müller et al., 2017 ; rapoff and pediatr, 2002). for the route of administration, the study concluded that the adherent group were used the parenteral rout more than the oral route in contrast to the non-adherent group. although, little data is available regarding the adherence and route of intake as most studies compared between oral and parenteral including drug safety, efficacy, disease response and tolerability but not adherence. it had been reported by some studies that parenteral intake is associated with higher bioavailability, bypassing the 1st pass hepatic metabolism, a reduced frequency and intensity of some gi side effects than oral mtx which may improve treatment compliance and reduce mtx discontinuation rates (de achaval and suarezalmazor, 2010 ; de cuyper et al., 2016 ; grijalva et al., 2007). other studies suggested that there was no difference in adherence between oral and parenteral mtx intake and increased adherence to oral intake was reported in certain ra patients; those who had phobia from the injection, elderly patients who didn’t have caregivers, those with severe involvement and deformity of hand joints in which they could not use their hand to inject themselves (harley et al., 2003). 627 global journal of public health medicine 2022, vol 4, issue 1 gggggglo studying the type of therapy, mono or in combination with anti-tumor necrosis factor, the results showed that patients who used mtx alone showed more adherence than those who used mtx in combination with anti-tumor necrosis factor. little data is available regarding the difference in patients’ adherence to mtx alone or in combination with other biological agents. however, few studies were directly assessed the patients’ adherence as most of them tried to assess treatment efficacy, toxicity, disease control, symptoms improvement, drugs' interaction and side effects (pascual-ramos et al., 2009 ; hovstadius and petersson, 2011 ; kromann et al., 2015 ; rutkowska-sak et al., 2009). some studies had been suggested that despite improvement in ra symptomatology, combination therapy might potentiate mtx side effects especially gi upset, liver toxicity, anemia and increasing the risk of recurrent infections (chest infection) which indirectly lead to treatment discontinuation and ultimately lead to non-adherence to combination therapy (curtis et al., 2016 ; boers et al., 1997). for the factors which tend to affect patients' adherence, the study showed that the most important factors that encourage the patients to take the drug are; 86.2% due to improvement of joint pain and 79.3% and 65.5% due to disease control and improvement of quality of life respectively. psychological factors are also seemed to be another candidate, 44.8% of adherent patients take the drug due to fear from ra complications, 31% fear from morbidity & mortality and 24% fear from disability. these results were in proximity with what was reported by a study which illustrated that more than 60% of adherent ra patients took the drug regularly due to improvement of pain, up to 30% due to improvement in quality of life and approximately 5% due to fear from disability and long term ra complications with a statistically significant difference (salt et al., 2010). for non-adherence group, there were many factors that affect adherence; some are related to the patients and others are related to the drug itself. the most common patients related causes of decreased patients’ adherence were forgetting the drug in 61.9%, lack of awareness of its importance in disease control in 57.1%, lack of awareness regarding its long term intake in 42.5%, and fear from its side effects in 38.09% which could be related to age and patients’ education. while the main drug-related factors were lack of availability in 42.58%, lack of affordability in 38.09% (which might be attributed to the low socio-economic state, expensiveness and the drug is not available in local pharmacies), intractable side effects in 23.8%, and 14.2% due to difficulty in the mode of intake, especially parenteral intake. 628 global journal of public health medicine 2022, vol 4, issue 1 gggggglo peoples’ negative advice and lack of family support are not uncommon, it constituted about 33.33% and 23.08% of causes of non-adherent respectively. fortunately, no one of the patients had a concept of mtx dependence in long term use. these were highly in agreement with the results of most of the studies had been done on mtx adherence in ra patients with some differences in the percentages (salt et al., 2010 ; calguneri et al., 1999 ; mottonen et al., 1999 ; keystone et al., 2010 ; keystone et al., 2014 ; breedveld et al., 2006 ). limitations: patients’ lost from follow up, mtx intake for less than 3 months, selfdiscontinuation of the drug and multi-drug usage. conclusion: non-adherence to mtx is noted frequently in ra patients, multiple factors encourage or discourage the continuity of mtx intake, some are related to patients themselves while others are related to the course of the disease or mtx itself. recommendations: future researches are highly recommended to study the effect of nonadherence on patient health outcomes, to provide a good patient education and counseling by doctor which might promote patients’ adherence. conflicts of interest the author declares no conflicts of interest. references • angelotti, f., parma, a., & cafaro, g. (2017). one year in review; pathogenesis of rheumatoid arthritis. clin exp rheumatol., 35(3):368-78. • arshad, n., ahmad, n.m., saeed, m.a., khan, s., batool, s., & farman, s. (2016). adherence to methotrexate therapy in rheumatoid arthritis. pak j med sci.,32(2):413-17. • boers, m., verhoeven, a., & marusse, h. (1997). randomized comparison of combined step-down prednisolone, methotrexate and suphasalazine with sulphasalazine alone in early rheumatoid arthritis. lancet.,350:309–18. • breedveld, f., weisman, m., & kavanaugh, a. (2006). a multi-center, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early aggressive rheumatoid arthritis who had not had previous methotrexate treatment. arthritis rheum.,54:26–37. • calguneri, m., pay, s., & caliskener, z. (1999). combination therapy versus monotherapy for the treatment of patients with rheumatoid arthritis. clin exp rheum.,17:699–704. • cannon, g.w., mikuls, t.r., hayden, c.l., ying, j., curtis, j.r., & reimold, a.m. (2011). merging veterans affairs rheumatoid arthritis registry and pharmacy data to assess methotrexate adherence and disease activity in clinical practice. arthritis care res.,63(12):1680–90. • curtis, j.r., bykerk,, v.p., aassi, m., & schiff, m. (2016). adherence and persistence with methotrexate in rheumatoid arthritis: a systematic review. j rheumatol.,43(11):1997-2009 . • curtis, j.r., xie, f., mackey, d. (2016). patient’s experience with subcutaneous and oral methotrexate for the treatment of rheumatoid arthritis. bmc musculoskelet disord.,17, 405. • de achaval, s, & suarez-almazor, m.e. (2010). improving treatment adherence in patients with rheumatologic disease. j musculoskelet med., 27(10). • de cuyper, e., de gucht, v., maes, s., van camp, y., & de clerck, l.s. (2016). determinants of methotrexate adherence in rheumatoid arthritis patients. clin 629 global journal of public health medicine 2022, vol 4, issue 1 gggggglo rheumatol.,35(5):1335-9. • de thurah, a., norgaard, m., johansen, m.b., & stengaard-pedersen, k. (2010). methotrexate compliance among patients with rheumatoid arthritis: the influence of disease activity, disease duration, and co-morbidity in a 10-year longitudinal study. scandinavian j rheumatol.,39(3):197–205. • escalas, c., dalichampt, m., combe, b., fautrel, b., guillemin, f., durieux, p., dougados, m., & ravaud, p. (2012). effect of adherence to european treatment recommendations on early arthritis outcome: data from the espoir cohort. ann rheum dis., 71(11):1803-8. • feist, e., & burmester. g.r.(2013). rheumatoid arthritis-clinical features. in: watts, r.a., conaghan, p.g., denton, c, et al, editors. oxford textbook of rheumatology. 4th ed. united kingdom: oxford university press.,858-63. • firestein, g.s. (2017). etiology and pathogenesis of rheumatoid arthritis. in: firestein, g.s, budd, r.c., gabriel, s.g., mcinnes, i.b., o’dell, j.r. kelley and firestein’s textbook of rheumatology, 10th edition. elsevier, inc., 69: 1115. • grijalva, c.g., chung, c.p., arbogast, p.g., stein, c.m., mitchel, e.f., & griffin, m.r. (2007). assessment of adherence to and persistence on disease-modifying antirheumatic drugs (dmards) in patients with rheumatoid arthritis. medical care.,45(10suppl 2): s66–76. • harley, c.r., frytak, j.r., & tandon, n. (2003). treatment compliance and dosage administration among rheumatoid arthritis patients receiving infliximab, etanercept, or methotrexate. am j managed care.,9(6 suppl): s136–43 . • hovstadius, b., & petersson, g. (2011). non-adherence to drug therapy and drug acquisition costs in a national population--a patient-based register study. bmc health services res., 11:326. • jeffery, r.c. (2014). clinical features of rheumatoid arthritis. medicine (baltimore)., 42: 231–6. • jeka, s., dura, m., zuchowski, p., zwierko, b., & waszczak-jeka, m. (2018). the role of ultrasonography in the diagnostic criteria for rheumatoid arthritis and monitoring its therapeutic efficacy. adv clin exp med. • keystone, e., genovese. m., & klareskog, l. (2010). golimumab in patients with active rheumatoid arthritis despite methotrexate therapy: 52-week results of the goforward study. ann rheum dis., 69:1129–35. • keystone, e., landewe, r,, van vollenhoven, r. (2014). long-term safety and efficacy of certolizumab pegol in combination with methotrexate in the treatment of rheumatoid arthritis: 5-year results from the rapid 1 trial and open-label extension. ann rheum dis.,73:2094– 100. • kromann, c.b., lage-hansen. p.r., koefoed, m., & jemec, g.b. (2015). does switching from oral to subcutaneous administration of methotrexate influence on patient reported gastro-intestinal adverse effects? j dermatolog treat.,26(2):188– 90 . • lin, j., he, y., & chen, j. (2016). datasets of yy1 expression in rheumatoid arthritis patients. data brief,. 9: 1034-38. • mcinnes, i.b., & schett, g. (2007). cytokines in the pathogenesis of rheumatoid arthritis. nat rev immunol.,7(6):429-42. • mottonen, t., hannonsen, p., & leiralalo-repoo, m. (1999). comparison of combination therapy with single-drug therapy in early rheumatoid arthritis: a randomized trial. lancet.,353:1568–73. • müller, s., wilke, t., & fuchs, a. (2017). non-persistence and non-adherence to mtx therapy in patients with rheumatoid arthritis: a retrospective cohort study based on german ra patients. patient prefer adherence.,11:1253-1264 . • neogi, t., aletaha, d., & silman, a.j. (2010). the 2010 american college of rheumatology/european league against rheumatism classification criteria for rheumatoid arthritis: phase 2 methodological report. arthritis rheum.,62(9):2582–91. • odells, j.r. (2016). rheumatoid arthritis. in: goldman l., schafer a. goldman-cecil medicine. 25th ed. philadelphia, pa.: saunders elsevier.,264:1755. • pascual-ramos, v., contreras-yanez, i., villa, a.r., cabiedes, j., & rull-gabayet, m. (2009). medication persistence over 2 years of follow-up in a cohort of early rheumatoid arthritis patients: associated factors and relationship with disease 630 global journal of public health medicine 2022, vol 4, issue 1 gggggglo activity and with disability. arthritis res therapy.,11(1): r26. • ranganathan, p., & mcleod, h. (2006). methotrexate pharmacogenetics: the first step toward individualized therapy in rheumatoid arthritis. arthritis rheum.,54:1366– 77. • rapoff, m.a., & pediatr, a.n. (2002). assessing and enhancing adherence to medical regimens for juvenile rheumatoid arthritis., 31(6):373-9. • rutkowska-sak, l., rell-bakalarska, m., & lisowska, b. (2009). oral vs. subcutaneous low-dose methotrexate treatment in reducing gastrointestinal side effects. reumatologia.,47(4):207–11. • salt, e., & frazier, s. (2010). adherence to disease-modifying antirheumatic drugs in patients with rheumatoid arthritis: a narrative review of the literature,.29(4):26075. • silman, a.j. (2016). rheumatoid arthritis, ed 4, st. louis, 2001, mosby elsevier. • singh, j.a., saag, k.g., & bridges jr, s.l. (2016). 2015. american college of rheumatology guideline for the treatment of rheumatoid arthritis. arthritis rheumatol.,68(1):1–26. key: cord-0032373-buo6r5p9 authors: bouhuys, marleen; lexmond, willem s.; van rheenen, patrick f. title: de-escalation of anti-tumor necrosis factor alpha agents and reduction in adverse effects: a systematic review date: 2022-04-29 journal: biomedicines doi: 10.3390/biomedicines10051034 sha: eb273e573f25e4408922af348dd33c88a7e80e21 doc_id: 32373 cord_uid: buo6r5p9 background: the long-term use of anti-tnf-α agents can lead to adverse effects, such as infections and immune-mediated cutaneous reactions. whether de-escalation by dose reduction or interval lengthening reduces these adverse effects is uncertain. this systematic review aims to compare the incidence of infections and skin manifestations after anti-tnf-α dose de-escalation with standard dosing. methods: medline, embase, and the cochrane central register of controlled trials were searched from inception to 14 january 2022. randomized controlled trials (rcts) and observational studies comparing anti-tnf-α de-escalation strategies with standard dosing among patients with inflammatory conditions, that report on infections, skin manifestations, or both, were included. the risk of bias was assessed with the revised cochrane risk-of bias tool (rcts) or the newcastle–ottawa scale (non-rcts). results: fourteen rcts and six observational studies (or 2706 patients) were included. eight rcts had low risk of bias or some concerns. four non-rcts were of good methodological quality. the studies described patients with axial spondyloarthritis (8 studies, 780 patients), rheumatoid arthritis (7 studies, 1458 patients), psoriasis (3 studies, 332 patients), or inflammatory bowel disease (2 studies, 136 patients). de-escalation strategies included interval lengthening (12 studies, 1317 patients), dose reduction (6 studies, 1130 patients), or both (2 studies, 259 patients). overall, the occurrence of infections and skin manifestations did not differ between standard treatment and de-escalation. the disappearance of infections or skin manifestations after de-escalation was only reported in two studies. the majority of studies focused on etanercept and adalimumab. heterogeneity in reporting of infections and skin manifestations precluded meta-analysis. conclusion: we found that anti-tnf-α de-escalation does not reduce infections or skin reactions. a de-escalation strategy should not be recommended for the sole purpose of reducing drug-related adverse effects. the meticulous documentation of adverse effects is recommended to further address this question. registration: prospero crd42021252977. anti-tumor necrosis factor alpha (tnf-α) agents are used in a variety of inflammatory diseases, including inflammatory bowel diseases (ibd), rheumatic diseases (rheumatoid arthritis (ra) and axial spondyloarthritis), and psoriasis [1] . antibodies targeting tnf-α inactivate the proinflammatory effect by direct neutralization. as a result, the tnf-αdependent cytokine cascade is interrupted, which leads to the downregulation of inflammatory pathways. adalimumab, infliximab, certolizumab pegol, golimumab, and etanercept all target the same epitope and are successfully used in the treatment of one or more of the above-mentioned inflammatory disorders [1, 2] . because of the effectiveness of anti-tnf-α agents, patient outcomes have greatly improved, and sustained remission has become a realistic treatment target [3] [4] [5] [6] [7] . the the current systematic review was performed according to the preferred reporting items for systematic reviews and meta-analyses (prisma) statement and checklist [21, 22] . this systematic review was included in the control group of the goodreports randomized trial (grreat) [23] . the grreat team anonymously and independently assessedthe manuscript for completeness by reporting against the prisma checklist. their feedback was incorporated in the manuscript before submission for publication. the completed prisma checklist can be found in table s1 . we searched medline (through pubmed), embase, and the cochrane library from inception to 14 january 2022. the search strategies were developed in collaboration with a medical information specialist and consisted of a boolean association of keywords, combining keywords for anti-tnf-α agents and de-escalation. the search strategies for each of the electronic databases are shown in table s2 . all searches were carried out on 14 january 2022. no language restrictions were applied. in addition, we hand-searched references of relevant publications to identify any additional studies that were missed in the database searches. to be included in this systematic review, studies had to be full-text articles based on randomized controlled trials (rcts) or observational cohort or case-control studies involving patients treated with standard-dosed anti-tnf-α agents (adalimumab, infliximab, certolizumab pegol, golimumab, or etanercept), undergoing anti-tnf-α de-escalation (dose reduction or interval lengthening), and had to contain information about the rate of disappearance and/or reduction in infections and/or skin manifestations, as well as the rate of occurrence of new infections and/or skin manifestations. with the inclusion of patients, regardless of their underlying inflammatory condition, we expected to identify a sufficient number of studies to reach an acceptably high level of evidence. studies in which de-escalation resulted in discontinuation without separate information about adverse effects during the de-escalation phase were excluded. case reports, case series, and studies with a cross-sectional design were also excluded. the search results were imported into endnote (version 20, clarivate, philadelphia, pa, usa) for de-duplication [24] , and subsequently imported into rayyan, an online tool used for systematic reviews [25] . two reviewers (m.b. and p.f.v.r.) independently reviewed titles and abstracts for eligibility. in case eligibility was unclear based on the title and abstract, the record was included for full-text assessment. disagreements were solved by referral to a third reviewer (w.s.l.). next, full-text articles were screened independently by the same two reviewers. again, disagreements were solved by referral to the third reviewer. the study selection process was summarized in a flow diagram. data were extracted by one reviewer (m.b.) and confirmed by another reviewer (p.f.v.r.). the following characteristics were extracted from each selected study: the first author, the year of publication, the report title, the name of the study, the corresponding author's contact information, the country of origin, the publication type, the study design, the in-and exclusion criteria, the allocation method, anti-tnf-α dosing (standard dosing and de-escalation method), the start date, the end date, the duration of participation, the sample size, baseline imbalances, withdrawals and exclusions, the patient characteristics at baseline (age, sex, diagnosis, anti-tnf-α agent(s) used, co-treatment), incidence/prevalence and/or disappearance/reduction in infections and/or skin manifestation at baseline and at each reported time point, as well as the type and severity of the adverse reactions. missing data were requested from the study authors via email. missing and unobtainable data were presented as 'not provided' and were not included in the syntheses. the risk of bias for the outcome of interest of rcts was assessed with the revised cochrane risk-of bias tool (rob 2) in microsoft excel (version 2016, microsoft corporation, redmond, wa, usa) [26] . the risk of bias was scored by two reviewers (m.b. and p.f.v.r.) as 'low', 'some concerns', or 'high' for each domain individually and overall. these results were displayed in a figure. the risk of bias of non-rcts was assessed with the newcastle-ottawa quality assessment scale (nos), converted to the agency for healthcare research and quality standards ('good', 'fair', or 'poor'), and displayed in a table [27, 28] . the occurrence of infections and skin manifestations was presented as the proportion of patients with at least one event for the standard treatment and the de-escalation group for each study individually. 95% cis were calculated for these proportions using the wilson method. difference in these proportions were considered to be statistically significant if there was no overlap in the 95% confidence intervals of the two groups or if the p-value, if provided, was below 0.05. a relative difference of ≥25% was considered numerically different. the narrative syntheses were grouped by adverse event type. results were summarized narratively and characteristics, and main results of included studies were presented in a table. in total, 2280 articles were identified, 128 of which were retrieved for full-text review. of these, 108 were excluded as they did not report the outcome of interest ( figure 1 ). table 1 lists the characteristics of 20 studies that compared any form of anti-tnf-α dose deescalation with standard dosing [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] . of these, 14 were rcts (2197 patients; eight trials with low risk of bias or some concerns, as shown in figure 2 ) [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] . four of six non-rcts (509 patients) were of good methodological quality (table 2 ) [43] [44] [45] [46] [47] [48] . biomedicines 2022, 10, 1034 4 of 17 summarized narratively and characteristics, and main results of included studies were presented in a table. in total, 2280 articles were identified, 128 of which were retrieved for full-text review. of these, 108 were excluded as they did not report the outcome of interest ( figure 1 ). table 1 lists the characteristics of 20 studies that compared any form of anti-tnf-α dose deescalation with standard dosing [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] . of these, 14 were rcts (2197 patients; eight trials with low risk of bias or some concerns, as shown in figure 2 ) [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] . four of six non-rcts (509 patients) were of good methodological quality (table 2 ) [43] [44] [45] [46] [47] [48] . eight studies reported on axial spondyloarthritis (including ankylosing spondylitis) (780 patients) [31, 33, 35, [38] [39] [40] [42] [43] [44] [45] , seven reported on rheumatoid arthritis (1458 patients) [30, 32, 34, 36, 37, 41] , three reported on psoriasis (332 patients) [29, 39, 47] , and two reported on inflammatory bowel disease (136 patients) [46, 48] . eight studies reported on axial spondyloarthritis (including ankylosing spondylitis) (780 patients) [31, 33, 35, [38] [39] [40] [42] [43] [44] [45] , seven reported on rheumatoid arthritis (1458 patients) [30, 32, 34, 36, 37, 41] , three reported on psoriasis (332 patients) [29, 39, 47] , and two reported on inflammatory bowel disease (136 patients) [46, 48] . minor infections: injection site reactions: occurrence of new skin manifestations: oral candidiasis: ci-confidence interval; de-de-escalation; iqr-interquartile range; pys-person years; rct-randomized controlled trial; sd-standard deviation; st-standard treatment. 1 requested data. 2 the de group continued on the standard dosing interval for 4 weeks, followed by the lengthened interval for 8 weeks. and then every four weeks, was evaluated in two studies [36, 40] . the intravenous administration of infliximab, which is usually carried out with three induction doses over 6 weeks (weeks 0-2-6), followed by maintenance therapy every 8 weeks, was evaluated in two studies [38, 44] . golimumab, commonly administered subcutaneously every four weeks, was evaluated in one study [38] . in table s3 , the standard regimen and the dose de-escalation strategy for each study are described in detail. etanercept, which is usually administered subcutaneously once or twice a week, was evaluated in 14 studies [29] [30] [31] [32] [33] [34] [35] [37] [38] [39] [42] [43] [44] [45] . adalimumab, normally administered subcutaneously every other week, was evaluated in eight studies [37] [38] [39] 41, 44, [46] [47] [48] . certolizumab, which is usually administered subcutaneously every other week until remission and then every four weeks, was evaluated in two studies [36, 40] . the intravenous administration of infliximab, which is usually carried out with three induction doses over 6 weeks (weeks 0-2-6), followed by maintenance therapy every 8 weeks, was evaluated in two studies [38, 44] . golimumab, commonly administered subcutaneously every four weeks, was evaluated in one study [38] . in table s3 , the standard regimen and the dose de-escalation strategy for each study are described in detail. * scored for outcome of interest of this systematic review (i.e., the occurrence of new infections and/or skin manifestations, or the reduction and/or disappearance of infections and/or skin manifestations). seventeen of twenty articles reported on the occurrence of new infections during the study observation period [29] [30] [31] [32] [33] [34] [35] [36] [38] [39] [40] [41] [42] [43] [44] [45] 47, 49] . eleven studies reported the occurrence of any infection [32] [33] [34] 36, 38, 39, [41] [42] [43] [44] 47] . there was no unequivocal evidence in favor of either de-escalation or standard therapy. the rct by raffeiner et al., which included 323 patients with ra, reported a higher incidence rate in the standard treatment group, as compared to the de-escalated group (17.2 per 100 person years (pys) versus 10.4 per 100 pys, respectively; p < 0.001) [34] . the risk of bias of this study was high due to missing data and a lack of information about the method of data collection. two studies reported numerically fewer new infections in de-escalated patients (defined as a relative group difference ≥25%); however, this difference did not reach statistical significance. the redes-tnf trial reported an incidence of 30.6% (95% ci 20.6-43.0) in patients on standard treatment and 18.0% (95% ci 10.4-29.5 vs. 30.6%) in de-escalated patients [38] . the cohort study by závada et al. reported an incidence of 10.8% (95% ci 5. 8-19. 3) in patients on standard treatment and 7.5% (95% ci 3.0-17.9) in de-escalated patients [44] . in contrast with the former two studies, the answers trial, a small rct with a high risk of bias, reported numerically more infections in de-escalated patients (43.5%, 95% ci 25.6-63.2 vs. 33.3%, 95% ci 17.9-53.3, requested data) [33] . five other studies reported no differences in the occurrence of new infections between standard treatment and de-escalation (i.e., relative difference <25%) [36, 39, [41] [42] [43] 47] . among them was one rct with a low risk of bias (c-early-2). in this study, a lengthened certolizumab pegol interval (200 mg every 4 weeks, n = 127) was compared to standard treatment (200 mg every 2 weeks, n = 83) in patients with ra and sustained low disease activity. after 52 weeks of follow-up, 38.6% (95% ci 30.6-47.3) of the patients in the deescalated group had at least one infection compared to 31.3% (95% ci 22.4-41.9) in the standard treatment group [36] . eight studies reported the occurrence of serious or severe infections [30, 32, [34] [35] [36] 41, 42, 45] ; however, a clear definition was only given in two [30, 34] . in six of these studies, no or few patients developed a serious infection, and there was no difference in the incidence rates between patients on standard treatment and de-escalated patients [32, 35, 36, 41, 42, 45] . the preserve study found higher incidences of treatment-emergent serious infections in the standard treatment group compared to the de-escalation group (1.5%, 95% ci 0.5-4.3 vs. 0.0%, 95% ci 0.0-1.9); however, this was not statistically significant [30] . on the contrary, the rct by raffeiner et al. found more severe infections in the de-escalation group (incidence rate 0.67 vs. 0.23 per 100 person years); however, this was also not significant [34] . five studies reported on specific infections, including upper respiratory tract infections, flu, urinary tract infections, oral candidiasis, and tuberculosis [29] [30] [31] 40, 42, 45] . none of the studies found significant differences between anti-tnf-α standard treatment and deescalation. neither was a difference in the type of infections reported between standard treatment and de-escalation. most infections were mild and opportunistic infections were uncommon [29] [30] [31] [32] 38, 39, 43, 45, 47, 49] . only one article included information about the disappearance of infections after anti-tnf-α de-escalation. in this retrospective case-control study by van steenbergen et al., frequent infectious symptoms disappeared in five out of seven (71.4%, 95% ci 26.2-69.0) ibd patients that underwent adalimumab interval lengthening from 40 mg every other week to every three weeks. in the standard treatment group, a disappearance occurred in none of the patients with frequent infectious symptoms at baseline (n = 5) [48] . eleven articles included information about the occurrence of new skin manifestations after anti-tnf-α de-escalation [29] [30] [31] 33, [35] [36] [37] 39, 43, 45, 47] . no study described a difference in the type of skin manifestations between standard treatment and de-escalation. five studies reported on the occurrence of any skin manifestation [33, 36, 37, 39, 47] . the c-early-2 trial, an rct with a low risk of bias (c-early-2), found numerically higher incidences of skin manifestations in the standard treatment group compared to the de-escalation group (9.6%, 95% ci 5.0-17.9 vs. 7.1%, 95% ci 3.8-12.9); however, this difference was not statistically significant [36] . the condor study, a somewhat smaller rct, showed a similar trend with more skin manifestations in the standard treatment group (7.3%, 95% ci 2.5-19.4 vs. 5.1%, 95% ci 1.4-16.9) (requested data) [39] . during the openlabel extension of this rct, none of the patients with a lengthened interval had a new skin manifestation, compared to 12.5% in the standard treatment group (requested data) [47] . in the answers trial, patients that underwent an etanercept dose reduction experienced more new skin manifestations than patients on standard treatment; however, this was not statistically significant, and the study had a high risk of bias [33] . in the opttira study that only reported the number of events instead of the number of incidences, a skin manifestation occurred 13 times in the standard treatment group (n = 19, no serious events) and 24 times in the de-escalated patient group (n = 44, two serious events) [37] . injection site reactions were reported in six studies [29, 31, 33, 35, 43, 45] . four studies looked at the incidence of etanercept-related injection site reactions with and without a dose reduction [29, 33, 43, 45] . two of them reported significantly fewer injection site reactions in de-escalated patients. in the cohort study by park et al., the incidence rate ratio was significantly smaller in de-escalated patients compared to patients on standard treatment (0.327, 95% ci 0.134-0.801, p = 0.014) [43] . the rct by papp et al. reported injection site reactions in 18.0% (95% ci 13.3-24.1) of the patients on standard treatment vs. 3.7% (95% ci 1.8-7.4) of the de-escalated patients [29] . however, we considered its 12-week follow-up (only 8 weeks after de-escalation) to be insufficient to draw meaningful conclusions. two studies reported numerically (but not significantly) fewer injection site reactions in de-escalated patients [31, 45] and one study found no differences [33] . surprisingly, the rct by li et al. reported more injection site reactions in patients that underwent interval lengthening (11.5% vs. 0.0%, not significant); however, in this study, follow-up after de-escalation also lasted for only 8 weeks [35] . two articles included information about the disappearance of skin manifestations after anti-tnf-α de-escalation. in the previously mentioned case-control study, de-escalation resulted in the disappearance of skin manifestations in 47.1% of the patients with skin manifestations at baseline. the disappearance of psoriasiform lesions was most common (two out of four, 50.0%), followed by xerosis cutis (three out of seven, 42.8%). no skin manifestations disappeared in the standard treatment group [48] . in a retrospective cohort study by pouillon et al., skin manifestations linked to anti-tnf-α therapy disappeared in four out of seven patients (57.1%, 95% ci 25.1-84.2) after adalimumab interval lengthening [46] . however, based on the newcastle-ottawa scale, this study was of poor methodological quality, mainly because of the absence of a control group. in this systematic review, we summarize results from 20 anti-tnf-α de-escalation studies (14 rcts and 6 non-rcts) on the reduction in infections and skin manifestation. we considered two de-escalation strategies (dose reduction and interval lengthening) and included all underlying inflammatory conditions and all currently available anti-tnf-α agents. after synthesizing the data, we conclude that anti-tnf-α de-escalation in patients treated according to the label does not reduce the occurrence of infections and skin manifestation compared to patients who continued standard dosing. however, the quality of evidence is low. it is unclear whether anti-tnf-α de-escalation improves existing infections and/or skin abnormalities. there are multiple reasons why both patients and healthcare professionals may wish to de-escalate anti-tnf-α therapy once remission is achieved. these reasons include a reduction in the number of hospital visits, the number of needle pricks, and costs. a reduction in anti-tnf-α-associated adverse effects is also often mentioned as a reason; however, we cannot confirm the validity of this approach. we suggest not to de-escalate standard-dosed anti-tnf-α medication solely for this reason. this advice does not apply to patients treated with a shorter dosing interval, a higher dose than standard, or both. our findings are consistent with a meta-analysis performed by vinson et al. they evaluated the incidence of serious infections and adverse events of specific interest in patients with ra or axial spondyloarthritis. thirteen studies were included in the metaanalysis, seven of which were also included in our systematic review. the de-escalation of the biological disease-modifying anti-rheumatic drug (predominantly anti-tnf-α agents) the or the jak inhibitor was not different from continuation of the initial regimen with respect to the incidence of serious infections (risk difference 0.01, 95% ci −0.00-0.02, p = 0.13, i 2 = 0%). in contrast to our systematic review, vinson et al. did not study dermatological adverse effects or non-serious infections [50] . likewise, a cochrane systematic review on the down-titration and discontinuation of anti-tnf-α agents in patients with ra also did not report on the occurrence of infections or skin manifestations. based on five studies, four of which are also included in our systematic review, the authors concluded that de-escalation has little to no effect on serious adverse effects; however, the evidence was also very uncertain [16] . although eight rcts and four non-rcts were of good overall methodological quality in our systematic review, measuring the outcome of interest was problematic. none of the studies defined adverse effects as their primary outcome. instead, data about infections or skin manifestations were at best presented as part of the obligatory reporting of adverse events. as a consequence, most studies were not powered to detect potential differences in the occurrence of adverse events. additionally, clear descriptions of the definitions and methods of measurement were lacking. in most studies, the occurrence of new infections was expressed as incidences. this may have resulted in an underestimation of the effect of anti-tnf-α de-escalation on adverse effects. for instance, if a patient in the standard treatment group had 10 infections during follow-up and another patient in the de-escalation group had only 1, different incidences cannot be attained, whereas the use of event rates can provide a more reliable estimate. we only included publications that reported on infections, skin manifestations, or both. because of the obligatory reporting of adverse events, it is likely that these data were also available for de-escalation studies that did not provide this information in their publication. in fact, 106 studies were excluded during the full-text selection because they contained no or insufficient information about the adverse events of interest. this may have caused selection bias; however, obtaining these missing data from such a large number of publications is not feasible. we included patients regardless of their underlying inflammatory condition and synthesized the data as if they were from one group. we anticipated to pool the data and perform subgroup analyses for each diagnosis and anti-tnf-α agent; however, due to limited data and heterogeneity, no valid results could be generated. further research is necessary for better quality data on the possible beneficial effect of anti-tnf-α de-escalation on anti-tnf-α-associated adverse effects. this is of particular importance for patients with ibd and psoriasis and the anti-tnf-α infliximab, certolizumab, and golimumab agents, which were underrepresented in the current review. to better address the question whether de-escalation reduces adverse effects, future studies should scrupulously register the adverse effects of interest, by sending out questionnaires specifically designed for this purpose, for instance [51] . another method to reduce exposure to anti-tnf-α agents and its associated adverse effects is the administration of this drug in cycles, with anti-tnf-α free periods in between. this concept is currently being investigated [52] . in conclusion, anti-tnf-α de-escalation does not seem to reduce infections or skin manifestations in patients on standard-dosed anti-tnf-α treatment; however, the available evidence is of low quality. we recommend against anti-tnf-α de-escalation for the sole purpose of putting an end to these adverse effects. adequately powered studies with meticulous documentation of adverse effects are likely to increase the certainty of the evidence. supplementary materials: the following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10051034/s1, table s1 : prisma 2020 checklist. table s2 : search strategies. table s3 : detailed description of standard treatment and de-escalation method used in the included studies. biology of anti-tnf agents in immune-mediated inflammatory diseases: therapeutic implications molecular mechanism of action of anti-tumor necrosis factor antibodies in inflammatory bowel diseases long-term efficacy of anti-tumor necrosis factor agents in pediatric luminal crohn's disease: a systematic review of real-world evidence studies vedolizumab and anti-tnfα real-world outcomes in biologic-naïve inflammatory bowel disease patients: results from the evolve study treating rheumatoid arthritis to target: recommendations of an international task force treating spondyloarthritis, including ankylosing spondylitis and psoriatic arthritis, to target: recommendations of an international task force sustained long-term efficacy and safety of adalimumab in paediatric patients with severe chronic plaque psoriasis from a randomized, double-blind, phase iii study systematic review and meta-analysis of dermatological reactions in patients with inflammatory bowel disease treated with anti-tumour necrosis factor therapy dermatological adverse reactions during anti-tnf treatments: focus on inflammatory bowel disease eczema as an adverse effect of anti-tnfalpha therapy in psoriasis and other th1-mediated diseases: a review review article: managing the adverse events caused by anti-tnf therapy in inflammatory bowel disease cutaneous side-effects in patients with rheumatic diseases during application of tumour necrosis factor-alpha antagonists risk of infections using anti-tnf agents in rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis: a systematic review and meta-analysis risk for overall infection with anti-tnf and anti-integrin agents used in ibd: a systematic review and meta-analysis difference in the risk of serious infections in patients with rheumatoid arthritis treated with adalimumab, infliximab and etanercept: results from the dutch rheumatoid arthritis monitoring (dream) registry down-titration and discontinuation strategies of tumour necrosis factor-blocking agents for rheumatoid arthritis in patients with low disease activity efficacy and safety of down-titration versus continuation strategies of biological disease-modifying anti-rheumatic drugs in patients with rheumatoid arthritis with low disease activity or in remission: a systematic review and meta-analysis flare rate in patients with rheumatoid arthritis in low disease activity or remission when tapering or stopping synthetic or biologic dmard: a systematic review dose adjustments and discontinuation in tnf inhibitors treated patients: when and how. a systematic review of literature reduction of biologics in rheumatoid arthritis: a systematic review and meta-analysis the prisma 2020 statement: an updated guideline for reporting systematic reviews prisma 2020 explanation and elaboration: updated guidance and exemplars for reporting systematic reviews does providing authors with customized article templates including items from reporting guidelines improve completeness of reporting? a randomized trial de-duplication of database search results for systematic reviews in endnote rayyan-a web and mobile app for systematic reviews rob 2: a revised tool for assessing risk of bias in randomised trials the newcastle-ottawa scale (nos) for assessing the quality of nonrandomised studies in meta-analyses extracorporeal membrane oxygenation (ecmo) in patients with severe covid-19 adult respiratory distress syndrome: a systematic review and meta-analysis. cardiothorac van de kerkhof, p.; the etanercept psoriasis study group. a global phase iii randomized controlled trial of etanercept in psoriasis: safety, efficacy, and effect of dose reduction maintenance, reduction, or withdrawal of etanercept after treatment with etanercept and methotrexate in patients with moderate rheumatoid arthritis (preserve): a randomised controlled trial duration of remission after halving of the etanercept dose in patients with ankylosing spondylitis: a randomized, prospective, long-term, follow-up study sustained remission with etanercept tapering in early rheumatoid arthritis is etanercept 25 mg once weekly as effective as 50 mg at maintaining response in patients with ankylosing spondylitis? a randomized control trial effects of half dose etanercept (25 mg once a week) on clinical remission and radiographic progression in patients with rheumatoid arthritis in clinical remission achieved with standard dose dose reduction of recombinant human tumor necrosis factor inhibitors (etanercept) can be effective in ankylosing spondylitis patients with synovitis of the hip in a chinese population a phase iii study evaluating continuation, tapering, and withdrawal of certolizumab pegol after one year of therapy in patients with early rheumatoid arthritis. arthritis rheumatol optimizing treatment with tumour necrosis factor inhibitors in rheumatoid arthritis-a proof of principle and exploratory trial: is dose tapering practical in good responders? the redes-tnf investigators non-inferiority of dose reduction versus standard dosing of tnf-inhibitors in axial spondyloarthritis comparison of tightly controlled dose reduction of biologics with usual care for patients with psoriasis: a randomized clinical trial maintenance of clinical remission in early axial spondyloarthritis following certolizumab pegol dose reduction adalimumab dose tapering in patients with rheumatoid arthritis who are in long-standing clinical remission: results of the phase iv predictra study tapering of etanercept is feasible in patients with rheumatoid arthritis in sustained remission: a pragmatic randomized controlled trial low dose etanercept treatment for maintenance of clinical remission in ankylosing spondylitis a tailored approach to reduce dose of anti-tnf drugs may be equally effective, but substantially less costly than standard dosing in patients with ankylosing spondylitis over 1 year: a propensity score-matched cohort study full dose, half dose, or discontinuation of etanercept biosimilar in early axial spondyloarthritis patients: a real-world study in china dose de-escalation to adalimumab 40mg every three weeks in patients with inflammatory bowel disease-a multicenter, retrospective, observational study. dig. liver dis two-year follow-up of a dose reduction strategy trial of biologics adalimumab, etanercept, and ustekinumab in psoriasis patients in daily practice dose de-escalation to adalimumab 40 mg every 3 weeks in patients with crohn's disease-a nested case-control study step-down strategy of spacing tnf-blocker injections for established rheumatoid arthritis in remission: results of the multicentre non-inferiority randomised open-label controlled trial (strass: spacing of tnf-blocker injections in rheumatoid arthritis study) impact of tapering targeted therapies (bdmards or jakis) on the risk of serious infections and adverse events of special interest in patients with rheumatoid arthritis or spondyloarthritis: a systematic analysis of the literature and meta-analysis efficacy of anti-tnf dosing interval lengthening in adolescents and young adults with inflammatory bowel disease in sustained remission (free-study): protocol for a partially randomised patient preference trial needs-driven, safer and cost-effective management of crohn's disease we thank sjoukje van der werf (information specialist, central medical library, university medical centre groningen) for her assistance in constructing the search strategy. we thank selma atalay, delphine bertrand, max yates, and karl gaffney for providing additional data. the authors declare no conflict of interest.biomedicines 2022, 10, 1034 key: cord-0042147-r04y1j22 authors: hedegaard, c. j.; bendtzen, k.; nielsen, c. h. title: the role of immune complexes consisting of myelin basic protein (mbp), anti‐mbp antibodies and complement in promoting cd4(+) t‐cell responses to mbp in health and multiple sclerosis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423k.x sha: ea3b1c4721836f94e19de86c26c6dbeb00153b5c doc_id: 42147 cord_uid: r04y1j22 multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti‐mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen‐presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp‐derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease‐associated anti‐mbp antibodies in ms patients, respectively. we have investigated the formation of mbp‐containing ic, the binding of mbp to b cells, the mbp‐elicited induction of th‐cell and b‐cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c‐inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4(+) t cells, we observed the production of tnf‐α, ifn‐γ and il‐10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il‐2, il‐4 and il‐5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp‐containing ic and thereby enhance the cytokine responses, by virtue of elevated anti‐mbp contents. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-0065962-lg0lsfbz authors: dessie, gashaw; ayelign, birhanu; akalu, yonas; shibabaw, tewodros; molla, meseret derbew title: effect of leptin on chronic inflammatory disorders: insights to therapeutic target to prevent further cardiovascular complication date: 2021-07-17 journal: diabetes metab syndr obes doi: 10.2147/dmso.s321311 sha: 0e5500da6e8dd226c95eabd4a5f28a07a92a8676 doc_id: 65962 cord_uid: lg0lsfbz in response to obesity-associated chronic inflammatory disorders, adipose tissue releases a biologically active peptide known as leptin. leptin activates the secretion of chemical mediators, which contribute to the pathogenesis of chronic inflammatory disorders, such as rheumatoid arthritis (ra), systemic lupus erythematosus (sle) and psoriasis. conversely, adiposity and obesity are the major aggravating risk factors in the pathogenesis of metabolic syndrome (mets), including type ii diabetes mellitus and obesity-associated hypertension. elevated level of leptin in obesity-associated hypertension causes an increase in the production of aldosterone, which also results in elevation of arterial blood pressure. hyperleptinemia is associated with the progress of the atherosclerosis through secretion of pro-inflammatory cytokines, like interleukin 6 (il-6), tumor necrosis factor α (tnf-α), il-17, and other cytokines to promote inflammation. the release of those cytokines leads to chronic inflammatory disorders and obesity-associated mets. thus, the aberrant leptin level in both mets and chronic inflammatory disorders also leads to the complication of cardiovascular diseases (cvd). therapeutic target of leptin regarding its pro-inflammatory effect and dysregulated sympathetic nervous system activity may prevent further cardiovascular complication. this review mainly assesses the mechanism of leptin on the pathogenesis and further cardiovascular risk complication of chronic inflammatory disorders. adipose tissue is the major organ to produce and release leptin. 1 leptin was discovered on animal models by friedman in 1994. 2 at the time of discovery, treatment of obesity was hopeful on this active adipokine molecule. however, hyperleptinemia was seen in obese individuals after it was found. 3 two years after its discovery, low energy signal transmission effect of the central nervous system was identified as its first physiological function. 4 neurons found in the ventral tegmental area of midbrain in the hypothalamus express leptin receptors. 5 energy homeostasis in the peripheral nervous system is regulated by highly complex interaction of neurons and leptin. 6 it can be maintained through interaction of leptin signaling pathways with neuropeptide y neurons. 7 leptin regulates the satiety, energy expenditure, inflammation, endothelial cells function, blood pressure, and insulin secretion. 8 it is proportional to the mass of adipose tissue. 9 impairment of adipose tissue results in the release of effector adipokines, including leptin and resistin 10 (figure 1 ). the signaling pathways of leptin can be impaired due to an increase in body mass. 11 thus, fat storage and energy homeostasis in adipose tissue could be impaired due to the effect of obesity-associated inflammation. 10 fatel et al 12 in 2018 mentioned leptin as a pro-inflammatory adipokine because it induces the activation and secretion of the pro-inflammatory cytokines, including tumor necrosis factor-α (tnf-α), lipopolysaccharide, and interleukin (il-1). in contrast, the inflammatory cytokines induce the generation and secretion of leptin to promote chronic inflammation. 12 the pro-inflammatory stimuli, il-1, tnf-α, and lipopolysaccharide lead to upregulation of expression of leptin messenger rna (mrna) to aggravate inflammation. 13 chronic inflammation may provoke leptin resistance through interruption of its receptor signaling cascade, which in turn leads to hyperleptinemia and obesity. 14 leptin is involved in the pathogenesis of various obesity-related inflammatory disorders, such as psoriasis, systemic sclerosis, diabetes, and hypertension. 15 psoriasis is an inflammatory skin disease, which is mainly associated with pro-inflammatory cytokines, including il-6, interleukin 17 (il-17), interferon-gamma (ifn-γ), and tnf-α. 16 obesity is an attributable risk factor for the complication of psoriasis. 17 the autoimmune inflammatory disorders, including systemic lupus erythematosus (sle) induce the release of leptin to modulate the immune system 18 that in turn leads to chronic inflammation. 19 although there are contradictory data, the majority of studies mentioned elevated level of leptin in sle patients. 20 rheumatoid arthritis (ra) is another chronic autoimmune inflammatory disorder, which leads to the synthesis both of leptin and cytokines. 21 leptin also induces the activation of macrophage, regulatory t-cells and th17 cells to release proinflammatory cytokines like interleukin 6 (il-6), tnf-α, , and other cytokines to promote inflammation. 22 the binding of leptin to its receptors induces an increase in arterial blood pressure 23 because obesity-related leptin for progression of atherosclerosis in t2dm. the synthesis of leptin is associated with atherogenic effect because leptin receptors are found on endothelial cells. thus, its elevation in obese individuals causes endothelial dysfunction. in this regard, obesity and secretion of leptin is a characteristic feature of t2dm. il-6 secreted from endothelial cells activate janus kinase/activators of transcription (jaks) after it binds to the cytoplasmic domain of gp130 within macrophage, followed by the phosphorylation of transmembrane tyrosine receptor motifs, such as tyr905, tyr814, tyr767, tyr705 and tyr915. this leads to increase expression of pro-inflammatory genes through stat3, which aggravates inflammation and atherosclerosis in t2dm. abbreviations: cvd, cardiovascular disease; il-6, interleukin-6; jak/stat, janus kinase/ activators of transcription; stat3, activators of transcription-3; t2dm, type ii diabetes mellitus. inflammation causes an impairment in leptin sympathetic activity that control the renin-angiotensin system. 23 the sensitizing effect of leptin on upregulation of the reninangiotensin system increases the risk of hypertension in obese individuals. 23 in addition to maintaining body weight, leptin is also involved in carbohydrate metabolism 24 and strengthening sensitivity of insulin in type i diabetes mellitus. 25 the expression of leptin receptor isoform, lep-rb in the peripheral tissue enhances the pathogenesis of various diseases, including immune dysregulation and type ii diabetes. 26 recently, leptin has gained the insight of the scientific community due to its association with obesity, cardiovascular risk, and insulin resistance. 27 evidence has suggested that obesity aggravates the development of chronic inflammation, which in turn leads to metabolic syndrome (mets). 28 characteristic features of mets, such as hypertension, atherosclerosis, insulin resistance and obesity are correlated with elevated levels of leptin. 29 studies revealed that obese individuals with hyperleptinemia are more likely to develop insulin resistance, type ii diabetes mellitus, degenerative disease and cardiovascular complications. 13, 30 studies revealed that insulin sensitivity became decreased in pre-diabetes because of a higher level of leptin. 31 the development of these chronic diseases are associated with obesityassociated complications. 32 in contrast, hypertension is one of the obesity-associated mets, which is caused by an abnormal secretion of leptin. 33 increased levels of il-6, tnf-α and leptin results in dysfunctional epithelial cells, proliferation of smooth muscle cells, and migration of macrophages toward the damaged endothelial cells, which also leads to the development of the cardiovascular risk factors, such as atherosclerosis and hypertension. 34 even if contradictory data has been shown in vascular diseases, 35 hyperleptinemia is the adverse effect of cardiovascular complications such as stroke, heart failure, and acute myocardial infarction. 36 although scientific evidence had argument on leptin effect in coronary artery disease, it has a correlation with intimamedia thickness and calcification of coronary artery among type ii diabetes mellitus patients. 37 coronary artery disease (cad) is associated with increased synthesis of the perivascular adipose tissue derived leptin and its atherogenic effect. consequently, the therapeutic target of this active adipokine molecule is recommended to treat obesity-associated cardiovascular complications. 38 hyperleptinemia is associated with the progress of atherosclerosis. thus, the therapeutic target of leptin may decrease the complication of cardiovascular diseases. 39 generally, hyperleptinemia acts as a major risk factor for the complication of cardiovascular disease (cvd) in addition to other traditional risk factors 40 (figure 1 ). this review article evaluates the obesity-associated chronic inflammatory diseases and correlates the effect of these disorders with cardiovascular complications. it focuses on the pro-inflammatory effect of leptin in various chronic inflammatory disorders and obesity-associated mets. according to a clinical and epidemiological global study in 2010, the prevalence of rheumatoid arthritis (ra) was estimated to be 0.24% and continues without change from 1990 to 2010. 41 it is characterized by a highly systemic inflammation, which leads to reduced life expectancy and increased mortality rate. 42 obesity-associated inflammation increases the burden of ra. 43 evidence argues about unknown plasma levels of leptin and its undefined effect in ra patients compared to healthy controls. 44 however, as early as 2006, a marked elevation of plasma leptin level was detected in ra patients. 45 recently, in 2018, de souza fatel et al 46 tried to confirm the association of ra and leptin. researchers have various insights about leptin and disease activity in ra. 47 studies showed that higher disease activity of ra is associated with hyperleptinemia. 48 in patients with ra undergoing anti-tnf therapy due to disease severity, there was a strong positive correlation between body mass index of the patient and serum levels of leptin. 49 a recent 12-month multicenter study done in 2020 revealed that the ratio of leptin/fat was elevated in ra patients treated with tocilizumab. 50 however, the alteration of serum leptin level was not evaluated in their study. it was also the case for ra patients undergoing intravenous therapy with the anti-il-6 receptortocilizumab. 51 moreover, a significant reduction of leptin levels was observed following one single intravenous infusion of the anti-il-6 receptor tocilizumab. 51 however, there was no a statistically significant differences in allele frequencies of leptin gene polymorphisms (lep rs2167270) between ra patients and controls. 52 leptin activated macrophage induces the release of il-6 and tnf-α 55 ( figure 2 ). in vitro, the chemotactic activity of macrophage is associated with induction of leptin. 55 in autoimmune diseases, deregulated immune response of cells are affected by alteration of metabolic process within these cells 55 because leptin binds to its long isoform receptor (ob-rb) to induce its biological and physiological effect through jak/stat signaling pathway. jak/ stat signal transduction is caused by the involvement of janus kinase 2 (jak2), activators of transcription (stat) and transducers found on longer receptor isoform (ob-rb). 13 in addition to leptin, this signaling pathway requires the interaction between complex molecules, including node-like receptor pyrin domain-containing protein 3 (nlrp3), micro rna-98 (mir-98), caveolin-1 (cav-1-nr2b) and il-33. 56 jaks are a group of tyrosine kinases, which bind to type i and ii tyrosine receptor family, 57 whereas stats are factors that are phosphorylated by cytokines activated tyrosine motifs. 58 leptinreceptor interaction begins after leptin binds to ob-rb, extracellular domain, later jak2 tyrosine kinase become activated. 59 this in turn causes auto-phosphorylation of jak2 and intracellular tyr1138, tyr1077, and tyr985 motifs. 13 physically associated receptor-jak complex activate phosphorylation of stats, including stat1, stat2, stat3, and other transcriptional signaling molecules. 60 in addition, phosphorylated tyr1138 induces the phosphorylation of stat3, which is exported to the nucleus for targeted gene transcriptional process. 59 this signaling cascade within macrophage induces synthesis of pro-inflammatory cytokines, including il-6 and tnf-α 61 ( figure 2 ). then, il-6 and tnf-α are released from macrophage to promote inflammation and contribute to the pathogenesis of ra. 62 although effective anti-arthritis therapies are designed from these first-class adipokines, drugs from leptin and other adipokines may also be formulated. 63 chronic inflammation resulted from jak/ stat signaling may be altered with a therapeutic drug (jakinibs) in rheumatoid arthritis patients. 56 in addition, leptin induces macrophage chemotaxis, synthesis and cumulative effect of leptin on both chronic inflammatory disorders and further cardiovascular complication. the homeostatic role of adipose tissue impaired due to obesity-associated inflammation. the impairment of adipose tissue results in the release of effector adipokines, including leptin. the adipokines secreted by adipose tissue involve in obesity related inflammatory disorders, such as psoriasis, sle and ra by activating of secretion of various cytokines. conversely, obesity induced inflammation causes an impairment in sympathetic activity that controls the renin-angiotensin system through secretion of aldosterone, which results in elevation in water and salt retention and leads to obesity-associated hypertension. the reduced physiological activity of leptin on adipose tissue to oxidize stored fat leads to a phenomenon known as leptin resistance. in this regard, leptin resistance occurs in diabetic and obese subjects. adiposity and obesity are risk factors for the occurrence of metabolic syndrome, such as t2dm and hypertension. in addition to this, ra, psoriasis and sle are developed due to adiposity and obesity, which in turn leads to cardiovascular complications. release of pro-inflammatory cytokine, il-12. 61 similarly, leptin activated macrophage induces synthesis of il-18 to mediate t-helper 1 immune response. 64 secreted il-18 and il-12 enable differentiation of th1 phenotype from t-helper cells and activate synthesis of interferon-γ (ifnγ) and il-2. converely, th1 cells synthesize leptin and induce the release of il-18, il-12, il-6 and tnf-α by stimulating macrophage. 65 the expression of glycoprotein (cd38), adhesion molecules, cd25, and cd69 also increased within activated macrophage. 61 in contrast, leptin contributes to the progression of ra by downregulating regulatory t-cell activity (cd4 + cd25 high ) and upregulating the activity of t-helper 1 (t h 1) cells. 66 diminished level of treg cells seen in an opposite effect to the level of leptin and bmi in obese individuals. 55 the experiment done in leptin-deficient mice showed the anti-inflammatory potential of leptin by inhibiting inflammatory agents. however, it elevates systemic inflammation in ra through t h 1-mediated immune response. 67 t-helper 1(t h 1) cells promote inflammation in different autoimmune disorders, including ra. 68 the elevated ratio of both regulatory t-cell and th17 cells (th17/treg) and th1/th2 promote the pathogenesis of ra 69 ( figure 2 ). even if there is no well-defined association among adipokines and ra, leptin is one metabolic risk factor. 70 in addition, elevation in leptin/adiponectin ratio and positive association of leptin with homeostasis model assessment of insulin resistance (homa-ir) indicates magnitude of atherosclerosis development and plaque formation. 70 therefore, leptin plays a significant role in the pathogenesis of ra and risk of cardiovascular complication. systemic lupus erythematosus (sle) is chronic autoimmune inflammatory disease characterized by inflammation of connective tissue. 71 versini et al 72 et al, in 2017, mentioned the availability of inadequate data regarding the association between obesity and sle through their crosssectional study. although studies argue regarding the association of leptin with sle, an increase in serum leptin concentration may elevate systemic inflammation in sle. 73 even though contradictory data are available, findings showed that the level of leptin increases in sle. 27, 66, 73 in contrast to this, the other meta-analysis done on leptin and sle revealed that diminished level of leptin is shown in sle. 74 alternatively, studies done in egypt in 2018 confirmed the presence of higher level of serum leptin among sle patients. 19 elevated level of leptin is correlated with mets and obesity, which act as an exposure risk factors for chronic autoimmune diseases, including sle. 74 in the pathogenesis of sle, the immune system is affected by leptin, which serves as a pro-inflammatory cytokine. 75 in this regard, macrophages synthesize tnf-α due to the stimulatory effect of leptin. 76 therefore, sle patients with cvd show an elevated level of il-1 and tnf-α. 77 at the onset of inflammation, leptin increases the synthesis of inflammatory mediators, including il-6 and tnf-α. 78 leptin contributes to the pathogenesis of sle by activating the synthesis of auto-antibody production and dysregulation of the immune system. 79 although the pathogenesis of sle is still undefined, the circulating auto-antibodies activate secretion of inflammatory cytokines, which in turn contribute to the progress of sle. 80 beyond leptin's significant enrollment, both auto-antibodies and cd4 + t-cells play another significant role in the pathogenesis of sle. 18 the abnormal activation of cd4 + t-cells and mediated inflammatory responses are seen in sle patients. 80 immune dysregulation associated with leptin is due to an increase in differentiation of th17 cells, 18 which in turn lead to tissue damage in autoimmune disorders, including sle. 79 the effector cell, th17 induces inflammation by the activating secretion of il-17 which also enhances tissue damage and inflammation in psoriasis, sle, and ra 81 ( figure 2 ). psoriasis is a chronic inflammatory disease in joints, nails and skin due to immune modulation, environmental, and genetic variation. 82 the world health organization describes it as a global health burden, and the prevalence of disease is expected to be 2% in western countries. 83 the complication of psoriasis is related to the effects both of mets and obesity. 82 profumo et al 84 in 2012 mentioned the development of obesity in psoriatic patients. obesity has an association with inflammatory skin cells by modulating their activity. 85 it also induces chronic inflammation by activating the secretion of cytokines and leptin. 86 the release of adipokines contribute to a chronic cutaneous inflammation in psoriasis. 87 leptin plays its own role in the promotion of psoriasis through secretion of proinflammatory mediators, which are recruited to cutaneous lesions. 88 although cutaneous psoriasis patients at moderateto-severe stages of the disease treated with anti-tnf biologics, leptin correlated with mets features and inflammation. in this regard, in these patients with moderate-to-severe psoriasis, leptin concentration is correlated with c-reactive protein and with systolic and diastolic blood pressure before the onset of the anti-tnf-adalimumab therapy. 89 a negative correlation with insulin sensitivity was also found. 89 although clarification on anti-inflammatory and pro-inflammatory effect of psoriasis is challenging, the expression of anti-inflammatory cytokines resolve th2 and th1/th17 unbalanced proportion in psoriasis. 16 however, studies done in mice showed that leptin induces and activates the differentiation of t-lymphocytes into t-helper-1 lymphocytes (th1 lymphocytes) to release proinflammatory cytokines, including tnf-α, il-6 and il-8 90 ( figure 2 ). researchers have confirmed that leptin increases the genetic expression of il-6 both in humans and rats. 91 regard to psoriasis pathogenesis, th1-cells are responsible for the synthesis of tnf-α, ifn-γ and il-2. 16 similarly, th17 cell becomes differentiated due to stimulatory effect of il-6 and this cell also induces the release of il-17, il-6, il-22 and il-21 16 (figure 2) . the systemic inflammation is due to a network of cytokines activation such as il-2, tnf-α, ifn-γ, il-6, il-17, il-21, il-22 and others. 16 the secreted cytokines cascade promote the development of accelerated atherosclerosis in psoriasis patients. 89 the il-17 deficient mice showed diminished level atherosclerotic plaque formation, hence il-17 lymphocytes may be involved in atherosclerosis development. 83 from a common perspective, inflammation mediated atherosclerosis has a common process similar to cvd. 83 similarly, leptin induces the secretion of pro-inflammatory cytokines like tnf-α and il-6 from macrophages and inhibits secretion of th2 cytokines. 92 in this context, the pro-inflammatory effect of tnf-α has been activated, 89 whereas the anti-inflammatory effect of th2 cytokines will be downregulated. 92 studies showed that th2-produced il-10 may assist psoriasis therapy because it diminishes synthesis of chemokines and pro-inflammatory cytokines from macrophage. 93 inflammation enhances pathophysiology of psoriasis, which in turn leads to the risk of cardiovascular complications. 83 inflammatory bowel disease (ibd) is a chronic inflammation of the digestive tract that includes ulcerative colitis 88 and crohn's disease. 76, 94 the incidence of ibd is higher in developed countries, but its prevalence is lower in developing countries compared to the former one. 94 intestinal microbiome difference, smoking habits, lifestyle modification, and a variation on dietary content are some of the factors, which contribute to variation in the prevalence of ibd. genetic, environmental factor, abnormality in immune response, and intestinal microbiome contribute to the development of ibd. 94 studies describe ibd as an inflammatory disorder, which is characterized by an increase in the level of pro-inflammatory cytokines, including, tnf-α, il-6, and il-1 94 (figure 2) . researchers have revealed that the pathogenesis of ibd is associated with activation of nuclear factor-κb (nf-κb), which enhances the genetic expression of pro-inflammatory cytokines. 95 binding of leptin to its receptors found on immune cells of lamina propria and small intestine enterocytes induce nf-κb activation, 96 which in turn leads to villi cell apoptosis phenomena within intestinal mucosa. migration of macrophage toward dead cells enhance and release pro-inflammatory cytokines, including il-6, il-1, and il-12. 97 neutrophils migrate toward the inflamed intestine 98 and synthesize or secrete leptin, but its proportion is not comparable to adipose tissue. 99 the cytokines secreted from macrophage are due to the stimulatory effect of leptin, especially in patients with crohn's disease (cd). 96 in contrast, proinflammatory cytokines, including tnf-α, il-6, and il-1 induce an elevated activity of leptin in inflamed tissue. 100 thus, neutrophils play a vital role in the pathogenesis of ibd by mediating intestinal inflammation. 98 during intestinal microbiome disturbance, adipocytes also involve in innate immune response through generation of leptin. 101 in addition, characteristic systemic inflammation shown in ibd is associated with elevated cd4 + cells polarization to th1 cells, which in turn lead to release of pro-inflammatory cytokines 97 (figure 2 ). in the us, 34% of the population are challenged with complication of type ii diabetes mellitus (t2dm). 102 this is because both obesity and t2dm are highly associated with each other. 102 obesity is the major risk factor for the pathogenesis of diabetes mellitus. 103 type ii diabetes mellitus is characterized by obesity and secretion of leptin. 104 the production of leptin is related with atherogenic effect, 104 its elevation in obese individuals causes endothelial dysfunction 103 (figure 3 ). leptin binds to its receptors on neutrophils 105 and activates the chemotaxis and phagocytosis process. 106 the effect of leptin on the generation of reactive oxygen species is still undefined, 105 but it causes the migration of leukocytes toward injured tissue. 106 at the onset of inflammation, leptin increases the synthesis of pro-inflammatory mediators like il-6 and tnf-α. 78 il-6 and tnf-α are synthesized from macrophage within endothelium and contribute to the atherogenic process. 107 in addition to macrophage, different types of cells are responsible for secretion of il-6, including endothelial cells, adipocytes, and skeletal muscle cells. 108 in contrast to pro-inflammatory effect, leptin activates synthesis of anti-inflammatory cytokines, including il-4 and il-10 from different types of immune cells. 65 however, in t2dm, the vascular complication promoted by the active pro-inflammatory mediator, il-6 through jak/ stat signal transduction pathway. 109 the interaction of this cytokine with its receptor induce activation of jak/stat signaling pathway. 60 the transphosphorylation of jak and stats is related with stimulation of receptors. 57 janus kinase/activators of transcription (jak) activation is caused by the binding of il-6 to the cytoplasmic domain of gp130 followed by auto-phosphorylation of jak and phosphorylation of transmembrane tyrosine receptor motifs, including tyr905, tyr814, tyr767 and tyr915. 99, 110 in addition, phosphorylation of both ser727 and tyr705 residues of ob-rb also occurred within macrophage. 111 mitogen activated protein kinases (mapks) are responsible for serine 727-phosphorylation of stat3 and stat1, which act as linkage sites for mapk and stats. 112 activators of transcription-3 (stat3) are the major activation transcription factor for leptin signaling cascade, which requires phosphorylation of tyr705. 109 in addition to tyr705, extracellular regulated kinase (erk)-activated phosphorylated ser727 also mediates stat3 stimulation during leptin signaling cascade. 111 then, stat3 dimerizes, phosphorylates and translocates to the nucleus to activate pro-inflammatory gene expression. 109 the translocated stat binds to gamma-activated sites 113 and interferon-stimulated response elements (isres) which figure 3 the impact of leptin on the pathogenesis of chronic autoimmune inflammatory disorders. leptin binds to its long isoform receptor (ob-rb) on macrophage to induce its biological and physiological effect through a jak/stat signaling pathway. this signaling cascade within macrophage induces the synthesis of pro-inflammatory cytokines, including il-6 and tnf-α. the synthesis and release of il-6 and tnf-α also involves the pathogenesis of ra. similarly, ibd is one of inflammatory disorders which is characterized by elevation in the level of pro-inflammatory cytokines, including tnf-α, il-6 and il-1 that leads to the development of diseases. in contrast, the immune dysregulation related to leptin is due to elevated differentiation of th17 cell, which in turn leads to tissue damage in autoimmune inflammatory disorders, including, sle, psoriasis, and ra. the effector cell, th17 induces inflammation by the activating secretion of il-17 which also enhance tissue damage and inflammation in sle. in the pathogenesis of psoriasis, studies done in mice showed that leptin induces and activates the differentiation of t-lymphocytes to t-helper-1 lymphocytes (th1 lymphocytes) to release pro-inflammatory mediators, including tnf-α, il-6 and il-8. are involved in the signaling cascade of ifn and acute phase response 109 (figure 3 ). in contrast, the lower level of leptin seen both in rodents and humans indicates the presence of normal metabolic activity of cells. 114 the higher insulin concentration in the circulation leads to the deposition of fat in adipose tissue in the form of triglyceride. 115, 116 in response to accumulated fat, leptin will be secreted by adipose tissue to oxidize it. but, the physiological activity of leptin does not act on adipose tissue to oxidize stored fat, which leads to a phenomenon called leptin resistance. 114 the demonstration done on rats showed that leptin resistance may be caused by downregulation of genetic expression of lep-rb in the hypothalamus. in addition to this, its signal transduction pathway can be inhibited through suppressor of cytokine signaling-3 (socs3) having counter regulatory effect. 117 studies done on mice confirm that obesity was inhibited by avoiding socs3 and protein tyrosine phosphatase 1b in the pro-opiomelanocortin (pomc) neurons. 118 elevation of counter regulatory signaling pathways and overexpression of leptin receptor cause inhibition of leptin signaling cascade, which in turn leads to leptin resistance. therefore, hypothalamus promotes the sensitivity of leptin through upregulation of stat3, jak2 and diminished genetic expression of socs3. 119 in addition to its receptor aberration, a defect in transportation of leptin also contributes to leptin resistance. 120 the expression of leptin receptor isoform (lep-rb) in the brain activates transportation of leptin to undergo its biological and physiological effect on hypothalamus. however, the blood-brain barrier inhibits transportation of leptin. 121 diminished response to biological and physiological action of leptin commonly occurs in obese individuals. 122 the observational study done by kennedy et al 123 in 2016 explained that elevated level of leptin was seen in hyperglycemia condition. in the hypothalamus, the jak-stat signaling pathway is induced through interaction of lep-rb and leptin, which leads to the biological effect of leptin action. 124 the glycolipid metabolism is regulated by leptin binding to its long isoform receptor, lep-rb which is found in the liver and hypothalamus. 125 lep-rb/stat3 signaling controls glycemic index, hence leptin regulates stat3 and phosphatidyl inositol-3 kinase (pi3k) activity. 126 glucose reduction activity of leptin through energy expenditure enhances insulin sensitivity effect. 127 however, in animal models, the mutated leptin receptor gene may be associated with leptin action, t2dm pathogenesis, and obesity 124 (figure 1 ). leptin resistance occurs both in diabetes and obese subjects. 128 an investigation done on an animal model showed that leptin replacement therapy decreased hepatic gluconeogenesis and hyperglycemia condition. 129 leptin therapy is not an adequate alternative method due to unresponsiveness to its physiological action. therefore, additional desensitizing molecules should be combined with leptin to strengthen its anti-obesity activity. 122 if the anti-obesity activity of leptin therapy is enhanced, the stored fat will be oxidized, as well as insulin sensitivity elevated. hence, the pathogenesis of diabetes might be decreased. globally, the obesity-associated hypertension increases at a higher rate. 130 several researchers confirmed that leptin leads to obesity related to hypertension. 131 a recent investigation tried to evaluate blood pressure in lipodystrophy patients through administration of leptin. although they identified elevated level of leptin, patients did not show a significant variation as their blood pressure is measured. 132 however, mutation of leptin and its receptor may cause severe obesity in human beings, but normal or lower arterial pressure and sympathetic tone may be detected. [133] [134] [135] obesity is a well-known risk factor for the development of hypertension. 136 in premenopausal women, obesity induces three-fold elevation for the progression of hypertension. 131 the abnormal secretion of biologically active peptide, leptin leads to aberration of the appetite regulation, insulin sensitivity, inflammation, and elevation of blood pressure (bp). 137 a recent investigation revealed that hypertension developed through elevation of the sympathetic nervous activity. 138 blood pressure is maintained through interconnected activity of the hormonal factors, such as tnf-α, angiotensin, melanocortin, and leptin. 130 high fat diet intake causes the secretion of effector molecules, such as leptin and tnf-α to induce their sympathetic activity on hypothalamus. 130 independent of food consumption, leptin regulates sympathetic activity and bp. 139 it affects bp by elevating sympathetic nervous system and aldosterone levels. 139 protein tyrosine phosphatase 1b (ptp1b) and socs3 have negative feedback on the effect of leptin by induction of leptin resistance. as leptin resistance occurred, the biological effect of leptin on sympathetic nervous system (sns) becomes disrupted, which contributes to arterial hypertension pathogenesis. 140 contrast, bp and heart rate decreased due to downregulation of leptin receptor in the proopiomelanocortinergic (pomc) neurons. 141 leptin activates sns to induce obesity related to hypertension 23 because it affects autonomic nervous system in lep-rb containing neurons. 142 in contrast to this, the activity of sns and bp can be impaired during intravenous administration of leptin in humans. 143 sympathetic nervous system and bp are regulated through the activation of melanocortin-4 receptors (mc4rs) in brain stem nuclei, hence the renal sns is altered. 144 the lysis of pomc protein induces the synthesis of α-melanocyte-stimulating hormone (α-msh), which in turn activates mc4rs. 144 the sensitization of hypothalamus mc4rs induces an increase in renal system activity, including elevated sodium retention and secretion of renin, further leading to an increase in bp. 144 in addition to this, receptors of leptin are mainly expressed in the hypothalamus and are responsible for elevated secretion of aldosterone, which also depends on the level of ca2+. 145 xie and bollag et al 146 in their focused review elaborate that leptin stimulates overproduction of aldosterone. the elevated level of aldosterone also leads to elevate retention of salt and water by the kidney, which results in an increase in bp 146 (figure 1) . consequently, elevated level of leptin among obese individual leads to hypertension through increased production and secretion of aldosterone. globally, cardiovascular diseases (cvd) are a major health burden, which accounts for up to 12.3 and 17.6 million deaths in 1990 and 2016, respectively. 147 different types of diseases are categorized under cvd, including coronary artery diseases, 148 myocardial infarction, heart failure, rheumatic heart diseases, stroke, and congenital heart diseases. 149 stroke and coronary artery disease (cad) are the major factors for the global death burden both in high and low-income countries. 150 in the usa, 15.4 million people were diagnosed with cad from 2007 to 2010 whose age was greater than 20 years old. 40 researchers are in contention whether leptin has a positive or negative impact on the heart and vascular system. 151 perivascular fat has a characteristic of atheroprotective vascular homeostasis and give a mechanical strength for vasculature through release of leptin. however, it loses its biological function during obesity. 152, 153 the expression of leptin has been elevated because of obesity-associated perivascular adipose tissue. 154 in contrast, investigations done on hypertensive rats showed the expression of perivascular adipose tissue (pvat)-derived leptin was decreased. 155 perivascular-derived leptin leads to vessel stiffness during obesity. 153 in addition, sns activity become elevated due to the release of leptin. 156 in addition to leptin, pvat activates the release of monocyte chemoattractant protein-1 (mcp-1), il-8, il-6, and tnf-α to promote the development of atherosclerosis through activation of smooth muscle cell migration. 157 perivascular-derived leptin activates macrophage migration, expression of adhesion molecule, synthesis of free radicals, secretion of il-6, and tnf-α. 158 obesity-associated perivascular adipose tissue activates p38 mitogenactivated protein kinases signaling pathways to induce phenotypic change on vascular smooth muscle cells. published scientific conclusions on the effect of leptin on vascular diseases differ. 35 several animal studies revealed that the pathogenesis of cardiac hypertrophy is associated with obesity. 159 although leptin causes increased ventricular thickness and cardiac mass, its effect on cardiac hypertrophy is still undefined. it may lead to cardiac remodeling through triggering its biological and physiological effect using pi3k, mitogen-activated protein kinase (mapk) and jak/stat3 signaling cascade mechanism. 160 however, cardiac hypertrophy may develop because of activation of sns and renin-angiotensin-aldosterone system. 161 stangl et al 162 in 2000 carried out a demonstration on mice, and they confirmed that infusion of leptin leads to an increase in sympathetic activity of certain organs. binding of leptin to its receptors induce the activation of sns. 163 receptors of leptin found on vascular cells indicate the potential role of leptin in the function of the vascular system. 164 therefore, it has autocrine and paracrine effects on the vascular modulation process. 36 investigations done on animal models showed that leptin leads to the development of atherosclerosis and thrombosis, which act as risk factors for cad. 27 even though animal investigations revealed that it contributes to atherosclerosis development, 115 findings from clinical investigations as well as experimental animal studies showed leptin's protective effect against atherosclerosis. 165, 166 with regard to this, the synthesis of reactive oxygen species (ros) increased rapidly after the treatment of leptin. 167 in addition to cad, evidence revealed that heart failure is caused by increased genetic expression of leptin and its receptors. 168 congestive heart failure causes an elevation in leptin plasma level. 167 the induction of leptin signal transduction pathway through pi3k, mapk, jak2/stat3 results in biological and physiological response in different tissues, including left ventricular, which is related to left ventricular hypertrophy (lvh). 160 moreover, angiotensin ii-associated myocardial remodeling may be due to the biological effect of leptin through its signal transduction. 160 in vitro investigation showed that rodent and human cardiomyocyte hyperplasia were activated by the effect of leptin. 169 adiposity and obesity are the major risk factors for the pathogenesis of mets, such as t2dm and hypertension, which in turn leads to cardiovascular complications, 170 because abnormality in adipose tissue and obesity induce a chronic inflammation. 55 adiponectin/leptin ratio will be diminished during systemic inflammation to induce pathogenesis of various mets. 10 in this regard, researchers agree with influence of dysfunctional adipokines on the pathogenesis of mets. 171 this is due to a positive correlation between the level of leptin and development of atherosclerosis 172 (figure 1 ). elevated mass of adipose tissue is characterized by increased secretion of adipokines, which contribute to the pathogenesis of atherosclerosis. 35 furthermore, the association between leptin and atherosclerosis is mainly associated with leptin resistance instead of hyperleptinemia. 173 leptin resistance occurs in diabetes and obese subjects. 128 on behalf of the obesity-associated hypertension, elevated level of leptin leads to an increase in the production of aldosterone. elevated aldosterone level also leads to increased retention of salt and water by the kidney, which results in an increase in blood pressure. 146 although it is difficult to clarify the mechanism of development of atherosclerosis, sle patients may develop atherosclerosis. 174 this is because of development of atherosclerosis, which may be associated with the deposition of cholesterol ester (ce) in foam cells during endothelial dysfunction. 175 concerning the atherosclerosis, a huge cohort study was done in systemic lupus international collaborative clinics (slicc) on 1249 patients and 31 of them developed atherosclerosis. 174 in addition to this cohort study, the population-based research done in sweden confirmed that higher incidence of cvd was seen in sle patients. 176 according to this study, sle patient's within the age range of 20-39 years have 16 times higher cardiovascular mortality risk than the general population. similarly, lewandowski and kaplan 177 in 2016 also describe that one third of deaths of sle patients are due to cardiovascular complications. therefore, the risk of developing cvd is higher in sle patients due to the development of atherosclerosis 174 (figure 1 ). in contrast, numerous investigations confirmed that ibd leads to the risk of cardiovascular complication, 178 which is associated with involvement of leptin in the development of atherosclerosis 178 (figure 1 ). regarding psoriasis, inflammation promotes pathophysiology of disease, which in turn leads to the risk of cardiovascular complication. 83 thus, cvd is one of the comorbidity effects of psoriasis. 83 similarly, patients with ra are 1.5 times more likely to develop cvd as compared to the general population. 179 adipokines modify the immune system and metabolic activity of cartilage, and bone which results in the occurrence of mets. 170 leptin induces the activation and release of pro-inflammatory cytokines, including il-6, tnf-α, il-17 and other cytokines to promote systemic inflammation in ra, sle and psoriasis. leptin-activated pro-inflammatory mediators contribute to complication of atherosclerosis in t2dm. conversely, it induces obesity-associated hypertension through activation of sympathetic nervous system in lep-rb containing neurons. it increases the secretion of aldosterone, which in turn causes an increase in arterial blood pressure. consequently, the development of both mets and chronic inflammatory disorders leads to the pathogenesis of cvd such as coronary artery disease and stroke. this review strongly suggests that the adverse individual effect of leptin on chronic inflammatory diseases in turn increases the risk of developing cvd. therapeutic target of leptin regarding its pro-inflammatory effect and dysregulated sympathetic nervous system activity may prevent further cardiovascular complication. therefore, we recommend that treating an elevated level of leptin has broad therapeutic potential to inhibit the pathogenesis of chronic inflammatory disorders and associated further cardiovascular complications. early therapeutic management of hyperleptinemia in obesity-associated inflammatory disorders has a cumulative therapeutic potential to manage complications of cvd. data sharing is not applicable to this article because no data sets were generated or analyzed during the review. we would like to acknowledge mr tadese asemamaw for his great support regard to proofreading, language and grammatical problems improving this review article. all authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. no funding source is available. the authors report no conflicts of interest in this work. leptin as an important link between obesity and cardiovascular risk factors in men with acute myocardial infarction is leptin coming back? a short introduction to the presentations in this symposium session at the 2015 annual meeting of the easd renaissance of leptin for obesity therapy leptin's physiologic role: does the emperor of energy balance have no clothes? leptin effects on dat neurons to control energy homeostasis the brain and energy homeostasis: from an apparently simple to a highly complex neuronal system energy partitioning between fat and bone mass is controlled via a hypothalamic leptin/npy relay adipokines in health and disease the regulation of aldosterone secretion by leptin: implications in obesity-related cardiovascular disease adiponectin-leptin ratio is a functional biomarker of adipose tissue inflammation interplay of circulating leptin and obesity in cognition and cerebral volumes in older adults adipokines in rheumatoid arthritis leptin in inflammation and autoimmunity role of leptin in inflammation and vice versa effect of psoriasis activity on serum adiponectin and leptin levels proinflammatory and anti-inflammatory cytokine profiles in psoriasis: use as laboratory biomarkers and disease predictors the role of leptin in the association between obesity and psoriasis la cava a. cutting edge: leptin-induced rorγt expression in cd4+ t cells promotes th17 responses in systemic lupus erythematosus serum leptin in systemic lupus erythematosus patients: its correlation with disease activity and some disease parameters circulating leptin level, soluble leptin receptor level and their gene polymorphism in patients with systemic lupus erythematosus: a systematic review and meta-analysis markers of inflammation are negatively correlated with serum leptin in rheumatoid arthritis an update on the role of leptin in the immuno-metabolism of cartilage leptin mediates high-fat diet sensitization of angiotensin ii-elicited hypertension by upregulating the brain renin-angiotensin system and inflammation the role of leptin in diabetes: metabolic effects euglycemia restoration by central leptin in type 1 diabetes requires stat3 signaling but not fast-acting neurotransmitter release polypeptides capable of inhibiting the binding between leptin and neuropilin-1. google patents adipokines: new therapeutic target for osteoarthritis? leptin-deficient (ob/ ob) mice are protected from t cell-mediated hepatotoxicity: role of tumor necrosis factor α and il-18 role of leptin in atherogenesis a novel leptin receptor antagonist uncouples leptin's metabolic and immune functions serum leptin in rheumatoid arthritis t-cell subsets in autoimmunity investigating the balance between th17/treg cells in rheumatoid arthritis and its association with disease activity adipokines, inflammation, insulin resistance, and carotid atherosclerosis in patients with rheumatoid arthritis fish oil n-3 fatty acids increase adiponectin and decrease leptin levels in patients with systemic lupus erythematosus smoking and obesity in systemic lupus erythematosus: a cross-sectional study emerging role of adipokines in systemic lupus erythematosus adipokine interactions promote the pathogenesis of systemic lupus erythematosus atherosclerosis biomarkers in female systemic lupus erythematosus patients with and without cardiovascular diseases adipokines, tumor necrosis factor and its receptors in female patients with systemic lupus erythematosus cardiovascular disease in systemic lupus erythematosus: a comprehensive update clinical associations of serum leptin and leptin/ adiponectin ratio in systemic sclerosis leptin facilitates the differentiation of th17 cells from mrl/mp-fas lpr lupus mice by activating nlrp3 inflammasome pathogenesis of human systemic lupus erythematosus: a cellular perspective regulatory t cells in systemic lupus erythematosus effects of treatment for psoriasis on circulating levels of leptin, adiponectin and resistin: a systematic review and meta-analysis systemic inflammation and cardiovascular comorbidity in psoriasis patients: causes and consequences biomarkers of subclinical atherosclerosis in patients with autoimmune disorders psoriasis: obesity and fatty acids biomarkers of subclinical atherosclerosis in patients with psoriatic arthritis serum levels of adipokines and cytokines in psoriasis patients: a systematic review and meta-analysis psoriasis in obesity: comparison of serum levels of leptin and adiponectin in obese subjects-cases and controls relationship of leptin with adiposity and inflammation and resistin with disease severity in psoriatic patients undergoing anti-tnf-alpha therapy new insights into different adipokines in linking the pathophysiology of obesity and psoriasis elevated leptin levels induce inflammation through il-6 in skeletal muscle of aged female rats metabolic syndrome and selective inflammatory markers in psoriatic patients il-10 improves skin disease and modulates endothelial activation and leukocyte effector function in patients with psoriatic arthritis combinatorial effects of diet and genetics on inflammatory bowel disease pathogenesis salidroside regulates the expressions of il-6 and defensins in lps-activated intestinal epithelial cells through nf-κb/mapk and stat3 pathways circulating leptin levels as a potential biomarker in inflammatory bowel diseases: a systematic review and meta-analysis the role of obesity in inflammatory bowel disease adiponectin and adiponectin receptor 1 overexpression enhance inflammatory bowel disease increased intestinal mucosal leptin levels in patients with diarrhea-predominant irritable bowel syndrome interaction of obesity and inflammatory bowel disease adipokines and the role of visceral adipose tissue in inflammatory bowel disease adipokines: potential therapeutic targets for vascular dysfunction in type ii diabetes mellitus and obesity activation of nrf2-antioxidant signaling by 1, 25-dihydroxycholecalciferol prevents leptin-induced oxidative stress and inflammation in human endothelial cells serum adipocytokines are associated with microalbuminuria in patients with type 1 diabetes and incipient chronic complications influence of exogenous leptin on redox homeostasis in neutrophils and lymphocytes cultured in synovial fluid isolated from patients with rheumatoid arthritis leptin is associated with disease activity but not with anthropometric indices in rheumatoid arthritis patients high insulin and leptin increase resistin and inflammatory cytokine production from human mononuclear cells il6r inhibits viability and apoptosis of pancreatic beta-cells in type 2 diabetes mellitus via regulation by mir-22 of the jak/stat signaling pathway therapeutic targeting of the proinflammatory il-6-jak/stat signalling pathways responsible for vascular restenosis in type 2 diabetes mellitus human leptin signaling in human peripheral blood mononuclear cells: activation of the jak-stat pathway leptin signaling: a key pathway in immune responses lipid metabolism of adipocytes in-silico drug repurposing study predicts the combination of pirfenidone and melatonin as a promising candidate therapy to reduce sars-cov-2 infection progression and respiratory distress caused by cytokine storm endospanin1 affects oppositely body weight regulation and glucose homeostasis by differentially regulating central leptin signaling leptin deficiency suppresses progression of atherosclerosis in apoe-deficient mice suppressing hyperinsulinemia prevents obesity but causes rapid onset of diabetes in leptin-deficient lepob/ob mice downregulation of lrb in vmh/dmh during the second half of gestation and upregulation of socs-3 in arc in late-pregnant ewes-implications for leptin resistance functional role of suppressor of cytokine signaling 3 upregulation in hypothalamic leptin resistance and long-term energy homeostasis diet-induced obesity and the mechanism of leptin resistance leptin-signaling pathways and leptin resistance. front eating weight regulation leptin resistance: underlying mechanisms and diagnosis leptin resistance in obesity: an epigenetic landscape leptin is associated with persistence of hyperglycemia in acute pancreatitis: a prospective clinical study intervertebral disc degeneration in mice with type ii diabetes induced by leptin receptor deficiency effect of konjac mannan oligosaccharides on glucose homeostasis via the improvement of insulin and leptin resistance in vitro and in vivo leptin receptor action and mechanisms of leptin resistance leptin induces fasting hypoglycaemia in a mouse model of diabetes through the depletion of glycerol obesity, diabetes, and leptin resistance promote tau pathology in a mouse model of disease pleotropic effects of leptin to reverse insulin resistance and diabetic ketoacidosis neural programmatic role of leptin, tnfα, melanocortin, and glutamate in blood pressure regulation vs obesityrelated hypertension in male c57bl/6 mice leptin induces hypertension and endothelial dysfunction via aldosterone-dependent mechanisms in obese female mice plasma leptin level is positively associated with blood pressure measures independent of gender and bmi leptin mediates the increase in blood pressure associated with obesity human leptin deficiency caused by a missense mutation: multiple endocrine defects, decreased sympathetic tone, and immune system dysfunction indicate new targets for leptin action, greater central than peripheral resistance to the effects of leptin, and spontaneous correction of leptin-mediated defects leptin as a mediator of obesity-induced hypertension association of two well-defined polymorphisms in leptin and leptin receptor genes with hypertension and circulating leptin: a meta-analysis leptin beyond the lipostat: key component of blood pressure regulation sympathetic nervous system as a target for aging and obesity-related cardiovascular diseases leptin is not essential for obesity-associated hypertension astragaloside iv prevents obesity-associated hypertension by improving pro-inflammatory reaction and leptin resistance role of leptin in energy expenditure: the hypothalamic perspective mechanisms of leptin action and leptin resistance the role of the autonomic nervous system in the pathophysiology of obesity melanocortin-4 receptors and sympathetic nervous system activation in hypertension adipocytes, aldosterone and obesity-related hypertension obesity, hypertension and aldosterone: is leptin the link? cardiovascular mortality attributable to dietary risk factors in 51 countries in the who european region from 1990 to 2016: a systematic analysis of the global burden of disease study dna vaccine protection against sars-cov-2 in rhesus macaques decreased circulatory microrna-4478 as a specifi c biomarker for diagnosing non-st-segment elevation myocardial infarction (nstemi) and its association with soluble leptin receptor leptin concentration and risk of coronary heart disease and stroke: a systematic review and meta-analysis plasma leptin levels and incidence of heart failure, cardiovascular disease, and total mortality in elderly individuals adipose tissue distribution, inflammation and its metabolic consequences, including diabetes and cardiovascular disease mechanistic links between obesity, diabetes, and blood pressure: role of perivascular adipose tissue perivascular adipose tissue regulates vascular function by targeting vascular smooth muscle cells roles of perivascular adipose tissue in hypertension and atherosclerosis nerve-perivascular fat communication as a potential influence on the performance of blood vessels used as coronary artery bypass grafts roles of cells from the arterial vessel wall in atherosclerosis perivascular adipose tissue (pvat) in atherosclerosis: a double-edged sword cardiac hypertrophy with obesity is augmented after pregnancy in c57bl/6 mice telmisartan improves myocardial remodeling by inhibiting leptin autocrine activity and activating pparγ lean heart: role of leptin in cardiac hypertrophy and metabolism elevated serum leptin in patients with coronary artery disease: no association with the trp64arg polymorphism of the β 3-adrenergic receptor effects of serum leptin and resistin levels on cancer cachexia in patients with advanced-stage non-small cell lung cancer influence of leptin on arterial distensibility: a novel link between obesity and cardiovascular disease? severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor fasting plasma leptin, tumor necrosis factor-α receptor 2, and monocyte chemoattracting protein 1 concentration in a population of glucose-tolerant and glucoseintolerant women: impact on cardiovascular mortality an autocrine role for leptin in mediating the cardiomyocyte hypertrophic effects of angiotensin ii and endothelin-1 role of nf-κb and p38 mapk activation in mediating angiotensin ii and endothelin-1-induced stimulation in leptin production and cardiomyocyte hypertrophy recent advances in understanding lipodystrophy: a focus on lipodystrophy-associated cardiovascular disease and potential effects of leptin therapy on cardiovascular function adipokines: linking metabolic syndrome, the immune system, and arthritic diseases adipokines, metabolic syndrome and rheumatic diseases association between leptin and il-6 concentrations with cardiovascular risk in patients with rheumatoid arthritis atherosclerosis in systemic lupus erythematosus association between leptin and systemic lupus erythematosus optimal monitoring for coronary heart disease risk in patients with systemic lupus erythematosus: a systematic review update on cardiovascular disease in lupus hypertrophic mesenteric adipose tissue may play a role in atherogenesis in inflammatory bowel diseases predictors of new atherosclerotic carotid plaque development in patients with rheumatoid arthritis: a longitudinal study original research, review, case reports, hypothesis formation, expert opinion and commentaries are all considered for publication. the manuscript management system is completely online and includes a very quick and fair peer-review system original�article abstract objective: to determine the frequency of dyslipidemia among patients suffering from rheumatoid arthritis. study design: descriptive study. place and duration of study: the study was conducted in medicine department in collaboration with pathology department of khalifa gul nawaz teaching hospital bannu, khyber pakhtunkhwa. the duration of study was two years from january 2016 to december 2017. materials and methods: a total of 187 cases were included. inclusion criteria were all diagnosed patient of rheumatoid arthritis of any age and sex. exclusion criteria were patients with history of diabetes mellitus, hypertension, endocrinopathies, alcohol intake and use of oral contraceptive. fasting blood samples were analyzed to measure various fractions of lipids, blood sugar, cholesterol, thyroid hormones, and liver and kidney functions. the data collected was analyzed in spss version 20 for various variables. the results were presented in tables and graphs where required. results: in this study male to female ratio was 0.5:1. the age range was from 22 to 60 years. average age was 43.49 years + 10.94. dyslipidemia was present in 48(25.67%) patients of rheumatoid arthritis; whereas 139(74.33%) patients didn't have dyslipidemia. conclusion: rheumatoid arthritis patients have significant dyslipidemias which may leads to increased risk of cardiovascular diseases. early diagnosis and treatment is mandatory to reduce morbidity and mortality. dyslipidemia must be considered an essential part of ra and may be managed appropriately and in time to avoid/ minimize complications of cardiovascular diseases. key words: cholesterol, dylipidemia, high density lipoprotein, low density lipoprotein, rheumatoid arthritis, triglycerides. 3 cardiovascular diseases. the pattern of cvd in ra is different from general population. they usually have silent ischemic heart disease, sudden death and heart failure. the inflammation of rheumatoid a r t h r i t i s i s a s s o c i a t e d w i t h a c c e l e r a t e d atherosclerosis. recent evidences show that high inflammatory status of ra is associated with lipid paradox especially serum cholesterol, inversely related to risk of cvd in untreated patients of ra. it is noted that inflammation influences lipid profile in ra patients and have a complex relationship between 4,5 inflammatory burden of ra and cvd risk. in general population evidences show that inflammation contributes to the onset and pathogenesis of atherosclerosis leading to cvd, where as inflammation underlies progression of atherosclerosis in ra. the impact of inflammation on dyslipidemia in ra is associated with inverse relationship between cvd risk and lipids levels. a similar relation is observed in other chronic inflammatory diseases like malignancy, sepsis and post myocardial infarction cases. the mechanisms introduction rheumatoid arthritis is a chronic systemic inflammatory disease which mainly manifest as synovitis of multiple joints. it has prevalence of 1% 1 with females more affected than males in 3:1 ratio. rd th 2 peak age of onset is between 3 to 5 decade. the frequency of dyslipidemia in general population is variable in different age groups, more in adults as compared to young. rheumatoid arthritis patients are at 2-3 fold higher risk of atherosclerosis leading to cardiovascular diseases (cvd). as high as 50% deaths in rheumatoid arthritis are due to dyslipidemia among patients of rheumatoid arthritis 1 2 3 4 naseeb ur rehman shah , mohammad sajjad khattak , sami ullah , asim muhammad correspondence: mohammad sajjad khattak associate professor department of pathology bannu medical college, bannu e-mail: sajjadkhattak66@gmail.com 1,3 department of medicine khalifa gul nawaz teaching hospital, bannu 2,4 department of pathology bannu medical college, bannu funding source: nil; conflict of interest: nil received: january 15, 2019; revised: august 10, 2019 accepted: august 12, 2019 dyslipidemia in rheumatoid arthritisjiimc 2019 vol. 14, no.3 146 involved in lipid changes in this inflammatory 6-9 process are not yet clear. the inflammation of ra is also associated with qualitative and quantitative changes of lipids profile. the hdl-c which is having a high protective role in cvd in general population is impaired in ra, leading to accentuation of cvd. genetic studies show that fractional composition of hdl-c isolated from ra 1 0 reveals significant changes. these findings regarding lipid paradox and qualitative and quantitative changes in ra needs further assessment to be addressed in future. in our country we have limited research in this field, and are lacking national consensus regarding lipid monitoring in rheumatoid patients. this study will provide us with local statistics of dyslipidemias in patients of rheumatoid arthritis. the objective of the study was to determine the frequency of dyslipidemia in patients suffering from rheumatoid arthritis patients in southern region of khyber pakhtunkhwa pakistan. materials and methods: this descriptive study was conducted in department of medicine in collaboration with pathology department of khalifa gul nawaz teaching hospital bannu pakistan. the duration of study was two years from january, 2016 to december, 2017. a total of 187 cases were included. inclusion criteria was all patients of rheumatoid arthritis both males and females of any age of more than five years disease duration as per acr criteria 2010 were included. exclusion criteria was patients with history of diabetes mellitus, hypertension, endocrinopathies, chronic kidney diseases, alcohol intake and use of oral contraceptive. all these diseases were excluded either by clinical manifestation or by performing relevant laboratory tests. data was collected after approval from hospitals ethical and research committee. all patients meeting the inclusion criteria were enrolled in the study after their written informed consent from the in and out patient department. aseptic blood sampling was performed from all patients after overnight fasting to measure total cholesterol, high density lipoprotein (hdl-c), low density lipoprotein (ldl-c) and triglyceride to confirm dyslipidemia. all these investigations were performed by a single laboratory under supervision of pathologist. all the relevant information was recorded in a pre designed proforma. the data collected was analysed for frequency with percentages and mean with standard deviation for different variables like age, total cholesterol, hdl-c, ldl-c and triglycerides. the results were presented in tables and graphs where required. operational definition: rheumatoid arthritis: american college of rheumatology (acr) criteria was used for diagnosis. 1. number and types of joints involvement. 2. serological tests like rheumatoid factor and anti citrulinated cyclic protein antibody (anti ccp antibody). 3. acute phase reactants i.e. er y throcy te sedimentation rate (esr) and c-reactive protein (crp). 4. duration of arthritis lasting six weeks or longer. dyslipidemia is defined as marked abnormal 11 concentration of lipoproteins or lipids in the blood. 1. total cholesterol >200 mg/dl 2. high density lipoprotein (hdl) cholesterol <40mg/dl 3. low density lipoprotein (ldl) cholesterol >130mg/dl 4. triglycerides >150mg/dl results in this study the age range of patients was from 21 to 60 years with mean age 43.49+ 10.94 years. male were 66 (35.29%) and female 121 (64.71%) with male to female ratio was 0.5:1. (table 1) the disease was more common in age group of more than 50 years 57(30.5%) followed by age group of 41 -50 years 55(29.4%), 31-40 years 48 (25.7%) and 27 (14.4%) in age group of less than 30 years. (table ii). dyslipidemia was present in 48 (25.67%) patients of rheumatoid arthritis, where as 139(74.33%) ra patients were free of dyslipidemia. (fig: 1).total cholesterol was ranged from 168-330 mg/dl with mean 212±34.96 mg/dl, hdlc was from 23-60 mg/dl with mean 39.9±12.57 mg/dl, ldlc was from 76-156 mg/dl with mean 99.2+ 29.60 mg/dl and triglycerides was 158-285 with mean 196+ 30.64 md/dl.(table iii). male suffered dyslipidemia relatively more common as compared to female. in male 21(31.8%) patients were suffering from dyslipidemia and in females 27(22.3%) patients were suffering from dyslipidemia and 94(77.7%) didn't have dyslipidemia. dyslipidemia in rheumatoid arthritisjiimc 2019 vol. 14, no.3 147 discussion: dyslipidemia is frequently associated with rheumatoid arthritis. in general, in inflammation including active ra have a lipid lowering effect on 12 blood lipid level. also ra patients have increased risk of cvd in relatively low blood cholesterol level in contrast to general population. rheumatoid arthritis increases mortality primarily due to cvd and can reduce life expectancy by about 8-15 years. this increase mortality is mainly due to atherosclerosis and dyslipidemia is a well major recognized risk 13 factor of atherosclerosis. in this study there is diffuse derangement of lipid levels in 25.67% cases. the most common was total cholesterol in 39.58% patients followed by ldl-c in 22.92% cases and hdlc and triglyceride both in 18.75% cases. the age range of patients was from 22 to 60 years with mean age 43.49 years + 10.94 years. in a study conducted 20 by erum et al in 2017 in karachi the age range is from 20-60 years with mean age of 36.31±10.46 21 years. another study conducted by attar et al in 2015 in saudi arabia the mean age is 40.49±12.19 years. the common age group was from 51-60 years 57(30.5%) followed by 41 -50 years 55(29.4%), 31-40 years 48 (25.7%) and 27 (14.4%) in age group of less 20 than 30 years. in a study conducted by erum et al the common age group is 31-40 years 37.5% followed by 20-30 years 32%, 41-50 years 24% and 51-60 years 6.5%. another study conducted by 19 hameed et al in 2017 in iraq the common age group is 50-59 years 345 followed by 40-49 years 22%, 3039 years 16%, 60-69 years 13% and less than 30 years 12%. male patients were 66 (35.29%) and female 121(64.71%) and male to female ratio of 0.5:1. in a 20 study conducted by erum et al male are 11.5% and female 88.5% with male to female ratio of 0.12:1. table i: gender distribu�on of dyslipidemic pa�ents suffering from rheumatoid arthri�s (n=48) fig 1: frequency of dyslipidemia in pa�ents suffering from rheumatoid arthri�s table ii: different age groups of rheumatoid arthri�s pa�ents suffering from dyslipidemia (n=48) table iii: lipid profile of dyslipidemic pa�ents suffering from rheumatoid arthri�s (n=48) table: iv. comparison of frequency of dyslipidemia in various studies this comparison of dyslipidemia in rheumatoid a r t h r i t i s s h ow a l m o st s a m e f re q u e n c y o f 14 dyslipidemia in study done by willerson et al and 15 scott et al. all other studies in above table show high frequency of dyslipidemia as compared to the present study. the reason may be difference in activity of disease during sample collection, different treatment modalities used, patients food habits, epidemiology and life style. amongst the dyslipidemic patients the total cholesterol was 212.3±34.96 mg/dl, hdl-c was 39.9±12.57 mg/dl, ldl-c was 99.2+ 29.60 mg/dl and 14 triglycerides was 196+ 30.64 mg/dl. erum et al show total cholesterol as 169.68±36.68 mg/dl, hdlc 40.02±10.23 mg/dl and ldl-c 93.28±26.17 mg/dl. 16 in hammed et al total cholesterol is 177.6±39.2 mg/dl, hdl-c 44.6±10.9 mg/dl, ldl-c 101.7±30.9 mg/dl and triglyceride is 148.8±70.9 mg/dl. these studies like present study show low level of hdl-c amongst the dyslipidemic patients of ra, which in dyslipidemia in rheumatoid arthritisjiimc 2019 vol. 14, no.3 148 high level is cardio protective. also these studies like present study show high level of ldl-c which is associated with increased risk of cvd in ra patients and the same is true for tc and tg in high level. the small sample size, current laboratory assessment tools and lack of causal relationship between lipid level and inflammatory activities of ra are the limitation of this study. it is suggested to incorporate modern laboratory technique in routine practice for assessment of various fractions of lipids. this study confirms dyslipidemia in ra patients, there might be differences in pattern of dyslipidemia amongst different racial and ethnic group as well as from country to country depending on food habits, religious belief and life style. conclusion: rheumatoid arthritis patients have significant dyslipidemias which may leads to increased risk of cardiovascular diseases. early diagnosis and treatment is mandatory to reduce morbidity and mortality. dyslipidemia must be considered an essential part of ra and may be managed appropriately and in time to avoid/ minimize complications of cardiovascular diseases. references 1. vinapamula ks, manohar sm, bitla ar, kanduri r, bhattaram sk, pemmaraju sr. evaluation of dyslipidaemia in patients with rheumatoid arthritis in south indian population.indian journal of rheumatology 2013; 8 (4):155-60. 2. bahlas s, ahmed mm. lipid levels and association with disease activity in ra and sle in saudi arabia 2013; 11 (7)16. 3. al-zaidi gh, abdulsamad t. serum lipids in patients with active rheumatoid arthritis and its relation to drug therapy: j fac med baghdad 2005; 47(1): 35-9. 4. david b hellmann, john b, imboden jr. musculoskelatal & immunologic disorders. in: stephen j mcphee, maxine a. papadakis. current medical diagnosis & treatment. 49th ed. new york: mc graw hill. 2010; 747. 5. choy e, ganeshalimgam k, semb ag, szekanecz z, nurmohamed m. cardiovascular risk in rheumatoid arthritis: recent advances in the understanding of the pivotal role of inflammation, risk predictors and the impact of treatment. rheumatology 2014; 53:2143-54. 6. tracey e, panoulas ve, smith jp, douglas kmj, metsios1 gs, stavropoulos-la;ompg as et al. rheumatoid arthritis susceptibility genes associate with lipid levels in patients with rheumatoid arthritis. ann rheum dis. 2011; 70:102532. 7. robert b. baron. lipid abnormalities. in: stephen j. mcphee, maxine a. papadakis. current medical diagnosis & treatment, 49th ed. new york: mc graw hill; 2010: 8. brown t. fda approves intravenous golimumab (simponi aria) for rheumatoid arthritis. medscape medical news [serial online]. july 18, 2013; accessed july 30, 2013. a v a i l a b l e a t : h t t p : / / w w w . m e d s c a p e . c o m / viewarticle/807965. 9. aletaha d, neogi t, silman aj, funovits j, felson dt, bingham co 3rd, et al. 2010 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative. arthritis rheum. 2010;62(9):2569-81. 10. shoenfeld y, gerli r, doria a, matsuura e, cerinic mm, ronda n, et al. accelerated atherosclerosis in autoimmune rheumatic diseases. circulation 2005; 112:3337-47. 11. national cholesterol education program (necp) expert panel on detection, evaluation and treatment of high blood cholesterol in adults (adult panel iii). final report. circulation2002; 106:3143-421. 12. turesson c, jacobsson lt, matteson el. cardiovascular comorbidity in rheumatic diseases. vasc health risk manag. 2008; 4:605-14. 13. toms te, symmons dp, kitas gd. dyslipidaemia in rheumatoid arthritis: the role of inflammation, drugs, lifestyle and genetic factors. curr vasc pharmacol. 2010a;8(3):301-26. 14. willerson jt, ridker pm. inflammation as a cardiovascular risk factor.circulation 2004; 109(21suppl 1):112-1110. 15. scott ic, ibrahim f, johnson d, scott dl, kingsley gh. current limitation in the management of cardiovascular risk in rheumatoid arthritis. clin exp rheumatol 2012; 30:228232. 16. hadda v, handa r, aggrawal p, lakshmiy r, umar uk, pandey rm. disease activity and lipid in rheumatoid arthritis: aprospective study. indian j rheum 2007;2:13740 17. soubrier m, zerkak d, dougados m. indication for lowering ldl cholesterol in rheumatoid arthritis an unrecognized problem. j rheumatol 2006; 33:1766-69. 18. nisar a, rasheed u, aziz w,farooqi az. prevalence of dyslipidemia in autoimmune rheumatic diseases.journal of the college of physicians and surgeons pakistan 2012;22 (4):235-9. 19. hameed sb, barzinjy nj. prevalence of dyslipidemia and its association with disease activity in patients with rheumatoid arthritis attending rizgary teaching hospital in erbil city. zanco j med sci 2017;21(2):1781-88 20. erum u, ahsan t, khowaja d. lipid abnormalities in patients with rheumatoid arthritis. pak j med sci. 2017; 33(1):22730. 21. attar sm. hyperlipidemia in rheumatoid arthritis patients in saudi arabia. saudi med j.2015; 36(6):685-90. 22. zrour sh, neffeti fh, sakly n, saoussen h,korbaa y, et al. lipid profile in tunisian patients with rheumatoid arthritis. 2011; 30 (10):1325-31. 23. toms te, panoulas vf, kitas gd. dyslipidemia in rheumatological autoimmune diseases.open cardiovasc med j. 2011;5:64-75. 24. shah sza, devrajani br, devrajani i, bibi i. frequency of dyslipidemia in obese verus non obese in relation to body mass index (bmi) and waist hip ratio (whr) and waist circumference (wc). pak j sci.2010; 61(2):27-31. dyslipidemia in rheumatoid arthritisjiimc 2019 vol. 14, no.3 149 s. de portu, l. g. mantovani, i. olivieri 19© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2008; 9(1) analisi economica (1)cirff centro di farmacoeconomia, facoltà di farmacia, università di napoli federico ii (2)dipartimento di reumatologia della lucania, ospedale san carlo di potenza e ospedale madonna delle grazie di matera abstract objective: a substantial number of patients with rheumatoid arthritis (ra) have an insufficient or unsustained response to tumor necrosis factor-α antagonists (anti-tnfs). the aim of the present study was to estimate the cost-utility of abatacept, a new selective t-cell co-stimulation modulator, in patients with moderately to severely active ra and an insufficient response or intolerance to anti-tnfs in the italian setting. methods: a probabilistic patient level simulation model was developed to estimate long-term costs and health outcomes of abatacept versus anti-tnfs (etanercept, adalimumab, infliximab) in ra patients. the model predicted patients’ haq (health assessment questionnaire) scores over time based on the initial response to treatment (% change in haq score at six months). responding patients continued treatment with a reduced rate of haq progression until long-term treatment failure. health-state utilities and use of health care resources (excluding ra therapies) were assumed to depend on haq scores. the model used data from a phase iii clinical trial of abatacept in patients with inadequate response to anti-tnfs (abatacept trial in treatment of anti-tnf inadequate responders [attain]) and various secondary data sources. the study was performed using the national health service (nhs) perspective. cost-utility of abatacept vs other anti-tnfs was derived in terms of incremental cost per quality-adjusted life-year (qaly) gained based on a lifetime horizon with costs expressed in euros. single-way sensitivity analyses were performed on key parameters. costs and health effects were discounted at 3% annually. results: abatacept therapy was estimated to yield 1.18 additional qalys per patient (5.02 abatacept vs 3.84 anti-tnfs) at an incremental cost of € 21,996.41 based on a 20 years time horizon. cost per qaly gained was € 18,567.24. these results were robust to variation of key model parameters and are well within the usual cost-utility acceptance ranges. conclusions: this study shows that in italy, compared to anti-tnfs, abatacept therapy is cost-effective in patients with moderately to severely active ra and with an insufficient response or intolerance to anti-tnfs. keywords: abatacept, rheumatoid arthritis, anti-tnfs, cost-utility, italy analisi di costo-utilità di abatacept nell’artrite reumatoide in italia simona de portu (1), lorenzo giovanni mantovani (1), ignazio olivieri (2) farmeconomia e percorsi terapeutici 2008; 9(1): 19-26 il trattamento di tale malattia dovrebbe portare a miglioramenti nei segni clinici e nei sintomi, con particolare attenzione alla funzione fisica e alla qualità di vita, cambiamenti che si auspicano duraturi nel tempo. le terapie attualmente in uso hanno come obiettivo colpire l’infiammazione generata dall’attivazione del sistema immunitario utilizzando agenti modificatori di effetto (dmard) come il metotressato o gli agenti biologici, che inibiscono la citochina proinfiammatoria quale il fattore di necrosi tumorale (tnf-α) [6]. nonostante l’efficacia di agenti come gli anti-tnf-α, una percentuale elevata di pazienti non hanno risposta [7-10], mentre in alcuni casi i miglioramenti raggiunti con il passare del tempo vanno riducendosi [11]. un certo numero di pazienti sviluppa tossicità [8-10], mentre altri sviluppano anticorpi verso gli stessi farmaci anti-tnf [12,13]. introduzione l’artrite reumatoide (ar) è una malattia infiammatoria cronica autoimmune ad eziologia sconosciuta [1]. la sua prevalenza è stimata pari allo 0,5-1% nel mondo [2,3], ma il suo carattere progressivo e la sua comparsa in giovane età comportano un elevato impatto economico. l’erosione delle articolazioni e la perdita progressiva della funzionalità con conseguente disabilità fisica e deformità sono associate ad un dispendio economico elevato non solo per i pazienti e le loro famiglie, ma anche per la società. a tale stato morboso, infatti, sono associati sia elevati costi diretti di gestione della malattia, sia costi indiretti dovuti alla perdita di produttività del paziente (circa il 50% dei pazienti dopo 10 anni dalla comparsa della malattia risulta inabile al lavoro) [4,5], sia costi intangibili legati alla compromissione della qualità della vita. corresponding author simona de portu sdeportu@unina.it analisi di costo-utilità di abatacept nell’artrite reumatoide in italia 20 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2008; 9(1) abatacept è il primo di una nuova classe di agenti per il trattamento dell’artrite reumatoide (ar) che modula il cd80 o il cd86-cd28 che costituiscono i segnali necessari per l’attivazione delle cellule t. l’efficacia di abatacept è stata riportata da due studi di fase due [14,15]. in pazienti con ar attiva e risposta inadeguata al metotressato, abatacept in combinazione con metotressato porta a significativi miglioramenti nei segni e sintomi della malattia, nella funzionalità fisica e nella qualità di vita per un periodo di 12 mesi [15,16], miglioramenti che perdurano a 3 anni [17] e a 5 anni [18], con un buon profilo di sicurezza. dato il nuovo meccanismo di azione, abatacept rappresenta un approccio terapeutico razionale nei pazienti con inadeguata risposta terapeutica ad un anti-tnf [19]. abatacept in combinazione con metotressato è, infatti, indicato per il trattamento dell’artrite reumatoide in fase attiva da moderata a grave in pazienti adulti che hanno avuto una risposta insufficiente o una intolleranza ad altri farmaci antireumatici modificanti la malattia, incluso almeno un inibitore del fattore di necrosi tumorale (tnf) [20]. l’obiettivo dello studio è quello di stimare il rapporto di costo-utilità di abatacept vs gli altri anti-tnfs in pazienti adulti con artrite reumatoide in fase attiva che hanno risposto inadeguatamente ai trattamenti con dmards, incluso un anti-tnf-α, in particolare stimando il costo per anno di vita guadagnato aggiustato per la qualità (qaly) di abatacept vs altri anti-tnfs. materiali e metodi descrizione del modello il modello utilizzato in questo studio, costruito con il software statistico r, è conosciuto come patient-level simulation model (figura 1). tale modello, costruito sulla base dei criteri metodologici approvati dal nice (national institute of clinical excellence) e adattato alla realtà italiana, simula il beneficio derivante dall’utilizzo di anti-tnf in termini di utilità in un’ipotetica coorte di 10.000 pazienti, che passano attraverso il modello uno alla volta. la simulazione probabilistica è stata effettuata sulla distribuzione dei valori di utilità associati al punteggio haq (health assessment questionnaire), mentre le altre caratteristiche dei pazienti sono state mantenute fisse. sono stati così stimati i costi e gli esiti in termini di qaly guadagnati per ognuno dei pazienti. le caratteristiche della popolazione considerata nel modello sono tratte dallo studio attain (abatacept trial in treatment of antitnf inadequate responders) [19] (tabella i). il modello contiene le seguenti assunzioni che riguardano la sospensione delle terapie che può avvenire a causa di: risposta clinica insufficiente, tossicità o altre cause (effetti avversi, co-morbidità, trattamenti chirurgici, etc.) si è considerata mancata risposta il non raggiungimento di un miglioramento clinico significativo dei sintomi definito come un miglioramento nel punteggio haq. in maniera conservativa si assume come miglioramento significativo un punteggio di haq ≥ 0,3 rispetto al basale, in quanto esso era l’endpoint primario nello studio attain [19]. la sospensione dovuta ad una risposta clinica inadeguata avviene a 6 mesi, mentre la sospensione per altre ragioni (es. eventi avversi, tossicità, morte) può avvenire in ogni momento durante il trattamento. essendo il metotressato nello studio attain il dmard maggiormente utilizzato [19] nel modello lo si è usato come rappresentativo del ventaglio di dmards convenzionali attualmente disponibili in italia per l’artrite reumatoide. i tassi di fallimento utilizzati e le altre assunzioni del modello sono riassunti in tabella ii. orizzonte temporale e prospettiva il modello, riflettendo l’indicazione di abatacept, assume che i pazienti entrino nella vafigura 1 patient-level simulation model mtx: metoressato haq: health assessment questionnaire haq baseline : punteggio haq al basale haq t : punteggio haq al ciclo precedente haq t+1 : punteggio haq al ciclo successivo ∆haq mtx , ∆haq bio : miglioramento nel punteggio haq dopo la terapia con metotressato o con biologico haq progression : progressione nel punteggio haq abatacept haq t+1 =haq t +∆haq bio +haq progression mtx haq t+1 =haq t +∆haq mtx +haq progression se ∆haq t+1;t ≥0,3 se ∆haq t+1;t <0,3 haq baseline valore riferimento bibliografico età 53 anni 19 sesso femmine 19 (perché in percentuale maggiore) haq basale 1,8 19 peso (kg) 63 data on file tabella i caratteristiche dei pazienti con artrite reumatoide al basale s. de portu, l. g. mantovani, i. olivieri 21© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2008; 9(1) lutazione una volta fallito il trattamento con un anti-tnf e ne escano solo alla morte, indipendentemente dal trattamento; i cicli temporali nel modello sono di 6 mesi. l’orizzonte temporale adottato è pari a 20 anni. la prospettiva è quella del servizio sanitario nazionale (ssn). alternative abatacept si assume somministrato come infusione endovenosa alla dose di 10 mg/kg; i pazienti con un peso inferiore ai 60 kg hanno ricevuto 500 mg di abatacept, quelli con un peso compreso tra i 60 e i 100 kg ne hanno ricevuti 750 mg e infine quelli con un peso superiore ai 100 kg ne hanno ricevuti 1.000 mg [19]. dopo la somministrazione iniziale, abatacept viene somministrato 2 e 4 settimane dopo la prima infusione e, successivamente, ogni 4 settimane. gli anti-tnfs inclusi nell’analisi si assumono somministrati come da scheda tecnica [27]: etanercept (25 mg 2 volte a settimana per iniezione sottocutanea), infliximab (3 mg/kg alle settimane 2 e 6 dalla prima infusione, quindi ogni 8 settimane) e adalimumab (40 mg in un’unica somministrazione ogni due settimane per via sottocutanea). efficacia il modello si focalizza sulla progressione del punteggio haq nel tempo e associa a tali punteggi una misura di utilità. cambiamenti nel punteggio haq il cambiamento nel punteggio haq dopo 6 mesi di trattamento con abatacept e anti-tnf-α è descritto nella tabella ii. tabella ii assunzioni del modello variabile [rif. bibliogr.] valore tasso annuale di fallimento del trattamento [19, 21]: abatacept e altri anti-tnfs (min-max) metotressato ­ ­ 8,2% (0,041-0,123) 0 moltiplicatore del tasso annuale di mortalità per ciascuna unità di incremento dell’haq [22] 1,8 cambiamento dell’haq in 6 mesi [21]: abatacept media (%) tnf-α media (%) ­ ­ -20,4 (33,14) -7,32 (11,89) soglia dell’haq richiesta per continuare il trattamento [19] 0,3 punteggio haq al baseline [19] 1,82 se un paziente discontinua la terapia con biologici, il punteggio haq peggiora di un ammontare uguale al guadagno avuto da quando è in trattamento [23] tutti i pazienti trattati hanno comunque una graduale progressione della malattia [23]: tnf-α metotressato ­ ­ 0,015 0,06 tasso di attualizzazione per costi ed esiti [24] 3% il punteggio haq è associato con la qualità della vita dei pazienti [25] costi diretti sanitari sono associati al punteggio haq [26] classe acr punteggio haq utilità media (ds) costi (€/paziente/anno) i <0,25 0,857 (0,16) 1.085,3 i 0,25-0,50 0,803 (0,13) 1.085,3 i 0,50-0,75 0,762 (0,14) 1.085,3 ii 0,75-1,00 0,713 (0,15) 1.835,9 ii 1,00-1,25 0,657 (0,15) 1.835,9 ii 1,25-1,50 0,590 (0,18) 1.835,9 iii 1,50-1,75 0,511 (0,19) 3.185,4 iii 1,75-2,00 0,427 (0,21) 3.185,4 iii 2,00-2,25 0,333 (0,24) 3.185,4 iv 2,25-2,50 0,229 (0,25) 4.594,1 iv 2,50-2,75 0,120 (0,27) 4.594,1 iv 2,75-3,00 0,034 (0,33) 4.594,1 tabella iii stima delle utilità per categoria del punteggio haq è stato assunto che il cambiamento percentuale medio nel punteggio haq a 6 mesi sia lo stesso di quello riportato a 12 mesi sulle evidenze che suggeriscono come il beneficio dei biologici sia costante nel tempo [9, 28, 29]. per riflettere il fatto che tutti i pazienti trattati con biologici hanno una graduale progressione della malattia è stato impiegato un tasso annuale di incremento dello 0,015 nel punteggio haq basato sulle stime riportate da brennan et al [23]. stima delle utilità la stima delle utilità è derivata dal database del national databank for rheumatic disease [30] (tabella iii). analisi di costo-utilità di abatacept nell’artrite reumatoide in italia 22 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2008; 9(1) costi i costi annuali della terapia farmacologica [31] sono riassunti nella tabella iv. i costi diversi da quelli dei trattamenti farmacologici sono stati tratti dallo studio di leardini et al. [32], l’unico reperibile nel contesto italiano (tabella v), dove le risorse riportate sono associate alle 4 categorie delle classi funzionali acr (american college of rheumatology) [33]. ad ogni classe acr si sono assunti associati i punteggi di haq secondo quanto riportato in tabella iii. i costi sono espressi in euro. analisi di sensibilità è stata eseguita un’analisi di sensibilità oneway su alcuni parametri chiave elencati quali (tabella vi): percentuale di cambiamento dell’haq a 6 mesi per abatacept; costo annuale del trattamento con farmaci biologici; tasso annuale della progressione dell’ haq per il trattamento con biologici; valore soglia di haq per la continuazione del trattamento; età al basale; haq al basale; costo annuale abatacept 11.256 biologici anti-tnfs: etanercept adalimumab infliximab ­ ­ ­ 10.663,01 * 12.448 12.205 7.249 metotressato 526,37 tabella iv costo annuale dei farmaci considerati nell’analisi (€) * media ponderata con i dati di consumo del 2007 forniti da ims tabella v costi diretti per classi funzionali acr classi acr i ii iii iv esami 61,4 79,0 105,3 114,6 esami strumentali 55,8 88,8 77,0 73,3 esami di laboratorio 130,6 162,6 177,6 206,5 fisioterapia 43,4 65,6 89,8 157,0 ospedalizzazione 399,6 751,7 1.466,7 1.968,5 riabilitazione 106,4 94,0 0 0 day hospital 60,9 101,7 59,4 13,4 trasporto 113,1 110,5 247,8 134,7 assistenza domestica 50,1 222,0 840,0 1.689,7 ausilio 64,0 160,0 121,8 236,4 totale 1.085,3 1.835,9 3.185,4 4.594,1 parametro valore testato % cambiamento dell’ haq a 6 mesi per abatacept 16,32%-24,48% costo annuale dei biologici anti-tnfs con sconto 5% -5% tasso annuale della progressione dell’haq per il trattamento con biologici 0-0,03 soglia haq per la continuazione del trattamento 0,22-0,7 età al baseline 40-60 haq al basale 2,05 moltiplicatore della mortalità 1-2,73 [34] orizzonte temporale (anni) 10-30 tasso di attualizzazione 0-5% sia costi che outcomes impatto economico del dose creeping di infliximab: costi diretti totali mg/kg somministrati ­ ­ +25% [35] 5mg/kg tabella vi parametri usati nell’analisi di sensibilità e valori testati s. de portu, l. g. mantovani, i. olivieri 23© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2008; 9(1) tabella vii analisi di costo-utilità (valori attualizzati al 3%) abatacept anti-tnfs differenza impatto del trattamento anni di vita 17,11280 16,79430 0,3185 haq finale 2,494433 2,693809 qaly 5,021909 3,837220 1,184689 costi del trattamento costi del trattamento 53.705,843 26.581,618 27.124,225 costi diretti 42.812,72 47.940,53 -5.127,81 costi diretti totali 96.518,56 74.522,15 21.996,41 costo per qaly 18.567,24 tabella viii risultati dell’analisi di sensibilità one-way: abatacept vs altri anti-tnfs descrizione risultati (€) variazione % % cambiamento dell’ haq a 6 mesi per abatacept 18.932,54-18.105,32 +1,96-2,48 costo annuale dei biologici anti-tnfs con sconto 5% 19.445,77 +4.73 tasso annuale della progressione dell’haq per il trattamento con biologici 20.808,18-17.088,48 +12,07-7,96 soglia haq per la continuazione del trattamento 16.819,63-17.698,75 -9,41-4,67 età al baseline 17.899,72-19.410,50 -3,59+6,35 haq al baseline 16.299,24 -12,21 moltiplicatore della mortalità 17.801,55-19,746,33 -4,12+6,35 orizzonte temporale 22.979.86-18.416,26 +23.77-0.81 tasso di attualizzazione 17.505,07-19.260,21 -5,72+3,73 impatto economico del “dose creeping” di infliximab: costi diretti totali mg/kg somministrati ­ ­ 17.701,37 dominante -4,66 moltiplicatore della mortalità; orizzonte temporale; tasso di attualizzazione impatto economico del dose creeping di infliximab. risultati l’analisi suggerisce che, con un orizzonte temporale di 20 anni, la terapia con abatacept permette in termini di efficacia di guadagnare 0,3185 anni di vita, che si traduce in 1,19 qaly guadagnati per paziente con un costo incrementale pari a € 21.996 (abatacept € 96.518 vs altri biologici € 74.522) (tabella vii). il rapporto di costo-efficacia incrementale (icer) per qaly guadagnato è pari a € 18.567,24. i risultati dell’analisi di sensibilità sono mostrati in tabella viii. discussione lo scopo dello studio è stato di determinare, attraverso un modello adattato alla realtà italiana nella prospettiva del ssn, il rapporto di costoefficacia di abatacept, dopo il fallimento terapeutico con dmards, verso gli altri anti-tnfs, quantificando il costo per qaly guadagnato. i risultati suggeriscono che abatacept, impiegato in pazienti adulti con artrite reumatoide in fase attiva che hanno risposto inadeguatamente ai trattamenti con dmards incluso un antitnf-α, è costo-efficace quando confrontato con gli altri farmaci biologici. il costo per qaly guadagnato di abatacept verso gli altri anti-tnfs è risultato pari a € 18.567,24, icer che rimane al di sotto della soglia di accettabilità (€ 45.000/50.000 per qaly) [36] per cui un farmaco viene ritenuto convenzionalmente costo-efficace, alla luce del quale il costo aggiuntivo del farmaco dovrebbe essere sostenuto a fronte di un reale beneficio per il paziente. l’analisi ha mostrato come il trattamento con abatacept porta ad una riduzione dei costi diretti a carico dell’ssn, anche se non tale da compensare interamente il costo addizionale dovuto al trattamento. analisi di costo-utilità di abatacept nell’artrite reumatoide in italia 24 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2008; 9(1) i risultati dell’analisi appaiono sostanzialmente stabili al variare dei parametri principali (tabella vi) e date le assunzioni del modello. infatti, il rapporto di costo-efficacia rimane sempre al di sotto della soglia dei € 45.000 per qaly. alcune delle assunzioni del modello base rendono la nostra analisi conservativa dal punto di vista farmacoeconomico. si è assunto, infatti, una soglia di efficacia in termini di punteggio haq pari a 0,3 quando attualmente la soglia di efficacia considerata è 0,22 [37-39]. tale soglia è stata testata nell’analisi di sensibilità (tabella viii) e porta ad un abbassamento del rapporto di costo-efficacia di quasi 10 punti percentuali (€ 16.819,63). inoltre, il modello assume conservativamente che non vi sia nessun incremento del dosaggio degli anti-tnfs, un fenomeno la cui ampiezza è descritta in numerosi studi internazionali [35,40-42]. poiché non sono ad oggi disponibili evidenze a livello italiano si è deciso di considerare tali conseguenze solo nell’analisi di sensibilità, assumendo dallo studio di gilbert et al. [35] un aumento pari al 25% nei costi diretti a seguito dell’aumento nel dosaggio di infliximab. tale considerazione porta una diminuzione di circa il 5% (€ 17.701,37) nel costo per qaly guadagnato di abatacept vs gli altri anti-tnfs. diversamente, se si considera un incremento della dose fino a 5 mg/kg, i costi della terapia con anti-tnf crescono fino a rendere la terapia con abatacept dominante (meno costosa e più efficace). l’analisi mostra comunque alcuni limiti, il principale dei quali è rappresentato dal fatto che le conseguenze indirette, quali la perdita o il guadagno di produttività legati ad una malattia cronica e invalidante come l’artrite reumatoide, non sono state incluse. sarebbe interessante valutare come la terapia con abatacept, data la sua efficacia, possa influenzare l’attività lavorativa dei pazienti: si può ipotizzare, infatti, che i pazienti in trattamento con abatacept abbiano una minore riduzione nella capacità lavorativa a fronte di un maggior beneficio clinico. per questo motivo l’introduzione nella valutazione economica anche dei costi indiretti potrebbe aumentare i benefici economici derivanti dall’utilizzo di tale farmaco. disclosure l’articolo è stato realizzato grazie al contributo di bristol myers squibb. bibliografia 1. harris ed jr. rheumatoid arthritis: pathophysiology and implications for therapy. n engl j med 1990;322: 1277-89 2. silman a, hochberg mc. epidemiology of rheumatic diseases. oxford: oxford university press, 1993 3. symmons d, silman a. the epidemiology of rheumatoid arthritis. in: wolfe f, pincus t, eds. rheumatoid arthritis: pathogenesis, assessment, outcome and treatment. new york: marcel dekker, 1994:131-50 4. fex e, larsson b-m, nived k, eberhardt k. impact of rheumatoid arthritis on work status and social and leisure time activities in patients followed 8 years from onset. j rheumatol 1997;25:44-50 5. barrett e, scott d, wiles n, symmons d. the impact of rheumatoid arthritis on employment status in the early years of disease: a uk community-based study. rheumatology 2000;39:1403-9 6. smolen js, steiner g. therapeutic strategies for rheumatoid arthritis. nat rev drug discov 2003;2:473-88 7. weinblatt me, keystone ec, furst de, et al. adalimumab, a fully human anti-tumor necrosis factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the armada trial. arthritis rheum 2003; 48: 35-45. errata, arthritis rheum 2003;48: 855 8. weinblatt me, kremer jm, bankhurst ad, et al. a trial of etanercept, a recombinant tumor necrosis factor receptor: fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. n engl j med 1999;340:253-9 9. lipsky pe, van der heijde dm, st clair ew, furst de, breedveld fc, kalden jr, et al. infliximab and methotrexate in the treatment of rheumatoid arthritis. anti-tumor necrosis factor trial in rheumatoid arthritis with concomitant therapy study group. n engl j med 2000;343:1594-602 10. maini r, st clair ew, breedveld f, furst d, kalden j, weisman m, et al. infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase iii trial. attract study group. lancet 1999; 354:1932-9 s. de portu, l. g. mantovani, i. olivieri 25© seed tutti i diritti riservatifarmeconomia e percorsi terapeutici 2008; 9(1) 11. buch mh, bingham sj, bryer d, emery p. long-term infliximab treatment in rheumatoid arthritis: subsequent outcome of initial responders. rheumatology (oxford) 2007;46:1153-6 12. wolbink gj, vis m, lems w, voskuyl ae, de groot e, nurmohamed mt, et al. development of antiinfliximab antibodies and relationship to clinical response in patients with rheumatoid arthritis. arthritis rheum 2006; 54:711-5 13. bartelds gm, wijbrandts ca, nurmohamed mt, stapel s, lems wf, aarden l, et al. clinical response to adalimumab: relationship to anti-adalimumab antibodies and serum adalimumab concentrations in rheumatoid arthritis. ann rheum dis 2007; 66:921-6 14. moreland lw, alten r, van den bosch f, et al. costimulatory blockade in patients with rheumatoid arthritis: a pilot, dose-finding, double-blind, placebo-controlled clinical trial evaluating ctla-4ig and lea29y eighty-five days after the first infusion. arthritis rheum 2002;46:1470-9 15. kremer jm, westhovens r, leon m, et al. treatment of rheumatoid arthritis by selective inhibition of t-cell activation with fusion protein ctla4ig. n engl j med 2003; 349:1907-15 16. kremer jm, dougados m, emery p, et al. treatment of rheumatoid arthritis with the selective costimulation modulator abatacept: 12-month results of a phase iib, double blind, randomized, placebo-controlled trial. arthritis rheum 2005;52:2263-71 17. kremer j, westhovens r, luggen m, et al. long term efficacy and safety of abatacept through 3 years of treatment in rheumatoid arthritis patients in the aim and attain trials. american college of rheumatology congress 2007 18. westhovens r, kremer j, moreland l, et al. maintained efficacy and safety of abatacept in rheumatoid arthritis patients receiving background methotrexate through 5 years of treatment. american college of rheumatology congress 2007 19. genovese mc, becker jc, schiff m, et al. abatacept for rheumatoid arthritis refractory to tumor necrosis factor α inhibition. n engl j med 2005;353:1114-23 20. scheda tecnica, autorizzazione emea. disponibile online su http://www.emea.europa.eu/humandocs/pdfs/epar/orencia/h-701-pi-it.pdf 21. clinical study report. october 2004 im101029 a phase iii, multi-centre, randomized, double-blind, placebocontrolled study to evaluate the efficacy and safety of abatacept in subjects with active rheumatoid arthritis on background dmards who have failed anti-tnf therapy. bristol myers squibb co 22. wolfe f, michaud k, geffeler o, et al. predicting mortality in patients with rheumatoid arthritis. arthritis rheum 2003; 48:1530-42 23. brennan a, bansback n, reynolds a, conway p. modelling the cost-effectiveness of etanercept in adults with rheumatoid arthritis in the uk. rheumatology 2004;43:62–72 24. capri, s, ceci, a, terranova, l, merlo, f, mantovani, l. guidelines for economic evaluations in italy: recommendations from the italian group of pharmacoeocnomic studies. drug information journal 2001; 35:189-201 25. hurst np, kind p, ruta d, hunter m, stubbings a. measuring health-related quality of life in rheumatoid arthritis: validity, responsiveness and reliability of euroqol (eq-5d). br j rheumatol 1997; 36:551-559 26. michaud k, messer j, choi hk and wolfe f. direct medical costs and their predictors in patients with rheumatoid arthritis. arthritis & rheumatism 2003; 48: 2750-62 27. http://www.emea.europa.eu/htms/human/epar/a.htm 28. maini rn, breedveld fc, kalden jr, et al. sustained improvement over two years in physical function, structural damage, and signs and symptoms among patients with rheumatoid arthritis treated with infliximab and methotrexate. arthritis rheum 2004;50:1051-65 29. olsen nj, stein mc. new drugs for rheumatoid arthritis. n engl j med 2004; 350: 2167-79 30. wolfe f. national data bank for rheumatic diseases. us, 2004 (unpublished data) 31. http://www.agenziafarmaco.it 32. leardini g, salaffi f, montanelli r, gerzeli s, canesi b. a multicenter cost-of-illness study on rheumathoid arthritis in italy. clinical and experimental rheumatology 2002, 20:505-15 33. hochberg mc, chang rw, dwosh i, lindsey s, pincus t, wolfe f. the american college of rheumatology 1991 revised criteria for the classification of global functional status in rheumatoid arthritis. arthritis rheum 1992; 35: 498-502 analisi di costo-utilità di abatacept nell’artrite reumatoide in italia 26 © seed tutti i diritti riservati farmeconomia e percorsi terapeutici 2008; 9(1) 34. sokka t, hakkinen a, krishnan e, hannonen p. similar prediction of mortality by the health assessment questionnaire in patients with rheumatoid arthritis and the general population. ann rheum dis 2004;63:494-7 35. gilbert td, smith d, ollendorf da. patterns of use, dosing, and economic impact of biologic agent use in patients with rheumatoid arthritis: a retrospective cohort study. bmc musculoskeletal disorders 2004; 5:36 36. national institute for clinical excellence. guide to the methods of technology appaisal.(2004). reference no515. disponibile online su http://www.nice.org.uk/tap_methods.pdf 37. wells ga, tugwell p, kragg gr, et al. minimum important difference between patients with rheumatoid arthritis: the patient’s perspective. j rheumatol 1993;20:557-60 38. kosinski m, zhao sz, dedhiya s, et al. determining minimally important changes in generic and disease-specific health-related quality of life questionnaires in clinical trials of rheumatoid arthritis. arthritis rheum 2000;43:147887 39. strand v, bombardier c, maetzel a, et al. use of minimum clinically important differences (mcid) in evaluating patient responses to treatment of ra. arthritis rheum 2001; 44: s187 40. ollendorf da, massarotti e, birbara c et al. frequency, predictors, and economic impact of upward dose adjustment of infliximab in managed care patients with rheumatoid arthritis. j manag care pharm 2005;11:383-93 41. sidiropoulos p, bertsias g, kritikos hd et al. infliximab treatment for rheumatoid arthritis, with dose titration based on the disease activity score: dose adjustments are common but not always sufficient to assure sustained benefit. ann rheum dis 2004; 63:144-148 42. ariza-ariza r, navarro-sarabia f, hernandez-cruz bet al. dose escalation of the anti-tnf-α agents in patients with rheumatoid arthritis. a systematic review. concise report rheumatology 2006; 1 of 4 maisaa final.doc j bagh college dentistry vol. 26(3), september 2014 salivary c-reactive orthodontics, pedodontics and preventive dentistry 138 salivary creactive protein in relation to periodontal health among a group of patients with rheumatoid arthritis in iraq maisaa i. abdul qadir, b.d.s, h.d.d. (1) athraa m. al-waheb, b.d.s., m.sc. (2) abstract background: rheumatoid arthritis is a chronic destructive inflammatory disease associated with destruction of joint connective tissues and bones, affecting 0.5%–1% of the population worldwide reporting higher prevalence of periodontitis among rheumatoid arthritis patients. the purpose of this study is to estimate level of salivary c-reactive protein in relation to the occurrence and severity of the periodontal disease and other oral parameters among group of patients with rheumatoid arthritis material and methods: fifty women patients with rheumatoid arthritis; twenty five on methotrexate treatment and twenty five on combination treatment of methotrexate and etanercept selected as study groups with an age range (30-40) years old and twenty five gender, age and body mass index matched healthy looking persons were selected as control. the diagnosis and recording of periodontal condition recorded through the application of community periodontal index according to who1997.collection of unstimulated salivary samples was carried out under standard conditions, in addition to estimation of salivary c-reactive protein. results: regarding count of sextants with community periodontal index, median count of sextant with cpi-score 0 was highest among controls (2) and lowest among both rheumatoid arthritis cases (0).the median count of sextant with cpi-score 3 was lowest among controls (0) and highest among both ra cases (2) and the difference observed in median count of sextant with cpi-score 3 between three groups was statistically significant (p< 0.01).the mean rank of salivary c-reactive protein was highest among controls 39.7mg/l however, the difference was not significant between three groups (p>0.05). conclusion: the results of the current research revealed that periodontal diseases were higher among rheumatoid arthritis patients without impact of both treatments on periodontal health without significant role of salivary creactive protein clinically in assessment of disease activity. key words: rheumatoid arthritis, salivary c-reactive protein, community periodontal index. (j bagh coll dentistry 2014; 26(3):138-143). introduction rheumatoid arthritis (ra) is a chronic systemic autoimmune inflammatory disease that affects all ethnic groups throughout the world. females are 2.5 times more likely to be affected than males because men may be protected by hormonal factors and require a stronger genetic component to develop disease (1,2). a prevalence survey for rheumatoid arthritis in iraq was carried out during the summer of 1975 and definite rheumatoid arthritis was observed in 1 % of the 6999 individuals studied (3). periodontal diseases (pd) are the second most common oral diseases next to dental caries, they are considered to be inflammatory disorder that damages tissue through the complex interaction between perio-pathogens and the host defense systems (4,5). periodontitis is one of common oral manifestation of ra, evidence that individuals suffering from ra are more likely to experience periodontitis was found by several studies (6-8). periodontitis and ra are both chronic destructive inflammatory disorders and result from deregulation of the host inflammatory response (9). (1)m.sc. student department of pedodontics and preventive dentistry, college of dentistry, university of baghdad. (2)professor, department of pedodontics and preventive dentistry, college of dentistry, university of baghdad. corgel et al. (10) reported thatinflammatory periodontal diseases exhibit an association with ra andthe effective treatment of periodontal infections is important to achieve oral health goals, as well as to reduce the systemic risks of chronic local inflammation and bacteremias. c reactive protein (crp) is a liver-produced, acutephase reactant; that serves as a systemic marker of inflammation; the name is derived due to the ability of the crp to react with c-polysaccharide isolated from pneumococcal cell walls (11); its levels can be used to monitor patients with overwhelming infections, and elevated crp levels have been demonstrated in persons with ischemia and myocardial infarction (12). alsopersistent elevations in crp are seen in chronic inflammatory states such as active ra, pulmonary tuberculosis, or extensive malignant disease (13). periodontal disease, being a low grade inflammatory disease of the tooth supporting structures, may increase blood levels of inflammatory markers including il-6 and crp (14). al-ghurabei found a significant elevation of mean serum level of high sensitive crp (hscrp) with periodontitis and hs-crp was showed significant positive correlation with each of probing pocket depth (ppd), clinical attachment j bagh college dentistry vol. 26(3), september 2014 salivary c-reactive orthodontics, pedodontics and preventive dentistry 139 loss (cal) and bleeding on probing (bop) among group of iraqi patients (15). slade et al. found an association between extensive periodontal disease and bmi with increased crp levels in otherwise healthy, middle-aged adults, suggesting the need for medical and dental diagnoses when evaluating sources of acute-phase response in some patients (12). saliva is a body fluid, its role as a diagnostic tool has advanced exponentially over the past decade assaliva sampling is safe for both operator and patient, making saliva possible to monitor several biomarkers when blood sampling is not available (16,17). components within saliva may provide additional clues on systemic health conditions. besides, the development of new technologies may promote a wider use of salivary assays in the near future (18). yet, no previous iraqi study could be found that investigates the salivary levels c-reactive protein and its relation to periodontal health among rheumatoid arthritis patients, for this research designed. materials and methods approval was achieved from the ministry of health in iraq for examining the patients with rheumatoid arthritis and collecting samples, in addition to that, the objectives of the study was explained for each participant. the study group composed of fifty ra women patients, with an age range (30-40) years. they were diagnosed clinically, by rheumatology specialist as rheumatoid arthritis depending on the seven criteria of the american rheumatism association with assessment of disease activity depending on disease activity score 28 (das28), they were divided in to two subdivisions groups: the first group (twenty five) under conventional treatment disease-modifying anti-rheumatic drugs dmard (methotrexate mtx) and the second group (twenty five) under combination treatment with anti-tnf-α (etanercept) and dmards (mtx). they were attending baghdad teaching hospital for their treatment. the control group composed of twenty five subjects and they wereapparently healthy according to their medical history matching with age, gender and body mass index bmi of the study group.the disease activity score 28 was calculated with the following equation using esr: das 28 = (0.56 × √tjc) + (0.28 × √sjc) + (0.70 × logn esr) + (0.014 × pga).esr = erythrocyte sedimentation rate.gas = global arthritis score, logn = log-normal, sjc = swollen joint count of 28 joints, tjc = tender joint count of 28 jointspga= patient global assessment. height and weight were determined with the subjects wearing light clothing without shoes using height and weight measurement mechanical scale. the body mass index (bmi) was calculated as (body weight/height 2) (kg/m2). the periodontal status was assessed by using specially designed light weight community periodontal index probe. the community periodontal indexdivide mouth into sextants defined by tooth numbers: (18-14), (13-23), (2428), (38-34), (33-43) and (44-48)with 10 index teeth(17,16,11,26,27,47,46,31,36,37), examination of four surfaces ofthe index teeth, and/or all remaining teeth in a sextant where there are no index teeth, was probed and the highest score was recordedfollowing the criteria of community periodontal index 1997.aspecific measure for periodontitis; plaque index was assessed (silness and loe 1964).the whole unstimulated salivary samples was collected for five minutes from the study and control groups and was performed under standardized conditions according to the instructions cited by tenovuo and lagerlöf (19).the salivary samples were then taken to the laboratory for biochemical analysis and centrifuged at 3000 r.p.m. for 10 minutes. the clear supernatant was separated by micropipette and was stored at (20°c) in a deep freeze and further detection of the crp presence in saliva was done by (huma tex crp). crp determination is based on the immunological reaction between human c-reactive protein of a patient specimen and the corresponding antihuman crp antibodies bound to latex particles, the positive reaction is indicated by a distinctly visible agglutination of the latex particles; positive reaction meaning that a content of crp in the sample equal to or greater than 6 mg/1. samples with positive results in the screening test were retested in the titration test. samples diluted with glycine-nacl buffer (gbs) ((ref 40037)) according to manufacture criteria of the kit, the titer read in the last dilution step with visible agglutination and multiplied the titer with the conversion factor 6 getting the result in mg/l. inter and intra examiner calibrations were performed to obtain the most critical consistency of diagnostic criteria.the results were analyzed using paired t-test and there were no significant differences (p<0.05) between the first and second observation of inter examiner calibration. statistical analysis data were translated into a computerized database structure. the database was examined for errors using range and logical data cleaning methods, and inconsistencies were remedied. an expert statistical advice was sought for. statistical analyses were done using spss j bagh college dentistry vol. 26(3), september 2014 salivary c-reactive orthodontics, pedodontics and preventive dentistry 140 version 20 computer software (statistical package for social sciences). compliance of quantitative random variables with gaussian curve (normal distribution) was analyzed using the kolmogorov-smirnov test. the correlation coefficient tests between the variables were done by using spearman’s rank linear correlation coefficient.p value less than 0.05 and 0.01 was considered statistically significant. results the study and control groups were comparable in age and body structure (table1).the mean values in addition to standard deviation and p values measured by the least significant difference (lsd) in mean of plaque index among study and control groups demonstrated in table (2).the mean plaque index was highest among ra cases on mtx treatment (1.9) and lowest among controls (1.1). the difference observed in mean plaque index between three groups was statistically significant. the mean plaque index was significantly lower (1.1) in controls compared to both ra on etanercept and ra on mtx (1.7 and 1.9 respectively).table (3) illustrates the median, mean rank and p (mann-whitney) for difference of sextants with cpi scores (0, 1, 2, 3 and 4) among study and control groups.the median count of sextant with cpi-score 0 was highest among controls (2) and lowest among both ra cases (0). the difference observed in median count of sextant with cpi-score 0 between three groups was statistically significant. the median count of sextant with cpi-score 0 was significantly higher (2) among controls compared to both ra on etanercept and ra on mtx (0 and 0 respectively).mean rank of sextants with cpi score 1 was obviously highest among ra cases on mtx (40.7) followed by ra cases on etanercept (37.8) and lowest among controls (35.5). the differences observed however, failed to reach the level of statistical significance.mean rank of sextants with cpiscore 2 was obviously highest among ra cases on etanercept (40.6) followed by ra cases on mtx (36.8) and lowest among controls (36.6). the differences observed however, failed to reach the level of statistical significance.the median count of sextant with cpi-score 3 was lowest among controls (0) and highest among both ra cases (2). the difference observed in median count of sextant with cpi-score 3 between three groups was statistically significant. the median count of sextant with cpi-score 3 was significantly higher among both ra on etanercept and ra on mtx (2 and 2 respectively) compared to controls (0). mean rank of sextants with cpiscore 4 was obviously highest among ra cases on etanercept (39.5) followed by controls (38.0) and lowest ra on mtx (36.5). the differences observed however, failed to reach the level of statistical significance.table (4) illustrates the difference in median and mean rank of salivary crp and between the two ra cases groups and control.mean rank of salivary crp was obviously highest among controls (39.7 mg/1) followed by ra cases on mtx (38.6 mg/1) and lowest ra cases on etanercept (35.7 mg/1). the differences observed however, failed to reach the level of statistical significance.table (5) clarifies the difference in median and mean rank of salivary crp between ra cases with high disease activity (das=5.1+) and those with low disease activity. there was statistically not significant difference between positive and negative of high disease activity in salivary crp.table (6) demonstrates the correlations coefficient between count of sextants with cpiscores and salivary crp for ra cases. there was weak and not significant linear correlation among count of sextants with cpi-scores and salivary crp for ra cases and in all direction of relation. table 1: age and body mass index among study groups. age in years apparently healthy controls n=25 rheumatoid arthritis treated with methotrexate n=25 rheumatoid arthritis treated with etanercept n=25 p value range (30-40) (30-40) (30-40) (ns) mean 33.8 34.1 35.8 sd 3.2 3.78 3.57 bmi (kg/m2) (ns) range (18.3-38.6) (16.9-44.4) (17.3-45) mean 28.4 30.1 28.5 sd 5.66 7.7 5.73 * d.f (between groups/within groups) = 2/72. j bagh college dentistry vol. 26(3), september 2014 salivary c-reactive orthodontics, pedodontics and preventive dentistry 141 table 2: comparison of mean plaque index among three groups (control, ra on mtx and ra on etanercept) variable control group (mean ±sd) n=25 methorexate group (mean ±sd) n=25 etanercept group (mean ±sd) n=25 p1 control + methotrxate p2 control + etanercept p3 methotrxate + etanercept f value p value for difference between 3 groups plaque index 1.1±0.81 1.9±0.78 1.7±0.73 < 0.001 0.007 0.46(ns) 6.919 0.002 * d.f (between groups/within groups) = 2/72. table 3: the difference in: median and mean rank of sextants with cpi-scores (0, 1, 2, 3 and 4) between the two ra cases groups and control group. count of sextant with cpi control group median (mean rank). n=25 methotrexate group median (mean rank). n=25 etanercept group median (mean rank). n=25 p1 (mannwtiney) control + mtx p2 (mannwtiney) control + etanercept p3 (mannwtiney) mtx + etanercept p value for difference between 3 groups score 0 2 (53.4) 0 (31.8) 0(28.8) <0.001 <0.001 (ns) <0.001 score 1 2 (35.5) 2 (40.7) 2(37.8) ** ** ** (ns) score 2 1 (36.6) 1 (36.8) 1(40.6) ** ** ** (ns) score 3 0 (23.6) 2 (43.1) 2(47.2) <0.001 <0.001 (ns) <0.001 score 4 0 (38.0) 0 (36.5) 0(39.5) ** ** ** (ns) ** not calculated, because the overall p values for difference between the three groups not reach the level of statistically significance. table 4: the difference in median and mean rank of salivary crp between two ra cases groups and control salivary crp (mg/1) apparently healthy controls n=25 ra treated with methotrexate n=25 ra treated etanercept n=25 p value range (0-48) (0-192) (0-48) (ns) median 0 0 0 mean rank 39.7 38.6 35.7 inter-quartile range (0-0) (0-0) (0-0) table 5: correlation coefficient between count of sextants with cpi-scores and salivary crp for ra cases variable linear correlation coefficient salivary crp (mg/l) count of sextants with cpi-score 0 r 0.083 p (ns) count of sextants with cpi-score 1 r 0.075 p (ns) count of sextants with cpi-score 2 r 0.161 p (ns) count of sextants with cpi-score 3 r 0.06 p (ns) count of sextants with cpi-score 4 r 0.089 p (ns) table 6: correlation coefficient between salivary crp with disease activity score among ra cases variables linear correlation coefficient salivary leptin(ng/ml) salivary crp(mg/l) disease activity score salivary crp (mg/l) r 0.242 0.004 p (ns) (ns) j bagh college dentistry vol. 26(3), september 2014 salivary c-reactive orthodontics, pedodontics and preventive dentistry 142 discussion rheumatoid arthritis (ra) is a common chronic systemic, inflammatory disease affecting the adult population; affected individuals experience significant morbidity, including loss of function, joint destruction, and permanent deformity, and have higher mortality than the general population (20). in recent years, studies reported remarkable epidemiological and pathological relationships between periodontal diseases and rheumatic diseases, especially rheumatoid arthritis (7,8). in present study the mean plaque index was significantly lower in controls compared to both ra on etanercept and ra on mtx. the results of current study come in agreement with findings obtained by recent iraqi study carried out by mahmood et al. in 2012, as plaque index differed significantly between ra (higher) patients and control (lower). the stiffness of hands muscles to achieve good oral hygiene among ra patients and changing in the life style of ra patients, as hands muscle function reduces leading to improper oral hygiene mechanism may explain elevation in plaque index among ra cases, also psychological well-being of patients with ra; they are more likely to suffer from anxiety, depression and low self-esteem which may affect their proper oral hygiene. in terms of periodontal diseases among the study and control groups, the community periodontal index used in which the median count for each score count the number of sextants containing that score only and then present the median for the group. the present study was found that the median count of sextant with cpi-score 0 was significantly higher among controls compared to both ra on etanercept and ra on mtx. gingival inflammation was obviously highest among ra cases on mtx (40.7) followed by ra cases on etanercept (37.8) and lowest among controls (35.5), but statistically not significant. although the prevalence of dental plaque was higher among study groups, gingival bleeding was not different between study and control groups. these results come in agreement with results obtained from previous studies (22, 23). the accepted explanation for these results that the chronic use (prolonged and continuous) of antiinflammatory drugs; non steroid antiinflammatory drugs (nsaids) and steroidal anti-inflammatory drugs (saids) which are used by majority of ra patients and cannot be excluded individuals who use these drugs may modulate plaque-associated gingivitis, however, results of present study are contradict findings of other studies regarding gingival bleeding and gingival inflammation which reported elevation among ra patients and explained by higher plaque index as a causative factor for gingival inflammation (24), this variation in the results may related to difference in age, type of treatment and index use for evaluation of gingival condition. the periodontal pocket is one of the most important clinical features of periodontal disease (25). pocket depth (4-5 mm) was evaluated in this study to be statistically significant higher among ra cases (both on mtx and etanercept) compared with healthy controls, these results come to confirm findings obtained from several studies; where most patients with ra study showed moderate-to-severe periodontitis (26) and also support the concept that patients with longstanding rheumatoid arthritis have substantially increased periodontal disease compared to healthy subjects. in the current study effect of both treatments (methotrexate and etanercept) on periodontal health was evaluated revealing no significant difference between two treatments regarding effect on periodontal health, this is consistent with findings of a previous study in ra subjects that anti-tnf-α therapy without periodontal treatment has no significant effect on the periodontal condition (27). in the same study the methotrexate effect on periodontal health was evaluated and also has no effect on periodontal condition without periodontal treatment confirming the inter-relationship between ra and pd. estimation levels of salivary biomarker crp among ra patients was one of aims of current study, as quantitative changes of specific salivary biomarkers could have significance in the diagnosis and management of both oral and systemic diseases; levels of salivary crp in present study reveals no significant difference was observed between the two ra study groups (mtx and etanercept) and controls (38.6 mg/l, 35.7 mg/l and 39.7 mg/l) respectively. serum crp in rheumatoid arthritis patients has been evaluated extensively but unfortunately, there was no study able to be found estimates its level in saliva. this non-significant difference between study and control groups may relate to effect of treatment, since hochberg et al.in 2011 reported that mtx rapidly decrease serum crp with the minimum value being noted on day 3 after onceweekly dosing and su et al. in 2009 demonstrated significant improvements in serum crp values at the end of the first 3 months treatment with etanercept and as saliva is mirror of the body reflects its concentration in serum, this may be an acceptable explanation. this study reveals weak correlation of salivary crp with das 28 among j bagh college dentistry vol. 26(3), september 2014 salivary c-reactive orthodontics, pedodontics and preventive dentistry 143 both groups of ra patients, this agree with keenan et al. (28) who concluded from his study regarding esrand crp values in ra andwhether or not they were correlated with outcomes in raby measures of disease activity;the study revealed that they were weakly correlated with disease activity measures.in conclusion periodontal destruction represented in pocket depth was statistically significant higher among both ra cases (on mtx and combination of mtx and etanercept) compared with healthy controls concluded severity of periodontal disease and that both treatments have no impact on periodontal destruction without periodontaltreatment among patients with ra, suggesting especial periodontal treatment and preventive programs, alsoanother outcome of current study that salivary crp using this assay is of low importance and not meaningfulclinically to assess the activity of the disease suggesting more specific and sensitive assays such as salivary high sensitivity crp assay. references 1. ollier w, harrison b, symmons d. what is the natural history of rheumatoid arthritis? best practclinrheumatol 2001; 15(1): 27-48. 2. klippel j, stone j, crofford l, white p. primer on the rheumatic disease. 13th ed. new york: springer; 2008. 3. al-rawi z, al-azzawi a, al-ajilli f, al-wakil r. rheumatoid arthritis in population samples in iraq. annals of the rheumatic diseases 1978; 37: 73-5. 4. detert j, pischon n, burmester g, buttgereit f. pathogenesis of periodontitis in rheumatic disease. z rheumatol 2010; 69:109-12. 5. khashu h, baiju c, bansal s, chhillar a. salivary biomarkers: a periodontal overview. j oral health comm dent 2012; 6(1): 28-33. 6. joseph r, rajappan s, nath s, paul b. association between chronic periodontitis and rheumatoid arthritis. rheumatol int 2013; 33:103–9. (ivsl). 7. detert j, pischon n, burmester g, buttgereit f. the association between rheumatoid arthritis and periodontal disease. arthritis research & therapy. 2010; 12: 218. 8. abdelsalam s, hashim n, elsalamabi e, gismalla b. periodontal status of rheumatoid arthritis patients in khartoum state. bmc research notes 2011; 4: 460. 9. berthelot j and le goff b.rheumatoid arthritis and periodontal disease. joint bone spine 2010; 77: 53741. 10. corgel j, pucher j, rethman m, reynolds m. state of the science: chronic periodontitis and systemic health. j evid base dent pract 2012; 1: 20-8. 11. pepys m, gideon m. hirschfield c-reactive protein: a critical update. j clin invest 2003; 111: 1805-12. 12. slade g, ghezz e, heiss g, beck j, riche e, offenbacher s. relationship between periodontal disease and creactive protein among adults in the atherosclerosis risk in communities study. arch intern med 2003; 163(10):1172-9. 13. firestein g, budd r, gabriel r, mcinnes i, o’dell j. kelley’s textbook of rheumatology. 9th ed. elsevier: 2013. 14. vidal f, figueredo c, cordovil i. periodontal therapy reduces plasma levels of interleukin-6, c-reactive protein, and fibrinogen in patients with severe periodontitis and refractory arterial hypertension. j periodontol 2009; 80(5): 786-91. 15. al-ghurabei b. evaluation of serum anti-cardiolipin antibody, hs-crp and il-6 levels in chronic periodontitis as possible risk factors for cardiovascular diseases. j bagh coll dent 2012; 24(2):161-5. 16. streckfus c, bigler l. saliva as a diagnostic fluid. oral dis 2002; 8: 69-76. 17. castagnola m, picciotti t, messana i, fanali c, fiorita a, cabras t, calò l, pisano e, passali g, iavarone f, paludetti g, scarano e. potential applications of human saliva as diagnostic fluid. acta otorhinolaryngologica italica 2011; 31: 34757. 18. tremblay m, loucif y, methot j, brisson d, gaudet d. salivary ph as a marker of plasma adiponectin concentration in women. d and ms 2012; 4: 4. 19. tenovou j, legerlöf f. saliva in: thylstup a, fejerskov o (eds). text book of clinical cariology. 2nd ed. copenhagen: munksgaard; 1996. pp.17-43. 20. heijde d, klareskog l, valverde v, codreanu c, bolosiu h, gomes j, molina j, wajdula j, pedersen r, fatenejad s. comparison of etanercept and methotrexate, alone and combined, in the treatment of rheumatoid arthritis. arthritis & rheumatism 2006; 54(4): 1063–74. 21. blaizot a, monsarrat p, constantin a, vergnes n, de grado f, nabet c, cantagrel a, sixou m. oral health-related quality of life among outpatients with rheumatoid arthritis. int dent j 2013; 63(3):145-53. 22. ishi p, bertolo b, rossa j, kirkwood l, onofre a. periodontal condition in patients with rheumatoid arthritis. braz oral res 2008; 22(1): 72-7. 23. abinaya p, kumar r. prevalence of periodontal disease among individuals with rheumatoid arthritis. jiads 2010; 1(4): 16-23. 24. mahmood a, shukri m. assessment of salivary elements (zinc, copper and magnesium) among groups of patients with rheumatoid arthritis and chronic periodontitis and its correlation to periodontal health status. j bagh coll dent 2012; 24(3): 87-92. 25. newman m, takei h, klokkevold p, carranza f. carranza’s clinical periodontology.10th ed. elsevier; 2010. 26. ziebolz d, pabel s, lange k, krohn-grimberghe b, hornecker e, mausberg r.clinical periodontal and microbiologic parameters in patients with rheumatoid arthritis. j periodontol 2011; 82(10): 1424-32. 27. ortiz p, bissada n, askari a. periodontal therapy reduces the severity of active rheumatoid arthritis in patients treated with or without tumor necrosis factor inhibitors. j periodontal 2009; 80(4): 535-40. 28. keenan r, swearingen c, yazici y. erythrocyte sedimentation rate and c-reactive protein levels are poorly correlated with clinical measures of disease activityin rheumatoid arthritis, systemic lupus erythematosus and osteoarthritis patients. clinical and experimental rheumatol 2008; 26: 814-9. key: cord-0070325-pd9e926j authors: mustafa, ghulam; mahrosh, hafiza salaha; salman, mahwish; sharif, sumaira; jabeen, raheela; majeed, tanveer; tahir, hafsah title: identification of peptides as novel inhibitors to target ifn-γ, il-3, and tnf-α in systemic lupus erythematosus date: 2021-11-13 journal: biomed res int doi: 10.1155/2021/1124055 sha: 4c01aab19d1ee43f12f0bdf4955eed084b1d6d31 doc_id: 70325 cord_uid: pd9e926j autoimmune disorder is a chronic immune imbalance which is developed through a series of pathways. the defect in b cells, t cells, and lack of self-tolerance has been greatly associated with the onset of many types of autoimmune complications including rheumatoid arthritis, systemic lupus erythematosus (sle), multiple sclerosis, and chronic inflammatory demyelinating polyneuropathy. the sle is an autoimmune disease with a common type of lupus that causes tissue and organ damage due to the wide spread of inflammation. in the current study, twenty anti-inflammatory peptides derived from plant and animal sources were docked as ligands or peptides counter to proinflammatory cytokines. interferon gamma (ifn-γ), interleukin 3 (il-3), and tumor necrosis factor alpha (tnf-α) were targeted in this study as these are involved in the pathogenesis of sle in many clinical studies. two docking approaches (i.e., protein-ligand docking and peptide-protein docking) were employed in this study using molecular operating environment (moe) software and haddock web server, respectively. amongst docked twenty peptides, the peptide dedtqammpfr with s-score of -11.3018 and haddock score of −10.3 ± 2.5 kcal/mol showed the best binding interactions and energy validation with active amino acids of ifn-γ protein in both docking approaches. depending upon these results, this peptide could be used as a potential drug candidate to target ifn-γ, il-3, and tnf-α proteins to control inflammatory events. other peptides (i.e., qepqesqq and frdehkk) also revealed good binding affinity with ifn-γ with s-scores of -10.98 and -10.55, respectively. similarly, the peptides khdrgdef, frdehkk, and qepqesqq showed best binding interactions with il-3 with s-scores of -8.81, -8.64, and -8.17, respectively. the innate and adaptive immune system controls the defense organization mediated by multiple components and molecules in an organism [1] . different organs, signaling pathways, and compounds collectively perform various tasks to protect the organism from external and internal damage. autoimmunity or production of autoantigens mainly brings disaster to the immune system due to inadequate immune tolerance [2] . the defect in the synergistic relationship between innate immunity and adaptive immu-nity causes severe consequences including autoimmune disorders, inflammations like systemic lupus erythematosus, rheumatoid arthritis, alzheimer's disease, multiple sclerosis, and many other complications [3] . systemic lupus erythematosus (sle) is the most common chronic autoimmune inflammatory disorder characterized by the presence of autoantibodies directed against own cells or tissues of the body. it is intermediated by b cells which generate autoantibodies against nuclear antigens, a type iii hypersensitivity reaction that causes chronic systemic inflammation and tissue damage in the joints, skin, brain, lungs, kidneys, and blood vessels [4] . the incidence of sle prevalence has been predominantly recorded in young middle-aged females. according to studies, the highest prevalence can be seen in certain ethnicities, reflected in prevalence rates of approx. 40/100,000 persons in northern european cohorts with comparison rates of 200/100,000 patients of african-american descent [5] . sle is multifactorial in its origin with a wide range of clinical and serological manifestations. there have been many efforts to elucidate the pathogenesis of sle with current recognition of genetic susceptibility, environmental triggers, and disruption in both the innate and adaptive immune systems [6] . adaptive immunity is an antigen-specific host defense that comprises of b and t lymphocytes and immunoglobulins. the defected immunity in sle results in a myriad of complications such as decreased t cell signaling, stimulation of autoaggressive t effector cells, and production of autoantibodies. apart from all these, any dysregulation in b cells that produces proinflammatory cytokines (i.e., il-1, il-3, il-6, il-23, and tnf-α) pushes the inflammatory events and drives the sle disease. thus, targeting these b and t cells could be proved as a therapeutic advantage to control sle [7] . the role of cytokines regarding sle has collected much interest of scientists. type i interferon family along with many other cytokines such as interleukins (il-3, il-6, il-10, and il-17) and tumor necrosis factor (tnf) is seen to be involved in sle. these interleukins play a significant role in diseases which are linked to inflammation and autoimmunity [4] . this disease has several variants which are genetically linked with pathogenic mechanisms. genetics and epigenetics are the factors which contribute directly to cause alterations in the cells of both innate and adaptive immune responses [8] . greater than 95% of sle patients have detectable serum antinuclear antibody (ana). anti-ds-dna antibodies are highly specific for sle and present in 65-70% of the patients (versus 0.5% of the healthy population). anti-ds-dna antibodies, anti-ro, anti-la, anti-c1q, and anti-sm antibodies have been demonstrated histologically in renal biopsy specimens. a number of specific antibodies have been associated with a particular expression of sle. anti-ro and antinucleosome antibodies are most strongly linked with cutaneous lupus [9] . many combination drug therapies have been in practice for the control of clinical manifestations of sle, but these have been recorded with severe side effects, and also, the patients of sle still show a higher standardized mortality rate (i.e., of 4.6-fold) with respect to the general population [10] . therefore, the current study was planned to reveal anti-inflammatory peptides from plant and animal sources using molecular docking approach. the study includes protein-ligand and peptide-protein docking of twenty anti-inflammatory peptides counter to ifn-γ, il-3, and tnf-α receptor proteins as targets for the treatment of sle. in the current study, we have investigated the potential of these peptides as drug candidates to attenuate the inflammatory response and tissue destruction due to activation of proinflammatory cytokines, b cells, and t cells which lead towards autoantibody production. the study includes the protein-ligand and peptide-protein docking of twenty anti-inflammatory bioactive peptides against three main receptor proteins (i.e., il-3, tnf-α, and ifn-γ) that play leading roles in the pathogenesis of sle. molecular operating environment v.2015.10 (chemical computing group ulc, montreal, qc, canada) [11] was employed for ligand-based docking, and haddock v.2.4 [12] an online server was used for peptide-protein docking. preparation. an extensive literature survey was performed to explore plant and animal derived bioactive anti-inflammatory peptides. the chemical structures of these ligands were prepared using acd chemsketch v.c40e41 (advanced chemistry development, inc., toronto, ontario, canada) [13] and saved in mol format in moe database as ready-to-dock compounds after energy minimization. proteins. the three-dimensional structures of il-3 (pdb id: 5uv8), ifn-γ (pdb id: 6e3k), and tnf-α (pdb id: 6op0) were retrieved from protein data bank (https://www.rcsb.org/). removal of solvent, addition of hydrogen atoms, energy minimization, and 3d protonation were performed using moe with default parameters, and the minimized structure of each protein was saved to use as receptor protein for docking studies. 2.3. ligand-protein docking. the active binding pocket of each receptor protein was selected using the site finder tool of moe. the prepared ready-to-dock library of twenty anti-inflammatory peptides was docked counter to il-3, ifn-γ, and tnf-α, and the top three ligands from each protein-ligand docking were selected on the basis of their interactions and s-scores. the algorithm of moe provides top conformations on the basis of binding patterns of ligands with active amino acids of the receptor protein, minimum energy structure, and maximum occupancy of the binding pocket. 2.4. peptide-protein docking. il-3, ifn-γ, and tnf-α have been extensively studied due to their roles in the pathophysiology of many autoimmune disorders. the dysfunctioning of the signaling pathways of b and t cells leads towards the production of autoantibodies against the body's own tissues and cells. in the literature, many anti-inflammatory compounds have been reported for autoimmune disease; however, in this study, for the first time, peptide-protein docking was employed to explore the inhibitory effects of peptides counter to selected receptor proteins for the treatment of sle. the most reported twenty anti-inflammatory peptides were used for docking studies against receptor proteins. for peptide-protein docking, the top peptides against each receptor protein were selected, which were obtained from the results of protein-ligand docking and used for further analysis of peptide-protein docking. the sequences of these peptides were retrieved from the literature and subjected to blastp to find their homologs. to predict three-dimensional (3d) structure of each peptide, the pdb database was used during blast analyses to find the best templates of each peptide using the homology modeling approach. modeller v.9.21 (ben webb, ucsf, ca, usa) [14] was used to predict 3d structures of selected peptides (i.e., dedtqammpfr and khdrgdef). haddock v.2.4 was used to carry out the docking analysis between the best selected peptides and selected target proteins. educational version of pymol molecular graphics system v2.0 (schrödinger, llc) was used to visualize the docked complexes and draw figures [15] . 2.4.1. molecular dynamic simulation. for 120 nanoseconds, desmond (schrödinger llc) was used to model molecular dynamics in triplicates [16] . the earliest phase of proteinligand complex for molecular dynamics simulation was used in the docking experiments. molecular docking studies can predict ligand binding state in static situations. docking is useful because it provides a static view of a molecule's binding pose at the active site of a protein [17] . by integrating newton's classical equation of motion, md simulations typically compute atom movements over time. simulations were used to predict the ligand binding status in the physiological environment [18, 19] . the protein-ligand complex was preprocessed using protein preparation wizard or maestro, which included complex optimization and minimization. all of the systems were prepared using the system builder tool. tip3p (transferable intermolecular interaction potential 3 points), a solvent model with an orthorhombic box was chosen. in the simulation, the opls 2005 force field was used [20] . to make models neutral, counter ions were introduced. to mimic physiological conditions, 0.15 m nacl was added. the npt ensemble with 300 k temperature and 1 atm pressure was chosen for the entire simulation. the models were relaxed before the simulation. the trajectories were saved for examination after every 100 ps, and the simulation's stability was verified by comparing the root mean square deviation (rmsd) values of protein and ligand over time. 3.1. protein-ligand docking. molecular docking predicts the intermolecular framework and binding interactions between ligand molecules and proteins. different docking approaches play a leading role in drug discovery. this study includes the protein-ligand docking and peptide-protein docking of twenty anti-inflammatory peptides counter to ifn-γ, il-3, and tnf-alpha. the top three hit peptides from each analysis were selected on the basis of energy structure and interactions with active amino acids of the respective receptor protein (table 1) . analyses. amongst twenty peptides as ligands, the peptide dedtqammpfr showed the best interactions counter to ifn-γ and tnf-α. the peptide with s-score of -11.31 showed interactions with lys34, arg42, gln46, and tyr53 amino acids of the binding pocket of ifn-γ ( figure 1 ). the second peptide, qepqesqq with s -score of -10.98, exhibited interactions with val22 and glu39 residues of ifn-γ as a receptor protein ( figure s1 ). the third peptide frdehkk with s-score of -10.55 showed interactions with amino acids val5, glu39, and tyr53 of ifn-γ ( figure s2 ). ifn-γ is a dimerized cytokine and known for its critical role in adaptive and innate immunity against variety of pathogens [21] . in addition to immunity response, it also activates a proinflammatory program in macrophages. the elevated level of ifn-γ has been observed in autoimmune complications including sle [22] . for the il-3, the peptide khdrgdef with s-score of -8.81 showed interactions with cys16, cys84, pro86, and leu87 amino acids of the binding pocket of il-3 ( figure 2 ). the peptide frdehkk with s-score of -8.64 showed interactions with asn15, asp21, and glu119 ( figure s3 ), and the peptide qepqesqq with s-score of -8.17 interacted with pro83, ala121, and glu119 residues of il-3 ( figure s4 ). interleukin-3 is a multispecific hemopoietin, a glycoprotein cytokine that is synthesized by t cells in response to antigen. il-3 has been involved in the proliferation and differentiation of many immune cells and both primitive multipotent and committed myeloid progenitors' cells [23, 24] . elevated levels and increased il-3-responsive progenitor cells have been reported in sle patients [4] . the other receptor protein tnf-α is an inflammatory cytokine and important for resistance to cancer and infection. the dysregulation and elevation of this protein has been associated with many autoimmune disorders. in our study, the peptide dedtqammpfr with s-score of -8.20 showed interactions with arg32 and leu142 amino acids of the binding pocket of tnf-α ( figure 3 ). other peptides (i.e., frdehkk and qepqesqq) with s-scores of -7.32 and -7.25 interacted with asp143, pro20 and glu23 ( figure s5 ), and arg32 amino acids ( figure s6 ) in the binding pocket of tnf-α, respectively. tnf-α is a proinflammatory cytokine, belongs to the super family of tumor necrosis factor, and secreted by macrophages in a defense mechanism to protect from damage by inducing inflammation against pathogenic stimuli. the elevated level or mutation in tnf-α signaling leads towards deleterious consequences including many autoimmune disorders [25] . collectively, with ifn-γ, il-3, and tnf-α, many other cytokines and different factors have been studied to understand the autoimmunity events. thus, there is an immediate need for the present age to understand the autoimmunity to control excessive inflammatory responses and balance cytokine signaling. 3.3. peptide-protein docking. peptide-protein interactions play a crucial role in a variety of regulatory and signaling pathways of the cell. the peptide-protein complex helps to open the key to elucidate important biological processes and to understand the underlying peptide-protein interactions. in this study, we used the top three ligands obtained from protein-ligand docking analysis for peptide-protein docking studies. the blastp was used to find suitable homologs and templates to build 3d structures of selected figure 5 (a). the plot showed that the complex reached stability at 10 ns. this was increase in rmsd of peptide at 50 ns. after that, for the length of the simulation, fluctuations in rmsd values for target remained within 1.5 angstrom which is absolutely acceptable [26] . the ligand fit to protein rmsd values fluctuated within 1.5 angstrom after they have been equilibrated. these findings indicate that the peptide stayed firmly connected to the receptor throughout the simulation period. on the rmsf graphic ( figure 5(b) ), peaks represented the portions of the protein that fluctuated the most during the simulation. protein tails (both n-and c-terminal) typically changed more than any other part of the protein. alpha helices and beta strands, for example, are usually stiffer than the unstructured section of the protein and fluctuate less than loop portions. according to md trajectories, the resi-dues with greater peaks belonged to loop areas or n and c-terminal zones ( figure s7 ). low rmsf values of binding site residues indicated that ligand binding to the protein is stable. alpha-helices and beta-strands are monitored as secondary structure elements during the simulation (sse). the graph above depicts the distribution of sse by residue index across the protein structure. throughout the simulation, the left graphic showed the sse composition for each trajectory frame while the right plot monitors each residue's sse assignment through time. protein interactions with the ligand can be detected throughout the simulation. these interactions were categorized and summarized by types ( figure s7 ). the four types of protein-ligand interactions (or "contacts") include hydrogen bonds, hydrophobic interactions, ionic interactions, and water bridges. the "simulation interactions diagram" panel in maestro was used to study the subtypes of each interaction type. over the course of the trajectory, the stacked bar charts were standardized: for example, a value of 0.7 indicated that the specific interaction was maintained for 70% of the simulation duration. because some protein residues may make several interactions of the same subtype with the ligand, values above 1.0 are feasible. the majority of the significant ligand-protein interactions discovered by md were hydrogen bonds and hydrophobic interactions ( figure 6) . a timeline has exhibited the interactions and contacts (h-bonds, hydrophobic, ionic and water bridges) as described above. in figure 7 , the top panel displayed the total number of specific connections the protein made with the ligand over the duration of the journey. the bottom panel of each trajectory frame showed which residues interacted with the ligand. some residues made many particular connections with the ligand which has been indicated by a deeper shade of orange color according to the scale to the right of the plot. over the course of the trajectory, the stacked bar charts were standardized: for example, a value of 1.0 signified that the exact interaction was maintained for 100% of the simulation duration. values exceeding 1.0 are possible because some protein residues may make several interactions of the same subtype with the ligand. the interactions of individual ligand atom with protein residues are showed in figure 8 . interactions that last more than 30% of the simulation period in the selected trajectory (0.00 to 100.0 nsec) are shown. the mmgbsa.py script from the desmond module of the schrodinger suite 2019-4 was used to perform the mm-gbsa analysis. every frame was collected from each md trajectory for binding free energy estimates of the receptor in combination with the peptide (figure 9 ). total energy was ranged from -996.226 to 5.056. the mean and median were -356.755 and -343.800, respectively, indicating good energy. total binding free energy (kcal/mol) was calculated using the law of additivity in which individual energy modules such as coulombic, covalent, hydrogen bond, van der waals, self-contact, lipophilic, solvation, and π-π stackings of the ligand and the protein were added together [27] . molecular docking is an elaborative approach to foresee the interactions between ligand and targeted amino acids in the binding pocket of the receptor protein [28] . computational approaches including molecular docking help scientists to predict the binding capacities of different small molecules and peptides as drug candidates against different receptor proteins [29] . in the current study, we have used some plantand animal-derived anti-inflammatory peptides ranging from 3 to 15 amino acid residues as ligands/peptides counter to proteins from different bacteria which are the leading cause of many autoimmune disorders. the autoimmune disease sle is a disorder of connective tissues with a wide range of clinical manifestations. the autoreactive b (bone marrow-or bursa-derived cells) and t cells (thymus cells) of adaptive and innate immunity play a leading role in the production of autoantibodies and lead 7 biomed research international towards autoimmune disorders including sle [6] . different factors such as il-3, il23, m-csf, ifn-α, ifn-γ, and il-6 have been reported in different studies to be involved in the production of autoantibodies but the main trigger for all these autoimmune complexities is still unknown [4, 30] . the elevated levels of proinflammatory cytokines and tnf-α have been reported in many studies as associated mainly with autoreactive b cells and t cells to produce autoantibodies [4, 6] . ifn-γ or type ii interferon is a pleiotropic cytokine that coordinates with a diverse array of cellular immunity processes [31] . ifn-γ is a homodimer and formed by noncovalent interactions of 17 kda polypeptide dimers and crucially known for early control of pathogen spreading. ifn-γ has 8 biomed research international been secreted mainly by cd4 t helper cells, cd8 cytotoxic t cells, and to a less extent by antigen-presenting cells (apcs) and natural killer cells [32] . the peripheral blood mononuclear cells (pbmcs) of sle patients showed high level of ifn-γ transcript, and the t cells of sle patients produce much more ifn-γ as compared to normal cells [33] . in the current study, strong interactions of three peptides (i.e., dedtqammpfr, qepqesqq, and frdehkk) were found with the active amino acids present in the binding pocket of ifn-γ which could be used as potential inhibitors of ifn-γ to treat sle. il-3 is a monomeric glycoprotein which is predominately produced by activated t cells in response to stimuli. it serves as a bridge between the immune system (t-lymphocytes) and the hemopoietic system that in response to foreign stimuli generates cellular elements for cellular defense [23] . the dysregulation of il-3 has been associated with various autoimmune diseases including arthritis and sle. in sle patients, elevated il-3 responsive progenitor cells have been observed in spleen, which show an association between il-3 and autoreactive cells [4] . the molecular docking approach used in this study exhibited strong interactions of three peptides (i.e., khdrgdef, frdehkk, and qepqesqq) with the active amino acids present in the binding pocket of il-3. these peptides could be potential inhibitors of il-3. tnf-α is a pleiotropic proinflammatory cytokine and contributes importantly to the development of b and t cells. tnf-α is a potent inflammatory mediator of chronic and acute inflammation and secreted by macrophages, t cells, and neutrophils [34] . the involvement of tnf-α in the pathogenesis of sle has been observed in many clinical studies due to overexpression and elevated levels of tnf-α in sle patients [35] . in current study, using molecular docking approach, three peptides (i.e., dedtqammpfr, frdehkk, and qepqesqq) showed strong interactions with the active amino acids present in the binding pocket of tnf-α and could be used in the treatment of sle. the overexpression and elevated levels of interferons, interleukins, and tumor necrosis factor point towards the relationship of these factors to the pathogenesis of sle and many other autoimmune disorders. the blockage of ifn-γ, il-3, and tnf-α could be proved as an effective strategy to control the tissue and organ damage in sle. in clinical trials, there are many ongoing therapies to control signaling pathways and targeted autoantibodies. till now, many drugs have been approved and marketized such as rituximab, epratuzumab, abetimus, sodium, obinutuzumab, lulizumab pegol, abatacept, and blisibimod for the treatment of autoimmune disorders. the drug blisibimod is a fusion protein which is an antagonist of baff with little encouraging outcomes obtained after a phase iii trial for the treatment of sle [30] . in spite of many drugs and combinational therapies, these drugs have been associated with severe after effects; so, there is a need of such types of drugs with maximum potency and minimum side effects. the proinflammatory cytokines, interleukins, and interferons make up the defense system of the cell and play a crucial role in generating different molecules in response to external pathogenic stimuli. any mutation and overexpression of any of these molecules lead towards the production of autoantibodies against body's own cells. the peptides reported in this study could be used as potential drug candidates counter to ifn-γ, il-3, and tnf-α as receptor proteins. further elaborative study is still needed to explore much more potential of these anti-inflammatory peptides. the docking analysis and s-scores of selected peptides have revealed the potential of selected peptides as drug candidates counter to inflammatory autoreactive proteins to control autoimmunity. in the current study, we used two types of docking analysis (protein-ligand and peptide-protein docking) to check the configuration and orientation of ligands/ peptides counter to selected receptor proteins. in our first approach, the peptide dedtqammpfr showed strong interactions with active amino acids of ifn-γ (s-score -11.31) and tnf-α (s-score 8.20) receptor proteins. the conformations showed the occupancy of the maximum binding pocket by ligand molecules. in our second approach (peptide-protein docking), the same peptide also showed strong bonding with the active amino acids of ifn-γ via haddock server. the ifn-γ-dedtqammpfr complex with haddock score of −10:3 ± 2:5 kcal/mol showed strong interactions amongst active amino acid residues of both peptide and receptor protein. further, the md simulation analysis also confirmed that the peptide stayed firmly connected to the receptor throughout the simulation period (i.e., 120 nanoseconds). the results of the current study have explored the potential of peptides of plant and animal sources as drug molecules to control autoimmunity. the study could be proved as an initial step for further use of these peptides after some required modifications as drug candidates against autoimmune disorders. figure s1 : interactions (a) and binding pattern (b) of qep-qesqq peptide with ifn-γ. figure s2 : interactions (a) and binding pattern (b) of frdehkk peptide with ifn-γ. figure s3 : interactions (a) and binding pattern (b) of frdehkk peptide with il-3. figure s4 : interactions (a) and binding pattern (b) of qepqesqq peptide with il-3. figure s5 : interactions (a) and binding pattern (b) of frdehkk peptide with tnf-α. figure s6 : interactions (a) and binding pattern (b) of qepqesqq peptide with tnfα. figure s7 : protein secondary structure element distribution by residue index throughout the protein structure (supplementary materials). (supplementary materials) the immune system molecular and cellular basis of autoimmune diseases sequence and structural characterization of toll-like receptor 6 from human and related species pathogenic inflammation and its therapeutic targeting in systemic lupus erythematosus systemic lupus erythematosus one year in review 2019: systemic lupus erythematosus systemic lupus erythematosus (sle): emerging therapeutic targets new insights into the immunopathogenesis of systemic lupus erythematosus from bilag to bilag-based combined lupus assessment-30 years on therapeutic advances in the treatment of sle the had-dock2.2 web server: user-friendly integrative modeling of biomolecular complexes personal experience with four kinds of chemical structure drawing software: review on chemdraw, chemwindow, isis/draw, and chemsketch comparative protein structure modeling using modeller pymol: an open-source molecular graphics tool scalable algorithms for molecular dynamics simulations on commodity clusters molecular docking and structure-based drug design strategies bringing molecular dynamics simulation data into view identification of lead compounds against scm (fms10) in enterococcus faecium using computer aided drug designing prediction of absolute solvation free energies using molecular dynamics free energy perturbation and the opls force field chapter 6-immunity and resistance to viruses, viruses the potential similarities of covid-19 and autoimmune disease pathogenesis and therapeutic options: new insights approach cytokines and cytokine receptors regulating cell survival, proliferation, and differentiation in hematopoiesis a 3d ipscdifferentiation model identifies interleukin-3 as a regulator of early human hematopoietic specification in silico characterization of growth differentiation factors as inhibitors of tnfalpha and il-6 in immune-mediated inflammatory disease rheumatoid arthritis significance of root-meansquare deviation in comparing three-dimensional structures of globular proteins additivity principles in biochemistry computer-aided prediction and identification of phytochemicals as potential drug candidates against mers-cov an in silico approach to target rna-dependent rna polymerase of covid-19 with naturally occurring phytochemicals targeted biologic therapy for systemic lupus erythematosus: emerging pathways and drug pipeline ifn-γ orchestrates tumor elimination, tumor dormancy, tumor escape, and progression interferon-gamma at the crossroads of tumor immune surveillance or evasion variety of endosomal tlrs and interferons (ifn-α, ifn-β, ifnγ) expression profiles in patients with sle, ssc and mctd potential roles of neutrophils in regulating intestinal mucosal inflammation of inflammatory bowel disease the use of anti-tnfalpha therapies for patients with systemic lupus erythematosus. where are we now? the authors would like to gratefully acknowledge department of biochemistry, government college university faisalabad, for providing space and facilities to accomplish this study. the data used to support the findings of this study are available from the corresponding author upon request. the authors declare that they have no conflict of interest. gm and hsm conceived and planned the experiments. gm, ss, and hsm carried out the protein-ligand and proteinprotein docking experiments. ht, ms, and tm drafted the manuscript. rj and gm helped conducting molecular dynamics and simulation study. gm supervised the project and proofread the article. all authors discussed the results and commented on the manuscript. 127 d o i: 1 0. 15 82 6/ ch im te ch .2 01 5. 2. 2. 01 3 k. v. savateev, s. s. borisov, e. k. voinkov, e. n. ulomsky, v. l. rusinov, o. n. chupakhin institute of organic synthesis ural division of ras, 19 mira street, ekaterinburg 6-aminotriazolo[1,5-a]pyrimidines as precursors of 1,2,4-triazolo[5,1-b]purines* triazolo[5,1-b]purines are rare structural analogues of natural nucleosides and nucleobases purine series. at the same time, prominent representatives of azolopurines exhibit a broad spectrum of antiviral effect, activity against of rheumatoid arthritis, psoriasis, alzheimer’s, parkinson’s and etc. despite the practical value azolo[5,1-b]purines extremely sparingly represented in the chemical literature, due to the complexity of their synthesis. we suggest a convenient way to synthesize triazolopurines with aminotriazolo[1,5-a]pyrimidines (2) as available starting compounds obtained in good yield by reduction of nitro derivatives (1). © savateev k. v., borisov s. s., voinkov e. k., ulomsky e. n., rusinov v. l., chupakhin o. n., 2015 * the work was supported by rfbr grant 13-03-0086 traditionally one of the most widespread way to turn azinons in aminazines is chorine deoxygenization of heterocycles followed by amination of chlorinated derivatives1,2. the need for blocking already existing amino group, for example with acyl moiety, is the peculiarity of this method. we have used several acyl protecting groups using corresponding anhydrides or acids, among which the most convenient one was trifluoracetyl protection, providing good solubility in organic solures of corresponding trifluoroacetyl derivative. as a model compound for chlorine deoxygenization, compound 3a was used. use of phosphoryl chloride and thionyl chloride in presence of tertiary amines or dmf gave no satisfactory results. pyridine usage as tertiary amine proved to be effective. the resulting chlorine derivative (4) was exposed to butylamine for 128 № 2 | 2015 chimica techno acta savateev k. v., borisov s. s., voinkov e. k., ulomsky e. n., rusinov v. l., chupakhin o. n. the purpose of obtaining of pyrimidine 7 aminotriazol straight away (5). however, spectroscopic data and elemental analysis showed the formation of triazolopurine (6). thus, we have shown that the use of cis-6-aminotriazole (2) is a promising way of synthesizing azolo [5,1-b] purines. 1. zhang n., ayral-kaloustian s., nguyen th., afragola j., hernandez r., lucas j., gibbons j., beyer c. synthesis and sar of [1,2,4]triazolo[1,5-a]pyrimidines, a class of anticancer agents with a unique mechanism of tubulin inhibition. j. med. chem. 2007;50(2):319-327. doi: 10.1021/jm060717i. 2. zhao x. l., zhao y. f., guo s. c., song h. s., wang d., gong p. synthesis and anti-tumor activities of novel [1,2,4]triazolo[1,5-a] pyrimidines. molecules 2007;12(5):1136–1146. doi: 10.3390/12051136. 129 у д к 5 47 .8 36 .3 к. в. саватеев, с. с. борисов, е. к. воинков, е. н. уломский, в. л. русинов, о. н. чупахин институт органического синтеза им. и. я. постовского уро ран 620002, екатеринбург, ул. мира, 19 6-аминотриазоло[1,5-a]пиримидины как предшественники 1,2,4-триазоло [5,1-b] пуринов* триазоло[5,1-b]пурины являются малораспространенными структурными аналогами природных нуклеозидов и нуклеиновых оснований пуринового ряда. в то же время известные представители азолопуринов проявляют широкий спектр противовирусного действия, активность в отношении ревматоидного артрита, псориаза, болезней альцгеймера, паркинсона и проч. несмотря на практическую ценность, азоло[5,1-b]пурины чрезвычайно скупо представлены в химической литературе, что обусловлено сложностью их синтеза. нами предложен удобный путь к синтезу триазолопуринов с участием аминотриазоло[1,5-a]пиримидинов (2) в качестве доступных исходных соединений, получаемых с хорошими выходами восстановлением нитропроизводных (1). © саватеев к. в., борисов с. с., воинков е. к., уломский е. н., русинов в. л., чупахин о. н., 2015 * работа была выполнена при поддержке гранта рффи 13-03-0086. традиционно одним из наи более распространенных способов превращения азинонов в аминоазины является хлордезоксигенирование гетероциклов с последующим аминированием хлорпроизводных [1,2]. особенностью синтеза диаминоазинов таким методом является необходимость защиты уже имеющейся аминогруппы, например, а цильным фрагментом. нами было использовано несколько ацильных защитных групп, с помощью соответствующих ангидридов или кислот, из которых наиболее удобной оказалась трифторацетильная защита, обеспечивающая хорошую растворимость в органических растворителях 130 № 2 | 2015 chimica techno acta саватеев к. в., борисов с. с., воинков е. к., уломский е. н., русинов в. л., чупахин о. н. соответс твующего трифторацетильного производного. в качестве модельного соединения для хлордезоксигенирования нами было использовано соединение 3а. использование фосфорилхлорида и тионилхлорида в присутствии третичных аминов или дмфа не дали удовлетворительного результата. эффективным оказалось использование в качестве третичного амина пиридина. полученное хлорпроизводное (4) сразу по выделении обработали бутиламином с целью получения 7-аминотриазолопиримидина (5). однако данные спектроскопии и элементного анализа показали образование триазолопурина (6). таким образом, нами показано, что использование 6-аминотриазолопиримидинонов (2) является перспективным путем синтеза азоло[5,1-b]пуринов. 1. zhang n., ayral-kaloustian s., nguyen th., afragola j., hernandez r., lucas j., gibbons j., beyer c. synthesis and sar of [1,2,4]triazolo[1,5-a]pyrimidines, a class of anticancer agents with a unique mechanism of tubulin inhibition. j. med. chem 2007;50(2):319-327. doi: 10.1021/jm060717i. 2. zhao x. l., zhao y. f., guo s. c., song h. s., wang d., gong p. synthesis and anti-tumor activities of novel [1,2,4]triazolo[1,5-a] pyrimidines. molecules 2007;12(5):1136–1146. doi: 10.3390/12051136. рекомендуем при цитировании данно статьи следующую ссылку: savateev k. v., borisov s. s., voinkov e. k., ulomsky e. n., rusinov v. l., chupakhin o. n. 6-aminotriazolo[1,5-a]pyrimidines as precursors of 1,2,4-triazolo[5,1-b]purines // chimica techno acta. 2015. vol. 2. № 2. p. 127–130. panacea journal of medical sciences 2021;11(1):31–36 content available at: https://www.ipinnovative.com/open-access-journals panacea journal of medical sciences journal homepage: http://www.pjms.in/ original research article joint involvement in rheumatoid arthritis: sonographic evaluation in comparison with radiography sharanayya1, shamrendra narayan2, vandana verma3, madhu sharma3, anjana pande3, vivek jirankali1,* 1dept. of radiology, navodaya medical college hospital & research centre, raichur, karnataka, india 2dr. ram manohar lohia institute of medical sciences, lucknow, uttar pradesh, india 3sarojini naidu medical college, agra, uttar pradesh, india a r t i c l e i n f o article history: received 14-08-2020 accepted 14-10-2020 available online 29-04-2021 keywords: erosions ultrasonography radiograph synovitis a b s t r a c t background: the aim of this study was to evaluate the role of ultrasonography (including power doppler) in assessing the joint involvement in rheumatoid arthritis (ra) and its comparison to radiographic changes. materials and methods: 55 patients with ra underwent ultrasound and radiographic examination of wrist and hand with laboratory investigations. 25 subjects were taken as controls. following points were specifically looked in ultrasound – synovitis, synovial hypertrophy, effusion, cartilage thickness, swelling of tendon, osteophytes, erosions. erosion sites were compared using radiographs. ultrasonography was performed by two radiologists and inter-observer agreement was calculated. results: out of total 55 cases, 44 cases were classified as early ra and 11 cases as advanced ra. out of 25 control subjects, 3 subjects had a lesion – atypical for ra, however – all 3 had previous trauma at that site. inter-observer agreement was excellent. intra-articular erosions were seen in all cases of advanced ra and in only 26/44 cases in early ra cases. tenosynovitis was seen in 21/44 cases of early ra while only one case of advanced ra showed tenosynovitis. there was reduced cartilage thickness in all patients of advanced ra while none was observed in early ra cases. synovial hypertrophy and synovial effusion were almost similar in both early and advanced ra cases. ultrasonography detected erosions in 37/54 cases while radiography detected erosions in only 11/54 cases. conclusions: sonography can be used as a primary modality to diagnose ra especially early ra, which helps in reducing disabilities by early aggressive treatment. it is more sensitive than radiography in detecting erosions. © this is an open access article distributed under the terms of the creative commons attribution license (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1. introduction wrists and hands are involved in many pathological conditions. their involvement is always associated with disability in performing regular activities on the part of the patient and difficulty in diagnosing on part of the treating clinician due to lack of specific diagnostic criteria. common causes of the chronic wrist pain include fractures, ligament tears, radioulnar subluxation, erosive * corresponding author. e-mail address: vivekjirankali@gmail.com (v. jirankali). and non-erosive arthritis, osteoarthritis, avascular necrosis of carpal bones, neoplasms, tendinopathy, neuropathy and various infections. rheumatoid arthritis (ra) is a chronic inflammatory disease of unknown aetiology marked by a symmetric, peripheral polyarthritis. the major abnormalities of ra appear in the synovial joints as soft tissue swelling caused by synovial hypertrophy, effusion, bursal and tendon sheath swelling. 1 marginal erosions are due to inflamed synovium destroying the cortex and underlying bone and they occur initially at the bare area: the margins where synovium is not https://doi.org/10.18231/j.pjms.2021.009 2249-8176/© 2021 innovative publication, all rights reserved. 31 https://doi.org/10.18231/j.pjms.2021.009 https://www.ipinnovative.com/ https://www.ipinnovative.com/open-access-journals http://www.pjms.in/ https://crossmark.crossref.org/dialog/?doi=10.18231/j.pjms.2021.009&domain=pdf https://creativecommons.org/licenses/by/4.0/ mailto:vivekjirankali@gmail.com https://doi.org/10.18231/j.pjms.2021.009 32 sharanayya et al. / panacea journal of medical sciences 2021;11(1):31–36 covered by cartilage. these findings can occur in unison or as an isolated entity depending upon the duration and the activity of the disease. presence of erosions in early ra is associated with bad prognosis and it may also guide in the choosing the appropriate therapy. 2 appropriate treatment of ra in its early stages helps in reduction of disabilities. 3 there is limited scope of physical examination and lab tests for early diagnosis of ra. and even radiographic changes are seen in the late course of the disease. 4 ultrasound is superior to clinical examination in detecting synovitis. 5 power doppler has been used to assess the disease activity and there is a good correlation between doppler hyperaemia and histologically detected pannus. 6 ultrasound is non-invasive, readily available, portable and inexpensive modality which can be used to examine multiple joints at a time. it can also be used to evaluate extra-articular entities such as enthesopathies and tenosynovitis. 7,8 the study has also compared the radiography and ultrasonography in detection of osteophytes and erosions. the aim of the current study was to evaluate the role of ultrasonography (including power doppler) in assessing the joint involvement in ra and its comparison to radiographic changes. 2. materials and methods 2.1. patients approval of institutional ethical committee was taken. 55 patients who were diagnosed with ra according to acr/eular criteria were included in the study. the study was conducted in a tertiary care hospital (navodaya medical college hospital and research centre, raichur) from march 2018 to january 2020. details regarding history and physical examination were collected. conventional radiograph of the affected hand was obtained in standard views. serological markers like rheumatoid factor (rf), c-reactive protein (crp) and anti-citrullinated protein antibody (anti-ccp) were estimated. 25 subjects were taken as controls who came for ultrasonography of the different part of the body (other than wrist). both the groups were age matched. radiographs were not taken for control groups as ethical committee did not grant permission. 2.2. ultrasonography ultrasonography was performed in all cases using samsung medison sa800 machine using high frequency (6-12mhz) linear array transducer. ultrasound was performed by two radiologists who were blinded to the clinical and laboratory data. scanning of wrist and hand was performed with the patient seated, with hands resting on the examination table. examinations were carried out on both sides for comparison. colour and power doppler were used as and when required to evaluate blood flow with standard settings of the machine. following points were specifically looked – synovitis, synovial hypertrophy, effusion, cartilage thickness, swelling of tendon, osteophytes, erosions etc. 2.3. statistical analysis after collecting the data, it was entered in microsoft excel. frequency and percentages were calculated for qualitative data. inter-observer agreement was calculated. data was analyzed by using “ibm spss statistics” (version 16.0). analysis was done by using student ‘t’ test and chi-square test. all statistical tests were applied at a significance level of “α=.05” (p value < 0.05). 3. results out of 25 control subjects, 3 subjects had a lesion – atypical for ra, however – all 3 had previous trauma at that site. the study subjects consisted of 55 individuals with 41 females and 14 males (females>males). majority of the patients belonged to the age group of 41-50 years. out of total 55 cases, 44 cases were classified as early ra and 11 cases as advanced ra. the sonographic findings that were observed were synovial hypertrophy, synovial effusion, intra-articular erosions, tenosynovitis and reduced cartilage thickness (tables 1 and 2). positive power doppler signals were seen in hypertrophied synovial tissues in few cases. interobserver agreement was excellent with kappa value of 0.87 (95% ci 0.77-0.93). intra-articular erosions were seen in all cases of advanced ra and in only 26/44 cases (59%) in early ra cases. tenosynovitis was seen in 21/44 cases (48%) of early ra while only one case (9%) of advanced ra showed tenosynovitis. tenosynovitis was most commonly seen in extensor group of tendons around the wrist (18/21 cases – 86%). it was seen in only 3/21 cases (14%) in flexor group of tendons. there was reduced cartilage thickness in all patients of advanced ra while none of the early ra case showed reduced cartilage thickness. synovial hypertrophy and synovial effusion were almost similar in both early and advanced ra cases. in both early and advanced ra, synovial hypertrophy and intraarticular erosions were found predominantly in wrist and inter-carpal joints. raised crp, ra factor and anti-ccp antibody was found in both early and advanced cases of ra (table 3). of the extensor group, extensor carpi ulnaris (ecu) was very commonly involved followed by extensor digitorum (ed). in the flexor compartment, flexor digitorum superficialis (fds) and flexor carpi radialis (fcr) were commonly involved. ultrasonography detected erosions in 37/54 (69%) cases while radiography detected erosions in only 11/54 (20%) of cases (table 4). ultrasonography detected osteophytes in sharanayya et al. / panacea journal of medical sciences 2021;11(1):31–36 33 8/54 (15%) cases while radiography detected osteophytes in only 3/54 (6%) of cases. table 1: sonographic findings in early ra (n=44) s.no sonographic feature no. of cases percentage 1. synovial hypertrophy (sh) 42 95.45% 2. synovial effusion (se) 04 9.09% 3. intra articular erosions (iae) 26 59.09% 4. tenosynovitis (tsyn) 21 47.72% 5. reduced cartilage thickness (rct) table 2: sonographic findings in advanced ra (n=11) s.no sonographic feature no. of cases percentage 1. synovial hypertrophy 11 100% 2. synovial effusion 00 00 3. intra articular erosions 11 100% 4. tenosynovitis 01 9.09% 5. reduced cartilage thickness 11 100% fig. 1: longitudinal ultrasound demonstrates synovial hypertrophy in mid inter-carpal joint with power doppler signal. 4. discussion early treatment and intervention of ra reduces the future possible occurrence of deformities and disabilities. 9 this led to the need of new diagnostic methods which can diagnose early ra with good accuracy. mri is one such modality, but is expensive and time consuming. thus, use of ultrasonography in early diagnosis of ra began and it showed to have good sensitivity. few previous studies have concluded that ultrasonography can be used to detect erosions in hands in ra. 10–12 only few studies among fig. 2: longitudinal ultrasound demonstrates synovial fluid effusion around the extensordigitorum tendon. fig. 3: longitudinal ultrasound demonstrates intra-articular erosions on ulnar side of inter-carpal joint withpannus. while plain radiograph of the same patient is normal. fig. 4: transverse ultrasound demonstrates tenosynovitis of extensor carpi ulnar is tendon. them have compared ultrasound with radiography for the detection of erosions. thus, aim of our study was to evaluate the role of ultrasonography in the diagnosis of ra (particularly early ra) and comparison with radiography in the detection of erosions. proliferative synovitis is the earliest pathologic change seen in rheumatoid arthritis and is usually but not exclusively bilateral and symmetric. 13 approximately 96% of our patients had this finding at presentation. it’s presence in symmetric distribution involving wrist, intercarpal, metacarpophalangeal (mcp), proximal interphalangeal (pip) joints in varying proportions increases the probability of the disease being ra. 14 this fact is even more reinforced by the chronicity of symptoms. 34 sharanayya et al. / panacea journal of medical sciences 2021;11(1):31–36 table 3: serological changes in early and advanced ra serological marker early ra percent advanced ra percent n=44 n=11 raised crp 34 77.27% 10 90.90% ra factor +nt 23 52.27% 07 63.63% anti ccp +nt n=27 48.14% n=8 62.50% table 4: x-ray and hrus (high-resolution ultrasound) comparison detecting osteophyte and erosions. modality erosions n=37 percentage osteophytes n=8 percentage x-ray 11 29.72% 3 37.5% usg 37 100% 8 100% the widely accepted method for synovial hypertrophy quantification with greyscale ultrasound is the semiquantitative scale. 15 0 indicates no intra-articular changes, and 1–3 indicates mild, moderate, and large synovial hypertrophy. we also found application of this quantification system in our group easier and effective. both dorsal and volar scans can be used to detect joint effusion and synovial hypertrophy (figures 1 and 2). backhaus et al found 86% of positivity when scanning volar side of the hand compared to dorsal one, with only 14% positivity of dorsal synovitis alone in clinically affected joints. 16 ostergaard et al found only a third of patients having synovitis on both volar and dorsal side of the fingers, in the majority of cases synovitis being limited to volar43% or dorsal27%. 17 in our study, we found it easier to demonstrate synovial hypertrophy (sh) on examining dorsal aspect of mcp, intercarpal, wrist joints and volar aspect of pip joints. the difficulty arose when there was no power doppler signal (inactive pannus). in such cases the abnormal thickened soft tissue lying in joint space was considered as sh. detection of bone erosions at the time of ra diagnosis is related to a poor long-term functional and radiographic outcome, 18 and the presence of erosions in early undifferentiated arthritis is a risk factor for developing persistent arthritis. 19 when compared with radiography, us is definitely more sensitive in identifying the presence of erosions during initial patient evaluation of ra patients. 20 the findings of our study is in agreement with findings proposed by bajaj et al. 2007. 20 out of the 37 patients, where erosions were evident on hrsg, x-ray was positive in only eleven cases. detection of erosions early in the disease is predictive of an aggressive disease course. 21 thus, us also helps in determining prognosis. intra articular erosions were greatest in the wrist followed by the metacarpophalangeal (mcp), proximal interphalangeal (pip), and distal interphalangeal (dip) joints respectively. 22 in agreement with study stated here, 18 and 17 patients had involvement of wrists and intercarpal joints (icj) respectively in our study, out of total 26 patients who had erosive early ra (figure 3). in fact, in our study, out of total 55 patients, 12 (8 in early ra and 4 in advanced ra) had only involvement of wrists and icj. mcp and pip were involved in only 4 and 2 patients respectively. and in advanced ra, wrists and icj were invariably involved (all the patients had involvement of wrists and icj). in the wrist, erosion distribution was concentrated in the radiocarpal and medial carpometacarpal complex. 22 our findings were also in agreement with the conclusions of j c buckland-wright. 22 probably least important were the pip joints when evaluation of erosions were concerned. thus, examination of wrists and icj for evaluation of ra cannot be overemphasised. it can be noted that erosions were detected more on ultrasound than radiograph as ultrasound is a three-dimensional modality while radiograph is a twodimensional one. the diagnostic accuracy of sonography in the detection of erosions could not be calculated as ultrasound detected more erosions than radiographs and radiography is the gold standard for the detection of erosions. therefore, it was just a comparison study between radiography and ultrasonography. tenosynovitis is commonly an accompanying sonographic finding in patients of early ra. various hand tendon abnormalities were described in early stages of the disease in ra: widening of the tendons sheaths, loss of normal fibrillar echostructure, irregularity of the tendon margins. 23 in our study, widening of the tendon sheath due to hypo-echoic irregular synovial thickening was the common abnormality seen as shown in figure 4 (76% of total tenosynovitis). it may or may not be associated with synovial effusion. alternatively, synovial effusion can be seen without synovial hypertrophy. least common sonographic finding was the altered tendon echotexture (seen only in 24%). although any tendon may be affected, the flexor digitorum, extensor digitorum, and extensor carpi ulnaris (ecu) were frequently involved. 24 extensor group of tendons were commonly involved in our study group (86%). one interesting observation in our study was the involvement of ecu. isolated ecu involvement without involving other tendons was seen in 6 patients (29%). and two of these patients had tenosynovitis of ecu as the sharanayya et al. / panacea journal of medical sciences 2021;11(1):31–36 35 only initial finding without any evidence of synovitis. on follow-up, these two were later diagnosed as ra. thus, we find the study conducted by siri lillegraven et al. 2011 agreeable. 25 as the disease progresses there will be cartilage destruction, there by reducing the joint space. tendon contractures which result in joint deformity are also common during this stage. however, role of sonography lies in preventing their occurrence rather than detecting them. 5. conclusions sonography can be used as a primary modality to diagnose rheumatoid arthritis, especially early rheumatoid arthritis, which helps in reducing disabilities by early aggressive treatment. it is more sensitive than radiography in detecting erosions. 6. limitations major limitation of the study is that ultrasonography is operator dependant. correlation with mri would have helped in better diagnostic outcome. 7. source of funding no financial support was received for the work within this manuscript. 8. conflict of interest the authors declare they have no conflict of interest. references 1. sharma b, sharma m. ultrasonography of hands in rheumatoid arthritis. indian j rheumatol. 2009;4:102–11. 2. heide avd, remme ca, hofman dm, jacobs jw, bijlsma jw. prediction of progression of radiologic damage in newly diagnosed rheumatoid arthritis. arthritis rheum . 1995;38(10):1466–74. doi:10.1002/art.1780381013. 3. breedveld fc, dijkmans ba. differential therapy in early and late stages of rheumatoid arthritis. curr opin rheumatol. 1996;8(3):226– 9. doi:10.1097/00002281-199605000-00010. 4. heijde dm. radiographic imaging: the ’gold standard’ for assessment of disease progression in rheumatoid arthritis. rheumatology. 2000;39(1):9–16. doi:10.1093/oxfordjournals.rheumatology.a031496. 5. farrant jm, connor pj, grainger aj. advanced imaging in rheumatoid arthritis. skeletal radiol. 2007;36(4):269–79. doi:10.1007/s00256006-0219-9. 6. walther m, harms h, krenn v, radke s, trutz-peter f, gohlke f, et al. correlation of power doppler sonography with vascularity of the synovial tissue of the knee joint in patients with osteoarthritis and rheumatoid arthritis. arthritis rheum . 2001;44(2):331–8. doi:10.1002/1529-0131(200102)44:2<331::aid-anr50>3.0.co;2-0. 7. hoving jl, buchbinder r, hall s, lawler g, coombs p, mcnealy s, et al. a comparison of magnetic resonance imaging, sonography, and radiography of the hand in patients with early rheumatoid arthritis. j rheumatol. 2004;31:663–75. 8. lopez-ben r, bernreuter wk, moreland lw, alarcon gs. ultrasound detection of bone erosions in rheumatoid arthritis: a comparison to routine radiographs of the hands and feet. skeletal radiol. 2004;33(2):80–4. doi:10.1007/s00256-003-0693-2. 9. molenaar et, boers m, heijde dvd, alarcon g, bresnihan b, cardiel m, et al. imaging in rheumatoid arthritis: results of group discussions. j rheumatol. 1999;26:749–51. 10. backhaus m, kamradt t, sandrock d, loreck d, fritz j, wolf kj, et al. arthritis of the finger joints: a comprehensive approach comparing conventional radiography, scintigraphy, ultrasound, and contrast-enhanced magnetic resonance imaging. arthritis rheum . 1999;42(6):1232–45. doi:10.1002/15290131(199906)42:6<1232::aid-anr21>3.0.co;2-3. 11. mcgonagle d, gibbon w, connor po, blythe d, wakefield r, green m. a preliminary study of ultrasound aspiration of bone erosion in early rheumatoid arthritis. rheumatology (oxford). 1999;38:329–31. 12. wakefield rj, gibbon ww, conaghan pg, o’connor p, mcgonagle d, pease c, et al. the value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: a comparison with conventional radiography. arthritis rheum . 2000;43(12):2762–70. doi:10.1002/1529-0131(200012)43:12<2762::aid-anr16>3.0.co;2-. 13. rowbotham el, grainger aj. rheumatoid arthritis: ultrasound versus mri. ajr am j roentgenol . 2011;197(3):541–6. doi:10.2214/ajr.11.6798. 14. ozgul a, yasar e, arslan n, balaban b, taskaynatan ma, tezel k, et al. the comparison of ultrasonographic and scintigraphic findings of early arthritis in revealing rheumatoid arthritis according to criteria of american college of rheumatology. rheumatol int. 2009;29(7):765– 8. doi:10.1007/s00296-008-0765-7. 15. szkudlarek m, court-payen m, jacobsen s, klarlund m, thomsen hs, østergaard m, et al. interobserver agreement in ultrasonography of the finger and toe joints in rheumatoid arthritis. arthritis rheumatism: official j am coll rheumatol. 2003;48(4):955–62. doi:10.1002/art.10877. 16. backhaus m, ohrndorf s, kellner h, strunk j, backhaus tm, hartung w, et al. evaluation of a novel 7-joint ultrasound score in daily rheumatologic practice: a pilot project. arthritis care res: official j am coll rheum. 2009;61(9):1194–201. doi:10.1002/art.24646. 17. østergaard m, szkudlarek m. ultrasonography: a valid method for assessing rheumatoid arthritis? arthritis rheum. 2005;52(3):681–6. doi:10.1002/art.20940. 18. heijde dmvd. joint erosions and patients with early rheumatoid arthritis. rheumatology. 1995;34:74–8. 19. visser h, cessie s, vos k, breedveld fc, hazes jmw. how to diagnose rheumatoid arthritis early: a prediction model for persistent (erosive) arthritis. arthritis rheum. 2002;46(2):357–65. doi:10.1002/art.10117. 20. bajaj s, ben rl, oster r, alarcón gs. ultrasound detects rapid progression of erosive disease in early rheumatoid arthritis: a prospective longitudinal study. skeletal radiol. 2006;36(2):123–8. doi:10.1007/s00256-006-0196-z. 21. der heijde dv, riel pv, leeuwen mv, hof mv, rijswijk mv, de putte lv, et al. prognostic factors for radiographic damage and physical disability in early rheumatoid arthritis. a prospective followup study of 147 patients. rheumatology. 1992;31(8):519–25. doi:10.1093/rheumatology/31.8.519. 22. buckland-wright jc, walker sr. incidence and size of erosions in the wrist and hand of rheumatoid patients: a quantitative microfocal radiographic study. ann rheum dis . 1987;46(6):463–7. doi:10.1136/ard.46.6.463. 23. grassi w, tittarelli e, blasetti p, pirani o, cervini c. finger tendon involvement in rheumatoid arthritis. arthritis rheum: official j am coll rheum. 1995;38(6):786–94. doi:10.1002/art.1780380611. 24. boutry n, lardé a, lapègue f, solau-gervais e, flipo rm, cotten a, et al. magnetic resonance imaging appearance of the hands and feet in patients with early rheumatoid arthritis. j rheumatol. 2003;30(4):671–9. 25. lillegraven s, boyesen p, hammer hb, ostergaard m, uhlig t, sesseng s, et al. tenosynovitis of the extensor carpi ulnaris tendon predicts erosive progression in early rheumatoid arthritis. ann rheum dis. 2011;70(11):2049–50. doi:10.1136/ard.2011.151316. http://dx.doi.org/10.1002/art.1780381013 http://dx.doi.org/10.1097/00002281-199605000-00010 http://dx.doi.org/10.1093/oxfordjournals.rheumatology.a031496 http://dx.doi.org/10.1007/s00256-006-0219-9 http://dx.doi.org/10.1007/s00256-006-0219-9 http://dx.doi.org/10.1002/1529-0131(200102)44:2<331::aid-anr50>3.0.co;2-0 http://dx.doi.org/10.1007/s00256-003-0693-2 http://dx.doi.org/10.1002/1529-0131(199906)42:6<1232::aid-anr21>3.0.co;2-3 http://dx.doi.org/10.1002/1529-0131(199906)42:6<1232::aid-anr21>3.0.co;2-3 http://dx.doi.org/10.1002/1529-0131(200012)43:12<2762::aid-anr16>3.0.co;2-## http://dx.doi.org/10.2214/ajr.11.6798 http://dx.doi.org/10.1007/s00296-008-0765-7 http://dx.doi.org/10.1002/art.10877 http://dx.doi.org/10.1002/art.24646 http://dx.doi.org/10.1002/art.20940 http://dx.doi.org/10.1002/art.10117 http://dx.doi.org/10.1007/s00256-006-0196-z http://dx.doi.org/10.1093/rheumatology/31.8.519 http://dx.doi.org/10.1136/ard.46.6.463 http://dx.doi.org/10.1002/art.1780380611 http://dx.doi.org/10.1136/ard.2011.151316 36 sharanayya et al. / panacea journal of medical sciences 2021;11(1):31–36 author biography sharanayya, assistant professor shamrendra narayan, assistant professor vandana verma, professor madhu sharma, assistant professor anjana pande, assistant professor vivek jirankali, senior resident cite this article: sharanayya, narayan s, verma v, sharma m, pande a, jirankali v. joint involvement in rheumatoid arthritis: sonographic evaluation in comparison with radiography. panacea j med sci 2021;11(1):31-36. introduction materials and methods patients ultrasonography statistical analysis results discussion conclusions limitations source of funding conflict of interest isds annual conference proceedings 2019. this is an open access article distributed under the terms of the creative commons attributionnoncommercial 4.0 unported license (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. online journal of public health informatics * issn 1947-2579 * http://ojphi.org * 11(1): e271, 2019 isds 2019 conference abstracts accuracy of icd codes for identification: review of chlamydia, gonorrhea and syphilis yenling a. ho1, saurabh rahurkar2, janet arno3, 4, brian e. dixon1, 2, 5 1 indiana university fairbanks school of public health, indianapolis, indiana, united states, 2 regenstrief institute, indianapolis, indiana, united states, 3 indiana university school of medicine, indianapolis, indiana, united states, 4 marion county public health department, indianapolis, indiana, united states, 5 roudebush va medical center, indianapolis, indiana, united states objective the purpose of this study is to review the extant literature for evidence on the validity of icd-9-cm and -10-cm codes for the purpose of identifying cases of chlamydia, gonorrhea, and syphilis. introduction administrative data refers to data generated during the processes of health care. these data are a rich source of patient health information, including diagnoses and problem lists, laboratory and diagnostic tests, and medications. established standards are used to code each data into the appropriate coding systems. the international classification of diseases, ninth and tenth revisions, clinical modification (icd-9-cm and icd-10-cm) codes are the coding standard for diagnoses and have been frequently used to identify cases for the creation of cohorts in examining care delivery, screening, prevalence, and risk factors [1,2]. however, while some studies have assessed the validity and reliability of icd-cm codes to identify various conditions such as cerebral palsy and rheumatoid arthritis [3,4], the evidence for using icd codes to accurately identify sexually transmitted infection (sti) cases is largely unexamined. the purpose of this study is to review the extant literature for evidence on the validity of icd codes for identifying cases of chlamydia, gonorrhea, and syphilis. our findings will inform efforts to improve the use of administrative data for sti-related health service and surveillance researches. methods our systematic review followed a protocol consistent with the preferred reporting items for systematic reviews and meta analysis (prisma). we comprehensively searched pubmed and scopus databases for peer-reviewed articles published before february 2018. articles were identified with search terms related to our stis of interest (chlamydia, syphilis or gonorrhea), pelvic inflammatory disease (pid), administrative codes, and validation studies. pid was included as 33% -50% of pid cases are due to chlamydia or gonorrhea [5]. only empirical publications appearing in peer-reviewed english language journals were included. further, we excluded articles classified as letters to the editor, policy briefs, perspectives, commentaries, summaries of fu ture research plans, and grey literature. additionally, articles without abstracts were also excluded. the screening process used by our review is outlined in figure 1. briefly, all articles were subjected to a two-step screening process. first, we reviewed articles based on title and abstract. we eliminated studies that did not focus on stis or on validation in the context of stis. articles were included if they focused on any combination of the stis of interest, or on pid, and were validation studies on diagnostic testing or administrative codes. second, selected articles were then reviewed in full to identify studies which included the stis of int erest, assessed and listed icd-9-cm or -10-cm codes, and measured validity. the snowball technique was used on included articles, whereby we reviewed all references found in the references of the included articles. results our search strategy identified 1,754 articles to be screened by title and abstract. of these, only five (0.29%) articles met the initial inclusion criteria. after full text review, only two articles [6,7] met the final inclusion criteria to be included in the systematic review. both articles focused on pid with no assessment of syphilis. they utilized icd-9-cm codes to identify cases with pid and performed chart reviews to determine true pid status. results of both articles found positive predictive value (ppv) of pid to be between 18%–79%. only one article [7] examined the ppv of chlamydia (56%; 5/9 cases) and gonorrhea (100%; 4/4 cases) separately. http://ojphi.org/ isds annual conference proceedings 2019. this is an open access article distributed under the terms of the creative commons attributionnoncommercial 4.0 unported license (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. online journal of public health informatics * issn 1947-2579 * http://ojphi.org * 11(1): e271, 2019 isds 2019 conference abstracts conclusions we identified just two studies that evaluated the validity of icd codes in identifying the stis of interest. both studies focused on pid cases in which chlamydia and gonorrhea diagnoses and tests might be documented. additionally, since both studies were published before 2015, neither evaluates the use of administrative data following the u.s. transition to icd-10 codes. given these findings, further studies are required to examine the predictive value of icd-9 and -10 codes for all three diseases in the general population. acknowledgement the work described is supported by the national library of medicine (nlm) training grant under grand number t15lm012502. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nlm. references 1. tao g, zhang cx. 2008. hiv testing of commercially insured patients diagnosed with sexually transmitted diseases. sex transm dis. 35(1), 43-46. pubmed https://doi.org/10.1097/olq.0b013e318148c35a 2. evans he, mercer ch, rait g, et al. 2009. trends in hiv testing and recording of hiv status in the uk primary care setting: a retrospective cohort study 1995-2005. sex transm infect. 85, 520-26. pubmed https://doi.org/10.1136/sti.2008.034801 3. oskoui m, ng p, dorais m, et al. 2017. accuracy of administrative claims data for cerebral palsy diagnosis: a retrospective cohort study. cmaj open. 5(3), e570-75. pubmed https://doi.org/10.9778/cmajo.20170013 4. sauer bc, teng c-c, accortt na, et al. 2017. models solely using claims-based administrative data are poor predictors of rheumatoid arthritis disease activity. arthritis res ther. 19(1), 86. pubmed https://doi.org/10.1186/s13075-017-1294-0 5. haggerty cl, ness rb. 2006. epidemiology, pathogenesis and treatment of pelvic inflammatory disease. expert rev anti infect ther. 4(2), 235-47. pubmed https://doi.org/10.1586/14787210.4.2.235 6. satterwhite cl, yu o, raebel ma, et al. 2011. detection of pelvic inflammatory disease: development of an automated case-finding algorithm using administrative data. infect dis obstet gynecol. 2011, 428351. https://doi.org/10.1155/2011/428351. 7. ratelle s, yokoe d, blejan c, et al. 2003. predictive value of clinical diagnostic codes for the cdc case definition of pelvic inflammatory disease (pid): implications for surveillance. sex transm dis. 30(11), 866-70. pubmed https://doi.org/10.1097/01.olq.0000087945.08303.38 http://ojphi.org/ https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=17724427&dopt=abstract https://doi.org/10.1097/olq.0b013e318148c35a https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=19564649&dopt=abstract https://doi.org/10.1136/sti.2008.034801 https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=28720597&dopt=abstract https://doi.org/10.9778/cmajo.20170013 https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=28482933&dopt=abstract https://doi.org/10.1186/s13075-017-1294-0 https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=16597205&dopt=abstract https://doi.org/10.1586/14787210.4.2.235 https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=14603097&dopt=abstract https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=14603097&dopt=abstract https://doi.org/10.1097/01.olq.0000087945.08303.38 isds annual conference proceedings 2019. this is an open access article distributed under the terms of the creative commons attributionnoncommercial 4.0 unported license (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. online journal of public health informatics * issn 1947-2579 * http://ojphi.org * 11(1): e271, 2019 isds 2019 conference abstracts figure 1. flowchart of the article selection process for the systematic review. http://ojphi.org/ raja f.doc j bagh college dentistry vol. 25(special issue 1), june 2013 the study of oral diagnosis 67 the study of tempromandibular joint disorders and anticyclic citrullinated peptide antibodies in serum and saliva of patients with rheumatoid arthritis raya m. khidhir, b.d.s. (1) raja h. al-jubouri, b.d.s., m.sc., ph.d. (2) abstract background: rheumatoid arthritis is an autoimmune disease that affects mainly the synovial membranes and articular structures and is characterized by chronic, systemic inflammation involving multiple joints.being a synovial joint, the temporomandibular joint is subject to the same disorders affecting other synovial joints, including ra.beside it was considered as a specific serological marker for diagnosing ra disease ,antibodies to cyclic citrullinated peptide have proven to be associated with joints destruction, though; it may play a potential role in the prediction of the disease severity. materials and methods: sixty nine individuals (69) were enrolled in this study, forty nine (49) were patients diagnosed with rheumatoid arthritis, and twenty (20) were healthy control subjects. blood and saliva samples were taken from each subject for immunological analysis of anti-cyclic citrullinated peptides antibodies by elisa. each patient with rheumatoid arthritis disease was examined by means of research diagnostic criteria for temromandibular disorders for the assessment of tempromandibularjoint involvement. results: frequency of positive serum anti-ccp antibodies was higher in rheumatoid arthritis patients compared to healthy controls (p=0.000).tempromandibular joint clinical findings were bilaterally involved except joint sounds, sometimes; it was unilateral. chronic rheumatoid arthritis patients associated with higher prevalence of tempromandibular joint disorders than newly diagnosed ra, except limited mouth opening which were prevalent in newly diagnosed ra patients, (p=0.012) was significant.positive serum anti-ccp rheumatoid arthritis patients were associated with higher frequency of tempromandibular joint disorderscompared with ra patients with negative serum anti-ccp, a non-significant difference was found. conclusions:anti-cyclic citrullinated peptide antibodies are considered as a biomarker of inflammation and disease activity. tempromandibular joint disorders are frequently involved in rheumatoid arthritis patients. rheumatoid arthritis patients with positive serum anti-cyclic citrullinated peptides antibodies associated with higher frequency of tempromandibular joint disorders. keywords: rheumatoid arthritis, tempromandibularjoint, anti-cyclic citrullinated peptide antibody. (j bagh coll dentistry 2013; 25(special issue 1):67-71). introduction rheumatoid arthritis (ra) is a systemic autoimmune disease that may affect many tissues and organs, but is mainly characterized by chronic inflammation of the joints. the inflammation leads to joint destruction, joint deformity with loss of function and increased mortality (1). rheumatoid arthritis causes chronic inflammation in joint tissues; it is usually seen in other joints prior to temporomandibular joint involvement. the common clinical tmj findings in rheumatoid arthritis are tenderness, pain, clicking, crepitation, stiffness, and limitation in jaw movements (2). the publication in 1992 of the research diagnostic criteria for temporomandibular disorders (rdc/tmd) provided clinicians and investigators with a precise and reliable tool for diagnosing the most common alterations of the tmj. group iii of axis i of this classification includes the sub-categories arthralgia, osteoarthritis and osteoarthrosis(3). (1) master student, department of oral diagnosis, college of dentistry, university of baghdad. (2)professor, department of oral diagnosis, college of dentistry, university of baghdad. until recently, assays detecting rheumatoid factor (rf), antibodies directed against the fc portion ofthe igg molecule, have been the primary serologic testin diagnosis ofra.however, rf antibodies are not very specificfor this disease and can also be detected in other rheumatic disorders, infections, and in 3-5% of apparently healthy individuals(4). recently, antibodies directed to citrulline-containing proteins, which appear to bea promising alternative of rf in the diagnosis of ra .anti-ccp antibodies is specific for ra and appear early in the disease, often even preceding the symptoms of ra . moreover, anticcp is a reliable predictor of a progressive and erosive course of ra (5,6). the aims of the study were: 1. assessment of anti-cyclic citrullinated peptide (anti-ccp) antibodies in serum and saliva of patients with rheumatoid arthritis and compare the results with those of healthy control subjects. 2. study the prevalence of tempromandibular joint disorders in rheumatoid arthritis patients. j bagh college dentistry vol. 25(special issue 1), june 2013 the study of oral diagnosis 68 materials and methods the study was conducted in al-kindi teaching hospital and baghdad teaching hospital.the study samples consist of forty nine rheumatoid arthritis patients sub grouped intofifteen newly diagnosed rheumatoid arthritis of a duration less than one year,and thirty four chronic rheumatoid arthritis patients. apparently healthy controls group were twenty subjects.tempromandibular joint disorders were evaluated according to the rdc/tmd (3),and the examination included:range of mouth opening(limitation),joint sounds,palpation of masticatory muscles, palpation of tmj sites.determination of serum and saliva anticyclic citrullinated peptide antibodies was done by means of enzyme linkedimmunosorbent assay, usingigg elisa (aeskulisa)2-kits. statistical analysis graphical presentation by using: cluster bar charts.inferential data analysis by using chisquare statistic, likelihood ratio test, fisher exact probability test, contingency coefficients test for the cause’s correlation ship of the association tables and odds ratio. results laboratory results of anti-cyclic citrullinated peptide antibodies results in table (1) reveal that from overall 49 ra patients, 35 (71.4%) were with serum positive anti-ccp antibodies and the remainder ra patients were 14 (28.6%) with serum negative anti-ccp antibodies. healthy controls have had non results of serum anti-ccp0 (0.00%), thus, a highly significant differences was found (p=0.000) .results in table(2) reveal that eleven 11(73.3%) of the newly diagnosed ra patients were with serum positive anti-ccp and remainder were with serum negative anti-ccp 4 (26.7%).twenty four 24 (70.6%) of the chronic ra patients were with serum positive anti-ccp and remainder were with serum negative anticcp 10 (29.4%), though, a non-significant differences was found (p=0.845). the prevalence of tempromandibular joint disorders among newly diagnosed and chronic rheumatoid arthritis patients figure (1) illustrates the prevalence of tmj clinical findings among the two groups of ra. temporalis muscle’s tenderness was found in 12(80.0%) of newly diagnosed ra patients, and in 24(66.7%) of chronic ra patients. masseter muscle’s tenderness was found in 2(13.3%) of newly diagnosed ra patients, and in 12 (35.3%) of chronic ra patients. lateral poles of tmj was found tender in 13 (86.7%)of newly diagnosed ra patients, and in 22 (64.7%)of chronic ra patients. posterior attachments was found tender in 7(46.7%) of newly diagnosed ra patients, and in 19(55.9%) of chronic ra patients, nonsignificant differences was found when p>0.05 regarding muscles and joint tenderness between newly diagnosed and chronic ra patients.limited mouth opening was significantly prevalent in 12(80.0%) of newly diagnosed ra patients compared with 14 (41.2%) of chronic ra patients, a significant differences was found p=0.013 between newly and chronic ra patients.joint sounds was the only tmj clinical finding that was detected unilateral in some ra patients 11(22.4%), they were 8 (23.5%) in chronic ra patients and 3 (20%) in newly diagnosed ra patients. bilateral joint sounds among ra patients was detected in 9 (18.45%), they were 8 (23.5%) in chronic ra patients and 1 (6.7%) in newly ra patients. statistically,a nonsignificant difference was found p=0.259 between newly diagnosed and chronic ra patients. the prevalence of tempromandibular joint disorders among ra patients with serum positive and negative anti-ccp antibodies figure (2) illustrates the prevalence of tmj clinical findings among serum positive and negative ra patients. the frequency of temporalis muscle’s tenderness was found in 25 (71.4%) of serum positive anti-ccp compared with 11 (78.6%) of serum negative ra patients. frequency of masseter muscle’s tenderness was found in 10 (28.6%) of serum positive compared with 4 (28.6%) of serum negative anti-ccp ra patients. frequency of tenderness of tmj’s lateral poles was found in 25 (71.4%) of positive serum anti-ccp compared with 10 (71.4%) of negative serum anti-ccp ra patients. frequency of tenderness of tmj’s posterior attachments was found in 20 (57.1%) of positive serum anti-ccp compared with 6 (42.9%) of negative serum anticcp ra patients.frequency of limited mouth opening was found in 20 (57.1%) of positive serum anti-ccp compared with 6 (42.9%) of negative serum anti-ccp ra patients. frequency of joint sounds was found in 17(48.6%) of positive serum anti-ccp compared with 3 (21.4%) of negative serum anti-ccp ra patients, a non-significant differences was found regarding all those findings between serum positive and negative ra patients when p>0.05. discussion positive serum anti-ccp antibodies among the studied groups high frequency of positive serum anti-ccp test was significantly found among ra groups j bagh college dentistry vol. 25(special issue 1), june 2013 the study of oral diagnosis 69 71.4% compared with 0.00% in healthy controls, these findings correlates with (7), who reported relatively similar results with a positive serum anti-ccp 79% among ra patients, and none of the healthy controls’ sera were positive for anticcp. on the other hand, the current study results are inconsistent with studies of (8), who reported that 58% of ra patients were positive for anticcp and (9) who suggested that citrullinated collagen-ii results in 40% positivity for anticcp, which is too low in comparison with results of the current study.in addition,results revealed high frequency of anti-ccp positivity among newly diagnosed ra 73.3% compared with 70.6% for chronic ra. a non-significant difference was found statistically. an iraqi study carried by (10)proposed 70% positivity for early ra and 95% for chronic ra patients compared with 0.0% for healthy controls. prevalence of tempromandibular joint disorders among the two groups of rheumatoid arthritis patients bilateral tmj findings were detected among the ra patients frequent muscle tenderness including the temporalis and masseter musclewas found prevalent in chronic ra more than newly diagnosed ra patients. ardic et al. reported muscle tenderness in ra patients secondary to tempromandibular joint involvement, and stated that if a joint is not functioning normally a patient will often develop muscle tenderness that can be experienced as facial pain (11) .frequent joint tenderness in the area of lateral pole and posterior attachment was found prevalent in chronic more than newly diagnosed rapatients.this is in agreement with studies (12, 13,14)they found bilateral tmj pain in patients with rheumatoid arthritis which might be due to increase in pressure within the joint capsule as the pannus extrudes itself into the inter bony spaces but before any significant bone resorption has taken place.mouth opening was reduced in 53.1% of ra patients, and furthermore it was prevalent in the newly diagnosed ra patients compared with chronic ra patients. previous studies report that complains of limitation in mouth opening is common in more than half of ra patients (15, 16, 17)and could be as a result of intra-articular fibrous adhesions or due to displacement of the disc(18). joint sounds was the only tmj finding that was sometimes detected unilateral, however it was frequently detected in the chronic more often than newly diagnosed ra patients, agreed with (18), they reported somehow similar findings and stated that a disturbance in the normal anatomic relationship between the disc and condyle that interferes with smooth movement of the joint and causes momentary catching, clicking or popping (19) . the prevalence of tempromandibular joint disorders among ra patients with serum positive and negative anti-ccp antibodies results revealed that ra patients with positive serum anti-ccp have been found to exhibit clinical tmj disorders more often than ra patients with negative serum anti-ccp after evaluating tmj findings by rdc/tmd which suggests that positive serum anti-ccp ra patients associated with significant prevalence of tmj disorders.correlation of positive anti-ccp and the degree of joints involvement, including tmj in ra disease, have been previously investigated by (20). they stated that ra patients with positive serum anti-ccp develop more progressive and significant disease course than ra patients with negative anti-ccp, and interestingly; tmj was the last joint with its prevalence rate among anti-ccp positive patients compared with joints such as meta carpophalangeal (mcp) or proximal interphalangeal (pip). for the best of knowledge this is the first study that proposed tmj clinical findings in relation to anti-ccp antibody status by means of rdc/tmd. references 1. alamanos y, drosos aa. epidemiology of adult rheumatoid arthritis. autoimmune rev 2005; 4(3):130-6. 2. helenius l, hallikainen d, helenius i, meurman j, könönen m, leirisalo-repo m, et al. clinical and radiographic findings of the temporomandibular joint in patients with various rheumatic diseases. a case control study. oral surg oral med oral pathol oral radiol endod 2005; 99:455-463. 3. dworkin sf, leresche l. research diagnostic criteria for temporomandibular disorders: review, criteria, examinations and specifications, critique. j craniomandib disord 1992; 6:301-55. 4. vallbracht i, rieber j, oppermann m, fo¨rger f, siebert u, helmke k. diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis. ann rheum dis 2004; 63:1079–1084. 5. schellekens g, visser h, de jong b de, et al. the diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. arthritis rheum 2000; 43:155-64. 6. nielen m, van schaardenburg d, reesink h, et al. specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. arthritis rheum2004; 50: 380–386. 7. vasishta a. diagnosing early-onset rheumatoid arthritis: the role of anti-ccp antibodies. am clin lab 2002; 21:34–6. 8. ronnelid j, wick m, lampa j. longitudinal analysis of citrullinated protein/ peptide antibodies (anti-cp) during 5 years follow up in early rheumatoid arthritis: anti-cp status predicts worse disease activity and j bagh college dentistry vol. 25(special issue 1), june 2013 the study of oral diagnosis 70 greater radiological progression. ann rheum dis 2005; 64: 1744-1749. 9. burkhardt h, sehnert b, bochermann r. humoral immune response to citrullinated collagen type ii determinants in early rheumatoid arthritis. eur j immunol 2005; 35: 1643-52. 10. el-saffar j. role of anti-cyclic citrullinated peptide antibodies and interleukins-2, 6 in diagnosis of rheumatoid arthritis in iraqi patients. a dissertation. college of science / university of baghdad. microbiology; 2008. 11. ardic f, gokharman d, atsu s, guner s, yilmaz m, yorgancioglu r. the comprehensive evaluation of temporomandibular disorders seen in rheumatoid arthritis. australian dental journal 2006; 51:(1):23-28. 12. tegelberg a, kopp s. clinical findings in the stomatognathic system for individuals with rheumatoid arthritis and osteoarthrosis. acta odontol scand 1987; 45:65 75. 13. movahedian b, razavi m, movahedian a, moeini m. assessment of manifestations of temporomandibular joint involvement in rheumatoid arthritis patients. j isfahan dental school 2006; 2(3): 1385. 14. hussein am. the study of oral manifestations, apoptotic activity and rheumatoid factor in serum and saliva of rheumatoid arthritis patients. a master thesis. department of oral diagnosis. college of dentistry/ university of baghdad, 2011. 15. kallenberg a, wenneberg b, carlsson g, ahlmen m. reported symptoms from the masticatory system and general well-being in rheumatoid arthritis. j oral rehab1997; 24: 342–349 (ivsl). 16. yamakawa m, ansai t, kasai s, ohmaru t, takeuchi h, kawaguchi t, takehara t. dentition status and temporomandibular joint disorders in patients with rheumatoid arthritis. cranio 2002; 20(3): 165–171. 17. lin y, hsu m, yang j, liang t, chou s, lin h. temporomandibular joint disorders in patients with rheumatoid arthritis. j chin med assoc 2007; 70:527 534. 18. aliko a, ciancaglini r, alushi a, tafaj a, ruci d. temporomandibular joint involvement in rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. int j oral maxillofac surg 2011; 40: 704– 709. 19. laskin dm. internal derangements. oral maxillofac surg clin north am 1994; 5: 217–222. 20. van der helm-van mil a, verpoort k, breedveld f et al. antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. arthritis research & therapy 2005; 7: 949-958. table 1: distribution of the studied samples according to positive serum anti-ccp with comparisons significant serum anti-ccp freq.’s &percents the studied groups total c.s. (*) p-value healthy control rheumatoid arthritis negative freq. 20 14 34 c.c.=0.544 p=0.000 hs % serum anti-ccp 58.8% 41.2% 100% % the studied groups 100% 28.6% 49.3% positive freq. 0 35 35 % serum anti-ccp 0.0% 100% 100% % the studied groups 0.0% 71.4% 50.7% (*) hs: highly significant at p<0.01 table 2:distribution of the ra groups according to positive serum anti-ccp with comparisons significant serum anti-ccp freq.'s &percents the studied groups total c.s. (*) p-value newly diagnosed ra chronic ra negative freq. 4 10 14 c.c.=0.028 p=0.845 ns % serum anti-ccp 28.6% 71.4% 100% % the studied groups 26.7% 29.4% 28.6% positive freq. 11 24 35 % serum anti-ccp 31.4% 68.6% 100% % the studied groups 73.3% 70.6% 71.4% (*) ns : non significant at p >0.05 j bagh college dentistry vol. 25(special issue 1), june 2013 the study of oral diagnosis 71 figure 1: tmj clinical findings among ra patient group figure 2: tmj clinical findings among positive and negative serum anti-ccp ra patients adenosine deaminase (ada) in rheumatoid arthritis in patients (ra) huda th. al-marsomi j fac med baghdad vol. 51, no. 2, 2009 198 adenosine deaminase (ada) in rheumatoid arthritis in patients (ra) huda th. al-marsomi* phd summary: background: rheumatoid arthritis (ra) is heterogenous syndrome. because the diversity of disease processes and formation of complex lymphoid microstructures that indicate the multiple t cell activation pathways are involved .affected patients have major abnormalities in the t cell pool with clonally expanded cd4 + t cell that lose expression of the cd28null molecule and lack the ability for profiliration. adenosine deaminase (ada) is an indicator of the proliferation and differenation of lymphocyte, in particularly the t cell subcells. patients and methods: total ada levels were measured in the sera of ra patients and healthy group according to giusti (1981). results: the mean value of ada was lower in patients with ra than control group with no significant differences. conclusion: the lower value of ada (which involved in the proliferation of lymphocyte) in ra patients may results from the predominance of cd4 + t cells in the peripheral blood key words: ra, ada, ir. introduction: rheumatoid arthritis (ra) is a sever chronic inflammatory auto-immune disorder of mysterious etiology, characterized by inflammation of synovial membrane, principally affecting peripheral joints in asymmetric fashion , extra-articular manifestations also occur, so ra is a disease of an aberrant immunresponse (ir) in a genetically predisposed host, both humoral and cellular ir are important in this disease(1,2).in ra patients have abnormalities in t cell function that are not restricted to the t cell participating in the synovial infiltrates . one aberration is the expansion of selected cd4 t cell to large colonel population. in patients with sever ra, the cd4 +cd28null t cells were initially identified(3,4) .adenosine deaminase (ada) (adenosine aminohydrolase, ec (3.5.4.4)) is the enzyme that irreversibly catalyzes the deamination of adenosine and deoxyadenosine to inosine and deoxyinosine respectively and ammonia(5) . ada is involved in the proliferation and differenation of lymphocyte, particularly the tcell subtype, which was found to play a crucial role in the metabolism of the immune system cells and it is essential for the proper development of both t and b lymphocyte in mammals(6). the aim of this study is to investigate the ada level among ra patients as indicator of ir. *department of microbiology, college of medicine, al-nahrin university. patients and methods: the study included two groups: a. rheumatoid arthritis (ra) patients: blood samples were collected from thirty patients, their ages ranged from (30-60) years who were attending to al – kadhmia teaching hospital from march to august in 2005 and diagnosed by doctors to be infected with ra( before any treatment),all patients have symptoms for two to six months. b .healthy control group: fifteen individuals from blood bank donors, who have no history of clinical evidence of ra or other clinical disease. sera were separated and stored at -20 ºc until use. total ada levels were measured in the serum of each patient by the method described by giusti ( 1981) (7). the method is based on measuring the rate of ammonia consumption at 620 nm following the reaction. results: as shown in table 1 results indicated that their were no significant differences between ra patients (14.63±2.4 u/l) and control group (18.9± 2.15 u/l). original article fac med baghdad 2009 vol. 51 no. 2 received june 2008 accepted jan. 2009 adenosine deaminase (ada) in rheumatoid arthritis in patients (ra) huda th. al-marsomi j fac med baghdad vol. 51, no. 2, 2009 199 table-1: mean± sd of serum adenosine deaminase (ada) level in u/l among ra patients and control group. t=3.09, p<0.05, sd=standard deviation discussion: ra is the most common inflammatory arthritis, affecting about 1% of the general population world wide (8) . pathogenesis of ra is till not fully understood , there is evidence that cd4 +t cells play a central role in initiating , perpetuating and precipitating chronic inflammation in synovial tissue(1,9). another role of activated cd4 +t cells is stimulation of b cells to differentiate into plasma cells producing rf (rheumatoid factor) and other autoantibodies(2,10). ada enzyme is one of the most essential immune enzymes. it is function gives a clear picture of the immune status of the body. it was found to play a critical role in proper development of the t and b-lymphocytes in mammals( 10,11). in this study, no significant difference was found regarding the mean value of serum ada among ra patients when compared with the control group and this may be because those in ra patients had marked difficulties in repopulating the t cell compartment. in ra patients, peripheral cd4+t cells counts remained depressed to lymphopenic levels for an extended period also t cell have a limited prolifirative life span and chronic immune activation could lead to accumulation of clonally expanded senescents cell (cd4+cd28null) which generally characterized by a limited or complete lack of proliferation that agreement with our resultas as that ada a marker for cell proliferation. it will be of interest to measure ada level in synovial tissue before and after treatment and compare it with ada level in serum to get more information about association between the immune response and ada level as parameter to response to treatment . references: 1. anderson, r.j; rheumatoid arthritis, clinical and laboratory features.in: keppel, j.h; crofford, l.j; ston, j.h.et al .primer on the rheumatic disease.12thedit.atlanta georgia-arthritis foundation .2001:218-225. 2. helen, ch. ,mansel, h. ,siraj, m. and neil, s.:joints and muscles. in: essentials of clinical immunology 5thed.blackwell publishing.u.s.a.;2006:p.180-183. 3. abbe,n.v., eduardo,d., cornelia,m.w. and jorg,j.g. :biology of t lymphocytes. rheum dis clin n am ,2004;(30 ) 135-157. 4. cornella,m.w.,bryl,e.,jorg, j.g.; the role of t cells in rheumatoid arthritis. archivum immunologiae et therapiexperimentalis,2000;48:429-435 . 5.dinjens,w.n.m., tenkate,j. and bosman,f.t.:distribution of adenosine deaminase complexing protein (adcp) in human tissue. j histochem cytochem ,1989;(37)1869-1875. 6.ammann, a.j.:immunodeficiency disease. in : stites, d.p.; stobo, j.and wells, j.v.editors. basic and clinical immunology.6thed. usa appleton&lange; 1987; p.339-341. 7. giusti, g.:adenosine deaminase.in: bergmeyer, h.v. ed. methods of enzymatic analysis.2nded . florid: verlag chemise international; 1981.2 p.1092-1099. 8. al-rawi, z.s., al-azzawi, aj., al-ajili, f.m., alwakilr.:rheumatiod arthritis in population samples in iraq. ann rheum dis,1978; 37:73-75. 9.kathleen ,b.and mason,i,:pathomechanisms in rheumatoid arthritis-time for a string theory? .the journal of clinical investigation ,2006 ;116(4):869871. 10. al-ubaide, a.h.; al-jeboori, t.i. &juma, a, s.adalevel in patients with hydatid disease (echinococcus granulosis).iraqi j med sci, 2003; 2(1):25-28. 11.juma, a., al-jeboori, t.i., tawfiq, m.s. &fadhil, r.s.:ada activity in the serum of patients with schistosoma haematobium & those with bladder carcinoma. iraqi j med sci, 2003; 2(3):24-28. group mean ±sd ra patients 14.63±2.4/l control group 18.9±2.15u/l philippine journal of otolaryngology-head and neck surgery vol. 23 no. 2 july – december 2008 52 philippine journal of otolaryngology-head and neck surgery practice pearls the past three years have seen an overwhelming increase in the number of dysphonic patients in our clinics. this phenomenon goes hand in hand with increased opening of call centers nationwide and increased demand for teachers, singers and performers abroad. this article discusses simple steps for the otolaryngologist interested in evaluating these patients with different voice demands. it is important to recognize these common voice problems and address them promptly, or to refer them accordingly to voice centers if necessary. chief complaint the most common chief complaint is change in the quality of the voice or hoarseness. hoarseness means a change in the perception of one’s voice, described as harsh, raspy, “paos” or “malat.” other complaints include breathiness, throat pain, neck pain, inability and unrealibility to reach high notes. inability to reach high notes suggests edema of the vocal folds making them more plump, as can be found in reflux laryngitis, allergies or smoking. lesions such as nodules, polyps and cysts cannot be discounted because they prevent vocal fold closure especially during high notes.1 throat and neck pain without an accompanying history of infection may suggest muscle tension dysphonia, especially in a voice professional who later develops maladaptive ways of talking that could strain other throat and neck muscles in an effort to speak.2 frequent throat clearing, a sensation of phlegm in the throat and cough are also important chief complaints that may lead the otolaryngologist to the cause of the voice problem. in the absence of upper respiratory tract infections and post-nasal discharge, these could be suggestive of laryngopharyngeal reflux.3 history does the hoarseness occur on and off? was it sudden? after shouting in a basketball event? is it becoming worse and permanent? what triggers or relieves it? intermittent hoarseness could be due to voice abuse and misuse especially in a voice professional. sudden hoarseness especially after watching a basketball event could be suggestive of vocal fold hemorrhage. a voice problem becoming worse and permanent could be a growing polyp or cyst, vocal fold paralysis in laryngeal cancer or thyroid cancer. a long lecture triggering the hoarseness and rest relieving it may suggest soft nodules, or reinke’s edema due to vocal fold trauma of voice abuse and misuse. to begin with, it is important to know the occupation of our patient. is our patient a voice professionalsomeone who uses his or her voice for a living? voice demands at work contribute to voice change significantly and voice abuse and misuse is one of the most common causes of hoarseness. what are the other associated symptoms? filipina t. cevallos-schnabel, md, mph1,2,3 1department of otolaryngology head and neck surgery east avenue medical center 2department of surgery, capitol medical center 3department of otolaryngology head and neck surgery faculty of medicine and surgery, university of santo tomas an easy guide for voice evaluation in the clinic correspondence: filipina t. cevallos-schnabel, md, mph rm. 1102 capitol medical center scout magbanua cor. panay ave , q.c. philippines 1103 phone : (632) 372-3825 loc 5102 email : pincevallosmd@yahoo.com reprints will not be available from the author. no funding support was received for this study. the author signed a disclosure that she has no proprietary or financial interest with any organization that may have a direct interest in the subject matter of this manuscript, or in any product used or cited in this article. philipp j otolaryngol head neck surg 2008; 23 (2): 52-54 c philippine society of otolaryngology – head and neck surgery, inc. philippine journal of otolaryngology-head and neck surgery vol. 23 no. 2 july – december 2008 practice pearls philippine journal of otolaryngology-head and neck surgery 53 medical problems like a recent bout of upper respiratory tract infection and allergies are among the most common causes of hoarseness and should not be discounted immediately. symptoms of hyperacidity are also significant.4 is there a history of breathiness and difficulty of breathing? voice fatigue, tremor, hypo or hypernasal voice? choking, globus, odynophagia or dysphagia? neck pain or head and neck trauma? these questions can give clues to the clinician regarding the possible cause of the problem. past medical history asthma, copd, pulmonary malignancy are associated with voice changes due to decreased airflow. gastric ulcers and gerd can be suggestive of associated laryngopharyngeal reflux disease changing the vocal fold mucosa leading to voice change.3 parkinsonism, myasthenia, traumatic brain injury and movement disorders can cause tremors, weakness or strained voice quality. rheumatoid arthritis, sle and other autoimmune disorders can cause voice changes such as paralysis in ra. endocrine problems such as hypothyroidism can cause edema of the vocal folds leading to decrease in pitch. thyroid cancer can cause vocal fold paralysis. a history of radiation secondary to malignancies in the head and neck can cause vocal fold scarring leading to voice change.1 personality and psychiatric disorders also lead to diagnosis. the outgoing, type a personality usually has vocal fold nodules while inhibited and shy persons have functional dysphonias.5 traumatic life events are also very important to take note of. history of surgery for neck trauma, thyroid nodules or malignancies, spine, cardiac, pulmonary and brain surgeries or previous endotracheal intubation can cause voice changes, usually related to vocal fold mobility problems. 1 medications such as inhalational steroids for asthma can cause fungal laryngitis. arb and ace inhibitors for hypertension can cause non specific vocal fold masses. antitussives, decongestants, antihistamines and vitamin c are known to cause dryness of the vocal folds. pills with sexual hormones can cause either elevations or decreases in pitch.6 smoking can cause polypoid conditions in the vocal folds, premalignant or malignant changes. intake of alcohol, diet and lifestyle can contribute to reflux problems and dysphonia. physical examination hearing the patient and forming a subjective impression of the patient’s voice should automatically be part of the interview process. ranking the voice according to a standard scale is subjective but becomes increasingly reproducible and precise with training and experience. voice can be evaluated according to pitch, loudness and vocal quality. pitch is the highness or lowness of the voice. is the speaking voice too low for the soprano? this could be the problem why a trained singer would have dysphonia. does the woman sound like a man over the phone? this could be reinke’s edema, maybe she is a smoker as well. does the adult male suddenly speak with elevated pitch? this could be vocal fold paralysis. loudness is the power of the voice. this is due to the source of power, the lungs. posture, type of breathing, technique or training can affect this. systemic problems like generalized weakness and cachexia are contributory. of course pulmonary problems can contribute to decreased power. voice quality can be evaluated using the grbas system.7 just hearing the voice and using this system is helpful in making an impression. ggrade rroughness bbreathiness aasthenia sstrain grbas uses a 0 to 3 scale (0= normal or absence of deviance; 1=slight deviance; 2=moderate deviance; 3= severe deviance). grade relates to the overall voice quality, integrating all deviant components roughness breathiness asthenia strain grbas sounds probable conditions grainy quality; diplophonic airy no voice tight quality vocal fold masses such as nodules, polyps, cysts, laryngitis unilateral paralysis, bowing, atrophy, abductor spasmodic dysphonia bilateral paralysis in paramedian position, vocal fold atrophy adductor spasmodic dysphonia, muscle tension dysphonia head and neck examination palpating the neck, especially the base of the tongue and neck muscles which are tense and tender can be suggestive of an ongoing muscle tension dysphonia as a cause of the voice change.8 thyroid masses, neck nodes, etc. can be helpful in leading the clinician to a diagnosis. visualizing the larynx has evolved as advances in technology have improved the understanding of vocal fold anatomy, physiology and voice production. at present, there is no single laryngeal examination tool that is superior to the others. what is important is that it gives a thorough visualization of the anatomy and a good functional evaluation of the larynx. selecting the appropriate instrumentation will be possible if we recognize the advantages and limitations of the diagnostic tool we are using.9 sometimes, a combination of these tools is important to make an accurate diagnosis. philippine journal of otolaryngology-head and neck surgery vol. 23 no. 2 july – december 2008 54 philippine journal of otolaryngology-head and neck surgery practice pearls references: 1. simpson cb, fleming d. medical and vocal history in the evaluation of dysphonia. otolaryngol clin n am 2000; 33(4): 719-750. 2. morrison md, rammage la, belisle gm. muscular tension dysphonia. j otolaryngol 1983; 12:302-306. 3. koufman ja. the otolaryngologic manifestations of gastroesophageal reflux disease (gerd): a clinical investigation of 225 patients using ambulatory 24-hour ph and an experimental investigation of the role of acid and pepsin in the development of laryngeal injury. laryngoscope 1991; 101: 1-78. 4. olson nr. laryngeal manifestations of gastroesophageal reflux disease: otolaryngol clin n am 1991; 24: 1201-1213. 5. rosen dc, sataloff rt. psychology of voice disorders. 2006. san diego (ca): plural publishing inc. pp. 729-50. 6. abaza mm, levy s, hawkshaw m, sataloff r. effects of medications on voice. otolaryngol clin n am 2007; 40:1081-1090. 7. dejonckere ph. perceptual and laboratory assessment of dysphonia. otolaryngol clin n am 2000; 33(4): 731-49. 8. sataloff rt, hawkshaw m, divi v, heman-ackah y. physical examination of voice professionals. otolaryngol clin n am 2007; 40: 953-69. 9. rosen c, murry t. diagnostic laryngeal endoscopy. otolaryngol clin n am 2000; 33(4): 751-57. 10. kaszuba s, garrett cg. strobovideolaryngoscopy and laboratory voice evaluation. otolaryngol clin n am 2007; 40: 991-1001. advantages and limitations of the different instruments to visualize the larynx indirect mirror laryngoscopy transnasal flexible laryngoscopy rigid 70 or 90 degrees laryngoscope videostroboscopy10 instrument advantages limitations readily available; inexpensive gives a gross idea of the anatomy; mobility; mucus; and mass (if big enough) helpful for hypergag patients; patients physiology involving the tongue, pharynx and palate are well visualized; can assess paresis from paralysis; can be recorded for review extremely clear and magnified view; less expensive; can be recorded for review provides a slow motion evaluation of vocal fold vibratory pattern, closure, mucosal wave; can differentiate benign vocal fold lesions limited in patients who are hypergag; patient is not in a normal physiologic position; hard to detect paresis and small lesions small lesions are hard to differentiate; color might not be reliable depending on the camera; may be expensive limited in patients who are hypergag; patient is not in a normal physiologic position; hard to detect paresis and muscle tension dysphonia expensive; requires additional training despite technological advances in laryngology, a good history and physical examination are still crucial in the diagnosis of voice disorders. certain clues can be provided by a good history that especially point to a hoarse patient. because no single instrument is superior for visualization of the larynx, it is important to recognize the advantages and limitations of each. some helpful vocal tasks when using a flexible scope: /ii/ sniff hee-hee-hee sniff then /ii/ /ii/ glide form low to high pitch task endoscopic findings adduction abduction either decreased adduction or abduction fatigues the vocal folds; detects paresis/ weakness ability to lengthen the vocal folds 06 26 proceedings s.z.m.c. vol: 36(1): pp. 26-30, 2022. pszmc-829-36-1-2022 colebrookea oppositifolia 1department of pharmacology, post graduate medical institute, lahore 2department of pharmacology, king edward medical university, lahore 3department of pharmacology, fatima memorial hospital, lahore 4department of histopathology, jinnah hospital, lahore 5department of medicine, sir ganga ram hospital, lahore colebrookea oppositifolia (co) colebrookea oppositifolia c. oppositifolia colebrookea oppositifolia , colebrookea oppositifolia c0 27 colebrookea oppositifolia anti-arthritic potential vs methotrexate in pristane induced rat arthritis colebrookea oppositifolia c0 c0 c0 c0 28 colebrookea oppositifolia anti-arthritic potential vs methotrexate in pristane induced rat arthritis c0 co co 29 colebrookea oppositifolia anti-arthritic potential vs methotrexate in pristane induced rat arthritis c. oppositifolia c. oppositifolia c. oppositifolia co co co co co 30 colebrookea oppositifolia anti-arthritic potential vs methotrexate in pristane induced rat arthritis colebrookea oppositifolia sm. pergularia daemia moringa rivae microsoft word gjphm-2019-hcq copy.docx 52 global journal of public health medicine 2019, vol 1, issue 2 gggggglo the role of hydroxychloroquine as monotherapy in managing early undifferentiated arthritis: a prospective hospital-based study ziryab imad taha mahmoud1,2 , mustafa mohamed ali hussein1,3, mohammed elmujtba adam essa adam1,3*,sherihan mohammed elkundi osman3,4, mutwaly defealla yousif haron1,3, mohammed altyb alshykh5,elnour mohammed elagib6, abdelkareem abdallah. ahmed1,7. 1 department of clinical medicine, medical and cancer research institute (mcri), nyala, sudan. 2 department of internal medicine and rheumatology, faculty of medicine, university of bahri, khartoum, sudan. 3 department of health, faculty of medicine, al fashir university, al fashir, sudan. 4 department of molecular medicine, institute of endemic diseases, university of khartoum, khartoum, sudan. 5 faculty of medicine, alzaim alazhari university, khartoum, sudan 6 department of internal medicine and rheumatology, omdurman military hospital, khartoum, sudan. 7 department of physiology and biochemistry, faculty of veterinary science, university of nyala, nyala, sudan. * corresponding author:awadali818@yahoo.com abstract introduction: early undifferentiated arthritis (eua) is a common form of arthritis comprising, joint pain, stiffness and swelling with no definitive diagnosis. patients of eua can progress to other forms of rheumatic arthropathies such as rheumatoid arthritis or remain in the same form or spontaneously disappear. the main focus of this study is to explore the potential effect of hydroxychloroquine (hcq) in management of eua as a monotherapy treatment. methods: this is a prospective hospital-based study which was conducted in almwada hospital in khartoum, sudan. the study included thirty patients of eua. full clinical examination and history were done by a rheumatologist, and all the related investigations were obtained, and they all received hcq after eua diagnosis has been established. result: the study shows that 96% of the patients responded well to the treatment and 10% had their duration of treatment doubled to show a favorable response. we also found that female patients were more commonly affected than male ones with higher incidence among middle aged as compared to others. after treatment with hcq, 86.6% of the patients showed average mean decrease in erythrocyte sedimentation rate (esr) by 44%, the other 13.4%, even though they were symptoms free after treatment they showed increased level of esr by 30% average. conclusion: in the present study we found out most of the eua patients are well responded to the hcq treatment, and most of them respond from the first course of treatment, the study also shows higher incidence among female in compared to male. key words: early undifferentiated arthritis, middle age group, hydroxychloroquine, monotherapy. 53 global journal of public health medicine 2019, vol 1, issue 2 gggggglo introduction early undifferentiated arthritis is an inflammatory polyarthritis or monarthritis with no definitive diagnosis that has a duration of less than three months and do not categorized under any connective tissues or rheumatic disease (combe et al., 2017; dixon & symmons, 2005; wevers-de boer, heimans, huizinga, & allaart, 2013), patients with early undifferentiated arthritis minimally should have one tender joint or swelling (aletaha et al., 2010), eua can be initial presentations of many rheumatic diseases including rheumatoid arthritis(combe et al., 2017; dixon & symmons, 2005; vaidya, baral, & nakarmi, 2018). after the 2010 eular/acr updated the classification criteria (aletaha et al., 2010) many cases of eua can be diagnosed as early rheumatoid arthritis (era). many patients of early scleroderma, rheumatoid arthritis or lupus are presented as eua and their diagnosis becomes more obvious after a period of one year and sometimes months (suresh, 2004), about one third of eua patients will achieved spontaneous remission(stockman et al., 2006), a third will remain as eua and the rest will progress to rheumatoid arthritis (ra)(hazes & luime, 2011). there is no standard management for eua, most of the treatments are off-label such as steroids or non-steroids anti-inflammatory drugs, until the patient exhibited apparent features of chronicity then others alternative drugs like disease modifying anti rheumatic drugs (dmards) are used(van dongen et al., 2007). current evidence proved that it is unjustifiable to wait until erosions or chronicity to initiate dmards (bosello et al., 2011; joshua, edmonds, & lassere, 2006; sudolszopinska et al., 2013), and the goal of management is to reduce the risk of chronicity and complications of the disease (combe et al., 2017; smolen et al., 2016), if the patient doesn’t reach significant improvement, then the therapeutic agent is upgraded as needed in a form of combination of methotrexate, hydroxychloroquine and sulfasalazine with dmerds according to the patient tolerance and side effect profile, in spite of the efforts and the studies on eua, until now no clear pathophysiology has been identified. the current study aims to explore the effectiveness of using hydroxychloroquine as monotherapy for treatment of eua patients. methods this is a hospital-based study which was conducted in almwada hospital, khartoum, sudan, where we screened and selected prospectively a number of 30 patients according to specific criteria in the outpatient department of medicine in the period of april 2017april 2018. all patients have been examined (musculoskeletal examination) with detailed history taken by specialist (dr. ziryab imad taha mahmoud) to achieve the final result after reviewing the lab investigation. the study is focusing on the effect of hydroxychloroquine (hcq) on undifferentiated arthritis patients after fulfill the inclusion criteria which includes only patients regardless of the age and gender, complaining of multiple tender joint pain in duration lees than six weeks , and the exclusion criteria is patients complaining of joints pain associated with any of the following: swelling, stiffness, deformity, fever, duration more than six weeks, any other systemic symptoms. we collected the data from the patients and they were given a discharge summary card for their regular follow up, the card contains the date of every follow up meeting for checkup of the renal function test, esr level and general wellbeing, the initial clinical presentation, all the relevant investigation such as the esr, rheumatoid factor (rf), anti-cyclic citrullinated peptide (anti ccp), antinuclear antibodies (anas), blood urea (bu), serum creatinine, liver enzymes and bilirubin level, complete blood count (cbc), and urine microscopic examination. finding of the clinical examining, duration of the disease, medication patterns, assessment various organs involvement, gender, age, ethnic and geographical distribution, were also been recorded to include in data sheet. modified disease activity score 28 (das28), is being used to establish the diagnosis and the treatment outcome. the therapeutic approach was initiated by using the hcq as monotherapyin management of eua at a dose of 400 mg daily, divided twice, in tablet form, for a period of 3 months, and another three months in the cases where a poor was noticed according to das28 criteria. 54 global journal of public health medicine 2019, vol 1, issue 2 gggggglo analysis the collected data were stored using the computer program. nominal data are expressed as frequency or proportion. all statistical analysis was performed using the statistical analysis package for social science (spss, v.22.0 chicago, illinois, usa), an independent t test and one-way anova are tested with a level of the significant set at (p<0.05) . results in this study we found that female has the higher prevalence among our patients in a percentage of 86.7% and male patients represented the remaining 13.3 % (fig 1). regarding age group, we classified the patients into three categories, age 20 years and less than 40years, 40 to less than 60 and 60-80years (table 1). the middle age group patients which are between 4059 years are the most affected group (46.7%), followed by 36.7% for the group between 20 years and less than 40 years, and the last age group which is the group between 60-80 years represent the remaining 16.7% (table 1). further analysis showed that response to treatment is 96.7%, while the remaining 3.3% of the patients shows no treatment response (fig2). also, we noted that 90% of the patients were responsive to treatment from the first course which is 3 month, while the other 10% had their duration of treatment doubled (fig 3). regarding the treatment effect on erythrocyte sedimentation test (esr), they were categorized into three groups, 86.6% of the patients had their average mean decreased to 44% after they receiving the treatment, 3.3% of the patients had their esr decreased to 13% after the treatment, the remaining 13.4% of the patients showed increase in esr level by 30% even after treatment but their complains disappeared (fig 4). the std deviation of the age and sex are 0.71 and 0.34 respectively (fig 5), the age groups, esr, gender, duration and treatment dosage, clinical presentation, outcome and investigations all have been showed in table (2). the initial das28 of the patients at the time of diagnosis shows that 73.3% of the patients has a high score and 26.7% with moderate score, after receiving treatment with hcq for three months, the high score group had 50% lower das28 score and 45.4% of them became moderate score and 4.6% achieved total remission. among those 45.4% with moderate das 28 score after receiving three months treatment, 20% of them received another three months treatment and achieved 100% remission. the second group with moderate das28 at the time of diagnosis after three months of treatment also showed 50% lower score, 37.5% remission and the remainder 12.5% showed no decrease in das28 score until they received another three months treatment to end by having 100% remission (fig 6) (table3). according to sheer's equation results showed that the mean size of zro2 nanoparticles molecules under study were 29.8 nanometers. the results were compared with (vasaikar et al., 2017). they showed that the size of the particles (20 nanometers) when measured according to the shearer equation and the highest value of the x-ray oxides measured by xrd. while (arefian et al., 2015) the xrd value of zro2 was 35 nanometers. the xrd measurement is used to identify the crystallization of molecules. in some cases, the crystallization of these molecules is not perfect, due to the insufficient thermal processor and time during the preparation process. the results of x-ray diffraction analysis show the crystallization or calcification of zirconium, and the removal of the protein improves the biopolymerization of zirconium (haghi et al., 2012). it is also used to detect the nature of particulate matter.(gowri, gandhi and sundrarajan., 2014) the results of this study showed that the molecules of zinc oxide have a crystalline nature. table 1: socio-demographic variables and response to treatment variables no. (%) pvalue gender male 4 13.3% <0.05 female 26 86.7% total 30 age groups (years) 20-39 11 36.7% <0.05 39-60 14 46.7% 60-80 4 16.7% response to treatment responded 29 96.7% <0.05 not responded 1 3.3% 55 global journal of public health medicine 2019, vol 1, issue 2 gggggglo table 2: patients variation age, gender, treatment duration, clinical presentation and clinical investigations a ge baseline esr gen der duration of treatment course of treatment clinical presentation outcome esr after 3 months from treatment accp, ug ana, rft lft, cbc 60 55 fem ale 3 months 1 tender multiple joint pain not responde d 86 nonsignificant 45 25 fem ale 3 months 1 tender multiple joint pain respond ed 30 nonsignificant 42 25 fem ale 3 months 1 tender multiple joint pain respond ed 26 nonsignificant 50 75 mal e 3 months 1 tender multiple joint pain respond ed 35 nonsignificant 36 52 mal e 3 months 1 tender multiple joint pain respond ed 45 nonsignificant 40 75 fem ale 3 months 1 tender multiple joint pain respond ed 45 nonsignificant 30 75 fem ale 3 months 1 tender multiple joint pain respond ed 60 nonsignificant 70 70 fem ale 3 months 1 tender multiple joint pain respond ed 55 nonsignificant 55 60 mal e 3 months 1 tender multiple joint pain respond ed 30 nonsignificant 38 35 fem ale 6 months 2 tender multiple joint pain respond ed 25/20 nonsignificant 45 25 mal e 3 months 1 tender multiple joint pain respond ed 30 nonsignificant 37 75 fem ale 3 months 1 tender multiple joint pain respond ed 50 nonsignificant 22 65 fem ale 3 months 1 tender multiple joint pain respond ed 55 nonsignificant 50 55 fem ale 3 months 1 tender multiple joint pain respond ed 40 nonsignificant 40 50 fem ale 3 months 1 tender multiple joint pain respond ed 40 nonsignificant 39 75 fem ale 3 months 1 tender multiple joint pain respond ed 65 nonsignificant 55 75 fem ale 3 months 1 tender multiple joint pain respond ed 65 nonsignificant 42 62 fem ale 3 months 1 tender multiple joint pain respond ed 30 nonsignificant 39 50 fem ale 3 months 1 tender multiple joint pain respond ed 30 nonsignificant 60 55 fem ale 6 months 2 tender multiple joint pain respond ed 48/30 nonsignificant 40 30 fem ale 3 months 1 tender multiple joint pain respond ed 10 nonsignificant 27 65 fem ale 3 months 1 tender multiple joint pain respond ed 40 nonsignificant 35 55 fem ale 3 months 1 tender multiple joint pain respond ed 35 nonsignificant 60 30 fem ale 3 months 1 tender multiple joint pain respond ed 15 nonsignificant 25 20 fem ale 3 months 1 tender multiple joint pain respond ed 5 nonsignificant 30 70 fem ale 3 months 1 tender multiple joint pain respond ed 20 nonsignificant 44 60 fem ale 3 months 1 tender multiple joint pain respond ed 40 nonsignificant 80 70 fem ale 3 months 1 tender multiple joint pain respond ed 30 nonsignificant 50 60 fem ale 6 months 2 tender multiple joint pain respond ed 40/10 nonsignificant 40 60 fem ale 3 months 1 tender multiple joint pain respond ed 40 nonsignificant 56 global journal of public health medicine 2019, vol 1, issue 2 gggggglo table 3: das28 score of the patients at the time of diagnosis and after treatment. no initial das28 severity das28 after three months treatment severity das28 after six months treatment severity 1 4.6 moderate 2.8 low 2 4.6 moderate 2.84 low 3 5.1 high 3.6 moderate 4 5.7 high 3.05 low 5 5.1 high 3.2 moderate 6 5.3 high 3.2 moderate 7 5.3 high 3.5 moderate 8 5.4 high 2.8 low 9 5.2 high 2.8 low 10 4.9 moderate 4.7 moderate 2.1 remission 11 4.6 moderate 2.8 low 12 5.6 high 3.3 moderate 13 5.6 high 3.5 moderate 14 5.51 high 3.2 moderate 15 5.3 high 3.14 low 16 5.8 high 3.6 moderate 17 5.2 high 2.8 low 18 5.09 moderate 2.8 low 19 5.16 high 5.09 moderate 2.38 remission 20 4.7 moderate 1,16 remission 21 5.27 high 3 low 22 5.16 high 2.9 low 23 4.59 moderate 1.9 remission 24 4.3 moderate 1.3 remission 25 5.6 high 2.1 remission 26 5.22 high 3.14 low 27 5.78 high 2.8 low 28 5.22 high 4.9 moderate 1.6 remission 29 5.22 high 3 low 30 5.12 high 2.96 low 57 global journal of public health medicine 2019, vol 1, issue 2 gggggglo figure 1: demonstrates male to female ratio figure 2: patients response to the treatment figure 3: patients response to the treatment 13.30% 86.70% 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% male femalet he p er ec en ta ge o f bo th s ex tr ea te d w it h h c q male to female ratio %96.7 %3.3 treatment responce responed to treatment resisted 90% 10% numbers of treatment course single course treatment double course tretment 58 global journal of public health medicine 2019, vol 1, issue 2 gggggglo figure 4: the average mean of the esr after treatment figure 5: age and sex statistics 86.60% 3.30% 13.40% -44% -13% 30% -100.00% -50.00% 0.00% 50.00% 100.00% a b c esr result after treatment patients esr 1.8 0.71438 0.13043 1.8667 0.34575 0.06312 0 0.5 1 1.5 2 2.5 mean std. deviation std. error mean statistical analysis of age and sex age sex 59 global journal of public health medicine 2019, vol 1, issue 2 gggggglo figure 6: modified das28 score percentage of the patients before and after treatment discussion: this study is the first of its kind to explore the use of hcq as a monotherapy in treatment of eua in sudan. many studies have shown that vast majority of eua are self-limiting especially those of viral etiology (cacoub et al., 1999; dendooven et al., 2006). we only focused on using hcq and no other drugs for treatment of eua. hcq is an anti-malarial drug used in combination with other drugs to treat a wide variety of rheumatologic diseases such as systemic lupus erythematosus (sle)(spinelli et al., 2018), ra(schapink, van den ende, gevers, van ede, & den broeder, 2019), primary sjogren's syndrome (wang, zhang, wei, & hua, 2017) and osteoarthritis (lee et al., 2018), and until now its therapeutic potential and pharmacokinetic has not been identified and explored in depth (hanaoka, iida, kiyokawa, takakuwa, & kawahata, 2019), records show no studies regarding the use and efficacy of hcq as monotherapy in eua as the drug carry less side effect and complications compared to other drugs used in treatment of the same disease such as methotrexate which has a negative impact on the bone marrow (yuncu, bukucu, bayat, sencar, & tarakcioglu, 2015) and gastro intestinal system (attar, 2010). biologic treatment has less side effects with good outcome (zavvar et al., 2019) but as most of the patients from the developing centuries have no affordability for the treatment and its need for regular follow up, hcq provides a better option. regarding male to female ratio there are only few studies that estimated the sex difference in eua, they also carry similar findings of this study as all of them showed that female constituted more than two thirds of total eua patients (o16 aetiology of early undifferentiated arthritis in india, 2009; shankar s, 2010), no clear explanation have been identified yet but as eua is an immunological disease (foocharoen, nanagara, suwannaroj, & mahakkanukrauh, 2011), recent evidence suggest that female hormones such as estrogen have strong role in development of autoimmune diseases(somers & richardson, 2014). the study shows patients at the age of forties and fifties are the most commonly affected by the disease, the findings are directly in line with previous result with same sample size of this study (shankar s, 2010), it remains unclear why exactly the middle age people are das 28 at the time of diagnosis 26.7% of the patients have moderate score three months hcq treatment 50% low 37.50% remission 12.5% moderate all received another three months treatment 100% remission 73.3% of the patients have high score three months hcq treatment 45.4% moderate 20% of them received another three months treatment 100% remission 50% low 4.6% remission 60 global journal of public health medicine 2019, vol 1, issue 2 gggggglo more likely to be affected rather than those at old age who tend to be vulnerable due to their weak immune system(merani, pawelec, kuchel, & mcelhaney, 2017), but we postulate that this might be due to female sex hormones(marder, vinet, & somers, 2015; somers & richardson, 2014) and also the menopausal change occured at this age (su & freeman, 2009). the management of eua is controversy as no standard treatment protocol is present, and around 30-60% of all the patients of eua has self-limiting disease(olivieri et al., 2012), so many questions been asked by us. should we treat every patient? however, our primary focus was on the effectively of hcq alone in the treatment of eua and deliver significantly good result with 96.7% fully response, no comparable studies have been found in the literature neither an explanation. das 28 score classify patient’s well-being into three categories, high for those who score above 5.1, moderate for the patients who score between 5.1 and more than 3.2, low between 3.2 and more than 2.6, remission for and 2.6.and below. the score calculation depends on the number of joints involved, swelling of the joints, the global health assessment of the patient, and either the crp or esr (van riel & renskers, 2016). half of the patients who received three months hcq treatment with high score das28 their score turned to low, the other half had complete remission or became moderate. those with six months duration of treatment their outcome ended by complete remission. as the used calculating score is modified to eua patients, no available reviews or similar study were founded with same result, and no clear explanation has found the limitation of this study is the sample size which is relatively low, early undifferentiated arthritis is not widely common, another limitation is the lack of the long term follow up for the patients to explore the total efficacy of the management and whether there is any recurrence of the previous complains and this lack of follow up is strongly due to patients factor as most of them were from rural and remote areas so establishing continuous communication with them somewhat is difficult. conclusion in conclusion, this study investigated the efficiency of hcq as monotherapeutic agent in treatment of early undifferentiated arthritis. although extensive studies have not been conduct in this matter, substantial evidence about the role hcq in rheumatologic diseases is already proved. 96.7% of the patients were completely cured and 90% of them responded from the first course of treatment. female are commonly affected by the disease and the age group from 40 to less than 60 have the higher incidence. ethical approval and consent to publish obtained from federal ministry of health (fmoh), khartoum, sudan. references aletaha, d., neogi, t., silman, a. j., funovits, j., felson, d. t., bingham, c. o., 3rd, . . . hawker, g. (2010). 2010 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative. arthritis rheum, 62(9), 2569-2581. doi: 10.1002/art.27584 attar, s. m. (2010). adverse effects of low dose methotrexate in rheumatoid arthritis patients. a hospital-based study. saudi med j, 31(8), 909-915. bosello, s., fedele, a. l., peluso, g., gremese, e., tolusso, b., & ferraccioli, g. (2011). very early rheumatoid arthritis is the major predictor of major outcomes: clinical acr remission and radiographic nonprogression. ann rheum dis, 70(7), 12921295. doi: 10.1136/ard.2010.142729 cacoub, p., poynard, t., ghillani, p., charlotte, f., olivi, m., piette, j. c., & opolon, p. (1999). extrahepatic manifestations of chronic hepatitis c. multivirc group. multidepartment virus c. arthritis rheum, 42(10), 2204-2212. doi: 10.1002/15290131(199910)42:10<2204::aidanr24>3.0.co;2-d capell, h. a., madhok, r., porter, d. r., munro, r. a., mcinnes, i. b., hunter, j. a., . . . ford, i. (2007). combination therapy with sulfasalazine and methotrexate is more effective than either drug alone in patients with rheumatoid arthritis with a suboptimal response to sulfasalazine: results from the double-blind placebo-controlled mascot study. ann rheum dis, 66(2), 235-241. doi: 10.1136/ard.2006.057133 combe, b., landewe, r., daien, c. i., hua, c., aletaha, d., alvaro-gracia, j. m., . . . van vollenhoven, r. (2017). 2016 update of the eular recommendations for the management of early arthritis. ann rheum dis, 76(6), 948959. doi: 10.1136/annrheumdis-2016-210602 61 global journal of public health medicine 2019, vol 1, issue 2 gggggglo dale, j., alcorn, n., capell, h., & madhok, r. (2007). combination therapy for rheumatoid arthritis: methotrexate and sulfasalazine together or with other dmards. nat clin pract rheumatol, 3(8), 450-458; quiz, following 478. doi: 10.1038/ncprheum0562 dendooven, a., de rycke, l., verhelst, x., mielants, h., veys, e. m., & de keyser, f. (2006). leflunomide and methotrexate combination therapy in daily clinical practice. ann rheum dis, 65(6), 833-834. doi: 10.1136/ard.2005.043620 dixon, w. g., & symmons, d. p. (2005). does early rheumatoid arthritis exist? best pract res clin rheumatol, 19(1), 37-53. doi: 10.1016/j.berh.2004.08.003 foocharoen, c., nanagara, r., suwannaroj, s., & mahakkanukrauh, a. (2011). clinical features and disease outcomes of undifferentiated arthritis in thailand. int j rheum dis, 14(3), e14-21. doi: 10.1111/j.1756-185x.2011.01606.x hanaoka, h., iida, h., kiyokawa, t., takakuwa, y., & kawahata, k. (2019). hydroxychloroquine improves the disease activity and allows the reduction of the corticosteroid dose regardless of background treatment in japanese patients with systemic lupus erythematosus. intern med, 58(9), 1257-1262. doi: 10.2169/internalmedicine.1999-18 hazes, j. m., & luime, j. j. (2011). the epidemiology of early inflammatory arthritis. nat rev rheumatol, 7(7), 381-390. doi: 10.1038/nrrheum.2011.78 hodkinson, b., magomero, k. r., & tikly, m. (2016). combination leflunomide and methotrexate in refractory rheumatoid arthritis: a biologic sparing approach. ther adv musculoskelet dis, 8(5), 172-179. doi: 10.1177/1759720x16664324 joshua, f., edmonds, j., & lassere, m. (2006). power doppler ultrasound in musculoskeletal disease: a systematic review. semin arthritis rheum, 36(2), 99-108. doi: 10.1016/j.semarthrit.2006.04.009 lee, w., ruijgrok, l., boxma-de klerk, b., kok, m. r., kloppenburg, m., gerards, a., . . . basoski, n. (2018). efficacy of hydroxychloroquine in hand osteoarthritis: a randomized, double-blind, placebocontrolled trial. arthritis care res (hoboken), 70(9), 1320-1325. doi: 10.1002/acr.23471 marder, w., vinet, e., & somers, e. c. (2015). rheumatic autoimmune diseases in women and midlife health. womens midlife health, 1. doi: 10.1186/s40695-015-0012-9 merani, s., pawelec, g., kuchel, g. a., & mcelhaney, j. e. (2017). impact of aging and cytomegalovirus on immunological response to influenza vaccination and infection. front immunol, 8, 784. doi: 10.3389/fimmu.2017.00784 o16 aetiology of early undifferentiated arthritis in india. (2009). (vol. vol. 4, no. 3 (suppl)): indian journal of rheumatology olivieri, i., sarzi-puttini, p., bugatti, s., atzeni, f., d'angelo, s., & caporali, r. (2012). early treatment in early undifferentiated arthritis. autoimmun rev, 11(8), 589-592. doi: 10.1016/j.autrev.2011.10.019 schapink, l., van den ende, c. h. m., gevers, laha, van ede, a. e., & den broeder, a. a. (2019). the effects of methotrexate and hydroxychloroquine combination therapy vs methotrexate monotherapy in early rheumatoid arthritis patients. rheumatology (oxford), 58(1), 131-134. doi: 10.1093/rheumatology/key275 shankar s, dhiman p, kumar pg. (2010). early undifferentiated arthritis in india: a six month follow up study (vol. volume 5): indian journal of rheumatology. shipman, w. d., vernice, n. a., demetres, m., & jorizzo, j. l. (2019). an update on the use of hydroxychloroquine in cutaneous lupus erythematosus: a systematic review. j am acad dermatol. doi: 10.1016/j.jaad.2019.07.027 smolen, j. s., breedveld, f. c., burmester, g. r., bykerk, v., dougados, m., emery, p., . . . van der heijde, d. (2016). treating rheumatoid arthritis to target: 2014 update of the recommendations of an international task force. ann rheum dis, 75(1), 3-15. doi: 10.1136/annrheumdis-2015-207524 somers, e. c., & richardson, b. c. (2014). environmental exposures, epigenetic changes and the risk of lupus. lupus, 23(6), 568-576. doi: 10.1177/0961203313499419 62 global journal of public health medicine 2019, vol 1, issue 2 gggggglo spinelli, f. r., moscarelli, e., ceccarelli, f., miranda, f., perricone, c., truglia, s., . . . conti, f. (2018). treating lupus patients with antimalarials: analysis of safety profile in a single-center cohort. lupus, 27(10), 16161623. doi: 10.1177/0961203318781008 stockman, a., tait, b. d., wolfe, r., brand, c. a., rowley, m. j., varney, m. d., . . . muirden, k. d. (2006). clinical, laboratory and genetic markers associated with erosions and remission in patients with early inflammatory arthritis: a prospective cohort study. rheumatol int, 26(6), 500-509. doi: 10.1007/s00296-005-0027-x su, h. i., & freeman, e. w. (2009). hormone changes associated with the menopausal transition. minerva ginecol, 61(6), 483-489. sudol-szopinska, i., kontny, e., maslinski, w., prochorec-sobieszek, m., warczynska, a., & kwiatkowska, b. (2013). significance of bone marrow edema in pathogenesis of rheumatoid arthritis. pol j radiol, 78(1), 57-63. doi: 10.12659/pjr.883768 suresh, e. (2004). diagnosis of early rheumatoid arthritis: what the non-specialist needs to know. j r soc med, 97(9), 421-424. doi: 10.1258/jrsm.97.9.421 vaidya, b., baral, r., & nakarmi, s. (2018). early undifferentiated arthritis: a developing country perspective from nepal. jnma j nepal med assoc, 56(214), 983-990. van dongen, h., van aken, j., lard, l. r., visser, k., ronday, h. k., hulsmans, h. m., . . . huizinga, t. w. (2007). efficacy of methotrexate treatment in patients with probable rheumatoid arthritis: a double-blind, randomized, placebo-controlled trial. arthritis rheum, 56(5), 1424-1432. doi: 10.1002/art.22525 van riel, p. l., & renskers, l. (2016). the disease activity score (das) and the disease activity score using 28 joint counts (das28) in the management of rheumatoid arthritis. clin exp rheumatol, 34(5 suppl 101), s40-s44. wang, s. q., zhang, l. w., wei, p., & hua, h. (2017). is hydroxychloroquine effective in treating primary sjogren's syndrome: a systematic review and meta-analysis. bmc musculoskelet disord, 18(1), 186. doi: 10.1186/s12891-017-1543-z wevers-de boer, k. v., heimans, l., huizinga, t. w., & allaart, c. f. (2013). drug therapy in undifferentiated arthritis: a systematic literature review. ann rheum dis, 72(9), 1436-1444. doi: 10.1136/annrheumdis-2012-203165 yuncu, m., bukucu, n., bayat, n., sencar, l., & tarakcioglu, m. (2015). the effect of vitamin e and l-carnitine against methotrexateinduced injury in rat testis. turk j med sci, 45(3), 517-525. zavvar, m., assadiasl, s., soleimanifar, n., pakdel, f. d., abdolmohammadi, k., fatahi, y., . . . nicknam, m. h. (2019). gene therapy in rheumatoid arthritis: strategies to select therapeutic genes. j cell physiol. doi: 10.1002/jcp.28392 iraqi j pharm sci, vol.31( 2 ) 2022 tnf-alpha gene polymorphisms doi: https://doi.org/10.31351/vol31iss2pp113-128 113 the effect of tnf-alpha gene polymorphisms at -376 g/a, -806 c/t, and -1031 t/c on the likelihood of becoming a non-responder to etanercept in a sample of iraqi rheumatoid arthritis patients. samer imad mohammed*.1 , munaf hashim zalzala** and faiq isho gorial*** *department of clinical pharmacy,college of pharmacy, university of baghdad, baghdad, iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. *** college of medicine, university of baghdad, baghdad, iraq abstract tumor necrosis factor-alpha (tnf-α) antagonists’ therapy are expensive and has a non-responsive rate between 30% to 40% in rheumatoid arthritis patients. genetic variation plays a vital role in the responsiveness to this type of therapy.the aim of this study is to investigate if the presence of genetic polymorphism in the tnf-α gene promoter region at locations -376 g/a (rs1800750), -806 c/t (rs4248158), and -1031 t/c (rs1799964) affects rheumatoid arthritis patient's tendency to be a non-responder to etanercept. eighty ra patients on etanercept (etn) for at least six months were recruited from the rheumatology unit at baghdad teaching hospital. based on the european league against rheumatism response (eular) criteria, patients were divided into two groups: responders and non-responders. after polymerase chain reaction amplification of their dna, the amplified dna was sequenced by sanger method to determine the polymorphisms at the positions -376g/a, -806 c/t, and -1031t/c. the results of this study found that equally phi correlation and binary logistic regression analysis revealed a nonsignificant association for all genotypes in the three polymorphic sites with the tendency for being non-responder. moreover, there was no significant difference in tnf-α mean level or the change in disease activity score for 28 joints (das28) after six months of etanercept therapy between all genotypes for each polymorphic site. the present study concludes that there was no correlation between the polymorphisms in the tnf-α promoter region at -376g/a, -806 c/t, and -1031t/c with the tendency for being non-responder to etn. key words: rheumatoid arthritis, etanercept, genetic polymorphism, response. 376g/a ,806c/tأثير تعدد األشكال الجينية في عامل نخر الورم الفا بالمواقع ت على الميل لعدم االستجابة لأليتانرسبت في عينة من مرضى التهاب 1031t/c-و المفاصل الرثوي في العراق *** ايشو كولايرو فائق **مناف هاشم زلزلة ،1،*سامر عماد محمد * فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق. بغداد ، العراق بغداد، جامعة الصيدلة، كلية والسموم، دوية فرع اال** العراق بغداد، بغداد، جامعة الطب، كلية *** الخالصة هي عالجات باهظة الثمن ،وتصاحب استخدامها نسبة عالية من عدم استجابة قد تصل (tnf-α)العالجات المضادة لعامل نخر الورم .يلعب التباين الجيني دوًرا حيويًا في تحديد نسبة االستجابة لهذا النوع من العالج .٪ في مرضى التهاب المفاصل الرثوي40٪ إلى 30بين ما األ لتعدد وجود هناك كان إذا ما لمعرفة هو الدراسة الفاهدف الورم نخر عامل لجين المحفزة المنطقة في الجيني المواقع شكال 376 -في (rs1800750) g/a 806-و (rs4248158)c/t 1031-و (rs1799964)t/c تأثير في مرضى التهاب المفاصل الرثوي بالعراق وهل هناك تم تسجيل ثمانين مريًضا من مرض التهاب المفاصل الرثوي أن يكون غير مستجيب لعقار االيتانرسبت. في التباين الجيني على ميل المريض الهذ ة الرابطة والمستخدمين لعالج االيتانرسبت لمدة ستة أشهر على األقل من وحدة عالج المفاصل في مستشفى بغداد التعليمي. وبناًء على معايير استجاب ، تم تقسيم مرضى التهاب المفاصل الرثوي إلى مجموعتين: المستجيبين وغير المستجيبين. وبعد تضخيم (eular) األوروبية لمكافحة الروماتيزم تفاعل البلمرة المتسلسل ، تم اجراء تسلسل للحمض النووي المتضخم للمرضى المشاركين بأستخدام تقنية باستخدام تقنية الحمض النووي الخاص بهم .اهم نتائج الدراسة كانت كشف كل من معامل االرتباط1031t/c-و c/t 806-و g/a 376-لجيني في المواضع سانغر لتحديد تعدد األشكال ا االستجابة وتحليل االنحدار اللوجستي الثنائي عن عدم وجود ارتباط ذو داللة احصائية لجميع األنماط الجينية للمواقع الثالثة مع ميل المريض لعدم فاي معدل مستوى معامل نخر المرض لك لم يكن لتغيير االنماط الجينية ألي من المواقع الثالثة تأثير ذو داللة احصائية على مقدارلعقار االيتانرسبت.كذ مفصل خالل ستة اشهر من العالج. 28الفا في المريض وال على مقدارالتغيير الحاصل لنتيجة نشاط المرض لـ ط بين تعدد األشكال الجيني في المنطقة المحفزة لعامل نخر الورم الفا في المواقع ملخص هذه الدراسه اوضح انه لم يكن هناك ارتبا 376g/a 806-و c/t 1031-و t/c .مع قابلية مريض التهاب المفاصل الرثوي إلى عدم االستجابة لـأليتانرسبت , االستجابة.الكلمات المفتاحية : التهاب المفاصل الرثوي , االيتانرسبت , تعدد االشكال الجيني 1corresponding author e-mail: samer.jameel@copharm.uobaghdad.edu.iq received:2 /10 /2021 accepted:5 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp113-128 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 114 introduction rheumatoid arthritis (ra) is a common chronic autoimmune joints disease. it is defined by progressive symmetric inflammation of the afflicted joints, which results in cartilage damage, bone erosion, and disability)1(. one influential proinflammatory cytokine that plays a critical role in controlling the inflammatory response and mediating ra pathogenesis is tumor necrosis factor-alpha (tnf-α) )2(. consequently, extensive research on tnf-α induced inflammatory processes has resulted in the conception of tnf-α antagonist medications to treat many inflammatory autoimmune diseases, including ra(3). they are produced from either a recombinant tnf-α receptor, like etanercept (etn), or a monoclonal antibody against tnf-α like infliximab and adalimumab(3). nowadays, tnf-α antagonists therapy has been widely used and is quite successful in treating ra(4). however ,they are prohibitively expensive, may induce some undesirable side effects, and not all ra patients respond well to these medications(5).in fact, between 30% to 40% of people with active ra do not respond successfully to tnf-α antagonists(6). the genetic disparity is a significant factor influencing tnf-α expression, and consequently, the response to tnf-α antagonists since the promoter region of the tnf-α gene is highly polymorphic (7). tnf-α antagonists are not all equally effective in all patients. as a result, being able to identify which tnf-α antagonist would be the most effective and should be taken initially would be tremendously beneficial in terms of reducing the time required to ensure an effective therapy, which has been repeatedly proved to be a significant factor in achieving long-lasting disease remission(8). etanercept , a recombinant human tnf-α receptor (p75) fusion protein that suppresses tnf-α competitively(9), is safe and effective in patients with active ra(10). as with other tnf-α antagonists, the clinical response to etn is variable, and one of the significant determinants of the response is genetic variability in the tnf-α gene(7). as a result, polymorphism testing aids in distinguishing patients with a high response rate from those with an insufficient response or even non-responsive (11). this information may be beneficial for some patients who concerned about their exposure to side effects or the associated high cost of biological therapy (11). several research articles published in the last few years have revealed conflicting outcomes about a possible association between tnf-α antagonists' response and polymorphisms in the tnf-α gene at several gene locations (12–14). due to the racial and ethnic variation in pharmacogenetics, which occurs as a result of the different in allele frequencies between populations, polymorphism analysis across populations is crucial for determining the genetic variables that may affect etn responsiveness. (15). earlier investigations did not examine the effect of single nucleotide polymorphisms (snps) in the tnf-α promoter region on the tendency to being non-responder to etn in iraqi ra patients; thus, the current study sought to determine whether the presence of snps in the promoter region of the tnfα gene at positions -376 g/a (rs1800750), -806 c/t (rs4248158) and -1031 t/c (rs1799964) may affect a patient's proclivity to be a non-responder. patients and methods this research article was a part of a large observational cross-sectional study conducted between october 12, 2020, and august 8, 2021. the study recruited a sample of eighty iraqi ra patients with established ra according to the revised 2010 american college of rheumatology (acr)/european league against rheumatism (eular) classification criteria for ra(16). the patients were recruited from the rheumatology unit of baghdad teaching hospital in baghdad, iraq. this unit provides services to a wide range of communities in iraq, including rural, urban, and inner-city districts from numerous governorates. the scientific and ethical committee of college of pharmacyuniversity of baghdad and rheumatology medical department at baghdad teaching hospital approved ethical permission with the number (recacpub-3102020b) on october 3, 2020. furthermore, written consent was obtained from all participants. patients selection ninety-seven patients with active ra received etn alone as a singular therapy during the study and met the inclusion criteria listed below. however, only eighty-six patients agreed to participate in this study, and only eighty completed the full requirements. the inclusion criteria: the patients must have been diagnosed with ra using the 2010 acr/eular ra classification criteria(16). also, patients with high disease activity according to disease activity score based on 28 joints and esr (das28-esr)(17) which is calculated as follows: das28 = 0.56 * sqrt(tender28) + 0.28 * sqrt(swollen28) + 0.70 * ln (esr) + 0.014 * patient global health. in order to be included, the das28-esr should be more than 5.1 at baseline. additionally, patients had to take etn subcutaneously and consistently for at least six months before enrollment, with no history of missed doses. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 115 the exclusion criteria: • patient who has used etn for less than six months. • patients who use additional diseasemodifying anti-rheumatic drugs (dmards) with etn. • patients with co-existent other connective tissue diseases • patient with inadequate data like missed laboratory data or initial das28. patients classification: as presented in figure 1, the patients were divided into two groups according to eular response criteria(17), which are based on the clinical response as determined by (das28) (18) following at least six continuous months of etn treatment. when the das28 value after six months was reduced from a high value of ≥ 5.1 to a value less than 5.1 and with a change in das28 of greater than 0.6, the patient was classified as an etn responder. on the contrary, the patient has categorized as a nonresponder if the das28 value did not fall below 5.1 or if the change in the das28 was less than 0.6. accordingly, the patients are distributed regarding their responses into two groups. the first group (group a) contained forty-one ra patients who responded clinically to etn. the second group (group b) contained thirty-nine ra patients who failed to respond to etn. figure 1. flow diagram for the study participants. data collection demographic data (age, weight, disease duration, tender and swollen joints, and the visual analog scale (vas) for the patients and the evaluator) were obtained via direct patient interviews utilizing a patient information chart designed explicitly for this study. sample collection and preparation five milliliters of venous blood were obtained from each patient's forearm vein. then, two milliliters of blood were transformed into an ethylene diamine tetraacetic acid (edta) tube for dna extraction. at the same time, the remaining three milliliters of blood were put into a gel tube and centrifuged for ten minutes (4000 rpm). the remainder of the serum was collected in an eppendorf tube and stored at (-20 co) until all samples were obtained. tnf-α was then measured using the eliza approach. measurement of serum tumor necrosis factoralpha (tnf-α) the serum tnf-α level was determined using a cusabio elisa kit (wuhan, china; cat. no. csb-e04740h). this assay employs the quantitative sandwich enzyme immunoassay technique(19). dna extraction: the “promega reliapreptm blood gdna miniprep system” for genomic dna enables the straightforward purification of dna from blood samples. enzymatic amplification was performed using pcr and a hybrid thermal cycler. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 116 quantitation of dna the "the quantus™ fluorometer" was used to determine the concentration of extracted dna and thus the sample's quality for downstream applications. (199 µl) diluted quantifluor dye was combined with 1 µl of dna, and then dna concentration readings were determined during a 5minutes incubation at room temperature. the primer: the tnf-α gene dna sequences were obtained from the ncbi genbank database. the primer premier 3 software was used to create pcr primers (table 1), with melting temperatures ranging from (58 to 62oc), primer lengths ranging from (18 to 23) nucleotides, and pcr amplicon lengths ranging from (800 to 1000) base pairs. table 1. the sequences, annealing temperature and size of the primers used in the study primer name sequence annealing temp. (°c) product size (bp) tnf-α_1-f 5`-tgtaaaacgacggccagtctcagagagcttcagggata-3` 60 966 tnf-α_1-r 5`-caggaaacagctatgaccgggacacacaagcatcaa-3` tnf-α_1-f: the forward primer. tnf-α_1-r: the reverse primer. primer preparation the forward and reverse primers used in this study were given in lyophilized form by the macrogen company. the primers were then dissolved in nuclease-free water to provide a stock solution with the highest concentration of (100 pmol/µl) that can be kept in the freezer at 20 °c. after that, a working solution for these primers was made by combining 10 µl of primer stock solution with 90 µl of nuclease-free water to yield a solution comprising (10 pmol /µl). primer optimization and pcr amplifications to determine the optimal annealing temperature for primers, the dna template was amplified using the identical primer pair (forward) (reverse) at annealing temperatures of 55, 58, 60, 63, and 65°c respectively. the best annealing temperature for the primer was 60°c as seen in figure 2. pcr amplifications were performed with 20μl volumes containing 10μl gotaq green master mix (2x); 1μl for each primer (10pmol); 6μl nuclease-free water and 2μl of template dna. pcr cycling was performed with pcr express (thermal cycler, biorad, usa) with the following temperature program: firstly, dna denaturation occurred at 94oc for 4 min followed by 30 cycles of denaturation at 94°c for only 30 sec; after that, annealing at 60°c for 30 sec; and extension at 72°c for 30 sec. a final extension incubation of 7 min at 72oc was included, followed by a 10 min incubation at 4oc to stop the reactions. figure 2. primer optimization at annealing temperatures of 55, 58, 60, 63, and 65°c. pcr products sequencing pcr product was sequenced by sanger method of sequencing using dna analyzer (abi3730xl) (macrogen corporation – korea). the results were obtained by email and analyzed with the use of geneious prime software (v 2021.1.1) (biomatters ltd., auckland, new zealand; www.geneious.com). iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 117 statistical analysis data were investigated using the spss for windows 26.0 software (spss, inc., chicago, il, usa) and graphpad prism version 7.04 (california, usa). continuous variables were stated as mean ± sem of the values. allele and genotypes are presented as percentages and frequencies. a probability that equals or less than 0.05 was considered significant. a shapiro–wilk test was used to test the normality of the results. the unpaired t-test was used for normally distributed data to determine if there is a significant difference in demographic characteristics and parameters between responding and nonresponding groups. a one-way analysis of variance (anova) test was used to analyze the difference between the means of more than two groups. then, a post-hoc analysis was used whenever a significant difference between three sample means was revealed by (anova). the chi-square test or fisher's exact test was used to test group differences of proportions. fisher's exact test was used if one of the expected values in a 2 x 2 comparison is < 5. phi correlation coefficient (phi) was used to measure the correlation between each genotype and the likelihood of being nonresponsive. the binary logistic regression analysis was used to estimate the relationship between tnfα level and the effect of genetic polymorphism on the likelihood of becoming a non-responder. results demographic data and clinical characteristics parameters of the study groups. table 2 summarizes the demographic characteristics of the study groups. in the current study, patients were matched. however, clinically, there was a significant difference in baseline das28 values between responder and non-responders (p. value <0.001). significant variations in das28 were also observed after six months of etn administration. in addition, after six months of etn medication, tnf-α levels were likewise shown to differ considerably between responders and nonresponders. table 2. demographic data and clinical characteristics parameters of the study groups. category responders group n=41 non-responders group n=39 p-value age(years) 49.46±10.54 51.18±11.95 0.497 a gender male n (%) 6 (14.6) 4 (10.3) 0.55 c female n (%) 35 (85.4) 35(89.7) 0.55 b weight(kg) 80.59±14.49 78.03 ±12.82 0.40 a disease duration (years) 10.10± 6.82 8.31±3.73 0.15 tnf-α (pg/ml) 78.63±34.1 113.35 ±54.54 0.001* a baseline das28 5.58± 0.343 6.06 ± 0.38 <0.001* a das28 after 6 months 3.34 ± 0.82 5.73 ± 0.54 <0.001* a results are reported as means ±sd or frequency (percentage). tnf-α: tumor necrosis factor-alpha. das28: disease activity score in 28 joints. a: independent 2 sample t-test. b: chi-square test. c: fisher-exact test dna concentration (µg/ml) the extracted dna concentration in all samples was found in a range of 20-30 µg/ml. pcr amplification result the amplification of the tnf-α genespecific region of human samples was presented in figure 3. the amplification of the tnf-α genespecific region of human samples was fractionated on 1.5% agarose gel electrophoresis stained with ethidium bromide (eth.br). figure 3. human samples' amplification of the tnf-α gene-specific region. the sample was fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 1-80 resemble 966bp pcr products. sanger sequences data analysis analysis of tnf-α ( -376 g/a) (rs1800750) snp figure 4 shows the analysis of (rs1800750) snp of the tnf-α gene using sanger sequencing. single "g" peak indicative of a (g) homozygous allele. the presence of the "g" and "a" peaks is indicative of the (g/a) heterozygous allele iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 118 figure 3. human samples' amplification of the tnf-α gene-specific region. the sample was fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 1-80 resemble 966bp pcr products. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 119 figure 4 analysis of rs1800750 snp of tnf-α gene using sanger sequencing. analysis of tnf-α (806c/t) (rs4248158) snp figure 5 highlights the analysis of (rs4248158) snp of the tnf-α gene using sanger sequencing. single "c" peak indicative of a (c) homozygous allele. presence of the "c" and "t" peaks indicative of (c/t) heterozygous allele. figure 5. analysis of rs4248158 snp of tnf-α gene using sanger sequencing. analysis of tnf-α (-1031 t/c) (rs1799964) snp analysis of rs1799964 snp of tnf-α gene using sanger sequencing is presented in figure 6. single "t" peak indicative of a t homozygous allele. single "c" peak indicative of a ( c) homozygous allele. the presence of the "t" and "c" peaks is indicative of the (t/c) heterozygous allele. figure 6. analysis of rs1799964 snp of tnf gene using sanger sequencing. prevalence of genotypes polymorphism table 3 highlights the high proportions of gg genotypes of -376 g/a. the tt genotype was the most prevalent in more than half of the patients with -1031t/c. almost three-quarters of patients had the cc genotype for the -806 c/t variant. table 3 also shows the allele frequency for all the snps. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 120 table 3. genotypes and alleles frequencies of tnf-α -1031 t/c, -806 c/t and -376 g/a gene polymorphisms in ra patients. n=80 patients genotypes -1031t/c genetic variant cc tc tt no. (%) 3(3.75) 22(27.5) 55(68.75) allele t c no. (%) 77(96.25) 25(31.25) -806 c/t genetic variant cc ct no. (%) 72(90) 8(10) allele c t no. (%) 80(100) 8(10) -376 g/a genetic variant ga gg no. (%) 4(5) 76(95) allele g a no. (%) 80(100) 4 (5) regarding the difference in alleles frequencies between the responders and non-responders, the results show no significant difference for all three polymorphic sites, as seen in table 4. additionally, the results of this study indicated that there was no significant difference in the distribution of all genotypes for all three sites in the tnf-α promoter region between responders and non-responders (table 4). table 4. difference in genotype frequencies of tnf-α -1031 t/c, -806 c/t and -376 g/a gene polymorphisms between responders and non-responders. genotypes responders group (n=41) no. (%) non-responders group (n=39) no. (%) p. value 1031 t/c cc 2 (4.9) 1 (2.6) 0.58 tc 12 (29.3) 10 (25.6) 0.71 tt 27 (65.9) 28 (71.8) 0.56 t 39 (95.1) 38(97.4) 0.97 c 14 (34.1) 11 (28.2) 0.40 -806 c/t cc 35 (85.4) 37 (94.9) 0.15 ct 6 (14.6) 2 (5.1) 0.15 c 41(100) 39(100) 1 t 6(14.6) 2(5.1) 0.17 -376 g/a ga 2 (4.9) 2 (5.1) 0.95 gg 39 (95.1) 37 (94.9) 0.95 g 41 (100) 39 (100) 1 a 2 (4.9) 2(5.1) 1 a chi-square test or fisher exact test was used to identify the statistical difference between the groups. association between genotypes and the likelihood of being non-responder table 5 shows the binary logistic regression analysis results, which are non significant for all genotypes of the three snps. this indicates that changing the genotype from the wild type to another polymorphic genotype cannot predict the tendency for being non-responder. table 5: binary logistic regression analysis of genotypes to predict the tendency of being non-responder for etn. parameter coefficient or p-value 95% ci. lower upper 1031 t/c 0.35 1.42 0.27 0.75 2.67 -806 c/t -0.88 0.41 0.10 0.14 1.21 -376 g/a 0.73 2.08 0.36 0.43 10.07 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 121 similarly, by using phicorrelation all the demonstrated genotypes were either correlated positively or negatively with the tendency to be a non-responder, but none were statistically significant as seen in table 6. table 6. correlation between each genotype and the likelihood of being a non-responder. genotypes phi-coefficient p. value 1031 t/c cc -0.061 0.58 tc -0.041 0.71 tt 0.064 0.56 -806 c/t cc 0.189 0.09 ct -0.189 0.09 -376 g/a ga 0.006 0.95 gg -0.006 0.95 phi-correlation coefficient was used to find the correlation between each genotype and the likelihood of being a non-responder. the correlations between the genotypes and the difference in das28 over six months . as shown in table 7, most of the differences in das28 after six months of etn treatment were not statistically significant. table 7. association of the change in das28 over six months between different genotypes in -1031 t/c, 806 c/t and -376 g/a tnf-α genotypes polymorphism. genotypes -1031t/c genetic variant cc tc tt p. value δdas28 1.71±1.24 1.59±1.31 1.19±1.18 0.37 a -806 c/t genetic variant cc ct δdas28 1.22 ± 0.14 1.38 ± 0.54 0.72 b -376 g/a genetic variant ga gg δdas28 1.29 ± 0.13 0.73± 0.48 0.36 b results are reported as means ±sd, δdas28: the change in disease activity score of 28 joints over six months, a = one-way anova used to find the statistical difference, b = unpaired t-test used to find the statistical difference. tnf-α level in different genotypes after six months of continuous etn therapy, there was no significant difference in tnf α level between all genotypes of each snp, as reported in table 8. table 8. tnf-α level in different genotypes genotypes -1031t/c genetic variant cc tc tt p. value tnf-α 121.3 ±63.57 80.14±40.06 99.95±48.93 0.17 a -806 c/t genetic variant cc ct tnf-α 93.51 ± 5.67 111.1 ± 16.79 0.30 b -376 g/a genetic variant ga gg tnf-α 98.03± 15.86 95.35±5.61 0.91b results are reported as means ±sd, a = one-way anova used to find the statistical difference, b = unpaired ttest used to find the statistical difference. discussion etn has been proven to promote remission, reduce disease activity, and delay clinical and radiological disease progression in patients with ra. this has resulted in significant improvements in symptoms, function, and quality of life (20). as well known, the effectiveness of etn fluctuates considerably amongst ra patients, with around onethird of patients failed to clinically respond (21). as a result, early identification of ra patients who will not respond to tnf-α antagonists, including etn, enables a swift switch to alternative biological medications, thus increasing the patient's likelihood of promptly attaining treatment goals(22). studying the effect of genetic variation in response to etn is essential because, in contrast to other variables that can influence and modify the etn response, genetic determinants will remain consistent throughout a patient's lifetime (23). numerous snps were studied in the promoter regions of the tnf-α gene for their potential to alter the production of tnf-α or other cytokines, hence influencing the susceptibility or severity of ra(24,25). regarding the study's demographic characteristics, the results were equivalent in terms of the mean of age and duration of ra disease to those of another iraqi study that examined beliefs about medications among a sample of iraqi ra patients(26). in terms of iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 122 male-to-female ratio variation, the findings were comparable to other iraqi studies that confirm a high female-to-male ratio in ra illness(27–29). the results of this study identified three snps in the promoter region of the tnf-α gene in a sample of eighty ra patients treated with etn: -376 g/a, 806 c/t, and -1031 c/t. three earlier studies in iraq(27,30,31) investigated the association between a polymorphism in the promoter region of tnf-α and ra in a cohort of iraqi patients. however, no study has evaluated the relationship between these snps and etn responsiveness. furthermore, previous studies focused on only one or two snps, in contrast to the current study. many studies have examined the effect of snp combination in the tnf-α gene on the response to tnf-α antagonists (32). for instance, the snps -857c > t, -308g > a, 238g > a, and +489g > a in the tnf-α gene and their association to therapeutic efficacy were evaluated in retrospective study that involve 58 greek ra patients taking infliximab(33). similarly, an association study of three tnf-α related snps (-308g/a, -238g/a, and -857c/t) was conducted in poland and involve 280 ra patients of caucasian origin who treated with tnf-α inhibitors for at least 6 months (34). additionally, meta-analyses have been conducted to examine the relationship between a variety of snps and tnf-inhibitor responsiveness, including the 308 g/a polymorphism, the -857 c/t polymorphism, and the -238 g/a polymorphism (3,35).nonetheless, the current study was the only study that investigated the association between three snps -376 g/a, -806 c/t, and -1031 c/t on etn responsiveness. concerning the prevalence of -1031t/c genotypes, the current study found that more than 68 % of patients have the tt genotype. while the cc genotype had the lowest prevalence 3.75%. moreover, 97.5 %of patients possessed the t gene, while only 30% possessed the c allele. similarly, in the 1000 genomes database, the -1031 t/c promoter polymorphism has a reported minor c allele frequency of 22%. also, the results of this study are similar to those of saudi patients with diffuse large b-cell lymphoma(36), patients with behçet's disease in western algeria (37), and japanese patients with crohn's disease(38), all of whom had a high frequency of the tt genotype and a low frequency of the cc genotype. recent pieces of evidence have highlighted the associations between the -1031 t/c polymorphism and immune-mediated illnesses such as ra (38,39). it can thus be reasonably assumed that the polymorphism in this site may influence the response to etn. however, the current study found no statistically significant correlation between etn responsiveness and polymorphism in -1031t/c. no previous studies investigated the effect of-1031t/c on response to etn in ra patients nevertheless, the -1031 t/t genotype was predictive of a favorable response to tnf-α antagonists in chinese han ankylosing spondylitis patients(40). likewise, a case-control study which performed in spain and include 109 patients with psoriasis indicated that those with the tnf-α -1031 tt genotype responded better to infliximab(41). along with the small sample size, the current study's inconclusive results may be due to the use of etn in ra, whereas prior trials involved infliximab or adalimumab in diseases other than ra. moreover, ethnicity differences play a significant role in the pharmacogenetic investigations since allele frequencies in the populations studied may vary(42). regarding the prevalence of -806 c/t genotypes, the cc genotype was detected in 90% of ra patients enrolled in the current investigation. though, the t allele was present in only 11.25 % of patients, the c allele was present in all patients, and there was no statistically significant difference in the availability of these genotypes or alleles between the responders and non-responders. although the number of studies investigating the role of -806c/t in various conditions is limited, the results are consistent with those of a recent study examining the putative role of tnf-α gene polymorphisms in patients with south indian systemic lupus erythematosus (sle)(43). also, these values correlate favorably with results from a south german caucasian cohort research (44). furthermore, the findings are comparable to a casecontrol study conducted in taiwan that looked at the connections between snps in the promoter region of the tnf-α and susceptibility to nasopharyngeal carcinoma(45). the current study is the first to investigate the role of -806 c/t in ra patients' responsiveness to etn. the findings clearly demonstrated a non-significant connection between any genotype and a proclivity for non-response to etn. unfortunately, no comparable study exists to which the findings may be compared. nonetheless, the only comparable result was from a study in south indian systemic lupus erythematosus (sle) patients, which discovered no correlation between the 806c/t genotypes and the tendency to develop sle disease (43). this result may suggest that this polymorphic site plays a minor role in autoimmune disease and, consequently, in response to medications used to treat these diseases. in the case of -376 g/a genotypes, the current study discovered that 95 % of patients possessed the gg genotype while heterozygotes ga were found in 5% of the cases, and there were no homozygotes aa genotypes. likewise, the a allele was only found in iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 123 5% of ra patients; however, the dominant g allele existed in all patients. the findings are similar to those of a turkish study that examined the association of tnf-α (-376 g/a) polymorphisms in turkish tuberculosis patients(46). also, the result was parallel to an iranian study which examined -376 g/a tnf-α gene polymorphisms in iranian celiac patients to healthy controls(47). while various experimental studies which tried to address the relation between tnf-α polymorphisms and its expression, and the mechanisms controlling its expressions in many cell types and diseases highlights that the tnf -376g/a snps may have functional significance and may influence tnf-α gene expression levels(41,48,49); however, the tnf-α 376 g/a polymorphisms have received scant attention in ra patients, and their consequence on increased susceptibility to ra is uncertain (50–52). for instance, the polymorphism -376 g/a was not associated with an increased risk of ra in egyptian ra patients(53) or mexican patients(54). correspondingly, the study of kang et al. (55) found that -376g/a was not polymorphic in korean ra patients. the current study's finding failed to find a significant correlation between tnf-α (-376 g/a) polymorphisms and response to etn in iraqi ra patients. no earlier studies have examined the connection between the -376 g/a and the response to etn or even other tnf-α antagonists, this made the comparison with other studies in applicable. the most plausible reason for the result mentioned above, in addition to the study's small sample size, is that the polymorphism at -376 g/a was very marginally associated with ra(50–52) , despite its effect on tnf-α gene expression(41,48,49). the das28 score is the most often used outcome measure in investigating therapeutic response in ra (56). concerning the associations between various genotypes and das28 change after six months of continuous etn treatment, the current study could not confirm a significant change in das28 for all three sites. although he uses different response measures in his study, kang et al.(55) found a nonsignificant change in acr20 or acr70 linked with -1031t/c and -863c/a snps in a sample of korean ra patients. the discrepancy between the current study and other studies in the lack of a significant change in das28 might be related to the vulnerability of subjective outcome scores to heterogeneity, such as das28, are vulnerable to heterogeneity depending on the reporting clinician. moreover, it is generally established that measuring joint tenderness and patient perceptions of ra disease activity are two areas that can be challenging because there is no objective metric to validate clinical assessment(56). in addition to the differences in research design, sex ratios, sample size clinical outcomes assessed (das28esr vs. das28crp), anti-tnf medicine utilized, and concomitant use of disease-modifying antirheumatic therapies (dmards) with the tnf-α antagonist in some studies. regarding the difference in tnf levels between different genotypes, the current study results indicated that, polymorphism did not cause any significant difference in all three sites. although no prior research has explored the association between these variants and tnf-α levels in ra patients after taking etn therapy, one study analyzed the -1031 t/c polymorphism and tnf-α levels in patients with the acute coronary syndrome(57). the results indicated that tc carriers had significantly greater tnf-α levels than tt homozygotes(57). in contrast, another investigation discovered that carriers of the uncommon tnf-α 1031 c allele tended to have a more significant blood tnf-α level in chronic obstructive pulmonary disease patients(58). in the current study, the cc of carriers the -1031 t/c had the highest mean level of tnf-α but not significantly different from tt and tc carriers. also, tt carriers have a slightly higher but not statistically significant than tc carriers. regarding the -376 g/a and -806 c/t polymorphisms, the current investigation found that the polymorphic genotypes of these snps (-376 ga, -806 ct) had a slightly higher level of tnf, but the difference was not statistically significant. two investigations indicated that the a allele of the -376 g/a is associated with higher tnf-α production and transcription(59,60). however, this contrasts with musa et al., who discovered no correlation between the -376 g/a genotypes and tnf-α levels in egyptian patients with ra(53). on the other hand, katkam et al.(43) found no association between -806 c/t polymorphisms with the tnf-α level in a patient with sle. limitations the current study has several limitations. first, this study had a small sample size, which could be attributed to the small number of patients who used only etn and met all other inclusion criteria. second, because this study was conducted in a single center, caution is required when generalizing the findings to all iraqi rheumatoid arthritis patients. however, the chosen center treated patients from a variety of iraqi governorates. moreover, the study was conducted during the covid-19 era, and the overall number of patients included was limited due to repeated curfews that resulted in the loss of several patients as a result of their inability to attend the rheumatology units and obtained their medications. although the study excludes smokers and patients taking etn in combination with another dmard to rule out any response effect that could bias the iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 124 results, one significant weakness is that the researchers focused exclusively on polymorphisms in the tnf-α promoter region, and ignored polymorphisms in other regions that could alter the response to etn. furthermore, because most of the patients were already on etn when they were enrolled, the baseline level of tnfwas not measured. the author relies on the difference in levels across genotypes to determine whether certain genotypes are associated with high levels of tnf-α, which may lead to non-responsiveness to etn. finally, the authors were unable to conduct a more powerful prospective cohort study by selecting only patients with specific genotypes and following them for six months due to the small number of patients who received only etn, insufficient financial resources, and the lengthy time frame required to recruit and conduct this type of study. conclusions this study has revealed that patients who do not respond to etn have a higher tnf-α level than respondents. sanger sequencing of the tnf-α gene promoter region revealed three polymorphic sites -1031 t/c, -806 c/t, and -376 g/a. the polymorphisms at these three locations did not affect the likelihood of iraqi ra patients being nonresponder. since the significance of these snps in ra patients' response to etn has not been established in the current study, and no previous study has inspected the effect of these snps in other populations. therefore, it is essential to examine this polymorphism in a larger group of populations with diverse ethnic backgrounds to confirm or refute the results of this study. references 1. smolen js, aletaha d, mcinnes ib. rheumatoid arthritis. lancet (internet) 2016 (cited 2021 mar 10);388(10055):2023–38. available from: http://www.thelancet.com/article/s014067361 6301738/fulltext 2. jang di, lee ah, shin hy, song hr, park jh, kang tb, et al. the role of tumor necrosis factor alpha (tnf-α) in autoimmune disease and current tnf-α inhibitors in therapeutics (internet). int. j. mol. sci.2021 (cited 2021 aug 11);22(5):1–16. available from: https://www.mdpi.com/14220067/22/5/2719/htm 3. kang cp, lee kw, yoo dh, kang c, bae sc. the influence of a polymorphism at position 857 of the tumour necrosis factor α gene on clinical response to etanercept therapy in rheumatoid arthritis. rheumatology 2005;44(4):547–52. 4. zamri f, de vries tj. use of tnf inhibitors in rheumatoid arthritis and implications for the periodontal status: for the benefit of both? front immunol (internet) 2020 (cited 2021 aug 11);11. available from: /pmc/articles/pmc7646519/ 5. bathon jm, martin rw, fleischmann rm, tesser jr, schiff mh, keystone ec, et al. a comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. n engl j med (internet) 2000 (cited 2021 aug 11);343(22):1586–93. available from: https://www.nejm.org/doi/full/10.1056/nejm2 00011303432201 6. wiens a, venson r, correr cj, otuki mf, pontarolo r. meta-analysis of the efficacy and safety of adalimumab, etanercept, and infliximab for the treatment of rheumatoid arthritis. pharmacotherapy (internet) 2010 (cited 2020 apr 4);30(4):339–53. available from: http://www.ncbi.nlm.nih.gov/pubmed/2033445 4 7. baseggio l, bartholin l, chantome a, charlot c, rimokh r, salles g. allele-specific binding to the -308 single nucleotide polymorphism site in the tumour necrosis factor-alpha promoter. eur j immunogenet (internet) 2004 (cited 2020 mar 18);31(1):15–9. available from: http://www.ncbi.nlm.nih.gov/pubmed/1500917 6 8. tao w, concepcion an, vianen m, marijnissen aca, lafeber fpgj, radstake trdj, et al. multiomics and machine learning accurately predict clinical response to adalimumab and etanercept therapy in patients with rheumatoid arthritis. arthritis rheumatol (internet) 2021 (cited 2021 aug 11);73(2):212–22. available from: https://onlinelibrary.wiley.com/doi/full/10.100 2/art.41516 9. moreland lw, baumgartner sw, schiff mh, tindall ea, fleischmann rm, weaver al, et al. treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)–fc fusion protein. n engl j med (internet) 1997 (cited 2020 mar 31);337(3):141–7. available from: https://www.nejm.org/doi/full/10.1056/nejm19 9707173370301 10. moreland lw, schiff mh, baumgartner sw, tindall ea, fleischmann rm, bulpitt kj, et al. etanercept therapy in rheumatoid arthritis: a randomized, controlled trial. ann intern med 1999;130(6):478–86. 11. guis s, balandraud n, bouvenot j, auger i, toussirot e, wendling d, et al. influence of 308 a/g polymorphism in the tumor necrosis factor α gene on etanercept treatment in rheumatoid arthritis. arthritis care res (internet) 2007 (cited 2021 jul 27);57(8):1426– 30. available from: https://onlinelibrary.wiley.com/doi/full/10.100 2/art.23092 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 125 12. scardapane a, ferrante r, nozzi m, savino a, antonucci i, dadorante v, et al. tnf-α gene polymorphisms and juvenile idiopathic arthritis: influence on disease outcome and therapeutic response. semin arthritis rheum 2015;45(1):35–41. 13. pavy s, toonen ejm, miceli-richard c, barrera p, van riel plcm, criswell la, et al. tumour necrosis factor α-308g→a polymorphism is not associated with response to tnfα blockers in caucasian patients with rheumatoid arthritis: systematic review and meta-analysis. ann rheum dis (internet) 2010 (cited 2021 jul 26);69(6):1022–8. available from: https://ard.bmj.com/content/69/6/1022 14. murdaca g, gulli r, spanò f, lantieri f, burlando m, parodi a, et al. tnf-α gene polymorphisms: association with disease susceptibility and response to anti-tnf-α treatment in psoriatic arthritis. j invest dermatol 2014;134(10):2503–9. 15. danila mi, hughes lb, bridges sl. pharmacogenetics of etanercept in rheumatoid arthritis. pharmacogenomics 2008;9(8):1011– 5. 16. aletaha d, neogi t, silman aj, funovits j, felson dt, bingham co, et al. 2010 rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative. arthritis rheum (internet) 2010 (cited 2021 mar 10);62(9):2569–81. available from: http://doi.wiley.com/10.1002/art.27584 17. fransen j, van riel plcm. the disease activity score and the eular response criteria. rheum. dis. clin. north am.2009;35(4):745–57. 18. prevoo mll, van’t hof ma, kuper hh, van leeuwen ma, van de putte lba, van riel plcm. modified disease activity scores that include twenty‐eight‐joint counts development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. arthritis rheum 1995;38(1):44–8. 19. gan sd, patel kr. enzyme immunoassay and enzyme-linked immunosorbent assay. j invest dermatol (internet) 2013 (cited 2021 sep 20);133(9):1–3. available from: www. jidonline .org1researchtechniquesmadesimple. 20. h.t. o, z. o. clinical efficacy of tnf-(alpha) inhibitors: an update. int j clin rheumtol (internet) 2010 (cited 2021 jul 26);5(1):101–15. available from: https://www.proquest.com/openview/88b12cc0 a1bd33949a3d3058082a24d8/1?pqorigsite=gscholar&cbl=55174 21. otten mh, prince fhm, armbrust w, ten cate r, hoppenreijs epah, twilt m, et al. factors associated with treatment response to etanercept in juvenile idiopathic arthritis. jama j am med assoc (internet) 2011 (cited 2021 jul 26);306(21):2340–7. available from: https://jamanetwork.com/journals/jama/fullarti cle/1104691 22. ma mhy, ma mhy, ma mhy, defranoux n, defranoux n, li w, et al. a multi-biomarker disease activity score can predict sustained remission in rheumatoid arthritis. arthritis res ther (internet) 2020 (cited 2021 jul 26);22(1):1–12. available from: https:// arthritis research. biomedcentral .com /articles /10.1186/s13075-020-02240-w 23. bergman mj, kivitz aj, pappas da, kremer jm, zhang l, jeter a, et al. clinical utility and cost savings in predicting inadequate response to anti-tnf therapies in rheumatoid arthritis. rheumatol ther (internet) 2020 (cited 2021 jul 26);7(4):775–92. available from: https://link.springer.com/article/10.1007/s4074 4-020-00226-3 24. martínez a, fernández-arquero m, pascualsalcedo d, conejero l, alves h, balsa a, et al. primary association of tumor necrosis factorregion genetic markers with susceptibility to rheumatoid arthritis. arthritis rheum 2000;43(6):1366–70. 25. miller sa, dykes dd, polesky hf. a simple salting out procedure for extracting dna from human nucleated cells. nucleic acids res (internet) 1988 (cited 2021 jul 27);16(3):1215. available from: http://nar.oxfordjournals.org/ 26. faiq mk, kadhim dj, gorial fi. belief about medicines among a sample of iraqi patients with rheumatoid arthritis. iraqi j pharm sci (internet) 2019 (cited 2021 sep 7);28(2):134– 41. available from: https://bijps.uobaghdad.edu.iq/index.php/bijps/ article/view/932 27. alanzy ak, altaee ah, alrubiae sj. serum tumor necrosis factor alpha and gene polymorphisms in rheumatoid arthritis patients in babylon province, iraq. j glob pharma technol (internet) 2018 (cited 2020 jul 15);10(3):387–95. available from: www.jgpt.co.in 28. alkazzaz a. incdence of rheumatoid arthritis ( 2001 to 2011 ). iraqi postgrad med j (internet) 2013 (cited 2021 nov 5);12(4):568–72. available from: https://ipmj.iraqiboard.edu.iq/article_85439.ht ml 29. getta, khoshnaw n, alwan af, sundus f, mirza rr. types of anaemia and its correlation with disease activity in patients with rheumatoid arthritis among kurdish population of iraq. iraqi j hematol (internet) 2021 (cited 2021 nov 5);5(1):114. available from: https:// www. ijhonline. org/ article .asp? issn=2072-8069; year=2016; volume =5;issue iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 126 =1 ; spage=114;epage=128; aulast=getta; type=0 30. mahmood as, al-kazaz a-ka, ad’hiah ah. a single nucleotide polymorphism of tumor necrosis factor alpha gene (rs1800629) is not associated with rheumatoid arthritis in a sample of iraqi patients. j gene c environ resour conserv (internet) 2017 (cited 2020 jul 15);5(2):59–63. available from: www.jgerc.com 31. ahmed z. alwaeli, ahmed c. albarqaawee, eman h. alsalami. joints’ changes in rheumatoid arthritis is reduced by polymorphism of tnf-α (-238 g/a -308 g/a) and il-1β (+3953 c/t) in najaf population. eurasian j biosci (internet) 2020 (cited 2021 jul 27);14:5405–12. available from: http://www.ejobios.org/download/jointschanges-in-rheumatoid-arthritis-is-reduced-bypolymorphism-of-tnf-238-g-a-308-g-a-and-il1-8239.pdf 32. pallio g, mannino f, irrera n, eid ah, squadrito f, bitto a. polymorphisms involved in response to biological agents used in rheumatoid arthritis (internet). biomolecules2020 (cited 2021 jul 27);10(9):1– 11. available from: https://www.mdpi.com/2218273x/10/9/1203/htm 33. chatzikyriakidou a, georgiou i, voulgari p v., venetsanopoulou ai, drosos aa. combined tumour necrosis factor-α and tumour necrosis factor receptor genotypes could predict rheumatoid arthritis patients’ response to antitnf-α therapy and explain controversies of studies based on a single polymorphism (1) (internet). rheumatology2007 (cited 2021 jul 27);46(6):1034–5. available from: https://academic.oup.com/rheumatology/article /46/6/1034/2899503 34. swierkot j, bogunia-kubik k, nowak b, bialowas k, korman l, gebura k, et al. analysis of associations between polymorphisms within genes coding for tumour necrosis factor (tnf)-alpha and tnf receptors and responsiveness to tnf-alpha blockers in patients with rheumatoid arthritis. jt bone spine (internet) 2015;82(2):94–9. available from: http://dx.doi.org/10.1016/j.jbspin.2014.08.006 35. zeng z, duan z, zhang t, wang s, li g, gao j, et al. association between tumor necrosis factor-α (tnf-α) promoter -308 g/a and response to tnf-α blockers in rheumatoid arthritis: a meta-analysis. mod rheumatol 2013;23(3):489–95. 36. al-khatib sm, abdo n, al-eitan ln, almistarehi ahw, zahran dj, kewan tz. lta, lep, and tnf-a gene polymorphisms are associated with susceptibility and overall survival of diffuse large b-cell lymphoma in an arab population: a case-control study. asian pacific j cancer prev (internet) 2020 (cited 2021 jul 27);21(9):2783–91. available from: /pmc/articles/pmc7779465/ 37. khaib dit naib o, aribi m, idder a, chiali a, sairi h, touitou i, et al. association analysis of il10, tnf-α, and il23r-il12rb2 snps with behçet’s disease risk in western algeria. front immunol 2013;0(oct):342. 38. negoro k, kinouchi y, hiwatashi n, takahashi s, takagi s, satoh j, et al. crohn’s disease is associated with novel polymorphisms in the 5’ flanking region of the tumor necrosis factor gene. gastroenterology 1999;117(5):1062–8. 39. qidwai t, khan f. tumour necrosis factor gene polymorphism and disease prevalence (internet). scand. j. immunol.2011 (cited 2021 jul 27);74(6):522–47. available from: https://onlinelibrary.wiley.com/doi/full/10.111 1/j.1365-3083.2011.02602.x 40. tong q, zhao db, bajracharya p, xu x, kong rn, zhang j, et al. tnf-α -857 and -1031 polymorphisms predict good therapeutic response to tnf-α blockers in chinese han patients with ankylosing spondylitis. pharmacogenomics (internet) 2012 (cited 2021 jul 15);13(13):1459–67. available from: https://www.futuremedicine.com/doi/abs/10.22 17/pgs.12.133 41. gallo e, cabaleiro t, román m, solano-lópez g, abad-santos f, garcía-díez a, et al. the relationship between tumour necrosis factor (tnf)-α promoter and il12b/il-23r genes polymorphisms and the efficacy of anti-tnf-α therapy in psoriasis: a case-control study. br j dermatol (internet) 2013 (cited 2021 jul 27);169(4):819–29. available from: https://onlinelibrary.wiley.com/doi/full/10.111 1/bjd.12425 42. hughes lb, morrison d, kelley jm, padilla ma, vaughan lk, westfall ao, et al. the hla-drb1 shared epitope is associated with susceptibility to rheumatoid arthritis in african americans through european genetic admixture. arthritis rheum (internet) 2008 (cited 2021 jul 29);58(2):349–58. available from: https://onlinelibrary.wiley.com/doi/full/10.100 2/art.23166 43. katkam sk, rajasekhar l, tasneem fsd, kutala vk. synergetic interaction of hladrb1*07 allele and tnf-alpha − 863 c/a single nucleotide polymorphism in the susceptibility to systemic lupus erythematosus. indian j clin biochem (internet) 2021 (cited 2021 jul 27);36(1):59–66. available from: https://link.springer.com/article/10.1007/s1229 1-019-00854-9 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 127 44. fürst d, zollikofer c, schrezenmeier h, mytilineos j. tnfa promoter alleles frequencies and linkage with classical hla genes in a south german caucasian population. tissue antigens (internet) 2012 (cited 2021 jul 17);80(6):502–8. available from: https://onlinelibrary.wiley.com/doi/full/10.111 1/tan.12025 45. ho s-yy, wang y-jj, huang p-cc, tsai s-tt, chen c-hh, chen hhhw, et al. evaluation of the associations between the single nucleotide polymorphisms of the promoter region of the tumor necrosis factor-α gene and nasopharyngeal carcinoma. j chinese med assoc 2006;69(8):351–7. 46. ates ö, musellim b, ongen g, topal-sarikaya a. interleukin-10 and tumor necrosis factor-α gene polymorphisms in tuberculosis. j clin immunol 2008;28(3):232–6. 47. nasiri z, nikzamir a, rostami-nejad m, sirati -sabet m, aghamohammadi e, chaleshi v, et al. serum level of tumor necrosis factor-α and its gene polymorphisms has no association with susceptibility to celiac disease in iranian population "serum level of tumor necrosis factor-α and its gene polymorphisms has no association with susceptibility to c. int j celiac dis (internet) 2018 (cited 2021 jul 18);6(2):42–6. available from: http://pubs.sciepub.com 48. ramírez-bello j, vargas-alarcón g, tovillazárate c, fragoso jm. polimorfismos de un solo nucleótido (snp): implicaciones funcionales de los snp reguladores (rsnp) y de los snp-arn estructurales (srsnp) en enfermedades complejas (internet). gac. med. mex.2013 (cited 2021 jul 27);149(2):220–8. available from: http://www.ncbi.nlm.nih.gov/projects/ 49. el-tahan rr, ghoneim am, el-mashad n. tnf-α gene polymorphisms and expression (internet). springeropen; 2016 (cited 2021 jul 27). available from: https:// springerplus .springeropen.com /articles/10.1186/s40064016-3197-y 50. waldron-lynch f, adams c, amos c, zhu dk, mcdermott mf, shanahan f, et al. tumour necrosis factor 5′ promoter single nucleotide polymorphisms influence susceptibility to rheumatoid arthritis (ra) in immunogenetically defined multiplex ra families. genes immun (internet) 2001 (cited 2021 jul 27);2(2):82–7. available from: https:// www. nature. com/ articles/ 6363738. 51. ates o, hatemi g, hamuryudan v, topalsarikaya a. tumor necrosis factor-alpha and interleukin-10 gene promoter polymorphisms in turkish rheumatoid arthritis patients. clin rheumatol (internet) 2008 (cited 2021 jul 27);27(10):1243–8. available from: https://link.springer.com/article/10.1007/s1006 7-008-0893-1 52. karray ef, bendhifallah i, benabdelghani k, hamzaoui k, zakraoui l. tumor necrosis factor gene polymorphisms and susceptibility to rheumatoid arthritis in regional tunisian population e. j infect dis immun (internet) 2011 (cited 2021 jul 27);3(2):30–5. available from: https:// academicjournals.org/ journal/ jidi/ article-abstract/ca0d0c33463 53. mousa ak. tnf-α genetic polymorphisms and its expression in egyptian rheumatoid arthritis patients. am j life sci (internet) 2014 (cited 2021 jul 27);2(4):234. available from: https:// www.researchgate.net/profile /ahmedghoneim-2/publication /275409587_ tnf-a_ genetic_ polymorphisms_ and_its_ expression_in_egyptian_rheumatoid_arthriti s_patients/links/ 553bf6220cf2c415bb0b1696/ tnf-agenetic -polymorphisms -anditsexpression -in-egyptian-r 54. cadena-sandoval d, alemán-ávila i, barbosacobos re, becerril-mendoza lt, fragoso jm, ramírez-bello j. tumor necrosis factor (tnf) and tnfr1 polymorphisms are not risk factors for rheumatoid arthritis in a mexican population. mol biol rep 2018;45(3):227–32. 55. kang c, lee k, yoo d, kang c, rheumatology sb-, 2005 undefined. the influence of a polymorphism at position− 857 of the tumour necrosis factor α gene on clinical response to etanercept therapy in rheumatoid arthritis. academic.oup.com (internet) (cited 2020 apr 1);available from: https://academic.oup.com/rheumatology/article -abstract/44/4/547/2899381 56. prajapati r, plant d, barton a. genetic and genomic predictors of anti-tnf response (internet). pharmacogenomics2011 (cited 2021 jul 18);12(11):1571–85. available from: https://www.futuremedicine.com/doi/abs/10.22 17/pgs.11.114 57. sandoval-pinto e, padilla-gutiérrez jr, valdés-alvarado e, garcía-gonzález ij, valdez-haro a, muñoz-valle jf, et al. association of the -1031t > c polymorphism and soluble tnf-α levels with acute coronary syndrome. cytokine 2016;78:37–43. 58. chen yc, liu sf, chin ch, wu cc, chen cj, chang hw, et al. association of tumor necrosis factor-α-863c/a gene polymorphism with chronic obstructive pulmonary disease. lung (internet) 2010 (cited 2021 jul 28);188(4):339– 47. available from: https:// link. springer .com/ article/ 10.1007/s00408-010-9236-5 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 128 59. cuchacovich m, ferreira l, aliste m, soto l, cuenca j, cruzat a, et al. tumour necrosis factor-α (tnf-α) levels and influence of -308 tnf-α promoter polymorphism on the responsiveness to infliximab in patients with rheumatoid arthritis. scand j rheumatol (internet) 2004 (cited 2020 mar 18);33(4):228– 32. available from: https:// www. tandfonline. com/doi/abs/10.1080/03009740410005863 60. mugnier b, balandraud n, darque a, roudier c, roudier j, reviron d. polymorphism at position -308 of the tumor necrosis factor α gene influences outcome of infliximab therapy in rheumatoid arthritis. arthritis rheum (internet) 2003 (cited 2020 mar 18);48(7):1849–52. available from: https://onlinelibrary.wiley.com/doi/full/10.100 2/art.11168 this work is licensed under a creative commons attribution 4.0 international license http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ j bagh college dentistry vol. 29(1), march 2017 serum level of tnfα oral and maxillofacial surgery and periodontics 104 serum level of tnf-α and il-17 in patient have chronic periodontitis associated rheumatoid arthritis munir nasr hamed, b.d.s.(1) basimagh. ali, b.d.s., m.sc.(2) abstract background: chronic periodontitis and rheumatoid arthritis are widely prevalent diseases and are characterized by tissue destruction due to chronic inflammation. recently, there is growing evidence that the two diseases share many pathological features the aims of the study to determine the periodontal health status in patient have chronic periodontitis with rheumatoid arthritis and compare it with those having chronic periodontitis without rheumatoid arthritis and determine the serum levels of interleukin -17(il-17), tumor necrosis factoralpha (tnf-α ) in both groups and compare with the control group (subject samples neither have periodontitis nor arthritis ) and correlate these immunological markers with the periodontal parameters plaque index , gingival index , bleeding on probing, probing depth, clinical attachment level and number of missing teeth. materials and methods: eighty (80) males and females subjects with age range (30-45) years were recruited in this study they were divided into three main groups the chronic periodontitis with rheumatoid arthritis group consist of thirty (30) subjects and second group consist of thirty (30) subjects have chronic periodontitis and third group consist of twenty (20) subject case control group. all subjects had normal weight and height range according to bmi (body mass index) that it value is (18.5-25), clinical periodontal parameters used in this study were plaque index, gingival index, bleeding on probing, clinical attachment level index, probing pocket depth and number of missing teeth was measured in all groups at four surfaces of all presented teeth blood samples were collected from all individuals and examined to determined serum level of interleukin -17 and tumor necrosis factor-a by mean of enzyme-linked immune–sorbent assay. results: the present study showed patients with chronic periodontitis and rheumatoid arthritis had higher prevalence of sites presenting dental plaque, a higher rate of gingival inflammation and bleeding on probing greater probing depth, greater attachment loss and high number of missing teeth compared to those had chronic periodontitis only and control subjects . also highly significant differences between studied group regarding serum level of il-17 and tnf-α atp < 0.001, as well as, it revealed that mean serum levels of il-17 were statistically higher in chronic periodontitis with rheumatoid arthritis group (607.9 ± 79.9) than chronic periodontitis group (421.4 ± 5.9) and control groups (15.9 ± 2.7) similarly serum level of tnf-α (402.2 ± 41.2 319.4 ± 526 85.3 ± 4.9) respectively at p < 0.001. regarding correlation, the current study observed strong positive correlation between serum levels of il-17 andtnf-α with pl.i, gi, bop, ppd cal and the number of missing teeth in the pra at p<0.001. also this study reveal significant correlation between the two immunological markers (tnf-α and il-17) in chronic periodontitis with rheumatoid arthritis group and in chronic periodontitis group. conclusion: it was concluded that there was higher potentiality to chronic periodontitis involvement among rheumatoid arthritis patients, that correlated positively with increase the level of serum levels of il-17 and tnf-α accordingly with high score of clinical parameters that had recorded. that mean tnf –a and il-17 may play an important role in increase the severity of periodontitis as well as rheumatoid arthritis. keywords: chronic periodontitis, rheumatoid arthritis, serum level ,tnf-α , il-17.(j bagh coll dentistry 2017; 29(1):104110) introduction the periodontal diseases range from the relatively simple form of gingivitis to more destructive form of periodontitis, periodontal disease are not only effect the dentition, but may also be a threat to general health(1) . periodontitis, the most common oral disease, is destructive inflammatory disease of the supporting tissues of the teeth and is caused by alveolar bone destruction due to a chronic inflammation (1). (1) master student, department ofperiodontics, college of dentistry, university of baghdad. (2) assistant professor, department ofperiodontics, college of dentistry, university of baghdad. group of specific microorganisms (2).so it was characterized by both connective tissue and rheumatoid arthritis (ra) was another form of a chronic destructive inflammatory disease which is characterized by the accumulation and persistence of an inflammatory infiltrate in the synovial membrane that leads to synovitis and the destruction of the joint architecture resulting in impaired function.it were also associated with inflammatory destruction of joint connective tissue and bone destruction (3). in particular, ra as a chronic inflammatory joint disease carries many characteristics and pathogenetic processes that have similarities to periodontitis. the relationship between rheumatoid arthritis and periodontitis were controversial (4). j bagh college dentistry vol. 29(1), march 2017 serum level oral and maxillofacial surgery and periodontics 105 periodontitis and ra represent an imbalance between pro-inflammatory cytokines and antiinflammatory cytokines, which considered the cause for tissue damage.(5) cytokines were the mean of communication between immune and non-immune cells. hence these cytokines are essential to the pathogenesis of several diseases, including periodontal disease and rheumatoid arthritis. (6) periodontitis had obviously cytokine profiles as that of ra, disease progression is due to continuous and persistence accumulation of proinflammatory cytokines as il-1β and tnf-α together with low levels of il-10 and transforming growth factor β (tgf-β). all might give a good picture for the active stages of both ra and pd (6). the role of pro-inflammatory cytokine, tnf-α, is of special interest for the understanding of immune responses in both ra and pd (7).because of the treatment with antitnf-α medication was commonly used to control the inflammatory process in ra; such therapy may also be relevant for the management of pd (8). the production of il-17 by th17 subset of cd4 t-cell identified in 2003 .it has been associated with the pathogenesis of numerous autoimmune and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel diseases, psoriasis and periodontitis (9). the present study was carry out to study the serum cytokine profile (il-17 and tnf-α) in chronic periodontitis subjects with ra compared to those without ra disease and the influence of the serum levels of these cytokines on clinical periodontal parameters in studied groups. materials and methods the sample in this study consisted of eighty (80) males and females subjects with age range (3545) years. the sample was divided into three groups (chronic periodontitis/rheumatoid arthritis) group(pra) thirty patients (30) diagnosed to have chronic periodontitis disease, and have rheumatoid arthritis. they were from attendants seeking treatment in the rheumatology clinic in baghdad teaching hospital. the rheumatoid arthritis state was diagnosed according to the revised criteria for the classification of rheumatoid arthritis of the american college of rheumatology (10) and also according to the laboratory investigation(esr,latex test). (chronic periodontitis / nonrheumatoid arthritis) group (cp) thirty patients (30) were recruited from the attendants to the clinic of the department of periodontics /college of dentistry /baghdad university.chronic periodontitis in patients was defined as the presence of at least four sites with probing pocket depth ≥4mm with clinical attachment level ≥1-2mm, this made according to the international classification system for periodontal disease (11). (control / systemically healthy) group (c) twenty patients (20) with clinically healthy periodontium and healthy systemic status. this group represents controls. clinical examination: clinical periodontal parameters include {plaque index (pli) silness&loe(12), gingivalindex (gi) loe&silness(13), bleeding on probing (bop), probing pocket depth (ppd), clinical attachment level (cal) and number of missing teeth}. also all subjects had normal range of bmi (body mass index) that it value is (18.5-25)(26). blood collection and biochemical analysis the blood was collected between (9 am-12 pm), the blood samples were taking from their arms from cubital fossa (cubital vein), and put it in a evacuated [ethylene diamine tetra acetic acid (edta)] tubes as anticoagulant tubes, after centrifuging plasma samples preserved immediately into other plain tubes and preserved in freeze (-15cº) until they have been assayed for il-17 and tnf-αby elisa according to manufacturer's protocol of instruction at the raybio il-17 elisa (enzyme-linked immunosorbent assay) kit and tnf-α (human) elisa kit protocol. results clinical analysis high significant differences were found between the mean (pli, ppd and cal) of pra group and cp group by using t-test at p < 0.001 and highly significant (gi) differences found between the same groups table (1). inter group comparison by median of the sites with positive bop presented in table (2) with each group there was high significant difference between sites with positive bop compare to non-bleeding sites. the number of missing teeth between pra group and cp group, using mann– whitney u test at-alpha p <0.05 reveal highly significant difference as shown in table (2). j bagh college dentistry vol. 29(1), march 2017 serum level oral and maxillofacial surgery and periodontics 106 table 1: statistical differences of the periodontal parameters (pli. gi, ppd and cal) among all studied groups. parameters chronic periodontitis rheumatoid arthritis (n=30) chronic periodontitis (n=30) control (n=20) p-value gingival index mean ± sd 1.9 ± 0.3 1.8 ± 0.4 0.5 ± 0.2 <0.001*,ª plaque index mean ± sd 2.1 ± 0.2 1.9 ± 0.3 0.6 ± 0.2 <0.001*,ª probing pocket index mean ± sd 5.2 ± 0.3 4.3 ± 0.5 <0.001*,t clinical attachment level mean ± sd 6.2 ± 0.7 4.1 ± 0.5 <0.001*,t ª anova test, t independent t-test, * significant at alpha level <0.05 table 2: median and significant differences of bleeding on probing and missing teeth for the studied groups. parameters pra group (n=30) cp group (n=30) cgruop (n=20) p-value bleeding on probing median (range) 63 (38-73) 53 (11-71) 8 (6-11) <0.001*, w number of missing teeth median (range) 5 (0 10) 1 (0 4) <0.001*,w kruskal-wallis nonparametric test, w mann-whitney u test * significant at alpha level <0.05 immunological findings the higher values of il-17 were in pra group (607.9 ± 79.9 pg/ml) compare to cp group (421.4 ± 5.9 pg/ml) and c group (15.9 ± 2.7 pg/ml). the current study pointed out that tnf-α reported higher increase in concentration in pra group (402.2 ± 41.2 pg/ml) as compared with cp group (319.4 ± 52.6 pg/ml) and c group (85.3 ± 4.9 pg/ml). using anova test to show significant of statistical difference. it appear that there was a high significant difference for both (il-17 and tnf-α ) among studied groups table (3). lsd test values between each two groups reveal a high significant difference in the level of il-17 and tnf-α p< 0.01 as shown in table (4). table 3: mean and significant differences of the levels of interleukin-17and tumor necrosis factor-alpha among included patients according to their group, n=80. parameters pra (n=30) mean ± sd cp (n=30) mean ± sd c (n=20) mean ± sd p-value level of interleukin17 (pg/ml) 607.9 ± 79.9 421.4 ± 5.9 15.9 ± 2.7 <0.001* tumor necrosis factor-alpha (pg/ml) 402.2 ± 41.2 319.4 ± 52.6 85.3 ± 4.9 <0.001* anova test, * significant at alpha level <0.05 j bagh college dentistry vol. 29(1), march 2017 serum level oral and maxillofacial surgery and periodontics 107 table 4: last significant differences of the levels of interleukin-17and tumor necrosis factoralpha between the included groups accordingly. groups level of interleukin17 (pg/ml) mean ± sd tumor necrosis factor-alpha (pg/ml) mean ± sd pra 607.9 ± 79.9 402.2 ± 41.2 cp 421.4 ± 5.9 319.4 ± 52.6 p-value <0.001* <0.001* pra 607.9 ± 79.9 402.2 ± 41.2 c 15.9 ± 2.7 85.3 ± 4.9 p-value <0.001* <0.001* cp 421.4 ± 5.9 319.4 ± 52.6 c 15.9 ± 2.7 85.3 ± 4.9 p-value <0.001* <0.001* lsd test, *significant at alpha level <0.01 correlations between the immunological parameters and clinical periodontal parameters to each group. the periodontal clinical parameters (pli, gi.ppd, cal) of pra group shown in table (5) have a high positive correlation with immunological parameters (il-17 and tnf-α ) using pearson’s correlation test significant at p <0.01 while the periodontal clinical parameters (bop and the number of missing teeth of pra interleukin -17 have high positive correlation to tnf-α within each of the studied group as shown in the table (6) group have a high positive correlation with immunological parameters (il-17 and tnf-α ) using spearman correlation test significant at p <0.01. accordingly the correlation that shown in cp group exhibit the same correlation between periodontal parameters and immunological parameters as in the pra group. for c group, (gi and pli) get a high positive correlation with serum level of il-17 and tnf-α . table 5: correlation of the levels of interleukin-17and tnf-α with periodontal health parameters according to each group. chronic periodontitis rheumatoid arthritis (pra)(n=30) (gi) p (pli) p (ppd) p (cal) p (bop) s (missing teeth) s il-17 (pg/ml) r 0.880** 0.853** 0.837** 0.922** 0.926** 0.876** p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 tnf-α (pg/ml) r 0.768** 0.711** 0.672** 0.801** 0.847** 0.797** p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 chronic periodontitis (cp) (n=30) (gi) p (pli) p (ppd) p (cal) p (bop) s (missing teeth) s il-17 (pg/ml) r 0.973** 0.958** 0.918** 0.868** 0.895** 0.942** p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 tnf-α (pg/ml) r 0.879** 0.906** 0.928** 0.885** 0.847** 0.906** p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 controls ( c ) (n=20) (gi) p (pli) p (ppd) p (cal) p (bop) s (missing teeth) s il-17 (pg/ml) r 0.889** 0.875** 0.845** p-value <0.001 <0.001 <0.001 tnf-α (pg/ml) r 0.922** 0.939** 0.897** p-value <0.001 <0.001 <0.001 p pearson’s correlation, s spearman’s rho correlation, ** correlation is significant at the 0.01 level (2-tailed). j bagh college dentistry vol. 29(1), march 2017 serum level oral and maxillofacial surgery and periodontics 108 table 6: correlation of between the levels of interleukin-17 and tnf-α within each of the study groups accordingly. groups il-17 (pg/ml) pra (n=30) tnf-α (pg/ml) r 0.844** p-value <0.001 cp (n=30) tnf-α (pg/ml) r 0.900** p-value <0.001 c (n=20) tnf-α (pg/ml) r 0.775** p-value <0.001 pearson’s correlation, ** correlation is significant at the 0.01 level (2-tailed). discussion chronic periodontal disease can be considered a potential focus of infection, which worsens the metabolic control of patients with ra. (14).the pathobiology of periodontal disease) and rheumatoid arthritis is similar, both are inflammatory chronic diseases, with activation of complement, production of cytokines and release of other inflammatory cell products (15, 16).the relationship between periodontal disease and rheumatoid arthritis still controversial (17, 18). current study revealed highly significant differences among the studied groups regarding pl.i; p.p.d; cal and b.o.p, significant level of gi and pli that is probably because patients with ra might be more likely to obtain temporomandibular joint involvement, severe hand dysfunction (caused by arthritis) which hinder the patient’s oral hygiene practices due to restriction of movements, at the same time, decreased saliva from secondary sjögren’s syndrome all enhances plaque accumulation as well as, ra patients may be emotionally depressed about their illness causing the deterioration of the attention to the personal hygiene (19,20). the elevated level of gi and pli reflects a higher inflammation in the pra group than the cp group and could be related to the increase in the plaque as the plaque is the causative factor of gingival inflammation. this result is agreed with (kässer) (20). the percentage of sites with bop was significantly higher in pra group than cp group. the potential altered abilities of ra patients to perform effective oral hygiene could result in an increased bop that exacerbates the risk for enhanced tissue destruction in periodontitis. moreover, interesting observations regarding the complexity of the oral and systemic challenge provide unique mechanisms by which dysregulation of host responses could occur (21). the mean value of ppd and cal in pra group was significantly higher compared to cp alone. this could be related to local and systemic factors. the local factor is the plaque which was significantly higher in the pra group and this has influenced ppd in this group. the systemic factor in the pra patients is the defect in the immune system which could result in inflammatorymediated destruction predisposing to periodontitis due to an unbalanced cytokine expression profile (22) clinical attachment level refers to the distance from the cementoenamel junction (cej) to the location of the inserted probe tip. thus, loss of fibers attachment expressed at the clinical level was due to the cumulative effect of destructive pathological processes in periodontal together with the protective and destructive effect of the immunological processes. the present study reported highly significant differences in mean il-17 values among the studied groups at p <0.001 also il-17 in pra group is highly elevated than clinically healthygroup. interleukin-17 plays a role in osteoclastogenesis via activation of rankl, causing bone destruction in inflamed joints the severity of ra increase by increase the serum level of il-17 in cp patient and significant increase of serum level of il-17 than healthy group and that showed in (23). il-17 induces cytokine and chemokine expression and may play a role in skeletal tissue destruction and inflammatory processes. patients with pra have markedly elevated in tumor necrosis factor-α levels compared with subjects of cp alone and healthy group at p <0.001. these findings suggest that anti-tnf-α may influence the destruction processes (as j bagh college dentistry vol. 29(1), march 2017 serum level oral and maxillofacial surgery and periodontics 109 reflected by the greater ppd and cal) these observations suggest that periodontal inflammation may be related to high levels of systemic and local tnf-α in patient with ra(24). tnf-α plays a central role in the host inflammatory reaction, which is related to the breakdown of alveolar bone as well as loss of connective tissue attachment that related to highly significant association between serum level of tnf-α and number of teeth lost in pra group and cp group (24). consequently, in chronic periodontal infection, bacteria and/or their components disseminate from the inflamed areas into the circulation to challenge the immune system, the circulating and resident immune cells of the body indicate that peripheral blood monocytes challenged by bacterial lps produce inflammatory mediators like il-1β and tnfα (25). references 1. destefano f, anda r.f, kahn h.s, williamson d.f, russell cm. dental disease and risk of coronary heart disease and mortality. br med j 1993; 306:688-691. 2. sainir,marawarpp,shete s, et al. periodontitis a true infection. j globalinfectdis.2009; 2:149-50. 3. weyand cm. new insights into the pathogenesis of rheumatoid arthritis.rheumatology 2000;39(1):3-8. 4. mercado, fb; marshall,ri; klestov, ac; et al. relationship between rheumatoid arthritis and periodontitis. j periodontol. 2001; 72(6):779-87. 5. eduardo de paula, carlos rossa, keith lough kirkwood,mirianaparecida: periodontal condition in patients with rheumatoid arthritis. (braz oral res 2008; 22(1):72-7. 6. cochran, d l. inflammation and bone loss in periodontal disease. j. periodontol. 2008;79: 15691576 7. preshaw pm, taylor jj. how has research into cytokine interactions and their role in driving immune responses impacted our understanding of periodontitis? j clin periodontol.2011; 38: 60–84. 8. orita s, koshi t, mitsuka t, et al. association between proinflammatory cytokines in the synovial fluid and radiographic grading and pain-related scores in 47 consecutive patients with osteoarthritis of the knee. bmc musculoskelet disord.2011; 12: 144. 9. silva n, dutzan n, hernandez m, dezerega a, rivera o, aguillon jc, aravena o, lastres p, pozo p, vernal r, gamonal j. characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells. j clinperiodontol 2008; 35: 206– 214. 10. arnett fc, edworthy sm, bloch da, mcshane dj,fries jf, cooper ns. the american rheumatism association 1987 revised criteria for theclassification of rheumatoid arthritis. arthritis rheum 1988; 31(3): 315 24. 11. lang np, bartold pm, cullinam. internationalclassification workshop: chronic periodontitis.annals of periodontology 1999; 4: 53. 12. silness j, löe h. periodontal disease in pregnancy ιι.actaodontolscand 1964; 24: 747-59. 13. löe h. the gingival index, the plaque index and theretention index system. j periodontol 1967;38: 610– 6. 14. slots j: casual or causal relationship between periodontal infection and non-oral disease? j dent res 1998; 77:1764-1765. 15. petty r.e, southwood t.r, manners p, baum j, glass dn, goldenberg j, et al. international league of associations for rheumatology classification of juvenile idiopathic arthritis: second revision, edmonton, 2001. j rheumatol 2004; 31:390-392. 16. smolik i, robinson d, el-gabalawy h.s. periodontitis and rheumatoid arthritis: epidemiologic, clinical, and immunologic associations.compendcontineduc dent 2009;30:188-190. 17. mercado f, marshall r.i, klestov a.c, bartold p.m: is there a relationship between rheumatoid arthritis and periodontal disease? j clinperiodontol. 2000;27(4):267-72. 18. mercado f.b, marshall r.i, klestov a.c, bartold pm: relationship between rheumatoid arthritis and periodontitis. j periodontol 2001;72:779-787 . 19. feldmann m, brennan f. and maini r: role of cytokines in rheumatoid arthritis. annual review of immunology 1996; 14, 397–440. 20. kässer u.r, gleissner c, dehne f, michel a, bolten ww. risk for periodontal disease in patients with longstandingm rheumatoid arthritis. arthritis rheum 1997; dec;40(12):2248-51 21. wegner n, wait r, sroka a, eick s, nguyen k. a, lundberg k, kinloch a. and venables p. j: peptidylarginine deiminase from porphyromonasgingivaliscitrullinates human fibrinogen and alpha-enolase: implications for autoimmunity in rheumatoid arthritis. arthritis and rheumatism (0202.) 22. bartold p.m, marshall r.i, haynes d.r. periodontitis and rheumatoid arthritis: a review. j periodontol 2005; 76:2066-2074. 23. gümüş p, buduneli e, bıyıkoğlu b, aksu k, saraç f, nile c et al. gingival crevicular fluid, serum levels of receptor activator of nuclear factor-kappa b ligand, osteoprotegerin, interleukin-17 in rheumatoid arthritis and osteoporosis patients with periodontal disease. journal of periodontology. 2013;1-13. 24. kobayashi t, yoshie h. host responses in the link between periodontitis and rheumatoid arthritis. current oral health reports. 2014; 2(1):1-8. 25. zahraa k, batool, h. study the role of proinflammatory and antiinflammatory cytokines in iraqi chronic periodontitis patients [internet]. repository.uobaghdad.edu.iq. 2012 [cited 7 may 2014]. 26. who expert consultation. appropriate body-mass index for asian populations and its implications for policy and intervention strategies. the lancet, 2004; 157-163. j bagh college dentistry vol. 29(1), march 2017 serum level oral and maxillofacial surgery and periodontics 110 الخالصة ى وجود امراض اللثة والروماتيزم الرثوي امراض شائعة في المجتمع وهي تتميز بتحطيم االنسجه بسبب االلتهاب المزمن ,الدراسات الحديثة تشير الالخلفية : صفات مرضيه مشتركه بين المرضين. المزمن لتحديد حالة اللثة الصحية لدى مرضى التهاب اللثة المزمن والمصابين بالروماتيزم الرثوي ومقارنتهم بالمرضى المصابين بالتهاب اللثة اهداف الدراسة : و تي ان اف الفا( في كال المجموعيتن ومقارنتهم مع المجموعه الغير المصابه 01ر لوكين وليس لديهم روماتيزم رثوي وايضا تحديد مستوى السيرم لكل من )انت عمق جيوب بأي من االمراض وربط عالقه بين الدالئل المناعيه مع دالئل التهاب اللثه السريري )مؤشر الصفيحة الجرثومية ,مؤشر التهاب اللثة,مؤشر سنان المفقود ( اللثة,مؤشرفقدان االنسجة الرابطة وعدد اال ( المعنين في هذه الدراسه ولقد تم تقسيمهم الي ثالث مجاميع .مجموعة 54-32اثنى وذكر يتراوح اعمارهم بين )تم اخذ عينات ثمانين شخص من المواد والطرق: شخص مصابين بألتهاب اللثة المزمن المجموعه الثالثه 32شخص. المجموعه الثانيه تتألف من 32م الرثوي تتألف من التهاب اللثة المزمن والمصابين بالروماتيز ياس في) بي تتألف من عشرين شخص والتي تعتبر المجموعه الغير مصابه بأي مرض . كل االشخاص يمتلكون معدل وزن وطول طبيعيان استنادا الى احكام الق ( ايضا لقد تم قياس مؤشر) الصفيحة الجرثومية,مؤشر التهاب اللثة,مؤشرعمق جيوب اللثة,مؤشرفقدان االنسجة الرابطة وعدد 04-4..0ه )ام اي( الذي قيمت يرم لكل من مستوى الساالسنان المفقوده( لكل المجاميع على االسطح االربعه لكل االسنان الموجود , عينات الدم التي جمعت من كل االفراد تم فحصها لمعرفة وتي ان اف الفا( بواسطه االنزيم الرابط بالمناعه. . 01 -)انتر لوكين ي االسنان هذه الدراسة اظهرت المرضى المصابون بالتهاب اللثة المزمن والمصابين بالروماتيزم الرثوي لديهم ميل اكبر لوجود الصفيحة الجرثومية ف النتائج: د مقارنتآ مع المصابين بالتهاب ومعدل اعلى اللتهاب اللثة,زياد كبيره في عمق جيوب اللثة وزياد كبيره في فقدان االنسجة الرابطة.وعدد عالي من االسنان المفقو اللثة المزمن فقط والمجموعه الغير مصابه. وتي ان اف الفا ( كذلك تكشف ان معدالت مستوى السيرم لل 01-ايضا هناك زياد واضحه وفرق في التركيز لكل المجاميع في مستوى السيرم) انترلوكين ( من مجموعه 16.6± 921.6مجموعه المرضى المصابون بالتهاب اللثة المزمن مع المصابين بالروماتيزم الرثوي ) ( احصائيا عاليه في 01-)انترلوكين (. وهذا يشابه مستوى السيرم لل )تي ان اف الفا( في بقية المجاميع 0.1± 04.6( والمجموعه الغير المصابه )4.6± 500.5المصابين بالتهاب اللثه المزمن ) ( و )تي 01-( . وبخصوص الترابط, الدراسه الحاليه تظهر ترابط قوي ايجابي بين مستويات السيرم لل )انترلوكين ±5.6 4.3. ±409 306.5 ±50.0 520.0) ( في مجموعه د ان اف الفا (مع )مؤشر الصفيحة الجرثومية ,مؤشر التهاب اللثة,مؤشر عمق جيوب اللثة,مؤشرفقدان االنسجة الرابطة وعدد االسنان المفقو المصابون بالتهاب اللثة المزمن مع المصابين بالروماتيزم الرثوي . ن اف الفا ( في مجموعة المرضى المصابون بالتهاب اللثة المزمن مع و تي ا 01-كذلك هذه الدراسه تكشف ترابط واضح بين الدالئل المناعيه االثنين ) انترلوكين المصابين بالروماتيزم الرثوي وفي مجموعة التهاب اللثة المزمن. . رم لل ان النتائج تشير الى ارتفاع معدل امراض اللثة لدى المرضى المصابين بالروماتيزم الرثوي والتي ترتبط ايجابيا مع زياده في مستوى السيالخالصة: ( تلعب دور 01-ان )تي ان اف الفا واالنترلوكين و تي ان اف الفا ( بالتتابع مع ارتفاع عالي في النتائج اللثويه السريره التي قد سجلت والذي يعني 01-)انترلوكين مهم في زياد شد امراض اللثة و كذلك في زياد امراض الرماتيزم الرثوي . . original�article abstract objective: the objective of this study was to determine the frequency of red cell alloantibodies in pregnant women of north west pakistan. study design: it was a descriptive study. place and duration of study. this study was conducted in one year november 2012 to october 2013 at the haematology department, army medical college, national university of sciences and technology (nust) in collaboration with department of gynaecology and obstetrics, military hospital, rawalpindi. materials and methods: a total of 600 samples were studied and it was a non probability convenience sampling. pregnant females of any age, parity and gestational age were included in the study and women with known autoimmune diseases (sle, rheumatoid arthritis) were excluded. data was collected through specifically designed proforma and was analysed by using spss version 20. descriptive statistics were used to describe the data. frequency and percentages were calculated for qualitative variables like blood group and alloantibodies. mean and standard deviation were calculated for quantitative variables like age, gestational age and parity. chi-square test was applied to find an association between all categorical variables. p-value <0.05 was considered significant. results: the frequency of alloantibodies in pregnant women in this study was 0.5% (3/600). prevalence of alloimmunization specifically in rhnegative blood group was 5.5% (3/54). all the antibodies detected were anti-d antibodies. conclusion: rh d antibodies are the only frequent antibodies in majority of pregnant women with rh negative blood group. so the practice of routine antenatal antibody screening for every pregnant woman should be avoided. key words: antibody formation, blood group antigens, pregnancy, pakistan. 3 newborn. antibody screening is done in pregnancy to identify these pregnancies which are at risk of fetal and neonatal hemolytic disease resulting from clinically 4 significant maternal alloantibodies. despite the introduction of rh ig this abo and rh incompatibility is still the major cause of haemolytic disease of the newborn in developing countries raising the importance of antibody screening in all antenatal 5 women irrespective of their blood phenotype. studies have shown variation in frequency of 6,7 alloantibodies among different countries. in asia although anti-d antibodies are the most frequent but the difference lies in the frequency of rest of the 8,9 clinically significant antibodies. developed countries like uk and netherlands do have their own guidelines for the antibody screening 10,11 in pregnant women. in pakistan only few case reports and prospective studies reveal the presence of some rare alloantibodies like anti-rh17, anti12,13,14 rhnull and anti-kell antbodies. these cases do raise the importance of such studies in order to know the prevalence of alloantibodies among pakistani introduction red blood cell alloantibodies are the unexpected immuneantibodies found other than the naturally occurring antibodies in the body, produced in response to the introduction of red cells possessing antigens that the subject lacks, as in cases of pregnancy, transfusion, transplantation or injection 1,2 of any immunogenic material. in pregnancy alloantibodies appear when fetal rbcs carrying a paternal antigen that is foreign to the mother, enters the maternal circulation and this incompatibility of blood groups between the mother and fetus can lead to alloimmune haemolytic disease of the frequency of red cell alloantibodies in pregnant women of north west pakistan 1 2 3 maria shafiq , ayesha noor , amer siddiq correspondence: dr. maria shafiq department of haematology army medical college, rawalpindi e-mail: khanmariashafiq@gmail.com 1 department of haematology army medical college, rawalpindi 2 3 department of haematology/ chemical pathology hbs medical college islamabad funding source: nil; conflict of interest: nil received: june 19, 2017; revised: aug 28, 2017 accepted: nov 30, 2017 red cell alloantibodies in pregnancyjiimc 2017 vol. 12, no.4 185 women which can enable us to set our own guidelines for the antenatal antibody screening. materials and methods it was a descriptive study and was conducted in the hematology department, army medical college, national university of sciences and technology (nust) in collaboration with department of gynaecology and obstetrics, military hospital, rawalpindi. it was completed in one year from nov 2012 to oct 2013. it was a non probability convenience sampling. a total of 600 pregant females were recruited from the out-patient department of gynaecology and obstetrics who came for routine antenatal checkups and were advised routine blood tests. all pregnant women were included irrespective of their age, parity and gestational age. women with known autoimmune diseases were excluded. history was taken from the females according to the structured questionnaire. their age ranged from 19 to 40 years. majority of females were in their third trimester and were primigravida. they were asked about their gravida status, transfusion experience and especially the blood group of their husbands. out of 600 women only 321(53.5%) were aware of their husband's blood group. obstetric history was also taken and those who gave history of one or more abortions were considered to have bad obstetric history. permission from the hospital ethical committee was taken. informed consents were obtained from all the patients. 5.5 ml of venous blood was withdrawn from antecubital vein using 10 ml syringe and was collected in two separate tubes. 2.5 ml blood was for the tube containing edta for abo and rh grouping and the other 3 ml blood was left to clot in the plain tube for antibody screening and identification. sample of each patient was given a laboratory number and record was maintained. each sample was then analysed. blood group was determined by forward and reverse blood grouping technique using commercially prepared blood grouping reagents (biotec) and freshly prepared pooled red cells. during rh blood typing, indirect antiglobulin test was performed on all negative results in order to confirm the weak d phenotype before reporting the sample as rh positive or negative. but we were not able to notice the presence of any weak d antigen among 54 rh negative blood samples. all samples irrespective of their blood group were screened for antibodies using 3 cell panel (diamed) by performing indirect antiglobulin test (iat). the samples which showed positive results on screening were identified using 11 cell panel (diacell). microscopy was done on all negative samples in all stages of iat. data was entered on a specifically designed proforma and was analysed using spss version 20. descriptive statistics were used to describe the data. frequency and percentages were calculated for qualitative variables like blood group, alloantibodies. mean and standard deviation (sd) were calculated for quantitative variables like age, gestational age, parity. chi-square test was applied to find an association between all categorical variables. pvalue <0.05 was considered significant. results a total of 600 women were screened for red cell alloantibodies. age range of patients included in the study was 19 years to 40 years. maximum number of patients 351(58.5%) presented in their third trimester. (fig i). fig 1: presenta�on of study group in various trimesters previous transfusion history was given by 123(20.5%) women. regarding the major blood group systems, figure ii shows the frequency of major blood group systems there were 546 (91%) d antigen positive and 54(9%) d antigen negative women in the study group. among the study group there were 348(58%) primigravida and 252(42%) multigravida females. jiimc 2017 vol. 12, no.4 186 red cell alloantibodies in pregnancy out of 348 primigravida 37(10.6%) were found to be d antigen-negative and the rest of 17 d antigen negative were multigravida (table i). obstetric history and in 0% (0/485) of antenatal women without an adverse obstetric history hence this study also shows a statistically significant association between adverse obstetric history and alloimmunisation rate (p<0.001) (table ii). fig 2: frequency of different blood groups among study group table i: rh phenotypes of primigravida and mul�gravida females a total of 3 irregular antibodies were identified in their blood samples, showing an overall frequency of alloantibodies as 0.5% (3/600). within the whole study group (n=600), anti-d was the only detected antibody, accounting for 100% of all the allantibodies. the husband's blood group was found to be d antigen-positive in all. among the 54 women in the d antigen-negative group, 3 developed antibodies, so the prevalence of alloimmunization in this group was 5.5% (3/54). no antibody was detected among the d antigen-positive group, showing an association of rh d antigen with antibody formation (p<0.001). an association is seen between antibody formation and the blood phenotype, as all the three women who were anti-d antibody positive belong to a negative blood phenotype. in this study, alloantibodies were found in 1.2% (3/249) of multigravida females and in 0% (0/348) of antenatal women who were primigravida showing statistically significant association between multigravida status and alloimmunization rate (p<0.041). as alloantibodies were found in 2.7% (3/112) of antenatal females with an adverse table ii: associa�on between an�body forma�on and rh phenotype (p<0.001), gravida status (p<0.041), adverse obstetric history (p<0.001) discussion this study with 0.5% frequency of red cell alloantibodies shows that throughout the world this frequency varies. a comparison of different study results are shown in table iii. in this study frequency is less as compared to the study carried out in iran(4.5%) and southern 15,16 pakistan(1.8%). reason behind it is that firstly the sample size in this study is smaller which increases the chances of missing rare antibodies which are reported in different case reports and secondly majority of females are primigravida in this study. it is also evident from this study that multigravida status, bad obstetric history and rh-negative phenotype are main risk factors for antibody formation which supports the studies carried out in 9,17 india and malaysia. in contrast to these studies, a nigerian study reveals comparatively higher alloimmunization rate of rh positive phenotype when compared to rh negative phenotype as 13 out of 17 detected antibodies were found in sera of rh 4 positive females. anti d antibodies are the most frequently seen in this study like europe, arab and asia with china as an exception showing anti e and anti mi antibodies 8,18 more common than anti d antibodies. recent studies carried out in america and australia also show anti e antibody more prevalent than anti d 19,20 antibodies. so these antibodies other than anti-d antibodies are the reason behind persistence of this hemolytic disease even after the introduction of rhig. the different red cell alloantibodies reported to jiimc 2017 vol. 12, no.4 187 red cell alloantibodies in pregnancy cause haemolytic disease worldwide are anti co (a), anti rh 17, anti diego, anti kell , anti c, anti cw, anti 21-,29 jk(b) and anti kpa. keeping in view the evidence of presence of these rare alloantibodies in pakistani women each and every blood group was screened for red cell alloantibody but none of these were found. many developed countries have formulated their own antenatal antibody screening guidelines in order to decrease the disease incidence. these countries include uk, netherlands, sweden, 10,11,30,31 australia and newzealand. studies conducted in china suggested that routine antenatal antibody screening of every chinese pregnant woman is not beneficial except those who are d antigen-negative or those having a previous history of haemolytic 8,18 disease of the newborn. guidelines for screening have also been laid down by the drug controller general, india (drugs and cosmetics act., 1989). in 2007, a case was reported in india wherein two women of rh (d) positive phenotype were found to be positive for alloantibodies has promulgated the need for antibody screening in rh (d) positive 32 women as well. unlike these countries, pakistan lacks the availability of proper antenatal antibody screening guidelines. this study can be helpful in formulating these guidelines by suggesting that regular antibody screening of each and every pregnant woman is not necessary and can be a burden on economy so it should remain limited to females with rh negative phenotype. females who have bad obstetric history, are multigravida should also be considered important candidates for antibody screening. there is a need of carrying this study in all regions of pakistan with large sample size so that we could be able to identify population which is at increased risk of developing haemolytic disease of newborn. conclusion rh d antibodies are the only frequent antibodies in majority of pregnant women with rh negative blood group. however, keeping in view the absence of nonrh d antibody in our setup, a guideline can be formulated about introduction of routine antenatal red cell alloantibody screening for just the women having rh-negative phenotype. however in order to lower the risk of haemolytic disease of the newborn, rh – positive women with bad obstetric history and table iii: frequency of red cell alloimmuniza�on jiimc 2017 vol. 12, no.4 188 red cell alloantibodies in pregnancy increased gravida status can also be selected for alloantibody screening test. references 1. hoff brand av. blood transfusion. in: essential haematology. 6th ed. uk: black well publishing limited, oxford. 2011; pp. 397-412. 2. jeremiah za, mordi a. evaluation of the clinical utility of maternal alloantibody screening as a surrogate to antiglobulin crossmatch procedures in resource limited settings. international journal of blood transfusion and immunohematology. 2011; 1: 1-6. 3. cohen dw. hemolytic disease of the newborn: rbc alloantibodies in pregnancy and associated serologic issues. 2012. 4. jeremiah za, mordi a, buser fi, adias tc. frequencies of maternal red blood cell alloantibodies in port harcourt, nigeria. asian j transfus sci. 2011; 5: 39-41. 5. basu s, kaur r, kaur g. hemolytic disease of the fetus and newborn: current trends and perspectives. asian j transfus sci. 2011; 5: 3–7. 6. hassan ma, al gahtani fh, gader am. the frequency of alloantibodies in pregnant and multi-transfused patients – a comparative study between the id-micro typing gel system and the conventional tube method. journal of applied haematology. 2011; 3: 234-42. 7. natukunda b, mugyenyi g, brand a, schonewille h. maternal red blood cell alloimmunisation in south western uganda. transfus med. 2011; 21: 262–6. 8. lee ck, ma es, tang m, lam cc, lin ck, chan lc. prevalence and specificity of clinically significant red cell alloantibodies in chinese women during pregnancya review of cases from 1997 to 2001. transfus med. 2003; 13: 227–31. 9. pahuja s, gupta sk, pujani m, jain m. the prevalence of irregular erythrocyte antibodies among antenatal women in delhi. blood transfus. 2011; 9: 388-93. 10. british committee for standards in haematology guidelines for blood grouping and red cell antibody testing during pregnancy. 2008. 11. semmekrot ba, de man aj, boekkooi pf, van dijk ba. irregular blood group antibodies during pregnancy: screening is mandatory. ned tijdschr geneeskd. 1999; 143: 1449–52. 12. salamat n, bhatti fa, hussain a. anti-rh17 (anti-hr0): a rare diagnostic and management problem. j pak med assoc. 2004; 54: 215-8. 13. qureshi a, salman m, moiz b. rhnull: a rare blood group phenotype. j pak med assoc. 2010; 60: 960-1. 14. anwar m, ali n, khattak mf, raashid y, karamat ka. a case for comprehensive antenatal screening for blood group antibodies. j pak med assoc. 1999; 49: 246-8. 15. shahverdi e, moghaddam m, gorzin f. maternal red blood cell alloantibodies identified in blood samples obtained from iranian pregnant women: the first population study in iran. transfusion. 2017; 57: 97-101. 16. karim f, moiz b, kamran n. risk of maternal alloimmunization in southern pakistan a study in a cohort of 1000 pregnant women. transfus apher sci. 2015; 52: 99102. 17. suria aa, nurudiyana mn, huik may l, alex lcs, noornabillah r, hud ma, et al. red cell antibody screening in pregnancy: a preliminary insight. med & health. 2012; 7: 41-6. 18. wu kh, chu sl, chang jg, shih mc, peng ct. haemolytic disease of the newborn due to maternal irregular antibodies in the chinese population in taiwan. 2003; 13: 311-4. 19. smith hm, shirey rs, thoman sk, jackson jb. prevalence of c l i n i c a l l y s i g n i f i c a n t re d c e l l a l l o a n t i b o d i e s i n pregnantwomen at a large tertiary-care facility. immunohematology. 2013; 29: 127-30. 20. pal m, williams b. prevalence ofmaternal red cell alloimmunisation: a population study from queensland, australia. pathology. 2015; 47: 151-5. 21. michalewska b, wielgos m, zupanska b, bartkowiak r. antico (a) implicated in severe haemolytic disease of the foetus and newborn. transfus med. 2008; 18: 71-3. 22. brumit mc, carnahan ge, stubbs jr, storry jr, reid me. moderate hemolytic disease of the newborn (hdn) due to anti-rh17 produced by a black female with an e variant phenotype. immunohematology. 2002; 18: 40-2. 23. haspl hz, marunic bs, susanj te, tomicic m, stipanovic vk. anti-diego a red blood cell alloantibody as a possible cause of anemia in a 3-week-old infant. arch med res. 2003; 34: 149-51. 24. tasic m, milosevic j, stefanovic m, janosevic rd, krstic m, antic v. kell immunization--a case report. med pregl. 2009; 62: 369-72. 25. singla s, kumar s, roy kk, sharma jb, kachhawa g. severe hydrops in the infant of a rhesus d-positive mother due to anti-c antibodies diagnosed antenatally: a case report. journal of medical case reports. 2010; 4: 57. 26. chu hp, kanagalingam d, chan dk. severe intrauterine hemolysis due to anti-c (w). am j perinatol. 2007; 24: 623-6. 27. kim wd, lee yh. a fatal case of severe hemolytic disease of newborn associated with anti-jk(b). j korean med sci. 2006; 21: 151-4. 28. costamagna l, barbarini m, viarengo gl, pagani a, isernia d, salvaneschi l. a case of hemolytic disease of the newborn due to anti-kpa. immunohematology. 1997; 13: 61-2. 29. jensen kg. haemolytic disease of the newborn caused by anti-kpa. vox sanguinis. 1962; 7: 476-8. 30. filbey d, hanson u, wesstrom g. the prevalence of red cell antibodies in pregnancy correlated to the outcome in the newborn: a 12 year study in central sweden. acta obstet gynaecol scand. 1995; 74: 687–92. 31. anzbt scientific subcommittee. guidelines for blood grouping and antibody screening in the antenatal and perinatal setting 2nd ed., australia: australian & new zealand society of blood transfusion inc, sydney. 2004. 32. thakral b, agrawal sk, dhawan hk, saluja k, dutta s, marwaha n. first report from india of haemolytic disease of the newborn by anti c and anti e in rh (d) positive mothers.haematology. 2007; 12: 377–80. 33. nabeel s, bondagi. rhesus alloimmunization in pregnancy. a tertiary care center experience in the western region of jiimc 2017 vol. 12, no.4 189 red cell alloantibodies in pregnancy saudi arabia. saudi medical journal. 2011; 32: 1039-45. 34. cakana az, ngwenya l. is antenatal antibody screening worthwhile in the zimbabwean population? cent afr j med. 2000; 46: 38-41. 35. heddle nm, klama l, frassetto r, o'hoski p, leaman b. a retrospective study to determine the risk of red cell alloimmunization and transfusion during pregnancy. transfusion. 1993; 33: 217-20. 36. rekik t, amor bi, louati n, rekik h, menif h, gargouri j. irregular antibodies testing during pregnancy in tunisia: study about 5369 women. transfus clin biol. 2012; 19: 6473. 37. gunduz e, akay om, teke hu, gulbas z. incidence of redcell alloimmunization due to non-anti-d antibodies during pregnancy: an experience from turkey. transfus apher sci. 2010; 43: 261-3. 38. paparizos fl, valsami s, bournas n, tsantes a, grapsas p, mantzios g, et al. alloimmunisation during pregnancy in greece: need for nationwide hdfn prevention programme. transfus med. 2013; 23: 254-9. jiimc 2017 vol. 12, no.4 190 red cell alloantibodies in pregnancy page 25 page 26 page 27 page 28 page 29 page 30 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 11 s afr fam pract issn 2078-6190 eissn 2078-6204 © 2016 the author(s) review introduction rheumatoid arthritis (ra) is the archetype of an immunemediated inflammatory disorder. numerous advances have been made in the understanding of the pathogenic mechanisms at a molecular level, which have allowed for novel efficacious therapies. an understanding of these pathogenic mechanisms is imperative, as therapies targeting specific molecular and cellular components of the inflammatory response are being used successfully to treat a diverse group of other immune-mediated inflammatory diseases such as psoriasis, spondylo-arthropathy, inflammatory bowel disease, and connective tissue disorders.1 pathogenesis of rheumatoid arthritis a normal immune response requires the innate and adaptive immune responses working together to protect against foreign organisms. the innate immune system consists of cellular and molecular components such as macrophages, neutrophils and complement that interact with foreign antigens in an immediate and non-specific manner and also present the antigens to the adaptive immune response. the adaptive immune system acts in a more delayed but specific response to get rid of or neutralise effects of foreign antigens, not handled completely by the initial response. the adaptive response consists predominantly of t and b cells that effect a response depending on the inciting antigen, environmental and host factors. this usually results in an increase in anti-body formation and molecular mediators of inflammation called “cytokines”. cytokines enable cross talk between various components of the immuno-inflammatory response with a host of cytokines such as tumour necrosis factor (tnf), interleukin (il) 1, il 6 and il 17 promoting inflammation, counter-balanced by cytokines such as interleukin il 4 and il 102 (fig 1). immune dysregulation in ra results in a host of auto-antibodies such as rheumatoid factor (rf) and anti-citrullinated peptide antibody (acpa). the inciting antigen is unknown, with infective agents such as parvo-virus b19 and an organism causing gingivitis (porphyromonas gingivalis) having been implicated as possible triggers.3 smoking has recently received much interest in the possible initiation of a chemical change in lung tissue – a process called “citrullination”. 4 citrullination involves a chemical change in certain peptides containing the amino acid arginine to citrulline. this citrullinated peptide is now exposed to the immune system and in genetically predisposed individuals results in the generation of acpa. citrullination also occurs in the rheumatoid synovium and interaction with these anti-bodies may not only be the trigger of disease but might also account for disease persistence. genetic factors play an important role in disease susceptibility, with specific amino acid sequence on certain human leukocyte antigens (hla) found to be associated with ra. the hla system is important for immune tolerance and response. for this reason certain specific genetically determined amino acid sequences may be associated with certain diseases.5 in ra this sequence is termed the “shared epitope” and is found in around 80% of patients, particularly in those that are also acpa positive. presence of the shared epitope varies in different ethnic groups, with some studies showing a lower prevalence in african patients; however, a recent south african study found a similar percentage as that seen in caucasians (83%).6 once the immune system is activated disease persistence is maintained by as yet unexplained mechanisms. phenotypic changes in synovial cells, particularly synovial fibroblasts, may play a critical role.7 these cells act in an autonomous almost tumour-like fashion, generating pro-inflammatory cytokines, which result in synovial proliferation and consequent local and systemic effects.8 abstract immune-mediated inflammatory disorders include a clinically diverse group of conditions sharing similar pathogenic mechanisms. conditions such as rheumatoid arthritis, psoriasis, spondyloarthropathy, inflammatory bowel disease and connective tissue diseases are characterised by immune dysregulation and chronic inflammation. this review will focus immuno-pathogenic mechanisms, aspects of early disease, co-morbidity and therapy in rheumatoid arthritis. south african family practice 2016; 58(2):11-17 open access article distributed under the terms of the creative commons license [cc by-nc-nd 4.0] http://creativecommons.org/licenses/by-nc-nd/4.0 rheumatoid arthritis mahmood m.t.m ally1, bridget hodkinson2, 1department of internal medicine faculty of health sciences, university of pretoria, pretoria, south africa 2department of medicine, groote schuur hospital and faculty of health sciences, university of cape town, cape town, south africa corresponding author, email tar@up.ac.za s afr fam pract 2016;58(2):11-1712 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 12 early rheumatoid arthritis immunological and biochemical changes occur even before the clinical onset of disease with differing immuno-biology before disease onset, in early disease, and in established disease. early disease represents a window of opportunity to get the best, most cost effective results. this window seems to be within the first 2-3 years of disease, with most authorities advocating aggressive therapy aimed at disease control within 3-6 months of symptom onset. the initial challenge is early diagnosis. the previous classification criteria (1987 ara criteria) proved to be very insensitive to the diagnosis of early ra. incorporated in these criteria is the presence of rheumatoid nodules and radiographic changes but these features are not seen in most patients with early disease. to this end the new 2010 ra criteria allow for making a diagnosis earlier, including in patients with disease duration of less than 6 weeks9 (table 1). these criteria are more sensitive and care needs to be taken to exclude viral infections, conditions such as osteoarthritis, psoriatic arthropathy and early connective tissue disorders. clinical features suggestive of early ra include morning stiffness lasting more than 30 minutes affecting multiple joints, symmetrical joint involvement particularly of the hands (fig 2) and feet (sparing the distal inter-phalangeal joints), associated soft tissue swelling (‘boggyness’), decreased range of motion (unable to make a fist) and metacarpal/metatarsal tenderness (positive squeeze test). characteristic deformities of ra are usually seen in patients with established, poorly controlled disease due to bone and soft tissue changes. typical deformities in the hand include radial deviation at the wrist, ulnar deviation of the fingers, z deformity of the thumb, finger swan neck and boutonniere deformities (figs 3 and 4). tendon rupture may occur in early or late disease and is related to tenosynovitis or attrition from adjacent bony deformities (fig 5). in patients with disease duration of less than 6 weeks viral aetiologies such as hepatitis b, hepatitis c and human immunodeficiency viral infections need to be excluded. in the presence of distal interphalangeal (dip) joint involvement, osteoarthris (oa) or psoriatic arthropathy should be considered. typical joints involved in oa (fig 6) are the dip, proximal inter-phalangeal (pip), first metacarpophalangeal (mcp) and first carpometacarpal joints of the hands, with associated bony swelling and morning stiffness of less than 10 minutes’ duration. patients with psoriatic arthritis may have inflammatory arthritis similar to ra often with dip joint involvement. systemic enquiry should include a family history of psoriasis and a search for characteristic nail and skin changes, noting that arthritic manifestations may precede skin involvement. early connective tissue disorders (ctds) such as systemic lupus erythematosus may have similar articular features to ra and a systematic review is essential to look for some suggestive manifestations such as malar or discoid rashes, photosensitivity, alopecia, recurrent oral ulceration, raynauds phenomenon, serositis or major organ involvement. extra-articular manifestations are less common in early ra and usually portend a poorer prognosis. rheumatoid nodules occur in about 1% of patients with early disease typically seen on the extensor surface of the forarm just distal to the elbow. ocular involvement includes dry eye syndrome, episclerits and rarely scleritis and keratitis. pulmonary fibrosis may occur from the disease or as a side effect of therapy. neurologic involvement is usually related to entrapment, such as carpal tunnel syndrome and myelopathy from cervical spine subluxation. investigations i) full blood count anaemia of chronic disease is mediated by cytokines such as il 6, inhibiting iron transport and utilization. associated thrombocytosis may reflect an acute-phase response or gastrointestinal bleeding. a low white cell count may be seen in patients with a more severe form of ra, namely felty’s syndrome (ra, splenomegaly, chronic vasculitic leg ulcers and neutropenia). ii) urea and electrolytes direct renal involvement in ra is rare but it is important to monitor renal function at least yearly as drug dosages may need to be adjusted or avoided altogether. iii) liver function tests (lft) evidence of chronic liver disease may be apparent but even if lft is normal viral hepatitis serology needs to be reviewed before starting therapy. drug-related hepatitis may occur therefore 3-4 monthly monitoring of aspartate transaminase/alanine transaminase is recommended. iv) erythrocyte sedimentation rate (esr) and c-reactive protein (crp) these non-specific measures of inflammation are usually, but not always, elevated in patients with active rheumatoid arthritis. up to 40% of patients may have a normal esr or crp at presentation despite active disease.10 the esr may be affected by multiple factors such as age, anaemia and immunoglobulin concentration. the crp may be more specific for inflammation but elevation may be related to smoking and associated insulin resistance.11 v) rheumatoid factor, anti-citrullinated peptide and anti-nuclear factor antibodies (anf) rheumatoid factor is an antibody to igg immunoglobulins seen in 80%-90% of patients with established ra. it is seen in only 50%-60 % of patients with early ra. patients with positive rf factor tend to have more aggressive disease and extra-articular manifestations. the test is used for diagnostic purposes only and has no role to play in monitoring response to therapy; hence if positive once there is no need to repeat. false positives may occur in the elderly and in numerous unrelated infective/ inflammatory conditions such as tuberculosis, hepatitis and interstitial lung disease. acpa has a specificity of around 98% for ra, but for rf it is seen in only around 50% of patients with early disease and used for diagnosis only. acpa positivity is associated with radiographic progression and a poorer prognosis. testing for acpa is appropriate if rf is negative in a patient with suspected ra or if rheumatoid arthritis 13 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 13 a false positive rf is probable, for example, in the elderly and in patients with hepatitis c. antinuclear factor antibodies are directed to nuclear antigens seen in a host of connective tissue diseases (ctd), with up to 30% of patients having a positive anf with no associated underlying ctd. in patients who have clinical features of ra and are seronegative (rf/acpa negative), the presence of anf may be the clue to the presence of an early ctd. an anf is considered positive if the titre is >1/160 and 5% of the normal population may have a positive test. the test should therefore be requested only in the presence of suggestive clinical features and not for non-specific arthralgia. vi) imaging plain radiographs of the hand and feet may reveal the presence of erosive disease and joint space narrowing. however, they are insensitive in early ra, as erosions are seen only after 1-2 years of disease. newer imaging modalities such as ultrasound and mri are able to detect synovitis and erosive disease very early in the course of the disease. internationally, ultrasound is gradually being incorporated into routine rheumatological practice for diagnosis and sonar guided infiltrations, with lack of expertise limiting widespread use locally. mri offers a more objective assessment of synovitis and bony involvement but cost and accessibility limits its use in routine clinical practice. co-morbidity of chronic inflammation uncontrolled chronic inflammation results in the obvious local effects of joint destruction, deformity and consequent disability. however, inflammation can have systemic effects, with ra having found to be associated with premature atherosclerosis, osteoporosis and depression. pro-inflammatory cytokine levels are elevated in the systemic circulation in patients with ra and this has several proatherogenic effects by affecting endothelial cell function, altering lipid metabolism and promoting insulin resistance. all of these effects results in ra being an independent risk factor for ischeamic heart disease comparable to the effect seen in type 2 diabetes.12 synovitis results in erosive bony changes mediated by activated osteoclasts at the junction of bone and the proliferating synovium called the pannus. normal bone turnover is a dynamic process maintaining homeostasis with a balance between bone resorbing (osteoclast) and bone-forming cells (osteoblasts). the pro-inflammatory cytokine milieu, both locally and systemically, enhances osteoclastic activity resulting in peri-articular and generalized osteopenia.13 patients with ra have a much higher prevalence of depression than the general population. depression in ra is associated with disease activity, increased work disability and poor compliance. the effect of chronic disease plays a significant role but abnormal pro-inflammatory cytokine profiles have also been demonstrated in patients with primary depression.14 therapy key principles in the management of ra are disease control within 3-6 months, aiming for remission or at least a low disease-activity state. an important pitfall in the management of patients with early ra is ‘reassurance’ of disease control with pain management. initially many patients’ symptoms improve significantly with pain management, especially if combined with corticosteroids, but a comprehensive review often reveals ongoing disease activity. ongoing disease activity is associated with joint damage, and studies in manual labourers with ra show loss of work ability within 2 years of disease onset. measuring disease activity is thus paramount to guiding therapy so as to bring the disease under control within 3-6 months. measures of disease activity unfortunately no single measure has been shown to correlate well with disease activity and consequent disease progression. the esr and crp are used as measures of activity in many inflammatory disorders but have significant limitations, as individual parameters in patients with ra. composite diseaseactivity measures have been validated to assess and monitor disease activity. these are various different scoring systems that incorporate: responses to patient questionnaires on function and pain; clinical assessment of the number of swollen tender joints; duration of morning stiffness; and esr or crp. a practical scoring system advocated by many for routine clinical use is the simplified disease activity index (sdai). the sdai is a summation of: i) the number of swollen and tender joints counts and the evaluation of bilateral shoulders, elbows, wrists, mcps, pips and knees (28 joint count); ii) physician global assessment of activity noted on a scale of 0-10 with 0 low activity and 10 high; iii) patient global activity score also out of 10; and iv) the crp in mg/dl. a score of >26 reflects high disease activity, >11 ≤26 moderate disease activity, >3 ≤11 low disease activity and ≤ 3.3 remission. ongoing disease activity is associated with radiographic progression, disability, morbidity and premature mortality. the objective is to achieve at least a low diseaseactivity score, with remission being the desired goal. with recent advances in the pharmacological management of ra this target is certainly achievable in the majority of patients. pharmacological therapy recommendations for the management of ra in sa have been published, and include an algorithm with appropriate therapies and appropriate timelines.15 disease-modifying anti-rheumatic drugs (dmards) remain the cornerstone of ra management. there have been tremendous advances in this class of drugs that have not only revolutionised management of ra but also therapy across the spectrum of immune-mediated inflammatory disorders. two sub-classes are now identified – synthetic(s) or biologic (b) dmards. synthetic dmards, also sometimes referred to as “traditional dmards”, include drugs such as choloroquine, salazopyrine, methotrexate and leflunomide. methotrexate is the anchor drug in the management of patients with ra. methotrexate is a folic-acid antagonist-blocking purine and pyrimidine synthesis required for nucleotide formation. inhibs afr fam pract 2016;58(2):11-1716 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 16 iting nucleotide formation results in the prevention of cellular proliferation in predominantly rapidly dividing cells, which include cellular components of the immuno-inflammatory response.16 inhibition of cellular proliferation also occurs in normal compartments such as the gastro-intestinal tract and bone marrow and accounts for some of the side effects seen, such as oral ulcers, diarrhoea and cytopaenias. co-administration of folic acid 5mg daily decreases these side effects with only a minor impact on efficacy. methotrexate exerts these effects only after undergoing an intracellular chemical change of polyglutamation. polyglutamated methotrexate can remain active intracellularly for days despite the drug being cleared from the systemic circulation within a few hours, hence the reason for the recommended weekly dosage. the usual initiating dose is 10mg-15mg weekly, with a maximum dose of 25mg weekly. side effects to monitor for include hepatitis, interstitial lung disease, mucositis, gi disturbances and bone marrow suppression. methotrexate should be avoided in patients with renal dysfunction, liver or pulmonary disease and it is teratogenic. in patients with poor prognostic features, triple therapy combined with chloroquine and salazopyrin may be considered. chloroquine and salazopyrin may be used as monotherapy in mild disease or if methotrexate is contraindicated. in patients on chloroquine, it is important to monitor for retinal toxicity with yearly opthalmological assessments. salazopyrine has a sulphur moiety and should be avoided in patients with a sulphur allergy. leflunomide is a pyrimidine antagonist, similar to methotrexate in blocking cellular proliferation and can be used as an alternative to methotrexate but is commonly used after methotrexate failure. it has a long half life and an enterohepatic circulation resulting in elevated serum levels months after the drug has been stopped. this is an important consideration in females planning a pregnancy as special washout regimens need to be followed. longer disease duration prior to starting sdmard therapy is associated with a poorer response. however, with the appropriate use of dmards in early ra, 70%-80% of patients will have an acceptable response to therapy. patients with an inadequate response will benefit from b dmards, albeit at a higher cost. biologic dmards have changed the landscape of ra therapy with marked improvement in up to 70% of patients that have failed the therapy referred to immediately above. these drugs target specific components of the immuno-inflammatory response. currently available drugs in south africa antagonise pro-inflammatory cytokines (tnf/il 6), t-cell function and b-cell proliferation. b dmards are generally well tolerated with specific precautions and patient screening required to minimise side effects, most importantly infections including reactivation tuberculosis; therefore, use of these agents is restricted to specialist care. despite the fact that pain management does not modify disease progression, it still remains an integral component in ra management. pain management includes prudent use of analgesics and non-steroidal anti-inflammatory drugs (nsaids), using the lowest dose required, combined with gastro-protective agents in patients at high risk of peptic ulcer disease. avoid nsaids in patients with renal dysfunction and use with caution in patients with ischemic heart disease, particularly if patients are on aspirin. the protective effect of aspirin on ischaemia may be lost if combined with some nsaids, as they compete with the same binding sites on platelets. if it is considered necessary to use the combination then separate the dosage times by at least 3 hours. on initiating an nsaid, it is also essential to monitor for changes in blood pressure, as some patients may develop hypertension. corticosteroids have a dual role in ra management, with excellent relief of symptoms especially in early disease and modified disease progression to a limited extent. corticosteroids are often given as a pulse either intramuscularly or orally. if orally then the lowest possible dose orally over a 6-month period, aiming to wean off completely thereafter. corticosteroids used in early ra may mask ongoing disease and joint damage, as they have the potential to delay appropriate therapy because their symptomatic effect is so great that patients markedly improve functionally. certainly there is no role for corticosteroids as mono-therapy for disease modification but they play rather an adjunctive role with other dmards. non-pharmacological therapy a multidisciplinary approach to patients with ra is vital. allied health disciplines such as physiotherapists, occupational therapists, podiatrists and nutritionists provide an important supportive role. educating patients about their disease improves compliance and outcome. specific counselling to deal with psycho-social matters and cessation of smoking should be incorporated in the management plan. challenges to the management of ra in south africa early diagnosis and management of patients with early ra especially in the public sector is of concern. hodkinson et al. in a recent study of 171 patients with early ra attending public sector rheumatology clinics demonstrated a high disease burden at presentation, with only 28% of patients achieving a low disease activity state at 12 months under routine care. over 60% of patients in this study had substantial functional disability and suboptimal mental health after 1 year of therapy.17 as shown in this and other studies, a low level of schooling was identified as one of the poor prognostic features. socio-economic factors and access to health services are obvious challenges affecting optimal management of ra. biologic therapies are expensive and may also predispose to specific infections such as tuberculosis. the direct cost of biological therapies needs to be balanced with the potential for decreasing the social and economic burden passed onto the public health system. guidelines have been established for the appropriate use of dmards including biological therapies in south africa and their use is steadily becoming available in the public sector. conclusion ra is a chronic inflammatory disorder with considerable morbidity. monitoring of co-morbidity such as increased cardiovascular risk needs to be incorporated into management strategies. early diagnosis and aggressive therapy have rheumatoid arthritis 17 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 17 improved the prognosis for most patients. the introduction of dmard therapy early in the disease course with frequent follow up aiming for rapid and measurable disease control is not only inexpensive but also effective in most patients. the judicious use of biologic therapies has added hope to all patients with ra. acknowledgements barbara english from the research office of the university of pretoria’s faculty of health sciences is thanked for language editing. recommended resource south african recommendations for the management of rheumatoid arthritis: an algorithm for the standard of care in 2013.4 references 1. kuek a, hazleman bl, et al. immune-mediated inflammatory diseases (imids) and biologic therapy: a medical revolution. 2007;83:251-260. 2. warrington r, watson w, et al. an introduction to immunology and immunopathology. allergy, asthma & clinical immunology. 2011;7(suppl 1):1-8. 3. mikuls t, payne j, et al. antibody responses to porphyromonas gingivalis (p.gingivalis) in subjects with rheumatoid arthritis and periodontitis. int immunopharmacol. 2009;9(1):38-42. 4. lee dm, phillips r, et al. quantifying anti-cyclic citrullinated peptide titres: clinical utility and association with tobacco exposure in patients with rheumatoid arthritis. ann rheum dis. 2009;68:201-208. 5. kurko j, besenyei t, et al. genetics of rheumatoid arthritis – a comprehensive review. clin rev immunol. 2013;45:170-179. 6. meyer pw, hodkinson b, et al. hla-drb1 shared epitope genotyping using the revised classification and its association with circulating autoantibodies, acute phase reactants, cytokines and clinical indices of disease activity in a cohort of south african rheumatoid arthritis patients. arthritis res ther. 2011;13(5):r160. 7. niedermeier m, pap t, et al. therapeutic opportunities in fibroblasts in inflammatory arthritis. best practice & research clinical rheumatology. 2010;24:527-540. 8. combe b. progression in early rheumatoid arthritis. best practice & research clinical rheumatology. 2009;23:59-69. 9. aletaha d, neogi t, et al. rheumatoid arthritis classification criteria. an american college of rheumatology/european league against rheumatism collaborative initiative. arthritis rheum 2010; 62: 2569-81. 10. keenan rt, swearingen cj, et al. erythrocyte sedimentation rate and c-reactive protein levels are poorly correlated with clinical measures of disease activity in rheumatoid arthritis, systemic lupus erythematosus and osteoarthritis patients. clinical and experimental rheumatology. 2008;26:814-819. 11. chandrashekara s, renuka p, et al. esr or crp, which inflammatory measure can accurately replace clinical measures in rheumatoid arthritis? indian journal of rheumatology. 2012;7(2):69-73. 12. sattar n, mccarey dw, et al. explaining how “high-grade” systemic inflammation accelerates vascular risk in rheumatoid arthritis. circulation. 2003;108:2957-2963 13. kleyer a, schett g. arthritis and bone loss: a hen and egg story. curr opin rheumatol. 2014;26:80-84. 14. mostafa h, radwan a. the relationship between disease activity and depression in egyption patients with rheumatoid arthritis. the egyptian rheumatologist. 2013;35:193-199. 15. hodkinson b, van duuren e, et al. south african recommendations for the management of rheumatoid arthritis: an algorithm for the standard of care in 2013. s afr med j. 2013 jun 14;103(8 pt 2):576-85. 16. chan esl, cronstein b. molecular action of methotrexate in inflammatory diseases. arthritis res. 2002;4:266-273. 17. hodkinson b, musenge e, et al. response to traditional disease-modifying anti-rheumatic drugs in indigent south africans with early rheumatoid arthritis. clin rheumatol. 2012;31(4):613-9. key: cord-0042121-10vhc3dn authors: astrinidou‐vakaloudi, a.; diamanti, i.; xytsas, s.; xatzidimitriou, d.; georgakopoulou, e. title: autoantibodies in patients with rheumatoid arthritis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423a.x sha: 8ec1d9ff81496d6badb553d83b2abdaf51d35e85 doc_id: 42121 cord_uid: 10vhc3dn aim: the aim of this study is to examine the diagnostic value of autoanitbodies in patients suffering from rheumatoid arthritis. we evaluated the presence of the following autoantibodies: rheumatoid factor (rf), antinuclear antibodies (anas), antibodies against cadiolipin (a‐cl) and antibodies against cyclic citrullinated peptide (anti‐ccp). methods: we studied the presence of rf, ana, a‐cl and anti‐ccp in 40 patients with rheumatoid arthritis. rheumatoid factor was measured using nephelometric method, while anas were examined by indirect immunofluorescence technique using hep‐2 cells as substrate. sera that reacted at 1/80 dilution were classified as ana positive. positive sera were studied up to 1/1280 dilution. a‐cl and anti‐ccp were measured by enzyme‐linked immunosorbent assay. results: rf was positive in 30 patients (75%), ana in 15 (37%), a‐cl in 10 (25%) and anti‐ccp in 36 (90%). predominant pattern of nuclear staining of ana‐positive sera was homogenous and speckled type. ana titres were particularly low; most patients (6) had ana titre equal to 1/80, and five patients had a titre of 1/160, while only four out of 40 had an ana titre of 1/320. conclusions: autoimmune disorders such as ra are characterized by various autoantibodies that usually are not specific, as they are present in many other diseases. however, rf and especially anti‐ccp are very often and show higher specificity for ra, being useful diagnostic serological markers. on the other hand, ana and a‐cl are less common in ra paitents; they may be useful in terms of prognosis and treatment, but they always should be evaluated in correlation with the clinical features and the rest of the laboratory findings of each patient. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. panacea journal of medical sciences 2021;11(3):401–405 content available at: https://www.ipinnovative.com/open-access-journals panacea journal of medical sciences journal homepage: http://www.pjms.in/ original research article a clinical study of surgical management of tendoachilles rupture by various modalities banna kiran kumar1, shenkeshi sirish kumar1, bolloju keshava rao1,*, k kishore kumar1, j venkateswarlu1 1dept. of orthopaedics, kakatiya medical college & mgm hospital, warangal, telangana, india a r t i c l e i n f o article history: received 12-01-2021 accepted 16-03-2021 available online 24-11-2021 keywords: achilles tendon paratenon raghu ram technique a b s t r a c t background: acute injuries to achilles tendon with open wounds in tendo achilles region is more common unlike the west, where the chronic ruptures and sport injury are more common. this is because most indians use indian squatting type of toilets which are common cause of open injury to achilles tendon (closet injury). aim & objective: this study aims to evaluate the outcomes of surgical management of tendo achilles rupture. materials and methods: it is a prospective observational study, of 21 patients with achilles tendon ruptures treated in the department of orthopaedics, mahatma gandhi memorial hospital, kakatiya medical college, warangal, telangana, india, between june 2019 to december 2020. results: achilles tendon ruptures are more common in males. in elderly people, tendo achilles ruptures occurred most commonly due to trivial trauma. most ruptures occur 2-6cm proximal to the calcaneal insertion of the tendon. risk factors for tendo achilles rupture are usage of quinolone antibiotics, steroid, and are also seen in patients with diabetes, gout and rheumatoid arthritis. primary surgical management if done well, produces good functional outcome without any functional deficit and chronic pain. raghuram technique is safe and good method for chronic ruptures. post operative evaluation is necessary in assessing final outcome with various scoring system including ultrasound and dynamometric studies. conclusion: 6 months after surgery following adequate immobilization and effective physiotherapy, unrestrained activity can be done. patients can stand on tip toes and complete plantar flexion is achieved. inadequate paratenon closure leads to delayed wound healing and increased rate of infection. this is an open access (oa) journal, and articles are distributed under the terms of the creative commons attribution-noncommercial-sharealike 4.0 license, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. for reprints contact: reprint@ipinnovative.com 1. introduction the achilles tendon formed by tendinous contributions from gastrocnemius and soleus, is the largest and strongest tendon in the body. it resists forces up to 2.6kn, but in spite of its tensile strength, it frequently gets injured. the loss of tendon leads to poor push off. it plays a crucial role in the bipedal human beings. 1–6 * corresponding author. e-mail address: drbkeshav@gmail.com (b. k. rao). achilles tendon rupture are the most common tendon rupture of lower extremity. etiology of tendon injury is multifactorial.75% of all the injuries occur during sporting (racquet sports dominates) given the broad array of critical functions that it helps to provide, injury to achilles tendon can be devastating. the profound disabilities like weak plantar flexion with deficit push off and flat foot gait will alter the energy expenditure. also flat foot leads to foot strain and tarsal arthritis. treatment of achilles tendon rupture was first reported in literature by ambrose pare in 1575. the primary goals of the management of acute https://doi.org/10.18231/j.pjms.2021.080 2249-8176/© 2021 innovative publication, all rights reserved. 401 402 kumar et al. / panacea journal of medical sciences 2021;11(3):401–405 achilles tendon ruptures are to ensure a rapid return to full function and to prevent complications. these are minimized by: following strict asepsis, atraumatic technique, use of less irritant suture material, and with meticulous soft tissue handling. 6–8 1.1. aims and objectives of the study to study the results of surgical management of tendo achilles rupture 1. time required for healing of ruptured achilles tendon. 2. range of plantar flexion of foot. 3. rate of delayed healing or re-rupture. 4. rate of functional deficit and chronic pain. 2. materials and methods in this prospective study, 21 patients, who attended the opd and emergency room of mgm hospital (affiliated to kakatiya medical college) warangal, and diagnosed as tendo achilles rupture were admitted and included in this study done between june 2019 to dec 2020. this study was undertaken to evaluate the cause, the clinical course, surgical management and functional outcomes of injury to tendo achilles. study included 15 males, 6 females with acute, neglected and compound tendo achilles rupture. the rupture was classified as early when rupture is less than 4 weeks and delayed when rupture is more than 4 weeks. typically patient present with pain, struck from behind, popping sound and inability to walk. signs of ruptured tendon include limping, altered gait, palpable gap in the continuity of the tendon, plantar flexion weakness. 2.1. diagnosis according to the aaos clinical practice guidelines, the diagnosis of acute achilles tendon rupture is established by two or more of the following physical examination test: 1. positive thompson’s test. 2. decreased plantar flexion strength. 3. presence of a palpable gap. 4. increased passive ankle dorsiflexion with gentle manipulation. other tests in vogue include matles test, o brien‘s needle test. 2.2. imaging diagnosis of acute achilles tendon rupture is primarily clinical, supported by imaging tests plain x-ray of the ankle with leg (lateral view) is taken to study for calcaneal exostosis, calcification and avulsed tendon and to rule out other ankle problems. 2.3. ultrasonography ultrasonography examination of heel is done to note the quality, integrity of tendon fibres, hematoma, calcification and gap. 2.4. pre-operative instructions the patient is explained about surgery, post-operative protocol and rehabilitation and taken up to surgery. routine blood investigations are performed for preparation of anesthesia. 2.4.1. surgical steps of raghuram et al technique 2.5. surgical procedures surgical technique we have followed depending on type of injuries whether injury is compound or simple, rupture is acute or neglected. at the time of surgery after the preparation, the patient is put in prone position. the incision is posterior central with slight medial deviation at the calcaneal tuberosity. the tendon is studied for the level of rupture, the length of distal stump and the gap in the tendon is noted. tendon ends are freshened and mobilized. in 15 tendon rupture cases, end to end repair with krackow sutures is done. in 4 cases, tendon repair is done with raghuram technique with aponeurosis taken and passed through calcaneum and in remaining two cases repair was done through pull through technique. after repair of tendon the paratenon surrounding the achilles tendon is repaired and the wound is closed in layers. sterile dressing is applied, a short leg cast with foot in 200plantar flexion is applied. 2.6. post-operative management in the immediate post operative period, the patient is administered parenteral broad spectrum antibiotics and analgesics. periodic wound inspection is done through the window in the cast. sutures are removed between 12th 14th post operative day. limb is immobilized in b/k (below knee) cast for a maximum duration of 3 weeks. follow up protocol : patients were instructed at the time of discharge to review at opd every 1 month for initial 3 months, and thereafter every 3 months for a period of 18 months. functional outcomes were evaluated using leppilahti score. 9 3. results total number of patients with rupture studied were 21 3.1. duration of study june 2019 to dec 2020 kumar et al. / panacea journal of medical sciences 2021;11(3):401–405 403 table 1: leppilahti score clinical factor points pain 15 none 10 mild, no limitations in recreational activities 5 moderate, limitations in recreational, but not daily activities 5 0 severe, limitations in recreational and daily activities stiffness 15 none 10 moderate, limitations in recreational, but not daily activities 5 5 severe, limitations in recreational and daily activities 0 calf muscle weakness none 15 mild, no limitations in recreational activities 10 moderate, limitations in recreational, but not daily activities 5 severe, limitations in recreational and daily activities 0 footwear restrictions none 10 mild, most shoes tolerated 5 moderate, unable to tolerate fashionable shoes, modified shoes tolerated 0 0 active range of motion difference between ankles normal (<6 degrees) 15 mild (6 to 10 degrees) 10 moderate (11 to 15 degrees) 5 severe (>15 degrees) 0 subjective results very satisfied 15 satisfied with minor reservations 10 satisfied with major reservations 5 dissatisfied 0 isokinetic muscle strength (score) excellent 15 good 10 fair 5 poor 0 leppilahti score excellent 90-100 good 75-89 fair 60-74 poor <60 table 2: sex incidence sex no. of cases percentage males 16 76.19 females 5 23.81 fig. 1: a: surgical steps in tendo achilles repair; a: surgical incision; b: exposed parts; c: isolated aponeurosis; d: making aponeurosis into cord; e: making hole in calcaneum; f: passing cord through calcaneum; g: suturing to proximal part table 3: age incidence: age no. of cases percentage 21-30 6 28.57 31-40 6 28.57 41-50 4 19.04 51-60 2 9.52 61-70 3 14.28 404 kumar et al. / panacea journal of medical sciences 2021;11(3):401–405 fig. 2: preoperative clinical photo fig. 3: postoperative clinical photograph table 4: aetiology aetiology no. of cases percentage stepping into commode hole 10 47.61 blunt and penetrating injury 4 19.04 trivial trauma 7 33.33 table 5: clinical evaluation clinical features no. of cases percentage skin changes 4 19.04 >20% difference in calf measurement 6 28.57 >3cm palpable gap in tendon 5 23.80 weakness of plantar flexion 18 85.71 table 6: site of rupture: zone no. of cases percentage zone 1 6 28.57 zone 2 14 66.66 zone 3 1 4.76 table 7: complications: complications no. of cases percentage wound dehiscence 1 4.76 infection 3 14.28 scar hypertrophy 2 9.52 table 8: 8: functional outcomes (leppilahati score) functional outcome no. o f cases percentage excellent 14 66.66 good 4 19.04 fair 1 4.76 poor 2 9.52 4. discussion in our series, majority of cases were seen in age group of 2140 years. there are 2 patients in age group between 51-60 years and 3 patients in >61 years age group. number of males were 16 (76.19%) and females were 5 (23.80%). the most common cause that led to tendo achilles rupture in our series was sudden forceful dorsiflexion at ankle due to stepping into the commode followed by trivial trauma in 7 (33.33%) patients. there were 7 cases of rupture due to trivial trauma above 40 years age group without any history of local steroid injection. in our study, there were 13 cases of compound tendo achilles rupture, of which 10 cases were due to stepping into the commode hole and 2 cases were due to road traffic accident and 1 due to sharp instrument injury. in our series, 15 cases presented within 4 weeks of trauma and 6 cases presented after 4 weeks. according to allen et al, 10 it was called early repair when presented before 4 weeks. most patients complained of pain and disturbances in gait which was mainly due to inability to plantar flex during gait cycle. kumar et al. / panacea journal of medical sciences 2021;11(3):401–405 405 thompson test was positive in almost all cases but it loses its efficacy after one week because plantar flexion may result from other plantar flexors or bridging fibrous tissue. there was difference in calf muscle measurement after prolonged period due to relative inactivity of gastrosoleus. weakness of plantar flexion was seen in almost all cases due to absence of gastrosoleus action. 11–14 in our series, rupture are more commonly (66.66%) seen in zone 2 (lindholm). in patients, who presented to the hospital more than 4 weeks after the injury, we have repaired with raghuram technique. among all the complications there was no re-rupture till date. there was increased incidence of infection and delayed wound healing in distal part of the wound. this was due to inadequate paratenon closure and increased plantar flexion postoperatively, which resulted in crumpling of skin which leads to delayed wound healing. 14 we achieved good to excellent results in 86% patients with surgical repair in all types of ruptures. 5. conclusion primary surgical management if done well produces good functional outcome without any functional deficit and chronic pain. raghuram et al technique is safe and good method for chronic ruptures. post operative evaluation is necessary in assessing final outcome including ultrasound and dynamometric studies. 6. acknowledgment the author is thankful to department of orthopaedics for providing all the facilities to carry out this work. 7. conflict of interest the authors declare that there are no conflicts of interest in this paper. 8. source of funding none. references 1. diab m. lexicon of orthopaedic etymology. singapore: harwood academic publishers; 1999. 2. kirkup j, chapter. mythology and history. in: helal b, wilson d, editors. the foot. edinburgh: churchill livingstone; 1999. 3. pare a. workes. (translated by t. johnstone.). london;. p. 1665. 4. the workes of that famous chirurgion ambrose parey. (translated out of latin and compared with the french byt. london: richard cotes;. p. 1649. 5. arner o, lindholm a. subcutaneous rupture of the achilles tendon; a study of 92 cases. acta chir scand suppl. 1959;116(239):1–51. 6. leppilahti j. achilles tendon rupture with special reference to epidemiology and results of surgery. thesis, university oulu, finland. of oulu; 1996. 7. maffulli n. rupture of the achilles tendon. j bone joint surg am. 1999;81(7):1019–36. doi:10.2106/00004623-199907000-00017. 8. hattrup sj, son kaj. a review of ruptures of the achilles tendon. foot ankle. 1985;6(1):34–8. doi:10.1177/107110078500600107. 9. leppilahti j, puranen j, orava s. incidence of achilles tendon rupture. acta orthop scand. 1996;67(3):277–9. doi:10.3109/17453679608994688. 10. allen e, turk jl, murley r. the case books of john hunter frs. london: royal society of medicine services limited; 1993. 11. stoïanovich qj. les ruptures du tendon achille. rev de chirurg. 1929;67:647–78. 12. platt h. observations on some tendon ruptures. br med. 1931;1(3666):611–5. 13. lawrence gh, cave ef, and ho. injury to theachilles’ tendon. am j surg. 1955;89(4):795–802. doi:10.1016/0002-9610(55)90146-x. 14. simmonds fa. the diagnosis of the ruptured achilles tendon. the practitioner. 1957;179(1069):56–8. author biography banna kiran kumar, assistant professor shenkeshi sirish kumar, assistant professor bolloju keshava rao, assistant professor k kishore kumar, associate professor j venkateswarlu, professor and hod cite this article: kumar bk, kumar ss, rao bk, kumar kk, venkateswarlu j. a clinical study of surgical management of tendoachilles rupture by various modalities. panacea j med sci 2021;11(3):401-405. http://dx.doi.org/10.2106/00004623-199907000-00017 http://dx.doi.org/10.1177/107110078500600107 http://dx.doi.org/10.3109/17453679608994688 http://dx.doi.org/10.1016/0002-9610(55)90146-x key: cord-0076398-nwbai6qr authors: zhang, lei; qin, ziguo; sun, han; chen, xiang; dong, jian; shen, siyu; zheng, liming; gu, ning; jiang, qing title: nanoenzyme engineered neutrophil-derived exosomes attenuate joint injury in advanced rheumatoid arthritis via regulating inflammatory environment date: 2022-03-23 journal: bioact mater doi: 10.1016/j.bioactmat.2022.02.017 sha: c9ce6969ee0fe78a1e26d3b3c57db4b661c086bb doc_id: 76398 cord_uid: nwbai6qr rheumatoid arthritis (ra) is a chronic inflammatory disease characterized by synovitis and destruction of cartilage, promoted by sustained inflammation. however, current treatments remain unsatisfactory due to lacking of selective and effective strategies for alleviating inflammatory environments in ra joint. inspired by neutrophil chemotaxis for inflammatory region, we therefore developed neutrophil-derived exosomes functionalized with sub-5 nm ultrasmall prussian blue nanoparticles (upb-exo) via click chemistry, inheriting neutrophil-targeted biological molecules and owning excellent anti-inflammatory properties. upb-exo can selectively accumulate in activated fibroblast-like synoviocytes, subsequently neutralizing pro-inflammatory factors, scavenging reactive oxygen species, and alleviating inflammatory stress. in addition, upb-exo effectively targeted to inflammatory synovitis, penetrated deeply into the cartilage and real-time visualized inflamed joint through mri system, leading to precise diagnosis of ra in vivo with high sensitivity and specificity. particularly, upb-exo induced a cascade of anti-inflammatory events via th17/treg cell balance regulation, thereby significantly ameliorating joint damage. therefore, nanoenzyme functionalized exosomes hold the great potential for enhanced treatment of ra in clinic. rheumatoid arthritis is an autoimmune disease with chronic systemic inflammatory disorder, which is characterized by chronic synovitis that induces damage to articular cartilage [1] [2] [3] [4] . immune cells (t cells, macrophages, and neutrophils) and nonimmune (fibroblasts and chondrocytes) cells play an important role in the progression and prognosis of ra. under the inflammatory microenvironment, immune cells participate in the secretion of many proinflammatory cytokines, chemokines and matrix metalloproteinases (mmps), thereby disturbing the immune balance and leading to cartilage and bone damage [2, [4] [5] [6] . the current therapeutic approaches for ra aim to suppress the inflammation process, for example, by inhibiting the generation of inflammatory cytokines or clearing inflammatory cells from inflamed sites [7, 8] . although these treatments have generated substantial success, there is still more than 30% of ra patients exhibiting insufficient response to the first-line therapy [9] [10] [11] . importantly, joint replacement has become the only choice for patients with advanced ra to restore joint function. therefore, it is essential to develop novel therapeutic strategy with the potential to improve the efficacy for ra treatments. peer review under responsibility of keai communications co., ltd. oxidative stress, a key component in the pathogenesis of chronic inflammation, implicated in the progression of ra [12] [13] [14] [15] . anti-oxidative agents, such as curcumin (cur), lycopene and vitamin e, have the ability to reduce cartilage damage in ra animal models [6] . unfortunately, their clinic applications were limited by low bioavailability, serious side effects, and expensive cost [16] [17] [18] [19] [20] . however, prussian blue nanoparticles (pbnps), as an antioxidative enzyme mimic, can effectively relief oxidative stress by catalyzing oxygen radicals [21, 22] . importantly, prussian blue has been approved by the u.s. food and drug administration (fda) as a commonly used dye and medicine, suggesting its good biocompatibility and broad biomedical application [22, 23] . particularly, pbnps can shorten the relaxation time of protons to enable their application as magnetic resonance imaging (mri) contrast agents. although pbnps has promising antioxidative activities in the treatments of ra, its clinical application has been restricted due to the lack of specificity [25, 26] . to overcome these defects of conventional drug treatment, it is necessary to develop a novel nanomedicine platform for effective ra therapy. currently, intelligent strategies for fostering restorative environment emerged as effective therapeutic interventions for the treatments of ra fig. 1 . schematic illustration of nanoenzyme functionalized neutrophil-derived exosomes (upb-exo) mediated restoring inflammation environment for effective treatment of advanced rheumatoid arthritis. (a) upb-exo were developed by surface engineered neutrophil-derived exosomes (nes-exo) with sub-5 nm ultrasmall pbnps (upb) via click chemistry. (b) upb-exo can selectively accumulate in inflamed joints, and inhibiting the production of pro-inflammatory factors and alleviating inflammatory stress in activated fibroblast-like synoviocytes (fls), macrophages and chondrocytes. in addition, upb-exo effectively targeted inflammation synovitis, penetrated deeply into the cartilage and real-time visualized inflamed joint in cia mice in vivo, leading to diagnosis of ra with high sensitivity and specificity. particularly, upb-exo showed significant therapeutic efficacy in ra by ameliorating joint damage and suppressing overall arthritis severity via inducing a cascade of anti-inflammatory events via th17/treg cell balance regulation. [27] . during the progression of ra, neutrophils accumulate in the inflamed joints of ra at the early-stage of ra responding to inflammatory signals, and release pro-inflammatory cytokines (e.g. tnf-α, il-1β, il-6) resulting in continuous production of increased reactive oxygen species (ros), leading to cartilage destruction and bone erosion [28] . inspired by the critical roles in the initiation and progression of the disease, neutrophil based broad-spectrum anti-inflammatory strategy has emerged as a potent therapeutic strategy for ra. zhang and the co-workers have developed neutrophil membrane-coated nanoparticles, which can neutralize proinflammatory cytokines, therefore inhibiting synovial inflammation and alleviating joint damage in inflammatory arthritis [29] . meanwhile, mauro perretti and his group reported that neutrophil-derived mvs could decrease the production of stress-adaptive homeostatic mediators, thereby, lessening cartilage degradation caused by inflammatory arthritis [30] . since neutrophil based anti-ra strategy have exhibited the therapeutic potential by decreasing proinflammatory cytokines, therefore, novel treatment strategy for attenuating inflammatory environments holds the great potential for the improvement therapy of ra. exosomes, 40-to 150-nm extracellular vesicles secreted by various kinds of cells, process the advantages of both synthetic carriers and cellmediated carriers [31] . previous studies demonstrate that exosomes can avoid the rapid clearance by the immune systems, minimize biotoxicity associated with synthetic vehicles, and even eliminate the unnecessary complexity in the applications of cell-mediated drug delivery systems in clinic [32] . therefore, exosomes have the great potentials as a next-generation drug delivery vehicle. in addition, many issues have reported that exosomes possessed of anti-inflammatory effects, making it an alternative therapeutic agent for autoimmune diseases treatment [33] . meanwhile, antioxidant therapy may offer novel complementary treatment options aiming at better controlling disease progression. for ra therapy, there is an unmet need to combine antioxidant and neutrophil derived exosomes, which can promote the synergistic effects of anti-inflammation for ra treatments. stimulated by our earlier successes in the development of neutrophilmediated drug delivery system [34, 35] , here we attempted to develop a nanoenzyme functionalized neutrophil-derived exosomes to restore inflammatory environment in the inflamed joints of ra. in this study, we prepared a neutrophil-derived exosomes engineered with sub-5 nm ultrasmall pbnps (upb) via click chemistry, shortened for upb-exo, which can effectively achieve anti-inflammatory therapy for ra (fig. 1a) . upb-exo selectively accumulated in activated fibroblast-like synoviocytes, subsequently neutralizing pro-inflammatory factors and alleviating inflammatory environment (fig. 1b ). in addition, upb-exo can target inflamed joints of ra in vivo, and generating mri images with good signal-to-noise. particularly, upb-exo inhibits the production of pro-inflammatory factors and alleviates inflammatory stress, and consequently relieving inflammation synovitis and ameliorating cartilage damage via inducing th17/treg cell balance regulation in advanced ra mice model. compared to current antirheumatic drugs, including anti-inflammatory drugs and biological antibodies [36] [37] [38] , our anti-ra strategy owns prominent targeting ability for inflamed joint, which could improve therapeutic efficacy, shorten administrations times, and decrease unwanted side effects. although nanotherapeutics have been developed for the treatment of ra with relatively reduced doses and side effects [39] [40] [41] , upb-exo processes of almost no potential side effects derived from nanoenzyme (pbnps) [22, 23] or endogenous exosomes [42] . most importantly, currently available ra mitigation drugs showed no obvious therapeutic effects for delaying the development of joint dysfunction in advanced ra [43, 44] , upb-exo could response to the increased inflammatory factors in the inflamed joints from ra mice with advanced arthritis, therefore scavenging reactive oxygen species and alleviating inflammatory stress. in summary, the engineered exosomes, as the inflammatory microenvironment regulator, has the great potential for facilitating ra diagnostic and therapeutic applications in clinic. the human peripheral blood samples, synovium, cartilage, and synovial fluid were obtained from three patients with late-stage ra patients (according to the american college of rheumatology criteria) undergoing joint replacement surgery. the normal synovium, peripheral blood samples, cartilage, and synovial fluid were obtained from three patients with no synovitis and no cartilage injury as confirmed by arthroscopic examination. all samples were collected from gulou drum tower hospital affiliated to the medical school of nanjing university. informed consent was obtained from all patients, and ethical approval was obtained from the ethics committee of gulou drum tower hospital affiliated to the medical school of nanjing university (approval no. 2020-156-01). lipopolysaccharide (lps, 1.5 mg/kg) was injected intraperitoneally into the mice to activate neutrophils in vivo. neutrophils were collected from bone marrow of mice after 6 h post injection, by percoll gradient method described previously [34] . neutrophils (10 7 ) were cultured in a 100-mm dish for 12 h in rpmi 1640 media and 10 ml supernatant were collected. then neutrophils-derived exosomes were isolated from the harvested supernatant by using a supercentrifuge rotor. briefly, the supernatant from neutrophils were centrifuged at 400 g for 10 min, 1250 g for 15 min, and 10,000 g for half an hour at 4 • c to remove cells and debris. after filtered using a 0.22-mm filter (millipore, billerica, ma, usa), the solution was centrifuged at 100,000 g for 70 min at 4 • c using an ultracentrifuge (beckman coulter, brea, ca, usa). the exosome pellet was collected and resuspended in pbs, and ultracentrifuged again at 140,000 g for 70 min. finally, the pelleted neutrophil-derived exosomes were obtained, and the protein quantification were determined by using a micro bca protein assay kit. to detect neutrophil marker (lfa-1, anti-il-1r, anti-tnf-αr) and exosome markers (cd9, cd63 and tsg 101), western blotting was carried out with anti-lfa, anti-il-1r, anti-tnf-αr, anti-cd9, anti-cd63 and anti-tsg 101 antibodies (abcam, cambridge, uk). pvp-modified ultrasmall prussian blue nanoparticles (upb) were fabricated according to the reported procedure [45] . for the preparation of azide modified upb, equal volume of upb (1 mg/ml) and paa solution (1.5 mg/ml) was mixed and stirred for 3 h. after removing excess paa molecules using filters (100 kda mwco, millipore), purified paa@upb was obtained. after stirring a mixture of paa@upb solution, pah solution (3 mg/ml) and edc solution (2 mg/ml) for 4 h at neutral environment (ph 7.4), the carboxyl groups from paa and the amino groups from pah on the surface of pb were chemically crosslinked. then, pah@paa@upb was obtained after purified with filters. finally, azide modified prussian blue nanoparticles n3@upb was obtained after mixing pah@paa@upb solution with azidoacetic acid nhs ester solution (2 mg/ml in pbs) under sonication overnight at room temperature. after purified with filters, transmission electron microscope (jeol, tokyo, japan) was employed to observe the morphology of pbs. the size distribution and zeta potentials of pbs were also measured using a nano zs90 device (malvern). the ultraviolet-visible (uv-vis) absorption spectra of pbs were then obtained by a uv-vis spectrophotometer (uv-3600, shimadzu, japan). in order to prepare upb-exo, dibenzylcyclootyne (dbco) groups were introduced on the surface of exosomes using a hetero bifunctional crosslinker [46] . briefly, dibenzocyclooctynesulfo-n-hydroxysuccinimidyl ester (dbco-sulfo-nhs) (sigma, st.louis, mo, usa) was mixed with exosomes (0.5 mg/ml) in pbs solution on a rotating mixer at room temperature (rt) for 4 h. after removing extra dbco-sulfo-nhs was through filters (millipore), dbco conjugated exosomes (dbco-exo) obtained for linking n3@upb via copper-free click chemistry. therefore, upb-exo were obtained, and the morphology was observed by transmission electron microscope (jeol, tokyo, japan). nta was performed using a nanosight ns300 system (malvern). the size distribution and zeta potentials of exosomes were measured using a nano zs90 device (malvern). ultraviolet-visible (uv-vis) absorption spectra of upbs were obtained by a uv-vis spectrophotometer (uv-3600, shimadzu, japan). the mri assay was performed using a 7.0 t mr scanner (biospec 7t/ 20 usr, bruker, germany). upb-exo (upb concentration: 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1.0 mm) and upb solution with equal upb concentration were used for t1 and t2 measurements. according to the t1 and t2 relaxation times, the values of longitudinal and transverse relativities (r1 and r2) were calculated to evaluate the contrast efficacy of samples. size distribution changes of upb-exo were evaluated after incubating in 50% fatal bovine serum at 37 • c. size and polydispersity index were measured at 1, 2, 4, 6, 8, 12 and 24 h by dynamic light scattering. primary mice chondrocytes, fbroblast-like synoviocytes (fls), or raw 264.7 (atcc) were cultured in dmem medium (both from cell applications) at 37 • c in a 5% co 2 environment. fls, chondrocytes or raw 264.7 cells were seeded in 6-well culture plates. after cultured overnight, the culture medium was changed and lps (2 μg/ml) was added for 6 h-stimulation, cells were washed with pbs and incubated with serum-free medium containing dio-labeled nes-exo or upb-exo (exosome 10 5 per well) for 2 h. after washed and stained by hoechst, the cells were immediately observed using clsm and the intracellular fluorescence intensity was measured using flow cytometry. fls, chondrocytes or raw 264.7 cells were seeded in 6-well culture plates. after cultured overnight, the culture medium was changed, and lps (2 μg/ml) or/and upb-exo (exosome 10 5 per well) was added for 48 h. the supernatant was collected from culture media, and analyzed for the secretion of tnf-α or il-1β using elisa kit. fls, chondrocytes or raw 264.7 cells were seeded and incubated with stimulations (lps 2 μg/ml) for 24 h upb-exo (exosome 10 5 per well) were then added and incubated for 6 h. the intracellular levels of ros in cells were stained by a ros probe dcfh-da. the cells were washed, and immediately observed using fluorescent microscope (olympus), and the intracellular fluorescence intensity was measured using flow cytometry. fls, chondrocytes or raw 264.7 cells were seeded and incubated with stimulations (lps 2 μg/ml) for 24 h upb-exo were then added and incubated for 12 h. the cell viability was measured through cck8 assay. fls, chondrocytes or raw 264.7 cells were seeded and incubated with stimulations (lps 2 μg/ml) for 24 h upb-exo (exosome 10 5 per well) were then added and incubated for 12 h. the live/dead viability assay kit was used to observe apoptotic cell death. fluorescence images were visualized under a confocal fluorescent microscope (olympus). fls, chondrocytes or raw 264.7 cells were seeded and incubated with stimulations (tnf-α 100 ng/ml) for 24 h upb-exo (exosome 10 5 per well) were pretreated for 4 h. the rates of cell apoptosis were determined using flow cytometry (fcm) analysis with an annexin v-fitc/pi apoptosis kit. after treated with tnf-α with or without upb-exo, total rna was harvested from fls, chondrocytes or macrophage. the total rna was extracted by trizol reagent (invitrogen, ca, usa), and paired-end sequencing were performed on an illumina hiseq 4000 at lc-bio technologies (hangzhou) co., ltd by following the vendor's recommended protocol. cdna library preparation and sequencing reactions were performed to unravel the crucial molecular regulator during upb-exo treatments in protecting fls, chondrocytes, or macrophage from cytokines. dba1/j mice were purchased from model animal research center of nanjing university. all mice were kept under pathogen free and 12 h light/dark cycle conditions with enough food and water. the animal use and the experimental protocols were reviewed and approved by the animal care committee of the nanjing university in accordance with the institutional animal care and use committee guidelines. cia mouse model was established by following a previously published protocol [47] . briefly, chicken cii (condrex inc., wa, usa) was emulsified with an equal volume of complete freund's adjuvant (sigma, mo, usa). dba1/j mice were immunized via an intradermal injection at the base of the tail with 50 μl of emulsion (cii 100 μg/mouse) at 0 day and 21 days. mice with 5 days of disease induction were stipulated as cia mice with early-stage arthritis and an advanced arthritis mice model was successfully developed 15 days after disease induction. cia mice were intravenously injected with dir-labeled exosome (dir@ne-exo and dir@upb-exo), fluorescence dye dir was taken as control. mice were anesthetized with isoflurane and images were taken by the ivis spectrum system (caliper, usa) at 1, 2, 4, 8, 12 and 24-h post injection. the mice were euthanized, and paws as well as major organs were harvested and subjected for ex vivo imaging. region-of-interests were circled around the organs, and the fluorescence intensity was analyzed by living image software. to further confirm whether dir@upb-exo was mainly accumulated in the cartilage region, the fresh joints were collected and immediately fixed in 4% pfa solution for 1 day, followed with the decalcification in 0.5 m edta at 4 • c with constant shaking for 5 days. then, the joints were infiltrated with 25% sucrose phosphate buffer for 2 days. all samples were embedded in the mixture of 25% sucrose solution and oct compound (leica) at the ratio of 1 : 1. the joints were cut into 10-μm-thick sagittal sections, and then stained with collagen ii antibody. nuclei were stained with hoechst (beyotime biotechnology), and images were taken by clsm. t1-weighted mr images were collected using a 7.0-t 20-cm-bore mri system (bruker biospin, ettlingen, germany) equipped with a mice brain surface coil (4 ch). t2-weighted mr images were acquired under inhalational anesthesia with isofluorane and at 1, 2 and 4 h after intravenous injection of upb-exo and upb solution (150 μl, 0.5 mg/ml in pbs). t1 color map images were processed with the image sequence analysis tool of bruker paravision 5.1 (bruker biospin, ettlingen, germany). dir-labeled engineered exosomes (dir@ne-exo or dir@upb-exo) or dir dye were incubated with the femoral heads pretreated with 10 ng/ml recombinant mouse il-1β at 37 • c for 24 h. after washed with pbs, femoral heads were embedded in oct compound for frozen sectioning. the joints were cut into 10-μm-thick sagittal sections, and then stained with collagen ii antibody. nuclei were stained with hoechst (beyotime biotechnology), and images were taken by clsm (zeiss, germany). to evaluate therapeutic efficacy with cia mice, 100 μl of engineered exosome upb-exo and nes-exo (exosome 10 7 per mouse) was injected into each tail vein on days 0 (first day of obvious clinical signs of latestage arthritis), 3, 6, 9, 12, 15, 18, and 21 days. sterile pbs and pbnps solution as negative control was injected intravenously to cia mice on the same days. the arthritis index, weight, paw thickness and paw volume of each group was recorded over time. after 25 days of treatment, the mice were sacrificed, and the paw joints were collected for micro-computed tomography (micro-ct) analysis. briefly, the paws of mice from the different groups were fixed for 24 h with 4% paraformaldehyde, then examined and analyzed using a micro-ct imaging system micro-ct scanner (scanco medical, bruettisellen, switzerland). at study endpoints, mice were euthanized and the joints were collected for h&e staining and safranin-o staining. in addition, the joint tissues sections were scored for changes in cell infiltration, bone erosion, and synovial proliferation, all on a scale of 0-4. the score was assessed and the average grades were calculated. to analyses fibroblast-like synoviocyte activation, knee joint sections were stained with a rabbit anti-il-6, rabbit anti-tnf-α or a rabbit anti-il-1β. alexa flour 594 labeled anti-rabbit igg was used as the secondary antibody. sections were counter-stained with to visualize cell nuclei. frozen sections were stained by dapi and imaged with confocal (zeiss, germany). the serum samples from cia mouse were collected on days 0 (first day of obvious clinical signs of late-stage arthritis), 5, 10, 15 and 20 days, and concentrations of il-1β and tnf-α were quantified with elisa according to the manufacturer's instructions. cells were isolated from the blood of arthritic mice after different treatments. the following fluorescence-conjugated mouse antibodies were used for flow cytometric analysis: from biolegend (san diego, ca, usa): mouse fitc-cd4, apc-cd25, pe/cy7-foxp3, pe-ifn-γ. cell subset was analyzed on a facs calibur flow cytometer using cell quest software (becton dickinson). for intracellular staining, including ifn-γ and foxp3, cells were first stained with surface marker cd4, and further fixed and permeabilized for intracellular staining. plot figures were prepared using flowjo software (tree star inc., ashland, or, usa). the safety of the exosomes was evaluated by examining the main organs via histological sectioning and h&e staining. samples from untreated mice were used as controls. spss 13.0 software (statistical package for the social sciences, usa) was used to analyze the obtained data, which are presented as the mean ± sd. comparison between two groups was accomplished via an unpaired two-tailed student's t-test. p < 0.05 indicated a statistically significant difference. rheumatoid arthritis (ra) is an inflammatory autoimmune disease, characterized by a highly coordinated inflammatory response which causes chronic synovitis and bone erosion [3] . at the initiation and progression of ra, neutrophils migrate into the articular cavity and become activated in response to inflammatory factors [30] . activated neutrophils sequentially interact with normal fls, and drive them into an inflammatory phenotype. to investigate the inflammatory microenvironment of ra patients, neutrophils associated inflammatory factors and chemokines in synovium tissue were measured by immunofluorescence analyses. as shown in fig. 2a , synovium tissue from ra patients exhibited higher expression of inflammatory factors (tnf-α and il-1β) and neutrophil chemokines (il-8) compared with normal patients (fig. s1 ). in addition, obvious expression of neutrophil surface maker cd11b was detected in synovium tissue from ra patients, however, synovium tissue from normal patients exhibited negative staining of cd11b by immunofluorescence analysis (fig. s1 ), suggesting that neutrophils play an important role in the development of ra. collected evidence have shown that the dysregulated inflammatory occurred in the synovium results in the destruction of both cartilage and bones of the joint [48] , we therefore identified the distribution of neutrophil in cartilage and synovial fluid in ra patients. as show in fig. 2b , noticeable number of neutrophils was observed in synovial fluid from ra patients compared to normal patients (fig. s2 ). in addition, neutrophils, stained with myeloperoxidase (mpo), a key neutrophil enzyme [49] , can penetrate into ra articular cartilage deeply (figs. 2c and s3). these results indicated that neutrophils can participate in the progression in ra patients with obvious cartilage destruction (fig. s4 ). since neutrophils act as mediators of inflammation, the concentration gradient of inflammation factor was further to explored in both serum and synovium in advanced ra patients by elisa kit. as shown in fig. 2d and e, the expression of inflammatory factors (tnf-α and il-1β) and neutrophil chemokines (il-8) in ra patients were more than 4-fold, compared to normal people samples, suggesting that neutrophils can migrate to the inflammation joint responding to chemotactic factor generation conditions. therefore, neutrophils associated dysregulated inflammatory occurred in the synovium, articular cavity, and cartilage of joints from ra patients. many issues have shown that the neutrophil activation could release il-1β, and stimulate macrophages and synoviocytes to express proinflammatory factors, which in turn amplify neutrophil recruitment. subsequently, neutrophil activation would produce extra ros and release mmps, which causes cartilage destruction and bone erosion [28] . therefore, the accumulation of extra neutrophil carriers in inflammation joints of ra has high risk for promoting the development and progression of ra. it has been reported that exosomes are nano-sized (30-100 nm) mammalian extracellular particles with essential properties in terms of cell communications [31] . exosomes are cell-created vesicles that inherit identical phospholipid membrane [50] . neutrophil membrane expressed the key surface antigens (including lfa-1, il-1r and tnf-αr). since the lfa-1 on the neutrophil membrane could bind with the intercellular activation molecule 1 (icam-1) overexpressed on activated fls [51] , chondrocytes [52] and macrophage [53] , the targeting capability of nes-exo is probably attributed to the expression of lfa-1 derived from neutrophils [54, 55] . therefore, we conjectured that neutrophil transfer key surface antigens to their exosomes, thereby, neutrophils-derived exosomes (nes-exo) can response to the appeals of inflammation. to explore the targeting ability of nes-exo in ra, a cia mouse model, a well-characterized model for assessing the stages of pathology in ra and exploring arthritis mechanisms, is established. we therefore investigated joint destruction and synovial inflammation in cia mice by using micro-computed tomography (micro-ct), immunofluorescence and elisa assay. after scanned the ankle joints of mice by using micro-ct, the smooth bone surface in ankle joints were observed in cia mice with early-stage arthritis compared to normal ankle joints. in the contrast, cia mice with advanced arthritis displayed apparent rough bone surfaces and significantly decreased bone mineral density (bmd) and bone surface density (bs/bv) (fig. 2f , g and 2h) compared to normal ankle joints. moreover, various chemoattractants, including tnf-α, il-1β and il-8, have been identified that promote neutrophil migration into inflammation ankle joint. compared to early-stage arthritis, significant increased expression of chemoattractants were observed in cia mice with late-stage arthritis ( fig. 2i and j) . these results indicated that nes-exo has the great potential to be a potent target carrier for advanced ra. to assess the recruitment ability of nes-exo (figs. s5 and s6) to advanced ra, in vivo imaging experiment were carried out in both early-stage arthritis and late-stage arthritis. as shown in fig. 2k , we found that apparent red signal of dir labeled nes-exo were observed in inflamed joints of cia mice after 1 h-i.v. injection. importantly, nes-exo exhibited notable accumulation in ankle joints region from advanced ra, generating a 2.4-fold higher in fluorescence intensity compared to early-stage arthritis (fig. s7 ). in addition, we found that more icam-1(green signal) were observed in late-stage arthritis compared to early stage (fig. 2l ). in addition, obviously dir labeled nes-exo (red signal) accumulated in icam-1(green signal) labeled inflamed ankle joints in late-stage arthritis, and the merged yellow signal were stronger than that in inflamed ankle joints in earlystage arthritis. these results indicate that nes-exo holds great potential as a target carrier for ra treatments. considering that nes-exo showed no apparently inflammation-promoting ability (fig. s8) , nes-exo therefore holds the great promising in the diagnosis and treatment of advanced ra. current ra mitigation drug show no obvious effects for delaying the development of joint dysfunction in advanced ra, hence, it is essential to develop novel therapeutic strategy with the potential to improve the efficacy for ra treatments. increasing evidence have reported that oxidative stress, which can lead to chronic inflammation, plays a key role in the pathogenesis of ra [12] . antioxidants therapies for ra were limited by low bioavailability, serious side effects, and expensive cost. since exosomes, possessing of good biocompatibility, have been widely applied as a drug delivery system. inspired by the excellent targeting ability of nes-exo for advanced ra and potential anti-inflammatory activity based neutrophil derived vesicles [30] , we therefore functionalized nes-exo with sub-5 nm ultrasmall pbnps (upb-exo) via "click chemistry", endowed it with mimic enzyme activities for ros scavenging and visualization capability for magnetic resonance imaging. as show in fig. 3a , upb modified nes-exo (upb-exo) were obtained by linking the dbco groups on the exosomes with azide-functionalized upb (fig. s9 , s10 and s11) to form stable triazole linkages using copper-free click chemistry. nes-exo with average diameter of 116 nm and zeta potential of − 25 mv displayed round and well dispersed appearance measured by tem images. after engineered with upb, the mean diameter of exosomes increased to 126 nm, and the zeta potential remained nearly unchanged for nes-exo ( fig. 3b and c) . in addition, the uv-vis spectra showed obvious upb peak at 694 nm, demonstrating that upb were successfully coating at nes-exo (figs. 3d and s12). meanwhile, the size distributions of upb-exo were evaluated using nta, which showed that the highest peak of both profiles were around 124 nm (fig. 3e) , which is consistent with dls measurement. more importantly, we implemented hsfcm to determine the grafting ratio of upb on nes-exo. cy3-n 3 were modified into the surface of nes-exo according to the same method of upb-exo, therefore cy3-exo were obtained. the particle concentration of cy3-exo (with an cy3 concentration of 0.1 mg/ml) was measured to be 1.98 × 10 10 particles/ml. based on the concentration and molecular weight of the cy3, it was calculated that the nes-exo contained an average of ~378 cy3-n3 per particle. these results indicated that nes-exo contained an average of ~378 upb per nes-exo. furthermore, the serum stability assay was conducted to investigate interactions between upb-exo and blood components. the average particle sizes remained nearly unchanged for nes-exo after 24 h of storage in 50% fetal bovine serum (fbs) at 37 • c (fig. 3f) , suggesting its good biostability. importantly, wb experiments demonstrated the presence and enrichment of key surface antigens, including tnf-α receptor (tnf-αr), il-1 receptor (il-1r) and lymphocyte function-associated antigen-1 (lfa-1) on both upb-exo and nes-exo (fig. 3g) , confirming that the modification of upb cannot affect the biofunction of nes-exo. in our previously work, upb exhibited good performance in podand cat-like catalytic activity [56] , we therefore assessed the antioxidative ability of upb-exo to relief ros. fig. 3h displays that upb-exo immunofluorescence analyses of the icam-1 (green) and the fluorescence images of dir labeled nes-exo (red) in the joint tissues from cia mice with early-stage arthritis and late-stage arthritis. scale bars indicate 20 μm. data represent the mean ± sd (n = 3 independent samples). statistical significance was determined by a two-sided student's t-test. ***p < 0.001. exhibits almost overlapped pod(fig. s13 ) and cat-like catalytic activity ( fig. s14) with upb, implying that upb-exos has superior pod-like catalytic activity similar to upb. as a multifunctional nanozyme, upb can also act as cat to catalyze the decomposition of h 2 o 2 into o 2 and h 2 o under neutral conditions. as shown in fig. 3h , s13 and s14, upb as well as upb-exo exhibited excellent catalytic activity even when its concentration (0.3 μg/ml). these results indicated that upb-exo exhibited an intensive ability of decomposing hydrogen peroxide to generate oxygen, scavenging free radicals, and protecting cells from being oxidized. therefore, upb-exo could effectively scavenge free radicals and owned great application potential in the antioxidative treatment for ra. prussian blue nanoparticles can influence the longitudinal and transverse relaxation times of protons from water as t1 contrast agent [56] . in this study, a 7.0 t mri system was used to measure a series of proton t1 relaxation times. the t1-weighted mri slices of phantoms consisting of upb and upb-exos with various upb concentrations are displayed in fig. 3i , the longitudinal and transverse relaxation rates (r1 and r2) could be measured with the linear slopes of 1/t1 and 1/t2 versus the upb concentration, respectively (fig. s15 ). in addition, the r2/r1 ratio of upb was calculated, which was similar as upb-exo. these results demonstrated that upb-exo could act as an effective t1 contrast agent for the diagnosis of ra in clinic. the activation of fibroblast-like synoviocytes (fls) in the inflamed synovium contribute to cartilage destruction through their production of inflammatory cytokines and chemokines [1, 2] . we therefore take activated fls as ra cell models to investigate the distribution behaviors of various prepared exosomes in inflamed joint in vitro. the fluorescent probe dio, was used to labeled upb-exo, and intracellular uptake were measured through confocal laser scanning microscope (clsm) and flow cytometry. as shown in figs. 4a and s16, upb-exo and nes-exo can selectively accumulate in cytokine activated fls compared to normal cells, suggesting that upb-exo could selectively target cytokine activated fls. in addition, flow cytometry measurements confirmed that significant increase in mean fluorescence intensity (mfi) in activated fls after incubated with upb-exo, which is 3.1-fold compared to normal fls (fig. s17) . the selectivity of upb-exo in activated chondrocyte and raw264.7 cells were also observed in fig. 4a and b. these results demonstrated the good targeting ability of upb-exo for inflamed ra-associated cells. the specific binding was probably attributed to specific interactions between lfa-1 on the surface membrane of upb-exo and the intercellular activation molecule 1 (icam-1) overexpressed on activated fls. the activated fls in ra joints play important roles in synovial inflammation and joint destruction [39] . to investigate whether upb-exo could effectively decrease the inflammation in fls, their expression profile of inflammation factors (tnf-α and il-1β) was determined by elisa kit. as shown in figs. 4b and s18, both nes-exo and upb demonstrated apparent anti-inflammation activities in activated fls compared to control group. particularly, upb-exo treatment exhibited the lowest expression of both tnf-α and il-1β in activated fls, compared to control groups. the anti-inflammatory ability of upb-exo were observed in activated chondrocyte and raw264.7 cells (fig. 4b) . these result indicating that upb-exo holds the great potential for ameliorating joint inflammation. ros regulate the migration and invasion of fls, and play a prominent role in the progression of ra. upb own outstanding pod-like and cat-like activities, thereby endowing upb-exo with the great antioxidant activities for relieving oxidative stress [36] . we next examined the ros level of activated cells pre-treated with upb-exo via flow cytometry assay (fig. s19 ) and fluorescence microscopic imaging (fig. 4c) , staining with a ros probe (dcfh-da). quantitative flow cytometry experiments showed that upb-exo demonstrated much better antioxidative effect than upb under the same concentration (fig. s19 ). in addition, the greatest decrease in green signals were observed in upb-exo treatments in activated fls, chondrocyte and raw264.7 cells, indicating that upb-exo has good ros elimination ability (fig. 4c) . moreover, the expression of antioxidative factors expression level was also assessed by immunofluorescence staining. superoxide dismutase 2 (sod-2) and nadph oxidase 2 (nox-2) protein are well known biomarkers of oxidative stress [57, 58] . sod-2 is localized within mitochondria and efficiently eliminates the superoxide generated from molecular oxygen in the respiratory chain [59] . many issues have reported that lps caused a time-and dose-dependent manner in up-regulating sod-2 protein expression, and the increased sod-2 conferred oxidative stress tolerance on cells [60] [61] [62] . in addition, lps treatment also could induce the production of ros [63, 64] . considering the increased nox-2 is an important indicator of ros generation [58] , these oxidative stress-related biomarkers (sod-2 and nox-2) were measured to further determine whether upb-exo could reduce oxidative stress in lps-treated fls. in previous studies, we found that lps upregulates sod-2 expression within 24 h in fls, and the sod-2 levels reached maximum values after 12 h-treatment (fig. s20) . these results indicated that elevated sod-2 protein levels could protect fls form subsequent challenges of oxidative stress induced by ros. fls were pretreated with upb-exo for 4 h, then cells were treated with 2 μg/ml lps for 12 h to induce profound elevation of sod-2 and nox-2 expression. as shown in fig. 4d , lps treatment induced apparently sod-2 expression in fls after lps treatment. however, pretreatment with upb-exo significantly reversed the improvement of sod-2 by lps treatment, which is 2.1-fold lower compared to ne-exo groups (figs. s21-s22) . these results suggested that upb-exo can act as sod-2 mimics with excellent protecting effects against oxidative stress. in addition, increasing expression of nox-2 protein was also observed in fls, chondrocyte and raw264.7 cells with only lps treatment. after pretreated with upb-exo, the protein expression of the nox-2 was notably decreased compared with upb or ne-exo groups, which are in good agreement with the ros assay data. these results indicated that upb-exo can be used as an effective ros scavenger to regulate oxidative microenvironment in inflamed joints. it has been generally accepted that cytokine induced cell apoptosis displays a key role in pathogenesis of ra [48] . pro-inflammatory cytokine tnf-α have been shown to induce the apoptosis of normal fls cells in vitro and in vivo [65] [66] [67] [68] , resulted in cartilage matrix degradation and joint inflammation, thereby contributing to ra progression. thus, our study used tnf-α to mimic an in vitro model of ra, and the cell apoptosis were measured via annexin-v/pi flow cytometry. as shown in figs. 4e and s23, after treated with upb-exo, the percentages of fls cells in both early and late apoptosis states were significantly reduced compared to tnf-α stimulated groups. since high level of proinflammatory cytokines activate apoptosis signaling and initiate cell death, we therefore evaluated the rescue of upb-exo for tnf-α-promoted cell death. upb-exo could significantly rescue the cell viability of fls induced by tnf-α (fig. s24) . in addition, a live/dead kit assay was conducted to evaluate joint protection ability of upb-exo in activated fls (fig. 4f ). our study further confirmed pre-treatment with upb-exo could remarkably reduce the cell apoptosis in stimulated fls induce by tnf-α. importantly, to determine whether upb-exo can modulate the expression of apoptotic marker, we examined tnf-α-stimulated fls with or without pre-treatment of upb-exo by western blot (fig. 4g) . the expression of cleaved caspase-3 was increased in stimulated fls compared to normal cells. however, upb-exo remarkably inhibited tnf-α-induced cleaved caspase-3 overexpression, indicating that upb-exo might have an anti-apoptotic effect on tnf-α-stimulated fls via suppressing cleaved caspase-3 expression. similar anti-apoptotic results were also observed in chondrocyte and raw264.7 cells (fig. 4e-g) . these results suggest that upb-exo has a joint protective effect on tnf-α-induced cell models via attenuating the oxidative stress in ra joints. since upb-exo has excellent protective effects against cytokine induced cell apoptosis, we therefore performed an rna-seq assay in combination with expression profiling using tnf-α stimulated fls, chondrocyte or raw264.7 cells pretreated with or without upb-exo to unravel the underlying mechanisms for regulating the inflammatory microenvironment. genome-wide analysis indicated that numerous modulatory genes were associated with fls cellular fate after upb-exo treatment. we found that the inflammation related genes, including colony stimulating factor 3 [69, 70] , matrix metallopeptidase 9 [71] , interleukin 1 alpha [72] , interleukin 1 beta [72] , interleukin 7 receptor [73] and von willebrand factor [74] , were markedly downregulated in upb-exo groups (fig. s25) . of note, their down-regulation indicated anti-inflammatory effects of upb-exo. collected evidences have shown that the downregulated genes, including colony stimulating factor 3 [75] , matrix metallopeptidase 9 [76] , interleukin 1 alpha [77] , interleukin 1 beta [78] , interleukin 7 receptor [79] and von willebrand factor [80] could be regulated by pi3k/akt pathway. meanwhile, kegg pathway enrichment analysis demonstrated that numerous down-regulated genes were significantly enriched pi3k-akt pathway in fls with upb-exo treatment (fig. 4h) . we therefore valuated the expression of pi3k/akt and related proteins in fls by western blot analyses after rna-seq screening. as shown in fig. 4i , tnf-α could induce the overexpression of p-akt and akt in fls compared with control group. after treated with upb-exo, inhibited p-akt and akt expression were apparently observed. in consideration of the same result observed in apoptosis analysis (fig. 4e) , these results demonstrated that upb-exo may inhibit the apoptosis of fls via regulating pi3k/akt signaling pathway. in addition, kegg pathway analysis showed that nf-kb pathway participated in raw264.7 cells after treated with upb-exo (fig. s26a ). as shown in fig. s26b , the expression of p-p65 were activated after tnfα treatment in raw264.7 cells, implying the detrimental effect of nf-κb activation on raw264.7 cells induced by tnf-α. however, the administration of upb-exo led to significant alleviation in terms of p-p65, indicating that the nf-κb pathway was inhibited by upb-exo to protect chondrocytes from tnf-α inflammation. furthermore, mtor pathway plays an important role in promoting the proliferation and survival of chondrocyte. to further clear the effect of upb-exo on the mtor pathway in chondrocyte, the phosphorylation level of mtor were determined. although the mtor phosphorylated activation was not obviously increased after treated with tnf-α, upb-exo indeed decreased the phosphorylation of mtor (fig. s26c) . hence, upb-exo protected joint from damage in ra through the effective inhibition of cytokinesinduced cell apoptosis, which making it a decisive option in the treatment against ra onset and development. neutrophils-derived exosomes (nes-exo) are nano-sized (50-150 nm) mammalian extracellular particles with essential properties similar to neutrophils. considering the inflammation targeting ability of nes-exo, the biodistribution of engineered exosomes in cia mice model were further evaluated. in this work, cia mice were i.v. injected with dir@nes-exo, dir@upb-exo or dir dye, followed by imaging with ivis spectrum imaging system. fig. 5a shows that both dir@nes-exo and dir@upb-exo exhibited the more accumulation into inflammation joints at every time point compared to dir group (fig. s27) , and reaching the maximum fluorescence accumulation with 4 h post injection in the region of inflamed paws (fig. s28) , indicating that the engineered exos have good ra targeting ability as well as nes-exo. in addition, dir@upb-exo group displayed significantly fluorescence within 24 h post injection, and generating a 11.5-fold higher fluorescent signal compare to dir group (fig. s28) , suggesting that upb-exo can improve inflammation joints accumulation with a long-period retention. furthermore, ex vivo imaging shows that obvious fluorescence was detected in inflammation paws after treated with dir@upb-exo, and lower distribution of dir signal in liver were observed in dir@upb-exo group compared to dir group (figs. 5b and s29), confirming that upb-exo has high selectivity toward inflammatory ankle joints. many studies have reported the good penetration ability of neutrophils in inflammation cartilage [33] , we therefore investigated the distribution of upb-exo in cartilage penetration. mouse femoral head explants were collected and cultured in medium containing il-1β to mimic inflammatory arthritis. dir labeled exosomes were administrated and cultured for 8 h of incubation. as shown in fig. s30 , notable accumulation of both dir@upb-exo and dir@nes-exo (red signal) were observed in coll ii labeled cartilage region (green signal) compared to dir group. despite the surface of cartilage, dir@upb-exo as well as dir@nes-exo penetrated cartilage matrix deeply. the enhanced cartilage penetration of dir@upb-exo is probably attributable to the adhesion interactions between nes-exo and chondrocytes. to further confirm whether dir@upb-exo could accumulate in the cartilage region deeply, the fresh joints were collected after injected with dir@upb-exo within 24 h. as shown in fig. 5c , the confocal images showed that more dir@upb-exo (red signal) was accumulated in the coll ii labeled cartilage region (green signal), exhibiting more yellow signal in ra cartilage region, as compared with dir group, which suggested that dir@upb-exo could penetrated cartilage matrix deeply in vivo. these results demonstrated the potential therapeutic effects of upb-exo for cartilage destruction. since upb-exo was a contrast agent for mri, we also analyzed mr images of the cia mice treated with upb-exo or upb (fig. 5d ). in the control group, slight signals of upbs were observed in the inflamed paws within 4 h after pbs injection. in contrast, the mice treated with upb-exo showed apparent amount of upb accumulated in the inflamed paw, and generating 8.6-fold higher in t1 signals compared to upb groups (fig. 5e) , which is consistent with the in vivo imaging experiments. these results confirmed that the modification of upb on nes-exo can promote delivery of upb to the inflamed paws and enhanced its retention, indicating that upb-exo has the great potential for effective treatment of ra. it has been reported that biodistribution determined the therapeutic efficacy of nanoscale therapeutics. inspired by the excellent inflammation ankle targeting capability and good cartilage permeability, we therefore investigated the therapeutic efficacy of upb-exo in cia mice model. saline or various exosomes were intravenously injected into cia mice (fig. 5f ). the cia mice in advanced-stages developed severe swelling in the ankles and paws after 15 days of arthritis induction. the extent of paw swelling was used as a direct indicator for the treatment of ra. thus, paw swelling was observed over time as an index of arthritis. as shown in fig. 5g , free upb and anti-tnf α group showed relatively low efficacy in decreasing the paw thickness and ankle diameters of cia mice with advanced arthritis. in addition, increased swelling was observed in free upb and anti-tnf-α group, compared to normal mice. in contrast, upb-exo and nes-exo showed higher efficacy in reducing swelling in ankle joints and paws ( fig. 5h and i) . significantly, upb-exo group showed an ankle diameter and paw thickness closer to that of the normal group at the study endpoint (figs. s31 and s32). these results indicated that upb-exo has excellent therapeutic effects for ra, holding great potential for the treatment of ra in clinic. to further confirm that upb-exo could relief joint inflammation and reduce cartilage destructions, the ankle joints of mice were sectioned for histological analysis at the study endpoint. as show in fig. 5j , he stained sections from the saline group displayed severe synovial hyperplasia, along with bone and cartilage destruction. the free upb and anti-tnf-α group exhibited a limited effect in reducing these symptoms, while upb-exo can not only reduce synovial inflammation but also decrease the loss of cartilage to some extent compared with the saline group (fig. 5j ). in addition, safranin-o staining indicated that the gag levels in upb-exo were higher than those control group, suggesting that the cartilage damage was efficiently reversed and prevented progressive. importantly, the upb-exo group had larger positive areas for safranin-o staining, which were closer to those of the normal group, suggesting effective therapeutic effects for ra. these results demonstrated that upb-exo can alleviate synovial inflammation and reduce cartilage destructions in cia mice with advanced arthritis, holding the great potential in the treatment of ra in clinic. collected evidence have shown that the therapeutic effects on rheumatoid arthritis have been positively associated with a decrease expression of proinflammatory cytokines. therefore, the changes of proinflammatory cytokine expression were evaluated after the treatments of upb-exo. as shown in fig. 5k , the expression of proinflammatory cytokines, including tnf-α, il-1β and il-6, were significantly elevated in the saline group compared with normal mice group, indicating the important involvement of these cytokines in the pathogenesis of ra. in addition, no significant differences in the expression of these cytokines were observed in both anti-tnf-α group and upb group compared with saline group. whereas, the expression levels of the proinflammatory cytokines, including tnf-α, il-1β and il-6, were decreased in the upb-exo group. in addition, the levels of proinflammatory cytokines in serum, including tnf-α and il-1β, were also monitored during the treatments of upb-exo. as shown in fig. 5l and m, upb-exo exhibited obviously anti-inflammatory ability compared to other treatments. these results demonstrated that the administration of upb-exo to cia mice with advanced ra could remarkably reduce the production of proinflammatory cytokines, presumably contributing to the therapeutic effect against ra. the relative fluorescent signal ratios between ra inflammation ankle joint regions and normal tissues were measured at different time points after injected with dir dye, dir@nes-exo or dir@upb-exo. data expressed as mean ± sd, n = 3. ***p < 0.001, compared with controls (two-tailed student's t-test). (c) representative confocal images of the knee joints of mice after systemic administrated with dir-labeled exosomes (red signal) or dir (red signal) for 24 h. the cartilage region was stained with collagen ii antibody (green signal), and nuclei were stained with hoechst (blue signal). the scale bars indicated 100 μm. (d). in vivo t1-weighted mr images of inflamed paws of cia mice injected intravenously with upb-exo or upb. (e) t2-weighted mr signal as a function of time for upb-exo or pb. data expressed as mean ± sd, n = 3. ***p < 0.001, compared with controls (two-tailed student's t-test). (f) the study protocol of a prophylactic regimen with a cia mouse model. (g) arthritis index in different groups over 25 days of treatment; n = 6, p-value, ***p < 0.001. (h) representative micro-ct images of ankle joints from mice with different treatments. (i) quantitative micro-ct analyses of bone mineral density (bmd). n = 6, p-value, ***p < 0.001. (j) representative images of h&e staining and safranin-o staining on ankle joints from mice with different treatments. images were acquired using a bright-light microscope (10 × magnification). (k) immunofluorescence analyses of the tnf-α, il-1β and il-6 in the joint tissues from cia mice treated with different groups. (l)(m) concentration profiles of tnf-α (l) and il-1β (m) in the serum of cia mice treated with different groups. (n)(o) the expression of ifn-γ (n), and cd25+foxp3+ (o) on cd4 + t cells were measured in serum by flow cytometry. data expressed as mean ± sd, n = 6. p-value, ***p < 0.001, compared with controls (two-tailed student's t-test). considering that th17/treg balances play a pivotal role in ra pathology [81, 82] , we next investigated the impact of upb-exo on these effector cells. to determine whether the th17 and treg cell populations were altered in upb-exo-treated cia mice, the ratios of cd4 + il-17 + cells (th17 cells) and cd4 + foxp3 + cells (tregs) in the cd4 + t cells were assessed by flow cytometry (figs. s33 and s34 ). compared to health mice, upb-exo-treated cia mice exhibited up-regulated cd4 + il-17+ cells and down-regulated cd4 + foxp3+ cells, suggesting the increased of th17 cell population and decreased treg cell population ( fig. 5n and o) , indicating that th17 cell/treg imbalances associated with arthritis were rescued by upb-exo treatment. importantly, upb-exo showed good biocompatibility, and no noticeable changes in the body weight and systemic toxicity were observed (figs. s35 and s36) . these results indicated that upb-exo induced a cascade of anti-inflammatory events via th17/treg cell balance regulation, thereby providing a promising strategy for ra in clinic. in summary, we have developed a neutrophil-derived exosomes engineered with sub-5 nm ultrasmall pbnps (upb) via click chemistry, termed as upb-exo, which could selectively neutralize pro-inflammatory factors and alleviate oxidative stress in activated fibroblast-like synoviocytes (fls), macrophages and chondrocytes. upb-exo effectively targeted to inflammation synovitis, and penetrated into cartilage deeply. in addition, upb-exo could significantly ameliorate joint damage and suppress overall arthritis severity in cia mice via th17/treg cell balance regulation in advanced ra mouse model. therefore, the engineered exosomes displayed considerable potential for the clinical diagnosis and treatment of ra. diagnosis and management of rheumatoid arthritis: a review evolving concepts of rheumatoid arthritis the pathogenesis of rheumatoid arthritis rheumatoid arthritis and depression: an inflammatory perspective anti-inflammatory and immuneregulatory cytokines in rheumatoid arthritis t cell-dependent affinity maturation and innate immune pathways differentially drive autoreactive b cell responses in rheumatoid arthritis medicinal plants used against various inflammatory biomarkers for the management of rheumatoid arthritis mif: a new cytokine link between rheumatoid arthritis and atherosclerosis methotrexate mechanism in treatment of rheumatoid arthritis optimising low-dose methotrexate for rheumatoid arthritis-a review the management of first-line biologic therapy failures in rheumatoid arthritis: current practice and future perspectives role of oxidative stress in rheumatoid arthritis: insights from the nrf2-knockout mice status of oxidative stress in rheumatoid arthritis myeloperoxidase and oxidative stress in rheumatoid arthritis rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases synergistic oxygen generation and reactive oxygen species scavenging by manganese ferrite/ceria co-decorated nanoparticles for rheumatoid arthritis treatment curcumin, inflammation, and chronic diseases: how are they linked? encapsulation and characterization of nanoemulsions based on an anti-oxidative polymeric amphiphile for topical apigenin delivery dijkmans, efficacy, tolerability and cost effectiveness of disease-modifying antirheumatic drugs and biologic agents in rheumatoid arthritis targeting cell signaling pathways for drug discovery: an old lock needs a new key mimicking peroxidase activities with prussian blue nanoparticles and their cyanometalate structural analogues prussian blue nanoparticles as multienzyme mimetics and reactive oxygen species scavengers prussian blue nanozymemediated nanoscavenger ameliorates acute pancreatitis via inhibiting tlrs/nf-κb signaling pathway catalytically synthesized prussian blue nanoparticles defeating natural enzyme peroxidase cxcr4 and cd44 dual-targeted prussian blue nanosystem with daunorubicin loaded for acute myeloid leukemia therapy metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis the multifactorial role of neutrophils in rheumatoid arthritis neutrophil membrane-coated nanoparticles inhibit synovial inflammation and alleviate joint damage in inflammatory arthritis neutrophilderived microvesicles enter cartilage and protect the joint in inflammatory arthritis exosomes: composition, biogenesis and function the biology, function, and biomedical applications of exosomes a novel nanoparticle drug delivery system: the antiinflammatory activity of curcumin is enhanced when encapsulated in exosomes neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence transforming weakness into strength: photothermal-therapy-induced inflammation enhanced cytopharmaceutical chemotherapy as a combination anticancer treatment strategies toward rheumatoid arthritis therapy; the old and the new development of anti-tnf therapy for rheumatoid arthritis the effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis targeted combination of antioxidative and anti-inflammatory therapy of rheumatoid arthritis using multifunctional dendrimer-entrapped gold nanoparticles as a platform targeted silver nanoparticles for rheumatoid arthritis therapy via macrophage apoptosis and repolarization reactive oxygen species-responsive dexamethasone-loaded nanoparticles for targeted treatment of rheumatoid arthritis via suppressing the irhom2/tnf-α/baff signaling pathway bioinspired exosome-like therapeutics and delivery nanoplatforms rheumatoid arthritis therapy reappraisal: strategies, opportunities and challenges immunometabolism in early and late stages of rheumatoid arthritis achieving ultrasmall prussian blue nanoparticles as high-performance biomedical agents with multifunctions surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy collagen-induced arthritis location, location, location: how the tissue microenvironment affects inflammation in ra myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity exosomes: cell-created drug delivery systems parallel comparison of fibroblast-like synoviocytes from the surgically removed hyperplastic synovial tissues of rheumatoid arthritis and osteoarthritis patients butyrate modulates inflammation in chondrocytes via gpr43 receptor emerging functions of icam-1 in macrophage efferocytosis and wound healing the multifactorial role of neutrophils in rheumatoid arthritis essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis progress in applications of prussian blue nanoparticles in biomedicine superoxide dismutases: role in redox signaling, vascular function, and diseases the role of nadph oxidases and oxidative stress in neurodegenerative disorders dual role of superoxide dismutase 2 induced in activated microglia: oxidative stress tolerance and convergence of inflammatory responses ulinastatin attenuates lps-induced human endothelial cells oxidative damage through suppressing jnk/c-jun signaling pathway over-expression of extracellular superoxide dismutase in mouse synovial tissue attenuates the inflammatory arthritis nitrodige analysis reveals inhibition of protein s-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells role of reactive oxygen species in lps-induced production of prostaglandin e2 in microglia mitochondrial ros govern the lps-induced pro-inflammatory response in microglia cells by regulating mapk and nf-κb pathways autophagy inhibitor regulates apoptosis and proliferation of synovial fibroblasts through the inhibition of pi3k/akt pathway in collagen-induced arthritis rat model effects of inflammatory cytokine il-27 on the activation of fibroblast-like synoviocytes in rheumatoid arthritis the protective effects of the gpr39 agonist tc-g 1008 against tnf-α-induced inflammation in human fibroblast-like synoviocytes (flss) il-35 ameliorates collagen-induced arthritis by promoting tnf-α-induced apoptosis of synovial fibroblasts and stimulating m2 macrophages polarization therapeutic blockade of granulocyte macrophage colony-stimulating factor in covid-19-associated hyperinflammation: challenges and opportunities gm-csf-dependent inflammatory pathways 4-o-carboxymethylascochlorin inhibits expression levels of on inflammation-related cytokines and matrix metalloproteinase-9 through nf-κb/mapk/tlr4 signaling pathway in lpsactivated raw264. 7 cells interleukin-1 and related cytokines in the regulation of inflammation and immunity glucocorticoids drive diurnal oscillations in t cell distribution and responses by inducing interleukin-7 receptor and cxcr4 inflammation, von willebrand factor, and adamts13 phosphatidylinositol 3 kinase/akt signal relay cooperates with smad in bone morphogenetic protein-2-induced colony stimulating factor-1 (csf-1) expression and osteoclast differentiation nicotine attenuates osteoarthritis pain and matrix metalloproteinase-9 expression via the α7 nicotinic acetylcholine receptor interleukin-1 receptor antagonist inhibits angiogenesis via blockage il-1α/pi3k/nf-κβ pathway in human colon cancer cell interleukin-1 beta increases activity of human endothelial progenitor cells: involvement of pi3k-akt signaling pathway interleukin 7 receptor activates pi3k/akt/mtor signaling pathway via downregulation of beclin-1 in lung cancer shear stress accumulation enhances von willebrand factor-induced platelet pselectin translocation in a pi3k/akt pathway-dependent manner maresin 1 improves the treg/th17 imbalance in rheumatoid arthritis through mir-21 progress in intra-articular therapy the authors declared that they have no conflicts of interest to this work. supplementary data to this article can be found online at https://doi. org/10.1016/j.bioactmat.2022.02.017. sa orthopaedic journal winter 2014 | vol 13 • no 2 page 31 atlanto-axial fusion: magerl transarticular versus harms instrumentation techniques rn dunn mbchb, mmed, fcs(sa) orth pieter moll and nuffield chair of orthopaedic surgery h stander mbchb(stell), fcorth(sa) registrar (at time of study) department of orthopaedic surgery, university of cape town correspondence: prof robert dunn email: robert.dunn@uct.ac.za tel: 021 404 5108 introduction atlanto-axial instability may occur following indirect trauma resulting in transverse ligament disruption or dens fracture. it may also occur insidiously in inflammatory conditions such as rheumatoid arthritis with progressive attenuation of the transverse ligament. less frequently it may be associated with dysplasias such as achondroplasia, down’s syndrome with a small dens or mucopolysaccharidosis such as morquio syndrome. traditional wiring techniques such as the gallie and brookes are technically simple but provide poor biomechanical control necessitating concomitant rigid external orthosis application and unacceptable non-union rates.1 magerl’s transarticular screw fixation offers high fusion rates but is not possible in 18% of patients due to a high riding vertebral artery.2 it also requires pre-instrumentation anatomical reduction which is not always possible. access can also be a problem due to thoracic kyphosis hindering drill direction. abstract transarticular screw fixation offers acceptably high fusion rates but is not possible in 18% of patients due to a high riding vertebral artery. it also requires pre-operative anatomical reduction which is not always possible. the harms technique utilises a posterior c1 lateral mass and c2 pedicle screw. this allows easier access due to the angle of drilling and has become an increasingly popular surgical technique. aim the aim of this study is to review and compare the above techniques with regard to surgery, complications and outcome. methods a retrospective case note and radiographic review of 42 patients undergoing posterior c1–2 fusion in a single institution during the period 2003 to 2011, identified on a prospectively maintained database, was performed. the indication for surgery was atlanto-axial instability with post-traumatic and rheumatoid arthritis the commonest aetiologies. there was no difference in age and gender between the two groups. results the harms method had a slightly higher mean blood loss compared to the transarticular method. the surgical time was no different. the transarticular technique was abandoned in three cases due to inability to place the screw safely. the harms technique was successfully completed in all cases. there were four unilateral vertebral artery injuries in the transarticular group and one in the harms group. there were three intra-operative unintentional durotomies in the transarticular group with one in the harms. all but one transarticular case fused, with five transarticular cases taking more than 9 months. conclusion although the harms technique had an increased blood loss, the incidence of vertebral artery and dura injury was lower. fusion was similar in both groups with the only non-union occurring in the transarticular group. the harms technique offers the advantage of intra-operative reduction and a smaller exposure due to the direction of access but at a higher instrumentation cost. key words: atlanto-axial fusion, harms, transarticular saoj winter 2014 bu_orthopaedics vol3 no4 2014/05/05 9:51 pm page 31 page 32 sa orthopaedic journal winter 2014 | vol 13 • no 2 the harms technique utilises a posterior c1 lateral mass and c2 pedicle screw.3 this allows easier access due to the more perpendicular direction of drilling and has become an increasingly popular surgical technique. the aim of this study is to compare the above techniques with regard to surgery, complications and outcome. material and methods following institutional research ethics committee approval, a retrospective case note and radiographic review was performed on all patients undergoing posterior c1–c2 fusion by the senior author. forty-two consecutive patients were identified from a prospectively maintained database. twenty-seven underwent transarticular fusion and 15 the harms technique. this largely represented a change in the surgeon’s choice of procedure, initially performing the transarticular technique as the default operation with transition to the harms procedure. the two groups were similar in terms of age and gender. the median age was 43 years (21–69 ± 15.7) in the transarticular group and 35 (12–74 ± 20.7) in the harms group. the difference was not statistically significant (p=0.14). there were 18 males and nine females in the transarticular compared to nine males and seven females in the harms group (p=0.53) in both techniques the patient was positioned prone on a relton hall frame with the skull held with a mayfield clamp. the neck was positioned to optimise access by slight flexion at the occiput but extended in the sub-axial spine. an attempt was made to reduce the c1–2 articulation during this positioning process. both procedures were performed with lateral fluoroscopy only. a midline incision was made over c1–2 but extended distally in the transarticular group to allow the required drill angulation. sub-periosteal exposure of the c1 arch and c2 posterior elements was done. a right-angled hook was used to run on the inner cortex of the c2 lamina in a lateral direction to palpate the medial isthmus, and thus the medial border of the screw placement. in the transarticular group, a starting point was based about 2–3 mm lateral to this medial isthmic palpation, inferiorly on the c2 lamina, directed in a cephalad direction and neutral in terms of medial–lateral angulation. this was done under lateral image guidance. the c1–2 joint was crossed and anterior c1 lateral mass perforated. titanium 3.5 mm cortical screws were placed (figure 1). in the harms group, the posterior aspect of the c1 lateral mass was visualised after sub-periosteal exposure of the posterior arch and a plane developed inferiorly onto the lateral mass. a watson-cheyne was placed into the c1–2 joint, after penetrating the capsule, to allow inferior retraction of the c2 root and improve access to the entry point. a burr was used to create a cortical breach in the lateral mass as it joins the arch superiorly. this allowed stable initial placement of the 2.7 mm drill bit. the path was drilled parallel to the arch with 10–15° convergence. this was observed on lateral image while drilling. a screw was placed with the tulip at the level of the arch, i.e. part of the proximal screw shaft remained visible. c2 pedicle screws were then placed under lateral image after medial isthmic/pedicle palpation (figure 2). cortico-cancellous bone graft was harvested from the iliac crest in all cases and placed from the c1 arch to c2 lamina. patients were managed in a philadelphia collar when up, for 6 weeks. patient demographics, surgical indications, intra-operative variables, complications and radiographic outcome were assessed. x-rays pre-op, post-op, and follow-up at 6 weeks, 3 months, 6 months, 12 months and 24 months were evaluated for successful instrumentation and subsequent fusion. results trauma predominated over aetiology by accounting for half the cases. the remainder was due to rheumatoid arthritis, os odontoidium, a pathological fracture from tuberculosis and neurofibromatosis-related destruction. this is tabulated in table i. there was a small statistically significant difference in blood loss between the two groups with a median of 150 ml in the transarticular group and 250 ml in the harms group. when considering the number of cases that bled more than 500 ml, it was clearly more frequent in the harms group (p=0.016). the cases that bled excessively were two rheumatoid cases (one converted from an aborted transarticular screw), two dens non-unions and the tuberculosis pathological fracture. there was no difference in surgical time between the two techniques (table ii). the transarticular technique was abandoned in three cases due to inability to place the screw safely. the harms technique was successfully completed in all cases. figure 1. ap and lateral x-ray of a well-fused transarticular instrumented fusion figure 2. ap and lateral x-rays of the harms technique saoj winter 2014 bu_orthopaedics vol3 no4 2014/05/05 9:51 pm page 32 sa orthopaedic journal winter 2014 | vol 13 • no 2 page 33 there were five unilateral vertebral artery injuries, four in the transarticular group and one in the harms group. these were easily controlled with screw placement in the former group but the other side not drilled. the harms injury occurred while tapping for the c1 screw and lacerating the artery as it passes on the c1 arch. the patients suffered no clinically evident effect from the single vertebral artery injuries (table iii). there were four intra-operative unintentional durotomies with csf leaks. three were in the transarticular group. these were successfully dealt with at the time of surgery with surgical, muscle and/or duroseal. there was one case of instrumentation failure with bilateral screw fracture in the transarticular group. despite this the patient went onto successful fusion. there were no cases of neurological deterioration or sepsis. median time to union was similar in both groups at around 5 months (table iv). all harms patients were fused by 9 months, with 18% of the transarticular group taking longer than this and one requiring revision for an established non-union. discussion atlanto-axial instability is a concern due to the risk of myelopathy and sudden death from proximal spinal cord compression.4 the decision to arthrodese the joint is based on the appreciation of this risk and the expectation that stabilisation will not occur spontaneously with conservative means. in the case of trauma, transverse ligamentous rupture recognised by an atlanto-dens interval of more than 5 mm on the lateral x-ray, is a clear indication as it is not expected to heal.5 dens fractures however may heal with restriction of motion but older patients, with marked displacement or angulation are recognised as a high non-union risk. theoretically, anterior dens osteosynthesis allows stabilisation without rotational motion sacrifice but this is often not the case due to spontaneous loss of motion.5,6 this may be due to associated c1–2 joint injury or the period of postoperative immobilisation and local inflammatory response to injury and surgery. in addition, reverse oblique fractures are not amenable to screw fixation. in non-compliant patients, rigid posterior fusion is the better option in our opinion. historically, uninstrumented and posterior wiring techniques (gallie and brookes) have been employed.7 although simple they provide poor biomechanical control of translation and necessitate rigid external orthoses and have a high non-union risk. magerl described the transarticular technique.2 it offers a stable construct which can be augmented with a posterior tension band between the c1 arch and c2 spine with socalled three-point fixation. this technique is demanding as it requires passage of the drill bit and screw medial and superior to the vertebral artery as it exits c2. up to 18% of patients have a high riding artery precluding this technique. we have also anecdotally noted that advanced rheumatoid patients tend to have enlarged vertebral artery foramina leaving less bone available for safe screw passage. the risk of vertebral artery injury is 5% per case.8 although a single vertebral artery injury is usually not clinically significant, it creates surgeon distress and necessitates an alternative technique if it occurs with the first screw as the second artery cannot be risked. the technique also demands anatomical reduction of c1 and c2 to allow a safe trajectory of the drill and subsequent screw. this is not always possible and can be difficult to maintain once reduced.9 the harms technique is initially more challenging to the surgeon as it requires visualisation of the c1 lateral masses which are a least a centimetre deep to the arch.3 the access can be difficult and excessive epidural bleeding can be encountered, which obliterates the surgeon’s view and prevents safe drill placement. this is due to the extensive venous plexus or epidural mesh of veins over the c2 root and posterior thecal sac. it is difficult to control with bipolar as once cauterised, the venous walls become hard and crack easily with further dissection increasing the bleeding. through trial and error, we have found the most efficient access is by sub-periosteal dissection on the inferior half of the c1 arch. the sharp side of the watson cheyne is used to lift to the periosteum inferiorly, creating a path on the posterior aspect of the c1 lateral mass. aetiology transarticular harms total trauma-related 52% transverse ligament rupture 5 2 7 acute odontoid fracture 5 1 6 odontoid non-union 6 2 8 atlanto-axial rotatory subluxation 1 1 inflammatory spondylopathy 38% rheumatoid arthritis 11 5 16 other 10% os odontoidium 2 2 tuberculosis with c2 fracture 1 1 neurofibromatosis 1 1 table i: aetiology of atlanto-axial instability table ii: blood loss and surgical time transarticular harms p-value blood loss (median) 150 ml (25–800 ± 153) 250 ml (50–800 ± 242.1) 0.02* surgical time (median) 90 min (45–150 ± 32.9) 95 min (70–225 ± 38.1) 0.22 blood loss > 500 ml 1 5 0.016* * indicates statistical significance the harms technique is initially more challenging to the surgeon as it requires visualisation of the c1 lateral masses saoj winter 2014 bu_orthopaedics vol3 no4 2014/05/06 12:12 pm page 33 page 34 sa orthopaedic journal winter 2014 | vol 13 • no 2 the watson-cheyne is then punctured into the c1–2 joint capsule bearing in mind the oblique nature form supero-medial to infero-lateral. this is then levered inferiorly by the assistant to retract the c2 root and create space over the c1 lateral mass. usually the surgeon retracts the space medially or laterally with a small sucker tip and creates a cortical notch under vision with a burr at the apex of the lateral mass where it joins the arch. there may be venous bleeding from laterally around the vertebral artery which can be controlled with a small patty. the drill is then placed in the starter hole and directed parallel to the c1 arch, assisted with lateral imaging. bicortical drilling is done to facilitate bicortical screw engagement. any residual bleeding can be controlled with small quantities of surgicel®. this is an extremely powerful screw and allows intraoperative reduction of c1 to c2. the c2 screws should be locked first, rods bent in lordosis and even a tool applied to the c2 screw head during reduction to ensure maximum c1 retro-translation without forward tilt of the rods at the c2 screw–rod interface. the downside of the harms is the increased expense of four polyaxial screws versus two cortical screws but the advantages outweigh this. the incision is smaller due to the required trajectories being less steep than the transarticular screws. the risk of increased blood loss is present, but with experience can be avoided with the aforementioned techniques. in our experience, the blood loss is minimal in most cases but if encountered, can be extensive due to the venous plexus over the screw insertion area. this was confirmed by hu in his review of manual screw position. their average blood loss was 450 ml but encountered a maximum of 1 200 ml. they had no vascular or neurological complications and post-op ct confirmed adequate placement of the freehand-inserted screws.10 conclusion although the harms technique had an increased blood loss, the incidence of vertebral artery and dural injury was lower. fusion was similar in both groups with the only non-union occurring in the transarticular group. the harms technique offers the advantage of intraoperative reduction and a smaller exposure due to the direction of access but at a higher instrumentation cost. the content of this article is the sole work of the authors, and no benefit of any form has been received or will be received from any commercial party. references 1. bransford rj, lee mj, reis a. posterior fixation of the upper cervical spine: contemporary techniques. the journal of the american academy of orthopaedic surgeons [internet]. 2011 feb;19(2):63–71. available from: http://www.ncbi.nlm.nih. gov/pubmed/21292929 2. magerl f, seeman p. stable posterior fusion of the atlas and axis by transarticular screw fixation. in: kehr p, weidner a, editors. cervical spine. 1st ed. wien, new york, springer verslag.; 1987. p. 322–27. 3. harms j, melcher r. posterior c1-c2 fusion with polyaxial screw and rod fixation. spine (phila pa 1976). 2001;26(22):2467–71. 4. dreyer sj, boden sd. natural history. clinical orth. 1999;(366):98–106. 5.. hsu wk, anderson pa. odontoid fractures: update on. j am acad orthop surg. 2010;18(7):383–94. 6. bormann rpb von, uct m. dens screw fixation: is it all it ’ s cut out to be? sa orthopaedic journal 2007 winter: 18–24. 7. santavirta s, konttinen y, laasonen e, honkanen v, anttipoika i, kauppi m. ten year results of operations spine for rheumatoid cervical disorders. j bone joint surg br 1991;73b:116–20. 8. low h, redfern r. c1-c2 transarticular screw fixation for atlantoaxial instability: a 6-year experience, and c1-c2 transarticular screw fixation-technical aspects. neurosurgery. 2002;50(5):1165–66. 9. dunn r, ombachi r. transarticular screw fixation of the atlanto-axial spine: a safe and effective option. saoj. 2006;(august):1–6. 10. hu y, kepler ck, albert tj, yuan z-s, ma w-h, gu y-j, et al. accuracy and complications associated with the freehand c-1 lateral mass screw fixation technique: a radiographic and clinical assessment. j neurosurg spine 2013 apr;18(4):372–7. available from: http://www.ncbi.nlm.nih.gov/pubmed/ 23373564 this article is also available online on the saoa website (www.saoa.org.za) and the scielo website (www.scielo.org.za). follow the directions on the contents page of this journal to access it. transarticular screws harms technique abandoned procedures 3 0 vertebral artery injuries 4 1 csf leaks 3 1 instrumentation failure 1 0 sepsis 0 0 neurological deterioration 0 0 table iii: summary of complications transarticular fixation harms technique p-value time to union (median) 5 (3–12 ± 3.2) months 4.5 (3–6 ± 1.5) months 0.04* delayed union 5 0 0.14 non-union 1 0 1.0 * indicates statistical significance table iv: time to union • saoj saoj winter 2014 bu_orthopaedics vol3 no4 2014/05/05 9:51 pm page 34 yefta 157 effect of range of motion and isometric strengthening exercises on grip strength and hand function in rheumatoid arthritis patients october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 universa medicina yefta daniel bastiana,*,**,a, angela bm tulaar**, surjanto hartono*** and zuljasri albar**** *department of anatomy, medical faculty, trisakti university **department of rehabilitation medicine, ***medical research unit, ****rheumatology division, department of internal medicine, faculty of medicine, university of indonesia correspondence adr yefta daniel bastian, sprm department of anatomy, medical faculty, trisakti university, jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2101 email: yeftabastian@yahoo.com univ med 2008; 27: 157-64 abstract in previous studies, duration of hand exercises in patients with rheumatoid arthritis (ra) had widely varying ranges, from 3 weeks to 4 months. an experimental study was conducted to evaluate the effect of range of motion (rom) and muscle strengthening exercises for 6 weeks on grip strength and hand function in ra patients. seventeen patients with chronic ra were randomly assigned to a treatment group and a control group. the treatment group (n=8) was given muscle strengthening exercises and heat therapy using paraffin baths 3 times a week at the hospital and rom exercises once a day at home for 6 weeks. the control group (n=9) was given only paraffin baths 3 times a week. after 6 weeks, there were significant differences in hand function (p=0.003), right and left grip strength (p=0.000 and p=0.001) and rom in the interventional group only. rom and isometric strengthening exercises significantly improved grip strength and hand function in patients with ra, while no impact was found when the patients were given paraffin baths only. in view of the small size of the study population, there is a need for further studies with larger populations. keywords: hand exercise, grip strength, rheumatoid arthritis introduction r h e u m a t o i d a r t h r i t i s ( r a ) i s a n inflammatory chronic systemic disease, which i s p a r t i c u l a r l y m a n i f e s t e d a t t h e s y n o v i a l membrane of diarthrodial joints and can result in destruction of the involved joints.(1) it is present in 0.5% to 1% of the general population, twice as often in women, and the age at disease onset is mainly between 45 and 65 years.(2) the clinical picture of ra is characterized by pain, fatigue, disability, and reduced quality of life. 158 bastian, tulaar, hartono, et al grip strength and hand function the course of the disease is often unpredictable, and the symptoms may vary from day to day. the main goals of treatment for ra are to prevent or control joint damage, prevent loss o f f u n c t i o n , a n d d e c r e a s e p a i n . ( 3 ) d e s p i t e substantial progress in the pharmacological and surgical interventions over the last decade, many patients with ra will still experience disability, pain, psychological distress, fatigue, and poor quality of life.(4) reduced levels of physical performance has been found to be associated with ra. patients with ra have been shown to have reduced muscle strength and aerobic capacity. impairments, disabilities, and h a n d i c a p s a s s o c i a t e d w i t h r a c a n b e devastating, leading to pain, activity restriction, and diminished quality of life, while placing a strain on the health care system and society.(5) besides pharmacological and surgical interventions, conventional therapies such as physical therapy, occupational therapy, and c o m p r e h e n s i v e r e h a b i l i t a t i o n a n d s e l f m a n a g e m e n t p r o g r a m s a r e c o m m o n l y a n d f r e q u e n t l y u s e d i n t e r v e n t i o n s . d e s p i t e d i f f e r e n t p a t h o p h y s i o l o g i c a l p r o c e s s e s , patients suffering from ra experience pain and a gradual decline in muscle strength, eventually resulting in loss of function and quality of life. increasing evidence shows that p h y s i c a l e x e r c i s e i m p r o v e s f u n c t i o n a n d prevents loss of function in ra.(6) owing to a fear of enh ancing joint inf lammation and accelerating cartilage destruction, it has been advocated that exercise in active ra should be restricted to gentle assisted range of motion (rom) exercises. on the contrary, exercise w a s f o u n d t o h a v e b e n e f i c i a l e f f e c t s o n f u n c t i o n , p a i n a n d m u s c l e s t r e n g t h . a n intensive exercise program consisting of rom strengthening and aerobic exercises is more effective than a conservative exercise program, a n d d o e s n o t h a v e d e l e t e r i o u s e ff e c t s o n disease activity.(7) in ra patients with active disease, an inpatient program with frequent exercise therapy was found to be superior to usual care regarding disease activity as well as muscle strength. long term high impact exercise has been proven to be beneficial regarding function and muscle strength in p a t i e n t s w i t h l o w d i s e a s e a c t i v i t y i n a n outpatient setting. in this study, exercise also did not increase disease activity.(8) hand function is recognized as being important to those diagnosed with ra, because reduction in muscle power and grip can lead t o i n c r e a s i n g d i f f i c u l t i e s i n p e r f o r m i n g activities of daily living. o’brien et al gave hand strengthening and stretching exercises as a home program for 6 months and showed significant results compared with stretching and joint protection.(9) theoretically, the effect of strengthening exercises can be expected after 2-3 weeks because of neural adaptation, but muscle adaptation itself can be seen after minimally 4 weeks of training.(10) the aim of this study was to examine the e f f e c t o f r o m i s o m e t r i c s t r e n g t h e n i n g exercises, combined with therapeutic heating using paraffin baths on grip strength and hand function in ra patients. methods research design this study was a single-blind, randomized controlled trial and conducted between january and may 2006. subjects subjects were ra patients in the subacute phase who had already been treated, aged 2070 years, male or female, meeting the criteria of the american college of rheumatology.(11) additional inclusion criteria were : (i) having joint involvement in the hand, which may be recognized from intrinsic muscle atrophy and/ 159 univ med vol.27 no.4 or a hand function index (hfi) score of 535.(12) (ii) never done structured rom and hand strengthening exercise before; and (iii) willing to be involved in the study. exclusion criteria were: (i) presence of peripheral nerve problems or muscle disease accompanied with atrophy; (ii) hand muscle weakness; (iii) sensory problems in the hand; (iv) finger amputation, open wound, fracture and contracture; (v) memory and cognitive problems. additional exclusion criteria were: (i) severe hypertension with systolic blood pressure of more than 150 mmhg and diastolic blood pressure of more than 100 mmhg; (iii) h a n d d e f o r m i t y r e l a t e d t o r a , i n c l u d i n g radioulnar or metacarpophalangeal (mcp) subluxation, boutonniere and swan neck; and (iii) doing strenuous grip activities in their activity of daily living, such as washing clothes. the study protocol was approved by the committee of medical research ethics of the faculty of medicine, university of indonesia. interventions all subjects meeting the inclusion and exclusion criteria, after having been given an explanation about the study program and the mechanisms of joint protection, were randomly assigned to the intervention group or the control group, using optimal allocation with a simple randomization. subjects in the intervention group were given heat therapy using paraffin baths 3 times a week at cipto mangunkusumo hospital, followed by isometric strengthening exercises. strengthening exercises were done b y o p p o s i n g t h e r e s i s t a n c e g i v e n b y t h e r e s e a r c h e r ’s h a n d o r b y p u t t y, w i t h o u t performing any movement of the joints. muscle contraction was sustained for 6 seconds and repeated up to 6 times for each joint, alternately for the right and left hands. every subject in this group also did rom exercises once a day at home. these were performed over the 6-week study period. subjects in the control group only had therapeutic heating using paraffin baths 3 times a week at the hospital and did not do any home exercises. all outcome assessments were undertaken at baseline and 6 weeks following randomization. outcome measures hand function was assessed using the hfi by giving a score according to the ability of the subject to do some finger activity then totally summed up. the minimum score is 4 and the maximum 42. the lower the score, the better the hand function. rom was measured using a goniometer, and grip strength (gs) of the right and left hands was measured using a modified sphygmomanometer. the sphygmomanometer was modified by rolling up the cuff and securing it within a bag made of nonstretch material so that when inflated to a specific point, the cuff attains a constant circumference of 6 inches. for each hand, gs was measured 3 times alternately. the best values were taken for each gs. gs measurements were done using a standardized protocol in which the subject had to sit with the shoulder in neutral rotation, the e l b o w f l e x e d 9 0 0, t h e f o r e a r m i n n e u t r a l position, the wrist extended 300, and the ulnar d e v i a t e d 1 5 0. t h e p r e s s u r e o f t h e sphygmomanometer should be adjusted to 20 mmhg before taking any measurements. statistical analysis descriptive statistics was done to know the distribution of the variables age, gender, education level, job and medication. changes i n o u t c o m e m e a s u r e s w e r e e x a m i n e d b y calculating 95% confidence intervals of the d i f f e r e n c e b e t w e e n b a s e l i n e a n d e n d l i n e scores. between-group differences in score changes were determined by student’s t test for unpaired samples. the significance level was set at 0.05. 160 bastian, tulaar, hartono, et al grip strength and hand function results subjects seventeen subjects were involved in this study, with eight subjects in the intervention group and nine in the control group. all subjects successfully followed the study to completion. subjects’ compliance in getting treatment in the hospital in the intervention group was 93%, while that in the control group was 79%. in the interventional group, subjects’ compliance to do rom exercises at home was 96.4%. all subjects were right handed. the age of the subjects ranged from 20 to 70 years (table 1). the majority of the subjects, totalling 14 p a t i e n t s ( 8 2 . 3 % ) w e r e f e m a l e a n d o n l y 3 patients were male. from the educational perspective, 8 subjects had an educational level of high school (47%) and 7 subjects had a master degree (41.2%). hand function h a n d f u n c t i o n b e f o r e a n d a f t e r intervention can be seen in table 2. hfi before and after intervention between the two groups showed non-significant results, in which the p value was more than 0.05. grip strength grip strength before and after intervention can be seen in table 2. gs before and after intervention between interventional and control group showed non-significant results in which the p value for right and left gs was more than 0.05. figure 1. flowchart of the number of subjects who completed the study ra patients attending rheumatology outpatient departmen (n=54) declined (n=33) : died (n=1) fail to be contacted (n=3) not interested (n=14) mobility difficulty (n=12) moved to another city (n=3) assessed for eligibility (n=21) participants randomized (n=17) not randomized (n=4) not meet inclusion criteria intervention group (n=8) control group (n=9) intervention group analysed after 6 weeks (n=8) control group analysed after 6 weeks (n=9) 161 univ med vol.27 no.4 range of motion r a n g e o f m o t i o n b e f o r e a n d a f t e r intervention can be seen in table 3. rom before and after intervention between the intervention and control group showed nonsignificant results for all joints. using the paired t-test, it was found that there was a significant improvement in rom for 12 areas of joints in the intervention group and 2 areas of joints in the control group. discussion the greatest prevalence of ra is in the 5059 year subgroup and 82.3% are female. this reconfirms the literature stating that ra is frequently found in the fourth and fifth decades and the incidence of ra is higher in females.(2) hfi was used in this study to measure hand function before and after intervention, because it has been proved to be a simple and fast tool for evaluating function and disease activity in ra.(12) between the two groups, there was no s i g n i f i c a n t d i f f e r e n c e b e f o r e a n d a f t e r intervention. in the interventional group, there was a significant difference in hand function before and after intervention (table 2), but there was none in the control group. this indicated that there were significant differences of hand function after intervention in both groups, but the difference was higher in the interventional g r o u p . h f i s h o w e d b e t t e r i m p r o v e m e n t characteristic intervention (%) n=8 control (%) n=9 age (year) 20-29 1 (12.5) 1 (11.1) 30-39 2 (25) 2 (22.2) 40-49 2 (22.2) 50-59 4 (50) 1 (11.1) 60-70 1 (12.5) 3 (33.3) sex female 7 (87.5) 7 (77.8) male 1 (12.5) 2 (22.2) education junior high school 1 (12.5) 1 (11.1) high school 4 (50) 4 (44.4) master degree 3 (37.5) 4 (44.4) occupation house wife 2 (25) 2 (22.2) retired 2 (25) 2 (22.2) civil servant 1 (11.1) teacher 3 (37.5) 2 (22.2) student 1 (11.1) unemployed 1 (12.5) 1 (11.1) medication methothrexate 8 (100) 6 (66.7) steroids 5 (62.5) 3 (33.3) nsaids* 5 (62.5) 5 (55.6) others** 2 (25) 4 (44.4) table 1. demographic and clinical data of 17 patients participating in the study at baseline *nsaids = non steroid anti inflammation drugs; **others (sulcolon and chloroquine) 162 bastian, tulaar, hartono, et al grip strength and hand function h fi = h an d fu nc tio n in de x; r g s= ri gh t g ri p st re ng th ; l g s= le ft g ri p st re ng th ; n s= n ot s ig ni fi ca nt (p >0 .0 5) in te rv en ti on g ro up c on tr ol g ro up p ar am et er b as e lin e e nd li ne m ea n di ff er en ce w it hi n gr ou p p b as e lin e e nd li ne m ea n di ff er en ce w it hi n gr ou p p m ea n di ff er en ce be tw ee n gr ou p p h fi 16 .8 8 ± 9. 46 14 .6 3 ± 10 .1 6 2. 25 ± 0 .7 0. 00 3 17 .1 1 ± 5. 75 15 .4 4 ± 4. 82 1. 67 ± 0 .9 3 0. 15 3 0. 58 ± 0 .2 3 ns r g s 89 .5 0 ± 18 .4 5 98 .0 0 ± 18 .7 3 8. 5 ± 0. 28 0. 00 0 89 .1 1 ± 15 .3 3 87 .5 6 ± 12 .7 2 1. 55 ± 2 .6 1 0. 72 5 6. 95 ± 2 .3 3 ns lg s 82 .7 5 ± 30 .6 3 91 .2 5 ± 29 .9 5 8. 5 ± 0. 68 0. 00 1 85 .3 3 ± 18 .1 9 88 .0 0 ± 13 .2 7 2. 67 ± 4 .9 2 0. 31 6 5. 83 ± 4 .2 4 ns ta bl e 2. h an d fu nc ti on a nd g ri p st re ng th b ef or e an d af te r in te rv en ti on ta bl e 3. j oi nt r an ge o f m ot io n be fo re a nd a ft er i nt er ve nt io n w =w ri st ; r =r ig ht ; l =l ef t; f= fl ex io n; e =e xt en si on ; m c p= m et ac ar po ph al an ge al ; p ip = pr ox im al in te rp ha la ng ea l; ns = no t s ig ni fi ca nt ( p > 0. 05 ) in te rv en ti on al g ro up c on tr ol g ro up p ar am et er b as e lin e e nd li ne m ea n di ff er en ce w it hi n gr ou p p b as e lin e e nd lin e m ea n di ff er en ce w it hi n gr ou p p m ea n di ff er en ce be tw ee n gr ou p p r w e 53 .7 5 ± 13 .8 2 60 .6 3 ± 15 .6 8 6. 88 ± 1 .8 6 0. 00 1 55 .0 0 ± 13 .9 2 52 .5 0 ± 10 .0 0 2. 5 ± 3. 92 0. 34 7 4. 38 ± 2 .0 6 ns r m c p1 f 61 .5 0 ± 14 .9 2 67 .5 0 ± 14 .5 3 6. 00 ± 0 .3 9 0. 01 3 58 .6 7 ± 15 .1 3 60 .3 3 ± 13 .9 6 1. 66 ± 1 .1 7 0. 60 5 4. 34 ± 0 .7 8 ns lm c p2 f 85 .0 0 ± 8. 42 90 .2 5 ± 5. 39 5. 25 ± 3 .0 3 0. 02 7 86 .0 0 ± 5. 57 88 .1 1 ± 4. 14 2. 11 ± 1 .4 3 0. 10 0 3. 14 ± 1 .6 ns lm c p2 e 20 .0 0 ± 9. 26 24 .2 5 ± 6. 45 4. 25 ± 2 .8 1 0. 08 5 20 .6 7 ± 8. 89 25 .3 3 ± 10 .0 5 4. 66 ± 1 .1 6 0. 01 9 0. 41 ± 1 .6 5 ns lm c p3 e 20 .0 0 ± 9. 26 24 .2 5 ± 6. 45 4. 25 ± 2 .8 1 0. 08 5 20 .8 9 ± 9. 06 24 .6 7 ± 9. 90 3. 78 ± 0 .8 4 0. 03 3 0. 47 ± 1 .9 7 ns lm c p4 f 84 .5 0 ± 8. 80 88 .2 5 ± 5. 90 3. 75 ± 2 .9 0. 03 5 83 .7 8 ± 9. 46 87 .2 2 ± 7. 14 3. 44 ± 2 .3 2 0. 06 0 0. 31 ± 0 .5 8 ns lm c p5 f 82 .5 0 ± 10 .9 9 88 .2 5 ± 4. 95 5. 75 ± 6 .0 4 0. 04 0 86 .4 4 ± 7. 67 86 .6 7 ± 8. 00 0. 23 ± 0 .3 3 0. 89 2 5. 52 ± 5 .7 1 ns r m c p4 e 84 .5 0 ± 8. 99 84 .7 5 ± 9. 07 0. 25 ± 0 .0 8 0. 04 4 20 .0 0 ± 8. 66 21 .7 8 ± 9. 08 1. 78 ± 0 .4 2 0. 08 6 1. 53 ± 0 .3 4 ns lm c p4 e 18 .7 5 ± 9. 91 24 .2 5 ± 6. 45 5. 5 ± 3. 46 0. 03 6 22 .0 0 ± 9. 54 24 .7 8 ± 10 .0 2 2. 78 ± 0 .4 8 0. 08 5 2. 72 ± 2 .9 8 ns r pi p3 f 99 .2 5 ± 10 .7 9 10 4. 00 ± 9 .7 2 4. 75 ± 1 .0 7 0. 03 2 10 2. 67 ± 8 .3 1 10 4. 56 ± 6 .9 1 1. 89 ± 1 .4 0. 17 6 2. 86 ± 0 .3 3 ns r pi p4 f 10 1. 00 ± 1 1. 95 10 6. 63 ± 1 1. 55 5. 63 ± 0 .4 0. 02 1 10 2. 67 ± 1 0. 10 10 7. 33 ± 5 .4 1 4. 66 ± 4 .6 9 0. 07 1 0. 97 ± 4 .2 9 ns r pi p5 f 99 .0 0 ± 8. 35 10 5. 00 ± 9 .4 4 6. 00 ± 1 .0 9 0. 01 2 10 2. 33 ± 1 0. 37 10 2. 44 ± 7 .4 5 0. 11 ± 2 .9 2 0. 96 9 5. 89 ± 1 .8 3 ns lp ip 3f 10 0. 25 ± 1 5. 51 10 4. 88 ± 1 4. 52 4. 63 ± 0 .9 9 0. 02 6 10 5. 67 ± 7 .2 8 10 8. 33 ± 4 .7 4 2. 66 ± 2 .5 4 0. 05 5 1. 97 ± 1 .5 5 ns lp ip 4f 10 0. 75 ± 1 3. 39 10 6. 75 ± 1 0. 31 6. 75 ± 3 .0 8 0. 00 2 10 5. 33 ± 7 .3 5 10 7. 44 ± 5 .5 0 2. 11 ± 1 .8 5 0. 13 0 4. 64 ± 1 .2 3 ns 163 univ med vol.27 no.4 especially by a combination of therapeutic exercises and heating. it is important to note that hfi measures the ability of the wrist and f i n g e r s t o m o v e i n t h e i r r o m , t h u s improvement in rom is indicated by a higher hfi. there was no significant improvement in right and left grip strengths between the two groups. there was a significant difference in right and left grip strengths in the interventional group (table 2), but not in the control group. it is known from the literature that strengthening exercises can have effect after a minimum of 4 weeks.(10) this was proved in the isometric strengthening of quadriceps muscle in patients with osteoarthritis of the knee.(13) other studies showed that exercise in ra patients needed a longer time to take effect, such as in the study by hakkinen for 2 years.(14,15) myositis in ra patients can result in muscle weakness, and can be confirmed by muscle biopsy, in which there is type ii muscle atrophy, acute myositis and focal necrosis. medications such as steroid agents also can result in myopathy.(16) table 1 indicates that 62.5% of subjects in the interventional group and 33.3% of subjects in the control group took steroid agents. in the control group, right gs decreased after intervention, while left gs increased. this may be due to the great variability in clinical manifestations, joint involvement, disease course a n d r e s p o n s e t o t r e a t m e n t . t h e r e w a s n o significant difference between the two groups. rom before and after intervention in the i n t e r v e n t i o n a l g r o u p s h o w e d s i g n i f i c a n t differences in 12 joints (table 3), while in the control group there were differences in only 2 joints.this may be due to the variability in baseline characteristics between the two groups. in the interventional group, rom improvement may have been the result of the heating and rom exercises that were done at home. in the control group, rom improvement could have been the result of therapeutic heating. as has been commonly recognized, the effect of heating is to improve tissue extensibility, decrease joint stiffness and pain, and help reduce infiltrate resolution in the inflammation.(17) there were no significant differences between the 2 groups in hfi, gs and rom, possibly as a result of the limited number of subjects in this study. the duration of this study which was only 6 weeks may have been one of t h e f a c t o r s r e s u l t i n g i n a n o n s i g n i f i c a n t difference in gs and rom between the two groups. moreover, rom exercises in this study were done only once a day. in this study, joint pain was not assessed. it is well-known that pain can stimulate reflex inhibition of muscular contraction, but the investigators attempted to reduce pain by giving the patient education about joint protection and b y m e a s u r i n g t h e g s u s i n g a m o d i f i e d sphygmomanometer.(18,19) conclusion r o m a n d i s o m e t r i c s t r e n g t h e n i n g exercises combined with therapeutic heating using paraffin baths for 6 weeks in ra patients can increase hand function. gs and rom better than therapeutic heating using paraffin baths alone. references 1. goronzy jj, weyand cm. arthritis rheumatoid: epidemiology, pathology, and pathogenesis. in: klippel jh, editor. primer on the rheumatic diseases. 12th ed. atlanta: arthritis foundation; 2001. p. 632-3. 2. stenstrom ch, minor ma. evidence for the benefit of aerobic and strengthening exercise in rheumatoid arthritis. arthritis rheum 2003; 49: 428–34. 3. american college of rheumatology subcommittee on rheumatoid arthritis. guidelines for the 164 bastian, tulaar, hartono, et al grip strength and hand function management of rheumatoid arthritis: 2002 update. arthritis rheum 2002; 46: 328–46. 4. astin ja, beckner w, soeken k. psychological interventions for rheumatoid arthritis: a metaanalysis of randomized controlled trials. arthritis rheum.2002; 47: 291–302. 5. kobelt g, eberhardt k, johansson b. economic consequences of the progression of rheumatoid arthritis in sweden. arthritis rheum 1999; 42: 347– 56. 6. de jong z, vliet vlieland tpm. safety of exercise in patients with rheumatoid arthritis. curr opin rheumatol 2005; 17: 177–82. 7. van den ende chm, breedveld fc, le cessie s, dijkmans bac, de mug aw, hazes jmw. effect of intensive exercise on patients with active rheumatoid arthritis: a randomised clinical trial. ann rheum dis 2000; 59: 615–21. 8. bulthuis y, drossaers-bakker kw, taal e, raskan j, oostveen j, van’t pad bp, et al. arthritis patients show long term benefits from 3 weeks intensive training directly following hospital discharge. rheumatology 2007; 46: 1712-7. 9. o’brien, av, jones p, mullis r, mulherin d, dziedzic k. conservative hand therapy treatments in rheumatoid arthritis – a randomized controlled trial. rheumatology 2006; 45: 577-83. 10. kisner c, colby la. resistance exercise. in: therapeutic exercise foundations and techniques. 4th ed. philadelphia; f.a. davis; 2002. p. 68-9. 11. arnett fc, edworthy sm, bloch da, mcshane dj, fries jf. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum 1988; 31: 315–24. 12. kalla aa, smith pr, brown gmm, meyers ol, chalton d. responsiveness of keitel functional index compared with laboratory measures of diseases activity in eheumatoid arthritis. br j rheumatol 1995; 34: 141-9 13. widjanantie sc. pengukuran fungsi lutut dengan time up and go test dan stair climbing test pada latihan isometrik otot kuadrisep pasien osteoartritis lutut (thesis). jakarta: program studi ilmu rehabilitasi medik, fakultas kedokteran universitas indonesia; 2006. 14. hakkinen a, sokka t, hannonen p. a home-based two year strength training period in early rheumatoid arthritis led to good long-term compliance: a fiveyear follow up. arthritis rheum 2004; 51: 56-62. 15. hakkinen a, sokka t, kautiainen h, kotaniemi a, hannonen p. sustained maintenance of exercise induced muscle strength gains and normal bone mineral density in patients with early rheumatoid arthritis: a five year follow up. ann rheum dis 2004; 63: 910-6. 16. hicks je, joe go, gerber lh. rehabilitation of the patient with inflammatory arthritis and connective tissue disease. in: delisa ja, gans bm, walsh ne, bockenek wl, frontera wr, et al, editors. 4th ed. philadelphia: lippincot williams & wilkins; 2005. p. 737. 17. basford jr. therapeutic physical agents. in: delisa ja, gans bm, walsh ne, bockenek wl, frontera wr, et al, editors. 4th ed. philadelphia: lippincot williams & wilkins; 2005. p. 255. 18. harris ed. clinical features of rheumatoid arthritis. in: ruddy s, harris ed, sledge cb, editors. kelley’s textbook of rheumatology. 6th ed. philadelphia: w.b. saunders; 2001. p. 969, 984. 19. harris ed. treatment of rheumatoid arthritis. in: ruddy s, harris ed, sledge cb, editors. kelley’s textbook of rheumatology. 6th ed. philadelphia: w.b. saunders; 2001. p. 1004-18. a profile of patients attending the physiotherapy department at the alexandra health centre and university clinic paulo ferrinhct huib cornielje** s g reinach ** key words: physiotherapy, community based rehabilitation, health information system. introduction planning the operations o f primary health care centres in south africa is ham pered by a lack o f the most basic data on the profile o f the patients seen at that level. a health information system, that has been partially discontinued1,2, provided us with inform ation ex­ tremely useful to gain insightson the patients attending a physiother­ apy d epartm ent at the alexandra h ealth c entre and university clinic (ah c), serving the population o f alexandra (alex). m ost o f the health services for the community are provided by the a h c, a privately funded non-profit facility. t h e a h c is the only provider o f rehabilitative care in alex3,4,5. rehabilitation services a t the a h c we are developing a community based r ehabilita­ tion (c b r ) program m e6. t h e c b r program m e at the a h c has 4 com ponents: research; mobilising the community; education and training; and clinical work. in this article we address the clinical com ponent only. comprehensive though basic clinical services have, for many years, been provided at the a h c on a part time basis. t hese services include: physiotherapy, speech and hearing therapy, podiatry, o p to ­ metry and psychology. they are being developed in the context o f the philosophy o f the cb r program m e. this m eans a strong com ­ m itm ent to outreach, to continuity o f care, to cost-effectiveness and for support o f appropriate referral centres. t h e only m ajor recent change in clinical work has been the impact o f the appointm ent o f a full-time physiotherapist to the p attern o f work. between may and d ecem ber 1989 part-tim e physiotherapists, working 10 hours a week, provided episodic clinical care to 417 patients. t h e age distribution was 4% : 0-4 years o f age, 5%: 5-14 years, 84%: 15-59 years and 8% over 60 years. f o u r percent w ere referred to hospital. two percent w ere patients with chronic p ro b ­ lems and 98% had acute problems. o f those with acute problems, 60% w ere first attenders and 40% repeat attenders. f or the equival­ en t period in 1990, a physiotherapist working 15. clinical hours a week, saw 740 patients with a similar age profile, but only 1% were referred to hospital; 39% w ere seen with chronic problem s and 61% with acute problems. o f the acute problems, 29% w ere first a tte n ­ ders and 71% w ere repeat attenders. t he m ajor changes w ere therefore in the direction o f m ore patients with chronic problems, m ore repeat attendances by patients with acute problem s and less hospital referrals6. d uring 1989, the a h c experim ented with a health information system to determ ine a profile o f the patients presenting a t the a h c 1,2. a lthough the system was reviewed in 1990, d ata collected in the physiotherapy departm ent between 1988 and 1990 have been recently analysed and this analysis is reported here. population and methods a standard form was used to collect data. d ata w ere both d e p a rt­ m ent and patient specific. each column has one patient’s data. t he data include age (in six categories), sex and diagnosis (categorised as acute new, acute repeat o r chronic and in nine m ore clinical ca­ tegories), source o f referral to the physiotherapy dep artm en t and referral from the physiotherapy d ep artm en t to o th er services. t he analysis was done using b m d p program m es7, on the ibm 4381 com puter o f the medical r esearch council. statistical signific­ ance was tested with pearson chi-square, y ates corrected chi-square o r the fisher exact test as most appropriate. results d ata w ere analysed for 1408 patients. t h e age distribution was 2% (n = 3 3 ): 0-4 years o f age, 5% (n = 7 4 ): 5-14 years, 84% (n = 1186): 15-59 years and 8% (n= 108): over 60 years (no data on 1% (n = 7 )). y oung children (0-4 years) w ere m ore commonly seen in ju n e and less frequently in october; older children (5-14years) w ere m ore commonly seen in august; the elderly w ere particularly com m on in d ecem ber and less com m on in f ebruary and a ugust (p=0,00). f rom the 923 patients with data on source o f referral, 78% (n = 7 1 7 ) were referred from casualty, 13% (n-124) from the adult outpatient departm ent, 4% (n-34) were self-referrals, 2% (n = 1 9 ) w ere from the paediatric outpatient d ep artm en t and 1% ( n = 7 ) w ere from o th er sources. thirty one percent (n = 4 3 9 ) had neck and back problems, 30% (n = 4 1 9 ) had problem s related to the arms, 22% (n = 3 0 3 ) had lower limb problems, 13% (n = 185) had hand problems, 4% (n = 5 6 ) had problem s associated with burns, 3% (n = 4 8 ) had chest related p ro b ­ lems, 1% ( n = 8 ) had osteoarthrosis, 0% ( n = 2 ) had rheum atoid abstract during 1989 the alexandra health centre and university clinic (ahc) experimented with a health information system to deter­ mine a profile of the patients presenting at the ahc physiother­ apy department. data were analysed for 1408 patients. the age distribution was 2% (n=33): 0-4 years of age, 5% (n-74): 5-14 years, 85% (n-1186): 15-59 years and 8% (n=108): over 60years (no data on 1%, n=7).31% (n=439) had neck and back problems, 30% (n-419) had problems related to the arms, 22% (n-303) had lower limb problems, 13% (n=185) had hand problems, 4% (n=56) had problems associated with burns, 3% (n=48) had chest related problems, 1% (n=8) had osteoarthrosis, 0% (n=2) had rheumatoid arthritis and 2% (n=33) had other unspecified problems. 94% (n=1323) had 1 diagnosis and 6% (n=85) had multiple diagnosis. 12% (n=172) were patients with chronic problems and 86% (n=1213) had acute prob­ lems (no data in 2%, n=23). of the 786 cases with data 1% (n=10) were referred to hospital, 96% (n=754) were not referred anywhere and 3% (n=22) were referred elsewhere. referrals were more common for bums and osteoarthrosis (p-0,00). the low rate of referral is a credit to the ability to cope with the work at local level. * alexandra health centre and university clinic and institute fo r urban p iim aiy health care, p o b ox 175, bergvlei 2012 ** alexandra health centre a nd university clinic and institute fo r urban primary health care *** institute fo r biostatistics o f the m edical research council bladsy 56 fisioterapie, november 1992, deel 48 no 4 r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) arthritis and 2% (n = 3 3 ) had other unspecified problems. t he occurrence o f bum s decreased with increasing age (p,00). lower limb problems are particularly com mon in the 5-14 years age group (p= 0,0). neck and back problems are uncommon before 15 years (p=0,00). ninety four percent ( n = 1323) had one diagnosis and 6% (n = 85) had multiple diagnosis. multiple diagnosis is relatively m ore common before the age o f 5 years (p=0,00). twelve percent ( n = 172) w ere patients with chronic problems and 86% (n= 1213) had acute problems (no data in 2% (n = 2 3 )). o f those with acute problems, 49% were first attenders and 51 % repeat attenders. o f 842 patients with data, 17% had problems related to traum a at work. chronic diagnoses are relatively m ore com mon over the age o f 60 years (p<0,05). o f the 786 cases with data, 1 % ( n = 1 0 ) were referred to hospital, 3% were referred elsewhere and 96% (n-754) were not referred anywhere. hospital referrals are m ore com mon before the age o f 15 years (p=0,00). referrals were m ore common for burns and os­ teoarthrosis (p=0,00). h and problems were m ore likely to be work related (p=0,00). t he prevalence o f the diagnosis varied with the m onth o f the year. in january, “o th er” was m ore common, osteoarthrosis and rheum a­ toid arthritis were m ore common in february and march, arm and chest problems were m ore common in june, burns in ju n e and july, hand problems in november and lower limb problems in january and o ctober (p < 0,00). chest and neck and back problems are more commonly referred from the adult outpatient departm ent, burns from the paediatric o utpatient departm ent, and hand problems from the casualty d e ­ partm ent (p<0,00). a cute repeat cases are m ore likely to be self referrals, and chronic cases a re m ore likely to be from th e adult and th e paediatric o u tp a ­ tients (p=0,00). discussion the method o f data collection is well accepted and adhered to, but it has become apparent that further changes in the forms used for data collection would be appropriate. t he age group 15-59 should be further split into finer categories; the chronic classification should differentiate between new and repeat chronic visits; the diag­ nostic classification should separate clinical/etiological diagnosis from anatomical siting of the disability. children under 15 years are under-represented in comparison with the age structure o f the population3. violence, an endem ic problem in alexandra3, and its associated traum a, are probably responsible for the bulk o f referrals to the physiotherapy d epartm ent via casualty. chest problems are m ore com m on in the winter m onth o f june. this is in-keeping with findings previously reported3,8. preventive work has its greater potential in relation to bum s and work related cases. t h e work related problems are already addressed by a different service o f the a h c 9. t h ere is a need to develop a burns prevention program m e that takes into account the winter peak incidence. t he low rate o f referral is a credit to the ability to cope with the work at local level. a t the sam e tim e the tem ptation to keep as much work at the primary level, although making clinical and financial sense, removes limited manpower from other, probably as o r more im portant, cb r functions. t h e primary care service m ust clearly define priorities, work o u t program m es and mobilise resources to achieve clearly spelt o u t objectives, keeping the tension between clinical and non-clinical work separate and under control. clinical professional care is part o f cbr. but, in the presence o f limited resources what can be achieved, what must be addressed and what is going to be neglected? these m ust be conscious decisions rather than the result o f pressure from dem and for clinical professional care from other m em bers o f the primary care team and patients th em ­ selves. t he results encourage us to recom m end that rehabilitation workers in primary care practice should collect this type o f inform a­ tion and share it in publications o r conferences. reference list 1. phakathi g, ferrinho p, robb d e t al. problems in the development o f a health information system at the alexandra health centre and university clinic. chasa journal o f comprehensive health 1992; in press. 2. ferrinho p, buch e, robb d et al. d eveloping a health information system for a prim ary health care centre in alexandra. s a fr m e d j 1991;80:400-403. 3. ferrinho p, robb d, m hlongo a et al. a profile o f alexandra. s a fr m ed j 1991;80:374-378. 4. ferrinho p, phakathi g. alexandra health centre and its patients: patient trends, age, sex and address profiles. s a fr fean practice 1991;12:50-55. 5. naylor cd. alexandra health centre: primary health care for an impoverished black township in south africa. annals o f internal m edicine 1988;109:73-75. 6. cornielje h, ferrinho p, kemp s et al. d evelopment o f a community based rehabilitation programme for alexandra. c onference paper presented at the 10th conference o f the epidemiological society o f southern africa, cape town 1991. 7. dixon w y . bmdp statistical software. berkeley, california. university o f c alifor­ nia press, 1981. 8. ferrinho p, reinach sg. a profile o f patients attending the adult outpatient department at the alexandra health centre and university c linic in alexandra. chasa journal o f comprehensive health 1992; in press. 9. rex g. launching a problem service. the organisation o f an occupational health service in the primary health care co n test the case o f the alexandra worker is health outreach programme. s a fr m e d j 1991;80:404-406. _________________book review________________ stroke: caring and coping edited by: vivian f ritz and c laire penn p ublished by: w itw atersrand university p ress stroke is one o f the leading causes o f death and disability in south africa at present. this locally produced resource book is a co m p re­ hensive guide for patients, family m embers, care givers and profes­ sionals o f stroke victims. the book includes chapters written by a m em ber o f each o f the professionals that offer skills and services to patients that have su f­ fered a cerebral vascular accident. contributions from th e disciplines o f neurology, nursing, neuropsychology, occupational therapy, physiotherapy, social work and speech and hearing therapy are infor­ mative and relevant to the south african situation. t he book is written in a way that allows the lay public easy access to information that will encourage holistic rehabilitation. t he chapter on gadgets and resources and the contribution from the multi-disci plinary team m ake this book the most practical and informative man ual on the treatm ent and rehabilitation o f such victims. an added attraction to this book is its reasonable price that will ensure a wide distribution. trish w allner case studies authors are invited to submit articles in a new category “case studies” to the sa jour­ nal o f physiotherapy. guidelines: articles should be not longer than 1,000 words or 3 pages typed in double spacing. the article should comprise • short abstract: 40 50 words • short background to the problem • description of case history assessment, treatment, results of treatment • conclusion summary and recommenda­ tions. physiotherapy, november 1992 vol 48 no 4 page 57 r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) microsoft word qasim mansoor 92 original article long-term follow-up of corneal and sclerocorneal grafting in severe eye perforations qasim mansoor, roa rashad qamar, s. biswas, h.p. adhikary pak j ophthalmol 2006, vol. 22 no.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . see end of article for authors affiliations …..……………………….. corrrespondence to: qasim mansoor 12-bluestone drive heaton mersey stockport, cheshire uk received for publication august’ 2005 …..……………………….. purpose: the objective of the present study was to assess the prognosis of grossly perforated eyeballs due to pathological condition by attempting a repair with corneal or corneo-scleral graft with anterior segment reconstruction. material and method: a retrospective analysis of 26 eyes of 23 patients who underwent free-hand corneal or sclero-corneal graft for perforated eyeballs over a period of 15 years was performed. the minimum follow up period was 3 years, except one patient who died after 4 months from an unrelated cause. the causes of perforations included corneal melt associated with rheumatoid arthritis, chronic non-specific corneal ulcer, rupture of acute hydrops of keratoconus (down’s syndrome), herpes zoster keratitis, trauma in a buphthalmic eye, sloughing of the cornea due to strep. pneumoniae infection and chronic ulcer due to chemical injury. results: all the patients were able to keep their eyes with a variable amount of vision, except one eye that became phthisical after three months. the patients who did not develop better vision were for various reasons, e.g., pre-existing poor vision, retinal problems and cataract still to be dealt with. conclusion: the long-term psychological benefits of retaining the eye, preserving a modicum of ocular motility, avoiding enophthalmos which might occur post-enucleation, and in the case of children, facilitation of orbital development make free-hand corneal graft worth considering even in grossly perforated eyeballs due to pathological condition. there is also an uncertain but distinct possibility of restoring a partial degree of vision that might be extremely useful to the patient. he management of grossly perforated eyeballs due to a pathological condition is a very difficult task. the choices of management are evisceration and enucleation or an attempt at corneal repair with or without anterior segment reconstruction, using a corneal or corneo-scleral graft. the major principles of penetrating keratoplasty in pathologically ruptured globe or perforated cornea are excision of devitalised and infected tissue, anterior chamber reconstruction including anterior vitrectomy if needed, cataract extraction if indicated, medical control of raised intraocular pressure or prophylaxis by filtering surgery and careful follow-up to control postoperative complications1. various adjunctive procedures like tarsorrhaphy, conjunctival flap, cyanoacrylate glue and amniotic membrane grafting have also been tried. t 93 although there is a high risk of not being able to save the eye in gross perforations, it might be worth availing of the small chance of preserving globe integrity, if not visual acuity, by attempting a repair and reconstruction instead of a primary evisceration or enucleation. corneal or corneo-scleral grafting in a perforated eyeball due to pathological condition is a daunting task to perform. the preparation of recipient bed, with the usual technique of trephination is neither safe nor possible, due to the collapse of globe. moreover, it is difficult to ascertain the exact extent of the pathological process in order to make a viable bed with minimum tissue loss; hence the graft is not always circular. so in these circumstances the only choice on many occasions is to consider a free hand corneal graft or corneo-scleral graft. there have been reports of free hand lamellar graft for marginal corneal degeneration2,3 penetrating graft in epithelial down growth4, or in otherwise healthy perforated cornea5. to our knowledge there are few reports on free hand graft in such grossly perforated eyeballs due to pathological conditions. material and methods this is a retrospective analysis of 26 eyes of 23 patients who underwent penetrating keratoplasty or sclerocorneal graft along with anterior segment reconstruction for perforated eyeballs over a period of 15 years from january 1984 to december 1999. of the 23 patients, 9 were male and 14 female. their age range was 13 years to 87 years, the mean age being 63 years. the pathological conditions leading to these perforations are shown in (fig. 1). all these patients had very soft eyeball and all the patients were conservatively managed by medical treatment prior to their referral for corneal graft, except the one with trauma in a buphthalmic eye that also had posterior synechae and seclusio-pupil. he had been receiving argon laser treatment for corneal and iris vessel, prior to planned corneal graft with triple procedure. he received a blunt injury leading to a rupture of the globe. corneal grafting was performed in 15 eyes. in another 11 eyes, along with corneal grafting various other associated procedures were performed as shown in (fig. 2). a single surgeon operated all the patients, as an urgent case. extensive corneal disease and perforation of the cornea needed free hand corneal grafting. for descriptive purposes, these patients were divided into two groups. in group i there were 15 eyes, whose associated pathological conditions are shown in (table 1). in this group it was possible to make a free hand circular bed on the recipient cornea. so it was not necessary for free hand cutting of the donor corneal disc, with the usual circular trephination being performed for watertight closure of the wound. the donor corneal disc was trephined through endothelial surface. in group ii there were eleven eyes whose donor cornea needed to be cut by free hand. the associated pathological conditions in this group are shown in (table 2). in these cases due to uncertainty and the extent of pathological condition, it was necessary for free hand cutting of both the recipient and donor’s disc for water tight closure of the wound. trephines were used to mark the area to be excised and the cornea dissected free hand, in view of the softness of the globe, to avoid the risk of collapse and excess tissue loss. an oversized donor button was trephined or fashioned to allow for adequate wound closure. viscoelastic substance (sodium hyalunorate) was used to reform the anterior chamber and delaminate the iris from the lens; part of it was left to reduce the risk of synechiae formation. the graft was sutured, using continuous 10/0 nylon. along with corneal grafting various other procedures were also carried out in some cases at the same sitting. the average follow up period of all the patients was 3 years (range 4 months to 4 years). the short follow-up period of 4 months was due to death of a patient from an unrelated cause. results all the patients were able to keep their eyes with variable amount of vision, as shown in fig. 3. the patients who did not develop better vision were for various reasons, such as pre-existing poor vision, retinal problems and cataract still to be dealt with. the patients with corneal melt associated with rheumatoid arthritis were all above eighty years, except one. summary of the complicated cases with highly unfavorable outcome is given below: case i: a sixty-seven years old female patient with poorly controlled rheumatoid arthritis presented with simultaneous bilateral melting of the cornea except in the areas of calcification, in her aphakic eyes. she had a free hand corneo-scleral graft, anterior vitrectomy and iris supported secondary implants. two years 94 later the same pathology started in both eyes together and a similar surgical procedure, involving anterior vitrectomy, was carried out. however, within three months one eye became phthisical while the other eye regained an unaided vision of 6/60. (fig. 4-5) case ii: a patient who had severe atrophy of the upper eyelid in the affected eye, following herpes zoster ophthalmicus, and presented with perforation of the cornea due to exposure keratitis. she had to have multiple procedures like corneal graft, cataract extraction and intra ocular lens implants, pupilloplasty and reconstruction of upper eyelid. she regained an unaided vision of 6/36. this patient died after four months from an unrelated cause. two of the 3 patients with down’s syndrome, who had hydrops of cornea, were severely mentally challenged. one patient also suffered from severe psoriasis of the eyelids, which was successfully managed by the dermatologists. table i: group i: free hand cutting of recipient bed only. (associated pathological conditions leading to perforation) n (%) corneal melt associated with rheumatoid arthritis 5 (33.3) herpes zoster ophthalmicus 2 (13.3) trauma on buphthalmic eye 1 (6.7) non-specific corneal ulcer 5 (33.3) keratoconus 1 (6.7) infective corneal ulcer 1 (6.7) total 15 (100) table 2: group ii: free hand cutting of both donor and recipient beds (associated pathological conditions leading to perforation) n (%) corneal melt associated with rheumatoid arthritis 5 (45.5) acute hydrops of ruptured keratoconus 3 (27.3) sloughing of cornea due to strep. pneumoniae 2 (18.2) chemical injury 1 (9.1) total 11 (100) discussion the major causes of destruction of corneal tissue are infections, dry eye syndrome, chronic exposure keratitis, trophic ulcerations, melting syndromes and trauma. the primary goal of therapeutic penetrating keratoplasty is to excise devitalised and infected tissue and to restore structural integrity1. corneal grafts undertaken in disorganized eyes are at risk of failure. kirkness et al6 in an analysis of the relative success and complications of penetrating keratoplasty in perforated eyes found no significant difference between sterile perforated eyes and infected perforated eyes, suggesting that perforation itself is a major risk factor. they recommend that when perforation is imminent, it is better to proceed to a penetrating keratoplasty as the angle may be better protected from peripheral anterior synechiae formation. the risk is increased by the presence of anterior synchiae that may exert traction at the site of attachment, and may perhaps expose the donor endothelium to blood vessels and increase the risk of rejection7. it has been shown that after anterior segment reconstruction and restoration of anatomical integrity, there is reduction of postoperative corneal oedema and also improvement of visual acuity8. corneal perforations due to herpetic keratitis and rheumatoid arthritis have been treated with temporising measures such as tissue adhesives provided the perforation is small, to be followed by therapeutic keratoplasty as soon as donor tissue is available9,13-15. in both these conditions, melting and perforations are associated with increased corneal collagenase activity. intensive topical steroid are widely used post-operatively to reduce anterior chamber inflammation, with an umbrella of antiviral coverage in herpetic infections9. the systemic immune mediated inflammation in the late stages of rheumatoid arthritis can cause a melting of host-graft junction, which might necessitate even systemic immunosuppression. it has also been suggested that an unstable corneal epithelium might trigger collagenase activity; hence tear supplements are helpful to stabilise the epithelium in these patients10,16,17. patients with down’s syndrome have a poorer outcome following keratoplasty, as there is less ability of patients with down’s syndrome to report graft reactions and infections, their tendency to selftraumatise and their increased susceptibility to 95 infections. post-operative results depend on severity of the disease itself, tendency of eye rubbing and selftraumatization11,12. however, careful post-operative follow-up with care provided by an attendant can improve outcomes as was also borne out by our study. in all of the cases presented here, it was possible to retain the eye following surgery. in addition to the psychological benefits gained in retaining the eye, associated pathological conditions 10 5 4 2 1 3 1 corneal melt associated with rheumatoid arthritis chronic non-specific corneal ulcer rupture of acute hydrops of keratoconus chronic ulcer herpes zoster ophthalmicus trauma on buphthalmic eye sloughing of cornea due to strep.pneumoniae infection chronic ulcer due to chemical injury fig. 1: associated pathological conditions of the perforated eyeballs. corneal graft with other procedures 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 corneal, anterior vitrectomy with secondary iris supported lens corneal graft, cataract with pc iol corneal graft, pupiloplasty cataract iol corneal graft, iol in situ vitrectomy corneal graft, pupiloplasty, cataract extraction,iol, reconstruction of the upper lid fig. 2: corneal graft performed with other associated surgical procedures. results 0 2 4 6 8 10 12 14 16 18 20 no perception of light hand movements cf to 6/18 6/9 or better series1 fig. 3: fig. 4: corneal melt and perforation associated with rheumatoid arthritis preoperative appearance of the right eye (the left eye having the same condition had a similar appearance). 96 fig. 5: postoperative appearance of the same eye after the first reconstructive keratoplasty with an unaided vision of 6/60 six months after the operation. ocular motility is preserved, thus avoiding enophthalmos, which may occur post-enucleation. in the case of children, retention of the globe may have an advantageous effect on future orbital development. conclusion surgical repair of a perforated eyeball with a free-hand corneal or corneo-scleral graft is a difficult proposition even in the best of circumstances. however, the possibility of retention of the eyeball, if not useful vision, makes repair and reconstruction a worthwhile first line of approach in preference to removal of the remnants of the globe. free-hand corneal or corneo-scleral graft with anterior segment reconstruction, even in cases with poor visual prognosis, is perhaps the desired procedure in perforated eyeballs due to pathological conditions, despite the sometimes small chance of retaining the eyeball. in addition to retaining the eye, there is also the prospect of restoring partial vision in a miniscule proportion of patients, which might still make a significant difference for them. there is always the option of removal of the eye at a later date if required. however, by attempting reconstruction and restoration of the normal anatomy as a first step, there is a possibility of a favourable outcome even in an apparently futile scenario. author’s affiliation dr. qasim mansoor 12-bluestone drive heaton mersey stockport, cheshire uk roa rashad qamar assistant professor bahawal victoria hospital bahawalpur s. biswas 12-bluestone drive heaton mersey stockport, cheshire uk h.p adhikary 12-bluestone drive heaton mersey stockport, cheshire uk references 1. taylor dm, stern a l. reconstructive keratoplasty in the management of conditions leading to corneal destruction. ophthalmology. 1980; 87: 892-902. 2. pettit th. corneo-scleral free hand lamellar keratoplasty in terrien’s marginal degeneration of the cornea long-term results. refract corneal surg. 1991; 7: 28-32. 3. brown ac, rao gn, aquavella jv. peripheral corneal graft in terrien’s marginal degeneration. ophthalmic surg. 1983: 14: 931-4. 4. friedman ah. radical anterior segment surgery for epithelial invasion of anterior chamber: report on three cases. trans. am aca ophthalmol otolaryngol. 1997; 83: 216-223. 5. kramer sg. simplified technique for freehand corneal grafting in traumatic cases. arch. ophthalmol. 1971; 86: 182-5. 6. collin m, kirkness, linda a, et al: the role of penetrating keratoplasty in the management of microbial keratitis. eye 1991; 425-431. 7. keneyon kr, strack t, hersch ps. penetrating keratoplasty and anterior segment reconstruction for severe ocular trauma. ophthalmology. 1992; 396-402. 8. waring iii, keneyon kr, gemmil md. results of anterior segment reconstruction for aphakic and pseudophakic corneal oedema. ophthalmology. 1988: 836-41. 9. james t, patten, cavanagh d. penetrating keratoplasty in acute herpetic corneal perforations. annals of ophthalmology. 1976. 10. bernauer w, ficker la. the management of corneal perforation associated with rheumatoid arthritis. ophthalmology. 1995; 102. 11. joanathan m, frantz, micheal s, et al. penetrating keratoplasty for keratoconus in down’s syndrome. am j ophthalmol. 1990, 109:143-7. 12. akova ya, dabil h, kavalcioglu o, et al. clinical features and keratoplasty results in keratoconus complicated by acute hydrops. ocul immunol inflamm 2000; 8: 101-9. 97 13. reed jw, joyner sj, knauer wj 3rd. penetrating keratoplasty for herpes zoster keratopathy. am j ophthalmol. 1989 15; 107: 257-61. 14. claerhout i, beele h, abeele kvd, et al. therapeutic penetrating keratoplasty, clinical outcome and evolution of endothelial cell density. cornea. 2002; 21: 637-42. 15. killlingsworth dw, stern ga, driebe wt, et al. results of therapeutic penetrating keratoplasty. ophthalmology. 1993; 100: 534-41. 16. palay da, stulting rd, waring go. penetrating keratoplasty in patients with rheumatoid arthritis. ophthalmology. 1992; 99: 622-7. 17. vanathi m, sharma n, jeevan s, et al. vajpayee. tectonic grafts for corneal thinning and perforations. cornea. 2002; 21: 792-7. skin september 2019 volume 3 issue 5 copyright 2019 the national society for cutaneous medicine 338 brief articles new-onset vitiligo following etanercept for ankylosing spondylitis carly dunn, ba1, stacy l. mcmurray, md1, allison jones, md1, debendra pattanaik, md2 1department of dermatology, university of tennessee, memphis, tn 2division of rheumatology, university of tennessee, memphis, tn tumor necrosis factor-alpha (tnf-α) inhibitors are used widely to treat a variety of chronic inflammatory conditions such as ankylosing spondylitis (as), crohn’s disease, rheumatoid arthritis, and psoriasis. although tnf-α inhibitors have been proven to be effective and safe for use in many conditions, reports of paradoxical immunological skin conditions, such as psoriasis, granuloma annulare, lichen planus, and vitiligo, are associated with their use.1,2,3 we report a case of as who initiated therapy with a tnf-α inhibitor and developed vitiligo shortly thereafter. a 19-year-old male with diagnosis of as was referred by rheumatology for evaluation of suspected vitiligo. the patient was initially treated for as with adalimumab which failed to control the disease. he was subsequently switched to etanercept with marked improvement in his as symptoms. however, within a month of starting etanercept, he developed depigmented macules and patches on his upper vermillion lip (figure 1), anterior base of the neck, dorsal hands (figure 2), volar wrists, and thighs. the patient had no personal or family history of vitiligo. despite the marked improvement in as symptoms, the decision was made per abstract tumor necrosis factor-alpha (tnf-α) inhibitors, are used widely to treat a variety of chronic inflammatory conditions such as ankylosing spondylitis, crohn’s disease, rheumatoid arthritis, and psoriasis. recently, there has been an increase in the number of reports of immunemediated skin diseases, such as lichen planus, psoriasis, or granuloma annulare, following the initiation of tnf-α inhibitors. although the exact mechanism is unknown, the proposed hypothesis is that tnf-α blockade results in a cytokine shift which activates autoreactive t cells and ultimately leads to an immunologic imbalance. we present a patient with ankylosing spondylitis previously treated with adalimumab who developed vitiligo shortly after switching to etanercept to achieve better control of his joint disease. there is no currently treatment algorithm for new-onset vitiligo following tnf-α inhibitor use. in many cases the drug is continued with a favorable outcome; however, withdrawal of the tnf-α inhibitor with initiation of an alternative tnf-α agent or biologic agent in a different class may be considered. introduction case report skin september 2019 volume 3 issue 5 copyright 2019 the national society for cutaneous medicine 339 patient and physicians to switch from etanercept to secukinumab due to the vitiligo. at his 3-month follow up the patient’s skin lesions were stable with no progression of disease. figure 1. depigmented patch on patient’s upper vermillion lip. figure 2. well-circumscribed depigmented macules and patches on patient’s dorsal hands. tnf-α has been linked to the pathogenesis of vitiligo. this cytokine has been shown to inhibit melanocyte differentiation and function and can cause apoptosis of melanocytes.4 simon et al showed that tnfα blockade can improve existing vitiligo.5 however, there have been an increasing number of reports of new-onset vitiligo following initiation of tnf-α inhibitors. since the first case report of de novo vitiligo under infliximab in 2005,6 there have been at least 17 other case reports as well as a retrospective study and a population-based study detailing cases from use of various biological agents.1,3,7,8,9 one study estimates one per 5437 patients on a biologic agent will develop de novo vitiligo.9 although the exact mechanism is unknown, it is postulated that tnf-α inhibition modifies the cytokine balance and affects downstream pathways, resulting in activation of autoreactive t cells and immunologic imbalance.4,7 a retrospective study by exarchou et al showed that 10 out of 183 (5.5%) patients with as who had been treated with tnf-α inhibitors had immune-mediated skin lesions (with one case of vitiligo).1 a 10-year population-based study demonstrated an increased risk of vitiligo in patients receiving tnf-α inhibitors, with an incidence rate of 5.9 vs 2.5 per 10,000 person-years in those on tnf-α inhibitors versus control, respectively. this translates to an overall increased risk of 1.99 in the treatment group. in subgroup analysis, they found that the risk of vitiligo was highest in female patients, those on etanercept, and patients with as.7 our patient adds to the growing number of reports of etanercept-induced vitiligo in as after prior treatment failure with adalimumab. as de novo vitiligo following tnf-α inhibitor use is rare, there is currently no treatment algorithm for this phenomenon. in a nationwide retrospective study by merybossard et al, 18 patients with de novo vitiligo were reported from july 2013 to january 2015.8 in most cases (66.6%) the drug was continued without any worsening of the vitiligo. however, in six of these cases discussion skin september 2019 volume 3 issue 5 copyright 2019 the national society for cutaneous medicine 340 patients underwent treatment with topical steroids, which may be a confounding factor. in three cases, the tnf-α inhibitor was changed, due to the underlying inflammatory condition, without progression of the patients’ vitiligo. the authors conclude that continuing anti-tnf-α therapy is appropriate if the underlying inflammatory condition is well-controlled. other therapy options include an alternative tnf-α inhibitor or another biological agent in patients whose underlying inflammatory condition had not improved or whose skin lesions progress.8 with growing literature describing immunemediated skin diseases following tnf-α inhibitors, dermatologists, rheumatologists, and gastroenterologists need to be aware of these potential side-effects. an interdisciplinary approach to a patient presenting with vitiligo following initiation of a tnf-α inhibitor can help determine whether the patient should continue the tnf-α inhibitor, switch to a different tnf-α inhibitor, or start an alternative biologic medication. conflict of interest disclosures: none funding: none corresponding author: carly dunn, ba department of dermatology, university of tennessee 1977 butler blvd, suite e6.200 houston, tx 77030 713-870-5413 carly.dunn@gmail.com references: 1. exarchou sa, voulgari dv, markatseli te, zioga a, drosos aa. immune-mediated skin lesions in patients treated with anti-tumor necrosis factor alpha inhibitors. scand j rheum. 2009;38(5):328-331. 2. ismail wa, al-enzy sa, alsurayei sa, ismail ae. vitiligo in a patient receiving infliximab for refractory ulcerative colitis. arab j gastroenterol. 2011;12(2):109-111. doi:10.1016/j.ajg.2011.03.001. 3. carvalho cldb, ortigosa lcm. segmental vitiligo after infliximab use for rheumatoid arthritis – a case report. an bras dermatol. 2014;89(1). doi: 10.1590/abd1806-4841.20142887 4. toussirot e, aubin f. paradoxical reactions under tnf-α blocking agents and other biological agents given for chronic immune-mediated diseases: an analytical and comprehensive overview. rmd open. 2016;2(2):1-12. doi:10.1136/rmdopen-2015-000239. 5. simon ja, burgos-vargas r. vitiligo improvement in a patient with ankylosing spondylitis treated with infliximab. case rep dermatol. 2008;216:234-235. doi: 10.1159/000112932 6. ramirez‐hernandez m, marras c, martinez‐ escribano ja. infliximab‐induced vitiligo. dermatology 2005; 210(1):79–80. doi:10.1159/000081494. 7. bae jm, kim m, lee hh, et al. increased risk of vitiligo following anti-tumor necrosis factor therapy: a 10-year population-based cohort study. j invest dermatol. 2018;138(4):768-774. doi:10.1016/j.jid.2017.11.012. 8. mery-bossard l, bagny k, charby g, et al. new onset vitiligo and progression of pre-existing vitiligo during treatment with biological agents in chronic inflammatory diseases. j eur acad dermatol venerol. 2017;31:181-186. doi:10.1111/jdv.13759. 9. webb kc, tung r, winterfield ls, et al. tumour necrosis factor-α inhibition can stabilize disease in progressive vitiligo. br j dermatol. 2015;173(3):641–650. doi:10.1111/bjd.14016. mailto:carly.dunn@gmail.com https://doi.org/10.1159/000112932 microsoft word 55-62 chemistry | 55 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 estimation of pentraxin-3(ptx3) in rheumatoid arthritis males’ patients (with and without) type ii diabetes mellitus in iraq nashwa s. sultan hamza2006n@gmail.com  anwar f. altaie dr.anwar1979@yahoo.com department of chemistry, college of education for pure science ibn al-haitham, university of baghdad, baghdad, iraq article history: received 30 august 2018, accepted 4 september 2018, published december 2018 abstract rheumatoid arthritis is a chronic inflammatory autoimmune disease its etiology is unknown. the classical autoimmune diseases, have adaptive immune genetic associations with autoantibodies and major histocompatibility complex (mhc) class ii such as rheumatoid arthritis (ra), diabetes mellitus type two (dm ii). serum of99 males suffering from ra without dmii as group (g1), 45 males suffering from ra with dm ii as group (g2) and 40 healthy males as group (g3) were enrolled in this study to estimation of alkaline phosphates (alp), c-reactive protein (crp) and pentraxin-3(ptx). results showed a highly significant increase in ptx3 levels in g1 and g2 compared to g3 and a significant decrease in g1comparing to g2. results also revealed a significant increase in crp levels in g1 and g2 when compared to g3, as well as a significant increase in g2 comparing to g1. results showed a significant decrease in alp levels in g1 and g2 while this phrase must no differences was observed betweeng1 and g2and there was no significant positive correlation between ptx3 and alp in sera of ra males’ patients with and without dm ii it be showed in our study. keywords: rheumatoid arthritis; diabetes mellitus; pentraxin; c-reactive protein; alkaline phosphates; methotrexate; folic acid omega 3 1. introduction pentraxin are a family of evolutionarily conserved pattern-recognition proteins that are acute phase protein made up of five identical subunits. based on the primary structure of the subunit that produced by immune and structural cells [1]. the pentraxin are structurally unrelated to the collections and include small pentraxin such as c-reactive proteins (crp), serum amyloid protein (sap) and such as ptx3 [14]. the multifunctional properties of ptx3 can be at least in part explained by its capacity to interact with a number of different ligands a characteristic shared with crp and serum amyloid protein (sap) [4]. recent studies have shown that ptx3 levels elevated in the presence of a bacterial infection [5]. it is a soluble inflammation and innate immunity [1]. study demonstrated that ptx3 have a role in allergic asthma [6] so several kinds of cell types are confirmed to produce ptx3—vascular endothelial cells, vascular smooth muscle cells and monocytes pattern recognition receptor with non-redundant functions in macrophages. [1]. acute-phase protein is a group of proteins that are synthesized in greater amounts include c-reactive protein (crp) in acute tissue damage, due to trauma chronic inflammation and malignant disease, [7, 8]. acute phase chemistry | 56 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 protein an include c-reactive protein (crp) which available marker led to. its concentration that increase 30-fold from a normal value of less than 5 mg/l during the acute phase response, rheumatoid arthritis and crohn’s disease [9]. its measurement appears to be both more sensitive and more specific than measurements of the erythrocyte sedimentation rate (esr) and plasma viscosity in this respect [9, 10]. alkaline phosphates (alp) comprise a group of enzymes that catalyze the hydrolysis of phosphate esters in an alkaline environment, generating an organic radical and inorganic phosphate [11]. alkaline phosphates are derived from a number of different tissues, including the liver, the osteoblasts in bone and the placenta. plasma activities rise in cholestatic liver disease because alp synthesis is increased and the enzyme within the bleary tract is regurgitated into plasma [12, 13]. this study aimed to evaluate ofpentraxin-3 (ptx3) in iraqi males’ patients suffering from ra with and without dm ii. in addition to found correlation relation for ptx3 with alp in ra patients with dm ii and without dm ii. 2. methods 2.1. patients study the samples of blood were collected from 188 males with age ranged between (18-67) years were enrolled in this study in medical city hospital in baghdad, general hospital in basra, teaching hospital and al-salaams hospital in mosul from october 2017 to april 2018.they were divided into three groups as follows: 1. rheumatoid arthritis patients without dm ii as group one (g1) that consist of (99) males. 2. rheumatoid arthritis patients with dm ii as group two (g2) that consist of (45) males. 3. healthy control groups as group three (g3) that consist of (40) males. all patients in groups (g1) and (g2) were taking methotrexate, folic acid and omega3 treatment, smoker patients were excluded from this study ptx3 were estimated by elasa kit no: yhb 2259hu from china. crp estimated by latex method kit l21-v3 from uk and alp estimated by a colorimetric method according to kit from france. ec 3.1.3. 2.2. statistical analysis the statistical analysis of this prospective study performed with the graph pad prism® 7 and microsoftexcel2013. numerical data with normal distribution were described as mean and standard deviation, analysis of variance (anova) used for multiple comparison using least significant difference. categorical data were described as count and percentage. chi-square test or fisher exact test used to estimate the association between variables. the lower level of accepted statistical significant difference is bellow or equal to 0.05. correlation of coefficient used for estimation of correlation between studied variables. alan c.2007). 3. results and discussion table 1. and figures 2. and 3. showed levels of ptx3, crp% and alp for the studies groups, that results showed a highly significant increase in ptx3 levels in g1 and g2 compared with g3 and no significant decreasing in ptx3levels in g1compared with g2. results also revealed a highly significant increase in crp levels in g1 and g2 when compared to g3, as well as a highly significant increase in g2 comparing to g1. results showed a highly significant decrease in alp levels in g1 and g2 when compared with g3, and there were a highly significant different between g1 compared with g2. chemistry | 57 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 table1. concentrations of pentraxin3, crp, and alp in males’ iraqi patients and healthy control. p value g3 no:40 g2 no:45 g1 no:99 groups parameters g1&g3 g2&g3 g1 & g2 0.001 h.s 0.001 s 0.129 n.s 0.68±0.09 3.02 ± 0.5 2.65 ± 0.78 ptx3 -µg/ml 0.001 h.s 0.001 h.s 0.001h. s 0% 37.8% 47.2% crp iu/ml 0.001 h.s 0.001 h.s 0.001h.s 48.95 ± 4.82 28.05± 5.61 22.04 ± 3.776 alp u/l g1 = ra without dm ii, g2=ra with dm ii, g3=healthy control s= significant p value, h. s=high significant p value., n. s=non-significant figure 1. pentraxin concentration -µg/ml in males patients and healthy control. chemistry | 58 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 figure 2. alp activity u/l, in males’ patients and healthy control. tables 2. and 3., figures 2. and 3. showed the level of pentraxin-3 in sera of the ra patients without and with dmп, respectively. groups gg (baghdad patients), gb (basra patients), and gm (mosul patients). the results of all groups in these tables and figures represent a highly significant different in ptx3 levels in ra patients without dmii in three cities (gg, gb, and gm), while there were a highly significant different in ptx3 levels in ra patients with dmii between (gg and gb) and (gb and gm) cities, while there were no significant different between (gg and gm) cities when compared with others. the results also show a highly significant different in crp levels in ra patients without dmii in three cities (gg, gb, and gm) when compared with them. in addition to no significant different in crp levels in ra patients with dmii between (gg and gb) and (gb and gm), while there was a significant different in crp levels in ra patients with dmii between (gg and gm) cities. table 2. concentrations of pentraxin-3, crp, and alp in three major cities of iraqi patients of ra without dmii. p value gm gb gg groups parameters gg vs. gm gb vs. gm gg vs. gb 0.001 h.s 0.001 h. s 0.001h.s 3.75±1.01 1.58±0.5 2.63±0.72 ptx3 µg/ml 0.001h.s 0.001h. s 0.001h. s 58.33% 43.75% 39.6% crp iu/ml 0.209 n.s 0.071n.s 0.002 s 22.23 ± 3.60 23.3 ± 4.4 20.59 ± 3.33 alp u/l gg=baghdad patients of ra. without dmп , gb=basra patients with ra. without dmп gm =mosul patients with ra. without dmп the concentration of ptx3 and crp were high in ra patient (with and without dmtt) this results lead to expect that (ptx3 and crp) may be a sensitive indicator of clinical arthritis. chemistry | 59 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 also, table 3. and figure 4. show no significant different in alp levels in ra patients with dm ii between (gg and gb) cities, and a highly significant decrease between (gb and gm), in addition to a significant different between (gg and gm) when compared with them. table 3. concentration pentraxin-3, crp and alk.p in three major cities of iraqi patients of ra with dm ii. p value gm gb gg groups parameters gg vs. gm gb vs. gm gg vs. gb 0.831 n.s 0.001h. s 0.0001 h.s 3.71 ± 1.05 1.76 ±0.91 3.59±0.71 ptx3 µg/ml 0.032.s 0.516n.s 0.663 n.s 26.7% 53.3% 33.3% crp iu/ml 0.021 s 0.001 h.s 0.153n.s 31.01 ± 4.77 25.31± 3.52 27.75 ± 6.7 alp u/l gg=baghdad patients of ra. with dmп , gb=basra patients with ra. with dmп gm =mosul patients with ra. with dmп figure 3. pentraxin concentration -µg/ml, in males patients of ra with and without dm ii and healthy control. chemistry | 60 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 figure 4. alp activity u/lin males’ patients of ra with and without dm ii in three iraqi cities. table 4. correlation between ptx-3 and alp in ra patients with and without dmtt. alp without dmtt 0.271 r ptx-3 0.072 p with dmtt 0.04 r ptx-3 0.633 p ra is an inflammatory disease characterized by chronic inflammation of the symposium, particularly of small joints, which of then leads to destruction of at ocular cartilage and juxta auricular one [14]. table 1. showed the level of pentraxin-3 (ptx3) in sera of g1, g2results of all groups represent significantly a high levels of ptx3 in g1 and g2 as a compared with g3.pentraxins are a group of highly conserved as a modulators of inflammatory processes that primarily produced and released by vascular cell wall study revealed that elevation in (ptx3) level in patients with ra due to the ptx3 belongs to a super family of phylogenically conserved multiservice proteins, with includes short and long pentraxin3. combination of ptx3 and crp could serve as better differential diagnostic markers for ra [15]. c-reactive protein a markers of system inflammation elevated crp levels have also been linked to an incensed risk of later development of diabetes [16]. and it being strongly predicting for the future development of ra in crp is a sensitive marker of systemic inflammation and is elevated in patients with ra [17]. as the have suggest alp are most effective in an alkaline medium [14]. infect the level of serum alp is increased in disorders characterized by accelerated bone turnover. for a long time, the diagnose is of ra was mainly based on clinical manifest a torn sand alp may add useful information for assessing fracture risk and for monitoring osteoporosis in ra patient [17]. ra is the main causes of increased level of alp because affect the wrist and small joints of the body besides the joints [18], but in and present study showing decrease in alp in patients of ra with and without dmп that is due to all patients taking a methotrexate a drug for treatment and taking (omega3 and folic acid) [19-20]. provide that omega 3 influence alp activities [19]. methotrexate is the most important drug modifying anti rheumatic for the treatment of ra and currently considered as the central drug for the standard care and the management of ra [21]. omega 3 is found primarily in fatty fish with high oil content. it's widely used in the treatment of chemotherapy chemistry | 61 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 as a common consist of multidrug regimens [22]. folic acid as an antagonist agonist which inhibits de novo synthesis of the nucleoside thymidine a prerequisite for dna synthesis. folic acid would be useful in reducing toxic manifestations that occur airing long term treatment with low-dose mtx for ra [23]. 4. conclusions this study is the first that observe the elevation of ptx3 levels in serum of iraqi male’s ra patients with and without dmп in three cities (baghdad, basra and mosel) therefore indicated that ptx3 may be a good biomarker for ra with and without dmп. references 1. shindo, a.; maki, t.; mandeville, et.; liang, ac.; egawa, n.; itoh, k.; itoh, n.; borlongan, m.; holder. j.c.; chuang, t.t.; mcneish, j.d. astrocyte-derived pentraxin-3 supports blood–brain barrier integrity under acute phase of stroke. stroke. 2016,47,4,1094-1100. 2. barbati, e.; specchia, c.; villella, m.; rossi, m.l.; barlera, s.; bottazzi, b.; crociati, l.; d’arienzo, c.; fanelli, r.; garlanda, c.; gori, f. influence of pentraxin 3 (ptx3) genetic variants on myocardial infarction risk and ptx3 plasma levels. plos one. 2012, 7, 12, 1-7. 3. yang, h.s.; woo. j.e.; lee, s.j.; park, s.h.; woo, j.m. elevated plasma pentraxin 3 levels are associated with development and progression of diabetic retinopathy in korean patients with type 2 diabetes mellitus. investig. ophthal mol. vis. sci. 2014, 55,9, 5989-5997. 4. george, s.j.; johnson, j. atherosclerosis: molecular and cellular mechanisms. john wiley & sons. 2010. isbn: 978-3-527-32448-4 5. thulborn, s.j.; dilpazir, m.; haldar, k.; mistry, v.; brightling, c. e.; barer, m.r.; bafadhel, m. investigating the role of pentraxin-3 as a biomarker for bacterial infection in subjects with copd. int j chron obstruct pulmon dis. 2017, 12,11991205. 6. zhang, j.; shan, l.; koussih, l.; redhu, n.s.; halayko, a.j.; chakir, j.; gounni, a.s. pentraxin-3 (ptx3) expression in allergic asthmatic airways: role in airway smooth muscle migration and chemokine production. plos one. 2012, 7, 4, 1-9. 7. whitby, l.g.; smith, a.f.; beckett, g.j. lecture notes on clinical chemistry. 4th ed. 1988. 8. dayoub, r.; buerger, l.; ibrahim, s.; melter, m.; weiss, t.s. augmenter of liver regeneration (alr) exhibits a dual signaling impact on hepatic acute-phase response. exp mol pathol suppl. 2017, 102, 3, 428-433. 9. du clos, t.w.; mold, c. pentraxins (crp, sap) in the process of complement activation and clearance of apoptotic bodies through fcγ receptors. current opinion in organ transplantation. 2011, 16, 1, 1-11.  10. rifai, n.; horvath, a.r.; wittwer, c. tietz. textbook of clinical chemistry and molecular diagnostics. elsevier. 2018. 11. najeeb, q.; aziz, r. comparison of alkaline phosphatase. lactate dehydrogenase and acid phosphatase levels in serum and synovial fluid between patients with rheumatoid arthritis and osteoarthritis. int. j. sci. res. 2015, 4, 4, 1069-1072. 12. crook, m. clinical chemistry & metabolic medicine. london: hodder arnold. 2006. chemistry | 62 ibn al-haitham jour. for pure & appl. sci. ihjpas https://doi.org/10.30526/31.3.2010 vol. 31 (3) 2018 13. syvolap, v.d.; nazarenko, e.v. evaluation data of laboratory and instrumental studies in gastroenterology. 2017. 14. abhishek, d.; daving, h.; suman, d.; laljem, h.; th ibetombi d. study of serum alkaline phosphatase level in rheumatoid arthritis. int j med r. 2017, 3, 3, 318-321. 15. sharma, a.; khan, r.; gupta, n.; zaheer, m.s.; abbas, m.; khan, s.a. acute phase reactant, pentraxin-3, as a novel marker for the diagnosis of rheumatoid arthritis. clin chim acta. 2018, 480, 65-70. 16. king, d.e.; mainous, a.g.; buchanan, t.a.; pearson, w.s. c-reactive protein and glycemic control in adults with diabetes. diabetes care. 2003, 26, 5, 1535-1539. 17. shadick, n.a.; cook, n.r.; karlson, e.w.; ridker, p.m.; maher, n.e.; manson, j.e.; buring, j.e. lee i.m. c-reactive protein in the prediction of rheumatoid arthritis in women. arch intern med. 2006, 166, 22, 2490-2494. 18. chandrakar, b.l.; harish, c. s.; chandrakar, k. s. activity of serum alkaline phosphatase in rheumatoid arthritis for diagnosis and management. int j med res prof. 2017, 3,3, 281-284. 19. shaimaa, s.k. evaluation of omega-3effect as adjuvant therapy to methotrexate on alkaline phosphates level in iraqi patients with active rheumatoid arthritis. tikrit journal of pharmaceutical. 2017, 12, 1, 9-16. 20. namiy, i. h.; essam n.; sana,a, h. a. effect of type 2 diabetes mellitus on extracellular 21. superoxide dismutase: without complications among iraqi patients. ihjpas. 2016, 29, 3, 132 145. 22. zhao, n.; zheng, g.; li, j.; zhao, h.y.; lu, c.; jiang, m.; zhang, c.; guo, h.t., lu, a.p. text mining of rheumatoid arthritis and diabetes mellitus to understand the mechanisms of chinese medicine in different diseases with same treatment. chin j integr med. 2018, 24, 10, 777-784. 23. karri, s.; vanithakumari, g. impact of methotrexate and leucovorin on hormonal regulated enzymes of carbohydrate metabolism in accessory reproductive tissues of ovariectomized rats. transl med. 2012, 2, 2, 1-9. 24. morgan, s.l.; baggott, j.e.; vaughn, w.h.; young, p.k.; austin, j.v.; krumdieck, c.l.; alarcon, g.s. the effect of folic acid supplementation on the toxicity of low-dose methotrexate in patients with rheumatoid arthritis. arthritis & rheumatology. 1990, 33, 1, 9-18. sudan journal of medical sciences volume 16, issue no. 2, doi 10.18502/sjms.v16i2.9294 production and hosting by knowledge e research article association between inflammatory cytokines and liver functions in rheumatoid arthritis patients mohamed abdelrhman eltahir1,2, kawthar abdelgaleil mohammedsalih3, elhaj noureldien mohamed3, faisal makki babekir4, and amar mohamed ismail5 1department of clinical chemistry, faculty of medical laboratory, sudan university of science and technology, khartoum, sudan 2department of clinical chemistry, faculty of medical laboratory, gadarif university, khartoum, sudan 3department of hematology, faculty of medical laboratory, sudan university of science and technology, khartoum, sudan 4blood bank and laboratory department, al-amal hospital, khartoum, sudan 5department of biochemistry and molecular biology, faculty of science and technology, alneelain university, khartoum, sudan orcid: mohamed abdelrhman eltahir: http://orcid.org/0000-0003-0172-8362 abstract background: rheumatoid arthritis (ra) is associated with abnormal liver tests, and the medications used for ra are often hepatotoxic. therefore, this study aimed to investigate an association between pro-inflammatory and anti-inflammatory cytokines and liver function tests in ra patients. methods: in this descriptive cross-sectional study, 88 ra patients were included, 84 of them were women and 4 men, aged 21–81 years. serum interleukin-10 (il-10), interleukin-17 (il-17), osteopontin (opn) were measured and liver function tests were conducted. results: the frequency of ra was higher among adults aged >41 years (72 [81.8%]) than young adults aged ≤41 years (16 [18.2%]). ra was more common in women (84 [95.5%]) than in men (4 [4.5%]) – approximately 21:1-fold. young adults had higher abnormal il-10 than adult ra patients (or = 3.72, p-value 0.044). abnormal il-17 (or = 5.67, p-value 0.034) was found to be increased in young-adult ra patients. no association was observed between age and opn and between the duration of disease and il-10, il-17, and opn. similarly, no association was noted between the types of treatment and il-10, il-17, and opn, nor between il-10, il-17, opn and liver parameters (ast, alt, alp, alb, tp, and ggt). conclusion: pro-inflammatory and anti-inflammatory cytokines are not associated with abnormal liver functions, as has been demonstrated in ra patients. keywords: rheumatoid arthritis, interleukin, liver function tests, cytokines how to cite this article: mohamed abdelrhman eltahir, kawthar abdelgaleil mohammedsalih, elhaj noureldien mohamed, faisal makki babekir, and amar mohamed ismail (2021) “association between inflammatory cytokines and liver functions in rheumatoid arthritis patients,” sudan journal of medical sciences, vol. 16, issue no. 2, pages 276–284. doi 10.18502/sjms.v16i2.9294 page 276 corresponding author: mohamed abdelrhman eltahir; department of clinical chemistry, faculty of medical laboratory, sudan university of science and technology, khartoum, sudan. email: alsahaf33@yahoo.com received 23 april 2021 accepted 6 june 2021 published 30 june 2021 production and hosting by knowledge e mohamed abdelrhman eltahir et al. this article is distributed under the terms of the creative commons attribution license, which permits unrestricted use and redistribution provided that the original author and source are credited. editor-in-chief: prof. mohammad a. m. ibnouf http://www.knowledgee.com http://orcid.org/0000-0003-0172-8362 mailto:alsahaf33@yahoo.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ sudan journal of medical sciences mohamed abdelrhman eltahir et al 1. introduction rheumatoid arthritis (ra) is a common autoimmune inflammatory disease. although the prevalence of ra is lower globally (0.5–1%), it is associated with socioeconomic burden and higher risk of mortality rate [1]. recent studies have demonstrated that the treatments used for ra improved outcome, and also accounts as a risk for hepatic complications [2]. the adverse effects of ra treatments include asymptomatic elevations of liver enzyme, fibrosis, and sometimes fatal hepatic necrosis [3]. on the other hand, liver disorders have been noted in untreated ra patients [4]. increasing amounts of interleukin-10 (il-10), a potent anti-inflammatory cytokine [5], can be detected in the synovium of ra patients. additionally, considering that the activity of ra cannot be attenuated by il-10 administration [6], many researchers suggest that il-10 plays an important role in chronic liver diseases [7]. interleukin-17 (il-17), a proinflammatory cytokine, is upregulated in many autoimmune diseases such as ra; high levels of il-17 have been reported to be produced in different samples of ra [8, 9]. some investigators suggest that il-17 plays a key role in many liver diseases and is also associated with the progress of the disease [10–12]. osteopontin (opn) is a proinflammatory cytokine that induces ra [13–15], and included in many liver diseases, despite its role in liver problems are still controversial [16]. therefore, this study was carried out to find out the association between pro-inflammatory, anti-inflammatory cytokines and liver function tests among ra patients. 2. materials and methods this descriptive cross-sectional hospital-based study was conducted on 88 ra patients who were clinically diagnosed according to the criteria of the american college of rheumatology (acr) 1987 and were examined at the common ra clinics in khartoum state (military, alamal hospital, and zain clinic). all patients received treatment; the demographic data, type of treatment, and duration of disease for each patient were recorded – 4 men and 84 women aged 28–90 years. non-sudanese patients with ra and those with unclear diagnosis were excluded. serum from each subject were centrifuged at 3000 g for 10 min after clotting for 30 min at room temperature and stored at –40°c until analysis. all samples were investigated for opn, il-17, and il10 by sandwich enzyme-linked immune sorbent assay (elisa) (elisa development; thermo fisher scientific systems, usa) according to the manufacturer’s instructions. in addition, liver functions tests (tp, albumin, ast, alt, ggt, and alp) were done using doi 10.18502/sjms.v16i2.9294 page 277 sudan journal of medical sciences mohamed abdelrhman eltahir et al fully automated mindray chemistry analyzer (bs 200). data were statistically analyzed by statistical software package, version 16. results were expressed as numbers and percentages. chi-square test was used to determine the level of significance (p-value of 0.05 was considered to be statistically significant). 3. results ra is more common in adults (72 [81.8%]) than young adults (16 [18.2%]), the frequency of ra was found to be higher in women (84 [95.5%]) than in men (4 [4.5%]). moreover, 52 (59.1%) patients were receiving steroids while 36 (40.9%) were on non-steroid treatment. the duration of disease for 62 (70.5%) patients was ≤6 years and for 26 (29.5%) was >6 years. abnormal il-10 was found in 63 (71.6%) patients, while 25 (28.4%) had a normal percentage. the results of characteristic data show that while 80 (91%) ra patients had normal il-17, 8 (9%) had abnormal. normal opn was observed in 76 (86.4%) ra patients and abnormal opn in 12 (13.6%) (table 1). chi-square analysis revealed that young adults group had a higher abnormal il-10 than adult ra patients (or = 3.72, p-value 0.044). also, abnormal il-17 (or = 5.67, p-value 0.034) was found to be increased in young adult ra patients while no association was seen between age and opn (or = 2.67, p-value 0.144; table 2). furthermore, no association was reported between the duration of the disease and il-10, il-17, and opn with p-values 0.410, 0.176, and 0.502 and or 0.77, 0.37, and 1.30, respectively (table 3). similarly, no association could be derived between the types of treatment and il-10, il-17, and opn with p-value 0.246, 0.286, and 0.351 and or 1.53, 2.21, and 0.65, respectively (table 4). pearson’s correlation analysis revealed that there were no association between il-10, il-17, opn and liver parameters (ast, alt, alp, alb, tp, and ggt; table 5). 4. discussion abnormal liver functions were observed in ra patients. the researchers further attributed the abnormality to immune aggregations and others justified it by drugs toxicity. accordingly, this study was carried out to assess whether the pro-inflammatory or anti-inflammatory cytokines are associated with liver functions in ra patients. the current study revealed that there is no association between interleukins and liver function tests. in fact, abnormal liver tests were noted in patients with ra [17]. concurrent with many previous studies, the frequency of ra is higher in elderly subjects [18, 19]. a possible explanation might be that the protective mechanisms in elderly population are doi 10.18502/sjms.v16i2.9294 page 278 sudan journal of medical sciences mohamed abdelrhman eltahir et al table 1: demographic and baseline characteristics of ra patients. variables frequency (%) age (yr) ≤41 16 (18.2%) >41 72 (81.8%) sex male 4 (4.5%) female 84 (95.5%) treatment steroid 52 (59.1%) non-steroid 36 (40.9%) duration (yr) ≤6 62 (70.5%) >6 26 (29.5%) cut-off il-10 abnormal 63 (71.6%) normal 25 (28.4%) cut-off il-17 abnormal 8 (9%) normal 80 (91%) cut-off opn abnormal 12 (13.6%) normal 76 (86.4%) total 88 (100%) table 2: association between interleukins il10, il17, opn and age groups. variables age (yr) or ci-lower ci-upper p-value ≤≤≤41 >>>41 il-10 abnormal 14 (23.0%) 47 (77.0%) 3.72 (0.78–17.7) 0.04 normal 2 (7.4%) 25 (92.6%) il-17 abnormal 2 (40.0%) 6 (60.0%) 5.67 (1.24–25.7) 0.03 normal 12 (15.0%) 68 (85.0%) opn abnormal 4 (33.3%) 8 (66.7%) 2.67 (0.69–10.2) 0.14 normal 12 (15.8%) 64 (84.2%) decreased, resulting in decreased immunotolerance and decreased cytokines synthesis and t cells proliferation [20]. the demographic data indicated that the prevalence of ra was found to be 21-fold higher in women than in men. in contrast to a previous study in sudan, the female-to-male ratio was 9:1 [21]. since the change in sex hormones after doi 10.18502/sjms.v16i2.9294 page 279 sudan journal of medical sciences mohamed abdelrhman eltahir et al table 3: association between interleukins il10, il17, opn and duration of ra. variables duration (yr) or ci-lower ci-upper p-value ≤≤≤6 >>>6 il-10 abnormal 42 (68.9%) 19 (31.1%) 0.77 (0.28–2.13) 0.41 normal 20 (74.1%) 7 (25.9%) il-17 abnormal 4 (50.0%) 4 (50.0%) 0.37 (0.08–1.65) 0.17 normal 58 (72.5%) 22 (27.5%) opn abnormal 9 (75.0%) 3 (25.0%) 1.30 (0.32–5.25) 0.50 normal 53 (69.7%) 23 (30.3%) table 4: association between interleukins il10, il17, opn and types of treatment. variables treatment or ci-lower ci-upper p-value steroid non-steroid il-10 abnormal 38 (62.3%) 23 (37.7%) 1.53 (0.61–3.83) 0.24 normal 14 (51.9%) 13 (48.1%) il-17 abnormal 6 (75.0%) 2 (25.0%) 2.21 (0.42–11.6) 0.28 normal 46 (57.5%) 34 (42.5%) opn abnormal 6 (50.0%) 6 (50.0%) 0.65 (0.19–2.21) 0.35 normal 46 (60.5%) 30 (39.5%) puberty is associated with high prevalence of ra in women, a woman’s immune system is potentially more reactive than that of a man. the current study reports that young adults are more likely to have abnormal il-10 and il-17. however, these results disagree with previous studies [22, 23]. no association was found between age and opn level. concurrent with this finding, a relationship between age and opn has been previously reported [24]. similar to other results, no associations between il10, il17, opn levels and the duration of disease have been demonstrated [22, 23, 25]. despite reducing il-17 after the use of steroids therapy, il-10 was increased [26]. the present study revealed no associations between il-10, il-17, opn levels and the types of treatment. it has become clear that steroids directly modulate the pro-inflammatory cytokine or suppress cytokines-producing cells [26, 27]. doi 10.18502/sjms.v16i2.9294 page 280 sudan journal of medical sciences mohamed abdelrhman eltahir et al table 5: association between cytokines and liver function parameters (pearson’s correlation results). parameters p-value r2 il-10 ast 0.62 0.12 alt 0.20 0.66 alp 0.80 0.05 alb 0.16 –0.13 tp 0.56 0.02 ggt 0.25 0.15 il-17 ast 0.18 –0.15 alt 0.82 0.02 alp 0.82 –0.02 alb 0.23 0.12 tp 0.59 0.05 ggt 0.17 –0.14 opn ast 0.50 0.07 alt 0.25 0.12 alp 0.89 0.01 alb 0.29 –0.11 tp 0.49 0.07 ggt 0.98 –0.02 5. conclusion the data of present study shows that women are at a higher risk to have ra. moreover, young adult ra patients are more likely to have abnormal il-10 and il-17. furthermore, pro-inflammatory and anti-inflammatory cytokines are not associated with abnormal liver functions as has been demonstrated in ra patients. acknowledgements the authors would like to express their sincere gratitude to the dr. mariam abbas for her consultation and wisdom. may god bless her. ethical considerations ethical permits for the study were obtained from the ethical review committees at the sites where patients were recruited, and all patients gave informed consent for their participation in the study. doi 10.18502/sjms.v16i2.9294 page 281 sudan journal of medical sciences mohamed abdelrhman eltahir et al competing interests the authors declare no known conflicts of interest in relation to this paper. availability of data and material the study data are available with the author upon reasonable request funding none. references [1] kwan ho, t. c., mok, c. c., cheung, t. t., et al. (2019). management of rheumatoid arthritis. clinical rheumatology, vol. 38, pp. 3331–3350. [2] sundbaum, j. k., eriksson, n., hallberg, p., et al. (2019). methotrexate treatment in rheumatoid arthritis and elevated liver enzymes: a long−term follow−up of predictors, surveillance, and outcome in clinical practice. international journal of rheumatic disease, vol. 22, no. 7, pp. 1226–1232. [3] conway, r. and carey, j. (2017). risk of liver disease in methotrexate treated patients. world journal of hepatology, vol. 9, no. 26, pp. 1092–1100. [4] rakuomi, o., go, o., kamau, e., et al. (2017). prevalence of abnormal liver function tests in rheumatoid arthritis. african journal of rheumatology, vol. 5, no. 1, pp. 70–75. [5] shikhpour, e., noorbakhsh, p., elnaz, f., et al. (2018). a survey on the role of interleukin 10 in breast cancer. report of biochemistry and molecular biology, vol. 7, no. 1, pp. 1–10. [6] holdsworth, s. r. and yi, g. p. (2015). cytokines: names and numbers you should care about. clinical journal of the american society of nephrology, vol. 10, no. 12, pp. 2243–2254. [7] zhang, l. j. and wang, x. z. (2006). interleukin-10 and chronic liver disease. world journal of gastroenterology, vol. 12, no. 11, pp. 1681–1685. [8] elvira, d., nasrul, e., sofyan, y., et al. (2018). increased serum levels of interleukin17 and transforming growth factor in patients with graves’ disease. earth and environmental science, vol. 125, pp. 1–4. doi 10.18502/sjms.v16i2.9294 page 282 sudan journal of medical sciences mohamed abdelrhman eltahir et al [9] mengesha, b. g. and conti, h. r. (2017). the role of il-17 in protection against mucosal candida infections. journal of fungi, vol. 3, no. 52, pp. 1–12. [10] du, w. j., zhen, j. h., zeng, z. q., et al. (2013). expressing of interleukin-17 associated with disease progression and liver fibrosis with hepatitis b virus infection: il-17 in hbv infection. diagnostic pathology, vol. 8, pp. 1–7. [11] tan, z., qian, x., jiang, r., et al. (2013). il-17a play acritical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation. journal of immunology, vol. 191, no. 4, pp. 1–11. [12] zheng, l., chu, j., shi, y., et al. (2013). bone marrow-derived stem cells ameliorate hepatic fibrosis by down-regulating interleukin-17. cell & bioscience, vol. 3, article 46. [13] shi, l., shi, l., wang, x., et al. (2018). regulatory roles of osteopontin in production of monocyte-origin mcp-1. cell transplantation, vol. 27, no. 8, pp. 1185–1194. [14] athanasiadous, d., jiang, w., goldbaum, d., saleem, a., et al. (2018). nanostructure, osteopontin, and mechanical properties of calcitic avian eggshell. science advances, vol. 4, no. 3219, pp. 1–13. [15] luukkonen, j., pascual, m. l., patlaka, c., et al. (2017). increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5b/5a ratio. plos one, vol. 12, no. 8, pp. 1–15. [16] iida, t., wagatsuma, k., hirayama, d., and et al. (2018). is osteopontin a friend or foe of cell apoptosis in inflammatory gastrointestinal and liver diseases? international journal of molecular sciences, vol. 19, no. 7, pp. 1–15. [17] dinić, r. b., rajković, t. s., zivkovic, v., et al. (2018). clinical connection between rheumatoid arthritis and liver damage. rheumatology international, vol. 38, no. 5, pp. 715–724. [18] mursal, t., elbager, s., fadl elmola, a., et al. (2016). differential diagnosis of anemia in rheumatoid arthritis sudanese patients. world journal of pharmaceutical and medical research, vol. 2, no. 4, pp. 1–4. [19] elsedig, m. i., elhag, w., elmak, m. j., et al. (2014). seroprevalence of human parvovirus b19 antibody among sudanese patients with rheumatoid arthritis. american journal of ethnomedicine, vol. 1, pp. 402–407. [20] kobak, s. and bes, c. (2018). autumn late: geriatric rheumatoid arthritis. therapeutic advances in musculoskeletal disease, vol. 10, no. 1, pp. 3–11. doi 10.18502/sjms.v16i2.9294 page 283 sudan journal of medical sciences mohamed abdelrhman eltahir et al [21] abdelsalam, s. k., hashim, n. t., elsalamabi, e. m., et al. (2011). periodontal status of rheumatoid arthritis patients in khartoum state. bmc research notes, vol. 4, article 460. [22] abd elazeem, i. m., mohammed, a. r., and abdallah, h. n. (2018). correlation of serum interleukin-10 with disease activity and severity in systemic lupus erythematosus. egyptian rheumatology and rehabilitation, vol. 45, no. 1, pp. 1–9. [23] akdeniz, s., akdeniz, o., sakalli, n., et al. (2018). the relationship between interleukin-17 and osteoporosis in patients with rheumatoid arthritis. turkish journal of osteoporosis, vol. 24, pp. 46–52. [24] iwadate, h., kobayashi, h., kanno, t., et al. (2013). plasma osteopontin is correlation with bone resorption marker in rheumatoid arthritis patients. international journal of rheumatic disease, vol. 17, no. 1, pp. 50–56. [25] al zifzaf, s. d., mokbel, n. a., and abdelaziz, m. d. (2015). interleukin-17 in bechet’s disease: relation with clinical picture and disease activity. egyptian rheumatology and rehabilitation, vol. 42, no. 2, pp. 34–38. [26] negera, e., walker, s., bobosha, k., et al. (2018). the effects of prednisolone treatment on cytokine expression in patients with erythema nodosum leprosum reactions. frontiers in immunology, vol. 9, pp. 1–15. [27] noack, m., ndongo, n., and miossec, p. (2016). evaluation of anti-inflammatory effects of steroids and arthritis-related biotherapies in an in vitro coculture model with immune cells and synoviocytes. frontiers in immunology, vol. 7, pp. 1–10. doi 10.18502/sjms.v16i2.9294 page 284 introduction materials and methods results discussion conclusion acknowledgements ethical considerations competing interests availability of data and material funding references 150 161 calidad de vida.indd 150 olga janneth gómez-ramírez1 arlen patricia gómez-ramírez2 calidad de vida, nivel de salud percibido y factores sociodemográfi cos en personas con artritis reumatoide 1 orcid.org/0000-0002-9160-4170. universidad nacional de colombia, colombia. ojgomezr@unal.edu.co 2 orcid.org/0000-0002-0429-4822. universidad nacional de colombia, colombia. apgomezr@unal.edu.co recibido: 22 de febrero de 2016 enviado a pares: 29 de abril de 2016 aceptado por pares: 18 de agosto de 2016 aprobado: 03 de octubre de 2016 doi: 10.5294/aqui.2017.17.2.4 para citar este artículo / to reference this article / para citar este artigo gómez-ramírez oj, gómez-ramírez ap. calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide. aquichan. 2017;17(2): 150-161. doi: 10.5294/aqui.2017.17.2.4 resumen objetivo: determinar la relación entre la calidad de vida, el nivel de salud y los factores sociodemográficos en personas con artritis reumatoide (ar). materiales y método: estudio correlacional explicativo. se realizó una entrevista directa en el momento de la consulta de enfermería antes del control médico de reumatología. a través de un muestreo intencional no probabilístico de sujetos tipo se contó con la participación de 635 personas. resultados: se encontró una frecuencia de presentación de ar mayor en mujeres (87%). también se evidenció una correlación positiva (0,596; p<0,001) entre calidad de vida y nivel de salud. sociodemográficamente, las mujeres con educación básica y bajo estrato socioeconómico presentan una menor percepción de calidad de vida y nivel de salud. estas variables se sometieron a un proceso de prueba con el uso de un modelo path de análisis de covarianza. conclusiones: las condiciones sociales que viven las personas con ar son factores que determinan la puesta en marcha de acciones de intervención dirigidas a solucionar la problemática de la calidad de vida en esta población. palabras clave artritis; calidad de vida; estado de salud; seguridad social; factores socieconómicos (fuente: decs, bireme). año 17 vol. 17 nº 2 chía, colombia junio 2017 l 150-161 151 calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide l olga janneth gómez-ramírez y otro año 17 vol. 17 nº 2 chía, colombia junio 2017 l 150-161 quality of life, perceived health status and sociodemographic factors among persons with rheumatoid arthritis abstract objective: determine the relationship between quality of life, health status and sociodemographic factors among persons with rheumatoid arthritis (ra). materials and methods: this is an explanatory correlational study. a direct interview was conducted during the nursing consultation prior to the medical check-up concerning rheumatic diseases. a non-probability intentional sampling of type subjects included 635 individuals. results: a higher frequency of ra was found among women (87%). there also was a positive correlation (0.596; p <0.001) between quality of life and health status. socio-demographically speaking, women with a basic education and a low socioeconomic status have a lower perception of quality of life and health status. these variables were subjected to a test process using a path model to analyze covariance. conclusions: the social conditions of persons with ra are factors that determine the implementation of action for intervention aimed at solving the problem this population faces in terms of quality of life. keywords arthritis; quality of life; health status; social security; socioeconomic factors (source: decs, bireme). 152 aquichan issn 1657-5997 eissn 2027-5374 qualidade de vida, nível de saúde percebido e fatores sociodemográficos em pessoas com artrite reumatoide resumo objetivo: determinar a relação entre a qualidade de vida, o nível de saúde e os fatores sociodemográficos em pessoas com artrite reumatoide (ar). materiais e método: estudo correlacional explicativo. realizou-se uma entrevista direta no momento da consulta de enfermagem antes da consulta médica de reumatologia. por meio de uma amostra intencional não probabilística de sujeitos tipo, contou-se com a participação de 635 pessoas. resultados: constatou-se uma frequência de apresentação de ar maior em mulheres (87 %). também se evidenciou uma correlação positiva (0,596; p<0,001) entre qualidade de vida e nível de saúde. no aspecto sociodemográfico, as mulheres com educação básica e baixa classe socioeconômica apresentam uma menor percepção de qualidade de vida e nível de saúde. essas variáveis foram submetidas a um processo de teste com o uso de um modelo path de análise de covariância. conclusões: as condições sociais em que as pessoas com ar vivem são fatores que determinam a manifestação de ações de intervenção direcionadas a solucionar a problemática da qualidade de vida nessa população. palavras-chave artrite; estado de saúde; fatores socioeconômicos; qualidade de vida; segurança social (fonte: decs, bireme). año 17 vol. 17 nº 2 chía, colombia junio 2017 l 150-161 153 calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide l olga janneth gómez-ramírez y otro introducción la artritis reumatoide (ar) es una enfermedad crónica autoinmune, de afectación sistémica, la cual genera un daño estructural en las articulaciones con consecuencias graves en la capacidad funcional de las personas (1). de acuerdo con lo reportado por seca et al. (2), la ar se considera un problema de salud pública porque afecta la calidad de vida de las personas y puede producir una mortalidad prematura. en colombia, el grupo latinoamericano para el estudio de la ar afirma que esta patología autoinmune es compleja en su manejo, por lo que se espera que cada vez se ofrezca un abordaje biopsicosocial a las personas y se realicen estudios de investigación a gran escala que permitan mostrar el impacto de esta enfermedad en la salud pública y en la productividad de la población (3). la calidad de vida y el nivel de salud percibidos por las personas con artritis reumatoide son fenómenos complejos de gran importancia porque implican el entendimiento de las variables que caracterizan la experiencia de vivir con una enfermedad catastrófica debido a la ausencia de cura y la condición de cronicidad de la misma (4, 5). de esta forma, al no existir un tratamiento efectivo que contrarreste los signos y síntomas de daño articular y de afectación sistémica, se hace imprescindible que los profesionales de la salud conozcan la complejidad del fenómeno y trabajen en el fomento del bienestar y de servicios de salud apropiados para estas personas. los estudios de mora et al. (6) y díaz-rojas et al. (7) reafirman que en colombia se desconoce cuál es la carga derivada de las enfermedades reumatológicas crónicas en términos de costos y afectación física y social. lo que se conoce de las variables psicosociales y de carga económica de la enfermedad se ha realizado en trabajos de muestras pequeñas no representativas de la población afectada. por lo anterior, el propósito del estudio fue conocer la relación entre las variables calidad de vida y nivel de salud percibido por las personas que viven con ar, así como establecer los factores asociados a la calidad de vida que aporten evidencias para el entendimiento de este fenómeno. materiales y métodos diseño del estudio. por medio de un tipo de estudio cuantitativo transversal y un diseño correlacional se determinó la relación entre la calidad de vida y el nivel de salud percibido en personas con ar. la elección del diseño correlacional se realizó con base en el análisis de los estudios previos a nivel internacional que brindan un sustento metodológico acerca de la importancia de estudiar la relación entre las variables que caracterizan la calidad de vida y el nivel de salud, incluidos factores asociados como los sociodemográficos y de acceso a los servicios de salud (8-10). análisis estadístico. la prueba usada para el análisis estadístico de correlación fue el rho de spearman; para el análisis inferencial se realizó un procedimiento de regresiones múltiples y path análisis para evidenciar las asociaciones más significativas entre las variables, las cuales han sido útiles en las ciencias de la salud para probar las variables que explicarían mejor ciertos fenómenos como la calidad de vida y la cronicidad de la enfermedad (11, 12). estas técnicas de análisis permiten predecir y explicar la variable dependiente, en este caso, la calidad de vida, a partir de variables explicativas o independientes, como el nivel de salud en ar y los factores sociodemográficos y de acceso a la seguridad social que puedan estar presentes. población y muestra. la recolección de datos se realizó durante en el primer semestre de 2013, previo cumplimiento de los requisitos y el aval del comité de ética correspondiente. la entrevista directa se realizó en el momento de la consulta de enfermería, antes del control médico de reumatología. a través de un muestreo intencional no probabilístico de sujetos tipo se contó con la participación de 635 personas. la estimación muestral se hizo teniendo como guía el estadístico para estudios con población finita, es decir, cuando se conoce el total de la población a la cual se busca inferir los análisis (13). lo que se indaga es cuántos adultos emergentes del total se tendrán que estudiar a partir de la prevalencia estimada de personas con ar para colombia con una proporción de 1 % (14): n = 36818; α = 0,05; ic: 95 %; z = 1,96 (a = 0,05); e = 4 error de aproximación de la media poblacional de acuerdo con la siguiente fórmula: los criterios de inclusión que se establecieron fueron: 1) personas mayores de 18 años de edad con diagnóstico de ar, de acuerdo con los criterios del american college of rheumatology (acr), que hacen parte de la guía basada en evidencia de la asociación colombiana de reumatología; 2) personas vinculadas a un servicio de seguimiento médico de reumatología, que no se encontraran hospitalizadas; y 3) personas que no estuvieran cursando un proceso agudo comórbido o de complicación de la enfermedad. 154 año 17 vol. 17 nº 2 chía, colombia junio 2017 aquichan issn 1657-5997 eissn 2027-5374 variables del estudio. las variables de respuesta que se plantearon para el estudio fueron: variables sociodemográficas: se tuvieron en cuenta las características de importancia que tuvieran relación con los participantes, como edad, género, estrato socioeconómico (según clasificación de estratos de la secretaría de hacienda para colombia, donde un mayor número de estrato constituye un mayor nivel de ingreso económico) (15), nivel educativo y ocupación. variables de seguridad social: se consideraron variables de acceso a la seguridad social relacionadas con el tipo de vinculación a la misma (régimen contributivo, subsidiado, régimen especial, plan complementario de salud), dificultades con la entidad promotora de salud (eps), y aspectos puntuales de acceso al tratamiento de la enfermedad (oportunidad en el tratamiento especializado, trámites y condiciones de servicio de salud). calidad de vida (variable dependiente): esta variable se considera como la satisfacción con importantes funciones y dimensiones de la vida. se midieron las variables física, social y psicológica, teniendo en cuenta la propuesta teórica de padilla y grant (16), que incluye también el apoyo de la familia. nivel de salud percibido (variable independiente): incluye dominios específicos de la afectación de la artritis en la movilidad, actividad física, destreza y dolor. el componente de desempeño social, actividad social, actividades cotidianas, y el componente psicológico que explora el nivel de depresión y ansiedad. estas variables fueron extraídas de la propuesta del instrumento de impacto de la enfermedad de meenan et al. (17). instrumentos de medición. el instrumento que se utilizó para medir la calidad de vida corresponde al desarrollado y validado al español por padilla et al. (18), específicamente diseñado para personas con artritis denominado qol-ra. se trata de un cuestionario de 8 ítems, en el que cada uno de ellos contiene una definición del elemento considerado, seguido por una escala horizontal numerada con 10 puntos entre 1 (muy mal) y 10 (excelente). los elementos sobre los que se interroga son: capacidad física, dolor, interacción con la familia y los amigos, ayuda de la familia y los amigos, depresión, ansiedad, ar y salud. el cuestionario es sencillo y demuestra validez, los coeficientes alfa de cronbach van de 0,87 a 0,90. la escala qol-ra se correlaciona significativamente con la arthritis impact measurement scale 2 (19). el nivel de salud se midió a través del instrumento diseñado por meenan et al. (17, 19) denominado arthritis impact measurement scales (aims), el cual valora las dimensiones física, mental y social en nueve dominios: movilidad, actividad física, actividades de la vida diaria, destreza, actividades del hogar, dolor, actividad social, depresión y ansiedad. cada escala contiene de 4 a 7 ítems, y entre 2 y 6 posibles respuestas. la fiabilidad test-retest de la validación al español fue estadísticamente significativa para todas las escalas. el coeficiente de confiabilidad es > 0,7 para todas las escalas. para controlar el sesgo de administración de pruebas se realizó una ensayo piloto con 36 personas con el fin de garantizar la validez aparente y de contenido de los instrumentos y encontrar aspectos por mejorar en el procedimiento de entrevista. resultados características sociodemográficas. en la tabla 1 se resumen los hallazgos obtenidos en la caracterización de las variables sociodemográficas del estudio. se destaca la mayor frecuencia de presentación de la ar en mujeres, así como en el rango de edad mayor a 36 años y en los estratos socioeconómicos dos y tres. tabla 1. frecuencia y proporción de personas con artritis reumatoide (ar) en cada una de las características sociodemográficas analizadas en el estudio característica frecuencia proporción ( %) género masculino 81 13 femenino 554 87 edad 18 a 35 años 57 9 36 a 59 años 325 51 mayores de 60 años 253 40 escolaridad primaria 130 20 secundaria 179 28 técnico 66 10 universidad 260 41 155 calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide l olga janneth gómez-ramírez y otro n = 635 personas que cumplieron con los criterios de inclusión del estudio. fuente: elaboración propia. acceso a la seguridad social. en general, el 45 % de los participantes refiere no tener dificultades en los servicios de salud; sin embargo, el 55 % informó algún tipo de dificultad de acceso a la seguridad social. análisis de correlación. los datos presentaron una correlación positiva (rho de spearman = 0,596) estadísticamente significativa (p < 0,001) entre la calidad de vida y el nivel de salud, tal como se muestra en la figura 1. figura 1. correlación entre calidad de vida y nivel de salud en pacientes con artritis reumatoide característica frecuencia proporción ( %) estado civil soltero 137 22 casado 321 51 separado 70 11 viudo 62 10 unión libre 45 7 ocupación hogar 263 41 empleado(a) 163 26 trabajo independiente 68 11 estudiante 14 2 pensionado(a) 127 20 estrato socioeconómico uno 13 2 dos 127 20 tres 328 52 cuatro 135 21 cinco 19 3 seis 13 2 tiempo con la enfermedad 0 a 6 meses 30 5 7 a 18 meses 48 8 19 a 36 meses 118 19 más de 36 meses 439 69 n = 635; rho de spearman = 0,596; p < 0,001. fuente: elaboración propia. en la tabla 2 se muestran las correlaciones entre las diferentes dimensiones del instrumento de percepción de salud y la calificación total de calidad de vida. se encontró una correlación positiva (0,662) y estadísticamente significativa (p = 0,001) entre los dominios de actividad física y movilidad, así como en la calificación total de nivel de salud con los dominios de actividad física (0,744), movilidad (0,763), destreza (0,730) y desempeño (0,703). realizar labores manuales y labores domésticas como lavar la ropa también presentó una correlación positiva (0,669). también se evidenció que la variable sociodemográfica de género, en la característica ser mujer, reduce significativamente el nivel promedio de calidad de vida en -4,116 puntos (valor beta) y el de nivel de salud en -19,83 puntos con respecto a ser hombre. en el análisis de regresión múltiple se encontró que ser mujer predice un nivel bajo de calidad de vida con respecto a ser hombre -17,413, el pertenecer a un estrato igual o inferior a cuatro reduce significativamente el nivel de calidad de vida en comparación con pertenecer a un estrato igual o superior a cinco. al analizar al interior de los estratos se observó que estas diferencias se acentúan cuando se compara el estrato dos o menos con el estrato cinco o más: 28.859. en cuanto a la escolaridad, tener una escolaridad menor o igual a bachillerato se asocia con una reducción en la 156 año 17 vol. 17 nº 2 chía, colombia junio 2017 aquichan issn 1657-5997 eissn 2027-5374 calificación promedio de la calidad de vida respecto de tener algún grado de educación superior (técnico, universitario y posgrado); esta diferencia se acentúa en las personas con un nivel de educación básica o menos -31,655. asimismo, presentar dificultades con la eps reduce el nivel de calidad de vida respecto a las personas que manifestaron no tener dificultades con la eps -13,494. construcción del diagrama teórico path y modelo de ecuaciones estructurales. la figura 2 presenta el diagrama path con la propuesta teórica de un modelo conjunto que esquematiza los resultados encontrados en el presente trabajo. en ella se asume que el nivel de salud percibido (nsp) está relacionado con el comportamiento de la variable movilidad de los individuos, así como con las variables: actividad física, destreza, dinámica social, actividad social, actividades cotidianas, dolor, niveles de depresión y ansiedad. también se asume que el nivel de salud está correlacionado con el constructo calidad de vida (cv). por otra parte, se presume que el comportamiento observado en el nsp y la cv de los pacientes estudiados se ve reflejado en aspectos sociodemográficos como: el género, la escolaridad, el estrato socioeconómico y las posibles dificultades que se han tenido con las respectivas eps (d_eps). es necesario aclarar que, adicional a las variables tabla 2. matriz de correlaciones entre atributos de calidad de vida y dimensiones del nivel de salud en pacientes con artritis reumatoide dominios tqol td1 td2 td3 td4 td5 td6 td7 td8 td9 taims tqol 1 td1 0,424 1 td2 0,399 0,662 1 td3 0,34 0,45 0,507 1 td4 0,301 0,545 0,588 0,477 1 td5 0,462 0,255 0,258 0,213 0,25 1 td6 0,317 0,399 0,4 0,396 0,451 0,266 1 td7 0,387 0,44 0,452 0,504 0,336 0,09 0,168 1 td8 0,458 0,376 0,323 0,397 0,252 0,229 0,203 0,4 1 td9 0,442 0,322 0,25 0,28 0,217 0,263 0,245 0,244 0,649 1 taims 0,596 0,744 0,763 0,73 0,703 0,485 0,536 0,607 0,649 0,578 1 tqol: total calidad de vida; td1: total dominio movilidad; td2: total dominio actividad física; td3: total dominio ansiedad; td4: total dominio desempeño social; td5: total dominio actividad social; td6: total dominio actividades de la vida diaria; td7: total dominio dolor; td8: total dominio depresión; td9: total dominio ansiedad; taims: total nivel de salud percibido. fuente: elaboración propia. tabla 3. regresiones múltiples de las variables socioeconómicas y demográficas que se asocian con el nivel de salud en pacientes con artritis reumatoide variable beta (b) et t p valor (constante) 62,858 2,409 26,092 0,000 género (mujer) -4,044 1,391 -2,907 0,004 nivel educativo primaria incompleta -7,976 2,057 -3,878 0,000 primaria completa -5,930 1,515 -3,914 0,000 bachillerato incompleto -4,156 1,725 -2,409 0,016 bachillerato completo -3,288 1,307 -2,516 0,012 técnico -4,029 1,630 -2,472 0,014 estrato socioeconómico estrato 2 o menos -4,711 2,410 -1,955 0,051 estrato 3 -4,416 2,205 -2,003 0,046 estrato 4 -6,108 2,300 -2,656 0,008 dificultades eps (sí) -2,147 0,952 -2,255 0,024 fuente: elaboración propia. 157 calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide l olga janneth gómez-ramírez y otro sociodemográficas, existen otras de igual importancia como el tipo de acceso al sistema de seguridad en salud, la ocupación (en especial el hogar) y, en una menor medida, el acceso a terapias biológicas las cuales, como se evidencia en los primeros planos factoriales de los análisis de correspondencias múltiples, están altamente correlacionadas con las variables sociodemográficas presentadas en la figura 2. a partir de los resultados del modelo de ecuaciones estructurales se encontró que un cambio significativo en el nivel de salud percibido afecta de manera significativa el comportamiento de los individuos en su capacidad de movilizarse independientemente, realizar actividades físicas y en sus niveles de destreza. figura 2. modelo path de calidad de vida y nivel de salud en pacientes con artritis reumatoide fuente: elaboración propia. discusión a través de esta investigación se determinó la relación entre la calidad de vida, el nivel de salud y los factores sociodemográficos en personas con ar, donde se destaca la mayor frecuencia de afectación en mujeres (20), siendo este dato similar al presentado por bautista-molano et al. (21), que establece una proporción del 85,3 %. este hallazgo se corroboró con los reportes que muestran una prevalencia alta en el género femenino, asociada con las características hormonales y genéticas propias de la mujer, las cuales hacen que las enfermedades autoinmunes sean más frecuentes en ellas (22-24). en la afectación por grupo de edad se evidenció que el mayor porcentaje está en la etapa laboral productiva. sin embargo, según lo reportado por gamal et al., en un estudio conducido en pacientes con ra en egipto se evidenció que los que trabajaban mostraban mejores condiciones tanto físicas como mentales (25). asimismo, se encontró que la frecuencia de la enfermedad aumenta con la edad, ya que el 40 % de las personas afectadas fueron mayores de 60 años, lo cual concuerda con los datos del estudio de francoaguirre et al., donde los pacientes tenían una edad promedio de 53 ± 12 años (26). los datos coinciden con lo reportado por branco et al. (27), donde se evidenció que el inicio más frecuente de la enfermedad está durante el cuarto y quinto decenios de la vida, de 158 año 17 vol. 17 nº 2 chía, colombia junio 2017 aquichan issn 1657-5997 eissn 2027-5374 forma que el 80 % de todos los pacientes presenta la enfermedad entre los 35 y los 50 años de edad. asimismo, el tiempo de la enfermedad mayor a 36 reafirma la consideración de la misma como una enfermedad crónica de alto costo. entre los problemas referidos por los participantes de esta investigación se incluyen los trámites excesivos para la atención especializada, así como la demora en la cita de reumatología. esta situación, de acuerdo con lo informado por pasma et al. (28) y emery et al. (29), expone a las personas a complicaciones y cierra la posibilidad de aplicar la ventana de oportunidad que establece que entre más oportuno sea el inicio del tratamiento ordenado por los especialistas es más probable retrasar el efecto sistémico y de daño articular. se plantea que el deterioro radiológico y funcional progresa lentamente durante todo el curso de la enfermedad, y la máxima velocidad de desarrollo de las lesiones estructurales ocurre durante los primeros dos años de manifestaciones clínicas (29); por esta razón, es prioritario el abordaje precoz de la enfermedad, ya que el pronóstico de daño articular mejora cuando se tiene un acceso temprano a los protocolos y esquemas de tratamiento más eficaces (30). la correlación positiva y estadísticamente significativa entre los dominios de actividad física y movilidad (31), así como en la calificación total de nivel de salud con los dominios de actividad física, movilidad, destreza y desempeño, está acorde con los resultados del estudio episer 2000 en españa, con 2192 participantes, en el que se evidenció la asociación positiva entre los dominios de actividad física, movilidad y la capacidad para trabajar (32-34). por otro lado, se observó que la variable sociodemográfica de ser mujer reduce significativamente el nivel promedio de calidad de vida y de nivel de salud con respecto a ser hombre. este hallazgo se complementa con lo reportado por pease et al. (35), en el que ser mujer se asocia a presentar incapacidad funcional 4 años después del comienzo de la enfermedad (or = 3,0). la correlación moderada observada bivariadamente entre los constructos nivel de salud percibido y calidad de vida tiende a diluirse en el modelo teórico planteado. esto llamaría la atención para estudiar de forma separada los constructos y de esta forma extraer las variables más significativas sin asumir relaciones causales a priori. al advertir el comportamiento de las variables sociodemográficas planteadas en el modelo teórico propuesto se encuentra que ser mujer afecta tanto el nivel de salud percibido como la calidad de vida observada; el pertenecer a un estrato menor o inferior al 1 o 2 afecta de manera negativa el nsp y la cv, y tiende a diluirse el efecto negativo observado en las personas pertenecientes a los estrato 2 y 3 sobre la salud y la calidad de vida percibidas; haber cursado estudios de primaria o poseer máximo este nivel de formación afecta de manera negativa el nsp y la cv. las dificultades percibidas con el acceso a las eps no se confirmaron en la propuesta teórica expuesta, hecho que exigiría a futuro un análisis a profundidad con el fin de verificar si esto se debe a problemas de tipo estadístico o a posibles correlaciones aún no observadas dentro del modelo propuesto. de esta forma, los resultados de este estudio demuestran que existe una influencia importante de algunas variables socioeconómicas y demográficas sobre la calidad de vida y el nivel de salud en las personas con ar. para ampliar esta argumentación, la revisión de la literatura realizada por anaya y calixto (36) presenta el concepto de estatus socioeconómico (socioeconomic status ses) como una clasificación social jerárquica asociada a diferentes resultados en la salud y la enfermedad. los factores más importantes que influyen en el ses son los ingresos económicos, el nivel educativo, el perfil laboral y la clase social. entre los factores que resalta la literatura está el estudio de bengtsson et al. (37), que encontró en personas con artritis de un nivel de escolaridad de diez años o menos una correlación positiva con un aumento de dos veces en un mal pronóstico. estos hallazgos son coherentes con otros autores que afirman que el menor nivel educativo está relacionado con una mayor probabilidad de mortalidad temprana y menor capacidad funcional. teniendo en cuenta el constructo ses, se infiere que las personas con un estatus socioeconómico bajo pueden presentar una situación que empeora la enfermedad, la salud física, la salud mental y calidad de vida (38, 39). conclusiones a partir de los resultados se confirmó la relación entre las percepciones del estado de salud y la calidad de vida en personas con ar; asimismo, con el uso de ecuaciones estructurales se describieron las variables que aportaron significativamente al entendimiento de las necesidades más sentidas por las personas y, con base en esta evidencia, se realizó un acercamiento teórico a un modelo que aporta a la comprensión del fenómeno de la calidad de vida y el nivel de salud para las personas que viven con ar. la afectación se da a partir de la tercera y cuarta década de vida, en una etapa muy importante de desarrollo del potencial laboral del ser humano; por esta razón, esta enfermedad se considera de 159 calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide l olga janneth gómez-ramírez y otro interés en salud pública, y a la disciplina de enfermería le corresponde tomar parte en el proceso de atención a fin de poner en marcha acciones para el cuidado de la salud y la promoción de la calidad de vida de estas personas. en cuanto a los métodos, se sugiere avanzar en estudios que combinen abordajes cualitativos y cuantitativos para conocer la experiencia y el significado de la enfermedad de manera más completa, que abarque el constructo de enfoque de género. igualmente, se motiva a realizar estudios posteriores que permitan validar y probar con mayor profundidad estadística el modelo teórico planteado. la problemática identificada por la inoportunidad y las fallas de acceso al tratamiento especializado que sufren las personas con ar en el actual sistema de salud colombiano constituye una alerta para los actores en salud y la comunidad en general, a fin de exigir un cambio en el modelo de atención en colombia que permita dar prioridad al tratamiento especializado, sin distinciones por la capacidad de pago o el nivel socioeconómico, de tal manera que el mayor número de personas se beneficien de la eficacia farmacológica, la prevención de complicaciones y las medidas que contrarrestan el daño articular que se produce cuando no se tiene un esquema de seguimiento y atención estructurado. un aporte de gran relevancia es la oportunidad de construir un rol de enfermería en reumatología, que permita abrir un campo nuevo a la disciplina para estructurar formalmente el cuidado para las personas con enfermedades autoinmunes. por esta razón, el presente estudio aporta a la justificación de la participación de enfermería en el cuidado de personas con ar en el contexto latinoamericano. se reconoce la limitación del uso de un muestreo no probabilístico en un estudio descriptivo cuantitativo, lo que condiciona la generalización de los resultados; sin embargo, se aclara la importancia que tuvo contar con una muestra grande y el procedimiento de entrevista en el contexto natural de la consulta de enfermería en reumatología, lo que otorgó al estudio la ventaja de ser trasladable a situaciones naturales de un fenómeno psicosocial como la percepción de calidad de vida. se invita a la realización de trabajos posteriores con muestras grandes como la presente investigación, con el uso de técnicas de muestreo aleatorio estratificado. agradecimientos este estudio fue financiado por la división de investigación de la universidad nacional de colombia sede bogotá, a través del grupo de investigación de cuidado y práctica, de la facultad de enfermería. referencias 1. ministerio de la protección social. resolución 3974 de 2009. por la cual se adoptan unas determinaciones en relación con la cuenta de alto costo. bogotá, d.c: diario oficial; 2009. 2. seca s, kirch s, cabrita a, greten h. evaluation of the effect of acupuncture on hand pain, functional deficits and health-related quality of life in patients with rheumatoid arthritis–a study protocol for a multicenter, double-blind, randomized clinical trial. integr med int. 2016;14(3):219. 3. delgado-vega am, martín j, granados j, anaya jm. epidemiología genética de la artritis reumatoide: ¿qué esperar de américa latina? biomédica. 2011;26(4):562-84. 4. klak a, raciborski f, samel-kowalik p. social implications of rheumatic diseases. reumatologia. 2016;54(2):73-8. 5. wang n, dai r, wang w, peng y, zhao x, bi k. simultaneous profiling of eicosanoid metabolome in plasma by uplc–ms/ ms method: application to identify potential makers for rheumatoid arthritis. talanta 161. 201;157-64. 6. mora c, díaz j, quintana g. costos directos de la artritis reumatoide temprana en el primer año de atención: simulación de tres situaciones clínicas en un hospital universitario de tercer nivel en colombia. biomédica. 2009;29:43-50. 7. díaz-rojas j, dávila-ramírez fa, quintana-lópez g, aristizábal-gutiérrez f, brown p. rheumatoid arthritis prevalence in colombia: an approach based on burden of disease study during 2005. rev colomb reumatol. 2016;23(1):11-6. 160 año 17 vol. 17 nº 2 chía, colombia junio 2017 aquichan issn 1657-5997 eissn 2027-5374 8. hyphantis t, et al. the relationship between depressive symptoms, illness perceptions and quality of life in ankylosing spondylitis in comparison to rheumatoid arthritis. clin rheumatol. 2013;32(5):635-44. doi10.1007/s10067-0122162-6. 9. ambriz murillo y, menor almagro r, campos-gonzález i, cardiel m. original: calidad de vida relacionada con la salud en artritis reumatoide, osteoartritis, diabetes mellitus, insuficiencia renal terminal y población geriátrica. experiencia de un hospital general en méxico. reumatología clinica. 2015;1168-72. 10. ackerman in, ademi z, osborne rh, liew d. comparison of health-related quality of life, work status, and health care utilization and costs according to hip and knee joint disease severity: a national australian study. phys ther. 2013;93(7):889-99. http://dx.doi.org/10.2522/ptj.20120423ly 30, 2014. 11. ryu e, west s, sousa k. distinguishing between-person and within-person relationships in longitudinal health research: arthritis and quality of life. ann behav med. 2012;43(3):330-42. 12. baillet a, gossec l, carmona l, wit md, van eijk-hustings y, bertheussen h, et al. points to consider for reporting, screening for and preventing selected comorbidities in chronic inflammatory rheumatic diseases in daily practice: a eular initiative. ann rheum dis. 2016;75:965-73. 13. tristán la. cálculo del tamaño de muestra y establecimiento de criterios y el problema del tamaño de la población. guía de usuario kalt criterial versión 2. méxico: instituto de evaluación e ingeniería avanzada san luis potosí; 2008. 14. juárez f, cadena j, alpi sv, anaya jm, contreras f. relaciones entre variables sociodemográficas, incapacidad funcional, dolor y desesperanza aprendida en pacientes con diagnóstico de artritis reumatoide. int j clin health psychol. 2004;4(1):91-103. 15. secretaría de hacienda distrital de la alcaldía mayor de bogotá. indicadores de impacto plan de desarrollo 2012-2016. disponible en: http://www.sdp.gov.co/portal/page/portal/portalsdp/informaciontomadecisiones/estadisticas/informes%20de%20ciudad/2014/dice161-indicadoresimpacacuerdo489-2014.pdf. 16. padilla g, grant m. quality of life as a cancer nursing outcome variable. advances in nursing science. 1985;8(1):45-60. 17. meenan r, gertman p, mason j. measuring health status in arthritis. the arthritis impact measurement scales. arthritis rheum. 1980;23:146-53. 18. padilla g, frank m, koresawa s. single instruments for measuring quality of life. instruments for clinical health care research. sudbury ma: jones and barlett publishers, inc.; 2004. p. 129. 19. pacheco-tena c, reyes-cordero g, mckenna sp, ríos-barrera va. adaptación y validación del rheumatoid arthritis quality of life scale (raqol) al español de méxico. reumatología clínica. 2011;7(2):98-103. 20. ahlstrand i, björk m, thyberg i, börsbo b, falkmer t. pain and daily activities in rheumatoid arthritis. disabil rehabil. 2012;34(15): 1245-53. disponible en: http://dx.doi.org/10.3109/09638288.2011.638034. 21. bautista-molano w, fernández-avila d, jiménez r, cardozo r, marín a, soler mp, et al. perfil epidemiológico de pacientes colombianos con artritis reumatoide evaluados en una clínica especializada de atención integral. reumatol clin. 2016. disponible en: http://dx.doi.org/10.1016/j.reuma.2015.11.009. 22. sapir-koren r, livshits g. rheumatoid arthritis onset in postmenopausal women: does the acpa seropositive subset result from genetic effects, estrogen deficiency, skewed profile of cd4þ t-cells, and their interactions? mol cell endocrinol. 2016; 431:145-63. 23. zhou x, zhu j, zhang h, zhou g, huang y, liu r. is the microrna-146a (rs2910164) polymorphism associated with rheumatoid arthritis? association of microrna-146a (rs2910164) polymorphism and rheumatoid arthritis could depend on gender. joint bone spine. 2015(3):166. 24. putrik p, ramiro s, keszei ap, hmamouchi i, dougados m, uhlig t, kvien tk, boonen a. extended report: lower education and living in countries with lower wealth are associated with higher disease activity in rheumatoid arthritis: results from the multinational comora study. ann rheum dis annrheumdis-20206737. 2014. 25. gamal r, mahran sa, el fetoh na, janbi f. quality of life assessment in egyptian rheumatoid arthritis patients: relation to clinical features and disease activity. the egyptian rheumatologist. 2016;38:65-70. 161 calidad de vida, nivel de salud percibido y factores sociodemográficos en personas con artritis reumatoide l olga janneth gómez-ramírez y otro 26. franco-aguirrea jq, cardona-tapias aa, cardona-arias ja. health related quality of life in patients with rheumatoid arthritis from medellín-colombia, 2014. rev colomb reumatol. 2015;22(3):153-61. 27. branco jc, rodrigues am, gouveia n, eusébio m, ramiro s, machado pm, et al. 2016 prevalence of rheumatic and musculoskeletal diseases and their impact on health-related quality of life, physical function and mental health in portugal: results from epireumapta national health survey. 2016;19;2(1):e000166. 28. pasma a, hazes j, busschbach j, van der laan w, appels c, van ’t spijker a, et al. psychosocial predictors of dmard adherence in the first three months of treatment for early arthritis. patient educ couns. 2016. 29. emery p, solem c, majer i, cappelleri j, tarallo m. a european chart review study on early rheumatoid arthritis treatment patterns, clinical outcomes, and healthcare utilization. rheumatol int. 2015;35(11):1837-49. 30. innala l, berglin e, möller b, ljung l, smedby t, umeå universitet m, et al. age at onset determines severity and choice of treatment in early rheumatoid arthritis: a prospective study. arthritis res. 2014. 31. listing j, kekow j, manger b, et al. mortality in rheumatoid arthritis: the impact of disease activity, treatment with glucocorticoids, tnf inhibitors and rituximab. ann rheum dis. 2015;74:415-21. 32. withall j, haase am, walsh ne, young a, cramp f. physical activity engagement in early rheumatoid arthritis: a qualitative study to inform intervention development. physiotherapy. 2016;102:264-71. 33. carmona l, villaverde v, hernández-garcía c, ballina j, gabriel r, laffon a. episer study group. the prevalence of rheumatoid arthritis in the general population of spain. rheumatology (oxford). 2002;41(1):88-95. 34. minichiello e, semerano l, boissier mc. time trends in the incidence, prevalence, and severity of rheumatoid arthritis: a systematic literature review. joint bone spine. 2016;5(16):30129-4. 35. pease ct, bhakta bb, devlin j, emery p. does the age of onset of rheumatoid arthritis influence phenotype? a prospective study of outcome and prognostic factors. rheumatology (oxford). 1999;38:228-34. 36. calixto oj, anaya jm. socioeconomic status. the relationship with health and autoimmune diseases. autoimmun rev. 2014;13(6):641-54. 37. bengtsson c, et al. socioeconomic status and the risk of developing rheumatoid arthritis: results from the swedish eira study. ann rheum dis. 2005;64:1588-94. 38. rogers hl, brothertona ht, olivera plaza sl, segura durán ma, pena altamar ml. depressive and anxiety symptoms and social support are independently associated with disease-specific quality of life in colombian patients with rheumatoid arthritis. rev bras reumatol. 2015;55(5):406-413. 39. baldassari a, cleveland r, luong m, jonas b, conn d, bridges s, et al. socioeconomic factors and self-reported health outcomes in african americans with rheumatoid arthritis from the southeastern united states: the contribution of childhood socioeconomic status. bmc musculoskelet disord. 2016; 171. original research 84 sajsm vol 23 no. 3 2011 introduction arthritis is one of the main causes of human disability, limiting everyday activities such as dressing, climbing stairs, getting in and out of bed, or walking. 1 rheumatoid arthritis (ra) is the most common type of chronic inflammatory arthritis. 2 ra is characterised by inflammation of the synovial lining of the joints, which ultimately results in cartilage and bone destruction. 3 ra can affect any joint, large or small; however, the small joints are the most commonly affected. since ra is a systemic disease, other parts of the body may be involved in the inflammatory process. 1 although there is no cure for ra the condition can be managed with various strategies. 4 the use of exercise in the treatment of patients with ra has been widely debated in the past. in the late 1800s, the concept of total bed-rest became the standard care. it was not until 1948 when the undesirable effects of prolonged bed-rest were described, that exercise resumed its role in arthritis therapy and rehabilitation. 5 over the past decades there has been growing evidence of the health benefits of physical activity for patients with ra. 6 in ra various factors may lead to a decline in functional ability. apart from the direct consequences of the disease on the function of joints and muscles, physical inactivity contributes further to stiffness of the joints, muscle weakness and cardiorespiratory deconditioning. 7 research shows that patients with ra are in general less fit and more at risk of comorbidities when compared with healthy, age-matched controls. 8-10 the primary goal of exercise therapy for ra is to improve joint mobility, muscle strength and aerobic and functional capacity. 11 however, there is a debate as to what type of exercise would be the best for ra patients. 5 hydrotherapy has been shown to increase muscle strength, increase joint range of motion, improve aerobic capacity, reduce pain and improve function. 12 the buoyancy of water and the ability to control its temperature make it favourable for patients with muscular and joint disease. although most research conducted suggests that exercises in water are beneficial for ra patients, numerous problems exist with the prescription of water therapy programmes. for example, proper water facilities for exercise therapy are not always available. heated pools designed for exercise therapy are expensive and maintenance is also time consuming and costly. home exercise programmes, usually consisting of land-based exercise, are often prescribed for ra patients. land-based exercises, specifically weight-bearing exercise, also have the advantage of strengthening the connective tissue surrounding the joints and stimulating bone formation. 5 these qualities are desirable because of the well-known complications of accelerated generalised osteoporosis induced by active inflammation, immobility and medication (cortisone) in ra. 11 in the past much research on ra patients and exercise has focused on water-based exercises. although recently more research has started focusing on land-based exercises, several questions remain unanswered. 13 the efficacy of land-based exercise intervention with respect to pain, disease activity, functional ability, quality of life and structural damage remain unclear. 14 therefore, the aim of this study was to determine the outcome of exercise therapy, specifically comparing the effects of a land-based exercise programme with that of a water-based exercise programme in ra sufferers. abstract objective. to compare the effects of a 3-month landand waterbased exercise programme among rheumatoid arthritis (ra) sufferers. methods. patients with ra functional class i and ii (n=10) were randomly assigned to a land-based exercise group (group l) (n=4), water-based exercise group (group w) (n=4) or a control group (group c) (n=2). testing parameters included swollen joint count (sjc), tender joint count (tjc), erythrocyte sedimentation rate (esr), haemoglobin (hb), 50-ft (15.2-m) walk test, grip strength, isokinetic strength of knee extensors and flexors, knee range of motion (rom) and aerobic capacity. results. there were individual improvements in most of the physical status parameters tested for the experimental groups (land-based exercise group and water-based exercise group) while the general trend for the control group was that of deterioration. appropriate land-based exercises did not appear to aggravate disease activity. however, the water-based exercise programme was superior in controlling the disease activity with regards to the tender and swollen joint counts. conclusion. both exercise interventions appeared to be beneficial in the treatment of ra. further research is required comparing various modes of exercises for the treatment of ra, using larger samples and evaluating the long-term effects. kim nolte (dphil)1 dina c janse van rensburg (mb chb, mmed, msc sports medicine)2 pieter e krüger (dphil)1 1 department of biokinetics, sport and leisure sciences, university of pretoria, south africa 2 section sports medicine, university of pretoria, south africa correspondence to: kim nolte (kim.nolte@up.ac.za) landand water-based exercises in rheumatoid arthritis patients: a series of case reports sajsm vol 23 no. 3 2011 85 methods ethical clearance for this study was obtained from the faculty of humanities research proposal and ethics committee of the university of pretoria, south africa. all subjects were required voluntarily to read and sign an informed consent. subjects patients with classical or definite ra and mild to moderate disease activity (american college of rheumatology functional class i and ii), were randomly assigned to the land-based exercise group (group l), water-based exercise group (group w) or non-exercise group (group c). all patients were on stable medication. exclusion criteria included the presence of unstable cardiopulmonary disease, acute joint symptoms and current participation in a physical fitness programme or organised sports activity. exercise intervention subjects in the exercise groups were required to exercise 2 3 times per week for a 3-month period. those in the control group were instructed to continue with their normal sedentary lifestyle. group l and w attended their rehabilitation sessions at the university of pretoria rehabilitation gymnasium and hydrotherapy pool, respectively. the same biokineticist instructed both groups throughout their participation in the study. both the landand water-based exercise programmes were aimed at improving range-of-motion, muscle strength and cardiorespiratory endurance. the exercise intervention consisted of warm-up exercises, strengthening exercises, aerobic exercises and cool-down exercises with stretches. initially, the duration of the warm-up and strengthening phases was longer in order to build muscle strength. aerobic exercise time was gradually increased as cardiorespiratory fitness improved. the total duration of an exercise session in each of the 2 programmes was approximately 45 minutes each. assessments each patient was assessed 3 times throughout the study to track progress: before the exercise intervention, 5 6 weeks into the exercise intervention and at the end of the exercise intervention (3 months). the results of only the preand post-exercise assessments are reported. table i. gender, age and anthropometry subject gender age (years) stature (cm) body mass (kg) w1 female 66 159.1 92.0 w2 female 64 161.7 85.8 w3 female 52 161.4 71.0 w4 female 52 162.7 68.1 l1 male 60 199.6 105.4 l2 female 41 168.8 52.1 l3 female 57 170.4 97.3 l4 female 53 169.2 60.2 c1 female 43 167.4 54.6 c2 female 53 170.0 60.0 average 54.1 ± 8.1 169.0 ± 11.5 74.7 ± 9.1 w = water-based exercise programme; l = land-based exercise programme; c = control. table ii. total tender and swollen joint counts total tender joint count total swollen joint count subject pre post difference clinically relevant pre post difference clinically relevant w1 6 2 -4 yes 5 1 -4 yes w2 24 10 -14 yes 14 10 -4 yes w3 11 8 -3 no 12 8 -4 yes w4 10 6 -4 yes 4 6 2 no l1 11 15 4 yes 13 13 0 no l2 9 6 -3 no 9 8 -1 no l3 10 10 0 no 10 9 -1 no l4 13 10 -3 no 13 11 -2 no c1 8 8 0 no 10 10 0 no c2 19 19 0 no 16 16 0 no w = water-based exercise programme; l = land-based exercise programme; c = control. 86 sajsm vol 23 no. 3 2011 clinical assessment a medical specialist rheumatologist performed all clinical assessments. joints were examined for soft-tissue swelling and tenderness and pain during motion, using the american college of rheumatology, rheumatoid arthritis clinical response criteria. 15 haematological assessment blood samples were drawn and standard laboratory procedures were used to estimate erythrocyte sedimentation rate (esr) (modified westergren, mm/h) and haemoglobin (hb)(gm/dl). 16 physical status assessment the following functional assessments were performed: • a 50-ft (15.2m) walk test. 3 • manual grip strength was measured with a sphygmomanometer cuff rolled up two turns and inflated to 20 mmhg. • the strength of the knee extensors and flexors was tested on an isokinetic dynamometer (cybex norm 7000). a speed of 60°/second was used, 3 trial repetitions and 5 test repetitions were performed. • knee range of motion was measured using a standard goniometer. • bicycle ergometer testing was performed to determine aerobic capacity. the astrand-rhyming protocol to obtain data for calculating the estimated vo2max of each subject was used. 3 the test was started with an initial load of 25 watts (w) at a cadence of 60 70 revolutions per minute with an increment increase of 25 w until exhaustion. statistical analyses computations to determine standard descriptive statistics (mean and standard deviation) for age, stature and body mass of participants were performed using the statistical package for social sciences (spss), microsoft windows release 9.0 (1999). due to the small sample the raw data and the difference between the pre and post data are presented for each subject. these changes are interpreted in the context of clinically meaningful results for each variable. results ten patients (9 females, 1 male) with classic or definite ra volunteered for the study. the mean (±sd) age, stature, and body mass of the subjects was 54.1±8.1 years, 169.0±11.5 cm and 74.7± 9.1 kg, respectively. subjects were randomly assigned to group l, w or c (table i). clinical and haematological assessment results an increase or decrease of 4 joints may be considered to be clinically significant or a meaningful change for the joint counts. the total tjc decreased in all the subjects in group w. the decreases were all deemed to be clinically significant. in group l, the total tjc decreased in two of the subjects, remained unchanged in one and increased in another. none of the changes was deemed to be clinically relevant except for the subject whose total tjc increased. there was no change in the total tjc of the subjects in group c. the total sjc decreased in a clinically meaningful way in 3 of the 4 subjects in group w. one subject’s sjc increased in group w; however, the increase was not deemed to be clinically relevant. there was a nonclinically relevant decrease in 3 of the subjects’ sjc in group l and 1 subject’s sjc was unchanged. there was no change in the sjc of the subjects in group c (table ii). the haemoglobin values remained unchanged in all 3 groups. there were changes in the esr in the groups; however, no specific trends were identified and values generally fell within normal clinical reference ranges (table iii). physical status assessments results various aspects of physical conditioning are shown in tables iv and v. there was an improvement in group w and group l’s physical condition as determined by the 50-ft (15.2-m) walk and aerobic capacity test for all subjects, while there were no improvements noted in group c. in general, for other variables there were trends of improvement for group w and group l but not group c. discussion there is a growing interest among health professionals in improving the care of patients afflicted with chronic disabling diseases such as ra. in particular there is interest on the effects of exercise training programmes on the measurements of improvement in joint function, mobility, strength, endurance and cardiovascular fitness. 17 one of the aims of this study was to determine whether exercise therapy is beneficial for ra patients. the positive changes produced by the landand water-based exercise programmes are evident in the results as far as the disease (total and swollen joint count) and physical status of the subjects were concerned. the exercise therapies appeared to assist in the control of the disease activity as both the tjc and sjc were reduced in most of the subjects in the experimental groups, but not in the control group. there was a decrease in 6 of the subject’s joint counts (tjc and sjc) in the experimental groups, 3 of which were clinically significant decreases. no specific trends could be identified in the esr. however, in the experimental groups, 3 subjects’ esr increased, 1 in group w and 2 in group l. despite the increases, values fell within acceptable ranges with the exception of 1 subject in group l. however, this subject’s esr was on the border of the acceptable range even before the start of the intervention. in the control group 1 subject’s esr remained unchanged and the other subjects decreased. it is important to note that esr can be influenced by factors other than ra, such as anaemia, pregnancy and age. therefore it may be useful to conduct other haematological assessments in addition to esr such as c-reactive protein (crp) to get a true reflection of the inflammatory status of the disease. table iii. erythrocyte sedimentation rate erythrocyte sedimentation rate (mm.h -1 ) subject pre post difference within clinical reference range w1 10 20 10 yes (pre and post) w2 20 10 -10 yes (pre and post) w3 50 26 -24 no (pre) yes (post) w4 2 2 0 yes (pre and post) l1 7 10 3 yes (pre and post) l2 9 7 -2 yes (pre and post) l3 30 42 12 yes (pre) no (post) l4 13 7 -6 yes (pre and post) c1 18 18 0 yes (pre and post) c2 30 14 -16 yes (pre and post) w = water-based exercise programme; l = land-based exercise programme; c = control. sajsm vol 23 no. 3 2011 87 most of the physical status parameters assessed were positively influenced by both exercise therapies. the improvements in aerobic capacity are especially noteworthy due to the fact that in the past exercise therapy in ra primarily aimed at maintaining joint mobility and muscle strength. 14 however, because of the increased risk of cardiovascular events such as atherosclerosis of the coronary artery, aerobic training and fitness should be given the sufficient attention it deserves with regards to exercise programming. other compelling evidence advocating the importance of aerobic exercises for ra sufferers was that of a systematic review and meta-analysis by baillet and colleagues. 14 the study found that aerobic exercises improve some of the most important ra patient outcomes: function, quality of life and pain. moreover, it appears that aerobic exercise decreases radiological damage and pain. 14 vo2max or vo2peak is considered a measure for aerobic fitness. in this study both the landand waterbased exercise groups similarly improved their relative and absolute vo2max as well as their 50-ft walk test time (table iv). another study has shown that vo2max of subjects improved by 12% after following a 12-week endurance training programme. 18 in contrast, in the nonexercise group there were no improvements in aerobic capacity parameters tested. in fact, one of the subjects showed deterioration. it is important to note that even small changes can be expected to have a detrimental impact on a ra patient, especially if one considers that the decline took place over a short period of time. due to the involvement of the joints of the hand and wrist, ra can influence grip strength. therefore grip strength is considered an important measurement of a ra patient’s functional status. poor grip strength can affect activities of daily living such as the ability to open and close small buttons, to write and to perform any function related to work or housework. 19 the exercise therapies appeared to have a positive effect on grip strength in comparison to the control group. in the experimental groups, 7 of the subjects’ left grip strength improved and 4 of the subjects’ right grip strength improved. in the control group there was a decrease in both left and right grip strength for both subjects except for 1 subject whose right grip strength minimally improved (table v). impaired muscle function is a common consequence in patients with ra. 8 it is important to maintain normal muscle strength, not only to maintain physical function, but also to stabilise the joints and prevent joint angulation and later osteoarthrosis. 16 in general, table v. physical parameters subject grip (mmhg) isokinetic flexion (nm/kg) isokinetic extension (nm/kg) range of motion (°) flexion range of motion (°) extension l r l r l r l r l r w1 138 162 29.3 21.7 64.1 64.1 84 79 0 0 w2 100 103 8.2 9.4 40.0 49.4 105 115 0 0 w3 80 102 65.0 66.2 125.4 140.8 130 125 0 0 w4 108 120 50.0 39.7 70.6 66.2 121 121 0 0 l1 265 265 83.8 64.8 168.6 140.0 120 125 9 14 l2 60 62 84.1 98.1 203.8 238.0 125 124 0 0 l3 81 85 47.4 48.5 101.0 89.7 112 115 0 0 l4 91 90 68.3 85.0 116.7 141.7 130 115 0 0 c1 59 41 92.6 85.2 175.9 188.9 131 130 0 0 c2 44 40 55.0 65.0 128.3 151.7 122 125 0 0 w = water-based exercise programme; l = land-based exercise programme; c = control. table iv. 50-ft (15.2m) walk test and relative maximal oxygen consumption 50-ft walk test (s) maximal oxygen consumption (ml.kg -1 .min -1 ) subject pre post difference pre post difference w1 10.1 9.1 -1.0 11.6 19.7 8.1 w2 10.5 7.5 -3.0 20.7 23.9 3.2 w3 11.4 9.7 -1.7 21.9 35.9 14 w4 9.6 8.0 -1.6 36.4 38.6 2.2 l1 8.0 6.8 -1.2 17.2 22.3 5.1 l2 8.3 6.9 -1.4 37.8 41.0 3.2 l3 9.2 7.8 -1.4 15.8 23.2 7.4 l4 8.0 6.9 -1.1 45.2 48.8 3.6 c1 7.5 7.3 -0.2 38.1 30.6 -7.5 c2 9.4 9.5 0.1 32.0 32.1 0.1 w = water-based exercise programme; l = land-based exercise programme; c = control. 88 sajsm vol 23 no. 3 2011 there was an increase in knee extensor and flexor strength in the experimental groups and a deterioration in the control group. there appeared to be a more consistent improvement in the knee flexor strength in relation to knee extensor strength in the experimental groups. this may be due to the fact that there were muscle strength imbalances between the knee extensors and flexors at the start of the intervention (table v). the maintenance of functional rom is necessary for daily activity and efficiency of movement. there were no significant deviations in knee extension rom in relation to normative data before the exercise interventions and therefore no large changes were expected. knee flexion rom did however improve for all the subjects in both experimental groups and stayed approximately the same for the subjects in the control group (table v). it appears that exercise therapy does indeed play an important role in the treatment of rheumatic disease and in the fight against rheumatic invalidism. however, the primary purpose of the study was to determine which exercise mode, water-based exercise therapy or land-based exercise therapy, would be more effective in the treatment of ra. importantly, results indicate that disease activity was not exacerbated by the land-based exercises. it is however worthwhile noting that there were more clinically significant decreases in the joint counts for the subjects following water-based therapy than landbased therapy. in addition, the tjc increased in one of the subjects following land-based therapy. the greater reduction in joint swelling and tenderness in the water-based exercise group may be attributed to the reduction of joint loading occasioned by the buoyancy. in addition, the hydrostatic pressure of water immersion is considered to reduce oedema. 20 concerning the physical status of the subjects in the experimental groups, both exercise therapies had a positive influence on most of the physical status parameters measured. however, it would appear that the improvements in the water-based exercise group seemed slightly more substantial than those of the land-based exercise group. conclusion the results indicate that the benefits derived from both landand water-based exercises are very similar. the prescription of landbased exercises is feasible, especially when hydrotherapy is not possible or contraindicated. the appropriate land-based exercises do not appear to enhance disease activity. therefore the importance and unique benefits of land-based exercises should be considered when prescribing exercises for ra patients. it is possible that the exercise of choice for ra patients should not be water-based exercises alone, as believed in the past, but an optimal combination of landand water-based exercises. ideally, the contribution of landand water-based exercises to the overall programme of the ra patient should be manipulated according to the patient’s needs and disease activity at that period of time. finally, it is important to emphasise the fact that this was a preliminary study and therefore the sample was small and any changes in disease symptoms must be viewed in the context of the natural course of ra, where daily fluctuations in joint motion, swelling, pain and tenderness are not uncommon. however, all patients were on stable medication, thus eliminating the possibility of confounding results due to changes in medication. thus the results from this study indicate that future research focusing on the effects of various exercise modalities as well as the long-term effects of exercise interventions in the treatment of ra patients could prove valuable. references 1. nieman dc. exercise soothes arthritis. joint effects. acsms health fit j 2000;4(3):20-27. 2. thompson jm. arthritis: everything you need to know about arthritis. south africa: zebra press, 1998:23-46. 3. norceau l, martineau h, ro l, belzile m. effects of a modified dance based exercise on cardiorespiratory fitness, psychological state and health status of persons with rheumatoid arthritis. am j phys med rehab 1995;74(1):19-27. 4. giannini mj, protas ej. exercise response in children with and without juvenile rheumatoid arthritis: a case comparison study. phys ther 1992;72(5):365-372. 5. kirsteins ae, dietz fd, hwang sw. evaluating the safety and potential use of a weight-bearing exercise, tai-chi chuan, for rheumatoid arthritis patients. am j phys med rehab 1991;70(3):136-141. 6. gaudin p, leguen-guegan s, allenet b, baillet a, grange l, juvin r. is dynamic exercise beneficial in patients with rheumatoid arthritis? joint bone spine 2008;75:11-17. 7. van den ende chm, hazes jmw, le cessie s, et al. comparison of high and low intensity training in well controlled rheumatoid arthritis. results of a randomized clinical trial. ann rheum dis 1996;55:798-805. 8. ekdahl c, broman g. muscle strength, endurance, and aerobic capacity in rheumatoid arthritis: a comparative study with healthy subjects. ann rheum dis 1992;51:35-40. 9. del rincon id, williams k, stern mp, freeman gl, escalante a. high incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors. arthritis rheum 2001;44:27372745. 10. bacon pa, townend jn. nails in the coffin: increasing evidence for the role of rheumatic disease in the cardiovascular mortality of rheumatoid arthritis. arthritis rheum 2001;44:2707-2710. 11. hazes jmw, van den ende chm. how vigorously should we exercise our rheumatoid arthritis patients? ann rheum dis 1996;55:861-862. 12. tork sc, douglas v. arthritis water exercise program evaluation. a selfassessment survey. arthrit care res 1989;2(1):28-30. 13. klepper se. effects of an eight-week physical conditioning programme on disease signs and symptoms in children with chronic arthritis. arthrit care res 1999;12(1):52-60. 14. baillet a, zeboulon n, gossec l, et al. efficacy of cardiorespiratory aerobic exercise in rheumatoid arthritis: meta-analysis of randomized controlled trials. arthrit care res 2010;62(7):984-992. 15. ritchie dm, doyle ja, mcginnis jm. clinical studies with an articular index for the assessment of joint tenderness in patients with ra. q j med 1968;37:393-406. 16. lyngberg kk, ramsing bu, nawracki a, harreby m. safe and effective knee extension training in rheumatoid arthritis. arthritis & rheum 1994;37(5):623-628. 17. komatireddy gr, leitch rw, cella k, browning g, minor m. efficacy of low load resistive muscle training in patients with rheumatoid arthritis functional class ii and iii. j rheumatol 1997;24(8):1531-1539. 18. janse van rensburg dc, viljoen m, coertzen c, et al. efficacy of an exercise programme on the functional capacity and disease activity in females with rheumatoid arthritis. saoj 2010;28:685-691. 19. minor ma, hewett, je. physical fitness and work capacity in women with rheumatoid arthritis. arthrit care res 1995;8(3):146-154. 20. hall j, maddison pj, chapman k. arthrit care res 2010;9(3):206-215. page 2 p h y s i o t h e r a p y march, 1972 the physiotherapist and juvenile rheumatoid arthritis jo a n b a l d w in , b.sc. phys. (r and) reprinted by kin d permission fro m journal o f the canadian physiotherapy association, vol. 23, n o. 3, june, 1971 the juvenile r heum atoid a rthritis u n it a t t he h ospital for sick children, t o ro n to , recently did physical assess­ ments on 33 children suffering from rheum atoid arthritis. many treatm ent program m es were revised in view o f the findings. r heum atoid arthritis is a systemic disease which is defined by jones as affecting four o r m ore joints an d lasting longer than three m onths. initially the disease attacks joint synovial membranes progressively destroying cartilage an d bone and producing disruption o f the joint. t he disease may encroach on surrounding muscles, nerves, tendons, bursae an d m ay directly attack supplying blood vessels. juvenile rheum atoid arthritis, often called still’s disease, djffers from the adult form in th a t it often presents with high spiking fevers and rashes, and also differs in the type o f deformity (see list o f com m on deformities). the onset in the adult is insidious and less dram atic. several groups consider it m ay even be a different disease. u nlike adults, immobilized for lengthy periods, children tend to be mobile once the initial fever (lasting one to tw o weeks) has been controlled and in spite o f acute jo in t involvement. because o f these differences the m anagem ent o f the disease has to be modified. t he peak onset ages were found to be between one to three and seven to ten years o f age, closely conform ing to the studies done by l aaksonen an d laine, an d ansell and bywaters. t he older th e child a t the onset o f the disease, the more severely his joints were affected and the slighter his chance th a t the disease would become inactive. in 70 per cent o f childhood cases, however, th e disease is thought to become inactive after a d uration o f one to eight years. influenced by such findings, a program m e was designed to help the child develop as norm ally as possible during the active phase o f the disease. a functional grading was also devised fo r b o th the upper and lower limbs. this to o k into account (a) the num ber of joints affected; (6) the num ber o f fixed deform ities; (c) the age o f onset; and (rf)the d uration o f the disease. the results o f these assessments indicated that, due to the greater num ber o f joints affected, th e lower limb usually suffered greater functional im pairm ent th a n the upper limb. in order o f frequency, th e m ost com m on deformities seen in 33 cases were: (a) wrist fixed in a flexed position (b) hip flexion contracture (c) knee flexion contracture (d) loss o f p ro n atio n and supination (e) lim itation o f neck extension ( / ) lack o f flexion at the m etacarpophalangeal joint and lack o f extension a n d /o r flexion at the proxim al inter phalangeal joints. i. t h e a im s o f p h y sio t h e r a p y in our program m es, m inim al bed rest an d maximal mobility are encouraged. m any children, however, especially m rs. baldwin graduated fro m the university o f ,the witwatersrand, johannesburg, south africa, in 1960. she worked in south africa, england and norway before coming to canada where she worked in two other hospitals before joining the h ospital fo r s ick children. she is now research therapist, juvenile rheumatoid arthritis unit. in th e early stages, may require m ore rest an d sleep than norm al children. o ur aims are: 1. t o relieve pain. 2. t o prevent deformities by stretching, strengthening a n d splinting. 3. t o record the process o f the disease (fig. 1). 4. t o help prevent em otional disturbances by ensuring th a t th e child participates in norm al, daily activities. 5. t o provide preand post-surgical treatm ent for children when necessary. these program m es vary w ith each patient and doctor. t he post-surgical treat­ m ent is intensive and specific for each individual. ii. m e t h o d s and r a t io n a l e o f treating a r t h r it ic c h ild r en i t should be emphasized th a t only a small percentage of children seen in the r heum atoid clinic need physiotherapy treatm ent. the children are assessed by a rheum atologist, physiotherapist an d social w orker before a treatm ent regimen is established. e ducation arid household routines are disrupted as little as possible, treatm ents being done after school and in the p atient’s hom e whenever possible. m uch has been w ritten o n the parents’ role in enforcing one h o u r o f exercise daily. f o r the large family this m ay be an unnecessary dem and and may create feelings o f guilt in the busy m other who has difficulty finding the time. specific exercises, such as quadriceps drill, are, however, taught to th e parents when possible. these are simple and should be kept to the minim um . r egular daily activities and, in some cases, periodic supervision by a physiotherapist should be adequate to ensure th a t muscle strength and jo in t mobility are m aintained. a. p ool therapy in a heated pool, g ro u p program m es th at stress strength­ ening an d stretching are popular with patients. the buoyancy o f the water facilitates m obility; the setting prom otes social mixing. m ore joints can be exercised simultaneously and m ore patients can be treated a t one time. b. exercise therapy exercises using proprioceptive neurom uscular facilitation (p n f ) technique a n d /o r isometric routines incorporate mass movem ent patterns and prove an-efficient, time-saving way o f exercising when several joints and muscle groups are affected. the adaptations fo r various age groups are dis­ cussed in th e section “ t reatm ent for d ifferent age g roups” . c . using daily activities e ach age group has certain abilities, skills and activities which should be used as exercise. d . special techniques 1. stretching techniques can be used in specific cases where ligamentous and capsular tightening restrict accessory movem ents. techniques using tractio n w ith minim al passive movem ents break th e small fibrinous adhesions. restoring these movem ents will in tu rn stim ulate the synovial mem­ brane an d secretion o f synovial fluid thus lubricating the jo in t. they are m ost effectively perform ed on affected finger joints but do not w ork in every case and should only be done by trained personnel. r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) march, 1972 p h y s i o t h e r a p y page 3 key ckffltbat. assessment chart ( juvenile rheumatoid project hospital for sick joint involvement (mark in red) affected joint ^ fixed deformity deformity can j— i be passively u corrected mobility planes of motion range of motion i , flexion & extension i i . abduction & adduction i i i . rotation (in t . & ext. orpron. & sup in) a."normal range b.-stretched range c.sshortened range d.°m id ranee max max shortened ^ ik stretched * >— < general acute tenderness on palpation tenderness on motion crepitus tenderness on extreme motion ' “5 ? # • v / f 1 \ \ v remarks hands feet 2 . splinting is used either to prevent or correct deformities. once a deformity has developed, an individual splint has to be devised to correct o r minimize it. in the past, splinting for children’s joints has been the same as that fo r adults. deform ities in children, however, qmer from those in adults (see earlier list o f com m on uetormities) and consequently the physiotherapist, occu pa l?na! t herapy d epartm ent and o rthopaedic w orkshop m tk ° i ic j-,epartm ent) are experim enting w ith new splinting tnk u s and raaterials. t he children’s splints have tended anh i, ’ included unaffected joints, increased deformities no have not been durable. we are experimenting with nlact^r’ coate^ p o ster, fiberglass im pregnated bandages and c materials such as sansplint and polycast. m ost o f these splints are m ade in the p atient’s home, an im portant factor when selecting splinting material. the wrist splint is applied after three m onths if the wrist remains swollen an d limited in m otion (especially extension) o r if there is a wrist flexion contracture. a plastic backslab (sansplint® ) is th e m ost com fortable type o f wrist splint because it “ pulls” rath er th an “ pushes” the wrist into slight extension. t he backslab extends from m id-forearm to one h a lf inch proxim al to the m etacarpal heads; an anterior b a r extends across the palm . fingers are not included in the splint since finger m otion should be encouraged especially a t the m etacarpo-phalangeal joint, which frequently lacks flexion. f urtherm ore, u lnar drifting o f the fingers is un­ com m on in children. t he splint is w orn m ost o f the day r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) page 4 p h y s i o t h e r a p y march, 1972 and night, the tim e being decreased as pain subsides and m ovem ent returns. the knee splint is applied to a swollen knee jo in t th a t has developed a flexion contracture. a bivalved stovepipe type o f cast is used fo r this purpose and the patient is encouraged to walk as m uch as possible. t he cast is rem oved for exer­ cises. w hen w alking w ith the knee extended, n o t only are th e quadriceps muscles being w orked statically against the resistance supplied by the body weight, b u t also th e collateral ligaments and capsule are taught, thus stretching these tightened structures. the p atient wears the cast day and night until the contracture has been corrected an d the quadriceps are strong enough to m aintain the knee in extension when walking. this may require three to four m onths. as this m ethod o f splinting differs so radically from th e conventional resting splints, we are docum enting all cases and studying the problem s o f m anaging knee flexion contractures in children. f o r th e children requiring a resting knee splint fo r pain, swelling and severe m orning stiffness, a bivalved stovepipe cast seems m ost satisfactory. t he foot is rarely enclosed in th e cast since usually th e knee can be fully extended w ithout it. since children are seldom immobilized in bed, there is no tendency to foot drop, even when the ankle is affected. c rutches are seldom used as th e m ajority o f children are able to bear weight and move well once th e initial morning stiffness is overcome. id . t r e a t m e n t f o r d iffe r e n t a g e g r o u ps a. one to three years tickling th e bottom s o f th e feet results in hip and knee flexion. placing a bracelet over the fo o t results in hip and knee flexion when th e child tries to pull it off. certain toys can be used to correct problem s. a tricycle stresses hip an d knee flexion as well as knee extension, ankle plantar, and dorsiflexion. d rum s o r xylophone encourage finger flexion and static w rist extension. blocks and building toys require finger movements. t he child should be encouraged to walk an d feed himself. binding techniques, whereby th e therapist grasps the child firmly and then encourages him to break free, results in the child unw ittingly exercising isometrically against the m anual resistance. f o r example when the therapist stretches th e child’s arm s above his head he tries to bring his arm s dow n producing isometric contractions o f shoulder extensors, elbow, wrist an d finger flexors. w hen a child’s legs are painful, he will prefer to sit, and his parents m ay have to lure h im in to w alking by placing a favourite toy beyond reach. the m other is show n w hat play techniques will help her child exercise in his m orning bath. t he w arm b ath also relieves early m orning stiffness. b. four to six years c hildren in this age group enjoy hitting a balloon thus exercising shoulder elevators, elbow, wrist and finger extensors. toys such as a bicycle o r skipping rope provide ad ditional exercise. should there be increased'swelling o r pain after an activity such as skipping, the activity is de­ creased and elim inated if necessary. pool therapy in w arm w ater can be started in this age group. isom etric exercises using binding techniques (see under o ne to three years) are helpful. t he m other should teach h e r child to dress an d undress. she should also encourage an d praise him , and report to th e physiotherapist any problem s such as increase o f pain and swelling after certain activities, inability to perform any daily activity, any new jo in t involvement, behavioural problem s o r problems with medication. c. seven to ten years play techniques such as ball throw ing will prom ote m obility o f th e joints. a lthough body contact sports such as football are discouraged, activities such as ice skating, bicycling and swimming are encouraged. o rganized resisted exercises using isometric and p n f techniques can be fun especially if the routine is changed frequently. participation in either brownies o r cubs is socially beneficial fo r the child. d om estic activities are also encouraged. pool therapy is popular. the parent should support an d encourage th e child an d report any problems to th e therapist. d . eleven years and over o rganized resisted exercises using p n f w ith isometric exercises, o r isometric exercises alone, are helpful. dancing and swimming are popular and foster cam araderie. pool therapy is excellent. c ooking and babysitting for girls and repair jo b s fo r boys prom ote n atu ral exercise. taking m edication and exercising are now th e responsibilities o f the child w ho can be taught exercises fo r specific problems. h e should be encouraged to rep o rt any problem s to the physio­ therapist. iv . g en era l tre a tm en t d isc u ssio n t he majority of. children w ith jo in t m obility loss and/or muscle weakness require only a weekly visit from th e thera­ pist. we found in com paring results o f the “ once weekly group pool therapy program m e” to those o f th e “once weekly hom e exercise program m e” th at hip and knee m obility as well as quadriceps strength was m aintained but n o t increased in 90 per cent o f th e patients in both groups. twelve children (six per group) to o k p art in these pro­ gram mes and it was felt th at, physically, the programmes were o f equal benefit. should specific muscle strengthening an d jo in t m obilization be required, a m inim um o f three treatm ents per week is necessary and should be done in the child’s hom e by either the parent or physiotherapist. when the child’s arthritis becomes inactive, regular check-ups are still essential to prevent deformities resulting from weaknesses and to modify treatm ents. such check-ups should continue until the child stops growing. i f the parents are unco-operative o r unable to assist, the physiotherapist may have to assum e the responsibility for the. child’s weekly treatm ent. the child o f unco-operative parents may have difficulty coping w ith arthritis in later life. i n addition, a specialized treatm ent centre provides vocational guidance, aptitude testing and physical tolerance assessments, orthopaedic research brace shops, research units, and com m unity physiotherapy. the physiotherapist is p a rt o f a team consisting o f paediatric rheum atologists; paediatric orthopaedic surgeons; paediatric opthalm ologists; social w orkers; occupational therapists; public health nurses. such a team is dedicated to caring fo r children suffering from juvenile rheum atoid arthritis. bib lio g r a ph y ansell, b arbara, and bywaters, e. g . : “d iagnosis o f 1 ‘p robable’ still’s disease and its o utcom e.” 1962, ann. rheum. dis., 21: 253. h ollander, j. l .: “ a rthritis an d allied c onditions,” 6 th e dition, 1960, lea and febiger, philadelphia. l aaksonen, a. l ., an d laine, u . a .: ‘‘c om parative study o f jo in t pain in a dult an d juvenile rheumatoid a rthritis.” 1961, ann. rheum. dis., 20: 386. c o r r e c t io n d ec e m b er , 1971 m odular lower extrem ity prosthetics by j. fo o r t , form erly technical director, manitoba rehabilitation hospital, winnipeg, canada. south african jo u rn al o f physiotherapy, 1971, 27(4): 2-5. the p hotograph labelled “ a m odular h ip d isarticulation prosthesis o f the w innipeg type,” on page 2, should be preceded by fig. 3. i t is an integral p a rt o f the article, “ m odular lower extrem ity prosthetics,” by j. f oort, pages 3-5. t he last line o f colum n 1, page 4, should read : “ . . the socket bolted on to the hip jo in t (fig. 3)” . r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) frequency of antineutrophil cytoplasmic antibodies (anca) in some autolmmune diseases iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 55 frequency of antineutrophil cytoplasmic antibodies (anca) in some autoimmune diseases falah s. manhal* ,1 and husam m. abbas ** * departement of clinical laboratories , college of health and medical technology,baghdad.iraq . ** baghdad teaching hospital , ministry of health , baghdad , iraq . abstract anti-neutrophil cytoplasmic antibodies (anca) are a heterogeneous group of autoantibodies with a broad spectrum of clinically associated diseases. the diagnostic value is established for proteinase 3 (pr3)-anca as well as myeloperoxidase (mpo)-anca. to estimate the frequency of anti-neutrophile cytoplasmic antibodies (anca) in sera from a group of iraqi patients with some autoimmune diseases compared with a healthy control group. serum samples were collected from one hundred patient, 74 males and 53 females; with age range of 16-70 years; 20 specimens from patients with systemic lupus erythematosus (sle), 30 from patients with ulcerative colitis (uc), and 50 from patients with rheumatoid arthritis (ra). a group of 40 apparently healthy blood donors was included as controls. anca were checked using enzyme-linked immunosorbent assay (elisa). positive anca was detected in sera of 81 (18%) patients with autoimmune disorders. anti-pr3 was detected in 6 (12%) patients with ra, and in 4(13.4%) patients with uc. anti-mpo was detected in 3(6%) patients with ra and in 5(16.6%) patients with uc. all serum samples of patients with sle showed negative anca. there were no ancas detected in sera from healthy individuals. mean of serum anti-pr3 (u/ml) among the studied groups was 2.057 in ra, 2.209 in sle, and 2.283 in uc, and 1.739 in control group. statistical analysis revealed that differences in the anti-pr3 between ra, uc and controls were highly significant (p > 0.01), whereas just significant with sle (p> 0.05). mean of serum of anti-mpo (u/ml) among the studied groups was 0.711 in ra, 0.695 in sle, and 1.170 in uc, and 0.652 in control group. statistical analysis revealed that the differences in the anti-mpo between ra and sle, controls were non significant (p < 0.05), whereas highly significance with uc (p> 0.01).it was concluded that anca markers might play a role in the inflammatory process and they are important factors for the clinical course, and prognosis in the patients with autoimmunity. however, anca in autoimmune disorders must be interpreted cautiously with particular attention paid to laboratory technique, the size, age and genetic background of the populations studied. key words: rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, antineutrophile cytoplasmic antibodies (anca). الخالصة هاام اوةم اام الىاناام ااا اذجساا ا ااطاولاام ان الااا ا اا ااا اذااا ا األجساا ا ااساا الىمية الم ااة اا الي االةالاا ااةى الااام اغاا وياااال ساابم والاا اس اذجساا ا 6004اغ الاام اااب ا 6002اء هااطا اااسا اام اانىاا ي ااا وااا ال اا اا م وااا اجاا .ااة وبطاام اا ال ال فام مااا م اوةم ام اا ااة لار اا ا اللل ااةاا يل ياب األاا ا اولام (anca) ااس الىمية الم ااة ا الي االةالا ااةى الاام 40-82م ا ي ية اام ةا 55 كامس 74 لنم اال ا ااة لر ) 800اا يطم األصح ء . وا جة مااةن م اي س م ي اةوةم لناام ااا 50ااا ااةااا يل ي اىااا اايمااامي ااىي اام 50 لناام ااا ااةااا يل يااااء ااااطو ااحةاا ام ااةوةاام م 60، اانم اااا كةوةم ام لا يطم . واا لنم اال ا ي ااةىبا ل األصاح ء فام ااا ا 70ااةا يل ي اىا ااةن صل اا ثم . جة ه 18. وااا وااالله هااطا اذجساا ا ااة اا الي فاام (elisa)ي ااىلااا وينلاام ااةي الساام ااةن لاام ا الةلاام ancaااىحاا اا جاامال % فام ا لار 85.7)7% اا ااةاا يل ي اىاا ااةن صال اا ثام فام 86) 2فام anti-pr3% ا كل ااة لر . ظاا 18) اا ا لار ااىاا اايماامي. 5(%16.6) % ا ا لر ااىا ااةن صل اا ثم 2) 5فم anti-mpoااىا اايمامي . ظا ك ه اانى وج ابم فم الاء ااطو ااحة ام ااةوةم م. اا وظا هطا اذجس ا فم ااةوةم م اا يطم . ظا ا خةم هاطا اااسا ام اي ) اااي / ااا anti-pr3اىم اا 5 فاام الاء ااااطو 2.209فاام ااىااا ااةن صااةا ثم 2.057ىاام الس ااه كاا ي ياال ااةواا ال اا فاام ااةوةم اام اا اا يطم . مظااا ااىحالاال ا ااا وم ياا ي 1.739فاام ااىااا اايمااامي ااىي اام 2.283ااحةاا ام ااةوةاام م > (pاا ا يطم كا ي ا نمالا يااالل ا م يال ااىاا ااةن صال ااىاا اايماامي ااىي ام اي س ام ي اةوةم ام anti-pr3اذخىة فام ) ااي / اا anti-mpo . ظا كاطا اي اىم ا p) <0.05 فم ل ك ي اذخىة ا نمال في فم الاء ااطو 0.08 5 كا ي ااةوةم ام فم 0.652فم ااىا اايمامي ااىي م 1.170فم الاءااطو ااحة ام 0.695فم ااىا ااةن صل 0.711 < (pاا يطم . ظا كطا اي اذخىة فم اانى وج يل ااىا ااةن صل الاء ااطو ااحة ام اوةم ام ااسالط ي كا ي لا ا نام . الساىنىج اا هاطا اااسا ام يا ي األجسا ا p) <0.08 فم ل ك ي اذخىة ا نمال ياالل م ا ااىاا اايماامي ااىي ام 0.05 لاا وا اد ال سا فام اا ةالام اذاىا يلام وحاالاا ااةسا س ااسا ال ااال ااة لار anca)الم ااة ا الي االةالا ااةى الاام )ااس الىمية ااةا يل ي ا ا ااةن م ااطاولم . االا جامال هاطا اذجسا ا ااة ا الي الواد اي الااله يحاطس فام ااا ا ااةن ام ااطاولام اا و كلا ة س ااة لر خانلىاا اامساثلم. اذ ىب ا ار وينلم اانحه اال ا 1 corresponding author e – mail : falahsalim@yahoo.com received : 3 / 3 / 2009 accepted : 26/ 9 / 2009 iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 52 introduction antineutrophil cytoplasmic antibodies (anca) are a heterogeneous group of autoantibodies with a broad spectrum of clinically associated diseases. antineutrophil cytoplasmic antibodies (anca) have specificity for constituents of neutrophil granules [1] . there are two different subtypes of anca identifiable by indirect immunofluorescence method. one subtype is an antibody against myeloperoxidase (mpo), which stains in a perinuclear pattern (panca) indirect immunofluorescence (iif) using a neutrophil substrate, and the other subtype is an antibody against proteinase3(pr-3), which stains in a diffuse granular cytoplasmic pattern (c-anca) by iif [2] . antineutrophil cytoplasmic antibodies (anca) are mostly known as a useful diagnostic tool in patients with small-vessel vasculitis. with the accumulating knowledge of these autoantibodies, however, it becomes clear that the role of anca may not be only limited to a diagnosis of such disorders [3] . the current review addresses, in addition to classical diagnostic associations, other diseases connected with anca positivity, both in adults and in children [4] . the etiology of anca remains unknown, but still, the importance of both genetic and environmental factors is undoubted. the role of infection and chemicals in the etiology of anca-associated diseases is stressed in particular. a pathogenetic role of anca is suggested because of clinical observations based on the correlation of the vasculitis activity and the titer of anca [5] . many experiments show the effects of anca in various steps of an inflammatory process, particularly on leukocyte microbicidal activity and oxidative burst. recent findings are analyzed in the experimental field and they are correlated with clinical significance [6] . many in vitro studies show that those anca have phlogistic potential, particularly at the interface of neutrophils and endothelial cells. a limited number of studies in experimental animals support their pathogenetic role. however, anca alone are not sufficient, as based on clinical and experimental data, and other, probably exogenous factors, seem necessary for disease induction and reactivation [7] . data concerning the investigation of pathological and/or diagnostic role of anca in autoimmune disorders from iraq are scarce. this study was designed to evaluate the frequency of anca in serum samples from a group of iraqi patients with certain autoimmune disorders compared with apparently healthy controls. materials and methods this prospective controlled study was conducted during the period from november 2006 to february 2007. blood samples were collected from one hundred patients with autoimmune disorders; 20 specimens from patients with systemic lupus erythematosus (sle), 30 from patients with ulcerative colitis (uc), and 50 from patients with rheumatoid arthritis (ra). the study patients were admitted with approved and documented autoimmune diseases. a questionnaire form was formulated that involved name, age, gender, clinical history, disease type, disease duration, family history, residence, socioeconomic status, general health condition, smoking, drinking, and any possible previous therapies. patient specimens were gathered from baghdad teaching hospital, and gastrointestinal and liver diseases center. a group of 40 apparently healthy blood donors was included as a control from national bank for blood transfusion. blood specimens from patients and healthy controls were properly conveyed to the location of processing and testing at the department of immunology, central laboratories for public health. the serum specimens were obtained and distributed in eppendrof vials and saved in deep freezing at -20 c° until testing. anti-pr3 testing anti-pr3 (elisa anti-pr3 kit, biomaghreb, tunisia) is an indirect solid phase enzyme immunometric assay. it is designed for the quantitative measurement of igg class autoantibodies directed against proteinase 3 (pr3). the microplate is coated with highly purified proteinase 3(pr3). when autoantibodies exist, it will bind to the pr3 and form a complex. by adding the enzyme conjugate solution and its substrate, an enzymatic color reaction occur which can be read later. four parameters fit with ling-long coordinates for optical density were used to calculate and convert the optical density readings. cut off value for the studied parameters were normal when anti-pr3 ab > 5 u/ml, and elevated when anti-pr3 ab < 5 u/ml. anti-mpo testing anti-mpo (elisa anti-mpo kit, biomaghreb, tunisia). it is designed for the quantitative measurement of igg class autoantibodies directed against myeloperoxidase (mpo). in case of the presence of autoantibodies to mpo, there will be a combination with the highly purified myeloperoxidase that coat the microplate, which in turn form an immune complex and amplified when add the enzyme conjugate iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 54 solution and its substrate resulting in an enzymatic reaction that produce color. four parameters fit with ling-long coordinates for optical density were used to calculate and convert the optical density readings. cut off value for the studied parameters were normal when anti-mpo ab > 5 u/ml, and elevated when anti-mpo ab < 5 u/ml. statistical analysis descriptive statistics was used for frequencies and percentages. inferential statistics was used in order to accept or reject the statistical hypotheses they include: binomial test for testing the difference between two ratios related to binary nominal responding with pointed their p-values; chisquare test for testing independency between the two categories factors in the contingency table with pointed their p-values, and anova test for less significant differences (lsd). results one hundred patients were included in this study, 47 (47%) males and 53 (53%) females. distribution of patients among age groups showed that 29 (29%) patients were in 16-30 yrs age group, 53 (53%) patients were in 31-50 yrs age group, and 18 (18%) patients were in 51-70 yrs age group. frequency of anti-pr3 and anti-mpo antibodies in sera from patients and apparently healthy controls was shown in table1. positive anca was detected in sera of 18 (18%) patients with all studied autoimmune disorders. anti-pr3 was detected in 6 (12%) patients with ra, and in 4(13.4%) patients with uc. anti-mpo was detected only in 5(16.6%) patients with uc. total serum samples of patients with sle showed negative anca. there were no ancas detected in sera from healthy individuals table 1: frequency of anti-pr3 and antimpo antibodies in sera from patients and apparently healthy controls 1:ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis . the results shown in table 2 indicate that mean of anti-pr3 antibody levels in sera from patients and healthy controls did not rise above normal cut off value, although there were remarkable differences between patient and control antibody levels. table 2: mean of anti-pr3 ab levels (u/ml) in sera from patients and healthy controls studied groups no. mean sd 4 min. max. anova (f-test) pvalue sig. control 40 1.739 0.618 1.20 4.30 0.024 p> 0.05 ra 1 50 2.057 0.836 0.15 5.70 sle 2 20 2.209 0.637 1.45 3.50 uc 3 30 2.283 0.912 1.10 5.20 total 140 1: ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 sd: standard deviation . in table 3, comparison of significance of differences in anti-pr3 antibody levels among patient and control groups indicates that were highly significant differences between controls and ra and uc. on the other hand, a significant difference only was shown between control and sle. table 3 : comparison of significance of differences in anti-pr3 ab levels among patients and control groups 1: ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 lsd: less significant difference in table 4, the mean of anti-mpo antibody levels in sera from patients and healthy controls did not rise above normal cut off value, although there were remarkable differences between patient and control antibody levels. studied groups no. positive anti-pr3 abs positive anti-mpo abs total no. % no. % control 40 ra 1 50 6 12% 3 6% 9 sle 2 20 uc 30 4 13.4% 5 16.6% 9 total 140 18 studied groups lsd 4 (f-test) pvalue significance control ra 1 0.048 highly sig. (p> 0.01) sle 2 0.024 sig. (p> 0.05) uc 3 0.003 highly sig. (p> 0.01) ra sle 0.449 non sig. (p< 0.05) uc 0.198 non sig. (p< 0.05) sle uc 0.735 non sig. (p< 0.05) iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 51 table 4 :mean of anti-mpo ab levels (u/ml) in sera from patients and healthy controls studied groups no. mean sd 4 min. max. anova (f-test) pvalue significa nce control 40 0.652 0.217 0.32 1.18 0.001 highly sig. (p> 0.01) ra 1 50 0.711 0.294 0.47 5.20 sle 2 20 0.695 0.278 0.32 1.50 uc 3 30 1.170 1.080 0.45 5.80 total 140 1: ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 sd: standard deviation . the results in table 5 show a comparison of significance of differences in anti-mpo antibody levels among patient and control groups that indicate non significant differences between controls and ra and sle. on the other hand, a high significant difference only was shown between control and uc . table 5: comparison of significance of differences in anti-mpo ab levels among patients and control groups 1:ra: rheumatoid arthritis, 2sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 lsd: less significant difference discussion antineutrophil cytoplasmatic antibodies (anca) are a group of autoantibodies found in several inflammatory disorders in which they supposedly amplify the inflammatory process [8] . anti-neutrophil cytoplasmic antibody (anca) tests are a routine clinical assay in most of western world. the anca tests are being widely applied with very poor return. guidelines for more effective usage are proposed [9] . although substantial studies have been carried out for investigation of autoimmune disorders, data concerning the pathological and/or diagnostic role of anca from iraq are scarce. these studies concerned with investigation of epidemiological and pathological aspects of autoimmune disorders rather than anca profiling. in this study, positive anca was detected in sera of 18 (18%) patients with autoimmune disorders. the low percentage of positive results detected by anca testing could be attributable to the limitations of our study, that's including: some of study patients were referred while they in remission stage rather than active phase of the diseases, the elisa test was not coupled with indirect immunofluorescence technique (iift) due to the hospital policy, anca levels were not measured serially by quantitative image analysis, and the clinical relevance of the studied cases might be doubtful. specific elisas for antibodies to pr3 and mpo are commercially available, and should be part of any standardized approach to the testing for anca. pr3-anca and mpo-anca are associated with substantially higher specificities and positive predictive values than the immunofluorescence patterns to which they usually correspond (cand p-anca, respectively) [10] . it was reported that the use of the immunofluorescence method coupled with identification of anca sub-specificities by elisa, is recommended for detection of anca in clinically suspected cases of small vessel and other systemic vasculitis [11] . several studies suggested that anca titers correlated with disease activity. serial measurements of pr3and mpo-anca titers in patients with anca-associated vasculitis during remission can help predict relapses, and preemptive increases in immunosuppression following fourfold titer rises reduces the risk of relapses [12] in this study, it was found that in patients with ulcerative colitis, antineutrophil cytoplasmic antibody titers do not correlate with disease activity and there was four patients (13.4%) showed titer elevation for anti-pr3 and five (16.6%) for anti-mpo. in a study conducted in baghdad on 2004 by albadry et al, approximately the same result was recorded [13] . several retrospective and prospective studies have suggested that the demonstration of anca lacks sensitivity and specificity, but these series have detected anca with neutrophil-indirect immunofluorescence alone and have included patients with inactive disease. in an iraqi study carried out in baghdad city it was confirmed that anca is not useful as a routine test for ulcerative colitis, rheumatoid arthritis and systemic lupus erythematosus and it is not specific for them [14] . the 'international consensus statement on testing and reporting anca' has been developed to optimize the clinical relevance of anca testing by the studied groups lsd 4 (f-test) p-value significance control ra 1 0.672 non sig. (p< 0.05) sle 2 0.814 non sig. (p< 0.05) uc 3 0.001 highly sig. (p> 0.01) ra sle 0.924 non sig. (p< 0.05) uc 0.003 highly sig. (p> 0.01) sle uc 0.014 sig. (p> 0.05) iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 55 adoption of standardized testing and reporting procedures. international collaborative efforts continue to focus on improving the tests for anca [15] . in ulcerative colitis, several studies reported that high titers of anca were particularly found in patients with active disease [16] . other studies failed to detect a relation between disease activity and anca titer [17] . in the present study, the main findings of systemic lupus erythematosus were the total absence of elevation in anca above normal levels. systemic lupus erythematosus known to be diagnosed with elevated antinuclear antibodies (ana) rather than anca. anca testing remains unstandardized and there are no references for normal ranges. in sizing up the utility of anca testing in clinical practice, it must be borne in mind that most studies of anca serologies have been performed at tertiary care centers using research laboratories focused on anca testing. translating the test characteristics from the environments of research laboratories to clinical practice must be done with caution. [18] . conclusion: anca markers might play a role in the inflammatory process and they are important factors for the clinical course, and prognosis in the patients with autoimmunity. however, anca in autoimmune disorders must be interpreted cautiously with particular attention paid to laboratory technique , the size , age and genetic background of the populations studied . the results of anca testing by elisa alone rema in controversial . indirect immunofluorescence test, known as the "gold standard" for screening of anca, can be further substantiated by elisa for confirmation and for identifying subspecificities like anti-myeloperoxidase (antimpo), anti-proteinase 3 (anti-pr3) and antilactoferrin (anti-lf.). references 1puszczewicz,-m; zimmermann-gorska,-i; bialkowska-puszczewicz,-g; tuchocka,-a. "prevalence and specificity of antineutrophil cytoplasmic antibodies (anca) in connective tissue diseases". pol-arch-med-wewn.; 109(1): 35-41, 2003. 2birck r; schmitt wh; kaelsch ia; van der woude fj. serial anca determinations for monitoring disease activity in patients with anca-associated vasculitis: systematic review. am j kidney dis. 2006 jan;47(1):15-23. 3seo p; stone jh. the antineutrophil cytoplasmic antibody-associated vasculitides. am j med 2004 jul 1;117(1):39-50. 4sinico ra; di toma l; maggiore u; bottero p; radice a; tosoni c; grasselli c; pavone l; gregorini g; monti s; frassi m; vecchio f; corace c; venegoni e; buzio c. prevalence and clinical significance of antineutrophil cytoplasmic antibodies in churg-strauss syndrome. arthritis rheum 2005 sep;52(9):2926-35. 5levy jb; hammad t; coulthart a; dougan t; pusey cd. clinical features and outcome of patients with both anca and anti-gbm antibodies. kidney int 2004 oct;66(4):1535-40. 6yang g; tang z; chen y; zeng c; chen h; liu z; li l. antineutrophil cytoplasmic antibodies (anca) in chinese patients with anti-gbm crescentic glomerulonephritis. clin nephrol 2005 jun;63(6):423-8. 7birck r; schmitt wh; kaelsch ia; van der woude fj. serial anca determinations for monitoring disease activity in patients with anca-associated vasculitis: systematic review. am j kidney dis. 2006 jan;47(1):15-23. 8bartunkova,-j; tesar,-v; sediva,-a. " diagnostic and pathogenetic role of antineutrophil cytoplasmic autoantibodies". clin-immunol. 106(2): 73-82, 2003. 9kallenberg,-c-g; rarok,-a; stegeman,-ca; limburg,-p. "new insights into the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis". autoimmun-rev.; 1(1-2): 61-6, 2002. 10csernok e; holle j; hellmich b; willem j; tervaert c; kallenberg cg; limburg pc; niles j; pan g; specks u; westman k; wieslander j; de groot k; gross wl. evaluation of capture elisa for detection of antineutrophil cytoplasmic antibodies directed against proteinase 3 in wegener's granulomatosis: first results from a multicentre study. rheumatology (oxford) 2004 feb;43(2):174-80. epub 2003 oct 29. 11stone jh; talor m; stebbing j; uhlfelder ml; rose nr; carson ka; hellmann db; burek cl. test characteristics of immunofluorescence and elisa tests in 856 consecutive patients with possible anca-associated conditions. arthritis care res 2000 dec;13(6):424-34. 12mclaren,-j-s; stimson,-r-h; mcrorie,-er; coia,-j-e; luqmani,-r-a. " the diagnostic value of anti-neutrophil cytoplasmic antibody testing in a routine clinical setting". qjm.; 94(11): 615-21, 2001. 13al-badry m.d., al-kubaisy w.a, al-naib k.t. and m. habib. mmj, vol. 12 no. 1&2 jan. 2006. iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 20 14hosam m abbas. measurements of various immunological markers in sera from patients with certain autoimmune diseases, a comparative study. m sc. thesis, college of health and medical technology, baghdad, july 2008. 15yu,-f; zhao,-m-h; zhang,-y-k; wang,-hy. "the relationship between subclassification of anti-myeloperoxidase igg by enzyme-linked immuno-sorbent assay analysis and vasculitis activity". zhonghua-nei-ke-za-zhi.; 42(1): 27-30, 2003. 16han,-w-k; choi,-h-k; roth,-r-m; mccluskey,-r-t; niles,-j-l. " serial anca titers: useful tool for prevention of relapses in anca-associated vasculitis". kidney-int.; 63(3): 1079-85, 2003. 17seibold f, weber p., klein r., et al,. "clinical significance of antibodies against neutrophil in patients with inflammatory bowel disease and primary sclerosing cholangitis". gut, 33:657-62, 1992. 18john h stone. clinical spectrum of antineutrophil cytoplasmic antibodies. upto-date online desktop 16.2, 2008. microsoft word 3. oasameera irfan 68 vol. 29, no. 2, apr – jun, 2013 pakistan journal of ophthalmology original article role of topical cyclosporin in scleritis: a case series sameera irfan, harris iqbal pak j ophthalmol 2013, vol. 29 no. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . see end of article for authors affiliations …..……………………….. correspondence to: sameera irfan 312, k1,wapda town lahore …..……………………….. purpose: to report on a series of cases of anterior, non-necrotizing scleritis, that were effectively treated with topical 0.075% 1% cyclosporine a and to provide a literature review on other treatment modalities of this condition. material and methods: this study includes eleven consecutive patients of acute anterior, non-necrotizing scleritis reporting to mughal eye hospital, lahore, during january to december 2012. there were 7 females and 4 males between the age 18 and 65 years. they all had mild to moderate, anterior scleritis, nodular in 6 cases and diffuse in 5 cases. all patients were thoroughly investigated regarding auto-immune disorders. only 2 cases had associated rheumatoid arthritis. they were treated with topical cyclosporine and preservative-free artificial tears for 2-3 months and followed-up for 1 year regarding recurrence. results: all patients showed a favorable response to treatment. the scleritis score improved remarkably within 2 weeks of cyclosporin therapy. the symptoms flared up in only 2 out of 11 cases (18%) when they abruptly stopped therapy. all cases remained asymptomatic over the one year follow-up after stopping treatment. conclusion: topical 0.075% 1% cyclosporine a is a safe and effective longterm treatment of anterior scleritis of mild to moderate severity. it should be considered as a steroid-sparing agent, particularly in recurrent disease and in those patients who experience adverse effects of systemic medications. cleritis is a severe, potentially sight threatening, inflammatory disease involving the ocular surface. it has been be classified by pg watson et al,1 into anterior and posterior. anterior scleritis can be diffuse, nodular, necrotizing with inflammation (necrotizing), and necrotizing without inflammation (scleromalacia perforans). posterior scleritis2 is characterized by flattening of the posterior aspect of the globe, thickening of the posterior coats of the eye (choroid and sclera), and retrobulbar edema. the author states that the anatomical site and clinical appearance of the disease at presentation reflected its natural history; majority of patients remain in the same clinical category throughout the course of their disease. diffuse anterior scleritis had a lower incidence of visual loss (9%) than either nodular scleritis (26%) or necrotizing disease (74%), patients with necrotizing scleritis were older than patients in the other groups and more frequently had an associated systemic disease than patients with either diffuse or nodular disease, 57%.3 according to another study, necrotizing scleritis is associated with rheumatoid arthritis in most cases and less often with sle, crohn's disease, behcet's disease and gout.4 diffuse and nodular anterior scleritis is less often associated with any systemic disease. an underlying infectious etiology has been found to be relatively less common. according to a study by gonzalez et al5, out of 500 patients presenting with scleritis, only 9.4% had an underlying infection with herpes virus infection (74%), tuberculosis (10%) and other infections in the remaining 14%. s role of topical cyclosporin in scleritis: a case series pakistan journal of ophthalmology vol. 29, no. 2, apr – jun, 2013 69 an autoimmune deregulation in a genetically predisposed host is presumed to cause scleritis. inciting factors may include infectious organisms, endogenous substances, or trauma. the inflammatory process may be caused by immune complex–related vascular damage (type iii hypersensitivity) and subsequent chronic granulomatous response (type iv hypersensitivity).6 ocular complications of scleritis, which cause visual loss and eye destruction, appear as a result of the extending scleral inflammation. these include peripheral ulcerative keratitis (13 – 14%), uveitis (about 42%), glaucoma (12 – 13%), cataract (6 – 17%), and fundus abnormalities (about 6.4%).7.they are most common in necrotizing scleritis, the most destructive type of scleritis. despite recent advances, its treatment remains a difficult problem. systemic immunosuppressive therapy with corticosteroids or immunosuppressive agents or both is usually required to control the disease. early therapeutic intervention is important to prevent ocular complications and to minimize the potential morbidity and mortality associated with underlying systemic disease. systemic corticosteroids in high dosage, either topical orally or in intravenous pulses, are widely accepted as an effective form of treatment in patients with severe scleritis.8 but this treatment is often associated with unacceptable side effects and does not always control scleral inflammation. side effects include adrenal suppression, vertigo, psychosis, pseudotumour cerebri, acne, osteoporosis, myopathy and delayed wound healing. the addition of immunosuppressive agents in patients with severe scleritis improves the ocular outcome and decreases the morbidity associated with systemically administered corticosteroids.9 azathioprine, cyclophosphamide, and cyclosporine have previously been reported to be effective and safe in the management of severe ocular inflammation.10 various studies have reported the successful use of topical cyclosporine in the treatment of patients with a variety of ocular inflammatory syndromes resistant to other immunosuppressive regimens.11,12 evidence obtained from these studies supports the efficacy of topical cyclosporine treatment through its immunomodulatory action, reversing inflammation of the ocular surface and lacrimal glands. in the present study we report the therapeutic effect of topical cyclosporine in the treatment of mild to moderate scleritis. material and methods this is a prospective case series of eleven consecutive patients with scleritis who attended the out patients department of mughal eye hospital, lahore, from january to december 2012. all cases were assessed by the same ophthalmologist. a detailed history was taken and physical examination performed; distinction was made between episcleritis and scleritis by the presence of eye pain, local tenderness over the area of nodule in 6 cases and diffuse purplish hue in 5 cases of scleritis; the conjunctiva could be freely moved over that area and no blanching of the lesion was achieved by a drop of 10% phenylephrine eye drops. the cases presenting with episcleritis were excluded from the study. all patients were graded to have an active anterior non-necrotizing scleritis of mild to moderate severity without associated anterior or posterior uveitis. appropriate investigations including cbc, esr, rh – factor, serum anca, ace, chest x-ray and mantoux test to exclude the presence of an associated auto-immune or infectious disease; hla autoantibodies and c reactive protein testing was not done due to economic constraints. the clinical features of the patients are summarized in (table 1). there were seven females between ages of 18 – 65 years and four males with the age of 45 years. a subjective grading system, analogous to that previously described for patients with uveitis and retinal vasculitis was used (table 2).13 a scleritis score was calculated when the patients first presented, then at each follow-up visit and at the end of the study (table 3). improvement was defined as a decrease in the total score of greater than 2 and resolution at a total score of less than or equal to 4. all patients were fully informed regarding the proper use and expected side-effects of topical cyclosporine which was then started as 1% twice /day (freshly prepared from cyclosporin capsules, 50 mg bioral; the water-miscible gel from 2 capsules mixed with 5cc distilled water) along with preservative – free artificial tears 4 x / day (biolan gel from stullin pharma, germany). treatment was started and patients were reviewed weekly to note their subjective and objective improvement. the therapy was continued for 2 months in mild cases (scleritis score 6 – 7) and 3 months in moderate cases (scleritis score = > 8). then it was gradually tapered by reducing the strength of cyclosporin drops to 0.075%, twice daily for 1 week, then once daily for one week, then on alternate day for a week and then it was finally stopped. sameera irfan, et al 70 vol. 29, no. 2, apr – jun, 2013 pakistan journal of ophthalmology results associated active rheumatoid arthritis was found in only 2 female cases who were 60 years old. no other systemic auto-immune or infectious disease was present in any other case. in all cases, the subjective improvement of eye pain was noted in the first week of therapy and objectively, reduction in scleral injection and tenderness improved within 2 weeks of therapy. the scleritis scoring system is detailed in table 2, and table 3 summarizes the scleritis score for each patient at presentation and at four weeks after the commencement of treatment. in all patients there was a significant decrease in the scleritis score at four weeks. this decrease in the scleritis score was maintained over the subsequent observation period. none of the cases developed scleral thinning, uveitis or a raised iop during or after the termination of therapy. the mean duration of treatment was 2 months in mild cases and 3 months in moderately severe cases. no recurrence of scleritis was noted in 9 cases after stopping the therapy. two cases (18%) stopped therapy abruptly after one month only; in role of topical cyclosporin in scleritis: a case series pakistan journal of ophthalmology vol. 29, no. 2, apr – jun, 2013 71 them, scleritis re-appeared which was again successfully controlled by starting the same therapy and educating them regarding their proper use and weaning. no recurrence of symptoms was noted in any case over one year follow-up after stopping the therapy. cyclosporin eye drops were well tolerated by all patients. table 4 summarizes the side effects looked for in the study. the only problem noted was stinging and a burning sensation on instillation of drops which subsided after instilling preservative-free tear drops after 10 minutes of instillation of cyclosporin eye drops. no other side effect was noted. discussion all eleven patients included in the study responded to the use of cyclosporine (freshly prepared at our hospital pharmacy). a subjective improvement of eye pain was noted within one week following the start of therapy and improvement of scleral injection and tenderness was noted after two weeks of therapy. it has been increasingly recognized as an effective therapeutic agent in the management of a variety of autoimmune diseases. it has been effectively used topically in ocular surface disorders like vernal keratoconjunctivitis, dry eye syndrome. inflammatory eye disease, particularly uveitis, is often well controlled by the regular use of systemic cyclosporin14. cyclosporin represents the prototype of a new class of drug that appears to work, at least in part, by acting at the level of cytokine production by immune cells.15 the selective ability of cyclosporine to interfere with the action of interleukin-2 makes it an appropriate agent for the treatment of diseases mediated by t cells. although the immunepathogenesis of scleritis is not fully understood, it is believed to be due to immune complex mediated vascular damage to scleral vessels, with the subsequent generation of a granulomatous reaction. t cells have an essential role in the formation of such granulomas, and cyclosporine may act in part by decreasing this component of the inflammatory response. multiple studies on the efficacy of topical cyclosporine for treating inflammatory ocular surface disorders have consistently shown a beneficial effect of the drug.14,17 the immune-pathogenic mechanism is complex and involves an ige mediated immediate hypersensitivity response as well as a t cell mediated immune reaction. animal studies have shown that cyclosporin has no intraocular penetration; it concentrates on the ocular surface which enhances its anti-inflammatory effect after long term use. even after a year of regular topical therapy, none to minimal blood concentration and in aqueous taps was found in rabbits. a large study16 of 392 patients with non-infectious anterior scleritis highlighted various therapeutic options available included nsaids, particularly cox-2 inhibitors (but they result in cardiovascular side effects) in 144 (36.7%), oral or topical steroids in 29 (7.4%), immune modulatory drugs (systemic cyclophosphamide, azathioprine, methotrexate) in 149 (38.0%), biologic response modifiers (brm) in 56 (14.3%), and none (n = 14). patients with idiopathic diffuse or nodular scleritis with a low degree of scleral inflammation or without ocular complications may respond to nsaids. patients with idiopathic diffuse or nodular scleritis with a high degree of scleral inflammation may respond to steroids. patients with diffuse or nodular scleritis with associated systemic disease may respond to imt or brms. patients with necrotizing scleritis may respond to imt, mainly alkylating agents. all these systemic therapies are associated with many side effects. similarly, topical steroids potentiate sameera irfan, et al 72 vol. 29, no. 2, apr – jun, 2013 pakistan journal of ophthalmology corneo-scleral thinning, raised intra-ocular pressure and cataract. in comparison, topical cyclosporin has minimal side-effects. only precaution to be used is that the beneficial affect is achieved after two weeks of therapy but the immune process is still active and the therapy has to be continued for at least two months and then gradually tapered, otherwise the disease flares up if treatment is stopped abruptly and too early as seen in our two cases. conclusion this study highlights the fact that topical cyclosporine is a potentially useful drug in the treatment of mild to moderate anterior scleritis; subjective and objective clinical improvement is achieved within 2-3 weeks of regular usage. hence, it is an effective steroidsparing agent. unfortunately, its use is complicated by frequent, mild side effects, like burning and stinging for a short while after instillation of eye drops and may be associated with recurrence of disease on suddenly stopping the therapy.14 patients need to be informed and educated regarding its appropriate use. to avoid recurrence of the disease, therapy has to be continued for at least 2 – 3 months and then gradually tapered. another problem with cyclosporine eye drops is that they have to be made fresh, without preservatives and have a shelf life of one week only. despite these limitations we consider that topical cyclosporine is a useful drug in the management of mild to moderate scleritis, and has a high therapeutic value in the treatment of this disease. author’s affiliation dr. sameera irfan mughal eye hospital (trust) lahore dr. harris iqbal mughal eye hospital (trust) lahore references 1. tuft sj, watson pg. progression of scleral disease. ophthalmology. 1991; 98: 467-71. 2. machado dde o, curi al, fernandes rs, bessa tf, campos wr, oréfice f. scleritis: clinical characteristics, systemic associations, treatment and outcome in 100 patients. arq bras oftalmol. 2009; 72: 231-5. 3. riono wp, hidayat aa, rao na. scleritis: a clinicopathologic study of 55 cases. ophthalmology. 1999; 106: 1328-33. 4. sousa jm, trevisani vf, modolo rp, gabriel la, vieira la, freitas dd. comparative study of ophthalmological and serological manifestations and the therapeutic response of patients with isolated scleritis and scleritis associated with systemic diseases. arq bras oftalmol. 2011; 74: 405-9. 5. sainz de la maza m, jabbur ns; foster cs. severity of scleritis and episcleritis. ophthalmology. 1994; 101: 38996. 6. sainz de la maza m, foster cs, jabbur ns. scleritis associated with systemic vasculitic diseases. ophthalmology 1995; 102: 687-92. 7. durrani k, zakka fr, ahmed m, memon m, siddique ss, foster cs. systemic therapy with conventional and novel immunomodulatory agents for ocular inflammatory disease. ophthalmol. 2011; 56: 474-510. 8. raizman m. corticosteroid therapy of eye disease. fifty years later. arch ophthalmol. 1996; 114: 1000-1. 9. sainz de la maza m, molina n, gonzalez-gonzalez la, doctor pp, tauber j, foster cs. scleritis therapy. ophthalmology. 2012; 119: 51-8. 10. carrasco ma, cohen ej, rapuano cj, laibson pr. therapeutic decision in anterior scleritis: our experience at a tertiary care eye center. j fr ophtalmol. 2005; 28: 1065-9. 11. machado dde o, curi al, fernandes rs, bessa tf, campos wr, oréfice f. scleritis: clinical characteristics, systemic associations, treatment and outcome in 100 patients. arq bras oftalmol. 2009; 72: 231-5. 12. gumus k, mirza ge, cavanagh hd, karakucuk s. topical cyclosporine a as a steroid-sparing agent in steroid-dependent idiopathic ocular myositis with scleritis: a case report and review of the literature. eye contact lens. 2009; 35: 275-8. 13. foster cs, forstot sl, wilson la. mortality rate in rheumatoid arthritis patients developing necrotizing scleritis or peripheral ulcerative keratitis. ophthalmology. 1984; 91: 1253-63. 14. utine ca, stern m, akpek ek. topical ophthalmic use of cyclosporine a. immunology and inflammation. 2010; 18: 352-61. 15. mccluskey pj, wakefield d. current concepts in the management of scleritis. aust nz j ophthalmol. 1988; 16: 169-76. 16. hillenkamp j, kersten a, althaus c, sundmacher r. cyclosporin a therapy in severe anterior scleritis. 5 severe courses without verification of associated systemic disease treated with cyclosporine a. ophthalmology. 2000; 97: 863-9. 17. kaçmaz ro, kempen jh, newcomb c, daniel e, gangaputra s, nussenblatt rb, rosenbaum jt, suhler eb, thorne je, jabs da, clarke ga, foster cs. cyclosporine for ocular inflammatory diseases. ophthalmology. 2010; 117: 576-84. numbering.indd 58 proceedings s.z.m.c. vol: 34(4): pp. 58-63, 2020. pszmc-772-34-4-2020 radiological analysis of hind paw joint in murine rheumatoid arthritis model treated prophylactically or therapeutically with cassia fistula versus naproxen 1hassan farooq, 2mariyam iftikhar piracha, 3muhammad usman, 1rabia tariq, 4saadia shahzad alam 1department of pharmacology & therapeutics, khawaja muhammad safdar medical college, sialkot, 2department of pharmacology & therapeutics, akhtar saeed medical college, lahore 3department of pharmacology & therapeutics, shalamar medical & dental college, lahore 4department of pharmacology & therapeutics, shaikh zayed medical complex, lahore abstract introduction: cassia fistula has anti-inflammatory activity. rheumatoid arthritis (ra) is an autoimmune disease, causing inflammation and disability of the affected joints. it is mostly treated symptomatically with nsaids, which themselves have a lot of adverse effects. in this study the anti-arthritic effect of cassia fistula versus naproxen has been observed radiologically. aims & objectives: the present study was radiological analysis of anti-arthritic effect of cassia fistula compared to naproxen in complete freund’s adjuvant (cfa) induced rat model of rheumatoid arthritis. place and duration of study: this study was conducted at the animal house of university of veterinary and animal sciences, lahore for a period of three months. material & methods: the study was carried out on 96 male rats divided into 12 groups of 8 rats each. single 0.2ml dose of complete freund’s adjuvant (cfa) was injected in the right hind paw of each rat in all groups except group 1 (negative control). group 2 was positive control. the prophylactic (3-7) and therapeutic (812) groups were given naproxen (25mg/kg), anthraquinone extract (250 &500mg/kg) or methanolic extract (250 & 500mg/kg) of cassia fistula orally bd on days 1,2 &3 (first dose preceding cfa injection by 30 minutes) and on days 9,10 &11 for therapeutic groups respectively. on day 15, x-rays of right ankle joints of all groups were taken as a confirmatory investigation on the final status of the arthritis. results: our results showed both anthraquinone and methanolic extracts of cassia fistula have dose dependent (500˃250mg/dl) prophylactic and therapeutic anti-arthritic potential albeit lesser than naproxen 25mg/kg, to reduce the swelling of hind paw and deformity of interphalangeal joints on radiological examination. conclusion: the efficacy of cassia fistula makes it a potential weaker candidate to naproxen in prevention and treatment of rheumatoid arthritis key words: cassia fistula, anthraquinone, ra, cfa, nsaid, anti-arthritic effect. introduction cassia fistula known as golden shower and amaltas, has great variety of uses ranging from constipation to glandular tumors in traditional medicine.1,2,3 each part of this plant has therapeutic potential but specifically its fruit pulp has antiinflammatory property and has demonstrated useful activity in various joint diseases.4 the common active principle found in all parts of the plant is a phenolic antioxidant: anthraquinone.5 rheumatoid arthritis is an autoimmune long lasting disease characterized by pain, swelling and stiffness of small joints which ultimately results in their deformity and disability.6,7 it has a better prognosis if treated early and militantly. the aim of treatment is to reduce pain and swelling, prevent bone deformity and improve quality of life. nsaids and disease modifying anti-rheumatic drugs (dmards) are usually used. nsaids are the most commonly prescribed drugs to subside pain and swelling throughout the world.1 naproxen (nsaid) inhibits tissue cyclo-oxygenase and decreases pro-inflammatory prostaglandins, the important mediators of pain and inflammation.8,9 thus, alleviates symptoms of rheumatoid arthritis but has some adverse effects.10,11 fruit pulp of cassia fistula has been used traditionally in many herbal medicines and shown to possess antiinflammatory activity.12 our previous experiments showed reduction in ra factor levels and ankle caliper measurement with cassia fistula given prophylactically and 59 radiological analysis of hind paw joint in murine rheumatoid arthritis model treated prophylactically... therapeutically.13 the present study was carried out to further verify those effects radiologically. this research was unique, as comparison of antiarthritic activity of the standard drug naproxen with cassia fistula methanolic fruit pulp extract and anthraquinone extract has not been done radiologically before to the best of our knowledge. material and methods after approval from the ethical review board committees of shaikh zayed federal postgraduate medical institute, lahore, this comparative study was completed over a period of six months at uvas, lahore. collection of cassia fistula bark and fruit pulp was done from botany department of university of punjab, lahore. cassia fistula extract preparation: the extraction process of cassia fistula bark and fruit pulp was performed in the labs of pcsir, lahore by following method.14 the extract was utilized after confirmatory anthraquinone test. anthraquinone extraction: methanolic extract preparation: confirmatory test for anthraquinones: cassia fistula bark and fruit pulp extract was boiled for 5 minutes after addition of 10ml of 1% hcl. sample was filtered and allow`ed to cool at room temperature. then using equal volumes of 10% ammonia and chloroform partition of the cooled filtrate was done twice and the layer was allowed to separate. the presence of combined anthraquinones was indicated by rose pink colour.13, 14 experimental setup: ninety six male wistar albino rats weighing 170200gm, acclimatized for a week, fed with standard laboratory diet, maintained at 25±2oc with relative humidity of 45-55% under 12 hours light and dark cycles were kept at uvas animal house, lahore. preparation of rat model of rheumatoid arthritis: to induce arthritis a single 0.2ml dose of cfa was injected in the right hind paw of each rat on day 1.15 a gradual increase in the swelling around injection site was noticed within few hours. the clinical evidence of arthritis was observed with gradual increase in the swelling around injection site and decreasing mobility of rats during 9th to 15th days of post cfa injection. 1% carboxymethyl cellulose was used as suspending agent for making water suspension of test extract and standard drug. extracts were given orally bd on days 1, 2 and 3 to the prophylactic groups (3-7) preceding cfa injection, and on days 9, 10 and 11 to the therapeutic groups (8-12).16 at the end of study on day 15 x-rays of right ankle joint were taken to evaluate bone deformity and disease progression. after careful numbering of rats, division into twelve groups was made with eight rats each. group 1: healthy male rats, not treated. given normal saline in equal amount. group 2: diseased control rats left for natural recovery after induction of rheumatoid arthritis with single 0.2ml cfa injection in right hind paw. prophylactic groups: group 3: tab. naproxen 25mg/kg group 4: anthraquinone extract at250mg/kg group 5: anthraquinone extract at 500mg/kg group 6: methanolic extract at 250mg/kg group 7: methanolic extract at 500mg/kg therapeutic groups: group 8: tab. naproxen 25mg/ kg group 9: anthraquinone extract at 250mg/kg group 10: anthraquinone extract at 500mg/kg group 11: methanolic extract at 250mg/kg group 12: methanolic extract at 500mg/kg radiological analysis: x-ray apparatus (siemens-60ma, germany) and industrial x-ray film (fuji photo film, japan) were used to take radioghraphs with operating conditions of 220v with peak of 40v, exposure time of 0.2 seconds and a 60cm tube to film distance for anterior-posterior projection. grading of radiographic images was done as follows: mild: diminished swelling of hind paw, no deformity of interphalangeal joints. moderate: minimal swelling of hind paw, low deformity of interphalangeal joints. intense: increased swelling of hind paw, highly deformed interphalangeal joints.17, 18 powdered 30gm fruit pulp of cassia fistula + ethanol 150ml (1:5) in soxhlet apparatus heated for 24hours at boiling point of solvent obtained 9% anthraquinone extract stored with desiccant after being concentrated and dried cassia fistula powdered bark extraction in soxhlet apparatus with double distilled water & methanol 9% concentrated extract was produced in rotary evaporator and refrigerated till further use 60 radiological analysis of hind paw joint in murine rheumatoid arthritis model treated prophylactically... results x-rays showed no swelling (ns) of hind paw and no deformity (nd) of interphalangeal joints of group 1, 3 and 8 (fig-1,3&8). swelling (s) of hind paw and deformity (d) of interphalangeal joints of group 2, 6 and 11 (fig-2,6&11). low swelling (ls) of hind paw and low deformity (ld) of interphalangeal joints of group 4, 7, 9 and 12 (fig-4,7,9&12). low swelling of hind paw and no deformity of interphalangeal joints of group 5 and 10 (fig-5&10). fig-1: group 1 healthy control shows no swelling (ns) of hind paw and no deformity (nd) of interphalangeal joints. fig-2: group 2 disease control shows swelling(s) of hind paw and deformity(d) of interphalangeal joints. fig-3: prophylactic group 3 naproxen treated rats shows no swelling (ns) of hind paw and no deformity (nd) of interphalangeal joints. fig-4: prophylactic group 4 anthraquinone 250mg/kg treated rats shows low swelling (ls) of hind paw and low deformity (ld) of interphalamgeal joints. fig-5: prophylactic group 5 anthraquinone 500mg/kg treated rats shows low swelling (ls) of hind paw and no deformity (nd) of interphalamgeal joints. fig-6: prophylactic group 6 methalonic extract of cassia fistula 250mg/kg treated rats showing mild swelling with less deformity of interphalangeal joints. fig-7: prophylactic group 7 methalonic extract of cassia fistula 500mg/kg showing low swelling of hind paw and low deformity of interphalangeal joints. improvement in joint space and cartilage deformity. ld l s n d n s s d 61 radiological analysis of hind paw joint in murine rheumatoid arthritis model treated prophylactically... fig-8: therapeutic group 8 naproxen 25mg/kg treated rats showing no bony destruction and swelling of joint with reduced edema and swelling of soft tissues. fig-9: therapeutic group 9 anthraquinone 250mg/kg treated rats showing reduced progression of ra with low joint swelling and less narrowing of joint space. fig-10: therapeutic group 10 anthraquinone 500mg/kg treated rats showing significant improvement of joint swelling by less soft tissue swelling. fig-11: therapeutic group 11 methanolic extract of cassia fistula 250mg/kg treated rats shows swelling (s) of hind paw and deformity (d) of interphalangeal joints. fig-12: therapeutic group 12 methanolic extract of cassia fistula 500mg/kg treated rats shows low swelling (ls) of hind paw and low deformity (ld) of interphalangeal joints. discussion rheumatoid arthritis, which is an autoimmune disease of progressive bones and cartilage damage, is associated with severe disability and has systemic effects as well.6 naproxen, a nonsteroidal antiinflammatory drug (nsaid) is used to treat rheumatoid arthritis but itself has many side effects.11, 12 plants have been used to treat different ailments including arthritis since ages.2 remarkable effects were produced by cassia fistula in earlier researches in murine model of arthritis.13,14 the current research was conducted to further verify this effect radiologically. radiological studies of hind paw joints: the radiological studies further verified the dominant anti-arthritic effect of methanolic and anthraquinone extracts of cassia fistula proven in our earlier experiments. clinically the diagnosis of rheumatoid arthritis, which requires therapeutic monitoring, is undeniable and it remains the standard method in assessing the disease progression. the reduced joint space, apparent in xrays, is a hallmark of arthritis. radiological studies of hind paw joints of disease control groups showed swelling of hind paw and deformity of interphalangeal joints as shown in fig 2. when treated by naproxen (both prophylactic and therapeutic groups; 3 and 8) there was almost complete reversal of radiological changes as compared to disease control group 2, with no deformity of joints and no swelling of hind paw as seen in fig-3 & 8. anthraquinone at the dose of 500mg/kg in group 5 and 10 had similar but lesser effects to that of naproxen group 3 & 8 with no joint deformity and low swelling of hind paw as shown in fig 5 & 10. anthraquinone has anti-inflammatory activity, inhibits production of superoxide anions from neutrophils and has antioxidant effect as well l s l d nd l s 62 radiological analysis of hind paw joint in murine rheumatoid arthritis model treated prophylactically... due to inhibition of lipid peroxidation.19,20,21 lowest or negligible improvement on rat hind paw joints radiological features was with that of 250mg/kg dose of methanolic extract of cassia fistula in group 6 & 11 as seen in fig-6 & 11. these radiological findings were novel and provided insight into the development of rheumatoid arthritis in the murine ankle joints and its resolution with cassia fistula extracts. conclusion in this innovative study, joint x-rays of both the prophylactic and therapeutic models were taken as a confirmatory investigation on the final status of arthritis which verified our findings regarding cassia fistula anthraquinone and methanolic extracts anti-arthritic potential in comparison to naproxen. therefore, the efficacy of cassia fistula make it a potential weaker candidate in prevention and treatment of rheumatoid arthritis. furthermore, anthraquinone extract in the dose of 500mg/kg was found to have more potent anti-arthritic effect. references 1. kala cp. ethnobotany and ethnoconservation of aegle marmelos (l.) correa. indian j traditional knowledge. 2006; 5(4): 537-540. 2. gupta ak, tondon n, sharma m. quality standards of indian medicinal plants, medicinal plants unit. indian council med res.2008;2: 47-53. 3. ayurvedic pharmacopoeia of india, part 1, vol.5, new delhi, government of india publication, 2001. page no. 8, 9. 4. indian herbal pharmacopoeia revised new edition, 2002, indian drug manufacturers association mumbai, page no.106-113. 5. nadkarni km. indian materia medica-2, 3rd ed. popular prakashan, bombay, gupta. pharm biol. 2009; 47(3): 195-202. 6. wollenhaupt j, zeidler h. undifferentiated arthritis and reactive arthritis. curr opin rheumatol 1998; 10(4): 306-13. 7. swash m, glynn m. hutchison's clinical methods. edinburgh. saunders elsevier, 2007. 8. zumora ra, billar tr. inducible nitric oxide synthase and inflammatory disease. mol med 2000; 6: 347-56. 9. lichtenberger lm, romero jj, dial ej, moore je. naproxen-pc: a gi safe and highly effective anti-inflammatory. inflammopharmacology. 2009; 17(1):1-5. 10. hoyle mg. naproxen and elevated liver enzyme [2013] available at traditional knowledge. 2006; 5(4): 537-540. 11. cooper k, bennett wm. nephrotoxicity of common drugs used in clinical practice. arch intern med. 1987; 147:1213-1218. 12. j.anitha and s.miruthula. anti inflammatory and phytochemical analysis of cassia fistula fruit pulp extracts. ijp. 2014; 1(3): 207-215. 13. farooq h, piracha mi, alam ss, sultan f. comparative anti-arthritic study of cassia fistula with naprexen in rheumatoid arthritis murine model. proceedings, szmc. 2018; 32(3): 35-39. 14. gupta v, agarwal a, tiwari hp (1990). isolation and characterization of two flavonol and a xanthone glycosides from the stem bark of cassia fistula linn. ind. j. chem. b. 28: 282-4. 15. a.m bendele. animal models of rheumatoid arthritis. j.musculoskeletal neuron interact. 2001; 1(4):377-385. 16. peter c. taylor, edward c. keystone et al. baricitinib versus placebo or adalimumab in rheumatoid arthritis. n engl j med 2017; 376: 652-62. 17. gupta a, singh s. evaluation of antiinflammatory effect of withania somnifera root on collagen-induced arthritis in rats. pharmaceutical biology. 2014;52(3):308-20. 18. fausto salaffi, m. c. m. d. c., 2016. conventional radiography in rheumatoid arthritis. int j clin exp med, 9(9), pp.17012-27. 19. kelly, s. et al., eur. j. neurosci of pharmacology. 2007. 20. siddhuraju p, mohan ps, becker k (2002). studies on the antioxidant activity of indian laburnum (cassia fistula l.): a preliminary assessment of crude extracts from stem bark, leaves, flowers and fruit pulp. j. agric. food. chem. 79: 61-7. 21. raju ilavarasan, moni mallika and subramanian venkataraman. antiinflammatory/ anti-oxidants activities c.fistula bark extracts. afr. j. trad cam. 2005; 2(1):70-85. the authors: dr. hassan farooq assistant professor, department of pharmacology & therapeutics, khawaja muhammad safdar medical college, sialkot. 63 radiological analysis of hind paw joint in murine rheumatoid arthritis model treated prophylactically... dr. mariyam iftikhar piracha assistant professor, department of pharmacology& therapeutics, akhtar saeed medical college, lahore. dr. muhammad usman senior demonstrator, department of pharmacology & therapeutics, shalamar medical & dental college, lahore. ms. rabia tariq pharmaceutical chemist, department of pharmacology & therapeutics, khawaja muhammad safdar medical college, sialkot. prof. saadia shahzad alam hod, pharmacology & therapeutics, shaikh zayed medical complex, lahore. corresponding author: dr. hassan farooq assistant professor, department of pharmacology, khawaja muhammad safdar medical college, sialkot. e-mail: hassan_oldravian@gmail.com key: cord-0049215-l46b5d2f authors: freites-núñez, dalifer; baillet, athan; rodriguez-rodriguez, luis; nguyen, minh vu chuong; gonzalez, isidoro; pablos, jose luis; balsa, alejandro; vazquez, monica; gaudin, philippe; fernandez-gutierrez, benjamín title: efficacy, safety and cost-effectiveness of a web-based platform delivering the results of a biomarker-based predictive model of biotherapy response for rheumatoid arthritis patients: a protocol for a randomized multicenter single-blind active controlled clinical trial (predira) date: 2020-08-31 journal: trials doi: 10.1186/s13063-020-04683-7 sha: b953b33cfc181e12932385d335ad4f3797bac65b doc_id: 49215 cord_uid: l46b5d2f background: rheumatoid arthritis (ra) is one of the leading chronic inflammatory rheumatism. first-line therapy with synthetic disease-modifying antirheumatic drugs (sdmard) is insufficiently effective in 40% of cases and these patients are treated with biotherapies. the increased use of these drugs each year is becoming a public health issue with considerable economic burden. this cost is 20 times higher than that of sdmard. however, among patients treated with biotherapies, clinical practice shows that about one third will not respond to the selected drug. in nonresponse cases, practitioners currently have no choice but to perform an empirical switching between different treatments, because no tool capable of predicting the response or nonresponse to these molecules is currently available. methods: the study is a prospective, phase iii, controlled, multicenter, and randomized, single-blind (patient) clinical trial, including ra patients with a previous failure to anti-tnf therapies. the main objective is the analysis of the clinical and pharmacoeconomic impact after 6 months of treatment. intervention arm: prescription of biotherapy (rituximab, adalimumab, abatacept) using sinnotest® software, a prediction software based on proteomic biomarkers. control arm: prescription of biotherapy based on current practice, without the sinnotest® software (any biotherapy). in addition, a substudy will be carried out within this trial to generate a biobank and further analyze the proteomic profile of the patients and their modification throughout the study. discussion: this clinical trial study will be the first validation study of a biotherapy response prediction software, bringing personalized medicine into the management of ra. we expect that the findings from this study will bring several benefits for the patient and the health care system. trial registration: clincaltrials.gov nct04147026. registered on 31 october, 2019. rheumatoid arthritis (ra) accounts for a large proportion of chronic inflammatory rheumatisms (cir), with a prevalence of 0.4% in the caucasian population [1] and an incidence of 9/100,000 subjects [2] . chronic inflammation is clinically manifested by pain and morning stiffness and can lead to joint destruction, often leading to major functional disability in the medium term [3] . ra is the result of the interaction between genetic and environmental factors leading to increased circulating levels of many cytokines. the inhibition of these cytokines, such as tumor necrosis factor (tnfα) or interleukin (il)-6, is a logical approach, which has introduced profound changes in the management of this condition. gradually since the 2000s, biological disease-modifying antirheumatic drugs (bdmards) were added to the existing therapeutic arsenal. there are currently available several bdmards, including tnfα antagonists, anti-il-6 receptor, anti-il-1, a t cell anti-activator, and a cd20+ anti-b lymphocyte [4] . in addition, new bdmards are expected to be approved for treatment of ra in the following years, making the therapeutic management of these patients even more complex. one of the major current challenges in ra management is being able to predict drug responsiveness before treatment initiation [5] , as there are no tools able to predict the response to a particular medication [6] . rheumatologists lack evidence-based knowledge to choose the most suitable dmard for their patients, and these drugs are prescribed in a "trial-and-error" manner. in case of a lack of response (which has been reported around 30-50% for bdmards [3, 7] ), the physicians have to perform an empirical rotation between the different drugs, until the correct drug, or combination of drugs for this particular patient is found. therefore, nonresponders are unnecessarily exposed to undesired adverse events along with the worsening of their physical condition. furthermore, the ineffective use of bdmards has a dramatic economic burden due to their important costs [4] . a study by meissner and colleagues [8] shows a greater overall economic impact on the health system of patients who changed bdmard following failure of a first biological compared to those who did not change. this economic burden is even greater when the patient has to change his/ her treatment for a second time. this phenomenon seems difficult to reverse in case of initial misdirection, since the probability of a patient who has already received an anti-tnf to respond to another biological treatment decreases gradually according to the increasing number of failures of previous treatments [9] . a tool able to provide a probability score of response or nonresponse to specific treatments could pose an important benefit for clinicians and patients. in order to improve the response rate to bdmards, research for predictive biomarkers of therapeutic response has been active for more than 15 years [10] . several approaches have been followed to identify biomarkers, including genomics, transcriptomics, epigenetics, and proteomics. however, not all of these strategies have contributed benefits at present. indeed, in the genomics field [11] , the search for candidate genes and the replication of results on different cohorts is fragile and tedious. the hypothesis of several associated genes each having a low impact is preferred over that where few genes each with a significant effect would be involved. its routine use remains complex. similarly, epigenetics has provided very preliminary data for the time being [12, 13] . finally, the study of the transcriptome makes it possible to identify a certain number of genes, but its widespread use by clinicians as part of their regular assessment of their ra patients is difficult on a daily basis [14] , and the study of the serum metabolome remains complicated [15] . the proteomic approach, on the other hand, is innovative and simpler to use in routine: it is focused on proteins, which are the terminal elements of cellular actions. it has been emerging in research for about 10 years in rheumatology for the theranostic side [16, 17] , but clinical applications are not routine. the latest developments in this area, especially those carried out by members of the predira consortium, could change the management of these patients [16, 18, 19] . these studies have made it possible to characterize biomarkers differentially expressed at baseline in patients who respond to a bdmard compared to weak or nonresponders, hoping to optimize a targeted prescription of bdmards [16, 17, 20] . in order to allow a rapid translation to clinical practice, our approach prioritizes the selection of biomarkers for which validated diagnostic assays exist, are routinely used, and are commercially available. several predictive algorithms aimed at 3 classes of bdmards have been developed: anti-tnfa (adalimumab), inhibitors of costimulation of t cells (abatacept), and anti-b-cell antigen cd20:(rituximab). based on identified biomarkers and predictive algorithms [16, 18, 19] , an application called sinnotest® has recently been validated [21] [22] [23] to determine a priori the most appropriate treatment for the patient with ra according to its clinical context (patients with a previous failure to bdmards), depending on its specific proteomic profile (predictive and personalized medicine). this medical device has also been ce marked/approved since august 2018. in practice, from a simple biological sample of a patient, such as blood (plasma or serum), specific protein biomarkers are quantified using routine techniques, allowing after combination through an algorithm, to associate the patient's profile at a personalized response or nonresponse status for these biotherapy rotational patients. an online interface allows the rheumatologist to access, for each patient, the results of biomarker analysis, as well as the probability of response to different biotherapies available on the market. we hypothesize that the sinnotest® software offers real-world clinical and pharmacoeconomic benefits by integrating the main biotherapies currently available. this study will target patients with ra in rotation of a first biotherapy due to inefficiency or toxicity. the expected economic impact is multiple: change in the use of care (hospitalizations, medicines, regular biological controls, outpatient stays, consultations, nursing, surgery, transportation, etc.); reduction of the costs related to the losses of production (reduction of the incapacities at work inducing absenteeism, long-term work stoppages, permanent disability ...); reduction of intangible costs, the assessment of which is difficult but which represents an expected social benefit of such a care strategy. in addition, we will try to show that sinnotest® also has a strong clinical impact in terms of early-adapted management via the responder rate. the primary objectives of this clinical trial is to study the clinical and pharmacoeconomic impact after 6 months of the use of the sinnotest® predictive tool in patients with rheumatoid arthritis who have failed to a first anti-tnf biologic agent compared to usual care. the secondary objectives will be: -pharmacoeconomic: to carry out a budget impact analysis (bia) at 6 and 12 months; -clinical: to describe the performance of the software's predictive model on new clinical data from the 6-month trial. we will also carry out a proteomics substudy with the objectives of comparing the variation of the proteomic profile between the m0 (date of inclusion) and the m6 (end of study date). the achievement of this objective will be based on the constitution of a biobank, which will serve as a basis for future studies focused on the therapeutic management of these patients. this project is a prospective phase iii randomized clinical trial in 2 parallel groups, multicenter, controlled (prescription of bdmard with or without the sinnotest® software), and single-blind (the patient will not know if his bdmard treatment has been prescribed with or without the help of sinnotest® software). the inclusion period will be 12 months. each patient will be followed up to 6 months (clinical evaluation) and up to 12 months (for the analysis of the budgetary impact at 12 months) post randomization. the population studied concerns patients with rheumatoid arthritis, who have failed a first anti-tnf medication (due to inefficiency or adverse events), and attend rheumatology outpatient clinics from tertiary care centers in the madrid region (spain). this situation corresponds to a very large majority of patients admitted to rheumatology consultations. patients meeting the criteria below will be eligible in the study: patients over 18 years old and under 70 years old with ra, defined according to the acr / eular 2010 or acr 1987 criteria; patients failing a first anti-tnf, defined as ineffectiveness (which is defined as a das28-esr ≥ 3.2 and an inadequate response to itnf according to the usual rheumatologist, which generally includes one or more of the following conditions: persistent swollen and tender joints, persistence of disease activity according to the overall evaluation of the patient, high levels of acute phase reactants, and/or dependence of analgesics, nonsteroidal anti-inflammatory drugs or corticosteroids), or toxicity (defined as the appearance of any adverse event that the habitual rheumatologist relates to the medication and requires discontinuation); effective contraception for patients of childbearing potential; patients able to read and understand the modalities of the protocol; patients who have dated and signed the informed consent form of the trial; stability of treatments (immunosuppressants, corticosteroids, nonsteroidal anti-inflammatory drugs) between the selection visit and the inclusion visit (m0). the exclusion criteria will be as follows: patients who do not meet the criteria below will be eligible in the study; patients with a contraindication to a bdmard or methotrexate; patients included in another therapeutic evaluation trial during the trial; surgical intervention programmed during the trial; patients with difficulties in understanding the spanish language; patients who cannot be followed up to 12 months; psychosocial instability incompatible with regular monitoring (homelessness, addictive behavior, antecedent of psychiatric pathology or any other comorbidity that would make it impossible for free and informed consent or limit adherence to the protocol); breastfeeding and pregnancy: although there are bdmards that can be used during pregnancy, since sinnotest may recommend a treatment not recommended for use in pregnancy, it is decided to exclude the recruitment of pregnant subjects. a previous failure to adalimumab does not constitute an exclusion criterion, and therefore those patients are eligible to participate in this trial. informed consent will be obtained by a practicing rheumatologist, able to prescribe any of the drugs that the study subject may receive, and a member of the study team. the investigator will be responsible for providing each patient with an information sheet about the trial and the objectives, methods, foreseeable benefits, and potential risks of the study, which should be read by the patient. the investigator must explain to the patients that they are totally free to refuse their participation in the study or to abandon it at any time and for any reason. the investigator will be responsible for obtaining the written informed consent of each of the participating patients before proceeding with any medical procedure of the study. the investigator will be responsible for not involving any patient in the study without having previously obtained their voluntary consent in writing. in addition, for the proteomics substudy, a second independent informed consent will be collected, which will also include the possibility of storing the samples not used in the present study in the biobank of the center where it is carried out in the study and in the biobank of the center hospitalier universitaire grenoble alpes (france). intervention description {11a} the patients will be randomized to an intervention group (sinnotest®) or an active-control group (usual care). patients allocated to the intervention arm will receive the prescription of bdmard (rituximab, adalimumab, abatacept) using sin-notest® software, while those allocated to the control arm will receive the prescription of bdmard without the sinnotest® software which corresponds to current practice (all bdmards). innovative procedure innovative medical device (imd): sinnotest® software version 2.0 sinnotest® is a therapeutic guidance device for patients suffering from chronic inflammatory rheumatism, in particular ra. sinnotest® consists of the following three elements: "routine" biological assay of biomarkers from a blood sample, calculation algorithm, and graphical user interface (software and online application). this device was designed by the company sinnovial. the assessment of its conformity was carried out by surgiqual institute. biomarkers are detected and quantified in blood samples of patients, using commercial tests aimed at determining the levels of these molecules. these results are sent to the sinnotest® secure server. these data are processed by algorithms to determine the probability of response to the treatments available in the therapeutic arsenal (biological therapies). these algorithms were developed in a population of patients with a previous failure to itnfs receiving the specific medication the algorithm was designed for. this data is sent to a secure server. the graphical user interface is an independent software, accessible through a web platform, which allows authorized health professionals to access the results of the analysis of individual patients. sinnotest® is a stand-alone imd-md software, accessible via a web platform. rheumatologists from the different centers will be contacted by email to register for the sinnotest® online platform. a link in this email will provide access to the registration part of the application. the rheumatologists will then be able to connect to the sinnotest® platform thanks to their personal identifiers. process of the innovative procedure the selection of the bio-drug is carried out based on the recommendations of sinnotest®. this test categorizes bdmard based on the probability of response. it will allow to prescribe both original molecules and biosimilars in an equivalent way. in the sinnotest® arm, the investigator prescribes the treatment defined as the most effective by sinnotest®, except in case of contraindication. if contraindicated, the investigator prescribes the second-choice treatment (if any) of sinnotest® in terms of efficacy. the bdmards possibly recommended by sinnotest® are adalimumab, rituximab, and abatacept. not being exhaustive in terms of biotherapies, it is possible that the sinnotest® cannot recommend any bdmard. in this case, the rheumatologist may prescribe one of the following other bdmards: etanercept, adalimumab, infliximab, certolizumab, tocilizumab, sarilumab, abatacept, anakinra, golimumab, or rituximab. reference procedure there is currently no evidence of treatment response in ra. the choice of the treatment by the rheumatologist is therefore empirical. in order to simplify the process and favoring blinding, regardless of which arm the patient is randomized to, the usual rheumatologist will record two treatment proposals in the clinical history. on the one hand is the treatment that he would prescribe if the patient was randomized to the control arm and, on the other hand, the one that he would prescribe if the patient was randomized to the intervention arm and if sinnotest® did not recommend any treatment (therefore, the proposed treatment in this case cannot be any of the 3 potentially recommended by sinnotest®). at the successive visit after 6-10 days in which the bdmard will be prescribed, if the patient was randomized to the control arm, or was randomized into an intervention group, but sinnotest® did not recommend any bdmard, the medication previously registered by the usual rheumatologist will be used. process of the reference procedure this is the current management of patients with ra, based on the eular recommendations. in case of rotation of biotherapy after prescription of a first biotherapy, the rheumatologist may prescribe the following bdmards: etanercept, adalimumab, infliximab, certolizumab, tocilizumab, abatacept, anakinra, golimumab, rituximab. all these bdmards can be prescribed as well as their biosimilars. in case of remission of more than 6 months, a reduction (dosage or spacing of the catch) of the biotherapy can be considered. concomitant medications authorized with anti-tnfα, anti-il-6r, and ctla-4 these include any conventional synthetic dmard that the patient was previously taking before being included in the study and that his rheumatologist deems necessary to continue. oral corticosteroids at dose ≤15 mg/day, intravenous corticosteroids, intraarticular, and peri-articular local injections of corticosteroids, salicylates, nonsteroidal antiinflammatory drugs (nsaids), analgesics, drugs necessary for the treatment of comorbidities, concomitant treatment with corticosteroids, nsaids, and analgesics showed no effect on the pharmacokinetics of these biotherapies. the discontinuing or modifying allocated interventions for a given trial participant in the study will be as follows: lack of response to medication in the intermediate visit (3 months after inclusion) using the eular response criteria; occurrence of adverse events, including malignancy; loss to patient follow-up; withdrawal of consent by the patient; decision of the investigator (inadequate follow-up of the instructions of the doctor or study staff); the rheumatologist or the promoter (medical monitor) decides that continuing the study can be harmful to the patient; in case of pregnancy (only if the patient is under treatment with a therapy that does not recommend pregnancy; in the event that the medication is compatible with the pregnancy, the patient can still be included in the study); in case the patient needs a treatment or has received some treatment not allowed in the study; erroneous inclusion in the study; circumstances not foreseen; cancelation of the study. patients discontinuing the intervention will not be followed up and will be withdrawn from the study. no treatment / procedure prohibited during the study. strategies to improve adherence to interventions {11c} no specials provisions will be put in place to improve adherence, besides those already carried out in usual care. provisions for post-trial care {30} all patients will return to standard care after the trial. clinical primary outcome measure therapeutic response (time frame: 6 months) the therapeutic response will be assessed using the eular response criteria [24] at 6 months, which is defined by a low disease activity (das28-esr < 3.2) and a decrease in the das28-esr > 1.2 points from baseline (further details regarding disease activity in the "clinical secondary outcome measures" section). pharmacoeconomic secondary outcome measures incremental cost utility ratio at 6 months (time frame: 6 months) this outcome will be calculated as the average differential cost per patient between both study arms (mean costs of the sinnotest® arm − mean costs of the control arm) divided by the difference in effectiveness between both study arms measured in the number of years of life weighted by the quality of life (qaly: quality-adjusted life year) generated by each of the strategies (mean qaly of the sinnotest® arm − mean qaly of the control arm). qaly will be measured using the euroqol-5d-5l. the spanish value set will be used to obtain the eq-5d index values. cost will be considered from a societal perspective, including both direct and indirect costs the ratio will be expressed in cost (2019 euros) per qaly earned, which represents the additional cost that will have to be spent to earn a healthy year of life. as direct costs, we will consider concomitant medications, procedures, outpatient consultations (both primary and specialized care), hospitalizations, and complementary test (laboratory, radiology…). regarding indirect costs, we will consider sickness absences, transportation, home assistance (both by professional and non-professional caregivers), and home modifications related to the disease. regarding the determination of unit costs, we will favor, where possible, a cost-of-production approach: -the prices of drugs dispensable through pharmacies will be obtained from the database of the spanish official college of pharmacists -the prices of hospital dispensing drugs will be obtained from the pharmacy services of each participating center. -the costs of the different medical acts (first visits or revisions, in primary or specialized care, by different medical professionals) will be provided by each participating center. -the costs of hospitalizations and the procedures that are carried out will be provided by each participating center. -the costs of the complementary tests will be obtained from each participating center. -the use of home assistance will be evaluated based on a standard average cost. the cost of employing professionals from the medico-social sector (for example, occupational therapist) will be estimated based on an average cost per hour. -the cost of improvements in the home will be estimated, as far as possible, in the billing data or in the data obtained by the professionals of the sector involved. -in relation to the costs of caregivers (nonprofessionals) and the loss of production of the patient, according to the recommendations, the direct costs will take into account the time of the people intervened and the time that the caregivers dedicate to the care of the patients. however, in relation to the valuation method, there is no agreement on the method to be used. we will favor the human capital method, where the "value" of an individual is estimated by its productive value in the labor market, that is, through the gross salary. these productive costs will be obtained through the data of age, sex, and professional sector of the individual through the national statistics available on the spanish social security website. -the cost of medical transport will be assessed through the rates established based on the distance and type of vehicle. we consider that 6 months is enough time to assess the performance of the intervention. according to the eular recommendations for biotherapy management in rheumatoid arthritis [25] , 6 months is the time to assess if the therapeutic objectives of the medication has been achieved (remission or at least low disease activity). based on those recommendations, if the patient is still active, treatment should be modified. incremental cost-effectiveness ratio at 6 months (time frame: 6 months) this outcome will be calculated as the average differential cost per patient between both study arms (mean costs of the sinnotest® arm − mean costs of the control arm) divided by the difference in effectiveness between both study arms measured as the percentage of patients achieving a good clinical response in each study arm (% in the sinnotest® arm − % in the control arm). good clinical response will be measured using the eular criteria of good clinical response. cost will be considered from a societal perspective, including both direct and indirect costs. the ratio will be expressed in cost (2019 euros) per increase in 1% of subjects achieving a good clinical response, which represents the additional cost that will have to be spent to earn a healthy year of life rates of treatment response patients associated respectively with the usual strategy without sinnotest® and with the strategy with sinnotest®. budget impact analysis at 6 and 12 months (time frame: 12 months) a budget impact analysis will be carried out if the innovation is deemed efficient, meaning, if the main outcome is achieved (sinnotest is cost-effective). a 12month horizon was chosen to make sure that the differences between both study arms were not compensated in the long term. this budget impact analysis will describe the resources consumed and the expenses generated by each scenario, a scenario with the use of sinnotest® and a scenario without sinnotest®. the same direct costs and the same method of reporting will be used as those used in the main outcome assessment. clinical secondary outcome measures disease activity, quality of life, and disability (time frame: 6 months) ra disease activity will be evaluated using both the das28-esr and crp indexes. they are composite scores derived from 4 measurements: number of swollen joints (out of 28), number of painful joints (out of 28), global assessment by the patient of ra activity on a scale visual analog of 0 to 10 cm (0 = no manifestation of ra, 10 = maximum severity that the patient can imagine), and either erythrocyte sedimentation rate (esr, in mm/h) or c-reactive protein (in mg/dl). quality of life will be measured with the eq5d-5 l. disability will be measured using the health assessment questionnaire (haq). software's predictive model performance (time frame: 6 months) sensitivity, specificity, and positive and negative predicted values of the predictive models using the biomarkers will be assessed on the new clinical data from the 6-month trial. proteomic secondary outcome measures description of the variation of the proteomic profile between m0 (biotherapy start date) and m6 (6 months visit) (time frame: inclusion and 6 months) based on shotgun and semi-quantitative proteomics, the differences between the proteomic profile at baseline and at m6 will be analyzed. this study will be conducted by rheumatologists who have the opportunity to follow patients with ra and to conduct this study in good conditions and in accordance with regulatory and legal recommendations. the only difference in follow-up between the 2 groups is the addition of the "sinnotest® protein biomarker" assay and the use of the results of this assay for therapeutic management. patients will be seen as part of their follow-up consultation in the rheumatology department: -screening: verification of the eligibility criteria. -inclusion or m0 visit: after information and signature of informed consent. all patients will have a sinnotest® blood sample. at the end of this visit, the patient is randomized to the sinnotest® group or the control group. in the control group, the sinnotest® sample is kept but not analyzed. -6-10 days after the consultation, the prescription of the biotherapy recommended by the rheumatologist is sent by post. this prescription uses sinnotest® results in the sinnotest® arm. -followed quarterly: m3 and m6. as part of the proteomics substudy, for those patients who agree to participate, blood samples will be collected from the 2 groups in the m0 and in the m6 to study the variations in the proteomic profile of the patients. in addition, the remaining samples will be deposited in a biobank (in the recruitment center and in the biobank of the center hospitalier universitaire grenoble alpes, france). the participant timeline is presented in table 1 . to re-evaluate the metrological properties of sinnotest®, including 90 patients per group will highlight a differential response rate between the software arm and the control arm of more than 18.2%, considering a current response rate of 65% (defined as an eular good response [24] ) with conventional care (control arm). the number of subjects required was estimated considering a bilateral alpha risk at 5% and a power of 80%. in total, 180 patients will be the estimated number of participants needed to achieve study objectives. due to the subject's and the study's characteristics, no allowance for loss to follow-up was considered in the sample size calculation: patients with a chronic disease, probably being followed up in the same center and by the same rheumatologist for some time (> 1 year), and the short follow-up time for the primary outcome. recruitment will be done among patients coming for rheumatology consultations in the 5 centers participating in the study. potentially eligible patients will be identified in the everyday clinical practice of the research staff, or referred to them for assessment of eligibility having been identified by rheumatologists who are not research staff. medical records will be checked to identify any other potentially eligible patients. the patient's eligibility will be confirmed by the responsible researcher. after confirmation, any patient who agrees to participate in the research must sign the informed consent to begin participation. it was centrally generated at the hospital clínico san carlos clinical research unit. only the person that has generated the list has access to it. the eligible patient will be randomized, with a 1:1 allocation, via an internet server (access by secure code 24/24; redcap) in one of two groups: sinnotest group or control group. a blood sample will be extracted from the patient randomized in the software arm for the determination of selected biomarkers for the use of sinnotest®. a patient randomized to the control arm will also have his/her blood drawn (but this one will not be analyzed) so that the two arms are identical and to maintain blinding for the patient. the coordinating center will manage its development and its availability on the internet. it will only be done information and informed consent: the patient will sign informed consent for the main study and additional consent (optional) if they accept 6-month and 1year inclusion samples, which will be retained for the creation of biobank c biomarkers: a blood sample will be taken for the determination of sinnotest® biomarkers in the intervention arm patients d patient booklet: the rheumatologist or cra investigator will deliver the booklet to the patient at the inclusion visit. the patient brings the notebook during each follow-up visit and will carry it with him after the visit. at the end of the follow-up, he will return his completed patient booklet to the cra / tec of the investigator center by mail using the envelope provided with the notebook e administration/dispensation of biotherapy: patients in the control group will receive their biotherapy prescription in current practice. for patients in the intervention group, the rheumatologist will prescribe the biotherapy selected by sinnotest®. the results of the pregnancy test should be negative f biobank: a blood test will be carried out in all included patients who signed the additional consent for the creation of a biobank after informing the patient and signing the consent. the randomization will be individual with blocks of random size, stratified by center. the investigator or cra will connect to the server after confirming that the patient meets all the inclusion/exclusion criteria. block randomization will ensure that an equal number of patients are randomized to each study arm and to each study site. the involvement of 5 centers in this study allows individual randomization without fear of the existence of a contamination bias. indeed, investigators may change their empirical management under study because of patient outcomes previously included with sinnotest®, but the relatively low inclusion counts per center suggest that this bias will be negligible. in addition, in the control arm, the treatments will be prescribed by the rheumatologist who usually treats the patient, so the number of patients who will have the opportunity to "learn" is even lower, because it is shared among all doctors that include patients in the study. this design also neutralizes the disappointment bias of investigators who can all use the innovation under study. by stratifying by center, about half the patients will have their treatment prescribed by sinnotest, so they can be sure they will have the opportunity to use the software and assess its functions. study subjects will be blinded to the intervention (who decides the medication prescribed). evaluators will also be blinded to the intervention where the subject was allocated. this is a single-blind study (single blind). only the doctor knows the details of how to prescribe treatment (i.e., the doctor knows if the patient is in the control group or the group using the sinnotest® software). lifting of the blind is not applicable. the investigator carrying out the aleatorization will be responsible for the blinding of the subject. the use of the sinnotest® v1 device is reserved for hospital rheumatologists. training in the use of the graphical interface will be dispensed. in addition, manuals have been created for the use of the device and for the collection, processing, and delivery of samples. in order to maximize the retention rate of the subjects included in the study, several strategies will be put in place: participants will be asked to provide both phone and email addresses, as well as the best time to contact them. study subjects will also be educated in the significance of research follow-up even if they decide to discontinue the study drugs. explaining how their experiences with the treatments, whether positive or negative, are critical to evaluating the intervention under study can increase their understanding of the research process and lay the groundwork for future discussions about the importance of completing research assessments. anticipated barriers to attending appointments such as transportation, work schedules, vacations, and childcare can be reviewed up front and resolved. phone calls regarding study visit reminders will be used for all appointments, and rapid follow-up of missed appointments will be incorporated into the protocol. the schedule of study visits will be flexible, ensuring subject retention. electronics case report forms (ecrfs) will be used that comply with the general and specific good practice standards, as well as the highest requirements for computer validation, with restricted access at the user level, provided with inconsistency detection filters and with traceability of all the information until the final closing of the study. crfs must be completed for each subject screened/enrolled in this study. this also applies to patients who do not complete the full follow-up planned in the trial. all crfs must be filled out by the personnel duly authorized to do so, who will have access codes to the application for entering personal and non-transferable data. the investigator will keep the records and data during the trial in compliance with all legal and regulatory provisions in force. all data must be supported by original documents in the test center. any record or document used as a source of information (which will be called the "original data of the subject") will be kept for review by authorized representatives of the promoter or regulatory bodies. the crfs will be filled in as soon as possible after the evaluation has been carried out. all the dates that appear in the crfs referring to analytical tests and other data must coincide with the dates in which the samples were obtained or the procedures were performed. to facilitate the statistical analysis, a computerized database will be created in which the integrity of the data from the crfs will be recorded, so that an exact replica of the information contained in them is created. a data management plan will be made before the beginning of the definition of the database in which the recording process and the errors and consistency controls that will be performed on the recorded data will be detailed. a dictionary of variables will be generated in which the correspondence between the data contained in the crfs and the variables of the database will be detailed, as well as the codifications used and the meaning of the recorded values. in the event of inconsistencies or errors in the data, requests for clarification will be generated for the researchers for their verification or correction, which will be treated in an equivalent way to the crfs. access to the database will be restricted to the data manager (design, input, and data cleansing) and the personnel in charge of data transcription (data entry). prior to the declaration of the definitive database, a verification is done of the consistency of the values of the inclusion/exclusion criteria, of the clinical evaluations, of the results of complementary explorations, of the dates of the visit, of the compliance, of the medication received, of the adverse events, of the information about dropouts, and of the evaluation of effectiveness. a definitive database will be declared that will be registered with signature and date. two protected copies of the same will be kept, and paper lists of the variables contained in the database will be generated for archiving. the final database will be used for statistical analysis. all the essential documentation of the clinical trial will be filed in a master file of the study, whose safe and complete conservation will be ensured for the time required according to the legislation in force and at the disposal of the authority that requests it. this documentation will include the following: work protocol (final version) and amendments; models of all the employed versions of information sheet and informed consent form; crec permits; authorizations of the health authorities; curriculum vitae of all the personnel participating in the study; random assignment list and treatment allocation codes; individual data collection notebooks; documentation related to the study monitoring procedures; documentation of the study database and definitive database; documentation of data management and clarification requests; statistical analysis; adverse event notifications; final report; certificates of audits; standardized work procedures applied in the study; study financing and payments; correspondence. the investigator will ensure the right to privacy of patients and must protect their identity against unauthorized third parties. the study monitor may have access to the patient's identity and data in relation to the study's monitoring procedures. the investigator will keep a patient identification list updated with the correspondence between the name, clinical history number, and the patient's identification number or code for the clinical trial, which will be kept together with the patients' informed consent forms in a file unique in the center. the full name of the patient should not appear in any other section of the data collection notebooks or study documentation. at the end of the study, a copy of the list in which the names of the patients will be hidden will be included in the file of the researcher of the study. in case that an audit of the study is conducted, the auditors who perform it, as well as the health authorities that may require it for regulatory purposes related to the study, may also have access to patient data. all participants in this research project expressly commit themselves not to disclose the identity of the treated patients and to respect the rules of confidentiality regarding the data and information to which they have access when participating in the trial. the personal data collected and stored for the purpose of this study will be treated in accordance with the provisions of the general data protection regulation (gpdr: regulation eu 2016/679). plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} in the case a subject agrees to participate in the proteomics substudy, after carrying out the planned experiments during the trial, the remaining biological samples will be donated to the biobanks belonging to the corresponding isciii biobank network of each participating center and the biobank of the center hospitalier universitaire grenoble alpes (france). these samples may be transferred to other researchers, in accordance with current regulations (biomedical research law 14/2007 and royal decree 1716/2011), to carry out studies related to their disease. any project for which samples are used will be previously approved by an accredited ethics committee. description of the population studied the study population will be described by a flow diagram according to the consort recommendations (consolidated standards of reporting trials). the different variables (demographic and biological) will be described by treatment arm: for quantitative variables, number of data analyzed, mean, and standard deviation, q1, q3, minimum, maximum, and median, and for qualitative variables, number of analyzed data, absolute frequencies, and percentage. clinical primary outcome measures therapeutic response therapeutic response at 6 months will be described using number of analyzed data, absolute frequencies, and percentage. comparison between the study arms will be performed using logistic regression models. models will be adjusted by baseline variables, and they will allow us to estimate an effect size for the difference between study arms regarding the clinical variables. clinical secondary outcome measures disease activity, quality of life and disability clinical parameters at 6 months will be described using number of analyzed data, mean, standard deviation, q1, q3, minimum, maximum, and median, for das28-esr, das28-crp, eq5d-5l, and haq. comparison between the study arms will be performed using linear regression models. all models will be adjusted by baseline variables, which will allow us to estimate an effect size for the difference between study arms regarding the clinical variables. in addition, as disease activity, quality of life, and disability will be assessed at baseline, 3 months, and 6 months, their evolution will be analyzed using generalized estimating equations models nested by patient [26, 27] and adjusted by study visit and study arm, using a gaussian family and identity as link function. different covariable structures will be tested (independent and exchangeable) and compared using the quasi-akaike information criterion [28] . time × study arm interactions will assess different effects of time in the evolution of das28-esr, das28-crp, eq5d-5l, and haq by study arm. a p value < 0.05 will be considered as a significant interaction. for each outcome, in case the interaction is not significant, we will consider that there is no statistically significant difference in the evolution of that outcome between study arms. software's predictive model performance sensitivity, specificity, and positive and negative predictive values, and likelihood ratio will be described. the performance of the sinnotest® will be calculated in the "sinnotest®" arm by comparing the response predicted by the software and the response observed at 6 months. it will be analyzed globally and for each of the biotherapies. pharmacoeconomic secondary outcome measures incremental cost utility ratio at 6 months the statistical analysis will proceed as follows: 1. calculating the 6-month incremental cost-utility ratio from the community perspective using the following parameters: difference of the averaged total cost per patient and difference in the average number of qalys generated by the strategies on both study arms. the ratio will be expressed in cost per qaly earned, which represents the additional cost that will have to be spent to earn a healthy year of life. qalys will be calculated from the answers provided by eq5d-5l, based on a weighting system reflecting preferences on the health status of the general spanish population (41). the eq5d questionnaire will be filled in by the patient at the inclusion visit and then at m3 and m6. the qalys will be calculated by the area under the curve assuming a linear evolution of the quality of life between the measurement times. the average number of qalys will be calculated for each of the strategies under study. in case of missing data, multiple imputation techniques will be carried out, in addition to sensitivity analysis, with the objective of checking that the assumptions made in the imputation were correct, and checking whether the conclusions of the study are modified, or not, according to the analysis strategy adopted. although subjects will be followed up to 12 months, icer will only be calculated at 6 months: as pointed out, according to the eular recommendations for biotherapy management in rheumatoid arthritis [25] , 6 months is the time to assess if the therapeutic objectives of the medication has been achieved (remission or at least low disease activity). based on those recommendations, if the patient is still active, treatment should be modified. 2. sensitivity analysis: deterministic sensitivity analysis: it allows to take into account the uncertainty on certain parameters that can influence the cost of the strategies. we will consider the best and worst scenarios. taking into account the variability in direct cost associated with treatment in ra [29] , we will consider a ± 20% cost increase/decrease. this analysis will take into account in particular the impact of the cost of sinnotest®, the impact of a change in the cost of treatments through biotherapy, the impact of the cost of work stoppages and the time of the caregivers, and probabilistic sensitivity analysis: it will include the calculation of the confidence intervals of the cost-utility ratio by the nonparametric bootstrap method and the plot of the associated acceptability curve. 3. in addition to the cost-utility ratio, differences in cost and utility will be analyzed separately: comparison of the cost of the two strategies: the difference in costs between the groups will be tested by a student test or a nonparametric test (mann-whitney tests) in case of non-gaussian distribution. the choice of the test will be made with regard to the distribution of costs in each group (shapiro-wilk test, on raw data or, if necessary, on transformed data. in addition, distribution will also be assessed using histograms). the construction of the average cost confidence interval will be based on the nonparametric bootstrap method and comparison of the utility: the number of qalys will be calculated taking into account the time elapsed between two successive measurements. if the initial utility level between the groups differs (> 10%), a comparison of the number of qalys between the groups will be made from a linear model to adjust for this level of utility [30] . given the time horizon, no updates will be made; those of the inclusion visit (m0) will be taken. 4. multivariable linear regression method will be used to determine the explanatory factors of the average cost per patient of the strategies (i.e., age, sex, socio-professional category). in a first step, bivariable analysis will be carried out. those variables with a p value < 0.15, plus age and gender, will be included in the multivariable analysis. different models will be compared using the akaike information criteria. incremental cost-effectiveness ratio at 6 months the incremental cost-effectiveness ratio at 6 months will be calculated from the cost differential of the strategies and the efficiency differential defined by the rate of good responder patients in each group. the icer and the 6 months will be subjected to a deterministic and probabilistic sensitivity analysis. protomic secondary outcome measures description of the variation of the proteomic profile the biomarkers of the proteomic profile will be compared between the inclusion visit (m0) and the 6month visit, as well as the differences in both the m0 and m6 visits between the two treatment arms. the comparison will be done by biomarker. the biomarkers will be described at each time (indicators and boxplot) globally, as well as by the observed response group. the risk of the first species α is fixed, by convention, at 5% for the different comparative analyses. no formal interim analysis will be carried out. methods for additional analyses (e.g., subgroup analyses) {20b} the influence of factors on the average cost per patient will also be analyzed by treatment arm. to carry out this analysis, multivariable linear regressions models will be developed and an interaction between demographic and clinical-related variables and study arm will be introduced to assess a significant difference between the association between factor and average costs by study arm. methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} analyses will be carried out by intention to treat (i.e., each patient will be considered to belong to the group to which they were randomized, regardless of the intervention they receive). for each patient that leaves the study before its completion, the cause will be collected, which will be classified as follows: lack of response to biotherapy, occurrence of adverse events, loss to follow-up, withdrawal of consent, the investigator's decision, use or need for medication not allowed, erroneous inclusion in the study, unforeseen circumstances, or cancelation of the study. to handle missing data, multiple imputation techniques will be applied [31] , in addition to sensitivity analysis with the object to check the assumptions made in the imputation, and check whether the conclusions of the study are modified, or not, according to the analysis strategy adopted. the multiple imputed dataset will be used to perform the primary and secondary outcome analyses. in addition, we also will carry out a per-protocol analysis of the primary and secondary outcomes. plans to give access to the full protocol, participant-level data, and statistical code {31c} a summary of the final version of the study protocol will be made available through the project's website (https: prevalence of rheumatoid arthritis in france long term structural effects of combination therapy in patients with early rheumatoid arthritis: five year follow up of a prospective double blind controlled study disease activity-guided dose optimisation of adalimumab and etanercept is a cost-effective strategy compared with non-tapering tight control rheumatoid arthritis care: analyses of the dress study rheumatoid arthritis therapy reappraisal: strategies, opportunities and challenges brief report: estimating disease activity using multi-biomarker disease activity scores in rheumatoid arthritis patients treated with abatacept or adalimumab should patients with recent-onset polyarthritis receive aggressive treatment? switching of biologic disease modifying anti-rheumatic drugs in patients with rheumatoid arthritis in a real world setting evaluating the efficacy of sequential biologic therapies for rheumatoid arthritis patients with an inadequate response to tumor necrosis factor-α inhibitors proteomics in rheumatoid arthritis research genetic and genomic predictors of anti-tnf response circulating mirna-125b is a potential biomarker predicting response to rituximab in rheumatoid arthritis prediction of treatment response to adalimumab: a doubleblind placebo-controlled study of circulating microrna in patients with early rheumatoid arthritis the potential use of expression profiling: implications for predicting treatment response in rheumatoid arthritis variations in the metabolome in response to disease activity of rheumatoid arthritis apolipoprotein a-i and platelet factor 4 are biomarkers for infliximab response in rheumatoid arthritis identification of 7 proteins in sera of ra patients with potential to predict eta/mtx treatment response protéines s100a8, s100a9 et s100a12 : marqueurs inflammatoires ou acteurs physiopathologiques de la polyarthrite rhumatoïde synovial fluid proteomic fingerprint: s100a8, s100a9 and s100a12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases a personalized approach to biological therapy using prediction of clinical response based on mrp8/14 serum complex levels in rheumatoid arthritis patients prealbumin, platelet factor 4 and s100a12 combination at baseline predicts good response to tnf alpha inhibitors in rheumatoid arthritis identification of cartilage oligomeric matrix protein as biomarker predicting abatacept response in rheumatoid arthritis patients with insufficient response to a first anti-tnfα treatment fetuin-a and thyroxin binding globulin predict rituximab response in rheumatoid arthritis patients with insufficient response to anti-tnfα development and validation of the european league against rheumatism response criteria for rheumatoid arthritis. comparison with the preliminary american college of rheumatology and the world health organization/international league against rheumatism cri eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update serum levels of vasoactive intestinal peptide as a prognostic marker in early arthritis contribution of the bone and cartilage/soft tissue components of the joint damage to the level of disability in rheumatoid arthritis patients: a longitudinal study akaike's information criterion in generalized estimating equations direct medical costs and their predictors in the emar-ii cohort: "variability in the management of rheumatoid arthritis and spondyloarthritis in spain estimating mean qalys in trial-based cost-effectiveness analysis: the importance of controlling for baseline utility the prevention and treatment of missing data in clinical trials long-term drug survival of biological agents in patients with rheumatoid arthritis in clinical practice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all clinicians and site investigators for their invaluable support for this project. this work has been supported by spanish clinical research network (scren), funded by institute of health carlos iii-general subdirectorate for evaluation and promotion of research, through project pt17/0017/0018 integrated in the state plan i+d+i 2013-2016, and cofinanced by european development regional plan ("fondo europeo de desarrollo regional"-feder). the combination of sinnotest® algorithms will optimize the selection of biotherapy for ra patients, and given the minimal risk for patients and the expected collective benefit, the benefit-risk ratio is very favorable. the study is in the data collection phase. recruitment started in december 2019 and was halted in march 19, 2020 , due to the sars-cov-2 pandemic. on june the 1, 2020, recruitment was restarted and it is estimated to end in january 2021. the current protocol is version 1.0, created in march 2019 and approved by the clinical research ethics committees (crec) of the participating centers, as well as the spanish agency of medicines and health products in december 2019 before starting the study. this study is registered at clinicaltrials.gov, identifier: nct04147026. registered on 31 october, 2019. authors' contributions {31b} pg and lrr were responsible for the study concept and definition of the scope. df, lrr, ig, jlp, ab, mv, pg, and bfg were involved in abstract and full-text screening. lrr and df were responsible for drafting of the manuscript. df, lrr, ig, jlp, ab, mv, pg, and bfg were responsible for critical revision of the manuscript. all authors read and approved the final manuscript. not applicable. this work is supported by the european institute of technology and innovation (eit-health) (#19577). all the funding sources are not involved in the study design and data collection and will not be involved in the analysis and interpretation of data. not applicable. this study will be carried out strictly respecting the ethical principles of biomedical research and current legislation in spain. all personnel participating in this study agree to follow, during the performance of the study, the standards of good clinical practice (guideline for good clinical practice e6 r2: https://bit.ly/2vuhzvd). the approval of the clinical research ethics committees (crec) of the participating centers will be obtained and documented, as well as the approval of the spanish agency for medicines and health products before starting the study. the local approvals corresponding to the participating centers will be obtained and documented before starting the study in the centers. the responsible researcher of each center will be the interlocutor of the crec corresponding to its center in everything related to the present study. it will keep the crec informed of the evolution of the study in the center and of the possible incidents and minor modifications that may occur. any relevant modification to the protocol after its approval must receive express approval from the reference crec and the spanish agency for medicines and health products before its implementation, unless there are risk circumstances for the participating subjects; in which case, they will be implemented with the precise measures to ensure the integrity of the patients immediately awaiting the corresponding approvals. a signed informed consent will be obtained from all study participants before any study-related procedures are undertaken. not applicable. the authors declare that they have no competing interests.author details key: cord-0042126-1tbk96e1 authors: bjoernhart, b.; svenningsen, p.; gudbrandsdottir, s.; zak, m.; nielsen, s.; bentzen, k.; müller, k. title: plasma tnf‐binding capacity and soluble tnf receptors in patients with juvenile idiopathic arthritis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423b.x sha: 60faef52bf6546591f62af9cb4a3a0e3fff99cbb doc_id: 42126 cord_uid: 1tbk96e1 background: unbalanced production of proinflammatory cytokines may be related to disease progression in rheumatoid arthritis and juvenile idiopathic arthritis (jia). within the tnf system, the two agonists, tnf‐α and tnf‐β, also called lymphotoxin‐α (lt), are bound by soluble tnf receptors (stnfr‐i and ‐ii) that act as natural inhibitors of tnf‐induced inflammation. we investigated the plasma levels of stnfr‐i in parallel with lt‐binding capacity (ltbc) in patients with jia. methods: the levels of stnfr‐i were measured by elisa (r&d). ltbc was determined by spiking diluted plasma samples with recombinant lt. detectable lt was measured by an in‐house elisa measuring unbound lt only. ltbc was expressed in arbitrary units (aus) as the percentage value of bound lt to added lt. result: in contrast to previous findings of elevated stnfr levels in patients with various chronic inflammatory diseases, we found slightly reduced stnfr‐i levels in jia patients (n = 123) compared with age‐matched healthy controls (n = 37): 1077 pg/ml (819–2280) versus 1185 pg/ml (625–2303) [median (range)], p = 0015. however, the stnfr‐i levels correlated positively with the number of active joints, physicians' global assessment and crp. in contrast, patient ltbc values were elevated compared to healthy controls: 44 au (36–52) versus 31 au (13–41), p < 0.0001. conclusion: despite overall slightly reduced plasma levels of stnfr‐i, the capacity to bind tnf was increased in plasma samples from jia patients. studies to identify the tnf‐binding substances in plasma are in progress. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. 1081vol. 10 | no. 4 | autumn 2013 |u r o lo g y j o u r n a l evaluation of sexual function in women with rheumatoid arthritis burhan coskun,1 belkis nihan coskun,2 gokhan atis,3 erbil ergenekon,4 kamil dilek5 purpose: to evaluate the link between rheumatoid arthritis (ra) and female sexual functioning. material and methods: a total of 32 women with ra and 20 healthy age matched controls were enrolled in this study. the participations are asked to complete female sexual function index (fsfi), the short form 36 (sf-36) health survey and beck depression inventory (bdi) questionnaires. results: the groups were comparable in terms of demographic characteristics. the women with ra represented significantly worse sexual functioning in category of desire, arousal, lubrication, orgasm, satisfaction domain and total fsfi score compared with healthy women (p = .0001, p = .0001, p = .0001, p = .0001, p = .022 and p = .0001, respectively). the mean bdi scores for the patients with ra were greater than control group (p = .036). women with ra also had significantly lower quality of life (qol) parameters: physical functioning, limitations due to physical health, pain, general health, vitality and limitations due to emotional problems compared with healthy women (p = .0001, p = .0001, p = .028, p = .002, p = .001 and p = .0001, respectively). conclusion: the present study shows that a significant percent of patients with ra had sexual dysfunction and also deterioration in qol. keywords: arthritis; rheumatoid; female; sexual dysfunction; quality of life. corresponding author: burhan coskun, md doburca cad, başak sitesi, c blok no 4, bursa, turkey. tel: +90 533 646 0323 fax: +90 216 521 8608 e-mail: drburhancoskun@ yahoo.co.uk received july 2012 accepted january 2013 1department of urology, inegöl hospital, bursa, turkey. 2department of internal medicine, school of medicine, uludag university, bursa, turkey. 3department of urology, göztepe training and research hospital, istanbul, turkey. 4department of urology, şişli training and research hospital, istanbul, turkey. 5department of rheumatology, school of medicine, uludag university, bursa, turkey. sexual disfunction and infertility 1082 | introduction the rheumatoid arthritis (ra) is a prevalent, idi-opathic, autoimmune disease, which is more com-mon in women with a prevalence of about %1 worldwide.(1) the characteristic feature of ra is persistent inflammatory synovitis in peripheral joints, which usually distributes symmetrically. the synovial inflammation may lead to cartilage damage, bone erosions and changes in joint integrity. finally, all these pathologies may cause various degrees of disability.(2,3) ra has deleterious effects on social, economic, psychological and sexual aspects of the patient’s life.(3) female sexual dysfunction (fsd) is a common health problem affecting 20% to 50% of population and prevalence of this condition correlates with age. it has a profound impact on quality of life (qol) and interpersonal relationships.(4-7) several factors such as endocrine, social, anatomical and psychological diseases may cause fsd and some specific causes like pain, fatigue, stiffness, functional impairment, depression, negative body image, reduced libido and drug treatment are also responsible in patients with rheumatic diseases.(8,9) in this study we aimed to evaluate sexual functions and qol of female subjects with ra and to compare them with voluntary healthy controls. materials and methods a total of 50 sexually active women with diagnosis of ra for more than one years and 20 age matched voluntary healthy women (controls) were included in the study. all of the subjects in both groups were married and patients were followed and treated at outpatient department of rheumatology clinic of uludağ university. the patients with ra were diagnosed according to the 1987 revised criteria for classification of ra of american college of rheumatology. (10) the local ethical committee approved the study. an informed consent was obtained from all subjects with ra and control group. demographic characteristics including age, educational attainment level, and occupational status were assessed in all women. participants who had coexisting diseases or condition resulting in sexual dysfunction (cardiovascular disease, neurological disease, major psychiatric disease, diabetes, previous pelvic surgery, menopause, hysterectomy, premature ovarian failure etc.) and had no sexual disfunction and infertility table 1. demographic characteristics of the patients with rheumatoid arthritis and control group. variables patients with ra (n = 32) control group (n = 20) p age* 38.43 ± 6.94 39.30 ± 5.52 .630 number of pregnancy* 1.93 ± 1.18 1.75 ± 1.25 .603 education status n (%) .383 primaryhigh school 12 (37.5) 5 (25) university 20 (62.5) 15 (75) occupational status n (%) .276 employment 29 (90.625) 20 (100) unemployment 3 (9.375) 0 (0) smoking history n (%) .747 current 8 (25) 4 (20) neverex-smoker 24 (75) 16 (80) alcohol consumption n (%) 1.000 yes 3 (9.375) 2 (10) no 29 (90.625) 18 (90) physical activity n (%) .754 daily-several times in a week 8 (25) 6 (30) once in a week –rarelynever 24 (75) 14 (70) data are presented as number/total (%), fisher’s exact test or *student’s t-test. key: sd, standard deviation; ra, rheumatoid arthritis. 1083vol. 10 | no. 4 | autumn 2013 |u r o lo g y j o u r n a l sexual activity within the past month were not included in the study. all women recruited into the study had a stable, heterosexual relationship, and were sexually active. five patients found to have coexisting disease, 6 patients did not have sexual intercourse in past one month, 4 patients did not want to reply the questions because of embarrassment, 3 of the responders did not reply the questions properly and 18 patients are excluded from study. turkish version of fsfi, which has been previously validated in the turkish language by turkish society of andrology(5,6) was used for evaluation of fsd. fsfi includes 19-item questionnaires, which assesses sexual functioning during the last 4 weeks. the specific subdomains including quality of desire, arousal, lubrication, orgasm, satisfaction and degree of pain is evaluated in fsfi. each domain scores and overall fsfi scores of the women were compared between two groups. the overall fsfi score was 2-36. sexual dysfunction were considered in a total score of less than 26.55.(11) beck depression inventory (bdi), a 21-item self-reported inventory, was previously adapted for the turkish population.(12,13) each of 21 questions is scoring between 0 and 3 (absent to severe), and the highest possible total score for the whole test is 63. psychiatric assessment was performed by the bdi and depression was diagnosed when the bdi score was 17 or greater. the qol in both groups was assessed with sf-36 questionnaire, which has been previously validated in turkish population by koçyiğit and colleagues. sf-36 questionnaire has been designed to evaluate following these concerns in global health: physical function, physical role, body pain, general health, vitality, social function, emotional role and mental health. the scores of the eight subscales range from 0 to 100. higher scores indicate less limitations or distress in the different dimensions.(14,15) the statistical analyses were performed using a computer with medcalc software version 11.4.3. data analyses were performed with the kolmogorov-smirnov test that was used for documenting the normal distribution. data were given as mean ± sd and median (minimummaximum). the mann-whitney u test and student’s t test were used for comparison of scores within the groups. categorical variables are presented as frequencies and percentages and were compared using fisher’s exact test. statistical significance was considered at p ≤ .05. results table 1 lists demographic characteristics of the patients with ra and control group. there was not any significant difference in age, educational level, occupational status, smoking history, alcohol consumption and physical activity between both groups. table 2 shows mean total and domain fsfi and bdi scores between the patients with ra and control group. the dofemale sexual function and rheumatoid arthritis | coskun et al table 2. the mean± sd, median (min-max) total and domain scores of fsfi with bdi scores in study groups. variables ra (n = 32) control (n = 20) p mean ± sd median (min-max) mean ± sd median (min-max) desire 3.47 ± 0.96 3.60 (1.20-4.80) 5.16 ± 0.74 5.40 (3.60-6.0) .0001* arousal 3.750 ± 1.44 3.75 (0.0-6.0) 5.37 ± 0.80 5.7 (3.30-6.0) .0001* lubrication 3.90 ± 1.34 4.05 (0.0-6.0) 5.40 ± 0.52 5.70 (4.206.0) .0001* orgasm 3.95 ± 1.34 4.0 (0.0-6.0) 5.46 ± 0.51 5.60 (4.0-6.0) .0001* satisfaction 4.41 ± 1.49 4.60 (0.0-6.0) 5.40 ± 0.54 5.60 (4.0-6.0) .022* pain 4.26 ± 1.77 4.80 (0.0-6.0) 5.50 ± 0.56 5.60 (4.0-6.0) .104 total 24.49 ± 6.00 25.0 (10.40-36.0) 32.31 ± 3.50 33.90 (24.50-35.0) .0001* bdi score 12.90 ± 8.87 11.50 (0.0-38.0) 7.82 ± 5.20 6.0 (2.0-21.0) .036** *mann–whitney u-test, **student’s t test. key: sd, standard deviation; (min-max), minimum and maximum values; ra, rheumatoid arthritis; bdi, beck depression inventory; fsfi, female sexual function index. 1084 | main scores of fsfi including desire, arousal, lubrication, orgasm, and satisfaction, in patients with ra were significantly lower than control group (p = .0001, p = .0001, p = .0001, p = .0001, p = .022 and p = .0001, respectively). the domain score of pain were not statistically significant (p = .104). the mean bdi scores for the patients with ra were greater than control group (p < .05). fsd was diagnosed in 22 out of 32 (68.75%) patients with ra and 3 out of 20 (15%) (p = .0001). table 3 shows mean total and domain fsfi scores of patients with ra according to they have morning stiffness more than one hour or not. the domain scores of fsfi including, satisfaction and total domain scores in patients who have morning stiffness more than one hour were significantly lower than the patients who do not have morning stiffness more than one hour (p = .05 and p = .026, respectively). the domain scores of fsfi including desire, arousal, lubrication, orgasm and pain were not statistically significant in both groups (p = .626, p = .182, p = .548, p = .097, and p = .609, respectively). table 4 shows mean scores of sf-36 questionnaire between patients with ra and control group. the scores of physical function, physical role, body pain, general health, vitality, emotional role were significantly lower in patient group than the control group (p = .0001, p = .0001, p = .028, p = .002, p = .001 and p = .0001, respectively). the scores of social function and mental health were not statistically significant (p = .4954 and p = .1192, respectively). discussion assessment of sexual functioning and qol in the present study revealed that women with ra had lower scores of total fsfi and also lower scores in all of the fsfi subdomains except pain subdomain, when compared with healthy women. women with ra also had lower scores of qol parameters except social function and mental health, when compared with healthy women. the impact of chronic disabling conditions like ra, on qol of the patients has been investigated in many studies; however sexual functioning in these patients remains a neglected area of qol.(16) therefore, we evaluated the link between ra and female sexual functioning by using fsfi, which is a validated and reliable self-report measurement. recently frikha and colleagues assessed sexual functioning in 10 women with ra by using fsfi.(17) they reported 7 women with fsd out of 10 and all subscales of fsfi were affected. in our study fsd rate was 68%, 75 out of 32 women with ra and 15% out of 20 women belonging to control group. to our knowledge, this is the first study comparing fsfi scores of patients with ra with healthy controls. fsfi is essential in evaluating different components sexual activity (like desire, arousal, lubrication, pain, orgasm, satisfaction and total disorders) rather than determination of sexual dysfunction.(5) therefore, we used cutoff score for sexual disfunction and infertility table 3. the mean ± sd, median (min-max) total and domain fsfi scores of patients with ra according to they have morning stiffness more than one hour or not. variables patients without morning stiffness (n = 23) patients with morning stiffness (n = 9) p mean ± sd median (min-max) mean ± sd median (min-max) desire 3.52 ± 0.91 3.60 (1.20-4.80) 3.33 ± 1.12 3.60 (1.20-4.80) .626 arousal 3.69 ± 1.51 4.2 (0.00-6.0) 2.93 ± 1.13 3.30 (1.20-4.50) .182 lubrication 3.99 ± 1.50 4.2 (0.00-6.0) 3.66 ± 0.80 3.60 (1.80-4.50) .548 orgasm 4.23 ± 1.57 4.40 (0.00-6.0) 3.24 ± 1.12 3.60 (1.20-4.40) .097 satisfaction 4.73 ± 1.50 4.80 (0.00-6.0) 3.60 ± 1.18 4.0 (1.20-4.80) .05* pain 4.36 ± 1.90 5.20 (0.00-6.0) 4.00 ± 1.45 4.0 (2.0-6.0) .609 total 25.94 ± 5.69 26.40 (11.90-36.0) 20.77 ± 5.37 22.20 (10.40-26.40) .026* *student’s t test. key: sd, standard deviation; (min-max), minimum and maximum values; fsfi, female sexual function index; ra, rheumatoid arthritis. 1085vol. 10 | no. 4 | autumn 2013 |u r o lo g y j o u r n a l the turkish population to evaluate fsd.(18) although the total score and all of the domain scores were lower in patients with ra control group, the domain score of ‘pain’ were not statistically significant. these results suggest, all domains of fsfi may contribute the fsd in patients with ra. interestingly pain domain of fsfi is found to be less effective on fsd when compared to healthy age matched controls in our study. the pain domain of fsfi is about the pain during vaginal penetration and it is apart from the pain that patients have routinely. sexual act may positively affect patients mood and most of them denote ease of their pain after sexual relation.(19) the percentage of patients with ra who experience sexual problems ranged in previous studies from 31 to 76% and these studies highlighted two main problems: difficulties in sexual performance and diminution of sexual desire and satisfaction.(16,20-26) in a study which hill and colleagues. investigated effects of ra on sexual activity they found that 56% limitations on sexual intercourse, 50-60% decrease in desire, 73% reduction of frequency of intercourse.(27) in the study of abdel-nasser and colleagues, it has been reported that women with ra had impaired sexual function with a prevalence of 60%.(28) in our study similar results observed with previous papers in terms of fsd. fsd is a common disorder, which has serious effects on women’s qol. aslan and colleagues evaluated the prevalence of and risk factors for fsd using the turkish version of fsfi in 1009 turkish women and found that fsd occurs in 43.4% of women.(29) similarly, in the study of cayan and colleagues it has been reported that 46.9% of women in turkey had fsd, which has been associated with increased age, lower education level, unemployment status, chronic diseases, multiparity and menopause status as important risk factors for fsd.(6) another important factor that effect female sexual functioning is depression and other mood disorders.(30) the present study revealed, the rate of depression in women with ra was significantly higher than healthy controls (p = .036). monga and colleagues assessed the relationship between sexual function and physiological measures in 70 patients with chronic pain and found that sexual problems are common in patients with chronic pain and in those with symptoms of distress and depression.(31) in our study we also evaluated fsfi in women with ra whether they have morning stiffness for a period more than one hour. the total score and the category of and satisfaction domain of fsfi in women who had morning stiffness more than one hour were significantly lower than the women who had morning stiffness less than one hour (p = .026 and p = .05, respectively). similarly gutweniger and colleagues reported that morning stiffness in women with ra plays an important in their feelings of being a handicap. female sexual function and rheumatoid arthritis | coskun et al table 4. the mean ± sd and median (min-max) scores of sf-36 questionnaire between patients with rheumatoid arthritis and control group. variables patients with ra (n = 32) control group (n = 20) p mean ± sd median (min-max) mean ± sd median (min-max) physical function 54.53 ± 23.08 45.0 (45.0-100.0) 86.75 ± 15.58 92.50 (45.0-100.0) .0001* physical role 32.03 ± 39.26 12.50 (0.0-100.0) 76.25 ± 30.85 87.50 (0.0-100.0) .0001* body pain 54.65 ± 18.28 52.0 (21.0-84.0) 68.80 ± 26.67 73.00 (12.0-100.0) .028* general health 44.93 ± 22.95 40.0 (15.0-97.0) 65.85 ± 20.96 74.50 (20.0-97.0) .002* vitality 39.21 ± 21.74 35.0 (0.0-80.0) 64.00 ± 26.08 65.0 (15.0-100.0) .001* social function 71.87 ± 23.54 68.75 (12.50-100.0) 76.25 ± 20.23 87.50 (37.50-100.0) .4954 emotional role 31.23 ± 41.42 0.0 (0.0-100.0) 74.95 ± 35.72 100.0 (0.0-100.0) .0001* mental health 54.25 ± 19.27 54.0 (8.0-100.0) 63.60 ± 22.80 62.0 (24.0-96.0) .1192 *student’s t test. key: sd, standard deviation; (min-max), minimum and maximum values; ra, rheumatoid arthritis; sf-36, the short form 36. 1086 | sexual disfunction and infertility they declared, women with substantial degree of morning stiffness had significantly more worries about their body image and lived more sexual dissatisfaction than females with lower degrees of morning stiffness.(32) the factors like physical disability, fatigue, altered body image and worries about partner interest are also reported to effect sexuality of the patients.(3,21,26) sf-36 has been used in several studies including the patients with ra and has been found to be reliable, valid, and responsive. the major advantage of this survey is its ability to compare the physical and mental status of ra patients with the overall population.(33) in a study, birtane and colleagues assessed the qol of patients with fibromyalgia, ra and healthy controls by using sf-36. the patients with ra and fibromyalgia syndrome had lower scores of all sf-36 subdomains except social function than the control subjects.(34) similarly we found scores of physical functioning, physical role, bodily pain, general health, vitality and emotional role significantly lower in patients with ra than healthy controls (p = .0001, p = .0001, p = .028, p = .002, p = .001 and p = .0001, respectively). although the scores of social functioning and mental health were also lower in patient group the results were not statistically significant (p = .4954 and p = .1192, respectively). our findings are relevant for patient communication, as physicians should be advised to address the sensitive subject of sexual dysfunction in women with ra. there are several limitations of the present study. first of all, the sample size was small and the results may not reflect general population. secondly, we conducted our study in cross-sectional method, which is less valuable than prospective cohort studies. thirdly, depression was not clinically diagnosed in patients and defined based on a self-rating questionnaire .also, we did not investigate whether the fsd was distressful or not for the enrolled women. this would bring more information regarding the relationship between depression and fsd. the last limitation was, lack of evaluation whether the treatment of ra has any effect on fsd. conclusion the present study shows that a significant percent of patients with ra had sexual dysfunction and also deterioration in qol. all domains of fsfi were affected except from the “pain” domain and this may reflect the pathophysiology of fsd in this group of patients. however, more studies in larger population comparing sexual functioning between the patients with ra and healthy controls is necessary to understand this relation. conflict of interest none declared. references 1. gabriel se. the epidemiology of rheumatoid arthritis. rheum dis clin north am. 2001;27:269-81. 2. lipsky pe. rheumatoid arthritis. in: braunwald e (ed) harrison’s 16th edition principles of i̇nternal medicine. new york: mc graw hill; 2005. p. 1968-76. 3. erlich ge. social, economic, psychologic, and sexual outcomes in rheumatoid arthritis. am j med. 1983;75:27-34. 4. laumann e, paik a, rosen rc. sexual dysfunction in the united states: prevalence and predictors. jama1999;281:537-44. 5. rosen r, brown c, heiman j, et al. the female sexual function index (fsfi): a multidimensional self-report instrument for the assessment of female sexual function. j sex marital ther. 2000;26:191-208. 6. cayan s, akbay e, bozlu m, canpolat b, acar d, ulusoy e. the prevalence of female sexual dysfunction and potentional risk factors that may impair sexual function in turkish women. urol int. 2004;72:5257. 7. basson r, berman j, burnett a, et al. report of the international consensus development conference on female sexual dysfunction: definitions and classifications. j urol. 2000;163:888-93. 8. salonia a, munarriz rm, naspro r, et al. women’s sexual dysfunction: a pathophysiologiacal review. bju int. 2004;93:1156-64. 9. østensen m. new insights into sexual functioning and fertility in rheumatic diseases. best pract res clin rheumatol. 2004;18:219-32. 10. arnett fc, edworthy sm, bloch da, et al. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum. 1988;31:315-24. 11. wiegel m, meston c, rosen r. the female sexual function index (fsfi): cross-validation and development of clinical cutoff scores. j sex marital ther. 2005:31:1-20. 12. onem k, erol b, sanli o, et al. is sexual dysfunction in women with obstructive sleep apneahypopnea syndrome associated with the severity of the disease? a pilot study. j sex med. 2008;5:2600-609. 1087vol. 10 | no. 4 | autumn 2013 |u r o lo g y j o u r n a l female sexual function and rheumatoid arthritis | coskun et al 30. berman jr, bassuk j. physiology and pathophysiology of female sexual function and dysfunction. world j urol. 2002;2:111-8. 31. monga tn, tan g, ostermann hj, monga u, grabois m. sexuality and sexual adjustment of patients with chronic pain. disabil rehabil. 1998;20:317-29. 32. gutweniger s, kopp m, mur e, günther v. body image of women with rheumatoid arthritis. clin exp rheumatol. 1999;17:413-7. 33. tugwell p, idzerda l, wells ga. generic quality-of-life assessment in rheumatoid arthritis. am j manag care. 2007;13:s224-36. 34. birtane m, uzunca k, taştekin n, tuna h. the evaluation of qol in fibromyalgia syndrome: a comparison with rheumatoid arthritis by using sf-36 health survey. clin rheumatol. 2007;26:679-84. 13. beck at, ward ch, mendelson m, mock j, erbaugh j. an inventory for measuring depression. arch gen psychiatry. 1961;4:561-71. 14. ware je jr, kosinski m, bayliss ms, mchorney ca, rogers wh, raczek a. comparison of methods for the scoring and statistical analysis of sf-36 health profile and summary measures: summary of results from the medical outcomes study. med care. 1995;33:as264-79. 15. koçyiğit h, aydemir ö, fişek g, ölmez n, memiş a. kısa form–36 (kf-36)’nın türkçe versiyonunun güvenilirliği ve geçerliliği. i̇laç ve tedavi dergisi. 1999;12:102-106. 16. tristano ag. the impact of rheumatic diseases on sexual function. rheumatol int. 2009;29:853-60. 17. frikha f, maazoun f, ben salah r, et al. sexual function in married women with rheumatoid arthritis. presse med. 2011;40:e521-7. 18. oksuz e, malhan s. reliability and validity of the female sexual function index in turkish population. sendrom. 2005;17:54-62. 19. katz wa. sexuality and arthritis. katz wa (ed). rheumatic diseases: diagnosis and management. philadelphia, toronto: jb lippincott co; 1973. p. 1011-20. 20. baldursson h, brattstrom h. sexual difficulties and total hip replacement in rheumatoid arthritis. scand j rheumatol. 1979;8:214-16. 21. blake dj, maisiak r, koplan a, alarcón gs, brown s. sexual dysfunction among patients with arthritis. clin rheumato 1988;l7:50-60. 22. brown gm, dare cm, smith pr, meyers ol. important problems identified by patients with chronic arthritis. s afr med j. 1987;72:126-8. 23. ferguson k, figley b. sexuality and rheumatic disease: a prospective study. sex disabil. 1979;2:130-38. 24. gordon d, beastall gh, thomson ja, sturrock rd. androgenic status and sexual function in males with rheumatoid arthritis and ankylosing spondylitis. q j med. 1986;60:671-9. 25. hill rh, herstein a, walters k. juvenile rheumatoid arthritis: followup into adulthood-medical, sexual and social status. can med assoc j. 1976;114:790-6. 26. kraaimaat fw, bakker ah, janssen e, bijlsma jw. intrusiveness of rheumatoid arthritis on sexuality in male and female patients living with a spouse. arthritis care res. 1996;9:120-5. 27. hill j, bird h, thorpe r. effects of rheumatoid arthritis on sexual activity and relationships. rheumatology (oxford). 2003;42:280-6. 28. abdel-nasser am, ali ei. determinants of sexual disability and dissatisfaction in female patients with rheumatoid arthritis. clin rheumatol. 2006;25:822-30. 29. aslan e, beji nk, gungor i, kadioglu a, dikencik bk. prevalence and risk factors for low sexual function in women: a study of 1,009 women in an outpatient clinic of a university hospital in istanbul. j sex med. 2008;5:2044-52. south african orthopaedic journal current concepts review doi 10.17159/2309-8309/2022/v21n4a7blair nr et al. sa orthop j 2022;21(4) citation: blair nr, van der merwe jf, matshidza s. truth or dair? a review of debridement, antibiotics and implant retention. sa orthop j. 2022;21(4): 228-235. http://dx.doi.org/10.17159/ 2309-8309/2022/v21n4a7 editor: dr jurek pietzrak, university of the witwatersrand, johannesburg, south africa received: may 2022 accepted: october 2022 published: november 2022 copyright: © 2022 blair nr. this is an open-access article distributed under the terms of the creative commons attribution licence, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. funding: no funding was received for this study. conflict of interest: the authors declare they have no conflicts of interest that are directly or indirectly related to the research. abstract debridement, antibiotics and implant retention (dair) is a viable treatment option in early postoperative and acute haematogenous periprosthetic joint infections (pjis) with a stable implant. despite lower success rates compared to oneand two-stage revisions, dair maintains satisfactory outcomes in selected patient groups and, if successful, has similar functional outcomes to primary arthroplasty. dair remains an attractive treatment option, providing satisfactory outcomes with decreased healthcare costs, reduced surgical burden on the patient and shorter hospital stays. with success rates of 37–90%, various factors need to be considered when deciding on dair as the appropriate treatment option for pji. the risk of dair failure needs to be weighed against the potential benefits of dair success. factors that increase success rates include an open dair procedure performed for a low-virulence, antibiotic-sensitive organism, within a short duration between symptom onset and/or index surgery and dair. the procedure involves intraoperative exchange of mobile components and copious wound irrigation, followed by an appropriate antibiotic regimen for a minimum of six weeks that can be administered either intravenously or orally in a well-optimised host, without significant soft tissue defects or contraindications to surgery. factors increasing the risk for dair failure include chronic/late pjis with resistant organisms, especially methicillin-resistant staphylococcus aureus (mrsa) in poor hosts with significant comorbidities, such as chronic obstructive pulmonary disease (copd), liver cirrhosis, rheumatoid arthritis, advanced age > 80 years, patients with fracture indications for arthroplasty and those who cannot tolerate rifampicinand fluoroquinolone-based antibiotic regimens. unfortunately, there is no definitive factor to serve as an indication of whether dair will be successful, but with recent data showing that a failed dair procedure does not lower success in future staged revisions, then even in the face of a 50% success rate, dair can maintain its role as an initial treatment option in the management of pjis. level of evidence: level 5 keywords: dair, debridement, antibiotics, implant retention, periprosthetic joint infection, pji, arthroplasty truth or dair? a review of debridement, antibiotics and implant retention neill r blair,* johan f van der merwe, steven matshidza department of orthopaedic surgery, school of clinical medicine, faculty of health sciences, university of the free state, bloemfontein, south africa *corresponding author: neillrblair@gmail.com introduction total joint arthroplasty is a common intervention to relieve pain from advanced joint disease. with improvements in joint replacement surgery and increasing life expectancy, total joint arthroplasties are expected to increase with time.1,2 with escalating joint arthroplasty procedures, increased periprosthetic joint infections (pjis) are expected.3 the incidence of pji is estimated at 0.5–2%, with knee arthroplasty at 0.8–1.9% and hip arthroplasty at 0.3–1.7%.4,5 pji is a devastating complication of total joint arthroplasty and significantly increases the burden on the patient, the surgeon and the healthcare system. prolonged hospitalisation, multiple surgical procedures, psychological stressors of progressive disease, increased healthcare costs, loss of income and physical disability all add to the burden of pji and reflect as diminished patient outcomes concerning morbidity, quality of life and mortality rates.2,4,6 pji is a leading cause of revision arthroplasty, as the management of the condition frequently requires a combination of surgical and medical intervention.7 eradicating a pji while retaining a viable and functional prosthesis remains a challenge due to multiple variables, including patient condition, the infective organism, surgical approach and antimicrobial use.3 debridement, antibiotics and implant retention (dair) has been identified as a viable treatment choice in pji when applied to selected patients, and its use has shown a significantly increasing trend of approximately 0.9–3.4% over a ten-year period.8 this review discusses the use of dair for pji and highlights its potential benefits and pitfalls. defining periprosthetic joint infection diagnosing pji with the decision to perform additional surgical procedures and selecting which one of the multiple available interventions to conduct is often difficult. differentiating superficial wound infection from deep joint infection can be challenging in early presentations, whereas only subtle signs of infection can be present in late pji, often leading to a delay in diagnosis. parvizi et al.9 released revised guidelines for the diagnosis of pji in 2018. the scoring system uses major and minor criteria to confirm pji in patients where infection is suspected. the new criteria have demonstrated a sensitivity of 97.7% and specificity https://orcid.org/0000-0002-7468-9969 page 229blair nr et al. sa orthop j 2022;21(4) of 99.5%.9 the criteria to take into consideration are illustrated in figure 1. once pji is diagnosed, it can be classified as either early/acute postoperative, late postoperative/chronic or acute haematogenous. various authors have suggested specific time cut-off values paired with these definitions. tsukayama et al. propose four types of pji: type i – positive intraoperative cultures; type ii – early postoperative infection within four weeks of index surgery; type iii – acute haematogenous infection that presents acutely after an asymptomatic period, with a suspected haematogenous origin; and type iv – late chronic infection presenting more than four weeks after the index procedure.10 zimmerli et al. defined pjis as early (within three months of surgery), delayed ( 3 to 24 months after surgery) and late (more than 24 months after surgery).11 in 2021, tarity et al. defined chronicity as acute postoperative (less than six weeks after surgery), chronic (more than six weeks after surgery and more than six weeks of symptoms) and acute haematogenous (less than six weeks of symptoms in a previously well-functioning prosthesis, more than six weeks after surgery).12 classification of pji assists with decision making when considering dair, as acute postoperative and acute haematogenous infections are more likely to be successful than chronic infections.13 treatment options the treatment goals in pjis are to eradicate infection and maintain pain-free joint function.3,14 options include prolonged suppressive antibiotics, dair, oneand two-stage revisions, resection arthroplasty, arthrodesis and amputation. chronic suppressive antibiotic therapy is a conservative approach to pji, usually reserved for patients who are unfit for or refuse further surgical management, and has poor success rates.5,15,16 oneand twostage revision arthroplasties show the highest success rates for pji eradication but are paired with more significant patient burden, prolonged hospitalisation, soft tissue and bony defects, and higher costs.4,8,14,15,17 resection arthroplasty can be considered in low-functioning, non-ambulatory patients with bony or soft tissue defects, those with resistant organism infections, and patients with failed two-stage revisions where antibiotic suppression and further implant intervention are unlikely to be successful.7 arthrodesis and amputation may be considered as last resort options for patients due to the severe impairment of functionality and quality of life, and are reserved for patients where other surgical options have failed.15 dair as a treatment option will be discussed in detail in this article. rationale behind dair two-stage revision arthroplasty is considered the most effective procedure for infection eradication and prevention of infection relapse. despite its success, it is not without challenges for both patient and surgeon. two-stage revisions require two extensive surgical procedures, each placing significant strain on the patient, who may already be frail or systemically compromised due to the infection. increased theatre time, blood loss, surgical difficulty and bone loss associated with implant removal and potential damage to surrounding soft tissues add to a procedure with significant morbidity, prolonged hospitalisation and costs to both patient and healthcare systems.3,14,18 when applied to selective patients, dair has shown itself as a cost-effective option in treating pji while maintaining implants and the surrounding soft tissue envelope. dair is associated with an overall decreased surgical demand on both the surgeon and patient, reduced hospital stay and improved functional outcomes when successful. it is regarded as similar to primary arthroplasty in uninfected cohorts with better results compared to two-stage revisions.4,6,15,17,19-21 major criteria (at least one of the following) intraoperative diagnosis (inconclusive preop score or dry tap) minor criteria (preoperative diagnosis) the same organism isolated from two separate positive cultures preoperative score 2–5 possible pji; ≥ 6 pji positive purulence score: 3 ↑ crp or d-dimer score: 2 ↑ synovial wbc count or le score: 3 ↑ synovial pmn (%) score: 2 sinus tract with observed link to the joint or the prosthesis visible on examination positive histology score: 3 single positive culture score: 2 ↑ esr score: 1 positive alpha-defensin score: 3 ↑ synovial crp score: 1 conclusion pji present conclusion pji present with score ≥ 6 inconclusive with score 4–5 no pji with score equal to or less than 3 conclusion pji present with score ≥ 6 possible pji with score 2–5 no pji with score 0–1 serum synovial fluid figure 1. criteria for the diagnosis of periprosthetic joint infections (pjis) crp: c-reactive protein; esr: erythrocyte sedimentation rate; wbc: white blood cell; le: leukocyte esterase; pmn: polymorphonuclear page 230 blair nr et al. sa orthop j 2022;21(4) dair approach although dair is considered less invasive than two-stage revision procedures, it is not to be thought of as a simple washout, and it is suggested that a senior surgeon perform the surgery.4 preand postoperative optimisation of the patient is required to minimise operative risks. dair is performed via the index surgery’s approach in open procedures. arthroscopic dair has been described and will be discussed later in the article. dair is a radical debridement of all potentially infected tissue from the skin to the prosthesis. previous scar tissue, sinus tracts and inflammatory tissue superficial and deep to the fascia are excised. debridement of the capsule, synovium and any sequestrum is required, with or without exchange of modular components. three to five wound samples are collected for microbiological culture, and empirical antibiotic therapy is started until the antimicrobial susceptibility profile of the causative organism has been determined. wound irrigation with normal saline or an antiseptic solution is advised and meticulous closure is undertaken with or without a suction drain. postoperative antibiotics and infection monitoring are continued until a satisfactory clinical response is achieved.18,20-22 success rates varying success rates for dair have been reported, while several confounding variables are at play. heterogeneity of cohorts, length of follow-up, inclusion and exclusion criteria, and definitions of success and failure are all factors adding to the overall rates of success reported.4 table i summarises the success rates for dair reported in the literature, but whether a success rate of 50% should discourage dair is debatable, as it should be decided on a caseby-case basis whether the potential benefits for a successful dair outweigh the consequences of failure.19 indications for dair debridement, antibiotics and implant retention is indicated in early postoperative and acute haematogenous infections with a stable, well-fixed prosthesis. some sources regard early postoperative infections as occurring within one to three months of the index procedure29,36 and acute haematogenous infections with symptoms no longer than three to four weeks,3,19 while others suggest dair is a viable option in all cases of pji with a well-fixed prosthesis, regardless of implant age,28 that is susceptible to anti-biofilm agents.35 contraindications the only absolute contraindication to performing dair is a loose implant. despite this, the procedure is discouraged when the odds of failure outweigh the potential benefit.19 therefore, dair is discouraged in chronic pji, patients with poor soft tissue cover where wound closure problems are expected, and virulent infections where antibiotic susceptibility is uncertain.36 current concepts in dair current concepts in dair are discussed under surgical and nonsurgical factors. these factors play a role in evaluating the risk of success/failure with dair, and despite the varying opinions raised, a pattern of successful traits and practices can be identified in the literature. surgical factors timing it is widely agreed in the literature that timing plays an essential role in determining the success of dair. many attempts have been made to determine a time cut-off when dair is no longer a viable option. however, the decision should not be solely based on the implant or symptom duration but rather on implant stability, organism virulence, the patient’s condition and soft tissue viability.37 three factors need to be considered when assessing the rate of success in dair. these factors include the type of pji, symptom duration and implant age or time from index surgery. table ii summarises the current literature. acute postoperative pjis have the best success rates for dair, followed by acute haematogenous infections. dair is often discouraged in late/chronic pji due to high rates of failure.1,12-14 a shorter duration of symptoms has also shown more favourable outcomes following dair, with some articles advising dair in patients with symptoms less than one week,4,33 while others say outcomes remain favourable if dair is performed within four weeks of symptom onset.15,37 despite varying time frames proposed, most authors agree that the sooner dair is performed after symptoms have developed, the greater the success rate can be expected to be. nevertheless, dair remains a favourable option in patients with an average duration of symptoms of less than three weeks.7,16,19,31 the time from index surgery or implant age is a heavily debated topic, with some literature suggesting that dair should only be performed if the duration from the index surgery is less than one month,7 whereas others have suggested that this time frame could be extended to three months3,8,16,19,34 or even a year.18 it has been suggested that when the implant is well fixed and the pji is caused by a less virulent organism in an otherwise viable joint, the implant age is not significant.4,33,37 again, when summarising the literature, the highest success rates in dair are seen with an implant age of less than three months. table ii summarises timing recommendations by various authors cited in this review. table i: reported success rates for dair published in the literature publication reported success rate bolduc et al., 20214 72.2% barros et al., 20196 89.5% boyle et al., 20208 37–88% (average of 50%) chalmers et al., 202123 21–36% failure rate chaussade et al., 201724 69.0% chung et al., 201925 86.7% deijkers et al., 202026 82.0% deng et al., 202121 67.3% gerritsen et al., 202127 67.0% grammatopoulos et al., 201728 84.0% horriat et al., 201813 34.1% failure rate kunutsor et al., 20181 61.4% lesens et al., 201816 76.0% lora-tamayo et al., 201729 57.0% ottesen et al., 201930 84.0% qu et al., 201931 57.11% rodríguez-pardo et al., 201432 68.0% tarity et al., 202112 47.6% failure rate tsang et al., 201733 64.7% van der ende et al., 202134 20.0% failure rate wouthuyzen-bakker et al., 201935 45% failure rate zhu et al., 202118 53.9% page 231blair nr et al. sa orthop j 2022;21(4) urgency dair is considered an urgent procedure but not an emergency. all efforts should be made to optimise the patient’s general condition prior to surgery without significantly delaying the procedure.1,4,8,19,20 number of surgeries consensus seems to have been achieved regarding the appropriateness of follow-up dair procedures, from the perspective that if an initial dair has failed, a second procedure is unlikely to improve the success rate.19 it has been shown in the literature that follow-up dair procedures have similar outcomes to the initial debridement, if not worse. the recommendation is that after one failed dair procedure, additional debridement procedures should be avoided and revision or resection arthroplasty considered.19,20,31 of note is that previous researchers have published a ‘double dair’ protocol, where a planned two-stage dair procedure was performed on all patients, with a planned re-examination at five days to maximise infection control.25,38 this protocol relied heavily on the short-term use of high-dose antibiotic-loaded beads and modular component exchange, with success rates of 86.7% and an average follow-up of 41.8 months.25,38 component exchange the practice of exchanging all mobile components and liners is considered to theoretically improve infection eradication by two means; first, to improve the exposure of the joint to aid debridement of all areas where a potential infectious focus is present, especially posterior aspects of the knee;19 and second, replacing mobile parts to remove a potential site of bacterial adherence that cannot always be achieved by debridement and irrigation, limiting the bacterial load present in the joint.15 despite some research showing that replacing mobile components does not relate to improved outcomes,27,30 a notable amount of literature reports significant improvements in implant survivability4,19,28 and infection eradication in both early and late pji.18,28 exchange of mobile components is an independent factor related to dair success.2,14,20,29,33,35 besides cost implications, the exchange of mobile components has no adverse effects. therefore, if possible, all mobile components should be exchanged.5 irrigation irrigation with normal saline with or without the addition of an antiseptic solution is strongly recommended by most sources, and the volume recommended ranges between six and nine litres.4,15,19 the addition of antiseptic solutions such as diluted povidone-iodine, chlorhexidine, peroxide and antibiotics to irrigation fluid has been described, but the concentrations used and efficacy over standard irrigation are unclear.19 pulsed lavage is practically convenient but has comparable efficacy to conventional irrigation.20 direct antibiotics direct intra-articular antibiotic loading using catheters or pumps, antibiotic-loaded beads, sponges and cement spacers has been described, but insufficient evidence supports the routine application of these methods.4 although antibiotic spacers can provide highdose local availability of a selected drug, it does not supply a consistent amount to remain therapeutic, and often concentrations fall to subtherapeutic levels within 72 hours, therefore reducing its efficacy.19 table ii: timing recommendations for the performance of dair published in the literature publication description of cases index surgery duration of symptoms additional comments argenson et al., 201919 < 3 months < 21 days the shorter duration between index surgery and symptom onset related to the best results bolduc et al., 20214 < 6 weeks < 1 week no limit in time from index surgery if the implant is well fixed boyer & cazorla, 202120 < 15 days boyle et al., 20208 < 90 days elkins et al., 201937 < 4 weeks < 4 weeks no time interval for a well-fixed prosthesis. implant, soft tissue, organism and patient health factors of more relevance grammatopoulos et al., 201728 < 13 weeks dair is viable in all pji regardless of duration from index surgery lesens et al., 201816 < 3 months < 3 weeks less dependent on duration, more dependent on the organism involved and implant stability lora-tamayo et al., 20213 < 3 months < 21 days the shorter duration between index surgery and symptom onset related to the best results. dair can still be of benefit in patients with a longer duration lora-tamayo et al., 201729 < 3 months osmon et al., 20137 < 1 month < 3 weeks ottesen et al., 201930 < 42 days qu et al., 201931 < 3 weeks tsang et al., 201733 < 4 weeks < 7 days duration from index surgery is less significant van der ende et al., 202134 < 3 months xu et al., 202015 < 4 weeks zhu et al., 202118 < 1 year duration of symptoms unreliable, especially in haematogenous infections horriat et al., 201813 acute postoperative pji > acute haematogenous > late/chronic pji kunutsor et al., 20181 acute postoperative pji > acute haematogenous > late/chronic pji qasim et al., 201714 acute postoperative pji > acute haematogenous > late/chronic pji tarity et al., 202112 acute postoperative pji > acute haematogenous > late/chronic pji page 232 blair nr et al. sa orthop j 2022;21(4) sinus tract a sinus tract is a pathognomonic feature of a pji,37 and many authors suggest that its presence is an independent risk for dair failure,20 or dair is contraindicated in patients with a draining sinus.14,15 one source has reported no difference in outcome between patients with or without the presence of a sinus tract, and that meticulous debridement and component exchange improved infection control in these patients.21 there is no consensus to suggest that a sinus tract is an absolute contraindication to dair. drain wound drains left in situ postoperatively are recommended in dair to prevent fluid accumulation and decrease potential dead space.4,14 some authors suggest high negative pressure drains, reporting improved outcomes, but they need to be used selectively due to increased hospital stay, costs and impaired mobility.15 stability the radical nature of soft tissue debridement in dair can contribute to postoperative instability. a thorough evaluation of joint stability intraoperatively is required, and when modular components are exchanged, more constrained components can be substituted to mitigate such issues. postoperative precautions, including patient education, splinting and structured physiotherapy, can also be implemented.28 arthroscopic dair arthroscopic dair without component exchange has been described, but many authors discourage arthroscopic dair, identifying it as a risk for failure with lower infection control rates.2,4,20,31 part of the rationale is that the arthroscopic approach does not allow for sufficient debridement or replacement of mobile components.14,15 dair in uniand mega-prosthesis the use of dair is supported in both uniand mega-prosthesis patients with pji. the unique feature of pji in a uni-knee arthroplasty is that both prosthesis and native cartilage are present, and in the event of failure, conversion to a total knee arthroplasty is required. mega-prosthesis pji is often a complex problem, and as revision options are usually limited, dair remains a viable initial approach.19 dair in revision arthroplasty debridement, antibiotics and implant retention should be considered carefully in pjis with prior revisions. poorer outcomes have been reported and revision surgery has been identified as an independent risk factor for failure in dair, with failure rates of 12–22% higher than in primary joint infections.4,15 dair effect on salvage two-stage revision despite its high rate of failure, the debate remains on whether dair is a viable option for patients with pji. a salvage two-stage exchange is often warranted when dair fails and opinions in the literature are still divided. some authors suggest no difference in functional outcomes between patients undergoing a direct twostage procedure versus those having first undergone dair, then a subsequent salvage two-stage procedure,3,19 leaving dair a promising initial option in managing pji. inferior outcomes of salvage two-stage procedures have also been reported, but more data are required to make a definitive decision.14 non-surgical factors organism involved identification of the organism involved in pji is not only valuable to guide antimicrobial therapy but may determine success rates in dair. staphylococcus aureus is the most common organism responsible and represents 27% of all pjis.16 s. aureus has been associated with early and late pji and is recognised as an independent risk factor for dair failure, independent of infection chronicity.12,14 infections with methicillin-resistant s. aureus (mrsa) bears the worst prognosis, with success rates reported as low as 30%. some authors discourage dair in cases of mrsa infection.4,14,19,24 following mrsa, enterococcal and fungal infections also carry a significant risk for dair failure due to its high virulence and frequent presentation with early treatment failure.3,20 mixed cultures or polymicrobial infections pose a moderate risk for failure.20 coagulase-negative staphylococcal (cons) infections are associated with late pji and due to their non-invasive nature and low virulence, present with a good overall prognosis.14 streptococci and fluoroquinolone-sensitive gram-negative bacteria present the best prognosis in pjis managed with dair, but the success rates decrease significantly once antibiotic-resistant gram-negative bacteria are cultured.1,32 when attempting dair in patients with mrsa, resistant gram-negative and fungal infections, careful consideration of the potential risks should be taken into account when deciding whether to perform the procedure. organism identification prior to dair laboratory-based identification of the causative organism in pji is a valuable tool to determine if dair is an appropriate treatment modality and estimate the probability of success, especially when highly virulent organisms, e.g. mrsa, are responsible for the infection.20 recommendations are to attempt identification of the organism prior to dair, as long as it does not delay definitive treatment.19 when requesting an urgent investigation, commonly used basic laboratory tests and microscopy can assist with a preliminary indication of the causative organism within a reasonably short period of time. choice of antimicrobial treatment microbial culture and sensitivity results are the basis on which antibiotic therapy in dair is based and should be tailored according to the causative organism’s antimicrobial susceptibility profile as soon as possible.2 knowledge of the organism preoperatively limits the transition time between empiric and organism-specific regimens.35 for staphylococcal infections, the addition of rifampicin to the treatment regimen is associated with higher success rates, especially in combination with fluoroquinolones.19,20,28 studies have shown that a fluoroquinolone and rifampicin combination decreases failure rates by up to 20% in staphylococcal infections.16,35 fluoroquinolones are also associated with higher success rates in gram-negative infections.3,19 beta-lactam antibiotics are the preferred treatment for streptococcal infections.29 in polymicrobial infections, a combination regimen directed against all cultured organisms is used.28 antimicrobial duration three schools of thought prevail in the literature regarding the duration of antibiotic therapy in dair, which include chronic suppressive antibiotics, intermediate course antimicrobial therapy and short-course antimicrobial therapy. after a dair procedure, chronic suppressive antibiotic therapy remains an appropriate option to prevent infection remission. however, treatment intolerances, potential resistance, and poor prognoses limit chronic suppression as an option for patients who either decline further surgery or are not medically fit to undergo revision surgery.4,14,19 intermediate course antimicrobial therapy has long been accepted as the ideal duration of therapy. many authors still recommend this approach which involves an initial course of page 233blair nr et al. sa orthop j 2022;21(4) intravenous therapy for two to six weeks, followed by three to six months of oral antibiotics.4,7,16,20 newer literature indicates that the duration of treatment does not influence the outcome of dair, with long-term treatment failing to show improved outcomes over short treatment schedules. furthermore, extended duration of antimicrobial therapy masks infective symptoms and postpones the diagnosis of treatment failure.1,15,19 newer recommendations advise a minimum of six weeks of antimicrobial therapy, with six to eight weeks of therapy being sufficient following the performance of dair. an initial course of intravenous therapy and conversion to appropriate oral therapy are proposed if allowed by the causative organism’s susceptibility profile.1,19,35,39 implant factors regarding implant indications, dair performed for pji in fracture arthroplasty showed a 20–30% increased failure risk compared to pji in primary joint arthroplasty.4,19 along with this trend, dair for revision arthroplasties also showed a 12–22% increased risk of failure, with revision arthroplasty being identified as an independent risk factor for dair failure.19,20 no consensus has been reached over cemented versus uncemented implants, with some sources citing cemented implants as having a greater failure risk.19,34 hip arthroplasty shows higher success rates for dair over knee arthroplasty,1,20 but shoulder arthroplasty shows a dair success rate of 75%, in keeping with hip and knee cohorts.20 patient factors despite optimal surgical and microbial conditions, the general condition of the host also plays a role in the success of dair. all efforts should be made to optimise host status prior to treatment. reversible conditions such as anaemia (haemoglobin < 10 mg/dl), malnutrition, coagulopathy and tobacco use should be addressed before attempting dair without causing a significant delay between presentation and treatment.19 chronic conditions such as rheumatoid arthritis, liver cirrhosis, renal failure, copd, diabetes mellitus and active malignancy should be considered prior to dair, as these conditions significantly increase failure rates, and alternative revision options may be more appropriate.16,19,31 patient factors such as advanced age (> 80 years), male sex, obesity (body mass index [bmi] > 30 kg/m2) and compromised immune status secondary to disease or steroid use have also been identified as risk factors for dair failure.15,19,26,34 lastly, a high american society of anaesthesiology (asa) score, elevated esr (> 40 mm/h) and elevated c-reactive protein (crp); (> 65 mg/l) on presentation are also predictors of failure.2,15,18,26,35 a multidisciplinary team, ideally including specialist nurses, therapists, infectious disease physicians and plastic surgeons in addition to the orthopaedic team, is recommended to treat the pji, optimise any associated comorbidities, manage reversible conditions, exclude any concurrent extrinsic infections and assist with postoperative rehabilitation.4 staging tools once surgical, microbial, implant and patient factors have been considered, the difficulty remains to decide whether dair is an appropriate treatment option and whether it will be successful. no system is available to take all these factors into account and most cases have to be assessed individually. however, staging tools are available to assist in decision making and the use of a scoring system is associated with improved outcomes.20 as summarised in table iii, the klic score (kidney, liver, index surgery, cemented prosthesis and crp value), ranging from 0 to 9.5, was developed to predict dair failure in acute postoperative infections.4,36 taking negative prognostic factors such as chronic renal failure, liver cirrhosis, fracture or revision index surgery, cemented prosthesis and an elevated crp (> 115 mg/l) into consideration, a score of 4 already suggests a success rate of less than 45%, which can aid in determining an alternative procedure for the patient.4,19,20,36 researchers have attempted to validate the klic scoring system and found that it shows reasonable sensitivity and specificity at higher scores (> 3.5) and facilitates the identification of high-risk patients, but its value at lower scores is uncertain.3,4,23,36 the crime80 score (based on the presence of specific predictive factors, namely copd and an elevated crp level of > 150 mg/l, table iii: summary of the klic and crime80 preoperative risk scores to predict failure following debridement, antibiotics and implant retention (dair) klic score: preoperative risk score development to predict failure following dair for early acute periprosthetic joint infections (pjis) klic score parameter individual parameter score total score failure rate (%) k chronic renal failure (kidney) 2 ≤ 2 4–5 l liver cirrhosis 1.5 2–3.5 19 i index surgery: indication: prosthesis: fracture or revision prosthesis 1.5 4–5 55 c cemented prosthesis 2 5.5–7 71 c-reactive protein (crp) > 115 mg/l 2.5 ≥ 7 100 crime80 score: preoperative risk score development to predict failure following dair for acute haematogenous periprosthetic joint infections (pjis) crime80 score parameter point(s) allocated per parameter total score failure rate (%) c chronic obstructive pulmonary disease (copd) 2 –1 22 c-reactive protein (crp) > 150 mg/l 1 r rheumatoid arthritis 3 0 28 i indication prosthesis: fracture 3 1–2 40 m male sex 1 3–4 64 e exchange of mobile components –1 ≥ 5 79 80 age ≥ 80 years 2 reproduced with minor changes under the terms of the creative commons attribution non-commercial no derivatives 4.0 international licence (cc by-nc-nd 4.0) (https:// creativecommons.org/licenses/by-nc-nd/4.0/) from tables 3 & 4 in bolduc et al.4 page 234 blair nr et al. sa orthop j 2022;21(4) rheumatoid arthritis, fracture-related indication for surgery, male sex, exchange of mobile components and more than 80 years of age) was developed as a prognostic tool for acute haematogenous infections.4 out of 12 points, a score of 3 or more is associated with a less than 40% success rate. the only positive predictive factor was the exchange of mobile components.19,20,35 the crime80 score showed an acceptable predictive value for dair failure on external validation.23 conclusion debridement, antibiotics and implant retention (dair) is a feasible treatment option in acute postoperative and acute haematogenous periprosthetic joint infections in healthy hosts with a well-fixed prosthesis. despite lower infection eradication rates compared to oneand two-stage revisions, dair provides a low-cost option with good functional outcomes and decreased surgical burden, limiting morbidity and mortality. with improvements in surgical techniques, antimicrobial therapy and a multidisciplinary approach, success rates for dair have been increasing with time and could continue to improve. any patient presenting with an acute pji who has a well-fixed prosthesis, culturing a low-virulence organism with adequate soft-tissue coverage and no significant comorbidities is eligible for dair with a mobile component exchange. as it is a far less invasive procedure and does not preclude revision surgery, an ideal treatment algorithm would primarily include dair, followed by a staged revision in unsuccessful cases. acknowledgement dr daleen struwig, medical writer/editor, faculty of health sciences, university of the free state, for technical and editorial preparation of the manuscript. ethics statement the authors declare that this submission is in accordance with the principles laid down by the responsible research publication position statements as developed at the 2nd world conference on research integrity in singapore, 2010. declaration the authors declare authorship of this article and that they have followed sound scientific research practice. this research is original and does not transgress plagiarism policies. author contributions nrb: contributed to the conception and design of the work, literature review and analysis, drafting of the manuscript, and final approval of the version to be published jfvdm: contributed to the conception and design of the work, revising it critically for important intellectual content, and final approval of the version to be published sm: contributed to the conception and design of the work, revising it critically for important intellectual content, and final approval of the version to be published orcid blair nr https://orcid.org/0000-0002-7468-9969 matshidza s https://orcid.org/0000-0003-0128-0385 references 1. kunutsor sk, beswick ad, whitehouse mr, et al. debridement, antibiotics and implant retention for periprosthetic joint infections: a systematic review and meta-analysis of treatment outcomes. j infect. 2018;77(6):479-88. 2. kuiper jw, willink rt, moojen djf, et al. treatment of acute periprosthetic infections with prosthesis retention: review of current concepts. world j orthop. 2014;5(5):667-76. 3. lora-tamayo j, mancheño-losa m, lumbreras c. to dair or not to dair: decision-making in the management of acute prosthetic joint infection – a narrative review. span j med. 2021;1(2):119-31. 4. bolduc m, fischman d, kendrick b, et al. contemporary outcomes of debridement, antibiotics and implant retention (dair) in hip arthroplasty. ann joint. 2021;6:34. https://doi. org/10.21037/aoj-20-87. 5. bezel p, fucentese s, burkhard j, et al. mini review on the impact of mobile parts’ exchange during the dair procedure (debridement, antibiotics, irrigation, retention) for infected total joint arthroplasties. j infectiol. 2019;2(4):13-17. 6. barros lh, barbosa ta, esteves j, et al. early debridement, antibiotics and implant retention (dair) in patients with suspected acute infection after hip or knee arthroplasty – safe, effective and without negative functional impact. j bone joint infect. 2019;4(6):300-305. 7. osmon dr, berbari ef, berendt ar, et al. diagnosis and management of prosthetic joint infection: clinical practice guidelines by the infectious diseases society of america. clin infect dis. 2013;56(1):e1-e25. 8. boyle kk, kapadia m, landy dc, et al. utilization of debridement, antibiotics, and implant retention for infection after total joint arthroplasty over a decade in the united states. j arthroplasty. 2020;35(8):2210-16. 9. parvizi j, tan tl, goswami k, et al. the 2018 definition of periprosthetic hip and knee infection: an evidence-based and validated criteria. j arthroplasty. 2018;33(5):1309-14. 10. tsukayama dt, estrada r, gustilo rb. infection after total hip arthroplasty. a study of the treatment of one hundred and six infections. j bone joint surg am. 1996;78(4):512-23. 11. zimmerli w, trampuz a, ochsner pe. prosthetic joint infections. n engl j med. 2004;351(16):1645-54. 12. tarity td, gkiatas i, nocon aa, et al. irrigation and debridement with implant retention: does chronicity of symptoms matter? j arthroplasty. 2021;36(11):3741-49. 13. horriat s, ayyad s, thakrar rr, haddad fs. debridement, antibiotics and implant retention in management of infected total knee arthroplasty: a systematic review. semin arthroplasty. 2018;29(3):244-49. 14. qasim sn, swann a, ashford r. the dair (debridement, antibiotics and implant retention) procedure for infected total knee replacement – a literature review. sicot j. 2017;3:2. 15. xu y, wang l, xu w. risk factors affect success rate of debridement, antibiotics and implant retention (dair) in periprosthetic joint infection. arthroplasty. 2020;2(1):4-9. 16. lesens o, ferry t, forestier e, et al. should we expand the indications for the dair (debridement, antibiotic therapy, and implant retention) procedure for staphylococcus aureus prosthetic joint infections? a multicenter retrospective study. eur j clin microbiol infect dis. 2018;37(10):1949-56. 17. fisman dn, reilly dt, karchmer aw, goldie sj. clinical effectiveness and cost-effectiveness of 2 management strategies for infected total hip arthroplasty in the elderly. clin infect dis. 2001;32(3):419-30. 18. zhu mf, kim k, cavadino a, et al. success rates of debridement, antibiotics, and implant retention in 230 infected total knee arthroplasties: implications for classification of periprosthetic joint infection. j arthroplasty. 2021;36(1):305-10. 19. argenson jn, arndt m, babis g, et al. hip and knee section, treatment, debridement and retention of implant: proceedings of international consensus on orthopedic infections. j arthroplasty. 2019;34(2s):s399-s419. 20. boyer b, cazorla c. methods and probability of success after early revision of prosthetic joint infections with debridement, antibiotics and implant retention. orthop traumatol surg res. 2021;107(1s):102774. 21. deng w, li r, shao h, et al. comparison of the success rate after debridement, antibiotics and implant retention (dair) for periprosthetic joint infection among patients with or without a sinus tract. bmc musculoskelet dis. 2021;22(1):895. 22. zhang c, he l, fang x, et al. debridement, antibiotics, and implant retention for acute periprosthetic joint infection. orthop surg. 2020;12(2):463-70. 23. chalmers bp, kapadia m, chiu yf, et al. accuracy of predictive algorithms in total hip and knee arthroplasty acute periprosthetic joint infections treated with debridement, antibiotics, and implant retention (dair). j arthroplasty. 2021;36(7):2558-66. 24. chaussade h, uçkay i, vuagnat a, et al. antibiotic therapy duration for prosthetic joint infections treated by debridement and implant retention (dair): similar long-term remission for 6 weeks as compared to 12 weeks. int j infect dis. 2017;63:37-42. 25. chung as, niesen mc, graber tj, et al. two-stage debridement with prosthesis retention for acute periprosthetic joint infections. j arthroplasty. 2019;34(6):1207-13. 26. deijkers rl, elzakker, pijls bg. debridement, antibiotics, and implant retention with the direct anterior approach for acute periprosthetic joint infection following primary tha. j bone joint surg open access. 2020;5(2):e0062. 27. gerritsen m, khawar a, scheper h, et al. modular component exchange and outcome of dair for hip and knee periprosthetic joint infection. bone joint open. 2021;2(10):806-12. 28. grammatopoulos g, kendrick b, mcnally m, et al. outcome following debridement, antibiotics, and implant retention in hip periprosthetic joint infection – an 18-year experience. j arthroplasty. 2017;32(7):2248-55. 29. lora-tamayo j, senneville é, ribera a, et al. the not-so-good prognosis of streptococcal periprosthetic joint infection managed by implant retention: the results of a large multicenter study. clin infect dis. 2017;64(12):1742-52. 30. ottesen cs, troelsen a, sandholdt h, et al. acceptable success rate in patients with periprosthetic knee joint infection treated with debridement, antibiotics, and implant retention. j arthroplasty. 2019;34(2):365-68. 31. qu gx, zhang ch, yan sg, cai xz. debridement, antibiotics, and implant retention for periprosthetic knee infections: a pooling analysis of 1266 cases. j orthop surg res. 2019;14(1):358. 32. rodríguez-pardo d, pigrau c, lora-tamayo j, et al. gram-negative prosthetic joint infection: outcome of a debridement, antibiotics and implant retention approach. a large multicentre study. clin microbiol infect. 2014;20(11):o911-9. 33. tsang stj, ting j, simpson ahrw, gaston p. outcomes following debridement, antibiotics and implant retention in the management of periprosthetic infections of the hip: a review of cohort studies. bone joint j. 2017;99-b(11):1458-66. https://orcid.org/0000-0002-7468-9969 https://orcid.org/0000-0003-0128-0385 page 235blair nr et al. sa orthop j 2022;21(4) 34. van der ende b, van oldenrijk j, reijman m, et al. timing of debridement, antibiotics, and implant retention (dair) for early post-surgical hip and knee prosthetic joint infection (pji) does not affect 1-year re-revision rates: data from the dutch arthroplasty register. j bone joint infect. 2021;6(8):329-36. 35. wouthuyzen-bakker m, sebillotte m, lomas j, et al. clinical outcome and risk factors for failure in late acute prosthetic joint infections treated with debridement and implant retention. j infect. 2019;78(1):40-47. 36. duffy sd, ahearn n, darley es, et al. analysis of the klic-score; an outcome predictor tool for prosthetic joint infections treated with debridement, antibiotics and implant retention. j bone joint infect. 2018;3(3):150-55. 37. elkins jm, kates s, lange j, et al. general assembly, diagnosis, definitions: proceedings of international consensus on orthopedic infections. j arthroplasty. 2019;34(2s):s181-5. 38. mcquivey kd, bingham j, chung a, et al. the double dair: a 2-stage debridement with prosthesis-retention protocol for acute periprosthetic joint infections. jbjs essent surg tech. 2021;11(1):e19.00071. 39. lora-tamayo j, euba g, cobo j, et al. shortversus long-duration levofloxacin plus rifampicin for acute staphylococcal prosthetic joint infection managed with implant retention: a randomised clinical trial. int j antimicrob agents. 2016;48(3):310-16. p-issn 1693-5683; e-issn 2527-7146 93 vol. 19, no. 2, november 2022, pp. 93-102 research article identification bioactive compound of ethanol-water fraction of coleus atropurpureus for anti-rheumatic rheumatism in cfainduced rats ipang djunarko1,4, nanang fakhrudin2*, arief nurrochmad3, subagus wahyuono2 1 postgraduate program of pharmacy, faculty of pharmacy, gadjah mada university, yogyakarta 55281, indonesia 2 department of pharmaceutical biology, faculty of pharmacy, gadjah mada university, yogyakarta 55281, indonesia 3 department of pharmacology and clinical pharmacy, faculty of pharmacy, gadjah mada university, yogyakarta 55281, indonesia 4 department of pharmacology and clinical pharmacy, faculty of pharmacy, sanata dharma university yogyakarta campus iii, 55282, indonesia. https://doi.org/10.24071/jpsc.004746 j. pharm. sci. community, 2022, 19(2), 93-102 article info abstract received: 09-06-2022 revised: 29-06-2022 accepted: 30-06-2022 *corresponding author: nanang fakhrudin email: nanangf@ugm.ac.id keywords: anti-rheumatic arthritis; coleus atropurpureus; complete freund's adjuvant nonsteroidal anti-inflammatory drugs are used for pain and to slow the progression of rheumatoid arthritis. accordingly, the discovery of rheumatoid arthritis active compounds from the ethanol-water fraction coleus atropurpureus (ewc) was conducted to characterize the isolated compounds as well as the anti-rheumatic effects of the ewc induced complete freund's adjuvant (cfa). we conducted in vivo study in rats which were randomly divided into 5 groups. group 1 was only given cfa as a negative control. group 2 as positive control was orally exposed to diclofenac potassium (9 mg/bw). three groups were given different ewcs orally as follows: 11.25 mg/bw, 22.5 mg/bw, and 45 mg/bw, respectively. rheumatism rates were then compared with positive controls using a visual arthritic scoring system. the compounds identified by isolation of the ewc of coleus atropurpureus predicted forskolin. the ethanol-water fraction coleus atropurpureus did not act as an anti-rheumatic arthritis agent in cfainduced rats. introduction one of the drugs of choice for rheumatoid arthritis are non-steroidal anti-inflammatory drugs (nsaids), which can relieve inflammation and reduce pain by slowing the progression of the disease. another group called disease modifying anti-rheumatic drugs (dmards) acts through immunological processes to decrease hyperactive inflammation. although the combination between the two groups could be more effective, however, they are rarely prescribed together due to the higher adverse side effects (hall et al., 2017). hence, due to the chronic arthritic nature, elderly patients tend to seek other alternative treatments. as one of the indonesian herbal medicines, coleus atropurpureus (c. atropurpureus) is known to have various health benefits including for inflammation disorders. however, strong scientific evidence is needed to confirm its antiinflammatory activity. flavonoids, saponins, polyphenols, and terpenoids were previously reported to be present in its leaves (fakhrudin et al., 2020). the distinct content of phytochemicals in the fractions (ethanol extract, n-hexane, and ethanol-water) was detected by using thin layer chromatography (tlc) analysis. the n-hexane fraction mostly contained terpenoids, while the ethanol-water fraction was dominated by flavonoids (djunarko et al., 2022). this study also http://issn.pdii.lipi.go.id/issn.cgi?daftar&1180428136&1&& http://issn.pdii.lipi.go.id/issn.cgi?daftar&1465346481&1&& https://e-journal.usd.ac.id/index.php/jfsk/index https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.24071/jpsc.004746 research article journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... 94 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 demonstrated that there are anti-inflammatory activities in the carrageenan model from all of the fractions. previous research confirmed that eugenol and thymol as terpenoid compounds from c. atropurpureus exhibited analgesic, antiirritant, antiparasitic, and antiseptic activities (lenny et al., 2013). isoflavones and flavones as flavonoids with substituents at c5, c7, and c4' were previously identified to be present in the leaves of c. atropurpureus (verawati et al., 2016). another study has shown that c. atropurpureus contains flavonoids and phenolics. these findings indicated the potential use of c. atropurpureus in anti-inflammatory study (fakhrudin et al., 2016; iqbal and singh, 2019). phenolics, flavonoids, and terpenoids are reported to be able to reactive oxygen species (ros) after carrageenan induction (heldin et al., 2016). carrageenan generates ros that induces initial tissue injury and tissue defects leading to inflammation (raker et al., 2016). in this present study, we conducted a deeper study to find a chemical entity from the ethanol-water fraction of c. atropurpureus that could be responsible for the potential activity as natural anti-inflammatory drug by in vivo evaluation using the complete freund’s adjuvant (cfa)-induced arthritis. the isolated compound was identified using spectroscopic methods including ftir, 1h-nmr, and 13c-nmr. methods material and chemicals fresh leaves of c. atropurpureus were provided by the herbal garden of sanata dharma university (yogyakarta, indonesia). the plant was identified by the department of biology at the faculty of pharmacy, sanata dharma university with voucher specimen 481/lkto/far-usd/05/13. the chemicals used for the isolation stage were n-hexane, ethanol, ethyl acetate (merck, germany), cerium sulphate (sigma-aldrich, germany), and silica gel f254 (merck, germany). during the in vivo study, the following material were used: carrageenan (sigma-aldrich, germany) and potassium diclofenac (novartis, indonesia). animal and ethics wistar rats (male, 150-200 g) were obtained from the imono laboratory, sanata dharma university, indonesia. the rats were housed in standard cages at 22± 3°c with 3070% in relative humidity and in a 12 h dark-light cycle. standard pellet diet and water were given ad libitum. the use of the animals was approved (number: ke/fk/0209/ec/2020) by the medical and health research and ethics committee of faculty of medicine, public health and nursing, universitas gadjah mada–dr. sardjito public hospital, indonesia. all animals were acclimatized for two weeks prior to the in vivo study. extraction and fractionation the fresh leaves were rinsed off and then dried in the oven (50°c, 48 h). dried leaves of c. atropurpureus (264 g) were macerated with ethanol (1:10). the macerate was filtered using vacuum filtration and was evaporated using a rotary evaporator (45oc) to give 32.36 g of a crude extract. the mixture 1:2:1 of ethanol, water and n-hexane was added to the crude extract (15 g) and shaken intensively. two separated layers were obtained; the upper layer was n-hexane fraction (8.84 g), and the lower fraction was ethanol-water fraction (5.88 g). a 2.36 g of the ethanolic extract was fractionated using separating funnels in ethanolwater-n-hexane (1:2:1). the ethanol-water fraction was then dissolved in ethyl acetate followed by centrifuging them at 3,000 rpm for 10 min. the solubilized phase was then collected and dried up using a porcelain dish and prepared for a preparative-tlc. the sample was dissolved in chloroform-methanol (1:1) and then spotted on the silica f254 glass plate. the plate was developed under ethyl acetate 100% as the mobile phase and the spot was detected under ultraviolet (uv)254 and uv365, and further identified using cerium-sulphuric acid. the desired bands were then scratched and collected, followed by dissolving them in methanol. the isolate was then concentrated to have a mass of 11.6 mg and characterized using ftir (thermo scientific nicolet is10) and -nmr (jeol 1703). the deuterated solvent used in the nmr was cdcl3. in vivo studies of anti-arthritic rheumatoid activity we divided twenty-five rats into five groups randomly. three groups were orally administered different ethanol-water fractions of c. atropurpureus leaves (ewc) as follows: group 1 (11.25 mg/bw); group 2 (22.5 mg/bw); and group 3 (45 mg/bw) (djunarko et al., 2022). furthermore, group 4 was orally given diclofenac potassium (9 mg/bw) as the positive control, whereas group 5 had no treatment as the negative control. arthritis was induced through cfa injection according to kumar et al. (2006). briefly, the paw of the right hind limb of each rat was given 100 μl of cfa containing heat-killed journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... research article 95 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 and dried mycobacterium tuberculosis (strain h37ra, atcc25177) on day 0. within those times, we measured the body weight using a digital balance every three days, sequentially. the arthritic level was then compared with the positive control using a visually arthritic scoring system. the poor arthritic status was measured using a digital caliper showing the paw edema associating with primary and secondary lesions. the lesion was measured on the days 0, 1, 3, 6, 9, 12, 14, 16, 19, 21, 24, 26, and 28 after cfa injection (bani et al., 2007; singh et al., 2003). the edema thickness, area under curve (auc), and percentage of inflammatory inhibition were calculated according to the previous study (djunarko et al., 2022). table 1. the overview of the isolated compounds 1h-nmr result (experimental) with the software prediction for forskolin structure proton type prediction experimetal δ (ppm) splitting pattern δ (ppm) splitting patern ch3 1.11 s 1.08 s ch3 1.11 s 1.13 s ch3 1.16 s 1.23 s ch3 1.31 s 1.31 overlapped ch3 1.41 s 1.41 overlapped ch2 1.24/1.49 d/t 1.26/1.38 d ch2 1.47/1.72 d/t 1.48/ 1.69 low intensities ch 1.47 d 1.47 overlapped oh 2.0 s (br) 2.0 overlapped (br) ch3 2.01 s 2.02 overlap ch2 2.45/2.70 s 2.28/2.32 overlap ch 3.15 t 2.98 low intensity ch 3.75 t 3.77 t ch 4.30 d 4.29 d ch2 5.23/5.24 d 5.27/5.33 d (overlapped) ch 5.89 t 5.36 t (overlapped) table 2. the overview of the isolated compounds 13c-nmr result (experimental) with the software prediction for forskolin structure carbon type predicted  (ppm) experimental δ (ppm) ch3 9.0 low ch3 11.6 14.35 ch3 21.0 22.92 ch3 26.0 low ch3 26.2 low ch2 27.4 29.59 cc 27.5 29.92 ch2 37.8 32.14 cc 43.0 low ch 43.9 low ch2 51.2 low coh 65.4 low coh 70.1 low cc 77.0 low ch 79.3 low cc 81.4 low cc 82.,7 low c=c 112.6 low c=c 145.7 low c=o 170.3 low c=o 209.6 low research article journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... 96 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 table 3. primary and secondary arthritic lesions at 28st day in cfa-induced arthritis in rats (n=5) treatment auc0-28 day (mm2.h) % induction of arthritis in the cfa cfa control 625.3 ± 56.8 0.00 diclofenac 561.0 ± 50.8* 10.28 ewc (11.25 mg/bw) 793.7 ± 71.6** -26.93 ewc (22.5 mg/bw) 830.6 ± 65.0** -32.83 ewc (45 mg/bw) 789.2 ± 63.2** -26.21 data represented in mean ± sem ∗p > .05, compared with control group. ∗∗p < .05, compared with control group. table 4. body and organ weights at 28st day in cfa-induced arthritis in rats (n=5). treatment body weight (g) thymus weight (g) spleen weight (g) cfa control 198.6 ± 14.9 0.48 ± 0.10 0,63 ± 0.06 diclofenac 232.0 ± 10.3* 0.47 ± 0.04* 0.88 ± 0.11* ewc (11.25 mg/bw) 211.6 ± 12.8* 0.49 ± 0.04* 1.01 ± 0.09* ewc (22.5 mg/bw) 217.2 ± 8.4* 0.53 ± 0.03* 1.12 ± 0.18* ewc (45 mg/bw) 206.0 ± 5.7* 0.48 ± 0.04* 0.98 ± 0.13* data represented in mean ± sem ∗p > .05, compared with control group. table 5. alterations in hematological parameters and crp in cfa-induced arthritis in rats treatment rbc (×106/mm3) wbc (×106/mm3) hb (mg%) crp (mg/dl) cfa control 7.6 ± 0.4 11.2 ± 4.3 12.4 ±0.6 <0,2 diclofenac 8.6 ± 0.4* 18.6 ± 4.7* 13.9 ± 0.7* <0,2* ewc (11.25 mg/bw) 8.0 ± 0.6* 14.4 ± 2.5* 13.0 ± 1.1* <0,2* ewc (22.5 mg/bw) 5.8 ± 1.5* 10.2 ± 1.7* 11.6 ± 1.3* <0,2* ewc (45 mg/bw) 7.6 ± 0.4* 12.8 ± 2.1* 12.1 ± 1.0* <0,2* data represented in mean ± sem (n = 5). rbc: red blood cell, wbc: white blood cell, hb: hemoglobin, crp: c-reactive protein ∗p > .05, compared with control group table 6. changes in various pain test scores in cfa-induced arthritis in rat. treatment flexion pain test score mobility score stance score cfa control 2 (2, 2) 2 (2, 2) 2 (2, 2) diclofenac 2 (2, 2)* 2 (2, 2)* 2 (2, 2)* ewc (11.25 mg/bw) 2 (1, 2)* 2 (2, 2)* 2 (2, 2)* ewc (22.5 mg/bw) 1 (1, 2)* 2 (2, 2)* 2 (2, 2)* ewc (45 mg/bw) 1 (1, 2)* 2 (2, 2)* 2 (2, 2)* data represented in median (minimum, maximum), n = 5. ∗p > .05, compared with control group ∗∗p < .05, compared with control group the arthritis score for evaluation of the pain associated with the arthritis was verified by a blinded observer using the visual arthritis scoring systems (kumar et al., 2006, laird et al., 2001). the arthritis score ranged from 0 to 4; wherein 0 indicates the least but definite swelling, while 4 represents the maximum swelling. this scoring system involves observations of all four paws giving a separate score for each limb. hematological parameters and serum c-reactive protein (crp) level were evaluated through routine laboratory methods. the animals were euthanized at the end of our study. we weighed the thymus and spleen of all the animals (cui et al., 2019). statistical analysis the results are presented as the mean ± standard error of the mean (sem) or median (minimum, maximum) for univariate analysis. statistical differences between the controls and the treatments were evaluated by one-way anova followed by dunnett’s multiple comparisons test and by the kruskal-wallis test followed by dunn’s multiple comparison test for normal data and for scored data, respectively. arthritic activity was analyzed using kolmogorov-smirnov test and mann-whitney (p < 0.05). journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... research article 97 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 results and discussion the ewc was produced in 5.88 g of yield appeared as thick gel, with leafy odor, and light brown-green color. further isolation was able to collect a single band from the tlc. we approached the structure of the isolated compound using ftir revealing some representative vibrated bands at the fundamental region. as seen in figure 1a, the ftir spectrum of the isolated compound demonstrates an -oh bonded stretching vibration at 3,399.69 cm-1, sp3 -ch stretching asymmetric vibration at 2,924.54 and 2854.33 cm-1, and -c=o stretching vibration at 1735.59 cm-1. looking at this ftir spectrum, it may lead to the compound’s identity of c. atropurpureus itself, i.e., coleonol or also named as forskolin (figure 1b). after comparing with the ftir spectrum of forskolin (figure 1c), we found a high similarity on both spectra. the forskolin ftir spectrum also shows -oh bonded stretching, sp3 -ch stretching asymmetric, and c=o stretching vibrations at the closed region with the isolated compound ftir spectrum. further support that the isolated compound could be forskolin was performed by the high similarity of their fingerprint region. further approach to characterize the isolated compounds were done by nmr. the 1hnmr shows some similar patterns with the 1hnmr spectrum of forskolin predicted by chemdraw ultra 8.0 software. they are ch3 protons showed at 1.08, 1.13, 1.23, 1.31, and 1.41 ppm. further proton signals such as ch2, ch, oh, and =ch can be overviewed in table 1 describing the individual chemical shifts and the splitting patterns. unfortunately, some proton signals show very weak intensities due to the small amount of the sample (figure 2). the 13c-nmr spectrum also shows some similarity with its software’s prediction. the carbon signals at 14.35 and 22.92 ppm were indicated as ch3 carbons. furthermore, the carbon signals at 29.59, 29.92, and 32.14 ppm were indicated as ch2, cc, and ch2 carbons, respectively. unfortunately, many carbon signals were not observed due to its less intensity in the electromagnetic irradiation performance (figure 3). the predicted as well as the experimental 13cnmr of the forskolin can be overviewed in table 2. previously, it had been reported that forskolin signatures have been found in another species of coleus, coleus forskholii (bhowal and mehta, 2017). figure 1. the presentation of a) ftir spectrum of the isolated compound from c. atropurpureus, b) forskolin structure, and c) the ftir spectrum of forskolin from saritha et al. (2015). research article journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... 98 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 figure 2. the 1h-nmr spectra of a) the isolated compound from c. atropurpureus, and b) forskolin predicted by software. observations of cfa-induced foot edema in rats and arthritis scores were recorded at days 0, 1, 3, 6, 9, 12, 14, 16, 19, 21, 24, 26, and 28 after adjuvant injection. the control group showed arthritis progression through increasing claw thickness in cfa-injected areas, thus indicating arthritic lesions in primary or secondary responses. weight loss and arthritis scores changing demonstrated arthritis induction in the cfa-treated control group. assessments performed on days 0 to 28 indicated that the treatment group, either diclofenac or ewc treatment, could reduce adjuvant-induced primary and secondary lesions significantly compared to the cfa control. notably, the secondary lesion reduction was not significantly different in the diclofenac and ewc-treated groups at either dose 11.25; 22.5; or 45 mg/bw (table 3). the cfa control group gained less weight than the group given ewc and diclofenac on day 28 (table 4). however, the effect on body weight was statistically distinct and not significantly different between the treatment groups. spleen and thymus weights at day 28 were not reduced significantly in the ewc-treated group. the ewc treatment could improve cfainduced hematological disorders (table 5). however, the changes between the treatment groups were statistically different but not journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... research article 99 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 significant. high serum crp levels (<0.2 mg/dl) as a marker of systemic inflammation was observed in the cfa control groups. however, in all of the doses of ewc and diclofenac treatments were not able to reduce the increasing serum crp levels. the effect of ewc was not statistically significant between the treatment groups. the flexion pain test scores, mobility scores, and attitude scores which were pain score parameters, did not change significantly in the mice treated with diclofenac and ewc doses 11.25; 22.5; or 45 mg/bw. the results indicated that ewc was not able to reduce the pain associated with adjuvant-induced arthritis (table 6). based on the spectrometric characterization, the isolated compound from ethanol-water fraction of c. atropurpureus leaves approaches forskolin. a study by chiadak et al. (2016) reported that lipopolysaccharideinduced modulation of mcp-1 and gpr120 in 3t3-l1 adipocytes through an inhibition of nfκb were inhibited by forskolin. another research by karthika et al. (2016) further supported the capability of forskolin isolated from solena amplexicaulis as an anti-inflammatory agent. cfa-induced arthritis is a chronic animal model that is the most widely used in rheumatoid arthritis experimental models (noh et al., 2021; patel et al., 2021). the increasing volume of the injected leg indicates chronic inflammation in the cfa model. however, the inhibitory effect of ewc (11.25; 22.5; 45 mg/bw) on the injected leg volume was not significantly different from that of diclofenac 9 mg/bw. figure 3. the 13c-nmr spectra of a) the isolated compound from c. atropurpureus, and b) forskolin predicted by software. research article journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... 100 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 an immune-mediated inflammatory reaction was related with cfa-induced arthritis (el-tanbouly et al., 2022). in this model, edema at the depot site were initial reactions due to the irritant effect of the adjuvant, whereas late-phase arthritis and flares in the injected foot are considered immunological events (biddle and sofat, 2020; yang et al., 2016). an un-injected leg swelling as secondary lesions is a manifestation of cell-mediated immunity. its immunosuppressive activity was obtained by suppression of these secondary lesions (bani et al., 2007; singh et al., 2003). ewc was less effective at reducing secondary lesions than diclofenac. this revealed that ewc caused less intense suppression in cell-mediated immunity. likewise, it decreases rheumatism scores and secondary leg swelling. the immunosuppressive effect of the anti-inflammatory drug was obtained by the selective reduction in arthritis scores (choudhary et al., 2014; faisal et al., 2018). the reduction in arthritis scores by ewc as observed in our study was less suggestive for its possible immunosuppressant activity. the insignificant reduction in thymus weight in the ewc-treated group further supports this observation. cfa-induced arthritis in mice is correlated with an increase in plasma levels of crp (he et al., 2022). the ewc did not significantly reduce the inflammation and autoimmune biomarkers in the ewc treatment groups. another arthritis pathology that was obtained during this study included hematological parameters, changes in body weight, organ weight and percentage of inhibition of rat paw edema. the results showed that treatment with ewc and diclofenac avoided joint changes associated with arthritis which was indicated by a decrease in rat foot edema although the results were not statistically significant and ewc was concluded to have no potential as an anti-rheumatoid arthritis agent. the diclofenac and ewc treatment groups had a recovery in body weight of rats along with observations until the 28th day. weight loss during inflammation is caused by a lack of nutrients absorption through the intestines and interestingly, the treatment using antiinflammatory drugs normalizes this absorption disturbance (maseda and ricciotti, 2020). the results show that the occurrence of this recovery was statistically different, however, they were not significantly different from the cfa arthritis induction treatment group. the ewc and diclofenac-treated groups led to a recovery in rat’s body weight which may involve the increased intestinal nutrient absorption and followed by a reduction in the suffering caused by the arthritis. in arthritic conditions, the moderate increase in white blood cells counts was observed, due to il-1bmediated increases in each colony-stimulating factor. this study revealed that ewc and diclofenac treatments tended to normalize white blood cells counts. other hematological changes such as a decreasing hemoglobin count and an increasing erythrocyte sedimentation rate (singh et al., 2003) were also reversed by ewc and diclofenac treatments. it is proposed that the decrease in hemoglobin count during arthritis is caused by the decreased erythropoietin levels, bone marrow erythropoietin response, and the premature destruction of red blood cells. the decreased spleen and the increased thymus weights are associated with the stimulation effect of the immune system (chen et al., 2019). the decreasing in spleen and thymus weights are observed in ewc-treated mice suggesting changes in the cell population of these organs, which are associated with the immune function. supposedly, diclofenac resulted in a reduction of the spleen and thymus weights, which could be attributed to its anti-proliferative action. however, it turned out that neither diclofenac nor ewc could show a similar effect on these organs. this finding shows that there is no immunosuppressant effect of both diclofenac and ewc, therefore evidence is needed to confirm the its immunosuppressant activity. arthritis is an inflammatory joint condition associated with hyperalgesia that is mediated by prostaglandins, other endogenous mediators and functional impairment (zhang and lee, 2018). treatment of diseases such as arthritis is expected to overcome changes in some of the mediators and/or their effects to obtain clinical benefits. evidence from this study proves that the plant-derived flavonoids ewc did not appear to exert valuable effects on various pathological manifestations of cfa-induced arthritis in rats. therefore, this molecule may be less proven to have clinical value if further systematic investigation and development were conducted. on the other hand, the terpenoid content of the hexane fraction is also proven as an acute antiinflammatory agent (djunarko et al., 2022). the activity of arthritis in our study was evaluated by arthritic scores and the dorsal flexion pain test visually. ewc was not effective to improve pain threshold and reduce flexion pain test score. moreover, the functional impairment in arthritis were determined by its journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... research article 101 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 mobility and stance scores. ewc treatment decreased the mobility score and improved the stance score, thus indicating pain reduction. it is proposed that the effects of endogenous pain mediators were affected by ewc significantly, even though actual quantification of the mediators of pain was not performed in this study (kumar et al., 2006). nowadays, drug development has been less relevant in many chronic diseases because of involving multiple organ systems and interdependent etiological factors. moreover, drug discovery is now changing from single-target to multiple-target approaches (ramsay et al., 2018). treatment of diseases such as arthritis is expected to overcome changes in some of the mediators and/or their effects in order to obtain clinical benefits. it is evident from this study, prediction of forskolin compounds isolated from ewc seems to have a beneficial effect on several pathological manifestations related to cfainduced arthritis in rats. accordingly, this molecule has not been able to prove clinical effects thus, it requires further investigation especially in chronic inflammatory and pain conditions. conclusions the compound identified by the isolation of the ethanol-water soluble fraction coleus atropurpureus using ftir, 1h-nmr, and 13c-nmr predicted forskolin. the ethanol-water fraction of c. atropurpureus did not act as an antirheumatoid arthritis agent in rats induced by complete freund's adjuvant. conflict of interest the authors affirm no conflict of interest in this study. references bani, s., kaul, a., khan, b., gupta, v. k., satti, n. k., suri, k. a., and qazi, g. n. 2007. anti-arthritic activity of a biopolymeric fraction from euphorbia tirucalli. journal of ethnopharmacology, 110(1), 92–98. bhowal, m., and mehta, d. m. 2017. coleus forskholii: phytochemical and pharmacological profile. international journal of pharmaceutical sciences and research, 8(9), 3599–3618. biddle, k., and sofat, n. 2020. rheumatoid arthritis: other perspectives towards a better practice. in intechopen, london. chen, c., su, x., and hu, z. 2019. immune promotive effect of bioactive peptides may be mediated by regulating the expression of socs1/mir‑155. experimental and therapeutic medicine, 18(3), 1850–1862. chiadak, j. d., arsenijevic, t., verstrepen, k., gregoire, f., bolaky, n., delforge, v., flamand, v., perret, j., and delporte, c. 2016. forskolin inhibits lipopolysaccharide-induced modulation of mcp-1 and gpr120 in 3t3-l1 adipocytes through an inhibition of nfκb. mediators of inflammation, 2016, 1431789. choudhary, m., kumar, v., gupta, p., and singh, s. 2014. investigation of antiarthritic potential of plumeria alba l. leaves in acute and chronic models of arthritis. biomed research international, 2014, 474616. cui, x., wang, r., bian, p., wu, q., seshadri, v. d. d., and liu, l. 2019. evaluation of antiarthritic activity of nimbolide against freund’s adjuvant induced arthritis in rats. artificial cells, nanomedicine, d biotechnology, 47(1), 3391–3398. djunarko, i., fakhrudin, n., nurrochmad, a., and wahyuono, s. 2022. in vivo anti-inflammatory activity of coleus atropurpureus leaves extract and fractions. tropical journal of natural product research, 6(1), 40–43. el-tanbouly, g. s. and abdelrahman, r. s. 2022. novel anti-arthritic mechanisms of transcinnamaldehyde against complete freund’s adjuvant-induced arthritis in mice: involvement of nf-кb/tnf-α and il-6/il23/ il-17 pathways in the immunoinflammatory responses. inflammopharmacology, 1, 1–12. faisal, r., ahmad, n., fahad, y. s., and chiragh, s. 2018. anti-arthritic effect of thymoquinone in comparison with methotrexate on pristane induced arthritis in female sprague dawley rats. journal of ayub medical college, abbottabad : jamc, 30(1), 3–7. fakhrudin, n., khairunnisa, s. y., azzahra, a., and ajiningtyas, r. j. (2016). study of radical scavenger activity, total phenol and flavonoid contents of artocarpus altilis leaves extracts. international journal of pharmaceutical and clinical research, 8(5), 352–356. fakhrudin, n., pertiwi, k. k., takubessi, m. i., susiani, e. f., nurrochmad, a., widyarini, s., sudarmanto, a., nugroho, a. a., and wahyuono, s. 2020. a geranylated chalcone with antiplatelet activity from the leaves of breadfruit (artocarpus altilis). pharmacia, 67(4), 173–180. hall, j. j., bolina, m., chatterley, t., and jamali, f. 2017. interaction between low-dose methotrexate and nonsteroidal antiinflammatory drugs, penicillins, and proton research article journal of pharmaceutical sciences and community identification bioactive compound of ethanol-water fraction... 102 djunarko et al. j. pharm. sci. community, 2022, 19(2), 93-102 pump inhibitors. the annals of pharmacotherapy, 51(2), 163–178. he, y., zhou, m., jian, z., fang, l., huang, l., and song, j. 2022. c-reactive protein knockout attenuates temporomandibular joint inflammation in rats. journal of immunology research, 2022, 8613986. heldin, c. h., lu, b., evans, r., and gutkind, j. s. 2016. signals and receptors. cold spring harbor perspectives in biology, 8(4), a005900. iqbal, h., and singh, d. 2019. antioxidants: a brief review. south asian res j med sci, 1(2), 36–39. karthika, k., jamuna, s., abinaya, g., venkatachalapathi, a., thenmozhi, k., and paulsamy, s. 2016. evaluation of antiinflammatory and antioxidant properties of crude extract and forskolin from solena amplexicaulis leaf. indian journal of pharmaceutical sciences, 78(3), 377–387. kumar, v. l., roy, s., sehgal, r., and padhy, b. m. 2006. a comparative study on the efficacy of rofecoxib in monoarticular arthritis induced by latex of calotropis procera and freund’s complete adjuvant. inflammopharmacology, 14(1–2), 17–21. laird, j. m. a., carter, a. j., grauert, m., and cervero, f. 2001. analgesic activity of a novel usedependent sodium channel blocker, crobenetine, in mono-arthritic rats. british journal of pharmacology, 134(8), 1742. lenny, s., barus, t., marpaung, l., and pandapotan nasution, m. 2013. structure elucidation of flavonoid compound from the leaves of coleus atropurpureus benth using 1d-and 2d-nmr techniques (elusidasi struktur sebatian flavonoid dari daun coleus atropurpureus benth menggunakan teknik 1d-dan 2d-nmr). the malaysian journal of analytical sciences, 17, 255–261. maseda, d., and ricciotti, e. 2020. nsaid–gut microbiota interactions. frontiers in pharmacology, 11, 1153. noh, a. s. m., chuan, t. d., khir, n. a. m., zin, a. a. m., ghazali, a. k., long, i., ab aziz, c. b., and ismail, c. a. n. 2021. effects of different doses of complete freund’s adjuvant on nociceptive behaviour and inflammatory parameters in polyarthritic rat model mimicking rheumatoid arthritis. plos one, 16(12), e0260423. patel, r., kadri, s., gohil, p., deshpande, s., and shah, g. 2021. amelioration of complete freund’s adjuvant-induced arthritis by calotropis procera latex in rats. future journal of pharmaceutical sciences 2021, 7(1), 1–11. raker, v. k., becker, c., and steinbrink, k. 2016. the camp pathway as therapeutic target in autoimmune and inflammatory diseases. frontiers in immunology, 7, 123. ramsay, r. r., popovic-nikolic, m. r., nikolic, k., uliassi, e., and bolognesi, m. l. 2018. a perspective on multi-target drug discovery and design for complex diseases. clinical and translational medicine, 7(1), 3. singh, b., bani, s., gupta, d. k., chandan, b. k., and kaul, a. 2003. anti-inflammatory activity of “taf” an active fraction from the plant barleria prionitis linn. journal of ethnopharmacology, 85(2–3), 187–193. verawati, v., aria, m., dira, d., maisa, s., and maharani, a. 2016. chemical characterization and anti-inflammatory activity of piladang leaf (coleus atropurpureus) extract. journal of chemical and pharmaceutical sciences, 9(4), 2496– 2499. yang, j., cai, h. da, zeng, y. l., chen, z. h., fang, m. h., su, y. p., huang, h. h., xu, y., and yu, c. x. 2016. effects of koumine on adjuvantand collagen-induced arthritis in rats. journal of natural products, 79(10), 2635–2643. zhang, a., and lee, y. c. 2018. mechanisms for joint pain in rheumatoid arthritis (ra): from cytokines to central sensitization. current osteoporosis reports, 16(5), 603610. page 6 p h y s i o t h e r a p y december, 1967 to be m et in a c o m p re h en siv e way. a t the sa m e tim e the tre a tm e n t p ro g ra m m e facilitate s a n d c o m p le m e n ts in every way th e e d u c a tio n a n d re sea rc h fu n c tio n s o f th e u nit. it is e v id e n t th a t th e re sp o n sib ility fo r de v elo p in g r h e u m a tic d isease u n its will fall u p o n v a rio u s pa rties, in clu d in g u n iv e rsity d e p a rtm e n ts o f m e dicine, t ea ch in g h o s p ita ls a n d g o v e rn m e n t h o s p ita l in su ra n c e c o m m issio n s, as well as local m edical societies. h ow ever, th e effort o f p lan n in g a n d seeking o u t m e th o d s o f a ch ieving th e d esired goal in e ach a re a , is a re sp o n sib ility to be m et, if a t all, only by in te rested lay g ro u p s such as the c a n a d ia n a rth ritis a n d r h e u m a tis m society. i n su m m a ry , th e ra tio n a le fo r a n d a im s o f th e r h e u m a tic d ise a se u n it c o n c e p t as well as o u r u n it a p p ro a c h to the m a n a g e m e n t o f r h e u m a to id a rth ritis have been described. w e h a v e b e en g reatly h e a rte n e d by o u r experiences to d a te a n d h o p e th a t th e e sta b lish m e n t elsew here o f sim ila r u n its will lead n o t only to b e tte r m e th o d s fo r c o n tro llin g the v a rio u s fo rm s o f a rth ritis , b u t will also e n h an c e e d u c a tio n a b o u t th e rh e u m a tic diseases, a n d lead to new know ledge a b o u t them . t h e a u th o rs wish to th a n k m iss r o se m ary j a c o b s o n o f jo h a n n e s b u rg , a re ce n t p h y sio th e ra p ist w ith th e u nit (see f ig u re 3, th ird fro m th e rig h t) for e n c o u ra g in g us to w rite th is a cc o u n t. r e f e r e n c e s 1. t h e c a n a d ia n a rth ritis a n d r h e u m a tis m society: ‘a rth ritis — p la n fo r a tta c k .’ canad. m e d . a ss. j. 62:34, 1950. 2. o g r y z l o , m . a ., g o r d o n , d . a. a n d s m y t h e , h . a. ‘t h e r h e u m a tic d isease u n it (r .d .u .) c o n c ep t a rth ritis a n d r h e u m a t.’ in press. 3. ‘h a r t , f . d . ‘c o m p lic a te d r h e u m a to id d ise a se,’ b rit. m e d . j., 2, 131, 1966. 4. r o p e s , m . w ., b e n n e t t , g . a ., c o b b , s ., j a c o x , r . f . a n d j e s s a r , r . a. ‘1958 r e v isio n o f d ia g n o stic c rite ria for r h e u m a to id a rth ritis ,' bull. r heum . d is., 9:175, 1958. 5 . b e l l , d . a ., g o r d o n , d . a., r a u m a l , r . a n d b r o d e r , i ‘c o rre la tio n betw een th e r h e u m a to id b iologically active f a c to r (r b a f ) a n d c linical f e a tu re s o f r h e u m a to id a rth ritis (r a ) a rth ritis a n d r h e u m a t.’ 10:266, 1967. 6. o g r y z l o , m .a . u n iv e rsity o f t o ro n to r h e u m a tic d ise a se u n it f iv e y e a r r e p o r t 1960-65. 7. c o h e n , b . s. b a u m , j . , l o g g i n s , b . a n d t e r r y , e. ‘h o m e c are p ro g ra m m e in th e m a n a g e m e n t o f a rth r itis .’ j. chronic dis. 19:631, 1966. 8. e n g e l m a n , e . t ., s e l l i n g e r , e . a n d m e t t i e r , s. r . ‘p ro b le m s in th e a d m in is tra tio n o f a n e xem p lary a rth ritis c linic in a t ea ch in g c e n tre ,’ a rth ritis an d r heum at. 6:78, 1963. place of physiotherapy in the treatment of rheumatoid arthritis by r. ja c o b s o n , b .s c .,p h y s.(r an d ) in 1964 t h e a m erican r h e u m a tis m a sso c ia tio n e s ta b ­ lished 6 -4 p e r c en t o f th e p o p u la tio n w ere re p o rte d to have a rth ritis a n d r h e u m a tism . t h e s ocio -ec o n o m ic im p a c t o f the rh e u m a tic diseases c an be a p p re c ia te d from d a ta o b ta in e d from the u .s. n a tio n a l h e a lth survey (1964)— th is show ed th a t o f th e one m illion p e rso n s c o nfined to th e house 17 per c ent a ttrib u te d th e ir re stric tio n to a rth ritis a n d rh e u ­ m atism a n d the sam e c o n d itio n s w ere blam e d fo r a w ork loss o f a p p ro x im a te ly 27 m illion days an n u ally . t h e m an a g em e n t o f rh e u m a to id a rth ritis is o f necessity so m e w h a t p ra g m a tic a n d the care o f e ach p a tie n t m u st be a d a p te d to his ow n needs. t h is a rtic le is a d e sc rip tio n o f th e general p rinciples e m ployed by the p h y sio th e ra p ist in the tre a tm e n t o f rh e u m a to id a rth ritis . t h e ideal s itu a tio n for such p a tie n ts is a u n it w here all the th e ra p is ts a re g e are d to th e e d u c a tio n a n d effective tec hniques o f m an a g em e n t. t y pes o f pa t ie n t s a d m it t e d t o t h e u n it ur) f irst tim ers— these p a tie n ts d e m o n s tra te th e active stage o f th e disease. (b) f la re u p ’s— reassessm ent o f the c o n d itio n a n d re ­ o rg a n is a tio n o f tre a tm e n t. (c) a d v a n ce d cases— these re q u ire m a in te n a n c e o f stre n g th a n d m axim al usage o f re m a in in g jo in t fu n c tio n . (cl) p o st surgical m an a g em e n t. before c o n sid erin g th e a p p ro a c h e s a v ailab le in th is c o n d itio n , it is e ssential to c o m p re h e n d th e forces p ro d u c in g th e p a in a n d d e fo rm ity . (see t a b le 1). j o in t d e f o r m it y j o in t fu n c tio n d e p en d s on th e a rc h ite c tu ra l in te g rity o f bones b e arin g surfaces a n d re stra in in g ligam ents, on m uscle p ow er a n d n e u ral re g u la tio n a n d free d o m fro m a dverse e x te rn al c irc u m s ta n c e s ; in th e rh e u m a to id all o f these m ay be involved. movement in th e n o rm a l p e rso n activities o f daily living re su lt in m ain ta in in g m uscle s tre n g th a n d a n a d e q u a te b lo o d su p p ly ; the n u tritio n o f th e c a rtila g e is d e p e n d e n t o n jo in t m o v em e n t a n d th e m o st effective stress on b o n e p re v en tin g disuse o ste o p o ro sis is fro m m uscle c o n tra c tio n . in th e p e rso n with changes c h a ra c te ris tic o f th e rh e u m a tic ty p e exercise c a n n o t be left to c h an c e b u t is re g u la te d by a th e ra p e u tic regim e c o n tro lle d for lo ad , d ire c tio n , d u ra tio n a n d frequency. capsule, ligament, cartilage and bone e ffusion in a jo in t will p ro d u c e a ra ised in tra -a rtic u la r pressure. t h is m ay re su lt e ith e r from a ctive disease process o r tra u m a tic in fla m m a tio n . d e a n d ra d e , g r a n t a n d d ixon suggested th a t stim uli from th e k nee jo in t reflexly in h ib it low er m o to r n e u ro n s su p p ly in g th e q u a d ric e p s . i n p a tie n ts w ith a rtic u la r disease p a in precedes w eakness. t h e highest pressures n o te d in th e knee a re d u rin g full k n e e b e n d w hereas th e low est p re ssu re n o te d w as w ith th e k n e e in a p o sitio n o f slight flexion. r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) decem ber, 1967 p h y s i o t h e r a p y page 7 c a r p a l t u n n e l t e n o s y n o v it is and t e n o v a g in it is v t r i g g e r t e n d o n s s t r e t c h in g o f c a p s u l e a n d l ig a m e n t s t e n d o n 1 d is p l a c e m e n t o f t e n d o n a r t h r i t i s a n o th e r effect o f effusion m ay be to stre tc h th e jo in t cap su le a n d ligam ents so th a t te n d o n h a v in g pulley a tta c h ­ m en ts will have its line o f pull shifted. p ro life ra tin g synovium m ay a lso cause d iste n tio n . s om e c apsules a re stre tc h ed o th e rs m ay be th ic k e n e d , re su ltin g in a loss o f jo in t m obility. p a n n u s invades a n d e ro d e s a rtic u la r c artila g e at the jo in t m argins, a t th e ju n c tio n o f c artila g e a n d sy n o v iu m a n d a t the a tta c h m e n ts o f c o lla te ral ligam ents b one e ro sio n occurs. m uscle, tendon and nerve m uscle w eakness is p o stu la te d to be d ue to in h ib itio n , disuse a n d reflex v a sc u la r c hanges. it is c o n sid ere d th a t b o th m uscle spasm a n d in h ib itio n m ay be m ed ia te d o n a n eural reflex basis by stim u la tio n o f th e p ro p rio ce p tiv e nerve en dings in th e in fla m m a to ry process. t e n d o n lesions interfere w ith th e m uscle a ctio n . dynamic aspects o f deformity (a) s tru c tu ra l re la tio n s h ip s a re c h an g e d . a n ta g o n istic m uscle p u ll is n o t o p p o se d a n d force is exerted a g ain st ligam ent a n d c apsule re su ltin g in a shift. (b) a new j o in t e q u ilib riu m is th u s estab lish e d . a s s e s s m e n t o f p a t ie n t emotional factors a s w ith a n y c o n d itio n it is necessary fo r th e th e ra p is t to u n d e rsta n d th e effect a c h ro n ic d e fo rm in g illness h as u pon a p a tie n t. e m o tio n a l fa c to rs c a n n o t be identified as real cause, how ever, lo n g p e rio d s o f c o n tin u a l p a in , a n d disability re n d e r p a tie n ts less a ble to c o p e w ith th e ir disease. an e d u c a tio n p ro g ra m m e is e ssential as th e p a tie n t u sually feels a n ta g o n istic a n d resentful. functioning o f the patient t h e p a tie n t is c arefully q u e stio n e d a b o u t his a b ility to cope in th e w ork, hom e a n d leisure situ a tio n s, e.g. a b ility to c o p e w ith physical re q u ire m e n ts a t w o rk , a b ility to cope w ith stairs, get in a n d o u t o f c h airs, bed, cars, etc. individual joint assessment e ac h jo in t is ex am in ed for ten d e rn ess, te m p e ra tu re , sw elling defo rm ity , active ra n g e o f m o v em e n t, forces re q u ire d to c o u n te ra c t d e fo rm in g forces. t o ta l lim b fu n c tio n is carefully n o te d a n d difficulties observed. m uscle strength g r o u p m uscle stre n g th a n d c o m p o n e n t s tre n g th a re b oth assessed, e.g. grip s tre n g th — a b o m a n o m e te r bag is u se d ; q u a d ric e p s se ttin g ability. t r ea tm en t p r o g r a m m e (a) e ducation b o th fam ily a n d p a tie n t m ust be e d u c a te d as to the n a tu re o f th e disease. t h e follow ing to p ic s sh o u ld be discussed: i. d e sc rip tio n o f jo in t stru c tu re . ii. a ffect rh e u m a to id a rth ritis has o n jo in ts . e m phasis th a t th e re is as yet no cure. iii. c o m m o n d e fo rm itie s e.g. flexion o f th e knees. p re ­ ven ta tiv e tre a tm e n t sh o u ld be discussed. iv. r e a s o n for rest, exercise a n d the b a la n ce betw een these. v. if p o ssible films a b o u t th e disease sh o u ld be show n. w o rk in g o n a rh e u m a to id u n it i fo u n d th a t m axim um c o -o p e ra tio n from th e p a tie n ts w as o b ta in e d if the a bove d iscussions w ere h e ld ; also th e p a tie n ts b e nefitted greatly from g ro u p discussion. (b) r e s t r e st is in d ividually p rescribed as a tre a tm e n t in th e acute p hase. i n th e later stages a b a la n ce o f rest a n d exercise is estab lish e d . m a n y p a tie n ts try to k eep as a ctiv e as possible to p re v en t d e fo rm ity a n d loss o f fu n c tio n . a t c e rta in stages in th e disease excess use o f jo in ts k eep th e m in a sta te o f su sta in ed a n d c o n tin u o u s in fla m m a tio n so accelerating d e stru ctio n . l ig h t rem oveable sp lin ts a re w o rn in te rm itte n tly d u rin g day a n d night. i. r est during the acute phase r e st m ea n s te m p o ra ry n o n w eight o r n o n use o f a jo in t, k e eping it in a n o p tim u m p o sitio n o f fu n c tio n . w h e n the r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) page 8 p h y s i o t h e r a p y december, 1967 join ts demonstrate acute sym ptom s total body rest and splinting o f specific jo in ts is valuable resulting in decreased inflam m ation and so increased m obility and function. t otal im m obilisation is not the answer, the patient must ac tiv e l y m ove each joint through as full a range as possible o n c e daily. r est is decreased progressively with improvement and subsidence o f pain. ii. sub acute phase r est periods are interspersed with periods o f physical therapy. the rest splints are worn at night and durmg rest periods. iii. chronic phase r ehabilitation is very active— the patient sets the pace. periods o f strenuous exercise are follow ed by rest. positions o f rest in bed. every patient must lie p r o n e for at least one hour daily to prevent hip flexion contractures. p a tie n t in supine (а) 1 in. plyw ood bed board to prevent flexion o f hip while supine. (б) f o o t board. (c) n o pillow or very sm all p illow under head. (d ) n o pillow under knees. (e) 6 in. blocks under casters to elevate bed— this permits the patient to get in and out o f bed with minimal joint strain. p rone position (а) h ips fully extended. (б) if possible feet at right angles. in a chair. i f possible chairs are raised 4 in. so as to prevent the hips being flexed to an angle greater than 90°. s p l in t in g i f possible aluminium splints or prenyl splints are used— if this is not possible p .o.p . splints serve this function equally. . splinting aiming at: reduction o f pain. prevention o f deformity, im provem ent o f function, correction o f deformity— as. in serial splints. leg splints. extend from m id thigh to m /p join ts o f foot. the knee must not be hyperextended, ankle in the mid position with the foot at right angles. i f the patient dem on­ strates a tendency to external rotation at the hip a rotator bar can be attached to the splint. velcro strapping is used to attach the splints— one strap is mid thigh, one directly over the patella (avoid pressure o n the tibial tubercle as the tibia tends to sublux on the femur due to the laxity o f the cruciate ligaments). arm splints. it has been stated that on e quarter o f patients suffering from rheum atoid arthritis dem onstrate major deformity o f the hands. a lthough types o f deformity are varied there is frequently a wrist flexion deformity with volar subluation o f the ulna, radial deviation o f the carpus and ulnar drift o f the fingers. t he wrist being the key joint in functional balance o f the hand. w hen flexion deformity occurs at this level an im balance o f long flexors and extensors to the digits occurs leading to finger deformity. c om m on hand deform ities include flexion deformities o f m .c .p. join ts with volar subluxation, ulnar deviation o f the p .i.p. join ts o f the fingers. a t the level o f the phalanges there m ay be swan neck deformities with hyperextension o f p.i.p. join ts and flexion o f d .i.p . join ts or the boutenniere deformity with hyperextension o f d .i.p . join ts and flexion o f p.i.p. jo in ts— this deformity is caused by the lateral slips o f the e xtensor d igitoru m slipping toward the volar side o f the finger and so acting as flexors. a hand splint m ust therefore be m ade to accom plish the follow ing: i. the wrist in the m id position (the patients com plained o f increased pain if wrist put into any degree o f extension). ii. splint must control radial deviation at the carpus. iii. support heads o f meta-carpals and lim it flexion at m .c .p . join ts to 30°. iv. splint must n o t extend beyond mid shaft o f proximal phalanges so as to allow flexion o f phalanges with support under metacarpal heads. v. ulnar deviation o f the fingers m ust be controlled. vi. velcro strapping is used— one directly over the wrist, one directly over the m .c .p . joints. ex e r c ise th e r a p y m uscle weakness and atrophy is a constant factor. individual attention is given to m uscles and joints. applicable principles passive m ovem ents are never given to a rheum atic joint. static exercise given where severe bon e o n bon e crepitus is present. resistance through range given if pain not to o severe. specific therapy as well as class work should be given. a n exercise routine should be established. class work— should be given with the aim o f putting each jo in t through as full a range as possible. a hand and foot class given daily follow in g wax as well as a general ward class should be given. the ward class should include postural exercises, quadriceps drill, hip exten sion exercises as well as general m obilising and strengthening exercises should be given. these classes were typed out and supervised w hile the patient was in hospital so that a similar routine could be follow ed at hom e. p .n .f . techniques proved m ost valuable when com bined with ice especially in reduction o f pain, spasm and increase in strength. r ou tin e exercises such as straight leg raising using sandbags were taught. r heum atic shoulders and knee joints responded exceptionally well to ice plus static con ­ tractions in various parts o f the range later progressing to m ovem ent through range in all p .n .f . diagonals. p o o l t h e r a py this proved m ost beneficial when individual supervision o f each patient was given. the technique which was m ost successful was dem onstrated to m e by a physiotherapist from bad r agaz in switzerland. n o individual m uscle was exercised but m ovem ent patterns were used. r e-ed ucation o f walking in the p ool post surgery (e.g. after synovectom y, osteotom y, cup replacements in the hip joint) allow ed patients to weight bear correctly and so retrain their pattern o f walking. r esisted walking in the p ool was given to re-educate, strengthen and increase endurance o f the patient. this can be done by giving direct resistance to the patient or by altering the speed o f m ovem ent in the water. r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) december, 1967 p h y s i o t h e r a p y page 9 p a in m axim um increase in m uscle strength is gained when the patient is pain free. each patient responds to each technique o f pain relief differently. (а) d rug therapy: the drugs vary with each group o f physicians supervising the patients therapy. (б) r est: described above. (c) h eat: can be used in either the dry or the m oist form. i did not find any dramatic relief o f pain when using s .w .d . or infra-red. h ow ever patients reported relief o f sym ptom s in the sub acute and chronic stage o f the disease w hen m oist heat was used: wax baths to hands and feet, p ool therapy, hydrocolator packs. in m any patients sym ptom s were aggravated by the application o f heat in any form even in the chronic stage o f the disease. (d ) ice: “in spite o f the fact that som e rheumatoid patients hate the cold weather, their natural prejudices against it have to be tactfully handled when introducing them to the treatment” (k n ott 1964). the application o f ice tow els proved m ost useful in com bination with isom etric exercise in the reduction o f pain in all the large joints. m any patients have obtained relief o f painful hands when immersing them in ice for few short repeated applications. n o t only was ice beneficial in reduction o f pain but g ood fast results were obtained when joint deformity was present the flexion contractures o f th e knees— ice packs applied to the hamstring group com bined with “ hold relax” to the contracted group with strength­ ening to the quadriceps mechanism. (e) exercise: described above. ( / ) w alking aids: w alking frames used in patients who are severely disabled. c anadian gutter crutches (fore­ arm support crutches) are preferable to canes for m any reasons: a patient invariably demonstrates pathology at the wrist— with the continuou s flexion and exten sion required at the wrist when using canes an increase in pain results as well as possible rupture o f extensor tendons o f the digits. a lso hand deformity does n o t allow patient to use cane efficiently. gutter crutches tend, if correct height to prevent flexion at the hip when walking. r e h a b il it a t io n a ll activities o f daily living m ust be checked and if necessary a dom iciliary visit sh ould be m ade to check height o f stairs etc. the patient must therefore be taught the easiest manner o f clim bing these stairs and the necessary adjustments to hom e made. discharge o f patients total re-assessment performed. regular reports to the arthritic clinic m ust be m ade. all patients m ust continue their hom e programme o f exercise. c o n c l u s io n in this paper i have only discussed the very general treatment o f the arthritic patient, also there has been no discussion o f the post-surgical approach to these patients. a physiotherapist can m ake a vital contribution to the habilitation o f the rheum atoid patient, especially when such therapy is supplem ented and com plem ented by all treating the patient. a c k n o w l e d g e m e n t (а) support and encouragem ent from mrs. m . kirschbaum, lecturer, departm ent o f physiotherapy o f the university o f the w itwatersrand. (б) the co-operation and instruction given to m e by the r heum atic d iseases u n it expecially from dr. d . g ordon and d r. j. stein. b i b l i o g r a p h y 1. b l a n d , j. h . ‘arthritis— m edical treatment and hom e care.’ 2 . b r o w n , m . e . ‘r h eum atoid arthritic h and s,’ american journal o ccupational therapy, 1966, v ol. x x . 3. e y r i n g , e . j. a rth ritis and rheumatism, 1963, v ol. 6. 4. h o l l a n d , arthritis. 5 . h a i n e s , j.‘a survey o f recent developm ent in cold therapy,’ p hysiotherapy, july, 1967. 6. r heum atic d iseases, am erican journal o f occupational therapy, sept., 1965. 7. r o b b , j. and r o s e , b. s. ‘r h eum atoid arthritis and m aternal d epravation,’ british m e d ica l journal o f m e dical psychology, 1965. 8 . s h a l i t , i. and d e c k e c k e r , j. lancet, 16 jan., 1965. a. c. miller & co. orthopaedic mechanicians m anufacturers and suppliers of: o r t h o p a e d ic a p p l ia n c e s, a r t if ic ia l lim b s, t r u s s e s , s u r g ic a l co rsets, u r in a l s , a r c h su pp o r t s, c o l o st o m y belts, e l a st ic st o c k in g s, a n k l e g u a r d s , w r ist g u a r d s , elbo w g u a r d s , k n e e g u a r d s , l ig h t d u r a l c r u t c h e s f o r c h il d r e n , w o o d e n c r u t c h e s , a n d m e t a l elbo w c r u t c h e s. • phone 23-2496 p.o. box 3412 312 bree street, johannesburg r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) key: cord-0029554-cm1j3vc9 authors: chen, ying; wang, yonglin; jiang, xianfang; cai, jinhong; chen, yuting; huang, hanji; yang, yuan; zheng, li; zhao, jinmin; gao, ming title: dimethylamino group modified polydopamine nanoparticles with positive charges to scavenge cell-free dna for rheumatoid arthritis therapy date: 2022-03-28 journal: bioact mater doi: 10.1016/j.bioactmat.2022.03.028 sha: 331c2e6c40c393df9eab7fb6785086594d7ff852 doc_id: 29554 cord_uid: cm1j3vc9 excessive cell-free dna (cfdna) released by damaged or apoptotic cells can cause inflammation, impacting the progression of rheumatoid arthritis (ra). cfdna scavengers, such as cationic nanoparticles (nps), have been demonstrated as an efficient strategy for treating ra. however, most scavengers are limited by unfavorable biocompatibility and poor scavenging efficacy. herein, by exploiting the favorable biocompatibility, biodegradability and bioadhesion of polydopamine (p), we modified p with dimethylamino groups to form altered charged dps to bind negatively charged cfdna for ra therapy. results showed that dps endowed with superior binding affinity of cfdna and little cytotoxicity, which effectively inhibited lipopolysaccharide (lps) stimulated inflammation in vitro, resulting in the relief of joint swelling, synovial hyperplasia and cartilage destruction in ra rats. significantly, dps with higher ds of bis dimethylamino group exhibited higher positive charge density and stronger cfdna binding affinity, leading to excellent ra therapeutic effect among all of the treated groups, which was even close to normal rats. these finding provides a novel strategy for the treatment of cfdna-associated diseases. cell-free dna (cfdna) is a dna fragment that mainly derived from nuclear and mitochondrial of apoptosis or necrosis cell [1] . increasing evidences suggested that cfdna played an active role in inflammatory diseases. it also tightly participated in immune responses in physiological conditions and lead to sterile inflammation in pathological conditions [2, 3] . specifically, in rheumatoid arthritis (ra) patients, the levels of cfdna in synovial fluid (sf) and peripheral blood were highly elevated [4] . genetic sequencing also identified that cfdna in sf of ra was rich in hypomethylated cytosine-phosphate-guanosine (cpg) and could cause severe inflammation both in vitro and in vivo through upregulation of tnf-α expression [5, 6] . therefore, cfdna is crucial for ra process. accumulating evidences indicated that cfdna was one of the main reasons for ra occurrence [7] [8] [9] . cfdna originated from mitochondrial peer review under responsibility of keai communications co., ltd. dna, similar to bacterial dna and would elicit high levels of oxidative damage. it could form complexes by binding with ll-37 [7] , hmgb [10, 11] , auto-ig [12] , or other proteins, leading to autoimmune response and driving the progression of variety inflammatory diseases [13] . stimulation of tlr9 would also activate nf-kb, which consequentially resulted in the production of inflammatory factors like tnf-α and il-6 [14, 15] . lack of dnase ii also digested the chromosomal dna from apoptotic cells so that mice with dnase ii deletion developed a chronic polyarthritis similar to human ra. in these mice, tnf-α was upregulated in the bone marrow, and rheumatoid factors exhibited high levels in serum [16, 17] . the above studies suggested that cfdna could be a potential target for ra. scavenging cfdna offers a new modality of treating ra. although it was difficult to specifically clear cfdna through the interaction of structural or complementary sequences [18] , the anions of cfdna were easy to be recognized by cationic polymers, which had been used as therapeutic targets recently [19] . different charge density of nucleic acid-binding nanoparticles (nabns) were used as cfdna scavengers, whose binding affinity increased with their charge density. as a result, nabns had strong attenuation capacity for cfdna derived pro-inflammation in vitro and in vivo [20, 21] . cationic nanoparticles (nps) could scavenge cfdna from patients and further block the activation of primary fibroblast-like synoviocytes and sf monocytes. these cfdna scavengers further relieved ra symptoms and achieved partial mobility recovery in a murine ra model [9, 22] . unfortunately, the above cationic nps were limited by high cytotoxicity for further application. recently, polydopamine (pda), a mussel-derived polymer, with minimal toxicity and favorable biodegradability has attracted tremendous attentions in biomedical application [23] . pda and its derivatives also had potential scavenging effects on excessive reactive oxidative stress (ros), further alleviating and treating ros derived diseases such as ischemic stroke, parkinson and osteoarthritis due to its abundant reductive groups like catechol and imine [24] [25] [26] [27] . pdas tended to target dopamine receptors, which were highly expressed in immune diseases including ra, and functions as immune system regulator as well [28] . significantly, it had unique property of bioadhesion similar to mussel-adhesive proteins that displayed high adhesion to most substrates [29] . pda-based therapy had been confirmed as a favorable gene carrier, which could effectively adsorb dna on the surface via polyvalent metal-mediated coordination [30, 31] . besides, pda reacted towards amino or thiol groups via schiff's base reaction and michael addition, which offered a simple way to modify the organic or inorganic materials. thus, pda nps had been conjugated with polyethyleneimines (pei) of different molecular weight (mw) to form efficient gene transfection agents, which had abundant positive charges to efficiently bind with anionic plasmid dna (pdna) to form nanocomplexes with low cytotoxicity [32] . furthermore, modified with pda, a positively charged magnetic nanoparticle (pda@fe 3 o 4 ) exhibited high adsorption capacity of dna [33] . therefore, pda based nanocomposites with high positively charge density can act as excellent nanocarriers for gene binding. in our study, to scavenge cfdna in the treatment of ra, the positively charged dps modified by 3-dimethylamino-1-propylamine (m) or 3, 3iminobis (n, n-dimethylaminopropyl) (b) were synthesized (fig. 1) . the abundant catechol and imine groups of p were firstly activated by 1, 1 ′ -carbonyldiimidazole (cdi) followed by the reaction with amino groups of m or b to form dimethylamino group modified p (dp-m or dp-b) with varied charge density to bind negatively charged cfdna. this novel cfdna scavenger may exhibit strong binding affinity of cfdna, inhibit the cfdna induced inflammation with high efficiency, and consequently promote the ra therapeutic efficacy. the positively charged pda nps may provide a novel strategy for ra prevention and therapy. 3-hydroxytyramine hydrochloride (da, ≥ 98%) was commercially obtained from aladdin (china). dimethyl sulfoxide (dmso, ≥ 99.9%), 1, 1 ′ -carbonyldiimidazole (cdi, ≥ 97.0%), 3-dimethylamino-1-propylamine (m, ≥ 99%), 3, 3-iminobis (n, n-dimethylaminopropyl) (b, ≥ 97%) were purchased from macklin (china). ammonium hydroxide and fig. 1 . schematic illustration of in vivo ra therapy effect of dimethylamino group (3-dimethylamino-1-propylamine (m) or 3, 3-iminobis (n, n-dimethylaminopropyl) (b)) modified polydopamine (dps). the dps were intra articular (ia) injected into the knee joint of cia rat and strongly bound with cfdna to lower the expression of inflammatory factors: mmp-13, tnf-α, il-6 and il-1β for ra therapy. ethanol was obtained from jinshan chemical reagent co., ltd (china) and sinopharm chemical reagent co., ltd (china) respectively. all reagents were used directly without further purification. p was synthesized according to previous literature [34, 35] , namely that da polymerized via oxidation under an ethanol-alkaline aqueous solution. firstly, ammonium hydroxide (3 ml), ethanol (40 ml), deionized (di) water (90 ml) were mixed and da (500 mg) was added into the solution with a dropwised manner. the mixture reacted for 24 h with stirring. next, the solution was centrifuged for 10 min at 12000 rpm with a centrifuge (eppendorf 5810r, germany) and then washed with di water for 3 times. finally, the p was obtained by freeze-drying in a freeze dryer and stored at 4 • c. p (0.1 g) was added into dimethyl sulfoxide (dmso, 20 ml, ≥ 99.9%) to obtain the mixed solution. and 0.2 g cdi was then added and stirred for 24 h. next, to obtain four types of modified p, 0.5 or 1 ml of m and 0.55 or 1.1 ml b was separately added into the mixture before stirred for another 24 h. the reacted mixtures were centrifuged for 10 min at 12000 rpm and washed for 3 times with di water. finally, the modified p was obtained through freeze-drying, named as m modified p (dp-m (l) and dp-m (h) ) and b modified p (dp-b (l) and dp-b (h) ) respectively (table s1 ). these pda nps (including p, and dps) were respectively characterized by using fourier transform infrared spectrometer (ftir, iraffinity-1s, shimadzu, japan), raman spectroscopy (jasco nrs-4500, japan), x-ray photoelectron spectroscopy (xps, thermo, uk) and x-ray diffraction (xrd, miniflex 600, japan). and the morphology and distribution of these nps were investigated by scanning electron microscope (sem, hitachi, japan) coupled with energy dispersive x-ray spectroscopy (eds), transmission electron microscopy (tem, hitachi, japan) and atomic force microscopy (afm, bruker, us) respectively. the zeta potential of pda nps in pbs buffer was characterized by nano-zs (malvern panalytical, china) as well. the dna binding capacity of p and dps were detected by agarose gel electrophoresis, where cpg 1826 (tccatgacgttcctgacgtt, gen-script, china) served as dna. details were: pda nps (100 μg) were added into cpg 1826 solution (1 μm in pbs, 2 ml) and incubated at room temperature (2 h). then, the mixture (10 μl) was loaded on 2% agarose gel and the gel electrophoresis was implemented at 100 v for 40 min. pure cpg 1826 was used as the control. and then the agarose gel was pre-stained with superred/gelred and imaged by ultra-sensitive multifunctional imager (amersham imager 6000). besides, the dna ladder (50-1500 bp, ameko life sciences, china) was applied as the dna template to investigate the binding ability of dps. briefly, dna ladder (5 μl) was mixed with different pda nps (50 μg/ml, 10 μl) or dp-b (h) with different concentration (0, 10, 20, 50 and 100 μg/ml, 10 μl) for 2 h before implementing dna gel electrophoresis (3% agarose gel, 100 v, 40 min). furthermore, to confirm their dna binding ability not affected by serum, the dna ladder (5 μl) was initially mixed with 5 μl serum from sd rats for 10 min, and then respectively mixed with pda nps (50 μg/ml, 10 μl) for 2 h. finally, the supernatant after centrifuge (500 ppm, 5 min) was collected for dna gel electrophoresis (3% agarose gel, 100 v, 40 min). raw 264.7 cells were commercially purchased from american type culture collection (atcc, usa). the chondrocytes were extracted from the knee joint of sprague-dawley (sd) rats (male, 3-5 day) and collected by centrifugation before cultured in dulbecco modified eagle medium (dmem, gibco, usa) containing 10% (v/v) fetal bovine serum (fbs, gibco, usa) and 1% penicillin/streptomycin (solarbio, china). and the osteoblasts were extracted from the frontal and parietal bone of sd rats (male, 3-5 day). after cut into pieces, the bones were digested in trypsin (0.25%, biosharp, china) at 37 • c for 10 min, and the precipitates were collected after centrifugation at 1000 rpm for 5 min and treated with collagenase ii (biofox, china) for further digestion. all cells were respectively cultured in dulbecco modified eagle medium. the cultured medium was replaced every 2 days. cells were passaged when reaching 85%-90% and collected for further research. the cell cytotoxicity of pda nps was evaluated by cell counting kit-8 (cck-8, dojindo, japan) assay, implemented as following: raw264.7, chondrocytes and osteoblast were respectively seeded in 96-well plates with the density of 5000 cells per well. and the cultured medium was replaced with fresh medium containing dps with different concentrations (0, 5, 10, 20, 50, 100 and 200 μg/ml). after incubation (24 h), the cells were washed for 3 times with pbs buffer and incubated in 100 μl medium (10% cck-8). the absorbance was recorded at 450 nm by spectrophotometer (thermo fisher, usa). and the viability of cells was calculated by comparing with control group (only fresh medium) and the experiment was repeated in triplicates. to establish the inflammatory cell model, raw264.7 cells (15 × 10 4 cells/ml) were stimulated with lipopolysaccharide (1 μg/ml) (lps, solarbio, china) following previously reported work, which also confirmed that lps stimulation could increase the amount of cfdna [36] . after 24 h' incubation, the stimulated cells were further co-cultured with 50 μg/ml pda nps for another 24 h. finally, the treated cells were used for further researches including live/dead staining, qrt-pcr and immunofluorescent staining. the live/dead staining was implemented using a calcein-am/pi assay kit (sigma, usa). briefly, pda nps treated raw264.7 cells were incubated with calcein and propidium iodide (pi) for 5 min in the darkness. after pbs buffer washing, the cells were imaged by microscope (olympus bx53, tokyo, japan). the gene expression levels of inflammatory factor including tnf-α, il-1β, inos and il-6 were separately quantified by qrt-pcr. total rna of raw264.7 cells was isolated by rna extraction kit (magen, china) following the protocol. the qrt-pcr reaction was conducted by the lightcycler® system (roche, switzerland). all primer sequences of these genes were presented in table s2 . the levels of relative gene expression were analyzed by the 2 − δδct method and normalized with glyceraldehyde-3-phosphate dehydrogenase (gapdh). besides, the supernatant of lps induced raw264.7 was collected to investigate the expression of inflammatory factors (il-6 and tnf-α) by elisa kit following the instruction of manufacturer [37, 38] . to further verify the change of inflammatory factors of raw264.7 cells after treated with pda nps, the protein expression of inos, tnf-α and mmp-13 was identified by immunofluorescent staining. at the beginning, the cells were firstly fixed with paraformaldehyde solution (4%, biosharp, china) for 10 min. after being treated with 3% h 2 o 2 for 15 min, the cells were blocked with goat serum to avoid nonspecific binding and incubated with primary antibody of inos, tnf-α or mmp-13 (1: 200 dilution) (boster, china) separately overnight at 4 • c. and then, the cells were added with fitc-anti-rabbit igg (boster, china) for incubation (1 h) in the dark before the nuclei was stained with dapi (5 min). the pictures were finally imaged by fluorescence microscope (olympus, japan). raw264.7 cells were initially cultured in 6-well plate with the density of 15 × 10 4 cells per well. then, 2 μl cy5-cpg 1826 (cy5-tccatgacgttcctgacgtt, 1 μm, genscript, china) was added into the cultured medium and incubated for another 4 h [39, 40] . the excessive cy5-cpg 1826 was removed by washing with pbs for 3 times before the addition of fitc labeled pda nps (50 μg/ml) or not. after incubation (24 h), the cells were stained with lyso-tracker red (beyotime, china) and dapi for observation by confocal microscope (leica sp8, germany). the mean fluorescent intensity of cy5-cpg 1826 was analyzed by image j. and the colocalization ratio of cy5-cpg 1826 and fitc-pda nps was calculated by the ratio of colocalization area (white) and foreground area (green and purple). the in vivo imaging systems (ivis, lumina lt, us) were applied to evaluate the in vivo retention and biodistribution of pda nps [41] . at the beginning, 20 mg dp-b (h) were dispersed in 100 μl dmso with stirring. later, 20 μl cyanine 5 nhs ester (cy5, 50 mg/ml, lumiprobe) was added into the solution followed by the addition of triethylamine (50 μl). the mixture reacted for 24 h before centrifugation and washing with ethanol for 3 times. after vacuum drying, the sample (cy5-dp-b (h) ) was isolated for further experiment. for in vivo degradation, cy5-dp-b (h) (50 μg/ml, 20 μl) was intra-articular (ia) injected into sd rat (180-200 g). the images were taken at predetermined time points (0, 4, 8, 24, 48, 72, 96 and 120 h) with the excitation and emission wavelength at 646 nm and 662 nm. after 120 h, the major organs including heart, liver, spleen, lung and kidney from sd rats were taken out for ivis imaging as well. five ~ six weeks' female sd rats (180-200 g) were chosen to implement the experiment in vivo. the in vivo studies were approved by the animal ethics committee of guangxi medical university with the guide of care and use of laboratory animals. the rheumatoid arthritis animal (cia) model was established by tail injection of type ii collagen (col2) solutions (initial immunization: 50 μl col2 and 50 μl complete freund adjuvant (cfa, 2 mg/ml), and booster immunization: 50 μl col2 and 50 μl freund incomplete adjuvant (fia) after one week, totally 100 μl per sd rat for every immunization) according to previous work [42] . at the 13th day, ra rats were randomly divided into four groups: ra (only pbs buffer injection), p (p injection), dp-m (h) (dp-m (h) injection), and dp-b (h) (dp-b (h) injection). all nps were ia injected into knee joints with a dose of 2.5 mg/kg every 3 day (totally 4 times till sacrificed). sd rats without any treatments were used as normal group. the swelling degree of joints was evaluated every 3-4 day (day 14, 17, 21, 24 and 27) from the onset of ra (day 13) until the rats were killed. the swelling of joints was evaluated based on the diameter of rats' ankle with vernier caliper (guanglu, china). and the clinical scores of hind paw of each rats were calculated according to the previous evaluation method [43] , where score 0, 1, 2, 3 and 4 corresponded to no swelling, mild swelling, mild swelling, moderate swelling and severe swelling respectively. the expression levels of inflammatory factors in serum and sf were investigated to evaluate the treatment effect. the whole blood was collected from all rats and treated with heparin for anticoagulation before being centrifuged at 1500 rpm for 10 min. after the plasma was separated, the inflammatory factors (il-6, mmp-13, tnf-α and il-1β) of serum were measured using the commercially available elisa kit (meimian, china) by the instruction of manufacturer. the absorbance at 450 nm was recorded by the microplate reader. similarly, the sf was collected from articular cavity after injecting 50 μl pbs buffer and further measured the expression of inflammatory factors (il-6, mmp-13 and tnf-α) by elisa. rats were sacrificed after treated with pda nps for 2 weeks. the ankles and knees were fixed with paraformaldehyde (4%) for 2 days. then, all samples were incubated in 10% ethylenediaminetetraacetic acid (edta, solarbio, china) buffer solution for decalcification using ultrasonic decalcifying unit (use 33, medite, germany) for 4 weeks. later, the tissues were embedded in paraffin and sliced into 5 μm thickness of tissue sections. the slides were finally stained with hematoxylin-eosin (he, solarbio, china) or safranin-o fast green (solarbio, china) before the images were obtained using a microscope (olympus bx53, japan). the immunohistochemical staining was applied to identify the expression of il-6, mmp-13 and tnf-α in the cartilage. in brief, the knee tissue sections were incubated with 3% h 2 o 2 for 10 min before blocked by goat serum for 30 min. subsequently, tissue sections were separately incubated with primary antibody of tnf-α, mmp-13 or il-6 (1: 200 dilution, boster, china) for 60 min with humidified chamber (37 • c). after pbs buffer washing for 3 times, the sections were incubated with secondary antibody (biotin-labeled goat anti-mouse/rabbit igg (zsgb bio, china)) for another 30 min. later, samples were stained with 3, 3 ′diaminobenzidine tetrahydrochloride (dab, boster, china) before counterstained with hematoxylin (solarbio, china). the sections were finally sealed with neutral resin before imaging (olympus bx53, tokyo, japan). the bone damage of knee joints was investigated by using ex-vivo micro-computed tomography (μct-100, swiss, scanco medical ag) and scanned with the resolution of 15 μm (70 kv and 200 μa) after fixation (4% paraformaldehyde). then, the dataset was reconstructed to obtain 3d images of joints and the bone mineral density (bmd) was also measured using inveon research workplace. and the body weight of each rats was weighed by animal weight balance (shanghai yu yan, china) during the ra therapy. meanwhile, the cfdna was extracted from peripheral blood sample of rats based on cfdna extraction kit (ezup, sangon biotech, shanghai) by following the manufacture's instruction. furthermore, the major organs including heart, liver, spleen, lung and kidney were fixed with 4% paraformaldehyde and cut into 2 μm thickness after h&e staining for histological evaluation. all experiments were carried out at least in triplicates. and the data were analyzed by spss 20.0 (spss inc., chicago, illinois, usa) and presented as mean ± standard deviation. the differences between two groups were analyzed with one-way anova followed by game howell analysis or least significant difference (lsd) analysis. p was prepared through polymerization of da under alkaline conditions via oxidative self-polymerization. next, four types of dimethylamino group modified p (dps), referred to as dp-m (l) , dp-m (h) , dp-b (l) , and dp-b (h) were successfully fabricated via modification with different concentrations of m or b ( fig. 2a) . table s3 illustrated the recipe of preparation of dps. after calculation, the degree of substitution (ds) was 1.2% and 7.6% for dp-m (l) and dp-m (h) with m modification, and 1.8% and 7.5% for dp-b (l) and dp-b (h) with b modification respectively. in fig. 2b , it displayed the zeta potential of pda nps before and after modification. the zeta potential of p was − 21.76 mv, which respectively increased to − 14.36 and + 1.35 mv for dp-m (l) and dp-m (h) after m modification. similarly, the zeta potential was − 12.86 mv for dp-b (l) and it increased to +10.33 mv for dp-b (h) . after interacted with m or b, the introduction of positively charged dimethylamino groups significantly improved the positive charge density of p. significantly, high ds of bis dimethylamino groups (dp-b (h) ) contributed to high positive charge density compared to dp-b (l) with low ds of bis dimethylamino groups and dp-m (h) with the same high ds of mono dimethylamino groups. meanwhile, ftir was performed to characterize pda nps (fig. 2c ). the wavelength bands observed at 3350-3450 cm − 1 corresponded to the ν (o-h) and ν (n-h) functions, which represented the phenolic hydroxyl and amino groups of p. and the characteristic bands of p were also obviously observed for all modified p due to the introduction of amine groups (n-h and n-c) . on the other hand, the peak at 1708 cm − 1 (c--o) was observed for dp-m (l) , dp-m (h) , dp-b (l) and dp-b (h) originated from cdi activation during the reaction, indicating the successful modification of m or b. besides, pda nps was also investigated by xps. as illustrated in figs. s1 and c, n, o elements were obviously observed for p and dps. for detailed c1s, n1s and o1s spectrum, no significant difference existed for p before and after modification (fig. s2) . the crystal structure of pda nps was characterized by xrd (fig. 2d ). as indicated in xrd patterns, a narrow and strong peak was observed at 2θ = 22.9 • , caused by the existence of amorphous carbon of biochar containing the aromatic carbon sheets, also a characteristic peak of p. no significant differences were observed for xrd patterns of all nps even after modification. the molecular structure of pda nps was also tested by raman spectroscopy. from fig. s3 , the apparent raman shift (1000-1750 cm − 1 ) was observed for p while the raman shift became weak after modification. significantly, the new shift existed at 250-750 cm − 1 for dps, more obvious observed with the increase of ds of dimethylamino groups. the morphology of pda nps was observed by tem and afm. as indicated in fig. 2e , p and dps remained the spherical shape with a size of 120-160 nm. besides, the height of all pda nps was around 100 nm by afm (fig. 2f ). in addition, pda nps was also investigated by sem-eds. as shown in fig. s4 , pda nps maintained the spherical shape even from the above, it confirmed that the morphology, molecular composition and crystal structure of dps was similar to the pure p, indicating that the introduction of dimethylamino groups did not affect the structural characteristics of pda nps but only contribute to the adjustment of positive charge density. the cytotoxicity on raw 264.7, chondrocytes and osteoblasts were detected through cck-8 assay. as shown in i of fig. 3a , within the concentration range of 0-50 μg/ml, the cytotoxicity of all nps was minimum with the cell viability above 95%. however, when the concentration was above 100 μg/ml, the cell viability of dps started to decrease, indicating that cells would suffer a certain of cytotoxicity. but the viability was still above 60%, attributed to the enrichment of positive charges. similarly, the chondrocytes displayed the same tendency of cell viability, with a little cytotoxicity at 200 μg/ml (ii of fig. 3a) . however, as indicated in iii of fig. 3c , almost no cytotoxicity existed for osteoblasts, with the cell viability above 95% during the concentration ranges from 0 to 200 μg/ml. yan et al. [13] had also found that although higher positive charge of cationic cfdna scavengers could be conducive to dna binding efficiency and showed longer retention time in psoriatic lesions, improving therapeutic outcome, it also increased cell cytotoxicity at the same time. therefore, 50 μg/ml was chosen for subsequent experiments. additionally, the live/dead staining was also applied to investigate the protection capacity of pda nps. compared to normal group, the quantity of live cells obviously decreased and a lot of dead cells were observed for control group (lps induced cells) with the live/dead ratio of 8.6%. however, the quantity of live cells increased and the number of dead cell decreased after pda nps (50 μg/ml) treatment so that the live/ dead ratio was above 80% for all other groups ( fig. 3b and c) . it suggested that pda nps exhibited almost no cytotoxicity and also had the protection capacity of lps induced cells by preventing inflammation if the concentration was below 100 μg/ml. the anti-inflammatory capacity of pda nps was evaluated by qrt-pcr via the inflammatory factors' expression levels. as illustrated in fig. 4a , lps stimulation remarkably increased the expression of inos, tnf-α, il-1β and il-6, slightly decreased by p treatment. it had already demonstrated that p could lower the expression of inflammatory factors. however, modified p displayed noticeable inhibition effects on lps induced inflammation, especially for dp-b (h) with the lowest expression of inflammatory factors among the groups, such as inos and tnf-α almost close to normal group. after calculation, the expression of inos, tnf-α, il-1β and il6 decreased significantly by 63.9%, 34.1%, 59.6% and 70.4% respectively for dp-b (h) compared to control group. meanwhile, the inflammatory factors expression of supernatant from lps stimulated cells was also evaluated by elisa. as shown in fig. s5 , lps stimulation obviously increased the expression of il-6 (a) and tnf-α (b) compared to normal macrophages. pure p could slightly decrease the expression of il-6 and tnf-α while dps significantly lower their expressions. among the dps, dp-b (h) with high ds of bis dimethylamino group presented the lowest expression of both inflammatory factors, close to normal macrophages. besides, the immunofluorescence staining of inflammatory factors: inos, tnf-α and mmp-13 was also illustrated in fig. 4b . for normal group, no fluorescent intensity was observed while the fluorescent intensity significantly increased after lps stimulation. especially, the fluorescent intensity of inflammatory factors decreased in p and dps. in accordance with the results of qrt-pcr, dp-b (h) manifested quite evident anti-inflammatory effects than the other nps with the significantly decreased expression of inos, tnf-α and mmp-13. after statistical analysis, the inos expression was 49% after lps stimulation and it decreased to 38% for p and below 26% for dps, which presented the same tendency as the expression of tnf-α and mmp-13. among the modified p, the tendency for lowering the inflammatory factors was dp-m (l) < dp-b (l) < dp-m (h) < dp-b (h) . significantly, the expression of tnfα and mmp-13 was 8% and 7% for dp-b (h) , infinitely close to normal group. as p itself displayed partly influence on the release of inflammatory factors from macrophages, the enhanced anti-inflammatory capacity of dps attributed to the rise of charge density, especially when the charge was converted from negative to positive, which facilitated the cfdna binding and scavenging [44] . in the meantime, dp-b (h) with the high ds of bis dimethylamino group performed the strongest binding affinity of cfdna, further most effectively reducing the expression of inflammatory factors. (bar = 100 μm) ("*" symbol compared with the control group, ****p < 0.0001). dna binding affinity of pda nps was initially evaluated through agarose gel electrophoresis. as shown in fig. 5a , the bands of p were similar to that of free cpg 1826, which indicated that there was no loss of dna after p treatment and almost no dna binding affinity of p. however, the bands became weak for dps with different ds, especially for dp-m (h) and dp-b (h) , suggesting that positively charged pda nps could effectively interact with negatively charged dna and prevented the migration of dna during gel electrophoresis. after statistic calculation, for 2 nmol cpg, the binding capacity of 100 μg p was 12.99% while it became 42.32%, 94.22%, 37.05% and 97.63% for dp-m (l), dp-b (l), dp-m (h) and dp-b (h) successively (fig. 5b) . meanwhile, dna ladder (50-1500 bp) was also applied as the dna template to investigate the dna binding ability. p had no binding ability of dna with the dna bands close to the dna ladder. however, the bands from (50-250 bp) obviously disappeared for dp-m (h) while most of bands disappeared in the whole range with the same amount (fig. s6a) . as shown in fig. s6b , the bands became lighter with the increase of dp-b (h) amount. when the concentration was 100 μg/ml, all bands disappeared. high ds of bis dimethylamino group was helpful to high dna binding ability. besides, it was expected that dna binding ability still maintained efficient even in the serum condition. compared to dna ladder without treatment, the bands of dna ladder changed a little after p incubation in the existence of serum, with darker bands observed at 550-600 bp. however, dps obviously decreased the intensity of bands. especially, the lighter bands (around 50-200 bp) was apparently observed for dp-m (h) and dp-b (h) (fig. s7) . the positively charged dps possessed strong dna binding ability, even in the existence of serum. and the binding ability was enhanced with the increase of positive charge density, contributed by the high ds of bis dimethylamino groups. furthermore, the dna binding affinity was also investigated by colocalization immunostaining of cpg 1826 and pda nps. cpg 1826 and pda nps were labeled with cy5 (purple) and fitc (green) respectively. in fig. 5c , it displayed that p was dispersed in the nucleus, lysosome and cytoplasm but not binding to cpg 1826, whereas no colocalization area (white area) was observed. however, the white area was observed for dp-m (l) and dp-b (l). most significantly, there were much more white areas for dp-m (h) and dp-b (h) . the overlay intensity of cy5 and fitc, corresponding to the colocalization of cpg 1826 and pda nps, was illustrated in fig. 5d . after statistical analysis, the ratio of cellular uptake was 58% for dp-b (h) and 43% for dp-m (h) , almost twice more than that of dp-b (l) (31%) and dp-m (l) (22%) while it was only 3% for p, indicating that the modified p could efficiently bind dna and high positive charge density always contributed to strong binding affinity. the above results also demonstrated that the positively charged dps exhibited favorable dna binding affinity. the in vivo degradation of pda nps was evaluated by ivis imaging. for unmodified dp-b (h) , almost no fluorescent intensity was observed ("*" symbol compared with control group, **p < 0.01, ***p < 0.001 and ****p < 0.0001, and " # " symbol compared between groups, # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001). from the beginning to the end. slightly intensity appeared in the ankle joint due to the noise of ivis (fig. s8a) . however, as illustrated in fig. s8b , the fluorescent intensity was obviously observed in the right knee joint of sd rat after cy5-dp-b (h) injection. the fluorescent intensity gradually decreased versus time. after 120 h, the fluorescent intensity almost disappeared. similarly, no fluorescent intensity existed in other major organs except for some noise observed in left knee or ankle joints. besides, the major organs of rats were also taken out for ivis. no fluorescent intensity was observed for all organs, indicating no cytotoxicity for organs (fig. s9) . from the above, pda nps presented a certain of retention time inside the articular cavity and gradually degraded versus time in vivo. therefore, pda nps possessed favorable biocompatibility and showed great potentials in clinic application. as dp could effectively bind to cfdna and further inhibit the lps stimulated inflammation of raw 264.7 cells, the therapeutic effects in cia model were evaluated through analysis of the remission of symptoms and protection of damaged cartilage and bone. fig. 6a illustrated ("*" symbol compared with ra group, **p < 0.01, ***p < 0.001 and ****p < 0.0001, and " # " symbol compared between groups, # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001). the establishment of cia model and experimental schedule of ra therapy. as known to all, the clinical findings of ra affected joints were painful and swollen joints due to the high levels of inflammatory cytokines and accumulation of excessive sf [45] . under microscope, ra was characterized by synovial inflammation and hyperplasia with pannus formation, causing bone and cartilage destruction [46] . in synovial tissues, lymphocytes and synoviocytes produced excessive inflammatory cytokines, such as il-6, il-1β and tnf-α, leading to synovitis [47] . furthermore, cytokine stimulated synoviocytes secreted mmp and receptor activator of nuclear factor kappa b ligand (rankl) into sf and consequently lead to cartilage degradation and joint destruction [48] . the pathobiology progression of cia rat model was similar to that in ra patients. not surprisingly, in fig. 6b , cia rats was accompanied by swelling and erythema in ankle and claw joints (ra group) compared to normal rats (normal group). meanwhile, the swelling thickness and erythema had not improved for ra + p group while the joints of ra + dp-m (h) and ra + dp-b (h) groups had obviously repaired. after quantification, the thickness of swelling maintained relatively stable around 8.7-8.9 cm for both ankles of ra group, much larger than those of normal group (around 5.1-5.8 cm). and the swelling thickness of ra + p group presented almost the same level as that of ra group, indicating that pure p had no effects on ra therapy. however, the swelling thickness of both ankles significantly decreased during the progression besides, the images of μct were illustrated in fig. 6d . the smooth surface of joint was observed for normal group where the joint surface became rough for ra group. however, the joint surface became relatively smooth after pda nps treatment. and the bmd was analyzed from μct results. as shown in fig. 6e , the bmd was 780 mg/cm 3 for normal group, which significantly decreased to 700 mg/cm 3 for ra rats. and pure p had no effects on bmd so that the bmd of ra + p group was 706 mg/cm 3 . nevertheless, the bmd became 721 mg/cm 3 and 725 mg/cm 3 for ra + dp-m (h) and ra + dp-b (h) groups respectively. the dps could effectively improve the bmd of ra rats. furthermore, the body weight was also a useful judgement of therapeutic effects. as shown in fig. 6f , the body weight maintained slightly increasing from 218 to 230 g versus time for normal group while it significantly went down below 200 g since day 21. conversely, the body weight did not decrease and maintained around 209-211 g for ra + p group. however, dps obviously increased the body weight, close to that of normal group since day 21. the inflammatory level of serum was also evaluated by elisa. as shown in i of fig. 6g , the gene expression of il-6 was 269.8 for normal rats while it significantly increased to 361.9 for ra group and 330.9 for ra + p group respectively. after the treatment of dps, the gene expression sharply decreased to 322.2 for ra + dp-m (h) group and 300.9 for ra + dp-b (h) group. the similar trend was also observed for the expression of mmp-13, tnf-α and il-1β (ii, iii and iv of fig. 6g ). in the meantime, high local cfdna concentration was detected for ra group (fig. 6h) , confirming that cfdna could act as a biomarker in ra. the concentration of cfdna from serum in normal group was 9 μg/ml, sharply increased to 52 μg/ml for ra group while it became 33 μg/ml after p treatment. significantly, the concentration of cfdna was 24 μg/ ml for ra + dp-m (h) group and 13 μg/ml for ra + dp-b (h) group respectively. besides, the expression levels of il-6, mmp-13 and tnf-α for sf were also investigated by elisa. all inflammatory factors significantly increased for ra group compared to normal group. and it was observed that p could decrease the expression of inflammatory factors to a certain extent. however, dps obviously reduced their expression levels, especially for dp-b (h) with the expression levels close to normal group (fig. 6i) . the bone erosion caused by synovial cavity inflammation became worse and worse during the progression of ra. the histological results of knee joints were illustrated in fig. 7 . as shown in h&e (fig. 7a) and safranin o (fig. 7b) images, the ra + p and ra + dp-m (h) groups presented various proteoglycan retention degrees, bone erosion attenuation, and tidemark integrity promotion, while these characteristics were deterioration over time in ra group. although ra + p and ra + dp-m (h) groups exhibited potential of cartilage repair, their performances were still far away from dp-b (h) group with significantly reduced synovial hyperplasia and cartilage destruction, which was close to the normal knee joints. besides, dps inhibited the inflammation in cia rats as the significantly decreased expression of il-6, mmp-13 and tnf-α. from fig. 7c , the immunohistochemical staining also confirmed the above results, as the protein expression of il-6, tnf-α and mmp-13 in cartilage was significantly inhibited by dp-b (h) treatment, which was evidenced by light and sparsely dying of these markers. however, the positive staining (dark brown) of these proteins was significantly observed on the cartilage surface for ra group and it slightly became light for ra + p group. in the meantime, although apparently decreased expression of il-6 and tnf-α was observed for ra + dp-m (h) group, its expression of mmp-13 still maintained a certain level, not better than that of ra + dp-b (h) group. thus, dps inhibited the progression of ra, especially for dp-b (h) , with highest charge density among them, exhibited an outstanding scavenging of cfdna owing to the high affinity of cfdna binding, which endowed it with superior anti-inflammatory and excellent effects of ra therapy. in the meantime, the histological results of ankle joints were also investigated to evaluate the ra therapy effects (fig. s11 ). similar to knee joints, the obvious hyperplasia and cartilage destruction were observed for the ankle joints of ra group while they almost maintained the same conditions for ra + p group. however, the alleviated hyperplasia and cartilage destruction significantly occurred for ra + dp-m (h) and ra + dp-b (h) groups. specifically for ra + dp-b (h) group, the apparent repaired effects were observed, infinitely close to normal group. in addition, the potential in vivo cytotoxicity of pda nps was still evaluated for biosafety concerns. the histological analysis was applied to investigate in vivo cytotoxicity after ra treatment for 2 weeks. as shown in fig. s12 , the major organs including heart, liver, spleen, lung and kidney maintained the normal state, and no obvious damaged tissue or inflammatory lesions were observed for all groups, indicating that these treatment strategies could act as an excellent ra therapy with minimal cytotoxicity. previous study of a novel polyamidoamine dendrimer (pamam) had discovered the potential advantages of nucleic acid scavenger on mitigation of pro-inflammatory effects [49] . furthermore, a highly cationic surface caused by methylation of the primary amines increased dna scavenging and significantly reduced inflammatory responses induced by cpg [50] . and inflammatory factors, such as mmp-3 was a protease produced by synovial tissues and played the important roles in the progression of joint destruction [51] . thus, the reduction of mmp-3 was a pleasurable marker for ra treatment. therefore, pda nps with huge amount of reductant groups could attenuated cartilage damage and synovial hyperplasia, exhibiting chondro-protection in cia rats, which provided anatomical structure for functional recovery. significantly, dps, especially dp-b (h) , with high cfdna binding affinity due to its high positive charge density, another reason for exhibiting the excellent effects of ra therapy. besides, the negatively charged surface in cartilage also contributed to the high efficacy of ra therapy by cationic scavengers. containing negative glycosaminoglycans (gags), cartilage provided binding affinity sites for the cationic agents, which facilitated the uptake of arthritis drugs and prolonged their retention time [52, 53] . to avoid the intrinsic cytotoxicity of positively charged cfdna scavengers and maintain their inhibitory function is important for designing therapeutic agents for ra therapy. herein, p was modified by m or b with different amount, forming dps including dp-m (l) , dp-m (h) , dp-b (l) and dp-b (h) with different positive charge density. the dps have superior binding affinity of cfdna and little cytotoxicity, which effectively inhibited lps induced inflammation in vitro, resulting in the relief of joint swelling, synovial hyperplasia and cartilage destruction in ra rats. particularly, dp-b (h) with high ds of bis dimethylamino groups exhibited superior cfdna binding affinity, leading to the best therapeutic effects among all groups. these findings suggested the novel dp nps could act as potential agents for ra therapy, which was conducive to design the safe and effective dna scavengers for other cfdna related diseases. ying chen: participated in the, investigation, data processing and, formal analysis, experiment management, participated in the data processing and, formal analysis, participated in revising the manuscript. yonglin wang: participated in the, investigation, data processing and, formal analysis, experiment management, participated in the data processing and, formal analysis. xianfang jiang: participated in the paper writing, participated in the data processing and, formal analysis. jinhong cai: participated in the, investigation, data processing and, formal analysis, experiment management, participated in the data processing and, formal analysis, participated in revising the manuscript. yuting chen: participated in revising the manuscript, all authors have read and agreed to the published version of the paper. hanji huang: participated in revising the manuscript. yuan yang: participated in revising the manuscript. li zheng: participated in the paper writing, acquired the, funding acquisition, participated in the study conception, data curation, formal analysis, and, writingreview & editing, and finalized this paper. jinmin zhao: acquired the, funding acquisition, participated in the study conception, data curation, formal analysis, and, writingreview & editing, and finalized this paper. ming gao: participated in the paper, writingoriginal draft, participated in the data processing and, formal analysis, participated in revising the manuscript, corresponding authors, participated in revising the manuscript, acquired the, funding acquisition, participated in the study conception, data curation, formal analysis, and, writingreview & editing, and finalized this paper. no potential conflicts of interest were disclosed. fig. 7 . histological results of in vivo therapeutic effects. a) representative images of h&e staining on knee joints. b) representative images of safranin o staining on knee joints. c) representative images of immunohistochemical staining (il-6, mmp-3 and tnf-α) on knee joints. the corresponding groups respectively were: normal (normal rats), ra (cia rats), ra + p (cia rats followed by p treatment), ra + dp-m (h) (cia rats followed by dp-m (h) treatment), ra + dp-b (h) (cia rats followed by dp-b (h) treatment). (bar = 100 μm) . guikead19254003). cell-free dna comprises an invivo nucleosome footprint that informs its tissues-of-origin cell-free dna and apoptosis: how dead cells inform about the living the nexus of cfdna and nuclease biology ab0248 circulating cell free dna; a marker to predict the therapeutic response for biological dmards in rheumatoid arthritis ab0032the adenosine deaminase isoforms activity in synovial fluid in patients with different arthritis identification of specific jointinflammatogenic cell-free dna molecules from synovial fluids of patients with rheumatoid arthritis a review of liquid biopsy as a tool to assess epigenetic, cfdna and mirna variability as methotrexate response predictors in patients with rheumatoid arthritis circulating free dna and its emerging role in autoimmune diseases cationic nanoparticle as an inhibitor of cell-free dna-induced inflammation cytosolic sensing of extracellular self-dna transported into monocytes by the antimicrobial peptide ll37 toll-like receptor 9-dependent activation by dnacontaining immune complexes is mediated by hmgb1 and rage targeting inflammation driven by hmgb1 topical cationic hairy particles targeting cell free dna in dermis enhance treatment of psoriasis tlr-9 and bcell antigen receptor triggering of primary b cells from mantle cell lymphoma induce cell proliferation and telomerase activity gc-rich extracellular dna induces oxidative stress, double-strand dna breaks, and dna damage response in human adipose-derived mesenchymal stem cells type i interferon-mediated autoinflammation due to dnase ii deficiency role of interferonγ-producing th1 cells in a murine model of type i interferon-independent autoinflammation resulting from dnase ii deficiency coordinated effects of sequence variation on dna binding, chromatin structure, and transcription nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond) treatment of severe sepsis with nanoparticulate cell-free dna scavengers long-acting nanoparticulate dnase-1 for effective suppression of sars-cov-2-mediated neutrophil activities and cytokine storm neutrophil membrane-coated nanoparticles inhibit synovial inflammation and alleviate joint damage in inflammatory arthritis mussel-inspired polydopamine: a biocompatible and ultrastable coating for nanoparticles in vivo a mussel-inspired persistent ros-scavenging, electroactive, and osteoinductive scaffold based on electrochemical-driven in situ nanoassembly polydopamine nanoparticles as efficient scavengers for reactive oxygen species in periodontal disease selfassembled polydopamine nanoparticles improve treatment in parkinson's disease model mice and suppress dopamine-induced dyskinesia polydopamine nanoparticles as dualtask platform for osteoarthritis therapy: a scavenger for reactive oxygen species and regulator for cellular powerhouses polydopamine nanoparticlemediated dopaminergic immunoregulation in colitis development of bioadhesives from marine mussels bioorthogonal dna adsorption on polydopamine nanoparticles mediated by metal coordination for highly robust sensing in serum and living cells polydopamine and its derivative materials: synthesis and promising applications in energy, environmental, and biomedical fields ultrahigh adsorption capacity of anionic dyes with sharp selectivity through the cationic charged hybrid nanofibrous membranes ph-responsive deoxyribonucleic acid capture/ release by polydopamine functionalized magnetic nanoparticles -sciencedirect mussel-inspired polydopaminepolyethylenimine conjugated nanoparticles as efficient gene delivery vectors for mammalian cells bioinspired polydopamine nanoparticles: synthesis, nanomechanical properties, and efficient pegylation strategy major involvement of bacterial components in rheumatoid arthritis and its accompanying oxidative stress, systemic inflammation and hypercoagulability cell-free dna release by mouse placental explants identification of a novel microrna-141-3p/forkhead box c1/β-catenin axis associated with rheumatoid arthritis synovial fibroblast function in vivo and in vitro cationic block copolymer nanoparticles with tunable dna affinity for treating rheumatoid arthritis topical cationic hairy particles targeting cell free dna in dermis enhance treatment of psoriasis cartilage-targeting peptide-modified dual-drug delivery nanoplatform with nir laser response for osteoarthritis therapy cationic block copolymer nanoparticles with tunable dna affinity for treating rheumatoid arthritis significantly improving the bioefficacy for rheumatoid arthritis with supramolecular nanoformulations topical nanoparticles interfering with the dna-ll37 complex to alleviate psoriatic inflammation in mice and monkeys situ synthesis of natural antioxidase mimics for catalytic anti-inflammatory treatments: rheumatoid arthritis as an example multiomics landscape of synovial fibroblasts in rheumatoid arthritis parsing multiomics landscape of activated synovial fibroblasts highlights drug targets linked to genetic risk of rheumatoid arthritis regulation of osteoclastogenesis by mast cell in rheumatoid arthritis cpg-free plasmids confer reduced inflammation and sustained pulmonary gene expression nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis ab0216overexpression of synovial lining peroxisome proliferator-activated receptor-gamma coactivator-1β involved in joint destruction through upregulating mmp-3 in rheumatoid arthritis cartilage-targeting drug delivery: can electrostatic interactions help? cartilage penetrating cationic peptide carriers for applications in drug delivery to avascular negatively charged tissues this study was financially supported by the national natural science supplementary data to this article can be found online at https://doi. org/10.1016/j.bioactmat.2022.03.028. microsoft word september 2020 bar 876 proof.docx skin september 2020 volume 4 issue 5 copyright 2020 the national society for cutaneous medicine 456 brief articles extensive tinea associated with tofacitinib therapy masquerading as new-onset scle connor r. buechler bs1, elena kurland md2, jesse veenstra md, phd2 1wayne state university school of medicine, detroit, mi 2department of dermatology, henry ford health system, detroit, mi fungi of the genera microsporum, trichophyton, and epidermophyton are frequent colonizers of the epidermis. each is normally readily controlled by the immune system, and therapies that dampen immune surveillance can predispose to fungal colonization. these infections, commonly referred to as tinea or dermatophytosis, can mimic a number of other cutaneous conditions, such as psoriasis, parapsoriasis, atopic dermatitis, mycosis fungoides, lupus and other connective tissue disorders complicating diagnosis in the absence of elevated clinical suspicion.1 tofacitinib is a small-molecule janus kinase inhibitor that is approved for treatment of adults with rheumatoid arthritis, psoriatic arthritis, or ulcerative colitis who have had inadequate response to or were intolerant of methotrexate.2 while infrequent cases of tinea pedis were observed in some clinical trials,3,4 there have as yet been no reports of more generalized dermatophytosis as a result of tofacitinib therapy. here, we report an interesting case of extensive tinea corporis and capitis developing in a woman shortly after starting tofacitinib treatment for rheumatoid arthritis. a 71-year-old african american woman presented with an intermittently pruritic rash on the upper body, beginning three months prior. she stated that this rash had spread from her stomach to her chest, back, shoulders, and scalp during this time. she denied recent fevers, chills, illnesses, unintentional weight loss, changing or bleeding lesions, or oral lesions. she did admit to recent weakness and hair loss, and emr review revealed a recent visit with chief complaint of unexplained weight loss. her past medical history was significant for a abstract rheumatoid arthritis is a chronic autoimmune disorder that often requires treatment with immunomodulatory agents. however, such interventions are not without risk of opportunistic infection. monitoring for such conditions is critical, though recognition and treatment can prove challenging, as they may manifest atypically or masquerade as another condition entirely. we present a case of extensive tinea corporis with concomitant tinea capitis masquerading as new-onset scle in a patient being treated for rheumatoid arthritis with the janus kinase inhibitor tofacitinib. introduction case presentation skin september 2020 volume 4 issue 5 copyright 2020 the national society for cutaneous medicine 457 three-year history of inadequately controlled rheumatoid arthritis, for which tofacitinib 5mg bid was recently added to her prior regimen of prednisone 10mg daily after she was unable to tolerate methotrexate. she started tofacitinib approximately 1-month prior to onset of the rash. autoimmune workup in the past had been significant for ana 1:640 (homogenous pattern) and negative dsdna, rnp antibody, smith antibody, ssa, ssb and complement c3 and c4 within normal limits. on exam there was diffuse poorly adherent white scale throughout the scalp (figure 1), scattered erythematous papules on the infraorbital medial cheeks and upper cutaneous lip (figure 2), large geometric to annular, well-demarcated, erythematous to hyperpigmented patches and thin plaques with erythematous raised plaques along the borders and diffuse fine scale on the upper chest (figure 3), back, shoulders, neck, conchal bowls, axillae, and abdomen. koh prep of scrapings from the chest showed fungal hyphae. biopsy of the right upper back was consistent with dermatophytosis, and fungal culture from her scalp grew trichophyton species. a six-week course of oral terbinafine 250 mg daily resulted in significant reduction in pruritis and normalization of the cutaneous physical findings, with the exception of a mild residual scaling on the scalp which again grew trichophyton on repeat fungal culture at three-month follow-up. a second course of oral terbinafine is in progress. the risk of fungal infection is thought to be increased in those on long-term tofacitinib therapy, largely due to inhibition of signaling via cytokine and chemokine cascades that are necessary for the destruction of fungi.5 clinical trial and long-term follow-up data of patients treated with tofacitinib show an increase in opportunistic infections (ois) with the median occurrence of non-tb ois at 40 weeks after treatment onset.6 while studies have shown increased incidence of dermatophytosis in those on anti-tnf-α therapy,7 there have been no reports of tofacitinib-associated dermatophytosis with the exception of infrequent tinea pedis.3,4 figure 1. hair loss and diffuse scaling on the scalp the present case highlights the need for frequent monitoring for ois in those receiving immunosuppressant therapy. our patient exhibited an extensive jak-inhibitor associated dermatophytosis that developed early in the treatment course, roughly four weeks from the first dose of tofacitinib and well before the median occurrence at 40 printed on 4/20/2020 04:57 pm page 1 of 1 peterson, brenda scan on 11/8/2019 by kurland, elena, md discussion skin september 2020 volume 4 issue 5 copyright 2020 the national society for cutaneous medicine 458 weeks. although it’s difficult to determine if the primary tinea infection pre-dated or occurred after initiation of tofacitinib treatment, she had no similar issues while on prednisone or previously with methotrexate. despite this, there have been reports of exuberant tinea corporis in the context of chronic oral steroid use.8 monitoring for ois is particularly important as the indications for tofacitinib continue to expand. additional treatment indications for other conditions, including plaque psoriasis, alopecia areata, vitiligo, and atopic dermatitis, are still under active investigation.9 although terbinafine and griseofulvin are both seen as first-line treatments for tinea capitis, terbinafine has been shown to be more effective for trichophyton-associated figure 2. erythematous papules on the face cases and requires shorter treatment durations to achieve similar effect.10 however, as our experience demonstrates, tofacitinib-associated dermatophytosis may require longer or repeated courses of treatment. close follow-up is warranted to ensure complete resolution, and a prolonged initial course of therapy may be helpful in these immunocompromised patients. if a prolonged or repeated course of therapy is indicated, cbc and liver function tests should be acquired at baseline and periodically during treatment.2 figure 3. well-demarcated erythematous thin plaques with fine overlying scale on the chest and neck in our patient with new-onset face and torso rash, several months of increasing fatigue, recent complaint of weight loss, a strongly positive ana and uncontrolled autoimmune disease, and known propensity of biologic therapies that alter immune function to paradoxically unmask autoimmune and connective tissue disease,11 it would have been easy to fixate on a diagnosis of newonset scle with a differential diagnosis also including dermatomyositis, photodermatitis, printed on 4/20/2020 04:59 pm page 1 of 1 peterson, brenda scan on 11/8/2019 by user: js523922 skin september 2020 volume 4 issue 5 copyright 2020 the national society for cutaneous medicine 459 or a nonspecific eczematous process. dermatophyte infections are infamous for being difficult to diagnose based on appearance alone, further emphasizing the importance of maintaining a high index of suspicion in patients on therapies that may make such infections manifest in ways they might not otherwise–such as extending across the torso, face, and scalp within mere weeks. by avoiding the temptation to anchor on the most straightforward explanation, we were able to perform the appropriate workup to arrive at the true diagnosis. misdiagnosis of another disease on the differential might have led to treatment with powerful topical steroids that would have exacerbated rather than treated our patient’s already extensive infection. frequent and early skin examinations along with rapid follow-up on cutaneous complaints is a necessary part of care for those undergoing immunosuppressive treatments. although not previously known to predispose to extensive dermatophytosis, initiation of tofacitinib should be considered a risk factor for such infections, which can be diagnosed with koh prep and treated with antifungals without the cessation of immunosuppressive therapy. conflict of interest disclosures: none funding: none corresponding author: dr. elena kurland, md 3031 w. grand blvd ste. 800 detroit, mi 48202 phone: 313-916-2151 fax: 313-916-5334 email: ekurlan1@hfhs.org references: 1. atzori l, pau m, aste n, aste n. dermatophyte infections mimicking other skin diseases: a 154person case survey of tinea atypica in the district of cagliari (italy). int j dermatol. 2012;51(4):410415. 2. xeljanz medication guide. united stated food and drug administration. updated may, 2018. https://www.accessdata.fda.gov/drugsatfda_docs /label/2018/203214s018lbl.pdf. accessed april 18, 2020. in. 3. hall s, nash p, rischmueller m, et al. tofacitinib, an oral janus kinase inhibitor: pooled efficacy and safety analyses in an australian rheumatoid arthritis population. rheumatol ther. 2018;5(2):383-401. 4. asahina a, etoh t, igarashi a, et al. oral tofacitinib efficacy, safety and tolerability in japanese patients with moderate to severe plaque psoriasis and psoriatic arthritis: a randomized, double-blind, phase 3 study. the journal of dermatology. 2016;43(8):869-880. 5. chen y, gong fy, li zj, et al. a study on the risk of fungal infection with tofacitinib (cp-690550), a novel oral agent for rheumatoid arthritis. sci rep. 2017;7(1):6779. 6. winthrop kl, park sh, gul a, et al. tuberculosis and other opportunistic infections in tofacitinibtreated patients with rheumatoid arthritis. ann rheum dis. 2016;75(6):1133-1138. 7. bardazzi f, balestri r, rech g, antonucci a, patrizi a. dermatophytosis during anti-tnf-alpha monoclonal antibody therapy. mycoses. 2011;54(5):e619-620. 8. sena lb, oliveira dc, paula cd, costa mc, franceschi le, costa im. trichophyton rubrum dermatophytosis in a patient under chronic use of systemic corticoids: an exuberant presentation. an bras dermatol. 2015;90(4):598-599. 9. montilla am, gómez-garcía f, gómez-arias pj, et al. scoping review on the use of drugs targeting jak/stat pathway in atopic dermatitis, vitiligo, and alopecia areata. dermatol ther (heidelb). 2019;9(4):655-683. 10. newland jg, abdel-rahman sm. update on terbinafine with a focus on dermatophytoses. clin cosmet investig dermatol. 2009;2:49-63. 11. her m, kavanaugh a. alterations in immune function with biologic therapies for autoimmune disease. j allergy clin immunol. 2016;137(1):1927. conclusion saban_fusion of axis 284 | saba et al fusion of axis with third cervical vertebra fusion of axis with third cervical vertebra: a case report n. saba1, a. rani1, g. sehgal1, rk verma1, ak srivastava1, m. faheem2 1department of anatomy, king george’s medical university, lucknow-226003, uttar pradesh, india 2department of neurosurgery, king george’s medical university, lucknow-226003, uttar pradesh, india abstract: introduction: fusion of one or more contiguous vertebral segments is usually the result of embryological failure of normal spinal segmentation. it may be associated with syndromes such as klippel-feil. fused cervical vertebrae (fcv) may also be acquired or pathologic. fcv is generally associated with disease like tuberculosis, other infections, juvenile rheumatoid arthritis and trauma. the commonest site of involvement is c2-c3. in condition of fusion the two vertebrae appear not only structurally as one but also function as one. this anomaly may be asymptomatic; however, it may also manifest in the form of serious clinical features such as myelopathy, limitation of the neck movement, muscular weakness, atrophy or neurological sensory loss. case report: we observed the fusion of axis with 3rd cervical vertebra. body, laminae and spines of c2 and c3 were completely fused on both anterior and posterior aspects, whereas the pedicles and transverse processes were not fused. foramen transversarium was present on both the vertebrae bilaterally. conclusion: this variation is noteworthy to neurosurgeons and radiologists in studying computed tomography (ct) and magnetic resonance imaging (mri) scans. key words: axis, 3rd cervical vertebra, fusion, block vertebra, foramen transversarium, variation introduction cervical vertebrae are seven in number of which c3-6 are typical, sharing similar features whereas c1 (atlas), c2 (axis), and c7 (vertebrae prominence) are atypical having different characteristic features. (1) vertebrae and intervertebral discs are one of the main manifestations of body segmentation or metamerism. (2) developmental and ossification process of c2 vertebra is the most complex among all the vertebrae. (3)fusion of one or more contiguous vertebral segments results from the embryological failure of normal spinal segmentation. the incidence is 0.4% to 0.7% with no sex predilection. (4) recent studies have documented associations between fusion of the cervical vertebral column and craniofacial morphology, romanian neurosurgery (2016) xxx 2: 284-288 | 285 including head posture in patients with severe skeletal malocclusions. this finding is expected to have importance for diagnostics and elucidation of etiology and thereby for optimal treatment. (5) skeletal abnormalities of cervical region or in cranio-cervical region are of interest to the anatomists, neurosurgeons radiologists and even orthodontists. (6) block vertebrae, formed after fusion of adjacent vertebrae is a condition which has embryological importance and clinical implications. since the fusion of cervical vertebrae 2nd with 3rd has a clinical importance, we need to emphasize the importance of multidisciplinary approach to help establish the precise occurrence of this congenital anomaly for preventing any serious damage such as osteoarthritis by early diagnosis and treatment. case report during routine survey of bones in the department of anatomy, king george’s medical university, lucknow, uttar pradesh, india, it was observed that the axis was fused with 3rd cervical vertebra. features of this block vertebra were analyzed and the specimen was photographed from different aspects. body, laminae and spines of c2 and c3 were completely fused on both anterior and posterior aspects. a ridge was noted on the dorsal surface of fused arches. joints between the inferior articular facet of c2 and superior articular facet of c3 displayed synostosis bilaterally (figures 1 and 2). diameters of foramen transversaria were measured from supero-lateral aspect of c2 and inferior aspect of c3. the diameters of foramen transversarium of c2 on right and left side were 5.5 mm and 5.0 mm respectively while of c3 vertebra was 6.0 mm on right side and 6.5 mm on left side. the pedicles and transverse processes were not fused (figure 3). figure 1 photograph showing complete fusion of body of c2 and c3 from anterior aspect figure 2 photograph showing complete fusion of laminae and spines of c2 and c3 from posterior aspect, presence of ridge on dorsal surface of fused arches (arrow 1), bilateralsynostosis between inferior articular facet of c2 and superior articular facet of c3 (arrow 2), foramen transversarium is seen in c2 vertebra superolaterally (arrow 3). 286 | saba et al fusion of axis with third cervical vertebra figure 3 photograph showing non-fusion of pedicles and transverse processes of c2 and c3 in left lateral aspect discussion in condition of the fusion of cervical vertebrae, two vertebrae appear not only structurally as one but also function as one. (7) it is important to identify the cause of fcv i.e. congenital, acquired or pathologic. embryologically, c2-c3 fusion was explained as improper separation of adjacent somites or their associated mesenchyme. (8) also it was found to be due to non-segmentation of primitive sclerotome.(9) in a condition known as chorda dorsalis, congenital fused cervical vertebra is one of the primary malformations believed to be due to defects during the development of the occipital and cervical somites. between 3rd to 8th weeks of embryonic life, cartilaginous framework of a vertebra is formed from paraxial mesoderm. (10) congenital fusion of two or more cervical vertebrae as seen in klippel-feil syndrome was believed to result from faulty segmentation along the embryos developing axis during weeks 3-8 of gestation. (11) cause of this anomaly is often a combination of environment and genetics which occurs during the third week post-conception. it has been described as an autosomal dominant condition. mutation in pax gene and notch signaling pathway and a chromosomal inversion inv (8) (q22.2q23.3) causes familial klippel-feil syndrome. (9) this syndrome was also hypothesized to result from embryological subclavian artery supply disruption sequence. (12) it was suggested that decreased local blood supply during third to eighth week of development may be a causative factor4. absence of the joints between articular facets in the fused vertebrae suggests failure of normal development and differentiation of vertebrae (i.e. fusion at the pre-cartilaginous stage of vertebral development). independent pedicle, transverse process and ridge on dorsal surface of fused arches suggest normal initial development followed by fusion. acquired fcv is generally associated with diseases like tuberculosis, other infections, juvenile rheumatoid arthritis and trauma. cause of these abnormalities may be multifactorial which also includes the role of certain drugs like thalidomide, lovastatin and progestin/estrogen on the developing fetus. the present case is very much similar to some previous studies in which the body, laminae and pedicles of c2 and c3 were completely fused on both anterior and posterior aspects. (1, 6) but in our case the pedicles were not fused while spines of the two vertebrae were fused on both anterior and posterior aspects. this anomaly may be romanian neurosurgery (2016) xxx 2: 284-288 | 287 asymptomatic, may appear with manifestations of serious clinical features such as myelopathy, muscular weakness, atrophy and neurological sensory loss or may be associated with syndromes such as klippelfeil in the form of limitation of neck movement. (7) the orthodontist may be the first person to detect cervical spine abnormalities as they are asymptomatic until adolescence or young adulthood and early diagnosis is based on incidental radiographic findings. symptoms vary according to the extent of pathology and may result in severe neck pain, decreased neck mobility, muscular weakness or sensory deficits of both upper limbs and sudden unexpected death. (6) normally aligned congenital synostosis of c23 is rarely associated with a junctional problem, whereas a kyphoticsynostosis is associated with a caudal junctional problem.(13) persons with klippel-feil syndrome and cervical stenosis may be at increased risk for spinal cord injury after minor trauma as a result of hypermobility of the various cervical segments. cervical spondylosis, disc herniation and secondary degenerative changes are more at levels adjacent to fused vertebra. there is increased incidence of osteophyte formation in the adjacent levels in cervical fusion. neurological signs and symptoms are variable, depending on the degree of pathology. another syndrome associated with this anomaly is wildervanck syndrome, characterized by klippel–feil (kf) deformity of the cervical spine in association with abducens palsy with retracted bulbi and hearing loss. the main clinical consideration of this deformity is a difficult airway due to the short, thick and immobile neck secondary to fused cervical segments, which compromises bag and mask ventilation if airway management is required. (14) persons with klippel-feil syndrome may be at high risk for developing a transient neurolgoic deficit due to cervical spinal cord injury following minor trauma, as these subjects are particularly prone to hyperextension trauma. conclusion fusion of axis and third cervical vertebrae is a rare and unusual finding which is clinically significant. this variation is noteworthy to neurosurgeons and radiologists in studying computed tomography (ct) and magnetic resonance imaging (mri) scans. it is of equal significance to the anaesthetists doing endotracheal intubation as in persons with block vertebra in cervical region hyperextension may precipitate disc prolapse. if cisternal puncture or lumbar puncture is to be done, we should look for possibility of block vertebrae in cervical and lumbar regions respectively. acknowledgement i would like to thank the faculty of the department of anatomy, king george’s medical university, uttar pradesh, lucknow, india and my husband for providing necessary help and valuable suggestions. correspondence dr. noor us saba department of anatomy, king george’s medical university, lucknow-226003, uttar pradesh, india e-mail: noorussaba83@gmail.com phone (mobile): 91-9368480101 288 | saba et al fusion of axis with third cervical vertebra references 1.wazir s, mahajan a. fusion of axis with third cervical vertebraea case report. indian journal of fundamental and applied life sciences. 2011; 1(4): 164-166. 2.standring s. gray’s anatomythe anatomical basis of clinical practice. 40thed. churchill livingstone; 2008.p 720-721. 3.pate d. normal radiographic differences between the adult and pediatric cervical spine.dynamic chiropractic.2012; 30(4). 4.solan s. fused axis vertebrae with a third cervical hemivertebrae a rare case report.iosr journal of dental and medical sciences. 2013; 10(4): 83-85. 5.sonnesen l.associations between the cervical vertebral column and craniofacial morphology.international journal of dentistry.2010; 2010. 6.yadav y, goswami p, bharihoke v. cervical vertebra synostosis (c2-c3) a case report, american journal of medical case reports. 2014; 2(6): 120-122. 7.erdil h, yildiz n, cimen m. congenital fusion of cervical vertebrae and its clinical significance.j anat. soc. india. 2003; 52(2): 125-127. 8.kaplan km, spivak jm, bendo ja. embryology of the spine and associated congenital abnormalities: review article. the spine journal.2005; 5: 564-576. 9.kulkarni v, ramesh br. a spectrum of vertebral synostosis. international journal of basic and applied medical sciences. 2012; 2(2): 71-77. 10.moore kl, persaud tvn, torchia mg. the developing human: clinically oriented embryology in skeletal system. 9th ed. elsevier.new delhi; 2013.p348350. 11.vaidyanathan s, hughes pl, soni bm, singh g, sett p. klippel-feil syndrome – the risk of cervical spinal cord injury: a case report. bmc fampract.2002; 3: 6. 12.tiwari a, chandra n, naresh m, pandey a, tiwari k. congenital abnormal cervical vertebrae–a case report.j anat. soc. india. 2002; 51(1): 68-69. 13.moon ms, kim ss, lee bj, moon jl, lin jf, moon yw. radiographic assessment of congenital c2-3 synostosis. journal of orthopaedic surgery. 2010; 18(2): 143-7. 14.schisler t, huttunen h, tang r,vaghadia h. ultrasound-assisted spinal anaesthesia in a patient with wildervanck syndrome and congenital abnormalities of the lumbar spine. british journal of anaesthesia.2012; 109(2): 290-291. the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 6 s afr fam pract issn 2078-6190 eissn 2078-6204 © 2016 the author(s) review introduction the arthritic diseases are progressive and debilitating and impair the quality of life of patients. it is now common practice to use disease modifying agents early after a diagnosis is made to prevent irreversible joint damage. in recent years the biological dmards have been proven to be efficacious against the progression of rheumatoid arthritis (ra) when there is no response or a poor response to traditional dmards.1,2 an overview of the biological disease modifying agents available for use in south africa is provided here. the biological dmards are effective when used as single agents but they are seldom used alone, since combinations with methotrexate have been shown to have clinical benefit.2 methotrexate use prevents the formation of antibodies to the biological agents, which extends their therapeutic lifespan. the prohibitive cost of the biological agents and their costeffectiveness remains an ongoing debate. in this context a recent cochrane review indicates that methotrexate based small molecule triple therapy may be as efficacious as combinations of biological dmards with methotrexate.3 what are biological agents? biological dmards are protein molecules and include monoclonal antibodies and antibody based fusion proteins. monoclonal antibodies bind to a specific molecular target, causing inactivation of the target and compete with the natural ligand for binding to the target, thereby preventing the function of the target molecule. once a monoclonal antibody has associated with its target it triggers the antibody dependent cellular cytotoxic immune response and/or the complement system causing the removal/destruction of the target by the abstract the past decade has seen a major change in the treatment options and strategies for rheumatoid arthritis (ra) and the other immune-mediated arthritic diseases. the disease modifying antirheumatic drugs (dmards) are now used in early stages of the disease in order to preserve joint architecture. there are two groups of dmards, the small molecules, like methotrexate, and the biological dmards, which are frequently referred to as “magic bullets” since they target specific cytokines and immune cells associated with arthritic conditions. they are monoclonal antibodies or fusion proteins designed to bind and inactivate immune targets. tumour necrosis factor-alpha (tnf-α) plays an important role in the pathogenesis of rheumatoid disorders and is the target of four biological dmards, etanercept, infliximab, golimumab and adalimumab. the other biological dmards include abatacept, rituximab and tocilizumab and these prevent t-cell costimulation, cause the depletion of mature cd20 positive b cells or prevent the activation of the interleukin-6 receptor molecule, respectively. ustekinumab, a monoclonal antibody against il12/il23 is effective in psoriatic arthritis. biological agents are indicated when patients do not respond adequately to the traditional dmards. numerous clinical trials have shown that the biological agents reduce joint inflammation and erosive damage, especially when used in combination with methotrexate. apart from their prohibitive cost, the biological agents are not without potentially serious adverse effects with infections being the main concern. the tnf-α inhibitors increase the risk for tuberculosis and other opportunistic infections, whereas the non-tnf-α immune inhibitors increase the risk for opportunistic viral, fungal and bacterial infections. this review provides an overview of the biological agents currently available in south africa. keywords: tnf-α inhibitors, non-tnf-α biological agents, anti-rheumatic drugs, monoclonal antibodies, fusion proteins, immune modulators south african family practice 2016; 58(6):6-10 open access article distributed under the terms of the creative commons license [cc by-nc-nd 4.0] http://creativecommons.org/licenses/by-nc-nd/4.0 an overview of the biological disease modifying drugs available for arthritic conditions in south africa leonie harmse,a* helmuth reuterb apharmacology division, school of therapeutic sciences, faculty of health sciences, university of the witwatersrand bdivision of clinical pharmacology, faculty of medicine and health sciences, stellenbosch university *corresponding author, email: leonie.harmse@wits.ac.za an overview of the biological disease modifying drugs available for arthritic conditions in south africa 7 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 7 immune system as shown in figure 1. in the treatment of the arthritis group of diseases there are two main groups of biological agents available, the tumour necrosis factor-α (tnf-α) inhibitors and the non-tnf-α inhibitors of the immune system. tnf-α as a drug target tnf-α, a pro-inflammatory cytokine, was identified by brennan et al in 1989 as an important target molecule in the inflamed synovium of rheumatoid arthritis patients.4 since then clinical trials have proven that a decrease in circulating tnf-α levels decrease disease severity and preserve joint architecture in ra. clinical trials proved tnf-α inhibitors to have equal efficacy to methotrexate when used alone and superior efficacy when used in combination with methotrexate. in addition, the combined use of tnf-α inhibitors and methotrexate prevents the formation of antibodies to the tnf-α inhibitors. the available tnf-α inhibitors bind to both soluble and membrane bound tnf-α.2,4 adverse effects associated with tnf-α inhibitors are associated with the role of tnf-α in the immune system. of particular concern is the increased risk of infections, especially with organisms causing the formation of granulomas, like tuberculosis (tb) and histoplasmosis. in south africa, the high tb burden makes it imperative to screen patients for latent and active infections prior to initiation of tnf-α therapy. it is advisable to immunize prospective patients against influenza, varicella zoster and pneumococcal disease.1 in addition, there is an increased risk for fungal viral and other bacterial infections. as with any immunosuppressive regimen, there is always a potential for the development of a malignancy, and patients require careful monitoring.5,6 the use of tnf-α inhibitors has been associated with a spectrum of cutaneous reactions. skin lesions range from injection site reactions to infections that are directly related to immune suppression, like bacterial cellulitis and herpes zoster. skin neoplasms and a host of immune mediated cutaneous diseases ranging from psoriasis to vasculitis and pemphigus are associated with tnf-α inhibition.7 tnf-α is secreted by monocytes, macrophages, t cells, b cells and fibroblasts and is important to the normal immune response. it is essential for both the formation and maintenance of granulomas in mycobacterial infections, explaining why latent infections are reactivated in patients treated with tnf-α inhibitors. tnf-α functions as a growth factor, induces the formation of proinflammatory cytokines and endothelial adhesion molecules needed for leucocyte accumulation in tissues. tnf-α is essential for the defence against intracellular pathogens and functions as a co-mitogen for t and b cells.8 on a molecular level, tnf-α causes the trimerization of tnf-α receptors which activates signal transduction cascades that trigger apoptosis and orchestrate the translocation of the transcription factors ap-1 and nf-κb to the cell nucleus, as shown in figure 2. ap-1 and nf-κb stimulate the transcription of numerous cytokine genes involved in the immune response.8 tnf-α inhibitors etanercept etanercept is a recombinant fusion protein consisting of the tnf-α p75 receptor dimerized on the fc portion of human igg1. the tnf-α p75 region binds to soluble and membrane bound tnf-α. the presence of mouse amino-acid sequences induces an antibody response that decreases it function, therefore coadministration of methotrexate is desirable.2,9 etanercept use is associated with an increased risk for bacterial infections and septic arthritis. compared to the other tnf-α inhibitors, reactivation of tuberculosis is less frequent with etanercept. the formation of anti-nuclear antibodies has been figure 1. a summary of the consequences of monoclonal antibody binding to its specific receptor molecules. r: antibody specific receptor, mcab: monoclonal antibody, fc: receptor binding to constant region of a monoclonal antibody, adcc: antibody dependent cellular cytotoxicity figure 2. the multiple roles of tnf-α in the immune response. tnf-α forms a trimeric molecule which initiates the trimerization of its receptor molecules. this stimulates signal transduction cascades which result in the translocation of cytoplasmic transcription factors like nf-κb and ap-1 to the nucleus. in the nucleus, this causes the transcription of more inflammatory and pro-inflammatory cytokine genes. it also induces the expression of endothelial adhesion proteins and chemokines in the synovium which attracts leucocytes to the synovium. in addition, activation of the receptor can cause apoptosis in cells harbouring intracellular pathogens. s afr fam pract 2016;58(6):6-108 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 8 reported with etanercept, but unless active lupus develops, cessation of treatment is not indicated.2,6 infliximab infliximab is an older generation chimeric human/mouse monoclonal antibody and binds soluble and membraneassociated tnf-α receptors. concurrent administration with methotrexate or glucocorticosteroids reduces formation of antichimeric antibodies and is recommended.2,6 golimumab this is a humanized monoclonal antibody reducing the risk of antibody formation and has a high affinity for soluble and membrane bound tnf-α. adverse effects are associated with the inhibition of tnf-α. it is used in moderate to severe rheumatoid arthritis in adults.2,6 adalimumab adalimumab is a human igg1 monoclonal antibody to tnf-α. it forms a complex with soluble tnf-α and prevents its interaction with tnfα p55 and p75 cell surface receptors. the use of adalimumab decreases macrophage and t-cell function.2,6 non-tnf-α biological immune modulators currently, four non-tnf-α biological molecules are available to use in the arthritic diseases. abatacept, rituximab and tocilizumab are used in the management of rheumatoid arthritis. ustekinumab is a new monoclonal antibody and is active against psoriatic arthritis and psoriasis but not against rheumatoid arthritis.5 abatacept abatacept is a genetically engineered fusion protein consisting of the extracellular domain of cytotoxic t-lymphocyte-associated antigen (ctla-4) fused to the hinge region of igg1. the activation of naïve t cells requires the cd80/86 costimulatory molecules on dendritic cells to interact with the cd28 molecule on naïve t cells. this interaction is required to complete antigen presentation. abatacept blocks t-cell costimulation by preventing the binding of cd80/86 to cd28 as shown in figure 3. this decreases the response of naïve t cells to antigen presentation.2,10 abatacept is used in both tnf-α naïve patients and in patients who have previously failed on tnf-α therapy.6,11 concurrent administration with tnf-α inhibitors is discouraged. abatacept increases the risk for re-activation of latent tb and increases the risk for upper respiratory tract infections. infusion reactions are rare. rituximab rituximab is a chimeric monoclonal antibody to the cd20 receptor found on mature b lymphocytes. its use leads to the long-lasting near depletion of b cells in the peripheral blood. rituximab is not used exclusively for the treatment of rheumatoid arthritis but also treat cancers like lymphoma. rituximab is indicated in rheumatoid arthritis when patients fail to respond to tnf-α inhibitors.12,13 cell mediated and complement dependent cytotoxicity and apoptosis are responsible for the depletion of b lymphocytes. table 1: tnf-α inhibitors kinetics and indications tnf-α inhibitor t½ (days) dose and interval indications etanercept (enbrel) 4.5 25 mg twice weekly or 50 mg once weekly subcutaneous rheumatoid arthritis, psoriasis, ankylosing spondylitis, juvenile chronic arthritis infliximab (revellex) 9–12 35 mg/kg every 4–8 weeks subcutaneous rheumatoid arthritis, psoriasis, ankylosing spondylitis golimumab (simponi) 14 50 mg/month subcutaneous rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis adalimumab (humira) 10–12 80 mg loading dose, thereafter 40 mg every 14–20 days subcutaneous rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, juvenile idiopathic arthritis, plaque psoriasis, crohn’s disease figure 3. a schematic diagram showing the binding of abatacept to cd80/86 and preventing its interaction with cd28 on naïve t-cells. apc: antigen presenting cell; mhc: major histocompatibility complex, tcr: t-cell receptor, ag: antigen, cd28: receptor molecule for cd80/86 an overview of the biological disease modifying drugs available for arthritic conditions in south africa 9 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 9 the high dose of rituximab causes an infusion reaction which can be managed by pre-treatment with a glucocorticosteroid. a skin rash is common with the first treatment but decreases with subsequent treatments. in the case of an urticarial rash or an anaphylactic reaction, the use of rituximab should be discontinued. rituximab is not associated with the reactivation of tb infections, lymphomas or other tumours. however, the reactivation of hepatitis b is a concern and concomitant antiviral therapy is recommended. although rare, multifocal leukoencephalopathy is a serious adverse reaction associated with rituximab.14 tocilizumab tocilizumab is a humanized monoclonal antibody that binds to soluble and membrane bound il-6 receptors, preventing il-6 mediated signalling. interleukin 6 is produced by various immune cells, including macrophages and fibroblasts, and is recognized as an important pro-inflammatory cytokine. il-6 regulates t-cell function, the acute phase reaction and terminal b-cell differentiation. binding of il-6 causes the recruitment of two molecules of glycoprotein 130 (gp130) to the receptor.15 the il-6 receptor is expressed on hepatocytes and a subpopulation of leucocytes and megakaryocytes. 15 il-6 combined with transforming growth factor β induces the differentiation of naïve t cells to inflammatory th17 cells. this promotes the maturation of b cells toward the production of igg. in hepatocytes it stimulates the production of acute phase reactants like c-reactive protein, serum amyloid a, haptoglobin, fibrinogen and α-anti-chymotrypsin. in addition, it decreases the hepatic production of transferrin and hepcidin, contributing to anaemia of chronic inflammation.15 used therapeutically, tocilizumab decreases rheumatoid factor, acute phase reactants and inflammatory th17 cells, and reduces bone resorption. however, tocilizumab is associated with several adverse drug reactions shown in table 2 and is not recommended for patients with hepatic disease. ustekinumab this monoclonal antibody inhibits the function of il12/il23 and is registered in south africa for the treatment of psoriasis. it is effective in treating psoriatic arthritis, psoriasis and crohn’s disease, but has no clinical benefit in rheumatoid arthritis.16 ustekinumab binds to the p40 subunit of both il-12 and il-23 and prevents them binding to their respective receptors. il-12 is produced by macrophages and promotes the th1 response, activates natural killer cells and together with il-18 causes interferon-γ release.16 the failure of ustekinumab in rheumatoid arthritis indicates that these processes are not important in the pathogenesis of rheumatoid arthritis. summary of key concepts in the use therapeutic use of biological dmards • biological dmards are indicated when therapy with small molecule dmards fails. • biological agents can be used with or without methotrexate, but methotrexate prolongs the therapeutic lifespan of the biological agents. • tnf-α inhibitors are used before the non-tnf-α inhibitors. • tuberculosis infections are common with the tnf-α inhibitors and latent infections can be reactivated. • it is advisable to vaccinate patients against common infections. • opportunistic fungal viral and bacterial infections are common. • biological agents do not effect a cure but contribute to achieving a state of remission. • withdrawal of biological agents or dmards in patients who are in remission causes reactivation of the disease. table 2. summary of the non-tnf-α biological dmards, their dosage, primary indications and common and serious adverse effects immune modulator dose and interval indications adverse effects abatacept (orencia) induction dose: week 0, 2 and 4  500 mg thereafter  monthly dose: < 60 kg: 500 mg 60–100 kg 750 mg > 100kg : 1000 mg intravenous rheumatoid arthritis, juvenile idiopathic arthritis infusion reactions, infections rituximab (mabthera) two iv infusions of 1000 mg two week interval repeat 6–9 months intravenous rheumatoid arthritis, haematological malignancies, anca associated vasculitis, non-hodgkin’s lymphoma, chronic lymphocytic lymphoma infusion reaction, infections, reactivation of hepatitis b, multifocal leuko-encephalopathy tocilizumab (actemra) 8 mg every two weeks intravenous rheumatoid arthritis, systemic juvenile idiopathic arthritis, juvenile idiopathic arthritis infusion reaction, infections, dyslipidaemia, gi perforation ustekinumab (stelara) 45 mg/100 kg /month (twice) then 45 mg/ 100 kg every 12 weeks intravenous psoriatic arthritis, plaque psoriasis, crohn’s disease infections, headache, pain – pharynx, larynx, myalgia, exfoliative dermatitis, hypersensitivity, malignancies s afr fam pract 2016;58(6):6-1010 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 10 conclusion the target-specific biological agents are clinically useful additions to the dmards and improve the chance to achieve remission in patients with rheumatoid arthritis. their crippling cost, however, limit their use in resource-poor countries. the risk for serious infections in patients on biological dmards is of great importance and patients require regular monitoring. conflict of interest i declare that i have no financial or personal relationship(s) which may have inappropriately influenced me in writing this paper. references 1. singh ja, saag kg, louis-bridges jr s, et al. 2015 american college of rheumatology guideline for the treatment of rheumatoid arthritis. arthritis rheumatol. 2016;68:126. 2. van vollenhoven rf. treatment of rheumatoid arthritis: state of the art. nat. rev. rheumatol. 2009;5:531-41. 3. hazlewood gs, barnabe c, tomlinson g, marshall d, devoe d, bombardier c. methotrexate monotherapy and methotrexate combination therapy with traditional and biologic disease modifying antirheumatic drugs for rheumatoid arthritis: abridged cochrane systematic review and network meta-analysis. br med j. 2016;353: i1777. 4. brennan fm, chantry d, jackson a, maini r, feldman m. inhibitory effect of tnf-alpha antibodies to synovial cell interleukin -1 production in rheumatoid arthritis. lancet. 1989;2:244-7. 5. tarr g, hogdkinson b, reuter h. superheroes in autoimmune warfare: biologic therapies in current south african practice. s afr med j. 2014;104(11):787-91. 6. scott dl. biologics-based therapy for the treatment of rheumatoid arthritis. nat rev clin phar ther. 2012;91(1):30-43. 7. hernández mv, meineri m, sanmartí r. skin lesions and treatment with tumor necrosis factor alpha antagonists. reumatol clin. 2013;9(1):53-61. 8. vasanthi p, nalini g, rajasekahr g. role of tumor necrosis factor-alpha in rheumatoid arthritis: a review. aplar journal of rheumatology. 2007;10:270-4. 9. emery p, breedveld fc, hall s, et al. comparison of methotrexate monotherapy with a combination of methotrexate and etanercept in active, early moderate to severe rheumatoid arthritis (comet ): a randomised, double blind, parallel treatment trial. lancet. 2008;372:375-82. 10. kremer jm, westhovens r, leon m, et al. treatment of rheumatoid arthritis by selective inhibition of t‑cell activation with fusion protein ctla4ig. n. engl. j. med. 2003;349:1907-15. 11. genovese mc, becker jc, schiff m, et al. abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. n. engl j med. 2005;353(11):1114-23. 12. emery p, fleischmann r, filipowicz-sosnowska a, et al. the efficacy and safety of rituximab in patients with active rheumatoid arthritis despite methotrexate treatment: results of a phase iib randomized, double-blind, placebo-controlled, dose-ranging trial. arthritis rheum. 2006;54:1390-1400. 13. cohen sb, emery p, greenwald mw, et al. rituximab for rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: results of a multicenter, randomized, double-blind, placebo-controlled, phase iii trial evaluating primary efficacy and safety at twenty four weeks. arthritis rheum. 2006;5:2793-2806. 14. clifford db, ances b, costello c, et al. rituximab associated progressive multifocal leukoencephalopathy in rheumatoid arthritis. arch. neur. 2011; 68:1156-64. 15. scheller j, garbers c, rose-john s. interleukin-6: from basic biology to selective blockade of pro-inflammatory activities. semin immunol. 2014;26(1):2-12. 16. gottlieb a, menter a, mendelsohn a, et al. ustekinumab, a human interleukin 12/23 monoclonal antibody, for psoriatic arthritis: randomised, double-blind, placebo-controlled, crossover trial. lancet. 2009;373:63340. name: original research article doi: 10.18231/2348-7682.2018.0013 panacea journal of medical sciences, may-august, 2018;8(2):51-53 51 serological profile of rheumatoid arthritis in a tertiary care hospital p.k surendran1 1assistant professor, p.k das institute of medical science, vaniyamkularn, kerala, india *corresponding author: email: surendranpkdr@gmail.com abstract rheumatoid arthritis (ra) is an autoimmune based inflammatory pathology with involvement of joints that show early morning stiffness alongwith pain resulting in loss of function. it usually affects females aged 30 years or above. a total of 150 patients of rheumatoid arthritis were included in the present study of which 120 were females and 30 males (f:m=4:1). serological tests were carried out in these patients. these test were analyzed for their sensitivity as well as specificity. combination of various test were also assessed for their results. in the present, study age and sex of patient affected by rheumatoid arthritis was analysed. combination of serological tests provided a high specificity as can be seen from the present study. quantitative rheumatoid factor estimation and anti rheumatoidantibodies assay gave the maximum specificity of 89.8%. a combination of serological tests is advisable rather than a single diagnostic test.this allows for the early diagnosis of rheumatoid arthritis so as to prevent complications by the disease. keywords: investigations, rheumatoid arthritis, serology, specificity, sensitivity. introduction rheumatoid arthritis (ra) has a prevalence of about 0.5% to 1% and an incidence of about 30 per 100 000 inhabitants, making it one of the commonest chronic inflammatory autoimmune disease.1-3 rheumatoid arthritis (ra) is an inflammatory autoimmune pathology involving joints mainly leading to pain, early morning stiffness and restriction of movements. cartilages, synovium, and certain systems of the body are affected with autoimmunity and inflammatory processes of ra. early diagnosis and aggressive treatment are the best means of avoiding joint destruction, damage to organs and disability. studies on the spectrum and expression of ra in asian population are limited in the literature.5-7 this prospective study was carried out to determine the relationship between ra and laboratory tests used in the diagnosis of ra.traditionally females are affected about three to four times more often than men. materials and methods this prospective study was carried out over a period of one year in a tertiary care teaching hospital between january 2017 and dec 2017. all patients who were clinicallysuspected to be suffering from ra were included in the present study. approval of the institutional ethical committee was taken.written consent was taken from all the patients who were included in the study. a total of 150 patients with 30 males and 120 females were included with a m:f ratio of 1:4. the age ranged from 23 years to 74 years. patients undergoing any treatment or previously diagnosed cases of ra were excluded from the study. all patients included in the study were subjected to all the investigations such as complete haemogram, esr, quantitative c-reactive protein (crp), rheumatoid factor (latex as well as quantitative), and anti ccp antibodies (anti-cyclic citrullinated peptide). esr was estimated using automated analyzer. crp and ra factor latex test was carried out using kits from tulip diagnostics. quantitative assays were performed using automated analysers as per the manufacturer’s instructions and protocol. statistical analysis was done using spss (statistical package for the social sciences) version 17.0 for windows software. p value of <0.05 was considered as statistically significant. results a total of 150 cases were included in the present study. the age and sex distribution of the cases is shown in table 1. table 1: age and sex distribution of patients with rheumatoid arthritis s. no. age group males females total n(%) 1 20 29 3 28 31(20.7%) 2 30 39 8 34 42(28.0%) 3 40 49 6 29 35(23.3%) 4 50 59 6 15 21(14.0%) 5 60 69 4 12 16(10.7%) 6 70 79 3 2 5(3.3%) total 30 120 150(100%) p.k surendran serological profile of rheumatoid arthritis in a tertiary care hospital panacea journal of medical sciences, may-august, 2018;8(2):51-53 52 the male: female ratio was 1:4. majority of the cases were in the age group of 30-39 years, followed closely by 40-49 years.the mean age was 45.2 years. results of the serological tests carried out in the patient are depicted in table 2. table 2: distribution of patients as per the results of the serological tests (n = 150) s. no. n % 1 raised esr 126 84.0 2 increased crp 132 88.0 3 anti ccp positive 112 74.7 4 ra factor positive 139 92.7 5 high quantitative ra factor 143 95.3 quantitative ra factor was raised in 143 of the 150 patients (95.3%). anti ccp antibodies were present in only 112 of the 150 patients (74.7%).there has been an observed overlap between the serological tests also. in some patients multiple parameters were positive or their values were more than the normal. we also studied the sensitivity and specificity of different serological markers as well as their combinations (table 3). table 3: sensitivity and specificity of different serological tests in the present study sensitivity specificity esr 92.1% 68.2% crp 84.1% 70.4% anti ccp antibodies 74.2% 88.5% r a factor (latex) 38.2% 87.8% quantitative r a factor 71.8% 64.6% anti ccp + q r a 71.4% 94.1% anti ccp + latex r a 52.4% 89.8% combination of test increases the specificity rates significantly. it was further observed that patients with multi joint involvement if tested negative for both latex ra factor as well as anti ccp antibodies indicate that the patient is not having rheumatoid arthritis. there was a positive correlation between anti-ccp antibodies and higher esr, crp. also there was a positive correlation between rf and increased esr and crp.in this study fifty percent of the patients were both anti-ccp and rf positive. the study (p<0.05) revealed significant positive correlation between rf and anti-ccp antibodies. discussion rheumatoid arthritis is a systemic disease, accompanied by progressive joint destruction and deformity. depending on the severity, there may also be extra-articular manifestations, involving skin, vascular channels, and internal organs as well. if inadequately treated, ra leads in the long term to a significant impairment of the quality of life; morbidity and increase in mortality. early diagnosis and suitable therapy are therefore of great importance in determining the prognosis of ra. the three pillars for the diagnosis of rheumatological disease are a good medical history, clinical findings (including radiological investigations) and serological laboratory tests. serological diagnostic testing is of growing importance in the early detection and differentiation of rheumatoid arthritis. apart from the traditional detection of rheumatoid factor, new specific autoantibodies to citrullinated antigens have made a crucial contribution to the diagnosis of ra. the rheumatoid factor is an autoantibody, which may be igm, igg or iga, and which was first mentioned in 1922.1 it recognizes domains ch2 and ch3 of the fc segment of human igg and is a component of the classification criteria for ra published by the american college of rheumatology.2 the classification criterion to define ra that is used internationally was defined by the american college of rheumatology (acr) in 1987. new criteria for rheumatoid arthritis classification were introduced in 2010.1 ra sera contained antibodies that reacted to the keratinized layer of epithelial cells. these were called antikeratin antibodies, and were reported only in ra patients. anti-ccp was included in the acr/eular (european league against rheumatism) ra classification criteria in 2010. erythrocyte sedimentation rate (esr), c-reactive protein (crp), and rheumatoid factor (rf) are the other serological tests used in the ra classification criteria. assessment of anti-ccp, rf, esr and crp in the serum was included in the acr/eular ra classification criteria in 2010. several studies have questioned the importance of anti-ccp antibody testing in distinguishing ra from other inflammatory diseases.1-2 joints that showed erosion or destruction showed strong association with anti ccp antibodies.3 ra has been associated with several autoantibodies, including rheumatoid factors (rf), anti‐perinuclear factor (apf), anti‐keratin antibodies (aka) and anti‐filaggrin antibodies (afa).4 p.k surendran serological profile of rheumatoid arthritis in a tertiary care hospital panacea journal of medical sciences, may-august, 2018;8(2):51-53 53 these autoantibodies bind antigenic determinants that contain the unusual amino acid citrulline, formed by a post‐transcriptional modification of arginine residues by peptidylarginine deiminase. to detect these autoantibodieselisa is used. this method uses as antigen a cyclic variant of a citrullinated peptide (ccp) derived from the sequence of human filaggrin. the anti‐ccp antibody test is now commercially available and its diagnostic accuracy is comparable with that of the rf test5. the diagnostic accuracy of this test in the detection of ra is still unclear, as sensitivities ranging from 41 to 68% have been reported.3,6,7 in the present study a comparison of the sensitivity and specificity of the anti‐ccp antibody test with those of other tests commonly used in the diagnosis of ra. role of these autoantibodies in assessing the severity of the disease is unknown.8-10 the present study establishes the value of anti‐ccp antibodies in the diagnosis of ra both singly as well as in combination. the specificity obtained for the anti‐ccp test (88.5%) was almost similar to that found by other groups: 91–98%.1,8-10 however, although there is consensus by different authors regarding specificity, there is significant variation in the sensitivity, rates ranging from 41–68%. this variation may be attributed to the different dilutions of serum used or, to the different cut‐off values as per the diagnostic test employed. the anti‐ccp antibody test has moderate sensitivity and excellent specificity, the aka test has poor sensitivity but excellent specificity, and the igm rf test has moderate sensitivity and specificity11-15. a positive test for anti‐ccp antibodies or aka practically establishes this diagnosis. hence these tests, may prove more useful in selected cases in clinical practice especially in cases where significant disagreement between these tests exist, true positives among ra patients and false positives among controls, suggests that there is considerable room for improvement in the serological diagnosis of ra.16 whether these tests are able to predict the occurrence of clinical or radiological manifestations of ra remains a mystery. the present study was cross‐sectional and therefore liable to possible selection bias. prospective studies would probably have produced more information than a cross‐sectional study of patients with definite ra. however, in prospective studies, it is mandatory to predict that treatments have not affected the results.17 conclusion in conclusion, our results suggest that anti‐ccp antibodies, aka and igm rf reflect clinically relevant disease processes in ra patients. however, in clinical practice, both igm rf and anti‐ccp antibodies may be useful, igm rf for their good sensitivity and as a marker of disease severity and anti‐ccp antibodies for their high specificity and their presence in some ra‐seronegative patients. references 1. tennakoon tmis, nissanka tm, bandaranayake bmvc. the spectrum of rheumatoid arthritis. a single unit experience. anuradhapura medical journal. 2015;9(2supp):s38. 2. aletaha d, neogi t, silman aj, funovits j, felson dt, bingham co, et al. an american college of rheumatology/ european league against rheumatism collaborative initiative 2010 rheumatoid arthritis classification criteria. arthritis rheum. 2010;62:25692581. 3. visser h, le cessie s, vos k, breedveld fc, hazes jmw. how to diagnose rheumatoid arthritis early: a prediction model for persistent (erosive) arthritis. arthritis rheum. 2002;46:357-365. 4. goldbach‐mansky r, lee j, mccoy a. rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset. arthritis res. 2000;2:236–43. 5. schellekens ga, visser h, de jong ba. the diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. arthritis rheum. 2000;43:155–63. 6. bizzaro n, mazzanti g, tonutti e, villalta d, tozzoli r. diagnostic accuracy of the anti‐citrulline antibody assay for rheumatoid arthritis. clin chem. 2001;47:1089–93. 7. gupta r, thabah mm, aneja r, chandrasenan pj. usefulness of anti-ccp antibodies in rheumatic diseases in indian patients. indian j med sci. 2009;3:92. 8. kashyap b, tiwari u, garg a, kaur ir. diagnostic utility of anti-ccp antibodies and rheumatoid factor as inflammatory biomarkers in comparison with c-reactive protein and tnf-y in rheumatoid arthritis. trop j med res. 2015;18:5-9. 9. lee dm, schur ph. clinical utility of the anti-ccp assay in patients with rheumatic disease. ann rheum dis. 2003;62:870-874. 10. swedler w, wallman j, froelich cj, teodorescu m. routine measurement of igm, igg and iga rheumatoid factors: high sensitivity, specificity and predictive value for rheumatoid arthritis. j rheumatol. 1997;6:1037-44. 11. arnett fc, edworthy sm, bloch da. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum. 1988;31:315-24. 12. schellekens ga, visser h, de jong ba. the diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. arthritis rheum. 2000;43:155-63. 13. ahmad a, singh tb, usha. an epidemiological study on clinical suspected rheumatoid arthritis rural patients of eastern uttar pradesh, india. irmjcr. 2014;2(1):48-54. 14. bharathi kv, sobharani yn, sridhar c. epidemiological study on rheumatoid arthritis. indian journal of multidisciplinary research. 2009;5(1):17-24. 15. malaviya a, kapoor s, singh r, kumar a, pande i. prevalence of rheumatoid arthritis in the adult indian population. rheumatol int. 1993;13:131-4. 16. ropes mw, bennett ga, cobb s, jacox r, jessar ra. revision of diagnostic criteria for rheumatoid arthritis. bull rheum dis. 1958;9:175. 17. vliet vlieland tp, buitenhuis na, van zeben d, vandenbroucke jp, breedveld fc, hazes jm. sociodemographic factors and the outcome of rheumatoid arthritis in young women. ann rheum dis. 1994;53(12):803-6. https://www.ncbi.nlm.nih.gov/pubmed/?term=vliet%20vlieland%20tp%5bauthor%5d&cauthor=true&cauthor_uid=7864687 https://www.ncbi.nlm.nih.gov/pubmed/?term=buitenhuis%20na%5bauthor%5d&cauthor=true&cauthor_uid=7864687 https://www.ncbi.nlm.nih.gov/pubmed/?term=van%20zeben%20d%5bauthor%5d&cauthor=true&cauthor_uid=7864687 https://www.ncbi.nlm.nih.gov/pubmed/?term=vandenbroucke%20jp%5bauthor%5d&cauthor=true&cauthor_uid=7864687 https://www.ncbi.nlm.nih.gov/pubmed/?term=breedveld%20fc%5bauthor%5d&cauthor=true&cauthor_uid=7864687 https://www.ncbi.nlm.nih.gov/pubmed/?term=hazes%20jm%5bauthor%5d&cauthor=true&cauthor_uid=7864687 https://www.ncbi.nlm.nih.gov/pubmed/7864687 cytokine profile in patients with rheumatoid arthritis falah s. manhal j fac med baghdad vol. 51, no. 4, 2009 433 cytokine profile in patients with rheumatoid arthritis falah s. manhal* phd summary: background: cytokines produced by inflammatory cells play a pivotal role in synovial inflammation and joint destruction in rheumatoid arthritis. patients and methods: the cytokine serum levels were measured by easia (enzyme amplified sensitivity immunoassay) in sera from 50 ra patients, and 40 healthy donors. cytokine levels were compared in different ra subpopulations (positive or negative rheumatoid factor (rf), long term or recent onset disease, high or low disease activity). in addition, the possible association with other demographic and clinical parameters (gender, age, etc) was also analyzed. results: it was demonstrated that il-2, il-6 and ifn-δ levels were elevated in serum samples of ra patients as compared with apparently healthy controls. maximum elevation of tnf-α was recorded in a few number of patient's sera. there were non significant differences between control and ra patient groups in serum tnf-α level. conclusions: assessing the serum il-2, il-6, ifn-δ and tnf-α levels may be helpful in the confirmation of the ra activity. due to the chronic course of this disease, other inflammatory markers must be identified in order to provide early therapeutic strategies to these patients. key words: rheumatoid arthritis, interleukine-2, interleukine-6, tumor necrosis factorα, interferonδ. introduction: rheumatoid arthritis is a chronic inflammatory disease characterized by synovial inflammation and structural damage of joints. although the cause of rheumatoid arthritis (ra) remains unknown, the excessive production of proinflammatory cytokines such as tumour necrosis factor-α (tnf-α), interferon gamma, interleukin-6 (il-6), interleukin-1 (il-1) and others by intra-articular macrophages occupies a critical pathogenic role in the development and progression of the disease [1, 2]. the key role of tnf-alpha led to the development of highly effective new therapies. tnf-alpha inhibitors, such as monoclonal anti-tnf-alpha antibody, infliximab (remicade), have demonstrated efficacy in clinical trials [3]. it is now clear that tnf-alpha blockade, in addition to reducing joint inflammation and leukocyte infiltration, also results in decreased formation of new blood vessels in the synovium. such mechanism of action is now paving the way for the development of the next generation of drugs for treatment of rheumatoid arthritis [4, 5]. recent data were presented mainly from laboratories illustrating the importance of il-15 and il-18 in the induction and perpetuation of chronic inflammation during experimental and clinical rheumatoid synovitis. these findings suggest that antagonists to these cytokines may have a potential therapeutic role against organ-specific autoimmune diseases [6]. the ability of il-1 to drive inflammation and joint erosion and to inhibit tissue repair processes has been clearly established in vitro systems and animal models [5]. although there are elevated levels of il17 in synovial fluid of patients with rheumatoid * dept. of clinical laboratories college of health and medical technology/ baghdad. arthritis, the pathogenic role of il-17 in the development of rheumatoid arthritis remains to be elucidated. there are observations suggest that il-17 plays a crucial role in t cell activation, downstream of il-1, causing the development of autoimmune arthritis [7]. the pathological roles of il-6 have also been clarified in various disease conditions, such as inflammatory, autoimmune, and malignant diseases. on the basis of the findings, a new therapeutic approach to block the il-6 signal using humanized anti-il-6r antibody for rheumatoid arthritis, castleman's disease, and multiple myeloma has been attempted [8]. sufficient evidence exists that establishes a key role for the kallikrein-kinin cascade in inflamed joints. in addition, there appears to be an inter-relationship between cytokines and kinins in the inflammatory process. kinins induce the release of cytokines, and cytokines have been shown to augment the effects of kinins. this may lead to an enhancement and perpetuation of the inflammatory process [8]. interleukin 7 (il7), a t cell growth factor and a regulator of th1 and th2 cytokine production, is produced by synoviocytes from patients with ra [9]. il-12 is a proinflammatory cytokine produced by different antigen presenting cells. it has been shown to exert a critical role in inducing th1 phenotype, thus initiating cellmediated immune responses. it was suggested that il-12, modulating cell and humoral immune responses, is involved in the pathogenesis of immune rheumatic diseases [10].little has been reported about the cytokine profiling in iraq, although data concerning the investigation of ra are substantial. this study was designed to investigate the serum levels of selected cytokines in a group of iraqi patients with rheumatoid arthritis (ra) compared with apparently healthy controls. original article fac med baghdad 2009; vol. 51, no. 4 received mar.2009 accepted june 2009 cytokine profile in patients with rheumatoid arthritis falah s. manhal j fac med baghdad vol. 51, no. 4, 2009 434 patients and methods: this case-control study was conducted during the period from march 2007 to june 2007. blood samples were collected from 50 patients with rheumatoid arthritis (ra). all patients have been submitted to a complete clinical and radiological examination by specialist doctors. the selection of study patients was achieved according to the original criteria of the american rheumatism association revised by klippel et al. [11]. pain intensity and functional disability were evaluated by visual analogue scale (vas) scored by a health professional. a questionnaire form was formulated that involved name, age, gender, clinical history, disease stage, disease duration, family history, residence, socio-economic status, general health condition, smoking, drinking, and any possible previous therapies. patient specimens were gathered from baghdad teaching hospital. a group of 40 apparently healthy blood donors was included as a control from national bank for blood transfusion. blood specimens from patients and healthy controls were properly conveyed to the location of processing and testing at the department of immunology, central laboratories for public health. the serum specimens were obtained and distributed in eppendrof vials and saved in deep freezing at -20 c° until testing. additional laboratory tests for general health assessment were conducted that's including: esr, crp, and rf tests. il-2 easia test: easia (enzyme amplified sensitivity immunoassay was applied using the biosource il-2 test (il-2 easia kits, biosource, europe, s.a, belgium) on microplates according to manufacturer instructions. reference interval of il-2 is 0-0.1 u/ml (detection limits: 0.05 u/ml) [12]. il-6 easia test: easia (enzyme amplified sensitivity immunoassay was applied using the biosource il-6 test (il-6 easia kits, biosource, europe, s.a, belgium) on microplates according to manufacturer instructions. standards or samples containing il-6 react with capture monoclonal antibodies (mabs-1) coated on the microtiter well. reference interval of il-6 is 3.08.5 pg/ml [13]. tnf-α easia test: the principle of biosource tnf-α test tnf-α easia kits, biosource, europe, s.a, belgium) on microplates is similar to il-6 according to manufacturer instruction. reference interval of tnf-α is 0-20 pg/ml. ifn-δ easia test: the principle of biosource ifnδ test ifn-δ easia kits, biosource, europe, s.a, and belgium) on microplates is similar to il-6 according to manufacturer instruction. reference interval of ifn-δ is 0.1-1.2 iu/ml. statistical analysis: data are presented as means ± sd for continuous variables or as number with percentage for categorical variables. differences between continuous variables were assessed by student's t test and differences between categorical variables were assessed by chi-square test. for all analysis, statistical significance was considered at highly significant level p values of < 0.01, significant level p values of < 0.05, and insignificant level p values of > 0.05. all the statistical analysis was done by using spss computer program version 10 and excel application. results: demographic and clinical characteristics of the study patients are shown in table1. fifty patients were included in this study, 22 (44%) males and 28 (56%) females. distribution of patients among age groups showed that 13 (26%) patients were in 16-30 yrs age group, 27 (54%) patients were in 31-50 yrs age group, and 10 (20%) patients were in 51-70 yrs age group. it was shown that patients with long term duration of illness were more than those with recent onset. all serum specimens obtained from study patients showed positive rf results. clinical presentation demonstrated equal number and percentage of study patients with high and low disease activity. table1: demographic and clinical characteristics of the study patients characteristics no. % gender: male -female 22 28 (44%) (56%) age in years: 16-30 31-50 51-70 13 27 10 (26%) (54%) (20%) duration of illness: long term recent onset 34 16 (68%) (32%) rheumatoid factor (rf): positive negative 50 0 (100%) 0 disease activity: high low 25 25 (50%) (50%) the results shown in table 2 indicate that mean of serum il-2 levels in sera from study patients was above normal reference interval, 0.845 iu/ml versus 0-0.1 iu/ml. comparison of differences in serum il-2 levels among patient and control groups indicated highly significant differences between control and ra patient groups. in the same table, it was shown that mean of serum ifn-δ (iu/ml) levels in sera from patients and healthy controls did not rise above normal reference interval, 0.605 (iu/ml) versus 0.1-1.2 iu/ml, although there were remarkable differences between patient and control levels. comparison of differences in serum ifn-δ (iu/ml) levels among patient and control groups indicated only significant differences between control and ra patient groups. cytokine profile in patients with rheumatoid arthritis falah s. manhal j fac med baghdad vol. 51, no. 4, 2009 435 table 2: mean of serum il-2 and ifn-δ (iu/ml) in sera from patients and healthy controls studied groups no. mean sd2 min. max. student's t test pvalue significance il-2 control ra1 40 50 0.767 0.845 0.127 0.183 0.40 0.37 0.99 1.17 0.043 highly sig. (p< 0.01) ifn-δ control ra1 40 50 0.231 0.605 0.054 0.427 0.26 0.31 0.76 2.48 0.013 sig. (p< 0.05) ra1: rheumatoid arthritis. sd2: standard deviation. table 3 indicates that mean of serum il-6 levels in sera from study patients was above normal reference interval, 30.50 pg/ml versus 3.0-8.5 pg/ml. comparison of differences in serum il-6 levels among patient and control groups indicated highly significant differences between control and ra patient groups. in the same table, it was shown that mean of serum tnf-α (pg/ml) levels in sera from patients and healthy controls did not rise above normal reference interval, although there were remarkable differences between patient and control levels. comparison of differences in serum tnf-α (pg/ml) levels among patient and control groups indicated no significant differences between control and ra patient groups. table 3: mean of serum il-6 and tnf-α (pg/ml) in sera from patients and healthy controls studied groups no. mean sd2 min. max. student's t test pvalue significance il-6 control ra1 40 50 6.471 30.50 2.05 19.19 3.04 9.21 9.98 85.20 0.00 highly sig. (p< 0.01) tnf-α control ra1 40 50 11.35 14.54 4.80 9.43 3.35 6.18 19.97 48.62 0.216 non sig. (p>0.05) ra1: rheumatoid arthritis. sd2: standard deviation. discussion: the role of cytokines in the pathogenesis of rheumatoid arthritis (ra) and their significance in clinical monitoring of the disease advancement has been attracting much attention in recent years [14]. the recorded elevations in our study for il-2 and il-6 were absolute and 94%, respectively. it was shown that concentration of il-6 in blood serum of ra patients is many folds higher than those in healthy individuals and might be correlated with the disease activity and its duration. these results were consistent with the observations made by madhok et al [15], and van leeuwen et al, [16]. our study showed that there were no significant differences between healthy control and ra patient groups in serum tnf-α (pg/ml) levels. this observation could be inconsistent with various studies and might be attributable to the limitations of our study, that's including: some of study patients were referred while they in remission stage rather than active phase of the disease, tnf-α levels were not measured serially by quantitative image analysis, or the clinical diagnosis of the studied cases might be doubtful. on the other hand, it was suggested from another study that tnf-α and ll-2 levels are upregulated in ra patients but did not significantly differ from the control group [17]. in contrast, substantial researches indicated that (tnf-α) plays a central role in rheumatoid arthritis (ra) pathogenesis [18]. however, its overproduction may also lead to pathologic changes. the latter situation occurs often in chronic inflammatory diseases such as rheumatoid arthritis. the concept suggesting tumor necrosis factor-alpha as a potential target emerged from experiments showing its key role in inducing many cytokines and mediators of inflammation. several clinical trials targeting this cytokine in rheumatoid arthritis patients with a novel group of antitnf-α factor agents demonstrated reduced synovial inflammation and inhibition of bone and cartilage degradation [19]. little has been reported about the cytokine profiling in iraq, although data concerning the investigation of ra are substantial. al-badry et al showed that there was a significant elevation in il-2 in a group of 80 iraqi patients with ra as compared with systemic lupus erythematosus (sle) and controls. it was concluded from al-badry study that elevation of il-2 was observed in active chronic ra cases rather than in recently diagnosed ra cases [20]. in our study, certain cytokines were selected due to clinical relevance of laboratory routine work in checking these cytokines in our hospitals. in conclusion: il-2, il-6 and ifn-δ levels were elevated in serum samples of ra patients. we suggest that assessing the serum il-2, il-6, ifn-δ and tnf-α levels may be helpful in the confirmation of the ra activity. due to the chronic course of this disease, other inflammatory markers must be identified in order to provide early therapeutic strategies to these patients. references: 1andersson u and erlandsson-harris h. high mobility group box chromosomal protein 1 (hmgb1) is a potent trigger of arthritis. j-internmed. 2007, 255(3): 344-50 2bencsath m, blaskovits a, and borvendeg j. biomolecular cytokine therapy. pathol-oncol-res. 2006, 9(1): 24-9. 3roux ch; brocq o; breuil v; albert c; eullerziegler l. safety of anti-tnf-alpha therapy in rheumatoid arthritis and spondylarthropathies with concurrent b or c chronic hepatitis. rheumatology (oxford). 2006 oct;45(10):1294-7. 4liew f y. the role of innate cytokines in inflammatory response. immunol-lett. 2003, 85(2): 131-4. 5wimett l. tnf-alpha inhibitors offer hope to ra patients. nurse-pract. 2005 28(10): 40-8. cytokine profile in patients with rheumatoid arthritis falah s. manhal j fac med baghdad vol. 51, no. 4, 2009 436 6tanaka f, migita k, kawabe y, aoyagi t, ida h, kawakami a, eguchi k. interleukin-18 induces serum amyloid a (saa) protein production from rheumatoid synovial fibroblasts. life-sci. 2004.13; 74(13): 1671-9. 7taylor p c. antibody therapy for rheumatoid arthritis. curr-opin-pharmacol 2006, 3(3): 323-8. 8 cassim b, mody g, bhoola k. kallikrein cascade and cytokines in inflamed joints. pharmacol-ther. 2002 apr-may; 94(1-2): 1-34. 9van roon j a, glaudemans k a, bijlsma j w, lafeber f p. interleukin-7stimulates tumour necrosis factor alpha and th1 cytokine production in joints of patients with rheumatoid arthritis. annrheum-dis. 2003 feb; 62(2): 113-9. 10spadaro a, scrivo r, rinaldi t, riccieri v, sili scavalli a, taccari e, valesini g. the role of interleukin-12 in immune-mediated rheumatic diseases. reumatismo. 2002 apr-jun; 54(2): 11321. 11klippel j h, grofford l j, stone j h, weyand c m. primer on the rheumatic diseases. 2001, 12th edit, arthritis foundation, atlanta,georgia, pp.6314. 12grau g. serum cytokines. immunology, 1998; 68:196-198. 13moscovitz h. plasma cytokine determination in emergency departmentclinical care medicine, 1994; 22:1102-1107. 14wimett,-l. tnf-alpha inhibitors offer hope to ra patients. nurse-pract. 2006 28(10): 40-8. 15madhok r, crilly a, watson j, cape ha. serum interleukine-6 levels in rheumatoid arthritis: correlation with clinical and laboratory indices of disease activity. ann rheum dis; 1993;52:232-234. 16van leeuween ma, westra j, limburg pc, van riel pl, van rijswijk mh. clinical significance of interleukin-6 measurement in early rheumatoid arthritis. an rheum dis, 1995, 54, 674-677. 17frode t s, tenconi p, debiasi m r, medeiros y s. tumour necrosis factor-alpha, interleukin-2 soluble receptor and different inflammatory parameters in patients with rheumatoid arthritis. mediators-inflamm. 2002 11(6): 345 9. 18mikuls t r, weaver a l. lessons learned in the use of tumor necrosis factor alpha inhibitors in the treatment of rheumatoid arthritis. curr-rheumatolrep. 2003 aug; 5(4): 270-7. 19-ziolkowska m, maslinski w. laboratory changes on anti-tumor necrosis factor treatment in rheumatoid arthritis. curr-opinrheumatol.2003,15(3): 267-73. 20al-badry m n, mohammed s. yousif, and shakir s. ramadhan. assessment of il-2 in some autoimmune disorders. brit. med. jour,jan.2006 vol.11 no.1&2, pp.133-137. meijer jg et al. sa orthop j 2020;19(4) doi 10.17159/2309-8309/2020/v19n4a4 south african orthopaedic journal http://journal.saoa.org.za foot and ankle citation: meijer jg, gräbe jc, greyling p. the outcome of first metatarsophalangeal joint arthrodesis using a locking compression plate. sa orthop j 2020;19(4):218-222. http://dx.doi.org/10.17159/2309-8309/2020/v19n4a4 editor: prof. n saragas, university of the witwatersrand, johannesburg, south africa received: march 2020 accepted: april 2020 published: november 2020 copyright: © 2020 meijer jg. this is an open-access article distributed under the terms of the creative commons attribution licence, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. funding: no funding has been received for this manuscript. conflict of interest: the authors declare they have no conflicts of interest that are directly or indirectly related to the research. abstract background: arthrodesis of the first metatarsophalangeal joint (mtpj) is a common and frequently performed procedure in the practice of orthopaedic foot and ankle surgery. fusion techniques and preferred surgical implants have significantly evolved during recent years. it is however still under debate which surgical device provides the best outcome. one of the modern techniques described includes the use of a dorsal anatomical locking plate. these plates are usually used in combination with an additional compression cross screw across the arthrodesis site. the aim of this study was to assess the outcome of arthrodesis of the first mtpj using a dorsal locking plate without making use of additional compression cross screw fixation. methods: we retrospectively assessed data at an orthopaedic practice specialising in foot and ankle surgery. all patients who had a first mtpj arthrodesis with an anatomical locking plate system between 2010 and 2016 were identified. no additional compression cross screw fixation was done in any of these cases. standard standing dorsoplantar and lateral x-rays of the foot were taken six weeks after surgery. as a primary objective, these radiographs were assessed to determine the rate of radiological union. as a secondary objective, any other complications that occurred in the post-operative period were recorded. results: we included 115 patients in the study. fifteen of these patients underwent bilateral first mtpj arthrodesis surgery, making the total number of feet included in the study 130. of these, 86% (n=99) were female and 14% (n=16) were male. the mean age at the time of surgery was 54.7 years (range 37–74). an observed radiological union rate of 97% at three months after surgery was recorded. a total of four cases (3%) presented with symptomatic non-union. two of these were successfully revised and progressed to union before the nine-month follow-up. one patient had bilateral surgery for severe rheumatoid arthritis with poor bone quality. no union was achieved even after a revision procedure with bone grafting. a keller-type resection arthroplasty was eventually performed in this patient. another secondary complication that was recorded is an overall infection rate of 2%. conclusion: the results of this retrospective study suggest that high union rates and a low incidence of complications can be expected when fusing the first mtpj with the use of a locking plate system without the need for an additional compression cross screw. level of evidence: level 4 keywords: first metatarsophalangeal joint, fusion, arthrodesis, locking plate, union the outcome of first metatarsophalangeal joint arthrodesis using a locking compression plate meijer jg1 , gräbe jc2 , greyling p3 1 mbchb(pret); orthopaedic registrar, department of orthopaedics, kalafong hospital, university of pretoria, pretoria, south africa ² mbchb(pret), fcs orth(sa), mmed orth(pret); orthopaedic surgeon, department of orthopaedics, steve biko academic hospital, university of pretoria, pretoria, south africa ³ mbchb(pret), fcs orth(sa), mmed orth(pret); orthopaedic surgeon, department of orthopaedics, steve biko academic hospital, university of pretoria, pretoria, south africa corresponding author: dr jg meijer, 165c lys street, rietondale, 0084; tel: +27 (12) 318 6400; email: jansiemeijer@gmail.com https://orcid.org/0000-0001-7773-6081 http://orcid.org/0000-0003-1054-0505 https://orcid.org/0000-0002-2535-4812 page 219meijer jg et al. sa orthop j 2020;19(4) introduction arthrodesis of the first metatarsophalangeal joint (mtpj) is a relatively common and frequently performed surgical procedure in the practice of orthopaedic foot and ankle surgery. it was first described by clutton in 1894 for the treatment of severe hallux valgus.1,2 a wide variety of conditions may affect the first ray of the foot, ultimately leading to significant pain and deformity of the first mtpj. in these conditions fusion of the joint has proven to be a successful procedure. common indications include hallux valgus, hallux rigidus, inflammatory arthropathy, salvage procedures after failed previous surgery and neuromuscular conditions.2-6 as noted in the literature, successful first mtpj arthrodesis has been attained with the use of a wide variety of implants ranging from crossed kirschner wires, cerclage wiring, sutures, staples, axially or obliquely placed compression screws, intramedullary steinman pins, external compression clamps, and dorsal plate and screw constructs. most of these methods have demonstrated successful union rates ranging from 70% to 100%.3,4,6,7 a review of the available biomechanical studies in the literature shows that fixation using a lag screw and a dorsal plate construct has the best overall mechanical stability.8,9 small fragment compression plates were frequently used in the past, but because of their size and prominence often caused local irritation and pressure symptoms. because of this complication, a high rate of hardware removal was seen.10,11 this has led to an evolution of the dorsal plate. more recently, anatomically contoured plates with locking and nonlocking screw options became available. these locking plates have a low profile and are pre-contoured to match the anatomy of the patient better, therefore aiming to negate the pressure symptoms as previously experienced with more bulky constructs. with the advances of these anatomical locking plates it is expected that outcomes with regard to union rate and patient satisfaction should also improve. currently these plate-and-screw constructs are mostly being used in conjunction with a compression cross screw across the arthrodesis site.3-5,11 as these constructs are biomechanically more rigid than previously used implants, the question arises whether an additional compression cross screw is still necessary in order to achieve an adequate union rate when fusing the first mtpj. the purpose of this study was to assess the outcome of arthrodesis of the first mtpj using a dorsal locking plate without making use of additional compression cross screw fixation. materials and methods this study was conducted at an orthopaedic practice specialising in foot and ankle surgery. ethical approval was obtained from the research ethics committee of the university of pretoria prior to the commencement of data collection. the data of all patients who underwent first mtpj arthrodesis with a dorsal anatomical locking plate between january 2010 and december 2016 were retrospectively reviewed. patients were included if they were above 18 years and underwent first mtpj arthrodesis with a dorsal locking plate without additional compression cross screw fixation. patients were excluded in the case of revision arthrodesis surgery and if any device other than a dorsal locking plate in isolation was used. surgical procedure all patients were operated by the same surgeon (senior author, jcg). the same surgical technique was consistently used in all cases. an above-ankle tourniquet was applied and inflated to 250 mmhg. the first mtpj was accessed through a standard dorsal approach. joint surface preparation was done with spherical mtpj reamers. all cartilage was removed in order to achieve congruent cancellous surfaces in a concave–convex configuration. multiple small holes were drilled into both surfaces with a 1.9 mm drill bit until adequate bleeding was observed. to determine the correct fusion position, we simulated weightbearing by applying a flat surface to the sole of the foot. we used either the flat bottom area of a large kidney dish or the cover of the plate set. we aimed at a position of slight valgus and slight dorsiflexion of the big toe. there had to be a space of approximately 8–10 mm between the flat surface and the plantar skin of the proximal phalanx. the mtpj was then temporarily stabilised with one kirschner wire, and a titanium dorsal locking plate (acumed®, oregon, usa) was applied. the same plate was used in all cases and had the following specifications: 10° of lateral angulation (hallux valgus angle) and a 4° dorsiflexion angle. for some of the cases, the plate had to be manually bent to either increase or decrease the dorsiflexion angle. this adjustment depended on the alignment of the foot, where slightly more dorsiflexion was required for a cavus foot and a lower dorsiflexion angle was preferred for a flat foot. first, the two distal screws were inserted into the proximal phalanx. manual compression was applied and a compression screw was then inserted into the metatarsal shaft. post-operatively all patients were placed in a protective shoe for six weeks. heel weight-bearing was allowed as tolerated. wounds were inspected and sutures were removed two weeks after surgery. standard standing dorsoplantar and lateral foot radiographs were reviewed to assess the rate of radiological union. x-rays were routinely taken six weeks after surgery. in most patients there were already signs of union at six weeks. if union was not evident at six weeks, we repeated x-rays at three months and again at six months if there was still any doubt. a cut-off of six months was used to allow for timely intervention in cases of symptomatic non-union. union was defined as the presence of osseous trabeculation across the joint interface involving at least three of the four bridging cortices f a ile d p re vi o u s su rg e ry ( 1 3 % ) h a llu x ri g id u s (3 2 % ) h a llu x va lg u s (2 7 % ) h a llu x va ru s (4 % ) n e u ro m u sc u la r in st a b ili ty ( 6 % ) o th e r* (2 % ) 45 40 35 30 25 20 15 10 5 0 figure 1. number of cases performed per surgical indication *hallux flexus, osteochondral lesions and post-traumatic arthritis page 220 meijer jg et al. sa orthop j 2020;19(4) (medial and lateral cortices on the dorsoplantar view and superior and inferior cortices on the lateral view).12 radiological analysis was done by two experienced orthopaedic foot and ankle surgeons (senior authors jcg and pg). general complications other than non-union were also recorded. these mainly included the prevalence of post-operative wound problems and infection. the different indications for arthrodesis were documented as shown in figure 1. statistical analysis continuous variables were described using mean with a range. categorical variables were described using frequency and proportions. the rate of complications was expressed as a proportion of all cases with a 95% confidence interval. all analyses were conducted in excel 2013. results a total of 115 patients were included in the study. fifteen of these patients had bilateral first mtpj arthrodesis surgery, making the total number of feet included in the study 130. the study population consisted of 99 females (86%) and 16 males (14%). fourteen of the 15 patients who underwent bilateral first mtpj arthrodesis were female. these cases were predominantly performed for severe hallux valgus in patients with rheumatoid arthritis or neuromuscular conditions. the mean age at the time of surgery was 54.7 years (range 37–74). the majority of cases were performed for hallux rigidus (n=42; 32%). a total of 35 surgeries (27%) were done for hallux valgus and 17 (13%) for failed previous surgery of the hallux. of the 20 surgeries performed for inflammatory arthropathy, rheumatoid arthritis was the most common indication (n=17). other indications in this group were systemic lupus erythematosus, gout and psoriasis. eight cases were done for neuromuscular conditions (6%) and five cases for hallux varus (4%). the remaining cases were performed for hallux flexus, post-traumatic deformity with secondary joint degeneration and osteochondral lesions (figure 1). a total of seven complications in six patients were recorded, making the overall complication rate 5% (95% ci = 2–9%). each complication in relation to the initial indication for first mtpj arthrodesis is outlined in table i. an overall union rate of 97% was recorded. a total of 126 out of the 130 cases progressed to radiological union by the threemonth follow-up visit. four cases in three patients had evidence of radiological non-union, making the overall non-union rate 3% (95% ci 0.1–6.0%). of these patients, one was operated for hallux rigidus and another for neuromuscular deformity. the third patient developed non-union in both feet following bilateral first mtpj arthrodesis for rheumatoid arthritis. all three patients presented with a symptomatic non-union. two of these cases (hallux rigidus and neuromuscular deformity) were revised with the additional use of bone graft and progressed to radiological union within nine months of the index surgery.  a cross screw was not used in the revision surgeries. the bone quality of the patient with rheumatoid arthritis was too soft and we thought that an additional screw would not have given any more stability. a different plate was used due to the bone destruction of her proximal phalanges. the revision surgery of this rheumatoid patient also failed, and she was eventually revised to a keller-type procedure in both feet. the rate of infection in our study was 2% (95% ci 0.3–4.9%). three patients presented with infection in the post-operative period. arthrodesis in these cases was performed for two patients who had previously failed surgery and for one diabetic patient with severe hallux valgus and charcot arthropathy. the diabetic patient presented with infection nine weeks after surgery and the other two patients at 10 and 14 weeks. the stability of the fusions was evaluated clinically and radiologically, and all three cases were found to be solidly united. successful surgical debridement, hardware removal and antibiotic cover was done in each case, resulting in complete resolution of infection at the six-month followup. no secondary surgeries were required after debridement and hardware removal in all three patients. discussion arthrodesis is defined as the surgical fusion of a diseased joint for the purposes of obtaining pain relief and stability.13 the aim of first mtpj arthrodesis is to achieve an immobile, pain-free joint that contributes to forefoot stability, improved gait and, ultimately, an improvement in quality of daily living.6,14 this procedure is frequently performed in the practice of orthopaedic foot and ankle surgery and has a multitude of indications. common indications include hallux valgus, hallux rigidus, inflammatory arthropathy, salvage procedures after failed previous surgery and neuromuscular conditions.2-6 similar indications for first mtpj arthrodesis were found in our cohort. in the present study we assessed the outcome of first mtpj arthrodesis with the use of a dorsal locking plate system without the use of additional compression cross screw fixation. we had a low overall complication rate of 5%, consisting only of cases that included non-union and infection. this rate is in keeping with reported overall complication rates in the literature ranging between 2.6% and 13.3%.4,5,15 the recorded radiological union rate in this study was 97% (126 of 130). this is consistent with various previous studies that have also demonstrated the reliability of arthrodesis when managing a variety of conditions affecting the first mtpj.5,6,11,14,15 an example of a case where successful union was achieved following arthrodesis for severe hallux rigidus is shown in figures 2 and 3. we recorded a low infection rate of 2%. this is in keeping with infection rates published in the literature. a recent study by fazal et al. showed a 3% infection rate following first mtpj arthrodesis in their cohort of 26 patients.16 two of the infection cases in our study table i: surgical indications in relation to specific complications indication for surgery non-union infection hallux rigidus 1 0 rheumatoid arthritis 2 0 neuromuscular conditions 1 0 failed previous surgery 0 2 hallux valgus & charcot arthropathy 0 1 total 4 3 figure 2. pre-operative dorsoplantar and lateral x-rays showing a case of severe hallux rigidus page 221meijer jg et al. sa orthop j 2020;19(4) had previous failed surgery to the hallux and one had diabetes with charcot arthropathy. these indications for arthrodesis are known to have a higher post-operative infection risk.17 a multitude of different fixation techniques have been described for arthrodesis of the first mtpj.3-6,11,14 these range from crossed kirschner wires, cerclage wiring, sutures, staples, axially or obliquely placed compression screws, and dorsal plate and screw constructs with or without additional lag screw fixation. the most commonly used methods have demonstrated successful union rates ranging from 70% to 100% with an average of 90%.5,11 the ideal fixation device should be mechanically strong, biocompatible, bone-conserving, and should be inserted with a reproducible surgical technique. it should also not require routine removal.11 in a study by denning and van erve it was noted that currently the most used methods of fixation are either dorsal plates or crossed screw constructs.18 claassen et al. compared the outcomes of fixation using either crossed screws or dorsal plates and a cross screw and found a significantly higher non-union rate in the crossed screws group.5 biomechanical studies also indicate that a dorsal plate-and-screw construct provides greater mechanical stability and rigidity.8,9 in a study by politi et al. it was determined that the dorsal plate and lag screw offers the biomechanically strongest fixation method for first mtpj arthrodesis.9 there has been considerable advancement in the design of dorsal plates, with modern constructs having a lower profile and an anatomical contour that is more specific to the first mtpj. recent literature showed that successful union and high patient satisfaction can be expected when fusing the first mtpj with these dorsal anatomical locking plates.3,4,11,12 this construct also allows for earlier weight-bearing and improved rehabilitation in the post-operative period.4 younger and more active patients have also shown to benefit from this form of arthrodesis and can generally expect satisfactory results allowing them to return to a wide variety of sports and activities post-operatively.19 flavin and stephens reported that the combination of a low-profile contoured titanium plate with an additional cross screw and a ball-and-socket bone-end preparation had both operative and biomechanical advantages over other fixation techniques. their patients had significant improvements in outcomes scores, union rates and other radiological parameters.20 due to a concern that these plates may not achieve adequate compression over the fusion site, they are often used in combination with a compression cross screw. the results of the present study, however, show that a very high union rate can still be expected without the addition of a compression cross screw. this rate is comparable or even superior to the fusion rates found in studies assessing the outcomes following arthrodesis with a compression cross screw and dorsal plate.3,4,11 in a more recent retrospective study by cone and his colleagues, it was determined if the addition of a lag screw to a dorsal locking plate would influence union rate and final alignment after fusion.6 they noted that post-operative dorsiflexion angles were better maintained in cases where additional lag screw fixation was used, but found no statistically significant difference in the union rate when comparing fixation with a lag screw and dorsal plate to fixation with a dorsal plate in isolation. their overall union rate, regardless of the fusion method, was 86%. this is significantly lower than the 97% noted in our study. the principle limitation of this study is its retrospective nature. it therefore depended on accurate and reliable data collection. the primary author (jgm) meticulously reviewed all data collected to address this issue. another limitation is the fact that a control group where compression cross screws were used in conjunction with a locking plate could not be added to the study. the rate of radiological union was the main objective in this study. other outcome measures like patient satisfaction, gait analysis and improvement of radiological parameters like dorsiflexion and hallux valgus angles were not assessed. we also did not collect and analyse outcome scores. the use of outcome scores and radiological parameters other than union rate could provide a more encompassing assessment of outcome. the large sample size is considered a strength of this study. conclusion the results of this retrospective study suggest that an overall low complication rate specifically with regard to non-union and infection can be expected when fusing the first mtpj with a dorsal locking plate system. high union rates can be expected without the need for additional compression cross screw fixation. ethics statement approval from the faculty of health science’s ethics committee (university of pretoria) was obtained prior to commencement of the study (protocol number: 504/2017). ethical principles were adhered to, as outlined by the world medical association declaration of helsinki. declaration the authors declare authorship of this article and that they have followed sound scientific research practice. this research is original and does not transgress plagiarism policies. author contributions jgm was responsible for data capture, data analysis, first draft preparation, manuscript preparation and manuscript revision. jcg and pg were study supervisors and responsible for study conceptualisation, manuscript preparation and manuscript revision. orcid meijer jg https://orcid.org/0000-0001-7773-6081 gräbe jc http://orcid.org/0000-0003-1054-0505 greyling p https://orcid.org/0000-0002-2535-4812 references 1. clutton hh. treatment of hallux valgus. st thomas hosp rep. 1894;22:1-12. 2. mahadevan d, korim mt, ghosh a, et al. first metatarsophalangeal joint arthrodesis: do joint configuration and preparation technique matter? foot ankle surg. 2015;21(2):103-107. 3. marudanayagam a, appan sv. first metatarsophalangeal joint fusion using a fyxis plate. j orthop surg. 2014;22(1):35-38. 4. mann jj, moon jl, brosky ta. low-profile titanium plate construct for early weightbearing with first metatarsophalangeal joint arthrodesis. j foot ankle surg. 2013;52(4):460-64. 5. claassen l, plaass c, pastor m, et al. first metatarsophalangeal joint arthrodesis: a retrospective comparison of crossed-screws, locking and non-locking plate fixation with lag screw. arch bone jt surg. 2017;5(4):221-25. figure 3. post-operative x-rays showing successful radiological union https://orcid.org/0000-0001-7773-6081 http://orcid.org/0000-0003-1054-0505 https://orcid.org/0000-0002-2535-4812 page 222 meijer jg et al. sa orthop j 2020;19(4) 6. cone b, staggers jr, naranje s, et al. first metatarsophalangeal joint arthrodesis: does the addition of a lag screw to a dorsal locking plate influence union rate and/or final alignment after fusion. j foot ankle surg. 2018;57(2):259-63. 7. fadel g, rowley d, abboud r. hallux metatarsophalangeal joint arthrodesis: various techniques. the foot. 2002;12:88-96. 8. neufeld sk, parks bg, naseef gs, melamed ea, schon lc. arthrodesis of the first metatarsophalangeal joint: a biomechanical study comparing memory compression staples, cannulated screws, and a dorsal plate. foot ankle int. 2002;23(2):97-101. 9. politi j, hayes j, njus g, bennett gl, kay db. first metatarsalphalangeal joint arthrodesis: a biomechanical assessment of stability. foot ankle int. 2003;24(4):332-37. 10. coughlin mj. arthrodesis of the first metatarsophalangeal joint with minifragment plate fixation. orthopedics. 1990;13:1037-44. 11. latif a, dhinsa bs, lau b, abbasian a. first metatarsophalangeal fusion using joint specific dorsal plate with interfragmentary screw augmentation: clinical and radiological outcomes. foot ankle surg. 2017;25(2):132-36. 12. sung w, kluesner aj, irrgang j, burns p, wukich dk. radiographic outcomes following primary arthrodesis of the first metatarsophalangeal joint in hallux abductovalgus deformity. j foot ankle surg. 2010;49(5):446-51. 13. parker l, singh d. the principles of foot and ankle arthrodesis. j orthop trauma. 2009;23(6):385-94. 14. hamilton ga, ford la, patel s. first metatarsophalangeal joint arthrodesis and revision arthrodesis. clin podiatr med surg. 2009;26(3):459-73. 15. roukis ts. nonunion after arthrodesis of the first metatarsalphalangeal joint: a systematic review. j foot ankle surg. 2011;50(6):710-13. 16. fazal ma, wong jh, rahman l. first metatarsophalangeal joint arthrodesis with two orthogonal two-hole plates. acta orthop traumatol turc. 2018;52(5):363-66. 17. boyd j, chmielewski r. prevention of infection in foot and ankle surgery. clin podiatr med surg. 2019;36(1):37-58. 18. dening j, van er ve rh. ar throdesis of the first metatarsophalangeal joint: a retrospective analysis of plate versus screw fixation. j foot ankle surg. 2012;51(2):172-75. 19. da cunha rj, macmahon a, jones mt, et al. return to sports and physical activities after first metatarsophalangeal joint arthrodesis in young patients. foot ankle int. 2019;40(7):745-52. 20. flavin r, stephens mm. ar throdesis of the first metatarsophalangeal joint using a dorsal titanium contoured plate. foot ankle int. 2004;25(11):783-87. _hlk31606137 _hlk31598127 _hlk30859234 medical/orthopaedic management of rheumatoid arthritis by rm . m a s o n , d .m ., f . r . c .p ., physician, d epartm ent o f p hysical m edicine, the london h ospital. r h e u m a t o i d a rth ritis p resents a n e xtrem ely difficult nroblem o f tre a tm e n t since it is a n in fla m m a to ry p oly­ a rthritis w hich pursues a very v a ria b le c o u rse. s p o n ta n e o u s remissions a re frequent and o c c u r in u p to 5 0 % o f h o sp ital o u t p a t i e n t s o ften leaving n o , o r on ly m in o r, re sid u a. t h e disease is associated with c o n s titu tio n a l d is tu rb a n c e — fever, loss o f w e i g h t , anaem ia, im p a irm e n t o f g ro w th in ch ild ren and w ith the presence o f n o d u les. t h e rh e u m a to id fa c to r is present in th e serum o f so m e 60 to 9 0 % o f a d u lt p a tie n ts. in p atien ts in w hich the disease p u rsu e s a p ro g ressiv e c o u rse the infla m m a to ry changes in th e jo in ts p ro d u c e erosive a nd destructive lesions— the re sulting disa b ility b e in g d u e to the two factors o f inflam m atory disease a n d d e stru ctiv e change. it is very im p o rta n t to assess the s e p a ra te c o n trib u tio n s o f i these tw o p h e n o m e n a before c o n sid erin g a p p ro p ria te tre a t­ ment. t h e inflam m atory c o m p o n e n t h as a ten d e n cy to dim inish in th e course o f tim e, w hilst th e disa b ility d u e to destructive changes tends to increase. t he three fu ndam ental a spects o f tre a tm e n t co m p rise physical th era p y , d rug th era p y a n d o rth o p a e d ic m easures. no single one o f these disciplines should be used in isolation and only a combined team can offer the p a tie n t a properly balanced and co-ordinated program m e o f trea tm e n t. ph y sic a l t h e r a p y d u rin g th e inflam m atory p h a se o f th e disease physical treatm ent sh o u ld consist m ain ly o f sp lin tin g , p re v e n tio n o f deform ity a nd m uscle re-e d u c atio n w ith o u t p ro d u c in g jo in t traum a. h e a t has no m ore th a n a p a lliativ e effect a lth o u g h this may, o f course, be useful. in th e la te r stages o f the disease, how ever, physical th e ra p y will be m o re co n ce rn ed with the m aintenance o f jo in t sta b ility a n d ra n g e o f m o tio n . it is only if a p ro p e r assessm ent o f th e in fla m m a to ry a n d structural changes has been m a d e t h a t p h y sic al th e ra p y can be ratio n alise d . rest as a basic fo rm o f tre a tm e n t should never b e overlooked a n d w h ilst th is m ay n o t influence disease activity, functional c ap a city c a n o ften b e im p ro v e d by prolonged, properly co n tro lled , rest. c o m p le te sp linting o f jo in ts in a p la ste r cast can be c arried o u t w ith o u t a n y risk o f loss o f ra n g e for perio d s o f u p to a t least a m o n th . drug t h e r a p y t hree gro u p s o f drugs a re a v a ila b le : (i) symptomatic. a spirin b utazolidin t an d e ril (ii) “ anti-rheum atic.” g old a nti-m alarials (iii) suppressive. c orticosteroids c o rtic o tro p h in symptomatic d rugs: a spirin is p ro b a b ly the safest a n d m o st v a lu a b le d ru g o f all, b u t it p ro d u ces a mild d egree o f g astric b lee d in g in a lm o st all subjects, usually a m ounting to 5m l. b lo o d loss p e r day. b utazolidin a n d tanderil d o n o t p ro d u c e re g u la r g a s tro ­ intestinal bleeding b ut toxicity is m o re fre q u e n t affecting some 2 5 % o f patients. toxic sy m p to m s d e velop e arly ra th e r th an late so th a t prolonged a d m in is tra tio n is p ossible in subjects w ho sh o w no evidence o f a n y idiosyncrasy. b lood dyscrasias m ay occur b ut if th e d o se is re stric te d to 300 to 400 mg. daily these are sufficiently ra re as n o t to fo rm a contra -in d ic atio n to the use o f th ese drugs. “ a nti-rhuem atic” drugs: m ulti-centre double-blind ciinical trials h a v e show n the value o f gold therapy in rh e u m a to id a rth ritis . t h e th e ra ­ peutic effect is m anifest a fte r 300 to 400 m g. o f g old h ave september, 1963 been a d m in iste re d a n d o n c e this h a s been achieved th e d o se c a n b e re d u ce d o r s p re a d o u t so t h a t p ro lo n g e d a d m in is ­ tra tio n c a n b e m a in ta in e d . a n ti-m a la ria ls such as th e c h lo ro q u in e g ro u p o f d ru g s h ave a ls o been sh o w n in sim ila r , tria ls to h a v e a d istin c t a n ti-rh e u m a tic effect. t h e o nly se rio u s toxic effect re p o rte d h a s been r e tin o p a th y w hich m ay c au se a p e rm a n e n t visual d e fec t. b u t th e risk o f th is c o m p lic a tio n is slight p ro v id ed th e to ta l d u ra tio n o f a d m in is­ tra tio n d oes n o t exceed tw o years. suppressive d rugs: t h e d e cision to use stero id s in rh e u m a to id a rth ritis is a m a jo r o n e a n d sh o u ld n o t be u n d e rta k e n lightly. t h ese d ru g s a re n o m o re th a n su p p re ssiv e a n d th e re is n o p lac e fo r s h o rt co u rses o f tre a tm e n t. u n less th e p h ysician is p re p a re d to c o n tin u e a d m in is tra tio n indefinitely these d rugs sh o u ld n o t be a d m in istere d . t h e d o se m u st be lim ited to o n e w hich c an be c o n tin u e d w ith o u t p ro d u c in g u n a c c e p ta b le side effects, i.e. 10 mg. d aily o f p re d n is o n e o r its e q u iv a le n t in a m ale a n d 7 -5 m g. daily in a p o st-m e n o p a u sa l fem ale. t h e p h y sic ia n m u st b e p re p a re d to a ccep t a m o d ic u m o f c o n tro l in o rd e r to achieve p ro lo n g e d a d m in istra tio n . t h e m o st im p o rta n t side-effect is th e d e v e lo p m e n t o f a rte ritis a n d p e rip h e ra l n e u ro p a th y w hich reflect a w ide­ sp re ad inv o lv e m en t o f th e a rte rio le s th ro u g h o u t th e b o d y a n d o ften h e ra ld a fa ta l o u tco m e . t h e fo u r c o m m o n serious m ista k e s in th e a d m in istra tio n o f ste ro id s a re as fo llo w s:— (i) in c o m p le te diagnosis. (ii) p re m a tu re a d m in istra tio n . (iii) s te ro id h o n e y m o o n . (iv) m e d d leso m e d o se m a n ip u la tio n . t h e fo u r fa ta l m ista k e s a re as fo llo w s:— (i) ineffective in d o c trin a tio n o f th e p a tie n t (including th e issue o f a ste rio d c ard .) (ii) e xcessive c o n tin u e d dosage. (iii) a b r u p t w ith d raw al o f the d ru g . (iv) i n a d e q u a te “ stre ss” cover. o r t h o p a e d ic t r e a t m e n t o rth o p a e d ic tre a tm e n t can o n ly be view ed a g ain st th e b a c k g ro u n d o f th e m edical m a n a g e m e n t o f th e disease. t h e m a jo r p ro b lem is th e b e h a v io u r o f the synovial p a n n u s w hich in v ad e s a n d d e stro y s th e j o in t a ctin g a lm o st as a locally m a lig n a n t lesion. in th e sm a ll jo in ts o f th e h a n d s e arly ra d io lo g ic a l c h an g e in d ic a te s severe d e stru c tio n o f th e jo in t. e a rly sy n o v e c to m y m ay well be a re w a rd in g p r o ­ c e d u re in c e rta in c irc u m stan c es in this s itu a tio n . surgical in te rfere n c e o f th e h a n d sh o u ld n o rm ally b e c arried o u t e ith e r very e a rly to p re v en t d e stru c tiv e c hanges, o r very late as a salvage p ro c e d u re , b u t it sh o u ld n o t be c arried o u t as a ru le d u rin g th e m id d le p h a se s o f th e disease w hen a d a p ta tio n is g ra d u a lly ta k in g place. t h e m o re d ra m a tic a ttr itio n ru p tu re s o f th e e x te n so r, o r occa sio n ally o f th e flexor te n d o n s sh o u ld n o t go un re co g n ise d a n d e a rly re p a ir c a n b e very sa tisfac to ry . surgery to th e w rist how ever c a n be c a rrie d o u t a t a ny stage. f u s io n o f th e ra d io -c a rp a l j o in t to g e th e r w ith excision o f th e lo w e r e n d o f th e u ln a is an o p e ra tio n a lm o s t w ith o u t pe n alty a n d re sto res to th e p a tie n t a sta b le , painless w rist w ith g o o d p r o n a tio n a n d su p in a tio n . surgical p ro c e d u re s to th e feet su c h as excision o f th e m e ta ta rsa l h e a d s a n d o p e ra tio n s o n the toes sh o u ld be c a rrie d o u t e arly ra th e r th a n late. t h is p a rtic u la rly applies if ste ro id s a re t o be a d m in iste re d a n d m ay, in d ee d , re n d e r th em u n n e ce ssa ry . in th e k n e e sy novectom y a n d pate lle cto m y is o fte n a very re w a rd in g p ro c e d u re , a lth o u g h these o p e ra ­ tio n s d o n o t solve th e p ro b le m o f th e u n s ta b le k n e e jo in t. f in a lly , th e p ro b lem o f th e h ip j o in t re m a in s u nsolved. in v o lv e m e n t o f this j o in t is th e m o st disa b lin g in rh e u m a to id a rth ritis a n d u su a lly h e ra ld s severe loss o f fu n c tio n a l capacity. e v en in th e h ip jo in t, h ow ever, s p o n ta n e o u s h e alin g m a y ta k e place. c o n c l u s io n t h e p h y s io th e ra p ist, th e p h y sic ian a n d th e o rth o p a e d ic s u rg e o n sh o u ld all m eet a t th e b e d sid e o f th e p a tie n t w ith c h ro n ic rh e u m a to id a rth ritis a n d w o rk o u t in c o lla b o ra tio n a p r o p e r p ro g ra m m e o f tre a tm e n t. o nly in th is w ay c a n th e p a tie n t w ith progressive rh e u m a to id a rth ritis receive a d e q u a te tre a tm e n t. page 3p h y s i o t h e r a p y r ep ro du ce d by s ab in et g at ew ay u nd er li ce nc e gr an te d by th e p ub lis he r (d at ed 2 01 3. ) sa journal of physiotherapy 2002 vol 58 no 2 15 abstract: objectives: muscle blood flow in the forearm of patients with rheumatoid arthritis and healthy volunteers following treatment with temperature increasing arm baths, mudpacks and shortor decimeter-wave diathermy was studied in this investigation. the aim of the study was to find out the difference of reactive hyperemia between the different temperature methods as well as the influence on the consensual reaction. subjects: eighty patients with rheumatoid arthritis, stage 3 according to steinbrocker, as well as 80 healthy human subjects had been assigned numerically in the four therapyand control groups. patients with diseases influencing the peripheral blood flow were excluded. design: blood flow was measured by venous occlusion plethysmography in both forearms with the subjects lying supine. the application of the local heat therapies had been excluded on the left forearm. the forearm blood flow was monitored before heat therapy, directly after as well as in two further 10 minutes intervals. an analysis of variance was used to determine the influence on blood flow of the response to the heat therapies in patients with rheumatoid arthritis and healthy subjects. results: under homogeneous starting conditions and a statistically uniformed high blood flow in rest the reactive values of blood flow on the left-hand side of application and the right consensual side showed high significant differences between all methods of therapy. differences between the patients and the healthy subjects only showed tendencies with partially lower reactions, concerning the patients with rheumatoid arthritis. all methods of heat therapy caused a statistically provable consensual reaction that turned out smaller after diathermic methods. here the post therapeutic reaction of the blood flow on the side of application was also lower or rather shorter. conclusion: greater differences of the blood flow in rest between the patients with rheumatoid arthritis and healthy subjects could not be observed. temperature increasing arm baths and mud packs induced a provable higher increase of local and consensual forearm blood flow than did diathermic methods. these results lead to the conclusion that there are differences in temperature distribution between the methods of therapy. increasing arm baths and mud packs seem to have a stronger influence on the thermo reflexive skin perfusion. key words: local heat therapy, muscle blood flow, patients with rheumatoid arthritis, healthy subjects. blood flow in the forearm in patients with rheumatoid arthritis and healthy subjects under local thermotherapy mucha c1 1 medizinische rehabilitation und prevention, deutsche sporthochschule, köln. correspondence to: c mucha medizinische rehabilitation und prevention deutsche sporthochschule carl-diem-weg 6 d-50933 köln e-mail: mucha@dshs-koeln.de r e s e a r c h a r t i c l e introduction although local heat treatment is often applied in patients with rheumatoid arthritis (ra), there are only few investigations (mucha, 1998) on the effect of thermotherapy on peripheral blood flow. such investigations are usually carried out in healthy subjects (silverman and pendleton, 1968; wessmann, 1971). oka et al (1971) investigated muscle blood flow in ra patients without clinical manifestations of vascular disease and observed a reduced resting blood flow with simultaneous intensification of the ischaemic reaction. betz (1955) described a more intense warming reacforearm of ra patients and healthy subjects? • what is the consensual reaction of the contralateral arm? • what differences are present in reactive blood flow between the thermotherapies applied? tion to temperature stimuli in patients with rheumatoid arthritis than in healthy subjects. in occasional cases, vascular lesions have also been described. their potential role in the pathogenesis of rheumatoid arthritis has been discussed (skrifvars et al 1969; bowman et al 1975; rothschild and masi, 1982). the following questions were to be investigated in the present study: • what blood flow reactions result after application of various local thermotherapies (rising-temperature arm bath, parafango pack, short-wave 27,12 mhz/11m and decimeter-wave 433,92 mhz/ 69 cm treatment) on the 16 sa journal of physiotherapy 2002 vol 58 no 2 test subjects eighty patients with rheumatoid arthritis in stage 3 according to steinbrocker (1949) and the same number of healthy test subjects were numerically allocated to eight comparison groups each comprising four treatment groups. the lack of an acute inflammatory episode, heart diseases not requiring treatment and polyneuropathies as well as normotensive blood pressure values were further inclusion criteria. in the healthy control groups, age classes and sex distribution were matched to the ra groups. all test subjects were instructed concerning the investigation procedure and method of measurement. methods the investigation took place in a laboratory with constant ambient temperature of 23 24˚c, a relative humidity of 36 38% and always at the same time of day. after an initial 20 minutes rest phase measurements and treatment took place in the supine position of the test subjects. in one patient group (n=20) and a test subject group (n=20) one of these thermotherapies was administered: rising-temperature arm bath, parafango wraps, short-wave diathermy, decimeter wave irradiation. the applications were carried out on the left side and for all modalities 15 minutes long. the arm bath was carried out in a tube bath. the application started at the thermoneutral zone of 36˚c. for the next 15 minutes of application the temperature was raised successively up to 44 45˚c adjusted by a thermostatic inflow. the parafango wrap was 4cm thick and was prepared at a temperature of 55˚c. after cooling down the surface to a tolerated temperature it was applied to the entire forearm from wrist to elbow. short-wave diathermy was applied with a diplode at dose level 4. the ultratherm(r) instrument (firm: siemens) was used. for the decimeter wave irradiation the siretherm(r) instrument (firm: siemens) was taken. the round-field emitter was placed over the forearm at a distance of 5cm at a power level of 4 = 80 watts. forearm blood flow was measured with venous occlusion strain gauge plethysmography simultaneously on both arms before application of heat, immediately afterwards as well as 10 and 20 minutes later. position and application conditions were maintained in accordance with the recommendations of graf (1964). the periquant 3800 (firm: gutman(r)) instrument used for blood flow registration automatically evaluated the volume fluctuations and expressed them as ml/100 ml tissue per minute. statistical analysis the blood flow values obtained at the defined measurement times were recorded. after completion of the measurement series, the data were subjected to statistical analysis together with the demographic data of the test subjects as well as the anamnestic data of the patients. the statistical package of social sciences (spss) program system was used. for descriptive statistics, the data were broken down into initial and progress criteria and their absolute and relative incidences were compared. multivariate analyses of variance were calculated for the group comparison over time. univariate analyses of variance were considered in significant f statistics. the newman-keuls test was used to compare group differences. the chi-square test served as a test of homogeneity for the evaluation for qualitative 25 20 15 10 5 0 rest after therapy 10 min. after therapy 20 min. after therapy [time in min] [m l/ 1 0 0 m l* m in ] ra patients – right side x x x decimeter wave mudpack increasing arm bath short wave figure 1: forearm blood flow after application of the different methods of thermotherapy in the patient groups (n = 20). 25 20 15 10 5 0 rest after therapy 10 min. after therapy 20 min. after therapy [time in min] [m l/ 10 0m l* m in ] healthy candidates – right side x x x decimeter wave mudpack increasing arm bath short wave figure 2: forearm blood flow after application of different forms of thermotherapy in the healthy reference groups (n = 20). sa journal of physiotherapy 2002 vol 58 no 2 17 and group semiquantitative initial data in contingency tables. the t-test was used to compare two linked random samples. the probabilities of error of 5% (p < 0.05) and 1% (p< 0.01) were laid down as significant levels. results the age range in the overall population of the ra patients was 51 60 years. the average age interval of manifestation of rheumatoid arthritis was 6 10 years. the distribution of these features did not show any statistically significant differences in the individual groups compared. the age and sex distribution was also homogeneous between the groups of ra patients and healthy test subjects. the blood flow values on the left (application) side in the therapy groups of ra patients and healthy test subjects at the defined measurement times are shown in figures 1 and 2. the values of the contralateral side are shown in figures 3 and 4. the initial values between 3.8 and 4.3 ml/100 ml tissue per minute in ra patients on the left and between 3.9 and 4.3 ml/100 ml tissue per minute on the right and in healthy subjects between 3.9 and 4.3 ml/100 ml tissue per minute on the left and 4.0 and 4.3 ml/100 ml tissue per minute on the right, do not show any significant differences. after application of thermotherapies, however, the forearm blood flow also shows a highly significant difference in the course between the methods of treatment employed (f9,682 = 267.824; p= 0,00). the post therapeutic reaction values of the blood flow between the patient and healthy subject groups are also different. this also applies to the comparison between the application side and the consensual changes in blood flow on the contralateral side (f1,54 = 54.868; p= 0,00). with isolated consideration of the blood flow reactions after rising-temperature arm bath and parafango packs, these do not differ between the patient and test subject groups up to ten minutes after application. the reactive blood flow in the ra patients remains demonstrably higher than in the healthy subjects only 20 minutes after application of the parafango packs. the consensual reaction after the parafango pack also only shows slight differences compared to the application side both in the patient and in the test subject group. the blood flow reactions after application of the diathermy method (short-wave 27,12 mhz/11m and decimeter-wave 433,92 mhz/69cm) show largely comparable interrelationships in their chronological development and in the consensual reaction. apart from the rise in blood flow immediately after application of the decimeter wave to the left side in healthy subjects, we attained an equally high level over time to that after rising-temperature arm bath. the reaction values of both highfrequency methods are significantly lower than those after parafango packs and rising-temperature arm bath. it is noticed that after application of decimeter waves, the highest blood flow reaction was immediately after application, whereas it was only 10 minutes later in short-wave therapy. the consensual reaction is very much less with both methods and is likewise only unequivocally demonstrable immediately after application of decimeter wave treatment. very much higher reaction values were also attained in the healthy test subjects than in the ra patients on the left (application) side. tables 1 and 2 summarize the post therapeutic mean value comparisons between the thermo25 20 15 10 5 0 rest after therapy 10 min. after therapy 20 min. after therapy [time in min] [m l/ 10 0m l* m in ] ra patients – left side x x x decimeter wave mudpackincreasing arm bath short wave figure 3: consensual blood flow reaction in the contralateral forearm after the individual thermotherapies in the patient groups (n = 20). 25 20 15 10 5 0 rest after therapy 10 min. after therapy 20 min. after therapy [time in min] [m l/ 10 0m l* m in ] healthy candidates – left side decimeter wave mudpack increasing arm bath short wave x x figure 4: consensual blood flow reaction in the contralateral forearm after the individual thermotherapies in the healthy references groups (n = 20). 18 sa journal of physiotherapy 2002 vol 58 no 2 therapy methods at the defined times of measurement on the application side. the corresponding values for the contralateral forearm are shown in tables 3 and 4. discussion venous occlusion plethysmography is a recognized, readily reproducible method of measurement for quantitative detection of segmental blood flow characteristics (greenfield, 1960; altenkirch et al 1989; le jemtel et al 1992). however, the resting blood flow is especially dependent on external influences so that the test must be conducted in accordance with the recommendations by graf (1964) in order to minimize these external influences. in this study, these recommendations include the observations of prior rest phases, positioning criteria, acclimatization, constant times of day. resting blood flow in the forearm is reported to be between 3 and 5 ml/100 l tissue per minute in the literature (bollinger, 1965; altenkirch et al 1989; joyner et al 1990). in this investigation, it was between 3.8 and 4.3 ml/100 ml tissue per minute. differences between the patient and test subject groups and the lateral location were very small and could not be detected statistically. on the other hand, oka et al (1971) found lower resting values and a more intense reactive hyperemia after ischemic stasis in ra patients compared to healthy subjects. although they used the clearance method, the divergencies from the present results cannot necessarily be explained by the technique of measurement. it is more likely that the patient groups were not directly comparable, since oka et al (1971) carried out the investigations in patients with manifest swelling in adjacent joints. in a further comparison, they observed that it is crucially affected by the soft-tissue conditions described. they did not observe dependence of blood flow on parameters of rheumatoid arthritis activity, duration of the disease or age of the patients. these variables were homogeneously distributed in the reference populations, so that they do not have to be taken into consideration for the cause of the reaction after local heat application. it is table 1: mean value comparisons (newman-keuls-test) of the post therapeutic reaction of circulation on the application side within the patients’ collectives. immediate post therapy method short-wave parafango decimeter incr. arm wave bath 7,06 8,00 10,19 13,96 ±s 0,99 1,17 1,16 1,73 parafango (p) 3,56 (0,05) decimeter wave (p) 11,92 (0,01) 8,36 (0,01) incr. arm bath (p) 26,31 (0,01) 22,75 (0,01) 14,38 (0,01) 10 minutes after therapy method decimeter short-wave parafango incr. arm wave bath 5,36 7,36 13,63 17,95 ±s 0,95 1,47 1,54 2,26 short-wave(p) 7,51(0,01) parafango (p) 31,11 (0,01) 23,59 (0,01) incr. arm bath (p) 47,34 (0,01) 39,82 (0,01) 16,23 (0,01) 20 minutes after therapy method decimeter incr. arm short-wave parafango wave bath 4,27 4,35 6,87 9,80 ±s 0,93 0,86 0,70 2,05 incr. arm bath (p) 0,29 short-wave(p) 9,90 (0,01) 9,61 (0,01) parafango (p) 21,07 (0,01) 20,77 (0,01) 11,16 (0,01) table 2: mean value comparisons (newman-keuls-test) of the post therapeutic reaction of circulation on the application side within the healthy collectives. immediate post therapy method short-wave parafango decimeter incr. arm wave bath 7,99 9,78 13,91 14,14 ±s 1,16 1,15 1,55 1,71 parafango (p) 6,86 (0,01) decimeter wave (p) 22,59 (0,01) 15,73 (0,01) incr. arm bath (p) 23,45 (0,01) 16,59 (0,01) 0.85 10 minutes after therapy method decimeter short-wave parafango incr. arm wave bath 7,49 9,19 15,30 17,98 ±s 1,33 1,43 1,92 2,13 short-wave(p) 6,47 (0,01) parafango (p) 29,74 (0,01) 23,26 (0,01) incr. arm bath (p) 39,97 (0,01) 33,49 (0,01) 10,22 (0,01) 20 minutes after therapy method decimeter short-wave incr. arm parafango wave bath 4,20 6,02 7,38 7,51 ±s 1,17 1,11 1,46 1,45 short-wave(p) 6,95 (0,01) incr. arm bath (p) 12,13 (0,01) 5,18 (0,01) parafango (p) 12,63 (0,01) 5,67 (0,01) 0.49 (0,01) [ ] [ ] [ ] [ ] [ ] [ ] sa journal of physiotherapy 2002 vol 58 no 2 19 a matter of discussion as to what extent the differences in resting blood flow described by oka et al (1971) and bowman et al (1975) result from differences attributable to vascular disorders in ra patients. for example, bollinger (1965) attaches no functional diagnostic importance to resting blood flow. on the other hand, reactive hyperemia results evidently reflect the functional capacity and efficiency of the vessels after arterial stasis or after muscular exercise (bollinger, 1965; joyner et al 1990; le jemtel et al 1992). the reactive blood flow values after application of all four thermotherapies in this investigation show distinct differences from resting blood flow and in the time course of the post therapeutic control intervals. the progress differences between the forms of therapy are significant (f9,682 = 267.824; p= 0,00). this applies both to the left (application) side and to the right (consensual) side. the reactive rise after rising-temperature arm bath is greater than after parafango packs, and both exceed the rises observed after diathermy techniques. the one exception is the blood flow value obtained immediately after application of decimeter wave irradiation. moreover, the blood flow reaction persists for the shortest time and the consensual reaction is least pronounced in the case of decimeter wave irradiation. however, the raised blood flow persists for 20 minutes after application of short waves, although there is no discernible tendency to reduction in the ra patients even then, in contrast to the effect observed in the healthy test subjects. it is probable that the greater concomitant warming of the skin/subcutaneous tissue by the short wave is responsible for this effect. since the plethysmographic measurements simultaneously register blood flow in skin and muscle, the especially high increases in blood flow after risingtemperature arm bath and parafango packs may be due to a greatly increased blood flow in the skin. besides intense direct warming, both forms of therapy lead to stimulation of the thermoreceptors in the skin, so that besides the direct effect of heat, an additional neuroreflex vascular reaction can be assumed immediate post therapy method decimeter short-wave parafango incr. arm wave bath 6,04 6,93 8,73 12,05 ±s 1,15 1,01 1,27 1,77 short-wave (p) 3,39 (0,05) parafango (p) 10,25 (0,01) 6,86 (0,01) incr. arm bath (p) 22,92 (0,01) 19,53 (0,01) 12,67 (0,01) 10 minutes after therapy method decimeter short-wave incr. arm parafango wave bath 5,12 7,08 9,45 14,89 ±s 0,89 0,99 1,14 1,56 short-wave (p) 7,59 (0,01) incr. arm bath (p) 16,76 (0,01) 9,16 (0,01) parafango (p) 37,85 (0,01) 30,25 (0,01) 21,08 (0,01) 20 minutes after therapy method decimeter short-wave incr. arm parafango wave bath 4,05 5,35 5,53 7,29 ±s 0,18 0,94 1,34 1,31 short-wave (p) 4,97 (0,01) incr. arm bath (p) 5,64 (0,01) 0,66 parafango (p) 12,34 (0,01) 7,37 (0,01) 6,70 (0,01) table 4: mean value comparisons (newman-keuls-test) of the consensual reaction of the circulation at defined intervals within the healthy collective. immediate post therapy method short-wave decimeter parafango incr. arm wave bath 5,31 5,36 7.08 9.45 ±s 0,97 0,91 1,00 1,14 decimeter wave (p) 0,19 parafango (p) 6,74 (0,01) 6,55 (0,01) incr. arm bath (p) 15,77 (0,01) 15,58 (0,01) 9,03 (0,01) 10 minutes after therapy method decimeter short-wave incr. arm parafango wave bath 4,28 5,84 7,82 12,93 ±s 0,95 1,73 1,21 1,60 short-wave (p) 6,04 (0,01) incr. arm bath (p) 13,74 (0,01) 7,69 (0,01) parafango (p) 33,55 (0,01) 27,50 (0,01) 19,81 (0,01) 20 minutes after therapy method incr. arm decimeter short-wave parafango bath wave 4,04 4,06 4,27 8,19 ±s 0,17 0,17 0,89 1,50 decimeter wave (p) 0,07 short-wave (p) 0,85 0,78 parafango (p) 15,81 (0,01) 15,74 (0,01) 14,96 (0,01) table 3: mean value comparisons (newman-keuls-test) of the consensual reaction of the circulation at defined control intervals within the patients’ collectives. [ ] [ ] [ ] [ ] [ ] [ ] 20 sa journal of physiotherapy 2002 vol 58 no 2 (peters et al 2000). this evidently led to the greater increase of blood flow. results of onabanjo (1978) showed that the warming of the sweat glands already leads to an enzymatically induced kininogen vasodilatation in the skin. this is moreover intensified by the effect of temperature on vasodilatory nerves. this interpretation is also supported by the vigorous consensual reaction which was attained under these forms of treatment. this fact as well as the comparatively smaller areas of application with the diathermy technique caused by technical factors could also explain their lower blood flow reactions, which would however also indicate a more direct warming of the musculature. presupposing that there was dystrophy of the forearm musculature due to ra in patients in an advanced stage of the disease with a long period of manifestation, the greater blood flow reaction attained in the healthy patients with the diathermy technique can also be seen in this context. the characteristics of the blood flow reactions over time after application of decimeter waves also supports the assumption that it is mainly muscle blood flow which is affected in the diathermy technique, since decimeter waves lead to an even more specific warming of the musculature than short wave therapy. according to lehmann et al (1974) an increase in temperature to 40˚c must be exceeded in order to attain a significant vasodilatation; moreover, this temperature must be maintained for three to five minutes. the blood values of the reactive blood flow attained in this investigation indicate that this temperature threshold was exceeded. the influence of all four methods of treatment on the rise of forearm blood flow could be demonstrated. these results have time-course characteristics specific to the form of therapy up to 20 minutes after application. at the same time, they bring about a consensual reaction on the contralateral side. the rising-temperature arm bath and parafango packs lead to a greater increase of blood flow both on the application side and consensually than the diathermy techniques. owing to the lack of differentiation between muscle and skin blood flow due to the techniques of measurement it may be assumed that their higher reaction values are brought about by a greater concomitant reaction of blood flow in the skin. the blood flow values attained after the diathermy techniques employed evidently represent mainly changes in muscle blood flow which are accompanied by a less pronounced consensual reaction. differences between ra patients and healthy subjects could only be demonstrated in part and if anything as a tendency. since they also represent the therapyspecific pattern of heat distribution, it may be assumed that they are a result of lower muscle volumes specific to the disease in ra patients and are less attributable to functional restrictions of the blood vessel that were referred to above all by skrifvars et al (1969), oka et al (1971) and bowman et al (1975). of all four thermotherapies risingtemperature arm baths caused the highest rise in blood flow followed by parafango wraps and decimeter wave irradiation. a comparably small rise in blood flow was registered after shortwave application. after both short wave therapy and parafango wraps the increase of blood flow was still detectable even 20 minutes after therapy though. this indicates a longer distribution of heat after those thermotherapies. the greatest consensual reaction was caused by the rising-temperature arm bath immediately afterwards. parafango wraps showed the greatest consensual reaction ten minutes later. no significant consensual reaction was recorded for decimeter wave irradiation. consequently decimeter wave diathermy causes selective muscular warming. references altenkirch h-u, fransson l, koch g 1989: assessment of arterial and venous circulation in upper and lower extremities by venous occlusion strain gauge plethysmography. normal values and reproducibility. journal of vascular diseases 18: 140-144 betz e 1955 die störung der peripheren durchblutung beim chronischen rheumatismus und deren beeinflussung durch balneotherapeutische maflnahmen. archiv für physikalische therapie 7 : 141-145 bollinger a 1965 bedeutung der venenverschlussplethysmographie in der angiologischen diagnostik. schweizerische medizinische wochenschrift 95 : 1357-1362 bowman s c, turner r, green h d 1975 peripheral vascular disease in rheumatoid arthritis. arthritis and rheumatism 18 : 389 graf k 1964 auswertung und messfehler okklusionsplethysmographischer durchblutungsregistrierungen. acta physiologica scandinavica 60 : 120-135 greenfield a d m 1960 venous occlusion plethysmography. methods in medical research 8 : 293-301 joyner m j, lennon r l, wedel o j, rose s h, shephard j t 1990 blood flow to contracting human muscles: influence of increased sympathetic activity. journal of applied physiology: 1453-1457 lehmann j f, warren cg, scham s m 1974 therapeutic heat and cold. clinical orthopaedics and related research 99 : 207-245 lejemtel th h, katz s, jondeau g, salomon s 1992 critical analysis of methods for assessing regional blood flow and their reliability in clinical medicine. chest the cardiopulmonary journal 101 : 219-222 mucha c 1998 einfluss lokaler wärmetherapie auf die unterarmdurchblutung von patienten mit chronischer polyarthritis. physikalische medizin, rehabilitationsmedizin, kurortmedizin 8 : 71-74 oka m, rekonen a, elomaa i 1971 muscle blood flow in rheumatoid arthritis. acta rheumatologica scandinavica 17 : 203-208 onabanjo a o 1978 studies on vasodilatation in the hand on exposure to heat. european journal of applied physiology 38 : 207-213 peters j k, vishiyasu t, mack g w 2000 reflex control of the cutaneous circulation during passive body core heating in humans. journal of applied physiology 88 : 1756-1764 rothschild b m, masi a t 1982 pathogenesis of rheumatoid arthritis: a vascular hypothesis. seminars in arthritis and rheumatism 12 :11-31 silverman d r, pendleton l 1968 a comparison of the effects of continuous and pulsed short-wave diathermy on peripheral circulation. archives of physical medicine and rehabilitation 49: 429-436 skrifvars b, laine v, wegelius o 1969 sclerosis of the arteries of the extremities in rheumatoid arthritis. acta medica scandinavica 186 : 1445-1472 steinbrocker o, traeger c h, battermann r c 1949 therapeutic criteria in rheumatoid arthritis. journal of the american medical association 140 : 659-662 wessmann h c 1971 effect of shortwave diathermy to the abdomen on peripheral circulation during pregnancy. physical therapy 51 : 43-47 11 49 proceedings s.z.m.c. vol: 36(1): pp. 49-55, 2022. pszmc-834-36-1-2022 1department of pharmacology, ayub medical college, abbottabad 2department of pharmacology, post graduate medical institute, lahore 3department of pathology, punjab institute of cardiology, lahore 4department of pharmacology, sahara medical college, narowal 5department of pharmacology, shalamar medical and dental college, lahore 50 therapeutic effect of berberine versus methotrexate on joint histopathology in a rat model… berberis lycium royle (blr), berberidiaceae group group name arthritis induction treatment day 15 onwards 51 therapeutic effect of berberine versus methotrexate on joint histopathology in a rat model… p p p p p p a-e 52 therapeutic effect of berberine versus methotrexate on joint histopathology in a rat model… a-e p p p p p p p 100 100 0 12.5 75 0 0 12.5 75 25 0 0 62.5 12.5 0 0 0 25 0 0 p value ### ### *** *** ### ^ ### 100 100 0 12.5 37.5 0 0 62.5 75 62.5 0 0 37.5 12.5 0 0 0 0 0 0 p value ### ### *** *** ** # 100 100 0 25 37.5 0 0 37.5 75 50 0 0 50 0 12.5 0 0 12.5 0 0 p value ### ### *** ** ## ** # 100 100 0 12.5 12.5 0 0 12.5 62.5 37.5 0 0 62.5 25 50 0 0 25 0 0 p value ### ### *** *** # *** # p p p p p p 53 therapeutic effect of berberine versus methotrexate on joint histopathology in a rat model… 54 therapeutic effect of berberine versus methotrexate on joint histopathology in a rat model… rhizoma coptidis 55 therapeutic effect of berberine versus methotrexate on joint histopathology in a rat model… kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 1 original research correlates of rheumatoid arthritis among women in albania julia kollcaku1, artur kollcaku2 1 ambulatory health service, polyclinic no. 3, tirana, albania; 2 rheumatology service, university hospital center “mother teresa”, tirana, albania. corresponding author: julia kollcaku, md; address: polyclinic no. 3, rr. “qemal stafa”, tirana, albania; telephone: 00355674039706; e-mail: artur_kollcaku@yahoo.com mailto:g@yahoo.com� kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 2 abstract aim: our aim was to assess the association of rheumatoid arthritis with socio-demographic characteristics and lifestyle factors among women in transitional albania. methods: a cross-sectional study was carried out in 2012-2013 including a sample of 2198 women aged 30 years and above who attended the rheumatology services at primary health care clinics in tirana municipality (mean age: 60.2±9.7 years; overall response rate: 95%). the diagnosis of rheumatoid arthritis was based on the american college of rheumatology/european league against rheumatism (acr/eular) 2010 criteria. in addition, a structured questionnaire was administered to all study participants including information on demographic and socioeconomic characteristics and behavioral factors. binary logistic regression was used to assess the association of rheumatoid arthritis with covariates. results: overall, 437 (19.9%) women were diagnosed with rheumatoid arthritis (both incident and prevalent cases). in multivariable-adjusted models, rheumatoid arthritis was positively and significantly related to older age (or=1.8, 95%ci=1.3-2.6), a lower educational attainment (or=1.4, 95%ci=1.1-1.9), smoking (or=1.5, 95%ci=1.1-2.0), alcohol intake (or=1.9, 95%ci=1.2-3.1) and overweight and obesity (or=1.5, 95%ci=1.22.0 and or=1.6, 95%ci=1.2-2.0, respectively). conclusion: this study provides useful evidence about selected correlates of rheumatoid arthritis among women attending specialized primary health care services in albania. health professionals and policymakers in albania should be aware of the magnitude and consequences of this chronic condition in the adult population. keywords: albania, behavioral factors, rheumatoid arthritis, socio-demographic factors, western balkans. conflicts of interest: none. kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 3 introduction rheumatoid arthritis is currently considered a clinical syndrome across several disease subsets (1), involving inflammatory flows (2), leading to an ultimate common pathway in which persistent synovial inflammation and associated damage to articular cartilage and underlying bone are present (3). overproduction of the tumor necrosis factor is the principal inflammatory process in the pathophysiology of the rheumatoid arthritis (1,4). this leads to overproduction of many cytokines such as interleukin 6, which causes persistent inflammation and joint destruction (1,5). regarding the etiology, genetic factors account for about 50% of the risk of developing rheumatoid arthritis (6,7). these factors are primarily related to either autoantibody-positive disease (acpa-positive) or acpa-negative disease (1). as for the lifestyle factors, smoking is considered the main environmental risk factor (1,8), doubling the risk for development of rheumatoid arthritis (9). rheumatoid arthritis affects 0.5%-1.0% of adults in developed countries (1). women are three times more affected than men (1). however, the prevalence of this condition is positively related to age in both men and women (1). in women, hormonal factors play an additional role as the prevalence of rheumatoid arthritis is highest among individuals over 65 years (10). regarding the incidence of rheumatoid arthritis in developed countries, it varies from 5 to 50 cases per 100,000 adults (11). on the other hand, the prevalence of rheumatoid arthritis displays significant geographical variations (12). the prevalence of this condition is higher in northern europe and north america compared to developing countries (13). such geographical variations have been linked both to different genetic inclinations as well as to different environmental factors which expose individuals from different regions to different levels of risk for rheumatoid arthritis (1). the information about rheumatoid arthritis in former communist countries of the western balkans including albania is scarce. in general, the burden of musculoskeletal disorders has increased in albania in the past few decades (14). the proportion of musculoskeletal disorders comprised only 8.5% of the total burden of disease in albania in 1990, whereas in 2010 it increased to 11.0% (14). there is evidence of a steeper increase in women than in men (3.7% vs. 2.0%, respectively) (14). in this context, the aim of our study was to assess the association of rheumatoid arthritis with demographic and socioeconomic characteristics and lifestyle/behavioral factors among women attending specialized primary health care services in transitional albania. methods this was a cross-sectional study which was carried out in 2012-2013. study population this study included a sample of 2198 women aged 30 years and over who attended the rheumatology services at primary health care clinics in tirana municipality. beforehand, the required sample size was estimated at 1870 women in order to obtain sufficient cases of rheumatoid arthritis among women who attended the rheumatology services in different polyclinics of tirana. in order to increase the study power and account for potential nonresponse, we decided to include 2500 consecutive women aged ≥30 years who attended t he rheumatology services. of these, 198 women were ineligible (too sick to participate), whereas 104 further women refused to participate. the final study sample consisted of 2198 eligible women who agreed to participate (overall response rate: 2198/2302=95%). of 2198 kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 4 women who participated in the study, 437 (19.9%) were diagnosed with rheumatoid arthritis (both incident and prevalent cases). data collection the diagnosis of rheumatoid arthritis was based on the american college of rheumatology/european league against rheumatism (acr/eular) 2010 criteria (15). these criteria consist of joint involvement, serology, acute-phase reactants and duration of symptoms (15). in addition, a structured questionnaire was administered to all study participants including information on selected demographic and socioeconomic characteristics and lifestyle/behavioral factors. socio-demographic factors included age (which in the analysis was dichotomized into: ≤50 years vs. >50 years), marital status (dichotomiz ed into: married vs. not married), employment status (employed and/or retired vs. unemployed) and educational attainment (trichotomized into: low, middle and high). conversely, lifestyle/behavioral factors included smoking, alcohol intake, coffee consumption and tea consumption – all dichotomized into: no vs. yes), as well as the body mass index (bmi, trichotomized into: <25, 25-29.9 and ≥30). statistical analysis independent samples t-test was used to compare the mean ages between women with and without rheumatoid arthritis. conversely, fisher’s exact test was used to compare the distribution of socio-economic characteristics and behavioral factors between women with and without rheumatoid arthritis. on the other hand, binary logistic regression was used to assess the association of rheumatoid arthritis (outcome variable) with socio-economic characteristics and behavioral factors (independent variables). initially, crude (unadjusted) odds ratios (ors) and their respective 95% confidence intervals (95%cis) were calculated. subsequently, multivariable-adjusted models controlling simultaneously for all covariates were run. multivariable-adjusted ors and their respective 95%cis were calculated. in all cases, a p-value of ≤0.05 was considered as statistically significant. statistical package for social sciences (spss, version 15.0) was used for all the statistical analyses. results overall, mean age of study participants was 60.2±9.7 years; median age was 60.0 years (interquartile range: 54.0-67.0 years). on the other hand, the age range was 30-92 years. women diagnosed with rheumatoid arthritis were older than those without rheumatoid arthritis (mean age: 62.0±9.8 years vs. 59.8±9.7 years, respectively; p<0.001) [not shown in the tables]. the distribution of socio-demographic characteristics and lifestyle/behavioral factors of women by rheumatoid arthritis status is presented in table 1. as expected, the proportion of older individuals (over 50 years of age) was higher among women with rheumatoid arthritis compared with their counterparts without this condition (91% vs. 85%, respectively, p<0.001). the proportion of a lower educational level was more prevalent in women with rheumatoid arthritis than in those without rheumatoid arthritis (20% vs. 16%, respectively, p=0.02). conversely, no differences were evident for marital status or employment between the two groups of women. regarding behavioral factors, the prevalence of smoking and alcohol intake were significantly higher in women with rheumatoid arthritis than in those without rheumatoid arthritis (for smoking: 15% vs. 11%, respectively, p=0.02; for alcohol consumption: 7% vs. 4%, respectively, p=0.01). similarly, the prevalence of coffee kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 5 consumption was higher among women with rheumatoid arthritis, but this finding was not statistically significant. the prevalence of tea consumption was similar in the two groupings. on the other hand, the prevalence of overweight and obesity were significantly higher in women with rheumatoid arthritis compared with those without this chronic condition (for overweight: 35% vs. 29%, respectively, whereas for obesity: 30% vs. 25%, respectively; overall p<0.001) [table 1]. table 1. distribution of socio-demographic characteristics and lifestyle/behavioral factors in a sample of albanian women by rheumatoid arthritis status variable total (n=2198) rheumatoid arthritis (n=437) no rheumatoid arthritis (n=1761) p † age-group: ≤50 years >50 years 298 (13.6)* 1900 (86.4) 37 (8.5) 400 (91.5) 261 (14.8) 1500 (85.2) <0.001 employment: employed and/or retired unemployed 1746 (79.4) 452 (20.6) 342 (78.3) 95 (21.7) 1404 (79.7) 357 (20.3) 0.509 marital status: married not married 1793 (81.6) 405 (18.4) 363 (83.1) 74 (16.9) 1430 (81.2) 331 (18.8) 0.408 educational level: low middle/high 364 (16.6) 1834 (83.4) 89 (20.4) 348 (79.6) 275 (15.6) 1486 (84.4) 0.021 smoking: no yes 1947 (88.6) 251 (11.4) 372 (85.1) 65 (14.9) 1575 (89.4) 186 (10.6) 0.015 alcohol intake: no yes 2105 (95.8) 93 (4.2) 407 (93.1) 30 (6.9) 1698 (96.4) 63 (3.6) 0.005 coffee consumption: no yes 758 (34.5) 1440 (65.5) 136 (31.1) 301 (68.9) 622 (35.3) 1139 (64.7) 0.103 tea consumption: no yes 1200 (54.6) 998 (45.4) 242 (55.4) 195 (44.6) 958 (54.4) 803 (45.6) 0.747 bmi: normal weight overweight obesity 971 (44.2) 655 (29.8) 572 (26.0) 155 (35.5) 151 (34.6) 131 (30.0) 816 (46.3) 504 (28.6) 441 (25.0) <0.001 * absolute numbers and their respective column percentages (in parentheses). † p-values from fisher’s exact test. table 2 presents the association of rheumatoid arthritis with demographic and socioeconomic characteristics and behavioral factors. in crude (unadjusted) models, there was a positive association of rheumatoid arthritis with older age (or=1.9, 95%ci=1.3-2.7), a lower educational attainment (or=1.4, 95%ci=1.1-1.8), smoking (or=1.5, 95%ci=1.1-2.0), alcohol intake (or=2.0, 95%ci=1.3-3.1) and overweight and obesity (or=1.5, 95%ci=1.22.0 and or=1.6, 95%ci=1.3-2.3, respectively). furthermore, there was a weak and kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 6 borderline statistically significant relationship with coffee consumption (or=1.2, 95%ci=1.0-1.5). on the other hand, there was no association with employment, marital status, or tea consumption. table 2. association of rheumatoid arthritis with socio-demographic characteristics and lifestyle factors variable crude (unadjusted) models multivariable-adjusted models or (95%ci)* p* or (95%ci)* p* age-group: ≤50 years >50 years 1.00 (reference) 1.88 (1.31-2.70) 0.001 1.00 (reference) 1.82 (1.26-2.62) 0.001 employment: employed and/or retired unemployed 1.00 (reference) 1.09 (0.85-1.41) 0.497 1.00 (reference) 1.08 (0.84-1.43) 0.522 marital status: married not married 1.00 (reference) 0.88 (0.67-1.16) 0.369 1.00 (reference) 0.94 (0.71-1.24) 0.654 educational level: middle/high low 1.00 (reference) 1.38 (1.06-1.80) 0.017 1.00 (reference) 1.44 (1.10-1.89) 0.008 smoking: no yes 1.00 (reference) 1.48 (1.09-2.01) 0.012 1.00 (reference) 1.46 (1.07-2.00) 0.017 alcohol intake: no yes 1.00 (reference) 1.99 (1.27-3.11) 0.003 1.00 (reference) 1.93 (1.22-3.05) 0.005 coffee consumption: no yes 1.00 (reference) 1.21 (0.97-1.51) 0.099 1.00 (reference) 1.16 (0.92-1.46) 0.210 tea consumption: no yes 1.00 (reference) 0.96 (0.78-1.19) 0.714 1.00 (reference) 0.92 (0.74-1.14) 0.421 bmi: normal weight overweight obesity 1.00 (reference) 1.54 (1.23-2.02) 1.59 (1.29-2.28) <0.001 (2)† 0.001 <0.001 1.00 (reference) 1.53 (1.18-1.98) 1.57 (1.22-2.02) <0.001 (2)† 0.001 <0.001 * odds ratios (or: rheumatoid arthritis vs. no rheumatoid arthritis), 95% confidence intervals (95%cis) and p-values from binary logistic regression. † overall p-value and degrees of freedom (in parentheses). upon multivariable-adjustment for all covariates entered simultaneously into the logistic regression models, rheumatoid arthritis was positively and significantly related to older age (or=1.8, 95%ci=1.3-2.6), a lower educational attainment (or=1.4, 95%ci=1.1-1.9), smoking (or=1.5, 95%ci=1.1-2.0), alcohol intake (or=1.9, 95%ci=1.2-3.1) and overweight and obesity (or=1.5, 95%ci=1.2-2.0 and or=1.6, 95%ci=1.2-2.0, respectively) [table 2]. discussion kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 7 this study provides evidence on selected socio-demographic and lifestyle correlates of rheumatoid arthritis among women seeking specialized primary health care in postcommunist albania. older age, low education, smoking, alcohol intake and overweight and obesity were strong and significant “predictors” of rheumatoid arthritis in this sample of adult women in albania. the positive association of rheumatoid arthritis with age which was found in our study is in line with several previous reports (1). on the other hand, the positive relationship with a lower educational attainment is appealing and deserves further investigation in populationbased samples. regarding the environmental factors, we found that, in multivariable-adjusted models, smoking was related to a 50% increase in the risk of rheumatoid arthritis. several studies have indicated that smoking is the main environmental risk factor which increases twice the risk of developing rheumatoid arthritis (9). it has been demonstrated that the effect of smoking is confined to patients with acpa-positive disease (8). nonetheless, at a population level, the risk associated with smoking is quite low and has limited clinical relevance regardless of the pathogenetic importance of this factor (1). in our study, we found a positive relationship between rheumatoid arthritis and alcohol consumption. the risk in women who reported to consume alcohol was about 90% higher than in those who did not report alcohol intake. this finding is generally compatible with previous studies conducted elsewhere (1,16). other potential environmental risk factors for development of rheumatoid arthritis may include coffee intake, vitamin d status, and oral contraceptive use (1,16). we did not assess the effect of vitamin d, or oral contraceptive use, but found a weak and borderline significant relationship with coffee consumption in unadjusted logistic regression models only. in any case, smoking excluded, the effect of environmental factors in the risk of rheumatoid arthritis is controversial (1). at present, there are many unresolved difficulties for individuals suffering from rheumatoid arthritis. yet, the constant introduction of innovative and ground-breaking treatments can overcome many of these difficulties and challenges (1). one of the main requirements involves the characterization of disease subsets in individuals with early onset of rheumatoid arthritis in order to target intensive treatment regimens for those who need them most and are also likely to respond (1). from this perspective, it is suggested that that the new direction of treatment and management of rheumatoid arthritis should be towards short intensive therapeutic courses that result in remission instead of the traditional approach which consist of long-term suppressive treatment strategies (1). this study may have several limitations. the study sample may not be representative of all women who attend rheumatology services at the primary health care level in tirana. nonetheless, we included consecutive women who fulfilled the eligibility criteria in order to ensure, to the extent possible, a representative sample of female primary health care users seeking rheumatology services in tirana municipality. yet, as our study was conducted in tirana only, the sample may not be necessarily representative of all the albanian women. assessment of rheumatoid arthritis was based on the acr/eular 2010 criteria (15), which is reassuring. however, the information related to lifestyle/behavioral factors of women included in this study may have been biased in the context of a traditional and patriarchal society such as albania. notwithstanding this possibility, there is no plausible reason to assume different reporting of behavioral factors in women with and without rheumatoid arthritis. kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 8 in conclusion, this study provides useful information about important correlates of rheumatoid arthritis among women attending specialized primary health care services in postcommunist albania. health professionals and policymakers in albania should be aware of the magnitude and consequences of this chronic condition in the adult population. references 1. scott dl, wolfe f, huizinga twj. rheumatoid arthritis. lancet 2010;376:1094-108. 2. van der helm-van mil ahm, huizinga twj. advances in the genetics of rheumatoid arthritis point to sub-classification into distinct disease subsets. arthritis res ther 2008;10:205. 3. van oosterhout m, bajema i, levarht ew, toes re, huizinga tw, van laar jm. differences in synovial tissue infiltrates between anti-cyclic citrullinated peptidepositive rheumatoid arthritis and anti-cyclic citrullinated peptide-negative rheumatoid arthritis. arthritis rheum 2008;58:53-60. 4. feldmann m, brennan fm, maini rn. rheumatoid arthritis. cell 1996;85:307-10. 5. choy eh, isenberg da, garrood t, et al. therapeutic benefit of blocking interleukin6 activity with an anti-interleukin-6 receptor monoclonal antibody in rheumatoid arthritis: a randomized, double-blind, placebo-controlled, dose-escalation trial. arthritis rheum 2002;46:3143-50. 6. van der woude d, houwing-duistermaat jj, toes re, et al. quantitative heritability of anti-citrullinated protein antibody-positive and anti-citrullinated protein antibodynegative rheumatoid arthritis. arthritis rheum 2009; 60:916-923. 7. barton a, worthington j. genetic susceptibility to rheumatoid arthritis: an emerging picture. arthritis rheum 2009; 61:1441-1446. 8. källberg h, padyukov l, plenge rm, et al, and the epidemiological investigation of rheumatoid arthritis (eira) study group. gene-gene and gene-environment interactions involving hla-drb1, ptpn22, and smoking in two subsets of rheumatoid arthritis. am j hum genet 2007;80:867-75. 9. carlens c, hergens mp, grunewald j, et al. smoking, use of moist snuff, and risk of chronic inflammatory diseases. am j respir crit care med 2010;181:1217-22. 10. charbonnier lm, han wg, quentin j, et al. adoptive transfer of il-10-secreting cd4(+)cd49b(+) regulatory t cells suppresses ongoing arthritis. j autoimmun 2010;34:390-99. 11. pedersen jk, kjaer nk, svendsen aj, hørslev-petersen k. incidence of rheumatoid arthritis from 1995 to 2001: impact of ascertainment from multiple sources. rheumatol int 2009;29:411-5. 12. costenbader kh, chang sc, laden f, puett r, karlson ew. geographic variation in rheumatoid arthritis incidence among women in the united states. arch intern med 2008;168:1664-70. 13. kalla aa, tikly m. rheumatoid arthritis in the developing world. best pract res clin rheumatol 2003;17:863-75. 14. albanian institute of public health. national health report: health status of the albanian population. tirana, albania; 2014. http://www.ishp.gov.al/wpcontent/uploads/2015/01/health-report-english-version.pdf (accessed: march 10, 2016). 15. aletaha d, neogi t, silman aj, funovits j, felson dt, bingham co 3rd, et al. 2010 rheumatoid arthritis classification criteria: an american college of https://www.ncbi.nlm.nih.gov/pubmed/?term=funovits%20j%5bauthor%5d&cauthor=true&cauthor_uid=20872595� https://www.ncbi.nlm.nih.gov/pubmed/?term=felson%20dt%5bauthor%5d&cauthor=true&cauthor_uid=20872595� https://www.ncbi.nlm.nih.gov/pubmed/?term=bingham%20co%203rd%5bauthor%5d&cauthor=true&cauthor_uid=20872595� kollcaku j, kollcaku a. correlates of rheumatoid arthritis among women in albania (original research). seejph 2016, posted: 29 august 2016. doi 10.4119/unibi/seejph-2016-127 9 rheumatology/european league against rheumatism collaborative initiative. arthritis rheum 2010;62:256981. 16. liao kp, alfredsson l, karlson ew. environmental influences on risk for rheumatoid arthritis. curr opin rheumatol 2009;21:279-83. __________________________________________________________ © 2016 kollcaku et al; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. skin november 2017 volume 1 issue 3 copyright 2017 the national society for cutaneous medicine 161 brief articles minocycline-induced agranulocytosis presenting as ecthyma gangrenosum katherine nolan mda, reema ishteiwy phda, john alexis mbchbb,c, martin n. zaiac mdd,e, anna j. nichols md phda a department of dermatology and cutaneous surgery, university of miami miller school of medicine, miami, fl, usa b department of pathology, mount sinai medical center, miami beach, fl, usa c department of pathology, herbert wertheim college of medicine at florida international university, miami, fl, usa d department of dermatology, herbert wertheim college of medicine at florida international university, miami, fl, usa e greater miami skin and laser center, miami beach, fl, usa a 51-year-old woman with a history of rheumatoid factor negative rheumatoid arthritis was admitted for tender abscesses, intermittent spiking fevers, chills, night sweats, malaise, shortness of breath and non-productive cough. she was diagnosed with rheumatoid arthritis 16 years prior to abstract a 51-year-old female with a history of rheumatoid arthritis was admitted for progressive fevers, chills and malaise. five weeks prior, she started minocycline for an ra exacerbation. two weeks after starting minocycline she developed an abscess on her right ankle that was treated at an urgent care facility with ceftriaxone and trimethoprim-sulfamethoxazole. she had minimal improvement so was switched to clindamycin. she developed additional abscesses on her right ankle and right axilla and spiking fevers so she was treated with incision and drainage under general anesthesia. routine blood work obtained prior to surgery revealed severe neutropenia (0.74 103/ul) and the patient was urgently referred to the emergency department. skin biopsy was obtained on admission and revealed ulceration, necrosis, acute and chronic inflammation, vasculitis with vascular thrombosis and rod-shaped bacteria in blood vessel walls and lumina consistent with ecthyma gangrenosum. the following day tissue and blood cultures confirmed the growth of pseudomonas aureginosa. bone-marrow biopsy showed decreased granulopoiesis and hematopoiesis, and a diagnosis of minocycline-induced agranulocytosis presenting as ecthyma gangrenosum was made. the patient had dramatic improvement with appropriate antibiotic therapy, discontinuation of minocycline and initiation of filgrastrim. she has remained healthy without recurrence for 17 months. case report skin november 2017 volume 1 issue 3 copyright 2017 the national society for cutaneous medicine 162 presentation. her rheumatoid arthritis was successfully treated with minocycline 100 mg daily for 6 months and subsequently her disease went into remission. five weeks prior to admission she developed worsening joint pain and swelling. given her prior treatment success with minocycline, her rheumatologist prescribed minocycline 100 mg daily. three weeks prior to admission she developed a tender violaceous papule with surrounding erythema on the right lateral leg. over the following 3 weeks the patient was treated at an urgent care center four times. she was diagnosed with an abscess and treated with intramuscular ceftriaxone, trimethoprimsulfamethoxazole ds twice a day, clindamycin 300 mg three times a day. she developed additional abscesses at distant sites and ultimately was treated with incision and drainage of the abscesses under general anesthesia because of the progression of symptoms despite oral antibiotics. the following day, the patient was notified that blood work drawn prior to surgery revealed an abnormally low white blood cell count. she was referred to the emergency department for further evaluation and admission for intravenous antibiotics. at the time of admission, review of systems was positive for intermittent spiking fevers, chills, night sweats, malaise, shortness of breath and non-productive cough. she denied unintentional weight loss. the patient had a complete blood count 3 months prior, which was normal. on admission the patient had temperature of 98.2f, heart rate 77, respiratory rate 20, blood pressure 113/77. she was initially treated with vancomycin but continued to develop intermittent episodes of fever (tmax 103.2f) a blood pressure of 79/50 so meropenem was added. pertinent laboratory analyses included decreased white blood cell count of 0.74 10 3 /ul, hemoglobin 11.2 g/dl, hematocrit 33.5% (mean cell volume 87.5 fl) and absolute neutrophil count of 0.16 10 3 /ul but increased platelet count 514 10 3 /ul, esr 72 mm/hr, crp 153 mg/l and lactic acid 3 mmol/l. chest x-ray showed peripheral prominent patchy airspace opacities more confluent in the bilateral upper lung fields and bilateral lower lung fields, blunting of the costophrenic angles consistent with multifocal pneumonia and small bilateral pleural effusions. dermatology was consulted and a skin biopsy was obtained for tissue culture and histology. histopathologic analysis of the edge of the right lateral leg ulcer showed ulceration, necrosis, acute and chronic inflammation, vasculitis with vascular thrombosis, and rod-shaped bacteria in blood vessel walls, lumina and the dermis. with this result, vancomycin and meropenem were discontinued, piperacillintazobactam, ciprofloxacin and filgrastrim were initiated. the following day, tissue culture and blood cultures grew pseudomonas aureginosa. peripheral blood flow cytometry failed to reveal a monoclonal b-cell proliferation or an aberrant t-cell immunophenotype and there was no evidence of a discrete blast population. bone marrow biopsy showed decreased granulopoiesis and hematopoiesis with adequate iron stores and without evidence of fibrosis, necrosis, granulomatous inflammation, lymphoma, leukemia, or cells extrinsic to the marrow. based on these findings, a diagnosis of minocycline-induced agranulocytosis presenting as ecthyma gangrenosum was made. skin november 2017 volume 1 issue 3 copyright 2017 the national society for cutaneous medicine 163 minocycline had been discontinued upon admission to the hospital. the patient had a dramatic improvement after piperacillintazobactam, ciprofloxacin and filgrastrim were initiated; vital signs and white blood cell count normalized and repeat blood cultures were negative for growth of p. aeruginosa. figure 1. clinical and histopathological images of ecthyma gangrenosum that developed in a patient treated with minocycline for rheumatoid arthritis. clinical progression of the initial lesion on the right ankle from a dusky, violaceous and tender papule to a necrotic ulcer in the weeks prior to hospitalization, images were taken by the patient (a-c). appearance of the lesions on the day of presentation to the hospital (d-e). histopathology revealed ulceration, necrosis and mixed acute and chronic inflammation (f-g) and vessels with vasculitis and rod shaped bacteria within the lumen and wall (h). minocycline is a semisynthetic tetracycline derivative antibiotic commonly used for the treatment of aerobic and anerobic gram positive and negative bacterial as well as fungal infections. interestingly, minocyline is involved with a wide variety of biological actions other than anti-microbial activity. for example, this drug decreases the production of substances causing inflammation, such as prostaglandins, metalloproteinases and leukotrienes and therefore is used to treat the inflammation associated with acne vulgaris as well as that inherent to autoimmune diseases such as rheumatoid arthritis. although minocycline has been used as a successful treatment for many diseases, serious but rare adverse events such as neutropenia associated autoimmune hepatitis and minocycline-induced lupus with neutropenia have been reported. 1-2 druginduced agranulocytosis occurs with a discussion skin november 2017 volume 1 issue 3 copyright 2017 the national society for cutaneous medicine 164 variety of classes of medications including dipyrone, diclofenac, ticlopidine, antithyroid drugs such as methimazole, carbamazepine, clozapine, and trimethoprim-sulfamethoxazole. a rapid decrease in agranulocytes often occurs within hours 1 to 2 days after administration of the drug. the recovery of the neutrophil count can take an average of 9 days to return to within normal limits (range: 9-24 days). 3 treatment of neutropenia usually consists of supportive care, including broadspectrum antibiotics for febrile patients. 4 delivery of granulocyte colony stimulating factors such as g-csf and gm-csf, has been shown to contribute to rapid healing by partial recovery of neutrophil production and function. 4 the neutropenia caused by minocycline therapy is thought to be immunologic in origin. although the mechanism(s) are unclear, it is thought that the unique metabolism of minocycline may be responsible for the increase in serious adverse events compared to other tetracycline antibiotics. minocycline reactive metabolites may bind tissue macromolecules causing direct cell damage or these metabolites may act as haptens and elicit immune responses in a secondary manner. 5 in conclusion, we present a rare case of minocycline-induced agranulocytosis presenting as ecthyma grangrenosum in a previously healthy patient with rapid resolution upon immediate discontinuation of minocycline and subsequent treatment with piperacillin-tazobactam, ciprofloxacin and filgrastrin. seventeen months after this illness, she remains in excellent health without any recurrences. although neutropenia due to minocycline appears to be a rare adverse event, it is important to draw attention to these known severe sequelae given the frequently with which minocycline is used. immediate discontinuation of the offending medication, initiation of broad-spectrum antibiotics and consideration of the use of granulocyte colony-stimulating factors may help improve outcomes in similar cases. conflict of interest disclosures: none. funding: none. corresponding author: katherine nolan, m.d. university of miami miller school of medicine 1600 nw 10th ave, rosensteil science medical building, room 2023a miami, florida 33136 305-243-4472 (office) 305-243-6191 (fax) katherine.nolan@jhsmiami.org references: 1. garrido-mesa n, zarzuelo a, gálvez j. review. minocycline: far beyond an antibiotic. br j pharmacol. 2013;169(2):337-52. 2. ahmed f, kelsey pr, shariff n. lupus syndrome with neutropenia following minocycline therapy a case report. int j lab hematol. 2008;30(6):543-5. 3. pick, a., nystrom k. nonchemotherapy druginduced neutropenia and agranulocytosis: could medication be the culprit? j pharm pract 2014;27(5):447-452. 4. gregorini m, castello m, rampino t, bosio f, bedino g, esposito p, borroni g., dal canton a. gmcsf contributes to prompt healing of ecthyma gangrenosum lesions in kidney transplant recipient. j nephrol. 2012;25(1):137-9. 5. ishikawa t, sakurai y, tanaka m, daikoku n, ishihara t, nakajima m, miyagawa s, yoshioka a. ecthyma gangrenosum-like lesions in a healthy child after infection treated with antibiotics. pediatr dermatol. 2005;22(5):453-6. http://www.ncbi.nlm.nih.gov/pubmed/23441623 http://www.ncbi.nlm.nih.gov/pubmed/18983308 http://www.ncbi.nlm.nih.gov/pubmed/18983308 http://www.ncbi.nlm.nih.gov/pubmed/18983308 http://www.ncbi.nlm.nih.gov/pubmed/16191001 http://www.ncbi.nlm.nih.gov/pubmed/16191001 layout 1 abughalya ms et al. saoj 2017;16(3) south african orthopaedic journal doi 10.17159/2309-8309/2017/v16n3a1 http://journal.saoa.org.za hip the radiological outcome of uncemented femoral stems in rheumatoid patients undergoing total hip arthroplasty: results at minimum eight years ms abughalya,1 p ryan,2 ie goga3 1 hdip(ortho), fcs ortho(sa), consultant orthopaedic surgeon, department of orthopaedic surgery, university of kwazulu-natal, durban, south africa 2 mbchb(uct), hdip(orth), mmed(ortho), fcorth(sa), specialist orthopaedic surgeon at arthroplasty and sports medicine units, department of orthopaedics, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa 3 md, frcs(edin), fcs orth(sa), professor and head of department orthopaedic surgery, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa corresponding author: dr ms abughalya, email: dr.abughalya@gmail.com, tel: +27 812 711935 abstract background: rheumatoid arthritis is a multi-systemic disease which affects all synovial joints. compromised bone quality may have a negative impact on prosthesis incorporation after total hip replacement, resulting in an increased risk of aseptic loosening and early implant failure. materials and methods: between 2002 and 2007, 49 patients (age 29–80 years) underwent total hip replacement. radiographs were evaluated for signs of loosening or failure. result: of the 49 hips, there was one case of stem subsidence, and one case of aseptic loosening. there were no revisions in the current series. complications included eight (16.3%) intra-operative calcar fractures, which healed uneventfully. conclusion: we report satisfactory radiological results, and revision rate in a group of rheumatoid patients at mid-term review following total hip replacement with uncemented femoral stems. key words: rheumatoid arthritis, uncemented stem, femoral stem, hip, total hip arthroplasty, radiological assessment citation: abughalya ms, ryan p, goga ie. the radiological outcome of uncemented femoral stems in rheumatoid patients undergoing total hip arthroplasty: results at minimum eight years. saoj 2017;16(3):22-26. doi 10.17159/2309-8309/2017/v16n3a1 editor: prof anton schepers, university of the witwatersrand received: march 2016 accepted: october 2016 published: august 2017 copyright: © 2017 abughalya ms et al. this is an open-access article distributed under the terms of the creative commons attribution licence, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. funding: no funding was received for the writing of this article. conflict of interest: the authors declare they have no conflicts of interest relating to this article. introduction rheumatoid arthritis (ra) is a chronic systemic inflammatory disorder characterised by multiple joint involvement. the hip joint is commonly affected (15–30% of patients).1 the disease process, medication and steroid use affect bone properties which lead to changes in bone bio-mechanics.2,3 destruction of articular cartilage, bony deformity and protrusion are commonly seen in rheumatoid hip disease. alterations to joint morphology, as well as bone quality contribute to an increased risk of intraand post-operative complications.4 there is a two-fold increase in the frequency of osteoporosis compared to the general population.5 this results in a decreased potential for bony in/on-growth to uncemented prosthesis, and a decrease in fixation strength, with early aseptic loosening. for this reason, cemented total hip replacement (thr) has been considered by many to be the gold standard for patients with ra.1 with modern cementing techniques, stem survival rates are increased as the relative risk for stem loosening decreases.6 saoj spring 2017 issue.qxp_layout 1 2017/08/06 2:01 pm page 22 abughalya ms et al. saoj 2017;16(3) page 23 the use of uncemented prostheses for hip arthroplasty continues to rise as shown in numerous national joint registries.7,8 although there have been some disappointing results for certain uncemented femoral stem designs used in this patient group – with high rates of subsidence and loosening – others have been more encouraging. with this in mind, we set out to perform a mid-term review of the radiological outcomes of a group of rheumatoid patients who had total hip replacements at our institution. methods and materials this study was conducted at the local arthroplasty unit in durban, kwazulu-natal. from 2002 to 2007, 61 cementless primary total hip replacements were performed in 46 rheumatoid patients. at the time of review, three patients had demised, and nine were lost to follow-up. in the remaining 34 patients, 49 uncemented thrs were performed. at the time of hip arthroplasty, the patients were aged between 29 and 80 years (mean of 58.4 years) (sd=12.12) (table i). there were ten males, and 24 females (graph 1), with 70.8% of the patients being female. fifteen patients had bilateral thrs and 19 patients had unilateral thrs (graph 2). all patients were operated on through a modified harding approach. all patients had index surgery without previous hip surgery. the femoral component was a collarless fully hydroxyapatite coated stem (corail, de puy international ltd) in 34 hips and a tapered grit blasted stem (cls zimmer, warsaw) in four. follow-up ranged from 8 to 13 years (mean of 8.8 years) (sd=1.70). the hips were evaluated radiographically pre-operatively and post-operatively by standardised anteroposterior view and lateral view of the hips. all measurements were taken by using siemens syngo.plaza software, and magnification errors were accounted for. the radiological assessment was made according to the following criteria: • stem subsidence defined as a >5 mm change in distance between the top of the stem and greater trochanter9 as in figure 1 • stem position in relation to the long axis of the femoral shaft in anteroposterior view as shown in figures 2a and b, valgus or varus more than 3 degrees10 figure 1. (a) immediate post-operative and (b) most recent film showing stem subsidence, but solid stem fixation a b figure 2. (a) ap view and (b) lateral view, demonstrating reactive bone formation from the tip of the femoral prosthesis towards the medial and anterior cortices a b table i: descriptive statistics mean (sd) n=51 age (years) 58.43 (12.12) years between surgery and final follow-up 8.82 (1.70) graph 1. sex of patients female male graph 2. uni and bilateral split 20 15 10 5 0 unilateral bilateral saoj spring 2017 issue.qxp_layout 1 2017/08/06 2:01 pm page 23 page 24 abughalya ms et al. saoj 2017;16(3) • loosening of the stem based on gruen zones11 as result of subsidence >5 mm, progressive change in the stem position >3 degrees, or continuous radiolucency more than 2 mm12 (figure 3) • stability of the femoral stem was graded as stable ingrown fixation or unstable according to engh’s criteria13 • femoral remodelling as evidenced by calcar resorption, cortical hypertrophy (figure 4) • stress shielding as evidenced by a decrease of bone quality of the trochanters due to unloading of the proximal femur (figure 5). data was analysed with stata v.12. descriptive statistics were calculated for the data, including mean and standard deviation for continuous data, and frequency and proportions for categorical data. chi-squared tests were used to identify significant associations between categorical variables. a significance level of p<0.05 was deemed significant. results the records and radiographs of 49 patients were retrospectively analysed. the stem position was central in 42 hips, varus in six hips, and valgus in one. there was one (1.9%) case of femoral subsidence (graph 3). this was noted in the six-week follow-up x-ray, and is considered to be due to undersizing of the femoral component (figure 1). there were eight cases (16.3%) of intra-operative calcar cracks, six of which were fixed with charnley wire, and two which were not. all fractures healed uneventfully, with no subsidence. reactive double line formation from bone remodelling was noted in zone 1 in five hips (figure 3), and at the distal tip of the component in 28 hips (57.14%). femoral calcar resorption was noted in two cases (graph 4), mild stress shielding was seen in 17 cases (33%), and more significant stress shielding in two hips (3.9%) (table ii). although, one hip was deemed to be radiologically loose, there were no revisions in this group. chi-squared tests (fischer’s exact) indicated a significant association between femoral calcar resorption and femoral osteolysis (p=0.001), femoral stress shielding (p=0.012) and femoral alignment (p=0.001). however, no significant association was observed between femoral osteolysis and femoral calcar rounding (p=0.12), and femoral alignment (p=0.072). the sex of the patient was not significantly associated with any of the variables. figure 5. bilateral thrs and bilateral proximal stress shielding figure 4. right hip replacement with medial calcar resorption/osteolysis figure 3. ap radiograph of right femoral stem demonstrating reactive double line formation in gruen zone 1 saoj spring 2017 issue.qxp_layout 1 2017/08/06 2:01 pm page 24 abughalya ms et al. saoj 2017;16(3) page 25 discussion total hip replacement has been shown to be a hugely beneficial and cost-effective procedure in rheumatoid patients, improving hip function, relieving pain, and improving quality of life.14,15 however, there remain concerns regarding periarticular bone stock in the rheumatoid patient. akesson et al.16 reported that compared to patients with primary osteoarthritis, rheumatoid patients going for hip arthroplasty showed an increase in bone turnover and a greater amount of unmineralised bone. this is postulated to be the cause of early implant loosening in rheumatoid patients. reported series of the use of cemented thrs in rheumatoid patients show varying degrees of success. poss et al.17 reported results at seven years’ follow-up: 96% of the patients in this study were happy with their outcome, and were clinically improved. despite 31% femoral stem subsidence, there was only a 1.6% revision rate.17 ranawat et al.18 reported 8% femoral loosening at an average 4.3 years’ follow-up with cemented stems. severt et al.19 reviewed 75 rheumatoid patients at an average of 7.4 years, and reported three loose stems, and one revision for aseptic loosening. creighton et al.20 found that cemented prosthesis survival in ra patients is comparable, if not better, compared to other diagnoses. with the introduction of modern cementing techniques, femoral stem survival rates have improved. rasquinha21 reported on 15 thrs at 15-year follow-up, with no aseptic loosening or stem revisions. when considering cemented thr one should remain cognisant of the risk of haemodynamic instability inherent during the cementing process.22,23 deep infection rates may also be increased due to the increase in operating time, and the local deep tissue injury from cement curing.17,19,24-27 the calculated cost of utilising cemented prostheses should include the increase in theatre time, which may be as much as 20 minutes longer on average than uncemented thr.28 cementless fixation of femoral stems has become more popular, and good long-term results can be expected if there is solid biological integration. however, subsidence rates as high as 80% have been reported.29 implant loosening does not necessarily correlate with implant survival. unger et al.25 reported an overall 16.7% revision rate in ra patients at 12.1 year follow-up, with a further 15.7% prostheses being radiologically loose. according to a study from the national finnish register, uncemented prostheses performed better in both juvenile and old rheumatoid patients when compared to matched groups of patients with cemented stems.30,31 in the swedish hip arthroplasty registry between 1992 and 2007, it was shown that the risk of revision for uncemented femoral stems for any reason was lower than that for cemented stems. revision for aseptic loosening was also lower in the uncemented group, after adjusting for age, sex, and underlying diagnosis. however, reasons for revision differed between the two groups, with a higher proportion of the revisions in the uncemented hips being for fractures (17%) compared to that of cemented stems (6% of the revisions in this group).27 table ii: categorical proportions n (%) sex male 14 (29.2) female 34 (70.8) type of femoral implant corail 44 (89.8) zimmer 4 (8.2) unknown 1 (2.0) femoral alignment central 38 (79.2) valgus 1 (2.1) varus 9 (18.7) femoral reactive double line formation none 45 (91.8) superolateral 4 (8.2) femoral stress shielding none 32 (65.3) mild 16 (32.7) moderate 1 (2.0) femoral calcar rounding no 47 (95.9) yes 2 (4.1) femoral calcar resorption no 43 (87.8) mild 5 (10.2) severe 1 (2.0) femoral cortical thickening no 46 (93.9) medial 3 (6.1) femoral osteolysis no 46 (98.0) yes 1 (2.0) femoral reactive bone formation no 20 (40.8) yes 29 (59.2) subsidence no 48 (98.0) yes 1 (2.0) femoral component fixation 49 (100.0) graph 3. indication of femoral subsidence 50 45 40 35 30 25 20 15 10 5 0 no yes subsidence graph 4. indication of femoral calcar resorption 50 45 40 35 30 25 20 15 10 5 0 no yes femoral osteolysis saoj spring 2017 issue.qxp_layout 1 2017/08/06 2:01 pm page 25 page 26 abughalya ms et al. saoj 2017;16(3) although we had one case of subsidence, and one case of femoral component loosening, there were no revisions in this series. several studies report the most common complication in uncemented total hip arthroscopy (tha) is intra-operative fracture, which may or may not need fixation. such fractures do not necessarily affect implant stability or long-term survival.32-35 in this series, there was a 16.3% fracture rate; however, none of these stems subsided, or were radiologically loose. conclusion in this current retrospective case series, we have demonstrated satisfactory radiological results at a minimum of eight years post-surgery. although, previous studies consider cemented femoral prostheses as the gold standard for rheumatoid patients, this study confirms that cementless femoral stem fixation is a safe alternative. compliance with ethics guidelines ethics clearance was approved by brec (biomedical research ethics cpmmittee) which is registered with the south african national health research ethics council (rec-290408-009) and has us office for human research protections (ohrp) federal-wide assurance (fwa678). references 1. thomason hc, lachiewicz pf. the influence of technique on fixation of primary total hip arthroplasty in patients with rheumatoid arthritis. j arthroplasty. 2001;16(5):628-34. 2. trancik t, mills w, vinson n. the effect of indomethacin, aspirin, and ibuprofen on the bone growth into a porous-coated implant. clin orthop. 1989;249:113-21. 3. wheeler dl, vander griend ra, wronski tj, et al. the shortand longterm effects of methotrexate on the rat skeleton. bone. 1995;16: 215-16. 4. zwartele re, witjes s, doets hc, stijnen t, poll rg. cementless total hip arthroplasty in rheumatoid arthritis: a systematic review of the literature. arch orthop trauma surg. 2012;132:535-46. 5. haugeberg g, uhlig t, falch ja, halse ji, kvien tk. bone mineral density and frequency of osteoporosis in female patients with rheumatoid arthritis. arthritis rheum. 2000;43:522-30. 6. onsten i, besjakov j, carlsson as. improved radiographic survival of the charnley prosthesis in rheumatoid arthritis and osteoarthritis: results of new versus old operative techniques in 402 hips. j arthroplasty. 1994;9:3-8. 7. no authors listed. the canadian joint replacement registry. http:// secure.cihi.ca (date last accessed 24 sep. 2015). 8. no authors listed. the swedish joint registry. http://www.shpr.se/ libraries/documents/annualreport_2013-04-1_1.sflb.ashx. 9. garcia-cimbrelo e, cruz-pardos a, madero r, ortega-andreu m. total hip arthroplasty with use of the cementless zweymuller alloclassic system. a ten to thirteen-year follow-up study. j bone joint surg am. 2003;85:296-303. 10. barrack rl, mulroy rd, harris wh. improved cementing techniques and femoral component loosening in young patients with hip arthroplasty. a 12-year radiographic review. j bone joint surg br 1992;74:385-89. 11. gruen ta, mcneice gm, amstutz hc. ‘modes of failure’ of cemented stem-type femoral components: a radiographic analysis of loosening. clin orthop relat res. 1979;141:17-27. 12. sanz-reig j, lizaur-utrilla a, llamas-merino i, lopez-prats f. cementless total hip arthroplasty using titanium, plasma-sprayed implants: a study with 10 to 15 years of follow-up. j orthop surg 2011;19:169-73. 13. engh ca, bobyn jd, glassman ah. porous-coated hip replacement. the factors governing bone ingrowth, stress shielding, and clinical results. j bone joint surg br. 1987;69:45-55. 14. liang m, cullen k, larson m, thompson m, schwartz j, fossel a. cost effectiveness of total joint arthroplasty in osteoarthritis. arthritis rheum 1986;29:937-43. 15. jonsson b, larsson s. functional improvements and costs of hip and knee arthroplasty in destructive rheumatoid arthritis. scand j rheumatol 1991;20:351-57. 16. akesson k, onsten i, obrant kj. periarticular bone in rheumatoid arthritis versus arthrosis: histomorphometry in 103 hip biopsies. acta orthop scand. 1994;65:135-38. 17. poss r, maloney jp, ewald fc, et al. six to 11 year results of total hip arthroplasty in rheumatoid arthritis. clin orthop. 1984;182:109-16. 18. ranawat cs, dorr ld, inglis ae. total hip arthroplasty in protrusio acetabuli of rheumatoid arthritis. j bone joint surg am 1980;62:1059-65. 19. severt r, wood r, cracchiolo iii a, amstutz hc: long-term follow-up of cemented total hip arthroplasty in rheumatoid arthritis. clin orthop 1991;265:13745. 20. creighton mg, callaghan jj, olejniczak jp, johnston rc. total hip arthroplasty with cement in patients who have rheumatoid arthritis: a minimum ten-year follow-up study. j bone joint surg am 1998;80:1439-46. 21. rasquinha vj, dua v, rodriguez ja, ranawat cs. fifteen-year survivorship of a collarless, cemented, normalized femoral stem in primary hybrid total hip arthroplasty with a modified third-generation cement technique. j arthroplasty. 2003;18:86-94. 22. ereth mh, weber jg, abel md, et al. cemented versus noncemented total hip arthroplasty embolism, hemodynamics, and intrapulmonary shunting. mayo clin proc 1992;67:1066-74. 23. nolan jp. arterial oxygenation and mean arterial blood pressure in patients undergoing total hip replacement: cemented versus uncemented components. anaesthesia 1994;49:293-99. 24. maric z, haynes rj. total hip arthroplasty in juvenile rheumatoid arthritis. clin orthop 1993;182:109-19. 25. unger as, inglis ae, ranawat cs, johanson na. total hip arthroplasty in rheumatoid arthritis: a long-term follow-up study. j arthroplasty 1987;2:191-97. 26. witt jd, swann m, ansell bm. total hip replacement for juvenile chronic arthritis. j bone joint surg br 1991;73:770-73. 27. hailer np, garellick g, kärrholm j. uncemented and cemented primary total hip arthroplasty in the swedish hip arthroplasty register. acta orthop 2010;81:34-41. 28. barrack rl, castro f, guinn s. cost of implanting a cemented versus cementless femoral stem. j arthroplasty 1996;11:373-76. 29. smilowicz m, kowalczewski jb. long-term results after total hip replacement; cemented and cementless in young rheumatic patients. chir narzadow ruchu ortop pol 2005;70:319-23. 30. eskelinen a, paavolainen p, helenius i, pulkkinen p, remes v. total hip arthroplasty for rheumatoid arthritis in younger patients. acta orthop 2006;77:853-65. 31. mäkelä kt, eskelinen a, pulkkinen p, virolainen p, paavolainen p, remes v. cemented versus cementless total hip replacement in patients fiftyfive years of age or older with rheumatoid arthritis. j bone joint surg am 2011;93:178-86. 32. turula k, savioja s, innes a, et al. early results of cementless total hip replacement in inflammatory joint disease. scand j rheumat 1987;67:61-63. 33. zwartele r, peters a, brouwers j, olsthoorn p, brand r, doets c. longterm results of cementless primary total hip arthroplasty with a threaded cup and a tapered, rectangular titanium stem in rheumatoid arthritis and osteoarthritis. int orthop 2008;32:581-87. 34. araujo j, gonzalez j, tonino a, international abg study group. rheumatoid arthritis and hydroxyapatite-coated hip prostheses. j arthroplast 1998;13:660-67. 35. arnold p, schüle b, schroeder-boersch h, jani l. review of the results of the aro multicenter study. orthopade 1998;27:324-32. saoj spring 2017 issue.qxp_layout 1 2017/08/06 2:01 pm page 26 south african orthopaedic journal knee doi 10.17159/2309-8309/2022/v21n4a3 nansook a et al. sa orthop j 2022;21(4) citation: nansook a, ryan p. a retrospective comparative study of complications after total knee replacement in rheumatoid arthritis and osteoarthritis patients. sa orthop j. 2022;21(4):207-211. http://dx.doi. org/10.17159/2309-8309/2022/ v21n4a3 editor: dr david north, paarl hospital, western cape, south africa received: february 2022 accepted: may 2022 published: november 2022 copyright: © 2022 nansook a. this is an open-access article distributed under the terms of the creative commons attribution licence, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. funding: no funding was received for this study. conflict of interest: the authors declare they have no conflicts of interest that are directly or indirectly related to the research. abstract background total knee arthroplasty (tka) rates have significantly increased over the past few decades; consequently, so too have the absolute number of complications. international literature expounds on complications in the rheumatoid arthritis (ra) and osteoarthritis (oa) subgroups from the developed context, but these findings cannot be generalised to the developing world, where access to medication, medical facilities and patient characteristics may differ. the purpose of this study was to determine the comparative rates and nature of complications that occur post total knee arthroplasty in ra and oa patients at a single south african quaternary hospital. methods this was a retrospective comparative study of complication rates in two groups following tka at inkosi albert luthuli central hospital (ialch) arthroplasty unit, between 1 january 2014 and 29 february 2020. the data was collected retrospectively, utilising the digitised patient management system at the hospital. data extraction included patient demographics, time to surgery, indication for surgery and early complication rates. descriptive analysis was performed to quantify complications, comparing the two groups. results the chart review yielded 332 cases, comprising 41 ra and 291 oa patients. the mean age of the combined participant group was 65 years (standard deviation [sd] 8). most cases were female (87%, 289 of 332), with males comprising 13% (43 of 332). concomitant human immunodeficiency virus (hiv) was present in 6% of patients (20 of 332), and 24% (80 of 332) had diabetes mellitus (dm). the absolute number of complications was greater in the oa group, where revision surgery was performed in 3% (8 of 291) of cases, infection occurred in 1% (3 of 291), mechanical complications in 3% (10 of 291), and deep vein thrombosis (dvt) in 1% (2 of 291) of cases. there was one complication, a dvt, in the ra group (2%, 1 of 41). conclusion in the current study, complications after tka occurred predominantly in the oa group, 8% (23 of 291) as compared to the ra group, 2% (1 of 41). complications included dvt, revision surgery, infection and mechanical complications. the study was underpowered to detect significant differences between the groups. further large-scale investigation will be required to determine if differences in complication rate are significant when low complication incidence is anticipated. level of evidence: level 4 keywords: arthroplasty, complications, osteoarthritis, rheumatoid, knee a retrospective comparative study of complications after total knee replacement in rheumatoid arthritis and osteoarthritis patients adisha nansook,* paul ryan school of clinical medicine, university of kwazulu-natal, durban, south africa *corresponding author: adisha08@gmail.com introduction the number of total knee arthroplasties (tkas) has increased significantly from 2003 to 2013, with studies in australia and the united states of america (us) estimating that by the year 2050, there will be a 276% increase in tkas being performed each year.1-3 the most common conditions contributing to this burden are primary osteoarthritis and inflammatory arthropathies, and much attention has been paid to comparing them. osteoarthritis (oa) and rheumatoid arthritis (ra) both require tka at the end stage of disease; however, patient factors including age, sex and comorbidities, in addition to the disease processes, vary.4 consequently, one would expect variability in outcomes between these groups. there has been an increase in the number of oa patients requiring tka (almost doubling over 14 years in one us study), compared to ra, which in the us has decreased from 21% in 1991, to as low as 2.4% in 2014.5-8 this could be accounted for by medical advances in understanding the disease process and improving pharmacological treatments available. however, research comparing complication rates has had conflicting findings, with some reports reflecting higher complication rates in ra, while others show no difference between the two.4-8 https://orcid.org/0000-0002-0365-6393 page 208 nansook a et al. sa orthop j 2022;21(4) although international studies have shown a decrease in ra patients requiring tka, these studies are generally based in developed countries.9 in finland for example, fewer ra patients required tka, suggesting aggressive medical therapy may alter the natural history of the disease progression to resemble oa.9 studies are lacking in lowand middle-income countries (lmics) with poorly controlled and late-presenting ra.10 a study based in africa and the middle east reported the incidence of ra was higher than figures quoted in studies internationally (0.06–3.4% as compared to 0.24%).10 this study concluded that lack of community education about the disease, and limited management options available, contribute to the late presentations with more advanced disease.10 the same concern was highlighted in a local study, which demonstrated that despite appropriate treatment, a large proportion of ra patients still develop significant functional impairment.11 no local studies have evaluated complications after tka comparing these two groups. the purpose of this retrospective study was to determine the comparative rates and nature of complications that occur post tka in rheumatoid and osteoarthritis patients at a single south african quaternary hospital. methods this study was a retrospective comparative study of complication rates in two groups of tka patients from inkosi albert luthuli central hospital (ialch) arthroplasty unit, between 1 january 2014 and 29 february 2020. the data was collected retrospectively, utilising the digitised patient management system at the hospital. the individual patient files were accessed to determine eligibility and for data extraction purposes. all patients with ra (identified based on seropositive markers or antibodies) or oa, receiving primary total knee replacement with a minimum two-year followup, were eligible for inclusion. patients presenting at the initial visit for a revision tka, post-traumatic oa, postinfective oa, or other indications for primary tka were excluded. those patients that had required joint-preserving procedures, such as high tibial osteotomies, prior to tka were also excluded. data extraction included patient demographics, comorbidities (human immunodeficiency virus [hiv] and diabetes mellitus [dm]), time to surgery (time from first visit to surgery), indication for tka, follow-up, and the documentation of early complications, defined herein as occurring within two years of tka. complications were subdivided into five categories. revision surgery included all noninfective causes for revision of components, such as malalignment due to subsidence of implants, aseptic loosening and periprosthetic fractures. the second category was infection: if revision was required for infection the case was counted as infection and not included in the revision surgery group, and infection was defined as early if it occurred within four weeks of surgery, or late, if it occurred after four weeks.12 mechanical complications included aseptic loosening awaiting revision or actively monitored cases, medial collateral ligament (mcl) insufficiency, stiffness post tka, and persistent knee pain. for the latter three examples, further surgery may have been performed but did not require component alteration. the final two categories were deep vein thrombosis (dvt), and death. no patients had more than one complication. jamovi (version1.6.23.0) was used for data analysis. categorical data were summarised using counts and percentages. numerical continuous variables were represented with means and standard deviations (sd) when normally distributed, or as medians with interquartile ranges (iqr) where non-parametric. associations between categorical variables were tested using chi-square with exact two-sided significance tests, or fisher’s exact test where counts were less than five in a sample. means were compared between two groups using t-tests. the non-parametric mann– whitney u test was used to compare time to surgery and follow-up between the two groups. table i: demographics of (n = 332) study participants, comparing osteoarthritis and rheumatoid arthritis groups combined oa ra count (%) count (%) count (%) p-value* sex 332 (100%) 291 (88%) 41 (12%) 1.00 female 289 (87%) 253 (87%) 36 (88%) male 43 (13%) 38 (13%) 5 (12%) mean (sd) mean (sd) mean (sd) p-value# age at time of operation (years) 65 (8) 66 (8) 59 (6) < 0.001 count (%) count (%) count (%) p-value* race 332 (100%) 291(88%) 41 (12%) 0.407 asian 96 (29%) 79 (27%) 17 (42%) black african 176 (53%) 157 (54%) 19 (46%) coloured 19 (6%) 18 (6%) 1 (2%) white 40 (12%) 36 (12%) 4 (10%) other 1 (0%) 1 (0%) 0 (0%) comorbidities count (%) count (%) count (%) p-value* hiv 20 (6%) 18 (6%) 2 (5%) 1.000 dm 80 (24%) 68 (23%) 12 (29%) 0.408 median (iqr) median (iqr) median (iqr) p-value** time to surgery (months) 21 (32) 19 (27) 33 (41) 0.003 follow-up (months) 32 (20) 31 (20) 34 (27) 0.533 ra: rheumatoid arthritis; oa: osteoarthritis; sd: standard deviation; hiv: human immunodeficiency virus; dm: diabetes mellitus; iqr: interquartile range * fisher’s exact test; # independent samples t-test; ** mann–whitney u test where significant differences occur between the groups, p-values are bolded. page 209nansook a et al. sa orthop j 2022;21(4) results demographics (table i) the database search yielded 530 patients who had undergone primary total knee replacement for oa or ra. no patients underwent simultaneous bilateral tka, and in patients who received a tka for both knees on separate occasions, each tka was considered a separate case. two hundred and twenty-nine patients were lost to follow-up at approximately six months. a further seven patients were erroneously coded as oa or ra but had post-traumatic osteoarthritis or postinfective osteoarthritis. combined, a total of 236 patients were excluded. a total of 332 patients were included in the study, comprising 41 ra and 291 oa patients. most cases were female (87%, 289 of 332), 13% were males (43 of 332), and this trend was reflected in both subgroups. the mean age of the combined groups was 65 years (sd 8). there was a significant age difference between the ra and oa groups (p < 0.001), with a mean age of 66 years (sd 8) in the oa group and 59 years (sd 6) in the ra group. the median time from initial visit to date of surgery was calculated for each group and was 33 months (iqr 27) for the ra group and 19 months (iqr 41) for the oa group, representing a statistically significant difference (p = 0.003). comorbidities the comorbid diagnoses of hiv and dm were captured to explore their potential confounding effect. six per cent of patients in this study (20 of 332) had concomitant hiv: 6% (18 of 291) in the oa group and 5% (2 of 41) of the ra group. twenty-four per cent of patients (80 of 332) had dm, 23% (68 of 291) and 29% (12 of 41) in the oa and ra groups respectively, but no statistical difference was found between the groups. no patients had both hiv and dm. complications one complication was found in the ra group (2%, 1 of 41): one patient developed a dvt which was treated successfully with oral anticoagulants. the remaining complications occurred in the oa group. revision surgery revision surgery was performed in 3% (8 of 291) of the oa cases. indications for revision were as follows: one patient was revised to a hinged implant due to a medial collateral ligament (mcl) rupture (discovered at the six-week postoperative visit, no documentation of mechanism); three patients were revised for aseptic loosening (the tibial tray only in two cases and in one case both tibial and femoral components); one patient sustained a periprosthetic tibial fracture with consequent tibial component loosening; one patient had postoperative coronal plane instability which was managed by replacing the polyethylene tibial insert component with a size bigger; and two patients with persistent anterior knee pain underwent patella resurfacing (not performed routinely with tka at our centre). infection infection occurred in 1% (3 of 291) of cases. all infections were defined as late infections and underwent surgical intervention. one patient developed infection more than two years post surgery, was treated with a poly exchange and had no recurrence. the remaining two patients required multiple surgeries, developed polymicrobial multidrug-resistant infection and each had a poor local soft tissue envelope. one patient required amputation and the other a knee fusion. mechanical mechanical complications occurred in 3% (10 of 291) of patients. aseptic loosening occurred in three patients: two are awaiting revision surgery and one is being actively monitored. four patients developed stiffness post tka and each required manipulation under anaesthesia (mua), two required arthroscopic release in addition to attain satisfactory range of motion (rom). the first patient had a rom of 0–110° prior to tka, developed stiffness 16 days after tka with a rom of 0–45°, and after mua a rom of 0–110° was restored. the second patient prior to tka had 0–80° rom, almost two years after tka had a rom of 0–45° which required mua and arthroscopic release to restore a 0–95° rom. the third patient initially had 10–90° rom, at 75 days post tka had a 5–30° rom, and following mua and arthroscopic release had a 0–90° rom. the fourth and final patient had a range of 0–90° prior to tka, 60 days post tka a rom of 10–45°, and after mua a rom of 0–110°. one patient was found to have an insufficient mcl post tka and underwent an augmentation with a tendo-achilles autograft. the final two patients reported persistent pain post tka; both patients had a biopsy and debridement performed, one patient had an anterior release performed at the same setting, and both patients had resolution of their symptoms. dvt dvt occurred in 1% of patients (2 of 291) in the oa group. both were successfully treated with oral anticoagulants. there was no statistical difference in the combined or individual complications between the ra and oa subgroups; however, the study was underpowered to detect significance (table ii). there were no reported mortalities in either of the groups. table ii: comparative table of complications between the osteoarthritis and rheumatoid arthritis groups combined (n = 332) oa (n = 291) ra (n = 41) count (%) count (%) count (%) p-value* combined complications 24 (7%) 23 (8%) 1 (2%) 0.335 complications requiring revision surgery 8 (2%) 8 (3%) 0 0.602 infection 3 (1%) 3 (1%) 0 1.000 mechanical complication 10 (3%) 10 (3%) 0 0.618 dvt 3 (1%) 2 (1%) 1 (2%) 0.327 death 0 0 0 ra: rheumatoid arthritis, oa: osteoarthritis *p-values representing results of fisher’s exact test no statistically significant differences were detected between the groups. post-hoc power analysis utilising overall complication rates between the two groups found a power of 17.9% for an alpha of 0.05. page 210 nansook a et al. sa orthop j 2022;21(4) discussion our study included 332 patients undergoing tka for either oa or ra: 89% were oa patients and the remaining 11% were ra. this was a much higher ra representation than a similar us populationbased study where ra represented only 3% of cases requiring tka.13 this could reflect the late-presenting and more advanced disease of rheumatoid patients requiring tka in south africa, as is the case in other lmics.10 it may also be as a result of the large number of patients lost to follow-up, potentially patients with oa. as in a us study, we also found that ra patients typically underwent tka earlier than oa patients (a difference in mean age of seven years in our study), and both studies found this difference to be statistically significant. this correlates with the natural history of ra as the pathological process within the knee generally progresses faster in ra than in primary oa.14 the age difference between the subgroups (59 years in ra vs 66 years in oa) could be a contributing factor to differing complication rates seen in this study; however, no significant difference in age was found for any of the complication groups. we explored dm and hiv as potentially contributory to our complication rate. the seroprevalence of hiv in patients undergoing tka was 6% (20 of 332) and dm was 24% (80 of 332). the hiv percentage was comparative to that of a local study by maharaj et al. who quoted a figure of 6% seroprevalence in their patient population of 1 007 who had undergone tka.15 of the 20 patients with hiv, only one complication occurred in a patient with oa who developed a dvt. this mirrors conclusions drawn in an article by boylan et al., who found that hiv did not increase their tka patients’ overall risk of complications.16 there is limited data on the concomitant diagnosis of dm and ra as factors potentially increasing tka complications rates; however, dm alone has been shown to increase the risk of complication post joint replacement.17 this study looked specifically at the complications following tka. we found an overall 7% (24 of 332) complication rate. in comparison to other studies, our overall complication rate was higher in some instances, likely due to comparative studies having greater patient volumes.18 in alternative studies our complication rates were lower; these studies, however, included complications that we did not investigate in our study, and other primary indications for surgery such as post-traumatic and postinfective oa, which could account for the difference.19 the demographics in these studies also differ, potentially contributing to a different complication profile. one such study showed a low overall complication rate compared to our study, a surprising finding as all patients in this study were over 80 years old. they reported higher mortality (asa > 2), congestive heart failure and chronic obstructive pulmonary disease.20 our overall complication rate was 7% (24 of 332). comparing the complications rates between oa and ra, 7% (23 of 332) of cases complicated and had oa, and 0.3% (1 of 332) complicated and had ra. for the overall complication rate and each of the five subcategories, there was no statistical difference between the groups; however, the study was underpowered to detect statistical significance in the findings. we found that revision surgery occurred exclusively in the oa group and comprised 33% of the overall complications (8 of 24). this finding is in contrast to several research papers that reported higher occurrence in ra subgroups, or no difference compared to oa.4,21 infection too was exclusive to the oa subgroup, accounting for 13% (3 of 24) of all complications in the study. all the infections reported were late infections (presenting after four weeks). we found no infections in the ra group, in contrast to the findings of goodman and hawker who explored the outcomes of total joint arthroplasty in ra patients. they found that tka in ra patients carries an overall risk for infection of 2%, a two-fold increased risk of infection as compared to their oa group.8 with respect to time from initial presentation to surgery, the oa group had a shorter time to surgery compared to the ra group, and this was found to be statistically significant (power of 79%, for an alpha of 0.05). the study was, however, underpowered to determine if this finding was contributory to the differing complication rates. the study had several limitations, most notably the small sample due to a large number of patients being lost to follow-up, with resultant exclusion. the small numbers limited calculation of significant differences between the ra and oa groups and the inability to determine the impact of many potential confounding variables. evaluation was also limited to the variables routinely captured within the digitised patient management system, which restricted comparison to existing studies that explored additional variables. to ascertain if there is a true difference in complication rate among these groups, a larger, prospective study would be required. there appears to be a deficit in research investigating ra patients that require arthroplasty in the south african literature. conclusion in the current study, the overall complication rate was noted to be 7% (24 of 332), predominantly affecting the oa group (7%, 23 of 332) as compared to the ra group (0.3%, 1 of 332). complications included infections, deep vein thrombosis, component revision surgery and mechanical complications. we were unable to detect statistically significant differences due to the small number of complications. further large-scale investigation will be required to determine if differences in complication rate are significant when low complication incidence is anticipated. ethics statement the authors declare that this submission is in accordance with the principles laid down by the responsible research publication position statements as developed at the 2nd world conference on research integrity in singapore, 2010. the study complied with the south african department of health ethics guidelines (2015), and the university of kwazulu-natal policy on research ethics. prior to commencement of this research, the appropriate ethical approval was obtained from the biomedical research ethics committee of ukzn (brec/00000013/2019). declaration the authors declare authorship of this article and that they have followed sound scientific research practice. this research is original and does not transgress plagiarism policies. author contributions an: study conceptualisation, study design, data capture and analysis, first draft manuscript preparation and finalisation of edits to manuscript pr: study conceptualisation, supervising of manuscript preparation and revisions orcid nansook a https://orcid.org/0000-0002-0365-6393 ryan p https://orcid.org/0000-0002-0957-6482 references 1. ackerman in, bohensky ma, zomer e, et al. the projected burden of primary total knee and hip replacement for osteoarthritis in australia to the year 2030. bmc musculoskeletal disorders. 2019;20(1):90. 2. ruiz d, koenig l, dall t, gallo p. the direct and indirect costs to society of treatment for end stage knee osteoarthritis. j bone joint surg am. 2013;95(16):1473-80. 3. inacio mcs, paxton ew, graves se, et al. projected increase in total knee arthroplasty in the united states – an alternative projection model. osteoarthritis cartilage. 2017;25(11):1797-803. 4. ravi b, escott b, shah ps, jenkinson r, et al. a systematic review and meta-analysis comparing complications following total joint arthroplasty for rheumatoid versus for osteoarthritis. arthritis rheum. 2012;64(12):3839-49. 5. mertelsmann-voss cls, pan tj, goodman sm, et al. us trends in rates of arthroplasty for inflammatory arthritis including rheumatoid arthritis, juvenile idiopathic arthritis, and spondyloarthritis. arthritis rheumatol. 2014;66(6):1432-39. https://orcid.org/0000-0002-0365-6393 https://orcid.org/0000-0002-0957-6482 page 211nansook a et al. sa orthop j 2022;21(4) 6. quam jp, michet cj, wilson mg, et al. total knee arthroplasty: a population-based study. mayo clin proc. 1991;66(6):589-95. 7. jonsson h, olafsdottir s, sigurdardottir s, et al. incidence and prevalence of total joint replacements due to osteoarthritis in the elderly: risk factors and factors associated with late life prevalence in the agesreykjavik study. bmc musculoskelet disord. 2016;17(14):8. 8. goodman sm, ravi b, hawker g. outcomes in rheumatoid arthritis patients undergoing total joint arthroplasty. int j clin rheumatol. 2014;9(6):567-74. 9. skyttä et, honkanen pb, eskelinen a, et al. fewer and older patients with rheumatoid arthritis need total knee replacement. scand j rheumatol. 2012(41):345-49. 10. almoallim h, saleh ja, badsha h, et al. a review of the prevalence and unmet needs in the management of rheumatoid arthritis in africa and the middle east. rheumatol ther. 2021(8):1-16. 11. hodkinson b, musenge e, ally m, et al. functional disability and health-related quality of life in south africans with early rheumatoid arthritis. scand j rheumatol. 2012(41):366-74. 12. coughlan a, taylor f. classifications in brief: the mcpherson classification of periprosthetic infection. clin orthop rel res. 2020;478:903-908. 13. stundner o, danninger t, chiu yl, et al. rheumatoid arthritis vs osteoarthritis in patients receiving total knee arthroplasty: perioperative outcomes. j arthroplasty. 2014(29):308-13. 14. lee jk, choi ch. total knee arthroplasty in rheumatoid arthritis. knee surg relat res. 2012;24(1):1-6. 15. maharaj z, pietrzak jrt, sikhauli n, et al. the seroprevalence of hiv in patients undergoing lower limb total joint arthroplasty in south africa. sicot-j. 2020;6:3. 16. boylan mr, basu n, naziri q, et al. does hiv infection increase overall risk of short term adverse outcomes following total knee arthroplasty? j arthroplasty. 2015;30(9):1629-32. 17. qin w, huang x, yang h, shen m. the influence of diabetes mellitus on patients undergoing primary total lower extremity arthroplasty: a systematic review and metanalysis. biomed res int. 2020;2020:6661691. 18. soohoo nf, lieberman jr, ko cy, zingmond ds. factors predicting complication rates following total knee replacement. j bone joint surg am. 2006;88(3):480-85. 19. claus a, asche g, brade j, et al. [risk profiling of postoperative complications in 17,644 total knee replacements]. der unfallchirurg. 2006;109(1):5-12. 20. yohe n, funk a, ciminero m, et al. complications and readmissions after total knee replacement in octogenarians and nonagenarians. geriatr orthop surg rehabil. 2018;9:2151459318804113. https://doi.org/10.1177/2151459318804113 21. ravi b, croxford r, hollands s, et al. increased risk of complications following total joint arthroplasty in patients with rheumatoid arthritis. arthritis rheumatol. 2014;66(2):254-63. https://doi.org/10.1002/art.38231 _hlk114557914 _hlk114558180 _hlk101179130 _hlk101179278 _hlk101179585 _hlk101180489 _hlk101595941 _hlk101595898 _hlk101180936 _hlk101595996 _hlk101596020 _hlk101181384 _hlk86399098 _enref_1 _enref_2 _enref_3 _enref_4 _enref_5 _enref_6 _enref_7 _enref_8 _enref_9 _enref_10 _enref_11 _enref_12 _enref_13 _enref_14 _enref_15 _enref_16 _enref_17 _enref_18 _enref_19 _enref_20 _hlk113966894 _enref_21 6 proceedings s.z.m.c. vol: 35(3): pp. 6-11, 2021. pszmc-801-35-3-2021 efficacy of telmisartan in pristane induced arthritis rat model 1quratulain mehdi, 2noaman ishaq, 3saba batool, 4kulsoom farhat, 5saman omer, 4arooj shahid 1department of pharmacology, quetta institute of medical sciences, quetta 2department of pharmacology, bakhtawar amin medical and dental college, multan 3department of pharmacology, cmh multan institute of medical sciences, multan 4department of pharmacology, army medical college, rawalpindi 5department of pharmacology, mohtarma benazir bhutto shaheed medical college, mirpur ajk abstract introduction: rheumatoid arthritis is one of the most common systemic inflammatory diseases characterized by progressive damage to the joints. there is rising evidence that renin angiotensin aldosterone system signaling is also involved in the inflammatory response in rheumatoid arthritis and its blockers possess anti-arthritic properties. telmisartan is an angiotensin receptor blocker and ppar-γ agonist and its anti-arthritic effects were evaluated. aims & objectives: this experimental study was designed to evaluate the anti-arthritic efficacy of telmisartan in pristane induced rat model of arthritis in adult female rats. place and duration of study: the study was conducted in the department of pharmacology, army medical college, rawalpindi, in collaboration with national institute of health and armed forces institute of pathology from july 2020 to august 2020. material & methods: twenty four (24) adult non-pregnant female sprague dawley rats were divided in three groups (n=8) designated as group a (normal control), group b (arthritic control) and group c (telmisartan group) & maintained on standard diet and water adlibitum. rheumatoid arthritis was induced in each rat of groups b &c by a single intradermal injection of 0.5ml pristane at the base of its tail on day 0 and the disease developed in two weeks. all 3 groups were given distilled water 2.5 ml/kg from 2-4 weeks and group c was additionally given dissolved telmisartan orally at 2 mg/kg/day. anti-arthritic efficacy was determined by assessing arthrogram score and total leukocyte count on day 0, 14 and 28 along with histological examination done at the end of the study. data analysis was done using spss version 25. results: healthy rats in group a maintained a unremarkable arthogram & histogram score & tlc count of 6675±350/μl during the entire study period. telmisartan administration in group c for two weeks after pristane induction resulted in significant reduction in arthrogram score (as) 9.5±3.66, total leukocyte count (tlc) 7350±550/μl and histological score (hs) to 6.88±1.24 as compared to (as) 14.50±2.07, wbc 10150±350/μl & (hs) 10.75±2.05 in group b, left untreated with pristane alone. the intergroup comparison showed significant p value < 0.05 respectively. conclusion: anti-arthritic effect was shown by telmisartan as it was able to ameliorate the changes induced by pristane. key words: anti-arthritic efficacy, telmisartan, pristane, rheumatoid arthritis introduction rheumatoid arthritis (ra) is one of the most common inflammatory disorders occurring due to multiple factors including environmental and genetic risk factors.1 clinical presentation of symmetrical joint involvement comprises of arthralgia, swelling, redness and limited mobility. the risk of development of extra articular manifestations such as keratitis, rheumatoid nodules, pericarditis and pleuritis is increased with poor control or severe disease. it is responsible for continuing disability, premature death and increased socioeconomic burden.2 several strategies are employed in managing the disease. surgical approaches help in improvement of functional activity and structural misalignment of joints but considered as a last resort. physical and occupational therapy helps in relieving the muscular spasm and related joint stiffness.3 current therapies include non steroidal anti-inflammatory drugs (nsaids), glucocorticoids (gcs) and biological agents which improve pain, fatigue and disability with more focus on controlling synovitis.4 treatment modalities with fewer side effects are required for improvement of therapy. 7 efficacy of telmisartan in pristane induced arthritis rat model the renin angiotensin aldosterone system (raas) is well known for its function as regulator of blood pressure but there is accumulating validation that its signaling is involved in reaction to inflammation identified in various disease states. angiotensin ii type 1 receptor (at1r) can be presumed as a potential therapeutic target as rodent models of arthritis have massively expressed its presence in cultured ra-fibroblast like synoviocytes (fls) as well as in hyperplastic synovium. the role of angiotensin converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs) in ameliorating the laboratory and clinical parameters has also been documented.5 telmisartan showed betterment in the parameters of arthritis in a rat model of ra. it exerts anti-inflammatory actions by restraining tumor necrosis factor α (tnf-α) production and inhibiting pro-inflammatory mediators such as interleukin-6 (il-6) and nuclear factor kappa beta (nf-κβ).6 various experiments and studies are being carried out to explore the anti-inflammatory properties of telmisartan with regards to various conditions. the current study is intended to demonstrate the antiarthritic effects of telmisartan in rat model of pristane induced arthritis (pia). material and methods study design: laboratory based experimental study. ethical approval: the ethics review committee of “center for research in experimental and applied medicine (cream)”, army medical college gave the approval for study. setting: it was carried out in department of pharmacology and therapeutics, army medical college (amc), rawalpindi in collaboration with national institute of health (nih), islamabad and armed forces institute of pathology (afip). study was conducted for duration of one month from july to august 2020 and the animals were kept in animal house of nih islamabad. sampling technique and size: twenty four (24) adult non-pregnant female sprague dawley rats weighing 250-300 grams were randomly divided into three groups of eight animals each. experimental setup: the rats were acclimatized for a week before the start of experiment. they were retained in wire topped cages and standard environmental conditions were maintained with temperature ranging between 25±5˚c and 50±10 % humidity. free excess to clean drinking water and standard rodent diet ad libitum was provided during the study duration. twenty four (24) adult nonpregnant female sprague dawley rats were divided in three groups (n=8) and were assigned as group a (normal control): comprised of healthy rats. intradermal injection of 0.1 ml normal saline was given at the base of tail at day 0 and rats were given distilled water 2.5 ml/kg/day orally from 14 to 28 days. group b (arthritic control): rheumatoid arthritis was induced with pristane on day 0 and rats were given distilled water 2.5 ml/kg/day orally from 14 to 28 days after induction. group c (telmisartan group): rheumatoid arthritis was induced with pristane on day 0 and rats were treated with telmisartan manufactured by pharm evo (pvt) ltd in a dose of 2 mg/kg/day orally for two weeks after induction.7 induction of rheumatoid arthritis: induction of ra was done by a single intradermal injection of 0.5 ml of pristane purchased from martin dow marker ltd at the base of tail of each rat in group b and group c.8 arthritis developed gradually over a period of two weeks. assessment of arthritic progression by arthrogram score: the assessment of arthritic progression was done by clinical scoring of joint inflammation on all four limbs on alternate days from week 0 to 4 weeks after pristane injection. it ranged from 0-4, where 0=no swelling and redness; 1=single joint involvement; 2=involvement of two joints; 3= involvement of more than two joints; 4=severe arthritis involving whole paw and digits. the collective minimum score for all four limbs would be 0 and maximum would be 16.9 the arthrogram score at day 0, 14 and 28 were statistically analyzed. hematological analysis: blood was collected from the lateral tail vein on day 0, 14 and 28 and was stored in ethylene diamine tetra acetic acid (edta) tubes for the analysis of total leukocyte count (tlc).10 it was done by using sysmex kx-21 automated analyzer. joint histopathology: all the rats were euthanized by chloroform at the end of the study on day 28. the proximal part of tibia was sliced by using 5.5" angled bone cutter and right ankle joints were removed. slides were prepared with eosin and hematoxylin stains. histological scoring was done according to a set criteria as described by alabarse and his colleagues. for synovial inflammation, five high‐power magnification fields were scored for the percentage of infiltrating mononuclear cells as follows: 0, absent; 1, mild (1-10%); 2, moderate (11-50%); 3, severe (51-100%); for synovial hyperplasia: 0, absent; 1, mild (5-10 layers); 2, moderate (11-50 layers); 3, severe (>20 layers); for 8 efficacy of telmisartan in pristane induced arthritis rat model extension of pannus formation: 0, absent; 1, mild; 2, moderate; 3, severe; for synovial fibrosis: 0, absent; 1, mild (1-10%); 2, moderate (11-50%); 3, severe (51-100%); for cartilage erosion, that is, the percentage of the cartilage surface that was eroded: 0, absent; 1, mild (1-10%); 2, moderate (11-50%); 3, severe (51-100%); and for bone erosion: 0, none; 1, minor erosion(s) observed only at high‐power magnification fields; 2, moderate erosion(s) observed at low magnification; 3, severe transcortical erosion(s).11 statistical analysis: the statistical package for the social sciences (spss) version 25 was used for data analysis with quantitative data expressed as mean + standard deviation (sd). one way of analysis of variance (anova) was applied to observe the mean difference among control and experimental group and compared by post hoc tukey’s test. the difference between two observations was considered significant if the p value was equal to or less than 0.05 (p ≤0.05). results the arthrogram score was zero in all the groups at day 0. it remained zero in group a throughout the study duration whereas in group b it reached a mean of 13.5±2.97 by day 14 and reached a mean of 14.5±2.07 by day 28. fig-1 shows a rat of group b with arthrogram score 12. arthrogram score of group c reached a mean of 12.5±3.96 by day 14 and was declined after treatment with telmisartan by day 28 with a mean of 9.5±3.66. fig-2 shows a rat of group c with arthrogram score 04. fig-1: rat of group b with hind paws showing edema and redness fig-2: rat of group c with improved arthrogram score. the mean tlc of group a, b and c was 6600±300/μl, 6562±450/μl and 6488±360/μl respectively on day 0. it remained almost constant in group a on day 14 and day 28 with a mean of 6600±310/μl and 6675±380/μl. tlc was increased on day 14 in the other two groups with mean of 9988±510/μl and 9987±710/μl. group b was left untreated and its mean tlc on day 28 was 10150±350/μl whereas a dosing schedule of 2 mg/kg/day of telmisartan led to a decrease in levels of tlc to 7350±550/μl. the histological examination of group a revealed minimum score as it was disease free and did not show any microscopic changes. the synovium lining was smooth with no penetration of infiltrating cells. maximum score was seen in the rats of group b which ranged from 8-13. marked percentage of infiltrating mononuclear cells was seen in four out of six slides while rest showed moderate infiltration. two slides showed severe synovial hyperplasia but was moderate in other slides. the extension of pannus formation was maximum in two slides while the others showed mild to moderate pannus formation. six out of eight slides showed moderate synovial hyperplasia. all the slides showed cartilage erosion. mild to moderate presence of bone erosion was seen in all slides except one. mean score of this group was 10.75±2.05. fig-3 shows a slide of group b with a score of 10 showing bone erosion and inflammation with infiltrating cells. slides of group c showed improvement. the score ranged from 5-9. two slides showed moderate infiltrating mononuclear cells, two showed no infiltration while four showed mild infiltration of mononuclear cells. mild to moderate synovial hyperplasia was present in all the slides. mild pannus formation and synovial fibrosis was a feature of all the groups. five slides showed mild cartilage erosion, whereas the remaining slides showed moderate erosion. mild bone erosion was seen in five slides and moderate in two slides. the mean score of this group was 6.88±1.24. fig-4 shows a score of 07 with surface corrugation. 9 efficacy of telmisartan in pristane induced arthritis rat model fig-3: photomicrograph of ankle joint of group b fig-4: photomicrograph of ankle joint of a rat of group c treatment with telmisartan for two weeks decreased the arthrogram score, tlc and also showed improvement on histological examination. the intergroup comparison of the three groups of all the parameters depicted a statistically significant p value ˂ 0.001 as shown in table-1. parameter group a (n=8) group b (n=8) group c (n=8) p value anova mean±sd mean±sd mean±sd arthrogram score 0 14.50±2.07 9.5±3.66 ˂ 0.001* total leukocyte count (per μl) 6675±380 10150±350 7350±550 ˂ 0.001* histopathology score 0 10.75±2.05 6.88±1.24 ˂ 0.001* * significant p value ≤0.05 table-1: intergroup comparison of arthrogram score, total leukocyte count and histopathology score on day 28. discussion ra is one of the common types of arthritis in the elderly population affecting the quality of life. the main pathological characters include persistent chronic synovitis, dysplasia of synovial membrane and pannus formation eventually causing cartilage and joint obliteration.12 pia is one of the extensively used models for research purpose due to appreciative resemblance to the chemical characteristics of ra.13 it is similar to the condition seen in humans due to the presence of symmetrical joint involvement, presence of rheumatoid factor, bone and cartilage destruction as well as dependence of specific immunity seen in such models.14 in the present study the results indicated that the development of arthritis correlated with the increase in arthrogram score, tlc and histological score in group b as compared to the group a (normal control) till day 14. these findings are backed up by a number of studies conducted by researchers. chen and his fellows in 2019 also observed the signs of severe arthritis spreading to ankle joints two weeks after injecting pristane.15 regarding tlc, tissue homeostasis is maintained by the monitored movement of leukocytes between lymphoid and peripheral tissue through various stromal compartments. this trafficking is disturbed in ra.16 feng and qiu in 2018 successfully created a model of ra by subcutaneous injection of emulsion. the results indicated higher leukocyte count as well as damaged cartilage and considerable infiltration of inflammatory cells.17 we followed a therapeutic model by commencing the treatment with telmisartan on day 14. it significantly lowered the aforementioned parameters when compared with the group b (arthritic control). group c (telmisartan group) displayed the absence of swelling, redness and an overall decrease in the mean arthritic score. these findings were in accordance with the work of hasanin and mohamed who studied the anti-inflammatory effects of telmisartan in a rat model and saw a significant decrease in arthritic parameters.6 two weeks treatment with telmisartan also displayed a decrease in tlc and histological score. angiotensin ii (ang ii) has a central role in leukocyte migration and infiltration at molecular and cellular level at the sites of inflammation.18 it is accountable for regulating proliferation of cells and apoptosis by binding to at1r. ang ii exhibited from synovial tissue regulates synovial perfusion and growth by its actions on synovial at1r.19 thus the decrease in tlc can be justified by its at1r blocking potential. other than this, olmesartan, an arb like telmisartan was used orally in a model of zymosan induced ra. it decreased the tlc with specific reduction in neutrophil count as well as showed improvement in histological score.20 this signifies that our experimental drug telmisartan may also have anti-arthritic effect against ra. limitations of study: this study has also some limitations. although the study showed that telmisartan has anti-arthritic properties, the preventive role of telmisartan should 10 efficacy of telmisartan in pristane induced arthritis rat model also be evaluated in such a model. more over future studies should also be conducted using this drug focusing on adverse effects. conclusion oral administration of 2 mg/kg/day of telmisartan for two weeks significantly lowered the mean arthrogram score, total leukocyte count and histological score in a rat model of rheumatoid arthritis. therefore, it can be concluded that telmisartan has anti-arthritic effects. acknowledgement: authors are thankful to dr. hussain ali, head of research animal facility, national institute of health, islamabad for his supervision regarding induction of rheumatoid arthritis. a special thanks to army medical college, rawalpindi for facilitating us to conduct this project. references 1. abhishek a, doherty m, kuo cf, mallen cd, zhang w, grainge mj. rheumatoid arthritis is getting less frequent-results of a nationwide population-based cohort study. rheumatology. 2017 may 1; 56(5):736-44. 2. guo q, wang y, xu d, nossent j, pavlos nj, xu j. rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies. bone res. 2018 apr 27; 6(1):1-4. 3. bullock j, rizvi sa, saleh am, ahmed ss, do dp, ansari ra, ahmed j. rheumatoid arthritis: a brief overview of the treatment. medical princpract. 2018; 27(6):501-7. 4. xu q, zhou y, zhang r, sun z, cheng lf. antiarthritic activity of qi-wu rheumatism granule (a chinese herbal compound) on complete freund’s adjuvant-induced arthritis in rats. evid based complement alternat med. 2017 jan 1; 2017:1960517 5. terenzi r, manetti m, rosa i, romano e, galluccio f, guiducci s, ibba-manneschi l, matucci-cerinic m. angiotensin ii type 2 receptor (at2r) as a novel modulator of inflammation in rheumatoid arthritis synovium. sci rep. 2017 oct 16; 7(1):1-1. 6. hasanin ah, mohamed rh. telmisartan alone or in combination with etanercept improves anemia associated with rheumatoid arthritis in rats: a possible role of anti-inflammatory and renoprotective effects. pharmacol rep. 2020 apr; 72(2):379-88. 7. yanagihara h, ushijima k, arakawa y, aizawa ki, fujimura a. effects of telmisartan and olmesartan on insulin sensitivity and renal function in spontaneously hypertensive rats fed a high fat diet. j pharmacol sci. 2016 jul 1; 131(3):190-7. 8. faisal r, gul m, anwar a. low doses of pristane in comparison with high doses in induction of arthritis in female-spraguedawley rats. khyber med univ j. 2016 dec 24; 8(4):185-8. 9. gul a, kunwar b, mazhar m, faizi s, ahmed d, shah mr, simjee su. rutin and rutin-conjugated gold nanoparticles ameliorate collagen-induced arthritis in rats through inhibition of nf-κb and inos activation. intimmunopharmacol. 2018 jun; 1(59):310-7. 10. faisal r, chiragh s, popalzai aj, rehman ku. anti inflammatory effect of thymoquinone in comparison with methotrexate on pristane induced arthritis in rats. j pak med assoc. 2015 may 1; 65(5):519-25. 11. alabarse pv, lora ps, silva jm, santo rc, freitas ec, de oliveira ms, almeida as, immig m, teixeira vo, filippin li, xavier rm. collagen‐induced arthritis as an animal model of rheumatoid cachexia. journal of cachexia, sarcopenia and muscle. 2018 jun; 9(3):603-12. 12. cai p, jiang t, li b, qin x, lu z, le y, shen c, yang y, zheng l, zhao j. comparison of rheumatoid arthritis (ra) and osteoarthritis (oa) based on microarray profiles of human joint fibroblast‐like synoviocytes. cell biochemistry and function. 2019 jan; 37(1):31-41. 13. wang b, zhao p, zhou y, meng l, zhu w, jiang c, wang l, cai y, lu s, hou w. increased expression of th17 cytokines and interleukin-22 correlates with disease activity in pristane-induced arthritis in rats. plos one. 2017 nov 28; 12(11):e0188199. 14. ruiz jt, luján l, blank m, shoenfeld y. adjuvantsand vaccines-induced autoimmunity: animal models. immunologic research. 2017 feb 1; 65(1):55-65. 15. chen q, zhang x, xiong y, chen c, lv s. the cd25+/cd4+ t cell ratio and levels of cii, cix and cxi antibodies in serum may serve as biomarkers of pristane-induced arthritis in rats and rheumatoid arthritis in humans. comparative biochemistry and physiology part c: toxicology & pharmacology. 2019 mar 1; 217:25-31. 16. buckley cd, mcgettrick hm. leukocyte trafficking between stromal compartments: lessons from rheumatoid arthritis. nature reviews rheumatology. 2018 aug; 14(8):476-87. 17. feng fb, qiu hy. effects of artesunate on chondrocyte proliferation, apoptosis and autophagy through the pi3k/akt/mtor signaling pathway in rat models with rheumatoid arthritis. biomedicine & pharmacotherapy. 2018 jun 1; 102:1209-20. 18. piqueras l, sanz mj. angiotensin ii and leukocyte trafficking: new insights for an old vascular mediator. role of redox-signaling pathways. free radical biology & medicine. 2020 feb11:157:38-54 19. wu y, lu x, li m, zeng j, zeng j, shen b, zeng y. renin-angiotensin system in osteoarthritis: a new potential therapy. international immunopharmacology. 2019 oct 1:75:105796. 20. guerra gc, de menezes ms, de araújo aa, de araújojúnior rf, de medeiros ca. olmesartan 11 efficacy of telmisartan in pristane induced arthritis rat model prevented intra-articular inflammation induced by zymosan in rats. biological and pharmaceutical bulletin. 2016 nov 1; 39(11):1793-801. the authors: dr. quratulain mehdi demonstrator, department of pharmacology, quetta institute of medical sciences, quetta. dr. noaman ishaq assistant professor, department of pharmacology, bakhtawar amin medical and dental college, multan. dr. saba batool assistant professor, department of pharmacology, cmh multan institute of medical sciences, multan. dr. kulsoom farhat associate professor, head, department & pharmacology, army medical college, rawalpindi. dr. saman omer demonstrator, department of pharmacology, mohtarma benazir bhutto shaheed medical college, mirpur ajk. dr. arooj shahid demonstrator, department of pharmacology, army medical college, rawalpindi. corresponding author: dr. quratulain mehdi demonstrator, department of pharmacology, quetta institute of medical sciences, quetta. e-mail: qurat.mehdi84@gmail.com microsoft word 48-56   48   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 study of il-33 and il-1r4 in iraqi rheumatoid arthritis female patient's with and without dyslipidemia prone to atherosclerosis. lara ali nazar laraali398@gmail.com eimanaa.abass drbiochem2007@gmail.com  department of chemistry,college of education for pure science ibn al-haitham , university of baghdad,baghdad,iraq. article history: received 31 august 2018, accepted 17 october 2018, publish january 2019 abstract this study is planned to find relationship between interleukin-33 (il-33) with its receptor interleukin-1 receptor 4 (il-1r4), and assurance il-33/il-1r4 proportion as biomarker to atherosclerosisin rheumatoid arthritis (ra) iraqi female’s patients with and without dyslipidemia. this study was attempted at baghdad teaching hospital included 60 female’s patients with ra that were isolated into: 30 patients with dyslipidemia(g2), 30 patients without dyslipidemia(g3) and 30 individuals as control group (g1). patients were experiencing treatment by methortexiene medication, analyzed by rheumatoid factor (rf) and erythrocyte sedimentation rate (esr) tests. all patients and control groups age ranged from (30-55) years. the results show an increase in esr, rf, il-33, and il-1r4 levels. in addition to decrease in il-33/il-1r4 ratio in the two patient’s groups when contrasted and control group. the momentum examine inferred that the level of esr, and il-33 in ra iraqi females patients with dyslipidemia were higher than that in ra iraqi females patients without dyslipidemia, while the level of il-33/il-1r4 ratio in ra iraqi females patients with dyslipidemia was lower than that in ra iraqi females patients without dyslipidemia patients; in this manner the il-33/il-1r4 ratio may be used as a biomarker in diagnostic early porn to atherosclerosis in ra females patients with dyslipidemia. keywords: rheumatoid arthritis, il-33, il-1r4, atherosclerosis disease, esr and rf. 1.introduction rheumatoid arthritis is a constant, incendiary, portrayed by joint torment, swelling, deformation, and eventually, handicap. lymphocytes intercedes the safe issue in ra. various cell writes take an interest in the pathogenesis. the exact pathogenic instrument isn't completely comprehended [1]. ra is a standout amongst the most predominant fundamental incendiary illnesses which include joints and additional articular tissues, subsequently causing organ harm. in light of the endless irritation and invulnerable dysregulation, the nearness of ra has been related with cardiovascular (cv) disease and an expanded cv related mortality [2]. the correct reason for ra has not been recognized but rather a few investigations called attention to that genius fiery cytokines, including tumor necrosis factor (tnf)α, interleukin il-1 family, il-6, il-17 and the arbiters delivered through downstream pathways in the ligament joints, constitute the milieu driving cartilage and bone destruction [3].   49   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 the il-33 initiates its signaling pathway by binding to the suppression of tumorigenicity2 (st2) receptor, or also known as il-1r4. il-33 is fundamentally associated with the t-helper cell type 2 (th2) safe reaction acceptance which gives defensive safe reaction towards parasitic disease, specifically helminths [4]. il-33 receptors are composed of st2 (also known as il-1r4), a ligand binding chain, and il-1 receptor accessory protein (il-1racp, also known as il-1r3), a signal transducing chain. il-1r3 is a common receptor for il-1a, and il-1b, il-33, and three il-36 isoforms [5]. the il-1r4 is an individual from the interleukin 1 receptor family. st2 remains for "suppression of tumorigenicity 2". it was found in 1989, yet just in 2002 weinberg et al. revealed that it could be communicated via heart cells in light of myocardial pressure, drawing the consideration of scientists to a part in the cardiovascular framework. st2 has two fundamental isoforms: trans membrane or cell (st2l) and dissolvable or circling (sst2) forms [6]. the association of il-33 and st2l has been turned out to be cardioprotective in exploratory models, lessening myocardial fibrosis, cardiomyocyte hypertrophy, apoptosis, and enhancing myocardial capacity. this cardioprotective activity happens only through the st2l receptor and not through the solvent receptor. the il-33/st2 framework is upregulated in cardiomyocytes and fibroblasts in light of cardiovascular damage. the collaboration of this dissolvable receptor with il-33 obstructs the il-33/st2l framework thus, disposes of the cardio defensive impacts depicted previously. the st2 framework acts not just as a middle person of il-33 work in its transmembrane full-length st2l isoform yet additionally as an inhibitor of il-33 through its dissolvable soluble suppression of tumorigenicity 2(sst2) isoform [7]. the il-33/st2 has emerged as an intercellular signaling system that participates in processes as varied as the antigen/allergen response, autoimmunity, organ fibrosis and cardiac injury. exploration of the effects of this pathway is underway. an effort to clarify the role of il-33/st2 in the crosstalk between end-organ effector cells and their environment, specifically the surrounding matrix and inflammatory cells, may lend itself to the discovery of novel therapeutic targets for the treatment of diseases such as asthma, rheumatoid arthritis, atherosclerosis and heart failure [8]. the esr is a nonspecific measure of inflammation and can be useful not only in diagnosing autoimmune diseases such as rheumatoid arthritis but also in monitoring the disease process. it can be an index observing the activity and severity of ra synovitis. the combination of esr and creactive protein (crp) may improve sensitivity and specificity of the diagnosis of ra [9]. this examination expected to estimation il-33 with it is receptor il1r4 levels, and assurance il-33/il-1r4 proportion as biomarker to atherosclerosis risk in rheumatoid arthritis iraqi females’ patients with and without dyslipidemia. 2. experimental 2.1. patients’ groups study this study was attemptedat baghdad teaching hospital, and included 60 female’s patients with rheumatoid arthritis that were partitioned into: 30 patients with dyslipidemia(g2) and 30 patients without dyslipidemia(g3), in addition to 30 individuals as control gathering (g1). patients were experiencing treatment by methortexiene medication, and they were analyzed by clinical examination by specialists, rf and esr tests. all patients and control bunches age extended from (30-55) years.   50   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 2.2. determination of serum rf the rf-latex test is a fast agglutination system, in view of change of the singer method1 created for the immediate recognition, the semi-quantitation on a slide of rheumatoid factor (rf) in serum. the test is performed by testing a suspension of latex particles covered with human gamma globulin against obscure serums. the nearness or nonattendance of an unmistakable agglutination, demonstrates the presence or nonappearance of rf in the examples tried [10].   2.3. determination of esr (mm/hr) the westergren technique is suggested as the reason for a satisfactory standard for esr test, which that measures the sedimentation rate of totaled red cells in plasma. the numerical incentive in mm is acquired by estimating the separation between the least purpose of the surface meniscus to the furthest reaches of the red cell dregs in a segment of anticoagulated and weakened blood that has remained in a chosen tube for a time of an hour [11]. 2.4. determination of serum interleukin 1 receptor 4(il-1r4) (ng/ml) the il-1r4 was controlled by utilizing the focused compound – connected invulnerable sorbent test (elisa) pack that was provided by my bio source, usa, which that in light of strategy using a polyclonal hostile to il-1r4 counter acting agent and an il-1r4-hrp conjugate. 2.5. determination of serum il-33 (pg/ml) serum il-33 levels were estimated utilizing particular protein connected immunosorbent test (elisa) unit (ray bio human il-33 for in vitro quantitative estimation of human il33 in serum), as indicated by the fabricates convention [12]. 2.6. statistical analysis data were communicated as (mean ± sem). the examination amongst patients and control group was communicated by t-test. t-test of < 0.005, < 0. 05, and < 0.05 were considered exceptionally highly significant, significant, and non-significant respectively. pearson's relationship coefficient (r) is utilized for depicting the relationship between the diverse examination parameters. 3. results and discussion the levels of rf, esr, il-1r4, il-33, and il-33/il-1r4 ratio levels in control (g1), rheumatoid arthritis female’s patients with dyslipidemia (g2) and without dyslipidemia (g3) groups were summarizes in table 1. the results which expressed as (mean± sem), showed a highly significant increase (p < 0.005) in rheumatoid factor rf and esr levels in both g2 and (g3) patients groups, respectively, when comparing with control group (g1), while there were no significant different (p > 0.05) in rf and esr levels between two patients’ groups. rf is autoantibodies coordinated against the fragment crystallizable (fc) region of igg. rheumatoid factor is an entrenched analytic and prognostic test in rheumatoid arthritis. high titer igm rf is moderately particular for the finding of ra with regards to an interminable polyarthritis, and was for quite a long time the sole serologic measure broadly utilized as a part of the determination of ra. patients with ra take after a variable infection course as to   51   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 outcome measures, for example, utilitarian status or radiological appraisal of joint damage [13]. aggravation in the synovium is reflected by a foundational provocative reaction. expanded esr and hepatocyte creation of intense stage proteins including crp and intense stage serum amyloid an (a-saa) are surrogate markers of this procedure [14]. in clinical management of ra, crp and esr are commonly ordered tests to guide diagnosis of ra besides the measurements of the mentioned auto antibodies. esr is a nonspecific measure of inflammation and can be useful not only in diagnosing autoimmune diseases, such as rheumatoid arthritis, but also in monitoring the disease process. it can be an index observing the activity and severity of ra synovitis [15]. the results in the current study show a highly significant (p < 0.005) elevation in il-1r4 and il-33 levels in both of g2 and g3 patients when compared with control group (g1), and there was no significant different (p > 0.05) in il-1r4 level and a significant different (p < 0.05) in il-33 level between g2 and g3 patients. also, the results in table 1 revealed to no significant decrease (p> 0.05) in il-33/il-1r4 ratio levels in both of g2 and g3 patients when compared with control group (g1). the present study showed a highly significant decrease (p < 0.005) in aip levels in both of g2 and g3 patients when compared with control (g1). il-33 can be discharged extracellularly as full-length protein through cell passing pathways and it can be cut to shorter structures with expanded cytokine movement. going about as a cytokine and authoritative to its receptor il-1r4 (previously known as t1/st2), il-33 drives compose 2-subordinate irritation and furthermore tissue repair, and it is associated with unfavorably susceptible and lung/mucosal aggravation.the dissolvable type of the receptor, sst2/sil-1r4, is a characteristic inhibitor of il-33 [16, 17]. in necrosis, the il-33/st2 pathway has been demonstrated to be a critical signaling pathway inducing related immune mechanisms, while il-33/sst2 appeared to be an interesting pathway in neutralizing the activation of il-33 [18]. numerous reports have discovered expanded il-33 and st2 generation in the serum and synovial tissue of patients with ra. the articulation level of il-33 and st2 are seems to be correspond with ra sickness movement.organization of il-33 prompts the generation of expert provocative cytokines (il-1β, mcp-1 and il-6) and fuels the advancement of cia in mice. this information show that revocation of il-33/st2 opened up incendiary reaction speaks to a restorative focus for ra [19]. the declaration of sst2 is corresponded with the continuous procedure of fibrosis and inflammation. after intense myocardial localized necrosis, the il-33 level is not quite the same as the sst2 level, which shows that the control of il-33 and sst2 is diverse after intense myocardial dead tissue. mineralocorticoid receptor enemies have been appeared to lessen cardiovascular fibrosis through balancing il-33/st2 and galectin-3 flagging.these outcomes show that il-33/st2 assumes a defensive part in cardiovascular fibrosis, and sst2 adversely directs this pathway as a bait receptor for il-33 [20]. it has been demonstrated that the receptor of il-33 assumes to be a critical part in aggravation, contamination, and immune system infections. recently, il-33has been appeared to change the manifestations of rheumatoid joint pain, foundational lupus erythematosus, and other immune system ailments. they may inspire gainful or hindering impacts relying upon the ailment setting. along these lines, ponders on il-33may give another thought and focus for the treatment of immune system illnesses [21]. as a kind of   52   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 pro-inflammatory cytokines, il-33 cause the immune pathological damage in some tissues. it plays the important role in chronic inflammatory and autoimmune diseases. it has been demonstrated that il-33/st2 flag transduction pathways are associated with different obsessive procedures.il-33 and its receptor st2 are the potential focuses for the treatment of hypersensitive and immune system fiery maladies [22]. large amounts of sst2 can predict worse prognosis in cardiovascular patients, which proposes that sst2 levels are adequate to sequester endogenous il-33 levels. in spite of the present absence of data on endogenous il-33 levels in people [23]. the essential site of irritation in ra is the synovial tissue, from which cytokines are discharged into the fundamental course. these flowing cytokines are in a situation to adjust the working of fat tissue, skeletal muscle, liver, and vascular endothelium and produce a range of proatherogenic changes that incorporates insulin opposition, a trademark dyslipidemia, prothrombotic impacts, ace oxidative pressure and endothelial brokenness [24]. ra patients will probably create atherosclerotic plaques, quiet ischemic sickness and they have an expanded danger of sudden heart passing, contrasted with the general population [25]. in patients with ra, the danger of cvd is expanded and screening of cvd risk factors and distinguishing proof of high-chance patients is justified [26]. study show that crp and esr have been related with cvd in ra and polyarthritis [27]. determinants of provocative action, other 'ailment related' markers have been recognized as potential novel hazard factors for cvd in ra, notably rf energy and anti-citrullinated protein antibody (acpa) positivity [28]. in this study, figures 1. and 3. were showed a highly significant positive correlation between il-1r4 with both of rf in g1(r =0.155), g2(r = 0.054), and g3(r = 0.008), and figure 4 show a highly significant positive correlation with il-33 in g1 (r =0.145), g2(r = 0.078), and g3(r = 0.148), respectively. also in figure 2., there were a highly significant negative correlation between il-1r4 with esr in g1(r = 0.147) and g3 (r = 0.441), and a highly significant positive correlation with esr in g2(r = 0.108). the results in figure 3. revealed no significant negative correlation between il-1r4 and il-33/il-1r4 ratio in both of g1(r = 0.345), g2(r = 0.338) and g3 (r = -0.688).   53   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 table 1. levels of rheumatoid factor (rf), erythrocyte sedimentation. rate (esr), interleukin 1 receptor 4(il1r4), il-33, and il-33/il-1r4 proportion in charge (g1), rheumatoid arthritisfemales patients with dyslipidemia (g2) and without dyslipidemia (g3) groups. hs: p <0.005 ; s: p < 0.05 ; ns: p> 0.05     figure 1. correlation between il-1r4 and rf in g1, g2 and g3. figure 2. correlation between il-1r4 and esr in g1,g2 and g3. groups parameters g1 no.(30) g2 no.(30) g3 no.(30) p-value g1&g2 p-value g1&g3 p-value g2&g3 rf ( iu/ml) 9.81±1.79 83.73±15.3 88.53±16.18 hs hs ns e.s.r (mm/hr) 5.2±0.95 41.83±6.64 18.25±5.86 hs hs ns il-1r4(ng/ml) 0.29±0.05 1.46±0.26 1.62±0.29 hs hs ns il33(pg/ml) 136.46±24.9 541.03±98.99 483.7±88.42 hs hs s il33 / il1r4 479.44±87.6 339.82±62.12 344.93±63.5 ns ns ns   54   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019     figure 3. correlation of il-1r4 and il-33/il-1r4 ratio in g1, g2, and g3.  figure 4. correlation of il-1r4 and il-33 in g1,g2, and g3. 4.conclusions in conclusion, current study proved a highly significant elevation in il-1r4 and il-33in addition to no significant decrease in il-33/il-1r4 ratio levels in iraqi women patients with rheumatoid arthritis. in addition to the level of esr, and il-33 in rheumatoid arthritis iraqi women patients with dyslipidemia were higher than that in rheumatoid arthritis iraqi women patients without dyslipidemia, while the level of il-33/il-1r4 ratio in ra iraqi women patients with dyslipidemia was lower than that in ra iraqi women patients without dyslipidemia patients; therefore the ra women patients with dyslipidemia may be they are more prone to atherosclerosis and heart diseases from ra women patients without dyslipidemia.although il-1r4 (st2) is a potentially useful biomarker in heart diseases such as heart failure and myocardial infarction. recently, physicians have begun to use sst2 as a biomarker of cardiomyocyte stress and fibrosis, and the level of sst2 is now used for additional risk stratification of acute or ambulatory heart-failure patients. here we showed that the recently described ligand il-33 was synthesized by cardiac fibroblasts and abrogated angiotensin ii– and phenylephrine-induced hypertrophy in cardiomyocytes in vitro. finally, it is possible that sst2 is not only a biomarker for poor outcome, but also a true pathophysiological mediator of disease progression. il-33 has now also been shown to participate in cardiovascular pathophysiology. furthermore, the il-33/st2 system may play a part in the progression of atherosclerotic vascular disease. references 1. malda, j.; boere, j.; van de lest, c. h.; van weeren, p. r.; wauben, m. h. extracellular vesicles—new tool for joint repair and regeneration. nature reviews rheumatology 2016, 12, 243-249. 2. picerno, v.; ferro, f.; adinolfi , a.;valentini, e.; tani, c.; alunno, a. one year in review: the pathogenesis of rheumatoid arthritis. clinical and experimental rheumatology. 2015, 33, 551-558. 3. alghasham, a.; rasheed, z. therapeutic targets for rheumatoid arthritis: progress and promises. autoimmunity. 2014,47, 77-94.   55   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 4. townsend, m. j.; fallon, p. g.; matthews, d. j.; jolin, h. e.; mckenzie, a. n. t1/st2-deficient mice demonstrate the importance of t1/st2 in developing primary t helper cell type 2 responses. journal of experimental medicine 2000, 191,6, 1069-1076. 5. jo, s.; kim, e.; kwak, a.; lee, j.; hong, j.; lee, j.; youn, s.; bae, s.; kim, b.; ryoo, s.; kang, t.b. reconstitution of st2 (il-1r4) specific for il-33 activity; no suppression by il-1ra though a common chain il-1r3 (il-1racp) shared with il-1. cytokine 2016, 83, 33–40. 6. weinberg, e.o.; shimpo, m.; de keulenaer, g.w.; macgillivray, c.; tominaga, s.i.; solomon, s.d.; rouleau, j.l.; lee, r.t. expression and regulation of st2, an interleukin-1 receptor familymember,in cardiomyocytes and myocardial infarction. circulation. 2002, 106, 2961–2966. 7. pascual-figal. d.a.; januzzi. j.l. the biology of st2: the international st2 consensus panel. the american journal of cardiology. 2015, 115, 3b-7b. 8. schmitz, j.; owyang, a.; oldham, e.; song, y.; murphy, e.; mcclanahan, t.k.; zurawski, g.; moshrefi, m.; qin, j.; li, x.; gorman, d.m. il-33, an interleukin-1-like cytokyne that signals via the il-1 receptor-related protein st2 and induces t helper type 2-associated cytokynes. immunity. 2005, 23, 479-490. 9. kakkar, r.; lee, r.t. the il-33/st2 pathway: therapeutic target and novel biomarker. nature reviews drug discovery. 2008, 7, 827-840. 10. singer, j.m.; plotz, c.m. the latex fixation test. i. application to the serologic diagnosis of rheumatoid arthritis. american journal of medicine. 1956, 21, 888-892. 11. boroviczeny, k-g.v.; bottiger, l. e.; chattis, a. j. b.; dawson. k.; fukutake. f. w.; gunz, s. m. lewis.; rewald, e. m.; roubicek, g.; ruhenstroth-bauer.; and westergren, a. reference method for the erythrocyte sedimentation rate (esr) test on human blood. journal of clinical pathology. 1973, 26, 301-302 12. burtis, ca.; ashwood, er.; bruns, de.; tietz. textbook of clinical chemistry and molecular diagnostics,4thed, elsevier saunders, 2006. 13. spartali, i.; kostantinos, h.; ioannis, k.;thrasivoulos, p. body fat percentage and body mass index as predictors of cadets’ physical performance. the open sports sciences journal. 2014, 7,53-59. 14. ingegnoli, f.; castelli, r.;gualtierotti, r. rheumatoid factors: clinical applications. disease marker.s 2013, 35, 727-734. 15. nielen, m.m.; van schaardenburg, d.; reesink, h.w.; twisk, j.w.; van de stadt, r.j.; van der horst‐bruinsma, i.e.; de gast, t.; habibuw, m.r.; vandenbroucke, j.p.; dijkmans, b.a. increased levels of c‐reactive protein in serum from blood donors before the onset of rheumatoid arthritis. arthritis & rheumatism: official journal of the american college of rheumatology. 2004, 50, 2423-2427. 16. shadick, n.a.; cook, n.r.; karlson, e.w.; ridker, p.m.; maher, n.e.; manson, j.e.; buring, j.e.; lee, i.m. c-reactive protein in the prediction of rheumatoid arthritis in women. archives of internal medicine. 2006, 166, 2490–2494. 17. gordon, e.d.; simpson, l.j.; rios, c.l.; ringel, l.; lachowicz-scroggins, m.e.; peters, m.c.; wesolowska-andersen, a.; gonzalez, j.r.; macleod, h.j.; christian, l.s.; yuan, s. alternative splicing of interleukin-33 and type 2 inflammation in asthma. proceedings of the national academy of sciences. 2016, 113, 8765-8770.   56   ibn al-haitham jour.for pure & appl.sci. ihjpas https://doi.org/10.30526/32.1.1920 vol. 32 (1) 2019 18. finlay, c. m.; stefanska, a. m.; walsh, k. p. helminth products protect against autoimmunity via innate type 2 cytokines il-5 and il-33, which promotes eosinophilia, the journal of immunology. 2016, 196, 703–714. 19. sanada, s.; hakuno, d.; higgins, l. j.; schreiter, e. r.; mckenzie, a. n.; lee, r. t. il-33 and st2 comprise a critical biomechanically induced and cardioprotective signaling system. the journal of clinical investigation. 2007, 117, 1538-1549. 20. hong, y.s.; moon, s.j.; joo, y.b.; jeon, c.h.; cho, m.l.; ju, j.h.; oh, h.j.; heo, y.j.; park, s.h.; kim, h.y.; min, j.k. measurement of interleukin-33 (il-33) and il-33 receptors (sst2 and st2l) in patients with rheumatoid arthritis. journal of korean medical science. 2011, 26, 1132-1139. 21. lax, a.; sanchez-mas, j.; asensio-lopez, m.c.; fernandez-del palacio, m.j.; caballero, l.; garrido, i.p.; pastor-perez, f.j.; januzzi, j.l.; pascual-figal, d.a. mineralocorticoid receptor antagonists modulate galectin-3 and interleukin-33/st2 signaling in left ventricular systolic dysfunction after acute myocardial infarction. jacc heart fail. 2015, 3, 50–58. 22. seltmann, j.; werfel, t.; wittmann, m. evidence for a regulatory loop between ifn‐γ and il‐33 in skin inflammation. experimental dermatology. 2013, 22, 102-107. 23. zhao, q.; chen, g. role of il-33 and its receptor in t cell-mediated autoimmune diseases. biomed research international. 2014, 2014, 1-10, doi.org/10.1155/2014/587376. 24. shimpo, m.; morrow, d.a.; weinberg, e.o.; sabatine, m.s.; murphy, s.a.; antman, e.m.; lee, r.t. serum levels of the interleukin-1 receptor family member st2 predict mortality and clinical outcome in acute myocardial infarction. circulation. 2004, 109, 2186-2190. 25. mcinnes, i.b. rheumatoid arthritis: from bench to bedside. rheumatic disease clinics of north america. 2001, 27, 373-387. 26. ogdie, a.; yu, y.; haynes, k.; love, t.j.; maliha, s.; jiang, y.; troxel, a.b.; hennessy, s.; kimmel, s.e.; margolis, d.j.; choi, h. risk of major cardiovascular events in patients with psoriatic arthritis, psoriasis and rheumatoid arthritis: a population-based cohort study. annals of the rheumatic diseases. 2015, 74, 326-332. 27. avina-zubieta, j.a.; thomas, j.; sadatsafavi, m.; lehman, a.j.; lacaille, d. risk of incident cardiovascular events in patients with rheumatoid arthritis: a meta-analysis of observational studies. annals of the rheumatic diseases. 2012, 71, 1524-1529. 28. aubry, m.c.; maradit-kremers, h.; reinalda, m.s.; crowson, c.s.; edwards, w.d.; gabriel, s.e. differences in atherosclerotic coronary heart disease between subjects with and without rheumatoid arthritis. the journal of rheumatology. 2007, 34, 937-942. orthopaedics vol3 no4 sa orthopaedic journal autumn 2016 | vol 15 • no 1 page 41 adult acquired flat foot deformity: the joint-preserving procedures in stage ii tibialis posterior tendon dysfunction np saragas mbbch(wits), fcs(sa)ortho, mmed(ortho surg)(wits) pnf ferrao mbchb(pret), fcs(sa)ortho netcare linksfield orthopaedic sports & rehabilitation centre (clinic), johannesburg and orthopaedic department, university of the witwatersrand, johannesburg, south africa corresponding author: dr np saragas tel: +27 11 485-1974/5 fax: +27 11 640-5313 email: saragas@global.co.za po box 1153 2037 highlands north johannesburg, south africa introduction the adult acquired flat foot (aaff) deformity is a chronic and progressive debilitating condition commonly associated with dysfunction of the posterior tibial tendon (ptt). the pathology is complex and consists not only of ptt insufficiency but also failure of the capsular and ligamentous structures of the foot, leading to a spectrum of deformity with varying degrees of hindfoot valgus, midfoot pronation and forefoot abduction. in later stages arthritic changes can develop in the hindfoot joints.1 ultimately this leads to a painful pes planus deformity and, with pathological changes in the deltoid ligament, associated ankle arthritis. initially there may not be a foot deformity at all, but aaff presents with medial foot pain and decreased function of the affected foot.2 unless the deformity is associated with a generalised medical condition such as rheumatoid arthritis, the diseased tendon is often overlooked and the diagnosis missed.3 kulowski initially described tibialis posterior tendinitis in 1936.4 it was not until 1983, however, that johnson discussed the condition in detail.3 anatomy and biomechanics the ptt originates from the posterolateral tibia, posteromedial fibula and interosseous membrane. it courses posterior to the medial malleolus and inserts into the abstract introduction: the adult acquired flat foot (aaff) deformity is a chronic debilitating condition commonly associated with dysfunction of the posterior tibial tendon (ptt). it is often missed unless it is associated with a generalised medical condition such as rheumatoid arthritis. surgical management is indicated when conservative treatment fails. the joint-preserving procedures have evolved over the years and are preferred for the flexible, non-arthritic deformity. materials and method: twenty-two patients were included in this prospective study. the mean age was 59.8 years with the majority being female. the average bmi was 28.7. the inclusion criterion was symptomatic aaff deformity due to stage ii ptt dysfunction. results: twenty patients were available for follow-up at one year. the mean aofas post-operative score of 89 was significantly improved from the pre-operative score of 42 (p value = <0.001). likewise most of the radiographic parameters also improved significantly. the complication rate was very low. conclusion: this prospective study shows that the joint-preserving procedures for aaff deformity due to stage ii ptt dysfunction are an effective and preferable option to arthrodesis. there is a low complication rate with high patient satisfaction. a certain amount of training and expertise though, is required. level 2 study. key words: adult acquired flat foot surgery http://dx.doi.org/10.17159/2309-8309/2016/v15n1a4 page 42 sa orthopaedic journal autumn 2016 | vol 15 • no 1 navicular tuberosity and the mid-point of the plantar aspect of the tarsus. blood supply to the tendon is poorest in the area behind the medial malleolus making it the most common site for rupture.5 the tibialis posterior tendon is the main supinator of the subtalar joint, an adductor of the midfoot and plantar flexor of the ankle. it is the primary dynamic stabiliser of the medial longitudinal arch and elevates it with its contraction. the midand hindfoot thus lock allowing the triceps surae to effectively push off the forefoot.6 the excursion of the ptt is only 1–2 cm and any ‘lengthening’ of this tendon has an adverse effect on its function.7 pathophysiology although acute/traumatic rupture of the ptt is an obvious cause of aaff deformity, the more usual cause is tendinosis (tendon degeneration) from repeated microtrauma. the weakness of this fibrotic ptt subsequently puts a repetitive load on the medial supporting structures, leading to eventual degeneration of the spring and deltoid ligaments. the consequent shortening of the gastrocnemius further aggravates the flattening of the medial longitudinal arch. with eversion of the subtalar joint forcing the heel into valgus and abduction at the talonavicular joint, the shortened gastrocnemius muscle1,8 further causes subluxation of the talonavicular joint as terminal dorsiflexion of the ankle is achieved by rotation around the talonavicular joint. the navicular thus moves superiorly, laterally and supinates further, rendering the medial column more unstable and collapses the arch.9 epidemiology this chronic debilitating condition typically occurs in obese middle-aged females,6,10-13 with up to 10% prevalence in this group14 and is known to increase with age,15,16 peaking at 55 years of age.8 other risk factors such as diabetes mellitus,15 hypertension,15 steroid injection around the tendon,17,18 and seronegative spondyloarthropathies6,19 have been implicated. mechanical factors must also be at play.20 abnormal forces arising from even mild flat-footedness may result in lifelong greater demands on the ptt.21-24 another possible mechanical cause is overpull of the opposing peroneus brevis muscle.25 classification johnson and strom first classified ptt dysfunction.26 myerson subsequently added to the classification when degenerative changes are present in the ankle joint or there is involvement of the deltoid ligament.6 johnson and strom’s original classification is based on the condition of the tendon, the position of the hindfoot and flexibility of the deformity.27 they identified three stages associated with dysfunction of the ptt, following a progressive course (table i). stage i is characterised by inflammatory changes of the ptt, but neither rupture of the tendon nor deformity. these patients present with tenderness and often oedema over the course of the ptt accompanied by pain and weakness with inversion of the foot,6,28 viz. single heel raise test. in stage ii disease patients present with an added flexible deformity, hindfoot valgus, collapse of the medial longitudinal arch and forefoot abduction, viz. ‘too many toes’ sign3 (figure 1). a fixed flat foot deformity is present in stage iii.26 myerson’s stage iv describes deltoid ligament insufficiency, valgus tilt of the talus and ankle arthritis.6 the ram (rearfoot [r] , ankle [a] , midfoot [m] ) classification further divides the aaff deformity into the individual components involved in the disease process (table ii).27 an even more ‘refined’ classification for stage ii disease has been adapted from bluman et al.8,29 (table iii). investigations plain erect ap, lateral and oblique radiographs of the foot and ankle are necessary to assess the degree of deformity and state of the ankle and hindfoot joints. mri, ct scan and ultrasonography are rarely required and only if the diagnosis is obscure. aaff deformity due to ptt dysfunction can be made clinically. table i: changes associated with various stages of tpt dysfunction26 stage 1 stage 2 stage 3 tpt condition peritendinitis and/or tendon degeneration elongation elongation hindfoot mobile, normal alignment mobile, valgus position fixed, valgus position pain medial: focal, mild to moderate medial: along tpt, moderate medial: possibly lateral, moderate single-heel-rise test mild weakness marked weakness marked weakness ‘too-many-toes’ sign with forefoot abduction normal positive positive pathology synovial proliferation, degeneration marked degeneration marked degeneration treatment conservative, 3 months: surgical, 3 months with synovectomy, tendon debridement, rest transfer fdl for tpt subtalar arthrodesis sa orthopaedic journal autumn 2016 | vol 15 • no 1 page 43 management the aim of treatment is to relieve pain, improve function, restore alignment and arrest the progression of the disease. the management of ptt dysfunction is dictated by the stage of the disease. the johnson and strom classification discussed earlier is commonly used to guide management decision-making. stage i is the early inflammatory stage. this is ideally managed conservatively. the treatment consists of rest, support and rehabilitation. the patient must refrain from all impact and strenuous activities for at least six weeks. the ptt can be supported with either a custom supportive orthotic or specialised brace (airlift brace). in severe cases boot or cast immobilisation is required. rehabilitation is addressed with physiotherapy, consisting of anti-inflammatory modalities, strengthening and proprioception. figure 1. ‘too many toes’ sign table ii: the ram classification27 rearfoot ankle midfoot ia tenosynovitis of ptt neutral alignment neutral alignment ib ptt tendonitis without deformity mild valgus (<5ᵒ) mild flexible midfoot supination iia flexible planovalgus (<40% talar uncoverage, <30° meary angle, incongruency angle 20° to 45°) valgus with deltoid insufficiency (no arthritis) midfoot supination without radiographic instability iib flexible planovalgus (>40% talar uncoverage, >30° meary angle, incongruency angle >45°) valgus with deltoid insufficiency with tibiotalar arthritis midfoot supination with midfoot instability – no arthritis iiia fixed/arthritic planovalgus (<40% talar uncoverage, <30° meary angle, incongruency angle 20° to 45°) valgus secondary to bone loss in the lateral tibial plafond (deltoid normal) arthritic changes isolated to medial column (navicular-medial cuneiform or first tmt joints) iiib fixed/arthritic planovalgus (>40% talar uncoverage, >30° meary angle, incongruency angle >45°) – not correctable through triple arthrodesis valgus secondary to bone loss in the lateral tibial plafond and with deltoid insufficiency medial and middle column midfoot arthritic changes (usually with supination and/or abduction of the midfoot) table iii: ‘refined’ classification for stage ii disease8,29 stage subdivision pathology recommendation ii a flexible hindfoot valgus with flexible forefoot varus medial posting/brace + tendoachilles lengthening if forefoot varus corrects only in equinus fixed forefoot varus mdco (arthroereisis) + fdl transfer + cotton osteotomy if fixed forefoot varus b forefoot abduction (at transverse tarsal, first tmt joint or both talar head uncovering <40% mdco + fdl transfer talar head uncovering >40% fdl transfer + lateral column lengthening ± dco/arthroereisis (if residual heel valgus) c medial ray instability persistent forefoot varus after correction of heel talonavicular/naviculocuneiform/ first tmt joint level fusion of appropriate joint if arthritic/cotton osteotomy abbreviations: cotton osteotomy: opening wedge medial cuneiform osteotomy; fdl: flexor digitorum longus; tmt: tarsometatarsal page 44 sa orthopaedic journal autumn 2016 | vol 15 • no 1 cortisone injections play no role in the management of this disease. if symptoms are refractory to conservative management for three months, surgical intervention is indicated. if the tendon integrity is maintained a tenosynovectomy is performed. stage ii by definition is a flexible flat foot deformity with intrinsic tendon pathology. these patients are candidates for the so-called ‘joint sparing (preserving)’ procedures. this is the group of patients this study investigated. in the presence of a positive silfverskiöld test a gastrocnemius recession needs to be performed. stage iia is managed with a medial displacement calcaneal osteotomy, spring ligament plication and flexor digitorium longus (fdl) tendon transfer. in stage iib the above is done with the addition of a lateral column lengthening to address the severe forefoot abduction. there is a high risk of the spring ligament being completely ruptured requiring reconstruction (this type of procedure is beyond the scope of this article). stage iic is the patient with fixed compensatory forefoot supination. this is corrected with a dorsal opening wedge osteotomy of the medial cuneiform (cotton osteotomy). stage ii will be discussed in detail. when the flat foot deformity becomes rigid and/or hindfoot arthritis develops, it gets classified as stage iii. the only surgical option at this stage is a triple arthrodesis of the hindfoot joints with correction of the deformity. stage iv with ankle arthritis is best treated with a pantalar fusion. stage iv with deltoid insufficiency can be managed with a deltoid reconstruction. a couple of techniques have been described with limited results in the literature. therefore a fusion of the ankle is still commonly performed. failure of conservative treatment is the commonest indication to consider surgery. surgery is indicated in patients who have failed nonoperative management for three months or more.30 materials and method the purpose of this prospective study is to report on the effectiveness of treating stage ii ptt dysfunction by jointpreserving procedures. non-surgical, surgical management of the other stages and the merits of certain operative procedures is beyond the scope of this article. between march 2013 and july 2014, 22 patients (16 female and six male) met the inclusion criteria. the inclusion criteria were symptomatic aaff deformity due to stage ii ptt dysfunction not responding to at least three months’ conservative treatment. all patients had weight bearing ap, lateral and oblique radiographs of the involved foot and ankle. as the disease is characterised by medial longitudinal arch depression, talar depression and abduction of the forefoot, the appropriate radiographic parameters were measured as follows:31,32 talonavicular coverage angle (tnca); percentage talar head uncoverage(%tu); talo-first metatarsal angle (meary’s)(tfma); lateral talocalcaneal angle (ltca); calcaneal pitch angle (cpa) and medial cuneiform to floor distance (mcfd) (figure 2). figure 2. view radiographic measurement31.32 ap a = talonavicular coverage angle (angle >7° indicates lateral talar subluxation) δ = percentage talar head uncoverage lateral b = talo-first metatarsal angle (meary’s) c = calcaneal pitch angle (normal: 17°–32°) d = lateral talocalcaneal angle (normal: 25°–45°. >45° indicates hindfoot valgus e = medial cuneiform to floor distance (normal: 18.38 mm ± 3.66 mm) sa orthopaedic journal autumn 2016 | vol 15 • no 1 page 45 ten patients had a pre-operative ultrasound, which showed varying degrees of ptt degeneration (moderate to complete rupture). all patients signed an informed consent form. surgical technique the patient is placed supine on the table with a bolster under the ipsilateral buttock as the calcaneal osteotomies are performed first. the procedure is performed under general anaesthesia with a regional block. a thigh tourniquet is used. medial displacement calcaneal osteotomy a 5 cm incision is made posterior to the peroneal tendons from the superior border of the calcaneal tuberosity distally to the inferior border of the tuberosity anterior to the inferior calcaneal spur (figure 3). the incision is parallel to the axis of the fibula and curves slightly anteriorly at its distal end. particular attention must be paid to the sural nerve branches, the peroneal tendons and the subtalar joint. it is prudent to use intra-operative fluoroscopy initially to identify the landmarks and the direction of the osteotomy. we have found that the larger the osteotomised calcaneal segment, the easier it is to displace it. once the osteotomy is complete (initially with a power saw and finished with fine osteotomes), two lamina spreaders are inserted in the osteotomy to prise it open and tear the medial periosteum (figure 4). the calcaneal tuberosity is shifted medially by approximately 8–10 mm. the osteotomy is fixed with either a 7.5 mm compression screw inserted from the posterior aspect of the tuberosity under image control or alternatively an edgelock™ plate (tornier sas, us, minneapolis, minnesota). the shelf of the anterior fragment created from the shift is ‘crushed’ with a punch so that the cortical continuity is maintained and no raw bony surface is left to cause adhesions of the soft tissue. lateral column lengthening if there is over 40% uncoverage of the talar head in the preoperative radiographs, a lateral column lengthening is performed. a second horizontal 4 cm incision is made, extending from the calcaneo-cuboid (c-c) joint, posteriorly across the neck. the c-c joint is identified with a hypodermic needle. the osteotomy is carried out approximately 15 mm from the c-c joint and extends between the anterior and middle facets of the calcaneus. the osteotomy is then distracted with either pin distractors or a lamina spreader, until the talus is adequately covered by the navicular as checked under image (figure 5). avoid overcorrection. once the desired distraction is achieved, the osteotomy is filled with a tricortical graft and staple or a maxlock extreme™ plate (tornier sas, us, minneapolis, minnesota) fixation (figure 6). we avoid c-c joint fusion. figure 3. skin incision for medial displacement calcaneal osteotomy figure 4. lamina spreader to prise osteotomy open figure 5. the lateral column lengthening figure 6. the lateral column lengthening with bone graft page 46 sa orthopaedic journal autumn 2016 | vol 15 • no 1 gastrocnemius recession the bolster is now removed from under the patient, allowing the leg to externally rotate. the gastrocnemius recession is indicated if the silfverskiöld test is positive pre-operatively. a small longitudinal incision is made posteromedially in the calf, approximately 15 cm from the achilles tendon insertion. the fascia of the gastrocnemius tendon is identified and dissected off the tendinous portion. the tendinous portion is horizontally transected starting from its lateral edge, extending medially under direct vision. be aware of the sural nerve laterally! flexor digitorum longus (fdl) tendon transfer an 8 cm curved incision is made over the ptt, extending from the medial malleolus to the naviculo-cuneiform joint (figure 7). the incision may extend more proximally, depending on the amount of ptt damage that will have to be debrided/resected. once the sheath of the ptt is opened, the tendon is inspected and debrided/repaired/excised (figure 8). peel off the ptt and roughen the insertion site on the navicular tuberosity by using a rongeur. the spring ligament is then inspected. it is identified as the tissue deep to the ptt attachment. it extends from the navicular tuberosity to the sustentaculum. the identification is easy when there is an obvious tear present (figure 9). more often it is attenuated. immediately behind and slightly posterior, at level of the medial malleolus, the sheath of the fdl is identified and opened (figure 10). the fdl is then delivered from its sheath and tugged with a tendon hook to make sure that the lesser toes move. a four-strand bioabsorable anchor, suturetak® #2 fiberwire® and two tigerwire sutures® (arthrex, naples, florida) is placed into the navicular tuberosity. the two strands of the anchor are used to repair/imbricate the spring ligament. this is secured first before the fdl (figure 11). the other two strands are used to secure the transferred fdl with the foot in full inversion and equinus (figure 12) (if there is no gastrocnemius recession), otherwise in neutral dorsiflexion under tension. if there is any good quality remainder of the ptt, this is attached to the fdl to further reinforce the transfer. do not leave any damaged ptt behind as it may be a pain generator. the distal portion of the transferred fdl need not be transected as it will ultimately stretch out and the lesser toe movement is not compromised. once all the repairs/transfers are secure, the ptt sheath is repaired with absorbable suture, as is the rest of the wound. the foot is maintained in full inversion and equinus/neutral dorsiflexion depending on whether a gastrocnemius recession was performed or not. a below-knee (b/k) non-weight bearing (nwb) cast is applied (figure 13). figure 8. damaged tibialis posterior tendon figure 9. torn spring ligament figure 10. the fdl tendon is identified figure 7. medial incision sa orthopaedic journal autumn 2016 | vol 15 • no 1 page 47 spring ligament reconstruction is beyond the scope of this article. post-operative protocol the patient remains in the nwb cast for six weeks. radiographs are taken once the cast is removed. the patient is then placed in a ‘moon boot’ allowing progressive weight bearing to full weight bearing over a period of six weeks while receiving physiotherapy. the physiotherapy/biokinetics continues until full strength and proprioception is achieved. the patient is advised that the average time to maximum improvement is 9–12 months. active/passive eversion beyond neutral is avoided for at least three months. of note, the patient is covered with oral anticoagulants for six weeks until the cast is removed. there is a high risk of deep vein thrombosis (dvt) with this type of surgery together with immobilisation and nwb status.33 results twenty patients were available for follow-up. there were 15 females and five males, with an average age of 59.8 years (range 49 to 70 years). the patients were examined and radiographs taken at least one year post-operatively. all radiographic measurements (preand final post-operative radiographs) were reviewed by the senior author (nps) so as to eliminate inter-observer error. the aofas questionnaire was completed by both authors. statistical analysis the data were analysed using stata 13 statistical software (stata corporation, college station, tx usa). the following variables were included in the analysis: mcfd, tfma, cpa, tnca, ltca, %tu and aofas score. exploratory data analysis included histograms, box graphs, mean (standard deviation) and range of continuous variables. the paired sample t-test was used to determine if there was a statistically significant difference in mean values recorded preand post-surgery for each measure; statistical tests were two-sided at α = 0.05. the average pre-operative aofas score was 42 (range 17 to 65). the average post-operative score was 89 (range 50 to 100). the improvement was statistically significant (p value = <0.001). the average bmi was 28.7 (range 19 to 53). the demographics in terms of bmi, age and gender of our cohort of patients were in keeping with the reported literature. the radiographic improvement was as follows: the average pre-operative medial cuneiform to floor distance was 12.4 mm (range 0 to 20) and the average post-operative score was 16.3 mm (range 6 to 24), (p value = <0.001). the average pre-operative talo first metatarsal angle (meary’s) was −20.5° (range −35 to −2) and postoperatively was −12.3° (range −32 to 6), (p value = 0.003). the average pre-operative calcaneal pitch angle was 17.2° (range 10 to 26) and post-operatively was 18° (range 8 to 23), (p value = 0.288). the average pre-operative talo navicular coverage angle was 27° (range 6 to 39) and post-operatively was 18.4° (range 4 to 43), (p value = 0.002). figure 12. the foot in full inversion and equinus figure 13. immobilise the foot in equinus and inversion figure 11. transferring the fdl tendon and repairing the spring ligament page 48 sa orthopaedic journal autumn 2016 | vol 15 • no 1 the average lateral talo calcaneal angle was 50° (range 35 to 62) and post-operatively was 47° (range 38 to 59), (p value = 0.155). the average pre-operative percentage talar head uncoverage was 35.6% (range 15 to 50) and post-operatively was 26.2% (range 10 to 45), (p value = <0.001) (table iv). for mcfd, tfma, cpa, tnca, ltca and %tu the data included 16 matched results; for aofas score the data included 20 matched results. there was an increase in the mean values from pre-op to post-op for mcfd, tfma, cpa and aofas score; these results are statistically significant for mcfd, tfma and aofas score, for cpa the difference is slight and not statistically significant. figure 14: box graphs of preand post-op values table iv: results: preand post-operative measurements measurement pre-operative mean (sd) range post-operative mean (sd) range p-value ap aofas score 42,0 (13.3) 17; 65 89,5 (12.3) 50; 100 <0.001 talonavicular coverage angle 26.9 (7.9) 6; 39 18.4 (11.6) 4; 43 0.002 percentage talar head uncoverage 35.6 (7.7) 15; 50 26.2 (10.8) 10; 45 <0.001 lateral medial cuneiform to floor distance 12.4 (4.7) 0; 20 16.3 (4.0) 6; 24 <0.001 talo first metatarsal angle (meary’s) −20.5 (8.6) −35; −2 −12.3 (11.4) −32; 6 0.003 calcaneal pitch angle 17.2 (4.3) 10; 26 17.9 (4.5) 8; 23 0.288 lateral talo calcaneal angle 50.2 (6.7) 35; 62 47.2 (6.3) 38; 59 0.155 sa orthopaedic journal autumn 2016 | vol 15 • no 1 page 49 there was a decrease in the mean values from pre-op to post-op for tnca, ltca and %tu; these results are statistically significant for tnca and %tu, for ltca the difference is slight and is not statistically significant. the data is further described in the box graphs, which compare median (iqr) of each set of matched values (figure 14). two patients had a lateral column lengthening (50% uncoverage of the talar head pre-operatively). no patients had a gastrocnemius recession. no patients had a cotton procedure. complications there were no wound complications. one patient had delayed union of the calcaneal tuberosity (radiologic union at three months). one patient took four months for the lateral column lengthening to unite (with fixatives failure – broken staple). thirty per cent of patients had transient numbness of the lateral heel. this became apparent only with direct questioning. there were no dvts. discussion multiple procedures have been described for the management of stage ii ptt dysfunction. initially, arthrodesis, either triple or double was the treatment of choice. joint-preserving procedures have evolved over recent years and a combination of soft tissue and bone procedures are now preferred34 yielding statistically significant improvement in the deformity, pain and function.34-41 we present a series of 20 patients with stage ii ptt dysfunction who underwent the joint-preserving procedures. these patients were followed up prospectively for one year. all patients were satisfied with respect to pain relief and functional outcome. only two patients considered reoperation for residual deformity. during the period of study no patient was converted to a fusion. this re-enforces the dictum ‘treat the patient and not the x-ray’. the individual procedures are technically demanding and should be carried out with good pre-operative planning and a solid foundation of the anatomy and aim of each individual procedure. it should not be undertaken by the occasional foot surgeon and a certain amount of training and expertise is required. since the above study, we are more prone towards gastrocnemius recessions and lateral column lengthening.35 conclusion in the surgical treatment of aaff deformity due to stage ii ptt, joint-preserving surgery which includes a combination of soft tissue and bone procedures, is preferred. most of these reconstructive procedures include a fdl tendon transfer and a calcaneal osteotomy with varying other components as required. our prospective study yielded a high patient satisfaction rate and minimal complications. we found that there is no correlation between general patient satisfaction and residual deformity. most of the radiographic parameters improved significantly so did the aofas score. although rehabilitation is lengthy, the procedure is functionally superior to arthrodesis of non-arthritic hindfoot joints. conflict of interest statement the content of this article is the original work of the authors. no benefits of any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. references 1. iossi m, johnson je, mccormick jj, klein se. short term radiographic analysis of operative correction of adult acquired flatfoot deformity. foot ankle int. 2013; 34(6):781-91. 2. mann ra. acquired flatfoot in adults. clin orthop rel res. 1983;181:46-51. 3. johnson ka. tibialis posterior tendon rupture. clin orthop rel res. 1983;177:140-47. 4. kulowski j. tendovaginitis (tenosynovitis): general discussion and report of one case involving the posterior tibial tendon. missouri state med assoc. 1936;33:135-37. 5. deland jt, de asla rj, sung ih, ernberg la, potter hg. posterior tibial tendon insufficiency: which ligaments are involved. foot ankle int. 2005;26(6):427-35. 6. myerson ms. adult acquired flatfoot deformity j bone joint surg. 1996;78a:780-92. 7. aronow ms. tendon transfer options in managing the adult flexible flatfoot. foot ankle clinics. 2012;17(2):205-26. 8. deland jt. adult acquired flatfoot deformity. j am acad orthop surg. 2008;16:399-406. 9. guha ar, perera am. calcaneal osteotomy in the treatment of adult acquired flatfoot deformity. foot ankle clinics. 2012;17(2):247-58. 10. kohls-gatzoulis j, woods b, angel jc, singh d. the prevalence of symptomatic posterior tibialis tendon dysfunction in women over the age of 40 in england. j foot ankle surg. 2009;15:75-81. 11. fuhrmann ra, trommer t, venbrocks ra. the acquired buckling flat foot. a foot deformity due to obesity? orthopaed. 2005;34:682-99. 12. mann ra, thompson fm. rupture of the posterior tibial tendon causing flat foot. surgical treatment. j bone joint surg am. 1985;67:556-61. 13. funk da, cass jr, johnson ka. acquired adult flat foot secondary to posterior tibial tendon pathology. j bone joint surg am. 1986;68:95-102. 14. kohls-gatzoulis ja, singh d. tibialis posterior dysfunction as a cause of flatfeet in elderly patients. foot. 2004;14:207209. page 50 sa orthopaedic journal autumn 2016 | vol 15 • no 1 15. holmes gbii, mann ra. possible epidemiological factors associated with rupture of the posterior tibial tendon. foot ankle. 1992;13:70-79. 16. pomeroy gc, pike rh, beals tc, manoli aii. current concepts review. acquired flatfoot in adults due to dysfunction of the posterior tibial tendon. j bone joint surg. 1999;81a:1173-82. 17. henceforth wd, 2nd, deyerle wm. the acquired unilateral flatfoot in the adult: some causative factors. foot ankle. 1982;2:304-308. 18. kohls-gatzoulis ja, angel jc, singh d, haddad f, livingstone j, berry g. tibialis posterior dysfunction: a common and treatable cause of adult acquired flatfoot. bmj. 2004;329(7478):1328-33. 19. myerson m, solomon g, shereff m. posterior tibial tendon dysfunction. its association with seronegative inflammatory disease. foot ankle. 1989;9:219-25. 20. mosier sm, pomeroy g, manoli aii. pathoanatomy and aetiology of posterior tibial tendon dysfunction. clin orthop rel res. 1999;365:12-22. 21. dyal cm, feder j, deland jt, thompson fm. pes planus in patients with posterior tibial tendon insufficiency: asymptomatic versus symptomatic foot. foot ankle int. 1997;18:85-88. 22. kohls-gatzoulis ja, singh d, angel jc. tibialis posterior insufficiency occurring in a patient without peroneii: a mechanical aetiology. foot ankle int. 2001;22:950-52. 23. mann ra, thompson fm. rupture of the posterior tibial tendon causing flatfoot. j bone joint surg 1985;67a:556-61. 24. yeap js, singh d, birch r. tibialis posterior tendon dysfunction: a primary or secondary problem? foot ankle int. 2001;22:51-55. 25. mizel ms, temple ht, scranton pe ii, gellman re, hecht pj, horton ga, et al. role of the peroneal tendons in the production of the deformed foot with posterior tibial tendon deficiency. foot ankle int. 1999;20:285-89. 26. johnson ka, strom de. tibialis posterior tendon dysfunction. clin orthop rel res. 1989;239:196-206. 27. raikin sm, winters bs, daniel jn. the ram classification. foot ankle clinics. 2012;17(2):169-81. 28. pomeroy gc, pike rh, beals tc, manoli aii. acquired flatfoot in adults due to dysfunction of the posterior tibial tendon. j bone joint surg am. 1999;81:1173-82. 29. bluman em, title ci, myerson ms. posterior tibial tendon rupture: a refined classification system. foot ankle clinics. 2007;12:233-49. 30. vulcano e, deland jt, ellis sj. approach and treatment of the adult acquired flatfoot deformity. curr rev musculoskelet med. 2013;6(4):294-303. 31. mehta sk, kellum rb, robertson gh, moore ar, wingerter sa, tarquino ta. radiographic correction of stage iii posterior tibial tendon dysfunction with a modified triple arthrodesis. foot ankle int. 2013;34(10):1355-63. 32. arangio ga, wasser t, rogman a. radiographic comparison of standing medial cuneiform arch height in adults with and without acquired flat foot deformity. foot ankle int. 2006;27(8):636-38. 33. saragas np, ferrao pnf, saragas e, jacobson bf. the impact of risk assessment on the implementation of venous thromboembolism prophylaxis in foot and ankle surgery. foot ankle surg. 2014;20:85-89. 34. hiller l, pinney sj. surgical treatment of acquired flatfoot deformity. what is the state of practice among academic foot and ankle surgeons in 2002? foot ankle int. 2003;24(9):701705. 35. silva mgan, tan shs, chong hc, su hcd, singh ir. results of operative correction of grade iib tibialis posterior tendon dysfunction. foot ankle int. 2015;36(2):165-71. 36. el-tayeby hm. the severe flexible flatfoot: a combined reconstructive procedure with re-routing of the tibialis anterior tendon. j foot ankle surg. 1999;38(1):41-49. 37. guyton gp. flexor digitorum longus transfer and medial displacement calcaneal osteotomy for posterior tibial tendon dysfunction: a middle term clinical follow-up. foot ankle int. 2001;22(8):627-32. 38. knupp mhb. the cobb procedure for treatment of acquired flatfoot deformity associated with stage ii insufficiency of the posterior tibial tendon. foot ankle int. 2007;28(4):416-21. 39. myerson ms. treatment of stage ii posterior tibial tendon deficiency with flexor digitorum longus tendon transfer and calcaneal osteotomy. foot ankle int. 2004;25(7):445-50. 40. parsons s. correction and prevention of deformity in type ii tibialis posterior dysfunction. clin orthop relat res. 2010;468(4):1025-32. 41. zaw h, calder jd. operative management options for symptomatic flexible adult acquired flatfoot deformity: a review. knee surg sports traumatol arthrosc. 2010;18:135-42. this article is also available online on the saoa website (www.saoa.org.za) and the scielo website (www.scielo.org.za). follow the directions on the contents page of this journal to access it. • saoj special issue 1220 icpasih ibn al-haitham journal for pure and applied science https://doi.org/ 10.30526/2021.ihicpas.2646 for more information about the conference please visit the websites: http://ihicps.com/ b i o l o g y | 23 estimation of some biomarkers and cholesterol / hdl ratio to predict the risk of cardiovascular disease in rheumatoid arthritis patients abstract rheumatoid arthritis (ar) is one of the chronic diseases resulting in many complications such as cardiovascular disease (cvd). any change in the lipid profiles and myocardial markers indicates cardiovascular disease risk, so this study is designed to monitor the pattern of lipid profiles and myocardial markers in newly diagnosed ra patients. blood samples were collected from 70 iraqi patients newly diagnosed with rheumatoid arthritis (male and female) and 30 healthy served as control. these individuals were aged 35-65 years. the serum samples were obtained to determine myocardial markers; included troponin, creatinine kinase (ck), lactate dehydrogenase (ldh), and glutamic oxaloacetic transaminase got; and lipid profiles; such as cholesterol, triglyceride, high-density lipoprotein (hdl), low-density lipoprotein (ldl), and very-low-density lipoprotein (vldl); using a kit from roch (germany), measured automatically with minividas, biomerieux (france). no differences between levels of all lipid profiles and myocardial markers in newly diagnosed ra patients compared with the healthy group were found as well as they were within the normal values, but interestingly, the cholesterol/hdl ratio increased significantly in ra patients comparing with healthy, so it could conclude that the risk of cvd could be increase also among the newly diagnosed of ra patients. moreover, the cholesterol/hdl ratio should be probably included in a model to predict the risk of cardiovascular disease for ra in addition to the gender, age at the disease onset, and severity markers of disease. keywords: cardiovascular disease risk, myocardial biomarkers, lipid profile, rheumatoid arthritis disease, tc/hdl ratio. 1. introduction one of the multiple systemic chronic diseases is rheumatoid arthritis (ra) which is usually characterized as heterogeneous symptoms, variable disease progression, and extracurricular manifestations [1]. one of the complications of this disease is the risk of developing cardiovascular disease (cvd), where studies indicate that the risk of accidental cardiovascular disease has increased by 48% in previously diagnosed patients with rheumatoid arthritis compared to the individuals without ra [2]. clinical reports define many markers that could detect the transaminases released from dying myocytes, help diagnosis of myocardial infarction, prognosis, and risk stratification of enas abdul kareem jabbar enaskareemjj0@gmail.com department of basic medical science, college of nursing, university of thi qar, iraq. shaymaa z. al-rumaidh shaymaaalrumaidh@gmail.com college of science,university of thi-qar, iraq. jamela jouda jamela.jouda@uomustansir iyaha.idu.iq department of biology, college of science, mustansiriyiah university, bagdad, iraq. http://ihicps.com/ mailto:enaskareemjj0@gmail.com mailto:shaymaaalrumaidh@gmail.com mailto:jamela.jouda@uomustansiriyaha.idu.iq mailto:jamela.jouda@uomustansiriyaha.idu.iq special issue 1220 icpasih ibn al-haitham journal for pure and applied science https://doi.org/ 10.30526/2021.ihicpas.2646 for more information about the conference please visit the websites: http://ihicps.com/ b i o l o g y | 24 patients with the potential cardiovascular disease. these markers included troponin, creatinine kinase (ck), glutamic oxaloacetic transaminase got, and lactate dehydrogenase (ldh). all these enzymes are released in different quantities by the death of muscle cells, but the research continued since the sensitivity and specificity for myocardial necrosis fuels are lacking [3]. many studies have reported lipid abnormalities in previously diagnosed ra patients [4, 5, 6] one of the traditional cardiovascular risk factors [7]. dyslipidemia is observed in these patients differently than in the general population, including total cholesterol and hdl [8, 9]. since the ratio of disproportionate between tc and hdl levels increases the index of atherogenic, it is an important predictive sign of cvd [1]. these pieces of evidence proved that the previously diagnosed patients with rd could have lipid and myocardial markers abnormalities while to our knowledge there is no research study on these markers in newly diagnosed patients with rd, so this study is designed to monitor the pattern of lipid profiles and myocardial markers in newly diagnosed ra patients. 2. materials method seventy newly diagnosed iraqi patients with rheumatoid arthritis and thirty healthy (male and female) are recruited from the al-husain hospital in thi-qar city at age of 35-65 years. five ml blood was collected from the antecubital vein of each of the patients and healthy and put in dry sterilized test tubes. the blood samples were centrifuged for 5min at 3000rpm to obtain the serum that used to determine myocardial markers; included troponin, ck, ldh, and got; and lipid profiles; included cholesterol, triglyceride, hdl, ldl, and vldl; using a kit from roch (germany), measured automatically with minividas, biomerieux (france). the results were computed as the mean ± standard error. in all statistical analyses, only p ≤ 0.05 was considered significant. differences among groups were analyzed by variance oneway analysis anova followed by fisher's test for multiple comparisons, using statview version 5.0. differences were considered significant when p<0.05. regression analysis was performed by analysis of covariance (ancova) also using stat view version 5.0. 3. results and discussion there are no differences between levels of myocardial markers in ra patients comparing with the healthy group included troponin, creatinine kinase, got, and lactate dehydrogenase. moreover, there are no differences between these enzyme levels in male and female groups. however, they were within normal values which are as following: (troponin=0-0.4ng/ml, ck=22-198u/l, got=8-45u/l, and ldh=140-280u/l) (figure 1). http://ihicps.com/ special issue 1220 icpasih ibn al-haitham journal for pure and applied science https://doi.org/ 10.30526/2021.ihicpas.2646 for more information about the conference please visit the websites: http://ihicps.com/ b i o l o g y | 25 figure 1. myocardial markers levels in rheumatoid arthritis patients and control groups. (a) troponin, (b) creatinine kinase (ck), (c) glutamic oxaloacetic transaminas got, (d) lactate dehydrogenase (ldh). figure 2 shows lipid profile levels in both ra patients and healthy groups (male and female). there were no differences in all lipid profiles levels of control and ra patients groups however they were within normal values which are as following: (cholestrol˂150mg/dl, triglyceride˂150mg/dl, hdl=40-59mg/dl, vldl=2-30mg/dl, and ldl˂100mg/dl). moreover, there are no differences between these enzyme levels in male and female groups. interestingly, the cholesterol / hdl ratio was also calculated. it increased significantly from (2.53±0.027) in control to (3.78±0.26) in ra patients. figure 2. lipid profiles levels in rheumatoid arthritis patients and control groups. (a) cholestrol, (b) triglyceridle, (c) high-density lipoprotein (hdl), (d) very-low-density lipoprotein (vldl), (e) low-density lipoprotein (ldl). the correlation between all these markers and age was also calculated. there were only significantly positive correlations between cholesterol and triglyceride with age whereas there http://ihicps.com/ special issue 1220 icpasih ibn al-haitham journal for pure and applied science https://doi.org/ 10.30526/2021.ihicpas.2646 for more information about the conference please visit the websites: http://ihicps.com/ b i o l o g y | 26 was no correlation between other markers and age (table 1). table 1. the correlation between lipid profiles and myocardial markers. parameters age r p. value myocardial markers troponin 0.134 0.198 ck 0.077 0.455 got 0.01 0.890 ldh 0.122 0.247 lipid profils cholestrol 0.219 0.049 triglyceride 0.305 0.002 hdl 0.01 0.946 ldl 0.055 0.687 vldl 0.045 0.687 4. discussion since ra patients have a higher rate of cardiovascular disease risk compared to individuals without autoimmune diseases [10], periodic examination of cardiovascular risk according to the national guidelines used for the care of ra patients [11]. however, the general risk calculators used do not succeed in capturing the increased risks in these patients [12, 13]. cardiovascular disease risk is associated with many mechanisms, for example, the adverse effects of pharmacological ra, ra treatment, and accelerated atherosclerosis due to inflammatory processes associated with long-term ra [14-17]. in our work, all patients were newly diagnosed with rheumatoid arthritis, so this could be the reason why there wasn’t a difference between cardiac parameters levels in ra patients compared with healthy groups. kremers et al and lindhardsen et al also stated that the cardiovascular disease risk in ra patients was similar to that of people without rheumatoid arthritis [7, 18]. but this result does not exclude the possibility of an increase in cardiovascular disease risk after 5–10 years of rheumatoid arthritis, although intensive treatment aimed at controlling inflammation in recent years is likely to decrease the cardiovascular disease risk rate that increases during the period of rheumatoid arthritis [19]. it is known that the lipid profile is one of the signs of cardiovascular disease because it makes rheumatoid arthritis patients triple cardiovascular disease risk. after all, this disease itself caused atherosclerosis (1). lipid levels seem to change due to rheumatoid arthritis activity. data of total cholesterol and low-density cholesterol concentrations differ in ra patients. some works show similar levels [20] or less [21] of total cholesterol, however other researches indicated high concentration of total cholesterol and ldl cholesterol in early ra patients [22] or indicate decreased total cholesterol, ldl, and hdl inactive untreated ra patients [6, 21, 23]. in our results, there was no difference in all lipid profiles between ra patients and the health group. regardless of the changes in total cholesterol and hdl in ra patients, several studies support the calculating importance of total cholesterol to hdl ratio in ra patients, which leads to a more fatty arteriosclerosis profile and is associated with disease activity and http://ihicps.com/ special issue 1220 icpasih ibn al-haitham journal for pure and applied science https://doi.org/ 10.30526/2021.ihicpas.2646 for more information about the conference please visit the websites: http://ihicps.com/ b i o l o g y | 27 improves after treatment with antirheumatic drugs [20, 21, 22, 24]. in our results, the cholesterol/hdl ratio increased significantly in the ra patients than in the control group. a similar gender-based pattern of cardiovascular disease risk observed in this study was reported by [25] and [7], who also found no general difference in cardiovascular disease risk between genders whereas there weren’t differences in lipid profile and myocardial markers between women and men with ra in this study. 4. conclusion in conclusion, the cardiovascular disease risk could be increase also in the patient with newly diagnostic rheumatoid arthritis so other patient characteristics, such as gender, age at the onset of the disease, disease severity markers, and tc / hdl ratio should be probably included in a model to predict the cardiovascular disease risk for ra, developing such a model, validating it and assessing its primary and secondary prevention strategies for cardiovascular disease in rheumatoid arthritis patients. references 1. erum, u.; ahsan, t; khowaja, d. lipid abnormalities in patients with rheumatoid arthritis. pak. j. med. sci. 2017, 33, 1, 227-230. 2. avina-zubieta, j.a.; thomas, j.; sadatsafavi, m.; lehman, a.j.; lacaille, d. risk of incident cardiovascular events in patients with rheumatoid arthritis: a meta-analysis of observational studies. ann. rheum. dis. 2012 ,71, 9, 1524-1529. 3. pant, s.; deshmukh, a.; neupane, p.; kumar, m.p.k.; vijayashankar, c.s. cardiac biomarkers. in: lakshmandoss u, editor. noval strategies in ischemic heart disease. india: mercy health 2012, 17-42. 4. gabriel, s.e.; crowson, c.s. risk factors for cardiovascular disease in rheumatoid arthritis. curr. opin. rheumatol. 2012 ,24, 2, 171-176. 5. boyer, j.f.; gourraud, p.a.; cantagrel, a.; davignon, j.l.; constantin, a. traditional cardiovascular risk factors in rheumatoid arthritis: a meta-analysis. joint bone spine. 2011 78, 2, 179-183. 6. choy, e.; sattar, n. interpreting lipid levels in the context of high-grade inflammatory states with a focus on rheumatoid arthritis: a challenge to conventional cardiovascular risk actions. ann. rheum. dis. 2009, 68, 4, 460-469. 7. lindhardsen, j.; ahlehoff, o.; gislason, g.h.; madsen, o.r.; olesen, j.b.; torppedersen. c. the risk of myocardial infarction in rheumatoid arthritis and diabetes mellitus: a danish nationwide cohort study. ann. rheum. dis. 2011 ,70, 6, 929-934. 8. steiner, g.; urowitz. m.b. lipid profiles in patients with rheumatoid arthritis: mechanisms and the impact of treatment. semin arthritis rheum. 2009 ,38, 5, 372-381. 9. amezaga-urruela, m.; suarez-almazor, m.e. lipid paradox in rheumatoid arthritis: changes with rheumatoid arthritis therapies. curr rheumatol rep. 2012 ,14, 5, 428-437. 10. naranjo, a.; sokka, t.; descalzo, m.a.; calvo-alen, j.; horslev-petersen, k.; luukkainen. r.k. cardiovascular disease in patients with rheumatoid arthritis: results from the quest-ra study. arthritis res. ther. 2008 ,10, 2, r30. 11. agca, r.; heslinga, s.c.; rollefstad, s.; heslinga, m.; mcinnes, i.b.; peters, m.j. eular recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update. ann rheum dis. 2017 ,76, 1, 17-28. http://ihicps.com/ special issue 1220 icpasih ibn al-haitham journal for pure and applied science https://doi.org/ 10.30526/2021.ihicpas.2646 for more information about the conference please visit the websites: http://ihicps.com/ b i o l o g y | 28 12. crowson, c.s.; matteson, e.l.; roger, v.l.; therneau, t.m.; gabriel, se. usefulness of risk scores to estimate the risk of cardiovascular disease in patients with rheumatoid arthritis. am j cardiol. 2012 ,110, 3, 420-424. 13. siontis, g.c.; tzoulaki, i.; siontis, k.c.; ioannidis, j.p. comparisons of established risk prediction models for cardiovascular disease: systematic review. bmj. 2012 ,344, e3318. 14. gonzalez, a.; maradit-kremers. h.; crowson. c.s.; ballman. k.v.; roger. v.l.; jacobsen, s.j. do cardiovascular risk factors confer the same risk for cardiovascular outcomes in rheumatoid arthritis patients as in non-rheumatoid arthritis patients? ann rheum dis. 2008 ,67, 1, 64-69. 15. solomon, d.h.; curhan, g.c.; rimm, e.b.; cannuscio, c.c.; karlson, e.w. cardiovascular risk factors in women with and without rheumatoid arthritis. arthritis rheum. 2004 ,50, 11, 3444-3449. 16. toms, t.e.; symmons, d.p.; kitas, g.d. dyslipidaemia in rheumatoid arthritis: the role of inflammation, drugs, lifestyle and genetic factors. curr vasc pharmacol. 2010, 8, 3, 301-326. 17. solomon, d.h.; kremer, j.; curtis, j.r.; hochberg, m.c.; reed, g.; tsao, p. explaining the cardiovascular risk associated with rheumatoid arthritis: traditional risk factors versus markers of rheumatoid arthritis severity. ann rheum dis. 2010 ,69, 11, 1920-1925. 18. kremers, h.m.; crowson, c.s.; therneau, t.m.; roger, v.l.; gabriel, s.e. high ten-year risk of cardiovascular disease in newly diagnosed rheumatoid arthritis patients: a population-based cohort study. arthritis rheum. 2008 ,58, 8, 2268-2274. 19. avina-zubieta, j.a.; choi, h.k.; sadatsafavi, m.; etminan, m.; esdaile, j.m.; lacaille, d. risk of cardiovascular mortality in patients with rheumatoid arthritis: a meta-analysis of observational studies. arthritis rheum. 2008 59, 12, 1690-1697. 20. park, y.b.; lee, s.k.; lee, w.k.; suh, c.h.; lee, c.w.; lee, c.h,. lipid profiles in untreated patients with rheumatoid arthritis. j. rheumatol. 1999 ,26, 8, 1701-1704. 21. boers, m.; nurmohamed, m.t.; doelman, c.j.; lard, l.r.; verhoeven, a.c.; voskuyl. a.e. influence of glucocorticoids and disease activity on total and high density lipoprotein cholesterol in patients with rheumatoid arthritis. ann rheum dis. 2003, 62, 9, 842-845. 22. georgiadis, a.n.; papavasiliou, e.c.; lourida, e.s.; alamanos, y.; kostara, c.; tselepis. a.d. atherogenic lipid profile is a feature characteristic of patients with early rheumatoid arthritis: effect of early treatment--a prospective, controlled study. arthritis res. ther. 2006 ,8, 3, r82. 23. myasoedova, e.; crowson, c.s.; kremers, h.m.; roger, v.l.; fitz-gibbon. p.d.; therneau. t.m. lipid paradox in rheumatoid arthritis: the impact of serum lipid measures and systemic inflammation on the risk of cardiovascular disease. ann rheum dis. 2011 ,70, 3, 482-487. 24. van halm, v.p.; nielen, m.m.; nurmohamed, m.t.; van schaardenburg, d.; reesink. h.w.; voskuyl, a.e. lipids and inflammation: serial measurements of the lipid profile of blood donors who later developed rheumatoid arthritis. ann rheum dis. 2007, 66, 2, 184188. 25. solomon, d.h.; goodson, n.j.; katz, j.n.; weinblatt, m.e.; avorn, j.; setoguchi, s. patterns of cardiovascular risk in rheumatoid arthritis. ann rheum dis. 2006,65, 12, 16081612. http://ihicps.com/ the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 38 s afr fam pract issn 2078-6190 eissn 2078-6204 © 2018 the author(s) review 1. fundamentals of rheumatoid arthritis rheumatoid arthritis (ra), an auto-immune disorder with unknown aetiology and a chronic and degenerative nature, is associated with synovitis (inflammation of the synovial membranes) and the eventual deterioration of cartilage and erosion of bone due to hyperosteoclastic activity1 extending even to extra-articular organs and tissue, resulting in inter alia vasculitis, rheumatoid lung disease, and pericarditis.2 a family history of ra has considerable value in predicting the development of the condition in first degree relatives, increasing an individual’s odds of suffering from ra up to fourfold3 and, combined with genetic (e.g. the hla drb1 alleles) and environmental (e.g. cigarette smoking, a diet lacking sufficient omega-3 fatty acids and exposure to infectious agents) risk factors, plays an important role in development and progression of the disease.4 early investigations into the pathophysiology of ra included the discovery of rheumatoid factor (rf) – an antibody in part produced by b-1 lymphocytes – in the blood of patients suffering from ra.5 another prominent characteristic of the autoimmune response in ra patients is the presence of anti-cyclic citrullinated peptide antibodies (acpas)6 which may precede the onset of disease by up to ten years7 – probably indicating that immune activity responsible for triggering the pathology occurs long before eventual symptom development and diagnosis.8 indeed, acpas have the most accurate predictive value among antibodies produced in ra.9 considering the presence of these factors, ra is characterised as an auto-immune disorder. however, although the presence of rf is usually predictive of a more severe disease progression (i.e. more severe joint damage and increased mortality,8,10,11 only 75% of patients (depending on disease stage) are seropositive for the presence of either rf or acpas, while up to 80% of patients test positive for both factors.5,10 regrettably, these factors may also be present in patients suffering from unrelated chronic inflammatory conditions (and even some healthy individuals). moreover, the possibility of seronegative conversion in patients previously presenting with rf is also highly likely subsequent to successful treatment strategies.8,10 as such, ra patients may or may not express these antibodies and the presence thereof is not mandatory or even definitive in the diagnosis of ra. they do however remain valuable tools in diagnostic and prognostic considerations. in addition to the presence of auto-antibodies, t-cell and b-cell infiltrate in the synovium also presents a prominent feature in ra.8 acpas may induce pathogenicity by indirectly activating macrophages (eliciting a pro-inflammatory cytokine response),12 and osteoclasts (via tumour necrosis factor (tnf); promoting bone resorption and explaining the increased presence of bone damage in acpa-positive patients)13 and form immune complexes that are able to interact with and potentiate the effects of rf, e.g. cytokine activation and direct macrophage activation. excessive pro-inflammatory cytokines, e.g. il-1 and tnf-α, drive the deleterious mechanisms involved in the pathology of ra and it is evident that cells belonging to both the innate (e.g. macrophages) and adaptive (e.g. b and t lymphocytes) immune systems not only interact with one another, but also with resident cells (i.e. synovial fibroblasts).14 subsequently, an array of cytokines are produced that affect the recruitment, retention, and differentiation of infiltrating leukocytes,15 producing the inflammatory processes observed in ra.1 aberrant cellular and humoral immune responses therefore forego the presence of autoantibodies, followed by synovial concentration of tand b-cells. with time, synovial hyperplasia develops due to macrophage invasion, promoting proliferation of synovial fibroblasts.16 continued erosion of cartilage promotes synovial hyperplasia, consequently leading to south african family practice 2018; 60(2):38-42 open access article distributed under the terms of the creative commons license [cc by-nc-nd 4.0] http://creativecommons.org/licenses/by-nc-nd/4.0 a quick and painless reminder: the pharmacotherapy of rheumatoid arthritis in primary practice linda brand,a de wet wolmarans,a* sarel j brandb a center of excellence for pharmaceutical sciences, division of pharmacology, faculty of health sciences, north west-university, potchefstroom, south africa b unit for environmental sciences and management, north-west university, potchefstroom, south africa *corresponding author, email: dewet.wolmarans@nwu.ac.za abstract rheumatoid arthritis, an auto-immune disorder, is characterised by chronic inflammation of the joints, synovial hyperplasia and bone erosion. these pathological features are promoted by a synovial microenvironment featuring b-cell and t-cell infiltrate, synovial fibroblasts and an intricate network of pro-inflammatory cellular messengers – prominent molecular role-players that represent critical targets in the pharmacotherapy of the disease. this review offers a brief overview of the etiopathology of rheumatoid arthritis while focussing on the practical aspects of methotrexate and glucocorticoid use that are of relevance for primary practice. a quick and painless reminder: the pharmacotherapy of rheumatoid arthritis in primary practice 39 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 39 the development of invading pannus tissue17 and increased fibroblast and osteoclast activity leads to the destruction of cartilage and surrounding bone.15–17 additionally, synoviocytes produce enzymes, e.g. collagenase and gelatinase, that lead to the detriment of structural matrices.18 as a result, ra is thus characterised by a complex interplay of several molecules creating a microenvironment in synovial tissue and/or joints that promotes on-going and debilitating chronic inflammation. several interactions, e.g. between haematopoietic cells (e.g. lymphocytes) and fibroblasts, t-cells and fibroblasts and t lymphocytes with macrophages and b-cells, contribute to the persistence of inflammation by producing signals that encourage white cell survival and retention.14 considering the above, it is important to note that therapies targeting band t-cell mediated mechanisms provide superior results in patients that are also acpaand rf-positive compared to seronegative patients. this, however, is often not the case for tnf-, il-6and il-1-targeted therapies. the adaptive immune response therefore mainly assists in the pathogenesis of seropositive patients while the intricate cytokine-involving processes are an almost universal feature in ra.19 2. current pharmacotherapeutic approaches to rheumatoid arthritis briefly, the goal of pharmacotherapy is to reduce the pain, swelling and stiffness of the joints, preserve joint function, slow destructive joint changes, prevent systemic complications and improve the patient’s ability to perform his/her daily activities. in this regard, the rapid diagnosis of ra is essential. indeed, a relatively narrow window of opportunity (3 – 6 months) from the onset of joint swelling and initiation of a treat-to-target approach with tight monitoring and control exists that may increase the likelihood of remission and lessen damage to the joints.20 the three main groups of drugs used for the treatment of ra are the non-steroidal anti-inflammatory drugs (nsaids), corticosteroids and disease-modifying antirheumatic drugs (dmards). nsaids reduce joint pain and swelling, but have no impact on disease progression, while short-term use (< 3months) of the corticosteroids can rapidly reduce joint inflammation. however, the only drugs that may actually slow disease progression are the dmards.21 that said, as the use of biological dmards is generally initiated by specialists, the current paper will only briefly summarise key aspects of this drug class while focussing on novel aspects pertaining to the use of glucocorticoids and methotrexate that are of relevance in primary practice. 2.1 a general overview of disease modifying anti-rheumatic drugs (dmards) contrary to earlier guidelines, treatment with the dmards is initiated concurrent with diagnosis of ra as they have been demonstrated to reduce the risk of permanent damage to the joints.20,22 dmards can broadly be divided into the traditional/ synthetic non-biological dmards and the recently introduced biological agents, i.e. monoclonal antibodies that still mainly target pro-inflammatory cytokines, e.g. tnf–α; however, antibodies not directed at tnf-α also exist. while the traditional dmards can be administered orally or parenterally, the biological dmards can only be injected under the supervision of qualified professionals. the biological dmards have a faster onset of action compared to the synthetic dmards and as they only target a specific element of the inflammatory response, they do not compromise the entire immune response. however, the high cost and potential risk of severe hypersensitivity reactions limit their use to patients not responding adequately to the traditional dmards.23,24 in this regard, a trial of at least 6 months on traditional dmards without adequate response may be an indication of therapeutic failure.24 first-line non-biological dmards include methotrexate, hydroxychloroquine, sulfasalazine and leflunomide. other dmards, now seldom used because of their higher toxicity and less than adequate efficacy, include gold (orally as auranofin®/ ridaura® or intramuscularly as myochrysine®), minocycline, azathioprine (imuran®) and cyclosporine (table 1).25 the biological agents with disease-modifying activity include the anti-tnf-α group of drugs viz. etanercept (enbrel®), infliximab (revellex®), adalimumab (humira®), certolizumab and golimumab (simponi®). non-tnf-α inhibitors of the immune system include abatacept (orencia®), that modulates t-cell activation, as well as tocilizumab (actemra®) and recently fda-approved sarilumab1 that act as il-6 receptor antagonists. rituximab, an anti-cd 20 monoclonal antibody that depletes peripheral b lymphocytes, and anakinra, an il-1 receptor antagonist, can also be used.23 another novel subset of dmards (targeted synthetic dmard) is the orally administered janus kinase (jak) inhibitors, e.g. tofacitinib1 (not yet registered in sa), that inhibit t-cell proliferation.26 nevertheless, the traditional dmards, e.g. methotrexate, are still the first-line choice of therapy with or without the concomitant use of glucocorticoids.27,28 however, although glucocorticoids have been in use for several decades, recent recommendations still do not provide clear evidence-based guidance with respect to dose, timing and optimal duration of use, tapering strategies and the determination of appropriate long-term benefit vs risk ratios.29 2.1.1 methotrexate despite the development and availability of a number of biological dmards since the 1990s and more recently the targeted synthetic dmards, methotrexate (mtx) is still regarded as the gold standard dmard. it is widely used in monotherapy or in combination with other synthetic or biological agents.30–32 however, as in the case of glucocorticoid (gc) therapy, a major clinical challenge associated with mtx use is treatment nonresponse. in fact, 40% of patients remain refractory to mtx treatment. as such, attempts to provide suitable alternatives currently focus on understanding the mechanisms involved in mtx-resistance and to identify putative biomarkers that may indicate adequate therapeutic response to mtx and other biologic dmards before such treatment is initiated.31,33-35 another recent recommendation regarding mtx therapy pertains to its optimal dosage. over the past few decades the recommended dosage of mtx has been gradually adjusted upwards and it is currently suggested that the mean dose prescribed in ra should range between 20–25 mg/week.25 1 not available in south africa s afr fam pract 2018;60(2):38-4240 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 40 however, as the oral absorption of mtx peaks at 15–20 mg/ week,25 parenteral mtx is also used more often.36 2.1.2 the glucocorticoids corticosteroids (administered either systemically or intraarticularly) are not only cost-effective but elicit potent antiinflammatory and immunosuppressive action. as such, they are still used routinely as part of the first-line strategy in the management of ra.37 the importance and efficacy of glucocorticoids (gcs) in ra therapy are also reflected in current guidelines for the initial treatment of ra where the concomitant use of gcs with a traditional dmard is recommended, followed by another synthetic or biologic dmard with/without a gc if the treatment target of remission is not reached within 3–6 months.27,28 further, irrefutable clinical trial evidence indicated that prednisone (7.5 mg daily) may slow the progression of the disease.22,38 in an attempt to provide clearer evidencebased guidelines for the use of gcs in ra, european researchers, supported by the gloria project, undertook a systematic literature review of clinical practice guidelines (2011–2015).29 the results of this review concluded that gcs at low doses and for a short duration of time are an appropriate treatment option of ra. considering the benefit vs risk ratio of gc therapy in ra, a positive ratio for long-term (3–6 months or more) low dose (≤ 5mg/day prednisone-equivalent) therapy has been demonstrated, while an increased risk of harm in most patients was found at dosages of > 10 mg/day.28 nevertheless, it is true that, at dosages between 5 and 10 mg/day, patient-specific characteristics (protective as well as risk factors) determine an individual’s risk of harm, being dose-dependent and patient-specific. therefore, overall and gc associated risk and protective factors, e.g. lifestyle and diet, should be taken into account when evaluating the actual current and possible future risk..28,39 among the most worrisome risks associated with long-term, high-dose gc use are the development of osteoporosis, diabetes mellitus, weight gain, cardiovascular disease, secondary infection, and myopathy.28 as such, in order to minimise adverse effects while preserving or even bolstering gc anti-inflammatory efficacy, two novel approaches are currently under investigation. though neither is currently advised as an appropriate therapeutic option, this may change soon. first, the development of liposomal gc molecules (prednisolone, methylprednisolone or dexamethasone) involves enfolding the drug molecules in a core of polyethylene glycol (peg) stabilised liposomes, thereby attempting to enhance tissue-specific drug accumulation in the inflamed joint following intravenous administration.37,40,41 clinical trials investigating the efficacy and safety of these liposomal gc preparations are ongoing.37,42 indeed, a successful phase ii clinical trial confirmed the safety and increased efficacy of liposomal prednisolone compared to traditional prednisolone formulations.43 second, the development of highly selective, dissociated, or modulating gc agonists (abbreviated segra, dagr, and segrm, respectively) is currently underway. such advances are based on the often oversimplified hypothesis that processes of transactivation (i.e. the synthesis of certain regulatory proteins, e.g. enzymes of gluconeogenesis) are responsible for the majority of adverse reactions, while transrepression (i.e. the synthesis of pro-inflammatory cytokines) is instrumental in the anti-inflammatory and immunomodulating effects of gcs.40 in this regard, segras will primarily induce the desired transrepression activity, but reduce the transactivation effects compared to conventional cgs.40,44,45 with respect to the dagrs, fosdagrocorat – a compound with potent anti-inflammatory activity and minimal effects on bone and glucose metabolism – is currently under clinical investigation for the treatment of ra.41,46,47 again, as is also true for mtx treatment, approximately 30% of ra patients remain resistant to gc therapy. a fact box for primary practice: when risk of prescribing mtx outweighs the benefit considering that the biological dmards are primarily reserved for use in specialist practice and that no clear guidelines exist that provide detailed algorithms for the initiation, continuation and cessation of mtx therapy in the primary care setting, a useful approach is to be aware of the signs and symptoms that indicate oral overtreatment and that necessitate immediate dose reductions or complete withdrawal from therapy. these are highlighted below:37 • acute renal failure – expect in 2–4% of patients. condition is often seemingly asymptomatic and will usually resolve following mtx withdrawal. • severe leukopenia – expect in 1–3% of patients. consider risk for secondary infection. condition resolves within 3 weeks following mtx withdrawal. • mucocutaneous pathology, i.e. ulceration, photosensitivity, erythema, urticarial and vasculitis. expect in all patients. dose reductions and folic acid supplementation are usually adequate to prevent exacerbation. • hepatotoxicity, especially in patients with comorbid hepatitis b or c, diabetes, obesity and alcohol abuse history. risk can be reduced, but not reversed with folic acid supplementation. • pneumonitis – occurs in 1% of patients. involves t-cell mediated hypersensitivity to mtx administration, resulting in pulmonary inflammation associated with a non-productive cough and dyspnoea. immediate mtx withdrawal is mandatory. a fact box for primary practice: glucocorticoids and comorbidity as is true for most auto-immune disorders, ra demonstrates a high degree of comorbidity with other conditions marked by aberrant auto-immune responses, especially type 1 diabetes mellitus, inflammatory bowel disease (ibd) and hashimoto thyroiditis (ht).50 as such, a few aspects must be kept in mind in the treatment of ra associated with established or possible comorbidities. prescribing glucocorticoids • due to its broad suppression of the immune system, gcs also form part of the first line therapy for ibd and ht. dosing schedules for these conditions are guided by the same principles as for ra. • an aspect that is often of great concern when gcs are initiated is established or possible comorbid type 1 diabetes mellitus. chronic gc administration is known to impair glucose tolerance. importantly however, high dose (i.e. 60–80 mg prednisone per day), short term (7–10 days) administration of gcs does not alter glucose metabolism to such an extent that coincidental pathology is triggered.51 in fact, patients will benefit from such dosing schedules with respect to its anti-inflammatory properties, without presenting with lasting and irreversible metabolic complications. moreover, as the inflammatory state underlying ra contributes significantly to impair glucose tolerance, the long term benefit of intermittent high dose gc administration most probably outweighs the risk for secondary metabolic complications.51 • the same principle as above applies with respect to the benefit vs risk ratio pertaining to all other pathophysiological constructs that may raise concern, e.g. bone-mineral density, hypertension, glaucoma, and cataracts. however, this does not apply to the risk of contracting infectious conditions, and great care needs to be taken in patients with, or who are at high risk for, infectious disease. a quick and painless reminder: the pharmacotherapy of rheumatoid arthritis in primary practice 41 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 41 3. concluding statement the current paper provides a brief overview of the currently available dmards and focuses on practical aspects of mtx and gc use for ra within the context of primary practice. for a complete review of ra, its etiopathology and treatment, the reader is referred to bester et al. (2016).52 table 1: summary of the traditional dmards, dosages, adverse effects, and monitoring advice (adapted from [48]) dmard trade name® dosage adverse effects comments monitoring c h lo ro qu in e nivaquine (150 mg) plasmoquine (150 mg) oral: initially 150–300 mg daily, reduced to 150 mg daily after 7–10 days. after remission is obtained, a 5-days-a-week regimen should be attempted. recommended dose 2.4 mg/kg macular damage, rash, diarrhoea onset of action may be delayed for up to 6 weeks, but should not be considered as therapeutic failure until after 6 months without response. use with caution in renal or hepatic impairment, alcoholism, blood disorders, neurological disorders, psoriasis, pre-existing visual disturbances ophthalmic examinations before treatment initiation, repeat every 6 months, regular full blood counts c ic lo sp or in sandimmun neoral (25, 100 mg) sandimmun iv infusion (50 mg/ml) oral: 2.5 mg/kg/day in 2 divided doses, may be increased gradually after 6 weeks to 4 mg/kg/day. discontinue if response is still inadequate after 3 months. maintenance: titrate to lowest effective dose, decreasing by 0.5 mg/kg monthly or bi-monthly dose-related nephrotoxicity, hepatotoxicity, neurotoxicity, gingival hyperplasia and hirsutism potential interaction with drugs metabolised by cytochrome p450 ciclosporin blood levels, kidney and liver function tests regularly, regular dental examinations le fl un om id e arava (10, 20 mg) rheumalef (10, 20 mg) active ra 10 – 20 mg once daily. a loading dose of 100 mg/day for the first 3 days may result in a therapeutic response within the first month hepatitis, gastrointestinal distress, alopecia, increase in blood pressure, headaches, dizziness, weight loss, skin rash contraindicated in liver disease alanine aminotransferase (alt) should be monitored monthly initially and periodically thereafter. complete blood cell count before therapy starts, 2 weekly for the first 6 months, then 2 monthly. risk of tuberculosis reactivation m et h ot re xa te methotrexatelederle (2.5 mg) abitrexate p&u methotrexate csv (25 mg/ml) oral: 7.5 mg weekly (as a single dose or 2.5 mg 12 hourly for 3 doses, max 25 mg/ week iv or im: 10 mg once weekly, increased as necessary up to 25 mg once weekly myelosuppression, hepatic fibrosis, cirrhosis, pulmonary infiltrates or fibrosis, hematologic, gastrointestinal irritation, stomatitis, rash relative rapid onset of action (2–3 weeks) lack of efficacy at 25 mg/week for 3 months is considered failure of therapy. may be given with folic acid 1–5 mg/day to reduce adverse reactions, without loss of efficacy. may precipitate acute attack of gout. patients with hyperuricaemia should maintain adequate fluid intake, alkalinisation of urine may be useful. full blood counts, urea and electrolytes, liver function tests before therapy starts and regularly thereafter su lfa sa la zi n e salazopyrin (500 mg) 500 mg daily, orally after food, maximum 40 mg/kg usual maintenance dose 1g twice daily myelosuppression, rash, gastrointestinal disturbances (take with food, divide the dose more evenly throughout the day). antirheumatic effects should be seen in 2 months. not recommended in renal or hepatic impairment megaloblastic anaemia may occur, folic acid supplementation should be prescribed. enteric coated tablets may be useful for long term full blood counts, urea and electrolytes, liver function tests before therapy starts and regularly thereafter s afr fam pract 2018;60(2):38-4242 the page number in the footer is not for bibliographic referencingwww.tandfonline.com/oemd 42 references 1. angelotti f, et al. one year in review 2017: pathogenesis of rheumatoid arthritis. clin exp rheumatol. 2017;35(3):368-78. 2. turesson c. extra-articular rheumatoid arthritis. curr opin rheumatol. 2013;25(3):360-6. 3. frisell t, saevarsdottir s, askling j. family history of rheumatoid arthritis: an old concept with new developments. nat rev rheumatol. 2016;12(6):335-43. 4. silman aj, pearson je. epidemiology and genetics of rheumatoid arthritis. arthritis res. 2002;4(suppl 3):s265-72. 5. nell v, et al. autoantibody profiling as early diagnostic and prognostic tool for rheumatoid arthritis. ann rheum dis. 2005;64(12):1731-6. 6. schellekens ga, et al. citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. j clin invest. 1998;101(1):273-81. 7. nielen mm, et al. specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. arthritis rheum. 2004;50(2):380-6. 8. scherer hu, burmester g-r. adaptive immunity in rheumatic diseases – bystander or pathogenic player? best pract res clin rheumatol. 2011;25(6):785-800. 9. rantapaa-dahlqvist s, et al. antibodies against cyclic citrullinated peptide and iga rheumatoid factor predict the development of rheumatoid arthritis. arthritis rheum. 2003;48(10):2741-9. 10. aletaha d, alasti f, smolen js. rheumatoid factor, not antibodies against citrullinated proteins, is associated with baseline disease activity in rheumatoid arthritis clinical trials. arthritis res ther. 2015;17(1). 11. gonzalez a, et al. mortality trends in rheumatoid arthritis: the role of rheumatoid factor. j rheumatol. 2008;35(6):1009-14. 12. clavel c, et al. among human macrophages polarised to different phenotypes, the m-csf-oriented cells present the highest pro-inflammatory response to the rheumatoid arthritis-specific immune complexes containing acpa. ann rheum dis. 2016;75(12):2184-91. 13. harre u, et al. induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. j clin invest. 2012;122(5):1791-1802. 14. buckley cd, et al. stromal cells in chronic inflammation and tertiary lymphoid organ formation. ann rev immunol. 2015;33(1):715-45. 15. buckley cd. why do leucocytes accumulate within chronically inflamed joints? rheumatology. 2003;42(12):1433-44. 16. burmester g, et al. rheumatoid arthritis: pathogenesis and clinical features. eular textbook on rheumatic diseases. london: bmj group; 2012:206-31. 17. redlich k, smolen js. inflammatory bone loss: pathogenesis and therapeutic intervention. nat rev drug discov. 2012;11:234. 18. smolian h, et al. secretion of gelatinases and activation of gelatinase a (mmp-2) by human rheumatoid synovial fibroblasts. biol chem. 2001;382(10):1491-9. 19. stoffer ma, et al. evidence for treating rheumatoid arthritis to target: results of a systematic literature search update. ann rheum dis. 2016;75(1):16-22. 20. burmester gr, pope je. novel treatment strategies in rheumatoid arthritis. lancet. 2017;389(10086):2338-48. 21. stark swrnad. rheumatoid arthritis. salem press; 2003. 22. weisman mh. rheumatoid arthritis. cary, united states: oxford university press; 2014. 23. harmse l, reuter h. an overview of the biological disease modifying drugs available for arthritic conditions in south africa. s afr fam pract. 2016;58(6):6-10. 24. tarr g, hodkinson b, reuter h. superheroes in autoimmune warfare: biologic therapies in current south african practice. samj. 2014;104(11):787-91. 25. jones g, nash p, and hall s. advances in rheumatoid arthritis. med j aust. 2017;206(5):221-4. 26. chatzidionysiou k, et al. efficacy of glucocorticoids, conventional and targeted synthetic disease-modifying antirheumatic drugs: a systematic literature review informing the 2016 update of the eular recommendations for the management of rheumatoid arthritis. ann rheum dis. 2017;76(6):1102-7. 27. smolen js, et al. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update. ann rheum dis. 2014;73(3):492-509. 28. cindy s, et al. defining conditions where long-term glucocorticoid treatment has an acceptably low level of harm to facilitate implementation of existing recommendations: viewpoints from an eular task force. ann rheum dis. 2016(6):952. 29. palmowski y, et al. “official view” on glucocorticoids in rheumatoid arthritis: a systematic review of international guidelines and consensus statements. arthritis care res. 2017;69(8):1134-41. 30. romao vc, canhao h, fonseca je. old drugs, old problems: where do we stand in prediction of rheumatoid arthritis responsiveness to methotrexate and other synthetic dmards? bmc med. 2013(1). 31. peres rs, et al. tgf-β signalling defect is linked to low cd39 expression on regulatory t cells and methotrexate resistance in rheumatoid arthritis. j autoimmun. 32. smolen js, et al. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update. ann rheum dis. 2017;76(6):960-77. 33. bansard c, et al. can rheumatoid arthritis responsiveness to methotrexate and biologics be predicted? rheumatology. 2009;48(9):1021-8. 34. carini c, et al. chromosome conformation signatures define predictive markers of inadequate response to methotrexate in early rheumatoid arthritis. j transl med. 2018;16(1):18. 35. hider sl, et al. can clinical factors at presentation be used to predict outcome of treatment with methotrexate in patients with early inflammatory polyarthritis? ann rheum dis. 2009;68(1):57-62. 36. gaujoux-viala ca, et al. optimal methotrexate dose is associated with better clinical outcomes than non-optimal dose in daily practice: results from the espoir early arthritis cohort. bmj publishing group ltd; 2017:2054. 37. gaies e, et al. methotrexate side effects: review article. j drug metab toxicol. 2012;3(4):1-5. 38. buttgereit f, bijlsma jwj, strehl c. will we ever have better glucocorticoids? clin immunol. 2017. 39. kirwan jr, t arthritis, rcl-dgs group. the effect of glucocorticoids on joint destruction in rheumatoid arthritis. n engl j med. 1995;333(3):142-7. 40. hoes j, et al. eular evidence-based recommendations on the management of systemic glucocorticoid therapy in rheumatic diseases. ann rheum dis. 2007;66(12):1560-7. 41. buttgereit f, burmester gr, lipworth bj. optimised glucocorticoid therapy: the sharpening of an old spear. lancet. 2005;365(9461):801-3. 42. vandewalle j, et al. therapeutic mechanisms of glucocorticoids. trends endocrinol metab. 2017. 43. barrera p, et al. long-circulating liposomal prednisolone versus pulse intramuscular methylprednisolone in patients with active rheumatoid arthritis. in arthritis and rheumatism. hoboken, nj: wiley-liss div john wiley & sons inc; 2008. 44. prasad lk, o’mary h, cui z. nanomedicine delivers promising treatments for rheumatoid arthritis. nanomedicine. 2015;10(13):2063-74. 45. schacke h, et al. dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. proc natl acad sci u s a. 2004(1):227. 46. schäcke h, et al. insight into the molecular mechanisms of glucocorticoid receptor action promotes identification of novel ligands with an improved therapeutic index. exp dermatol. 2006;15(8):565-73. 47. huh j, et al. treatment of generalized anxiety disorder: a comprehensive review of the literature for psychopharmacologic alternatives to newer antidepressants and benzodiazepines. prim care companion j clin psychiatry. 2011;13(2). 48. stock t, et al. improved disease activity with fosdagrocorat (pf-04171327), a partial agonist of the glucocorticoid receptor, in patients with rheumatoid arthritis: a phase 2 randomized study. int j rheum dis. 2017;20(8):960-70. 49. rossiter d. (ed). the south african medicines formulary. cape town: health and medical publishing group; 2016:650. 50. cooper gs, bynum mlk, somers ec. recent insights in the epidemiology of autoimmune diseases: improved prevalence estimates and understanding of clustering of diseases. j autoimmun. 2009;33(3-4):197-207. 51. den uyl d, et al. metabolic effects of high‐dose prednisolone treatment in early rheumatoid arthritis: balance between diabetogenic effects and inflammation reduction. arthritis rheumatol. 2012;64(3):639-46. 52. bester fcj, bosch fj, van rensburg bjj. the specialist physician’s approach to rheumatoid arthritis in south africa. korean j intern med. 2016;31(2):219-36. biology, medicine, & natural product chemistry issn 2089-6514 (paper) volume 9, number 1, april 2020 | pages: 1-6 | doi: 10.14421/biomedich.2020.91.1-6 issn 2540-9328 (online) antioxidant activities of the leaf extract and fractions of dryopteris filix-mas (l.) schott could be attributed to the abundance of polyphenol compounds earnest oghenesuvwe erhirhie1,2,*, emmanuel emeka ilodigwe1, daniel lotanna ajaghaku3, blessing ogechukwu umeokoli4, peter maduabuchi eze5, festus basden chiedu okoye4 1department of pharmacology and toxicology, faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, nigeria. 2department of pharmacology and toxicology, faculty of pharmaceutical sciences, chukwuemeka odumegwu ojukwu university, igbariam, nigeria. 3department of pharmacology, faculty of pharmaceutical sciences, enugu state university of science and technology, enugu state, nigeria. 4department of pharmaceutical and medicinal chemistry faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, nigeria. 5department of environmental health science, faculty of health sciences and technology, nnamdi azikiwe university, nnewi campus, anambra state, nigeria. corresponding author* erhirhieochuko@yahoo.com, tel: +234-7060434974 manuscript received: 17 august, 2019. revision accepted: 06 february, 2020. published: 10 april, 2020. abstract dryopteris filix mas (d filix-mas) is wildly used in ethnomedicine for the management of rheumatoid arthritis, wounds and other diseases. we investigated the anti-oxidant activities of its leaf extract, and chromatographic fractions. the ethanol leaf extract was partitioned into four fractions; n-hexane, ethyl acetate, n-butanol and water. ferric reducing anti-oxidant power (frap), 1, 1-diphenyl-2-picrylhydrazil (dpph) and nitric oxide (no) scavenging in vitro assays were carried out on the extract and fractions at 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml. the most active fraction (ethyl acetate fraction) was further purified using chromatographic techniques to isolate its major compound whose structure was elucidated using id nuclear magnetic resonance (nmr) and mass spectrometry. the ethyl acetate fraction produced the highest free radical scavenging activity among the other fractions. the fraction (vlc-e7) from which the bioactive compound, quercetin-3-o-αl-rhamnopyranoside, was isolated had the best frap and dpph scavenging activities with ec50 and ic50 values of 88.81 ± 3.41 and 26.87 ± 0.24 respectively more than the ethyl acetate fraction. this study revealed that the polyphenol flavonoid, quercetin-3-o-αl-rhamnopyranoside could be responsible for antioxidant activity of ethno-medicinal property of d filix-mas leaf. keywords: dryopteris filix-mas; wounds; rheumatoid arthritis; quercetin-3-o-αl-rhamnopyranoside; anti-oxidant properties introduction free radicals are constantly produced in all normal living cells (kumar et al., 2014). imbalance between the production of these radicals and endogenous antioxidants creates a state of oxidative stress with its associated damaging effects on endogenous molecules such as lipids, proteins and nucleic acids (yan et al., 2015). unbridled oxidative stress has been reported to be implicated in several chronic and degenerative diseases such as diabetes, rheumatoid arthritis, cancer, neurodegenerative diseases, atherosclerosis, ischemic heart disease, ageing among others (khatoon et al., 2013). in attempt to ameliorating oxidative damages caused by free radicals, synthetic antioxidants such as butylated hydroxytoluene (bht), butylated hydroxyanisole (bha), and tert-butylhydroquinone (tbhq) had been developed. however, these synthetic antioxidants left much to be desired due to their side effects, such as liver toxicity, carcinogenicity as well as their high cost and inaccessibility (deepa et al., 2014). these limitations have inspired the search for more effective antioxidant from natural source, especially medicinal plants, which are biodegradable, less toxic, affordable and accessible (ezeja et al., 2015; ahlem et al., 2015). d filix-mas is an evergreen fern belonging to the family of dryopteridaceae. male fern, worm fern, aspidium and shield fern are common names assigned to it. it grows between 60-150 cm high and it is habitat to stream and moist environments (uwumarongi et al., 2016). it is native to europe, asia, and north america. its leaves are bipinnated and consist of 20-35 pinnae on each side of the rachis (fig 1). its stalks are covered with orange-brown scales (bafor et al., 2017). the leaf decoction is ethno medicinally used in rheumatoid arthritis, wounds, bleeding disorders, ulcers, worm infestation and malaria (tagarelli et al., 2010; erhirhie et al., 2019). it is also reported as one of the ferns with https://doi.org/10.14421/biomedich.2020.91.1-6 2 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 useful secondary metabolites against chronic diseases and aging (valentyna et al., 2017). previous studies have found that it possesses antihelmintic activity (urban et al., 2014), antimicrobial activity (mandal and mondal, 2014), antidiarrheal (uwumarongi et al., 2016), uterine relaxant (bafor et al., 2017) and anti-inflammatory (erhirhie et al., 2019) activities. a preliminary study by sekender et al, (2012) revealed that it possesses anti-oxidant activity. in this present study, we evaluated the antioxidant activity of the leaf extract and fractions. the bioactive compound responsible for its antioxidant activity was isolated and characterized. figure 1. d. filix-mas photograph. methodology materials visible spectrophotometer (721g, zhejiang, china), thermostatic water bath (equitron mumbai india), analytical weighing balance (ohaus corp. nj usa), quercetin (institute of pharmaceutical biology and biotechnology, heinerich-heine university, dusselforf, germany), 2,2-diphennyl-1-picrylhydrazyl, dpph (sigma aldrich), ascorbic acid (sigmaaldrich). procedure plant collection, extraction, fractionation and purification of active compound plant collection, authentication, extraction, fractionation, chromatographic separation, and structural elucidation techniques were carried out using similar method as described in our earlier study (erhirhie et al., 2019). the purification and structural elucidation was based on bioactivity guided antioxidant screening of liquid-liquid fractions (n-hexane, ethyl acetate, butanol, and water fractions), vacuum liquid chromatographic fractions and sephadex fractions. dpph scavenging assay the test was carried out based on the method described by ajaghaku et al (2017). the reaction mixture contained 0.1 ml of various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml) of sample, 0.1 ml of 0.6 mmol of dpph and 0.8 ml of methanol. the mixture was incubated in the dark for 30 minutes at room temperature. the absorbance of the sample was measured at 517 nm against blank (methanol) using a spectrophotometer. ascorbic acid and quercetin were used as standards. a tube containing 0.1 ml of dpph solution and 0.9 ml of methanol served as control. experiments were carried out in duplicate. free radical scavenging activities of each sample were determined as follows: 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (𝐴𝐶– 𝐴𝑆)/𝐴𝐶) × 100 ac : average absorbance of control as : average absorbance of sample a graph of percentage inhibition against concentration was plotted and the concentration that produced 50% inhibition (ic50) was extrapolated using a regression analyses equation. ferric reducing antioxidant power (frap) assay frap assay was carried out following the method described by habibur et al, (2013). two hundred and fifty microliter (0.25 ml) of various concentrations, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml of samples were mixed with 0.625 ml of phosphate buffer and 0.625 ml of 1% potassium ferricyanide [k3fecn6]. the mixtures were heated at 50oc for 20 minutes. then, 0.625 ml of 10% trichloroacetic acid (tca) was added and the mixtures were centrifuged at 3000 rpm for 10 minutes. from the upper layer, 0.625 ml was pipetted and mixed with 0.625 ml of distilled water and 0.125 ml of 0.1% (w/v) ferric chloride (fecl3) solution. absorbances of the mixtures were measured at 700 nm against air using a spectrophotometer. ascorbic acid and quercetin were used as standards. tests were performed in duplicate and percentage inhibition was calculated using the formula below. % inhibition = average absorbance of sample−average absorbance of blank 1 x 100 1 erhirhie et al. – antioxidant activities of the leaf extract and fractions … 3 a graph of percentage inhibition against concentration was plotted and the effective concentration (ec50) was extrapolated using a regression analyses equation. nitric oxide scavenging activity this assay was carried out following the method described by ezeja et al., (2015). two milliliter of 10 mm sodium nitroprusside prepared with phosphate buffer saline (ph 7.4) was mixed with 0.5 ml of samples at various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml). the mixture was incubated at room temperature for 150 minutes. thereafter, 0.5 ml of the reaction mixture was withdrawn and mixed with 0.5 ml of griess reagent (1% sulphanilamide + 0.1% naphthylethylenediamine dichloride + 3% phosphoric acid) was added to each test tube. the absorbance of the pink chromophore formed was measured at 540 nm. ascorbic acid and quercetin were used as standards. assays were carried out in duplicates. percentage inhibition was calculated using the formula below abs control−abs test abs control × 100 1 inhibitory concentration, ic50 was estimated from graph of % inhibition against concentration using regression analyses equation statistical analysis results were presented as mean ± standard error of mean (sem) using statistical package for social science (spss, version 20). calculation of fifty percent inhibitory concentration (ic50) of the extracts and fractions was carried out using regression equation in microsoft excel, 2010. results dpph, scavenging activity of extract and fractions in dpph scavenging assay, 50% inhibitory concentration (ic50) of 134.12 ± 2.88, 94.97 ±0.14, 63.91 ± 1.29, 62.09 ± 0.21and 45.72 ± 0.16 µg/ml were recorded in n-hexane, water fractions, extract, butanol and ethyl acetate fractions respectively. the ethyl acetate fraction produced the lowest ic50 value among other samples, except ascorbic acid and quercertin which showed ic50 values of 12.26 ± 0.20 µg/ml and 10.46 ± 0.15 µg/ml respectively (table 1). inhibition against dpph at 100 µg/ml by vlc fractions ethyl acetate vlc fractions, vlc-e1 (78.68%), vlce3 (52.33%), vlc-e7 (72.38%), vlc-e6 (73.84%), vlc-e14 (65.60%), vlc-e8 (73.08%), vlc-e10 (74.81%), vlc-e12 (72.09%), vlc-e16 (69.67%) produced more than 50 % inhibition against dpph (table 2). table 1. free radical (dpph) scavenging activities of extract and fractions of d filix-mas. dpph, scavenging activity, ic50(ug/ml) extract 63.91 ± 1.29 n-hexane fraction 134.12 ± 2.88 ethyl-acetate fraction 45.72 ± 0.16 butanol fraction 62.09 ± 0.21 water fraction 94.97 ±0.14 ascorbic acid 12.26 ± 0.20 quertcetin 10.46 ± 0.15 values are expressed as mean ± standard error of mean. table 2. dpph scavenging activities of ethyl acetate vacuum liquid chromatographic fractions of d filix-mas. solvent ratio sample code inhibition against dpph (%) ethyl acetate fraction 73.26 n(500): e (0) vlc-e1 78.68 n(450): e (50) vlc-e2 19.38 n(400): e(100) vlc-e9 18.12 n(350): e(150) vlc-e13 23.06 n(300):e(200) vlc-e4 28.00 n(250): e(250) vlc-e11 36.34 n(200):e(300) vlc-e3 52.33 n(150):e(350) vlc-e15 16.86 n(100):e(400) vlc-e5 12.79 n(50): e(450) vlc-e17 37.79 n(0):e(500) vlc-e7 72.38 d(500):m(0) vlc-e6 73.84 d(450):m(50) vlc-e14 65.60 d(350):m(150) vlc-e8 73.06 d(250):m(250) vlc-e10 74.81 d(100):m(400) vlc-e12 72.09 d(0):m(500) vlc-e16 69.67 n = n-hexane, e = ethyl acetate, m = methanol. ferric reducing antioxidant power (frap), nitric oxide (no) and dpph scavenging activities of extract selected fractions. frap activity in frap assay, quercetin and ascorbic acid produced the lowest ec50 values followed by vlc-e7, ethyl acetate fraction, extract and butanol fraction. in nitric oxide scavenging assay, ascorbic acid exhibited the lowest ic50 value (14.74 ± 0.11 µg/ml) followed by ethyl acetate fractions (15.38 ± 3.65 µg/ml) and butanol fractions (15.45 ± 2.72 µg/ml), followed by vlc-e7 fraction (16.66 ± 0.48 µg/ml), quercetin (18.14 ± 2.57 µg/ml) and extract (1854.60 ± 200.25 µg/ml). in dpph assay, quercetin and ascorbic acid produced the lowest ic50 values (11.50 ± 0.08 and 14.19 ± 0.43µg/ml) followed by vlc-e7 (26.87 ± 0.24 µg/ml), ethyl acetate fraction (50.25 ± 0.40 µg/ml), butanol fraction (62.09 ± 0.21 µg/ml) and crude extract (63.91 ± 1.29 µg/ml) (table 3). 4 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 dpph, scavenging activity of sephadex fractions dpph scavenging activities of samples tested decreased in the following order; quercetin (10.46 ± 0.15 µg/ml) > ascorbic acid (12.26 ± 0.20 µg/ml) > sph-e3 (26.35 ± 1.38 µg/ml) > vlc-e7 (26.87 ± 0.24 µg/ml) > sphe6 (33.44 ± 0.38 µg/ml) > sph-e5 (37.30 ± 0.30 µg/ml) > sph-e7 (46.95 ± 0.61 µg/ml) > sph-e4 (86.10 ± 7.69 µg/ml) (table 4). table 3. ferric reducing antioxidant power (frap), nitric oxide (no) and dpph scavenging activities of extract and fractions of d filix-mas. sample frap, ec50 (µg/ml) nitric oxide, ic50 (µg/ml) dpph, ic50 (ug/ml) extract 1854.60 ± 200.25 38.51 ± 2.23 63.91 ± 1.29 ethyl acetate fraction 461.75 ± 22.91 15.38 ± 3.65 50.25 ± 0.40 butanol fraction 1911.03 ± 137.44 15.45 ± 2.72 62.09 ± 0.21 vlc-e7 88.81 ± 3.41 16.66 ± 0.48 26.87 ± 0.24 ascorbic acid 69.34 ± 2.41 14.74 ± 0.11 14.19 ± 0.43 quercetin 30.81 ± 0.86 18.14 ± 2.57 11.50 ± 0.08 frap: ferric reducing antioxidant power. values are expressed as mean ± standard error of mean. table 4. dpph scavenging activity, of sephadex (sph) fractions of d filix-mas. dpph, scavenging activity, ic50( µg/ml) vlc-e7 26.87 ± 0.24 sph-e1 sph-e2 22.10 ± 0.62 sph-e3 26.35 ± 1.38 sph-e4 86.10 ± 7.69 sph-e5 37.30 ± 0.30 sph-e6 33.44 ± 0.38 sph-e7 46.95 ± 0.61 ascorbic acid 12.26 ± 0.20 quercetin 10.46 ± 0.15 values are expressed as mean ± standard error of mean. discussion it is well established that by-products of oxygen metabolism produce free radicals such as reactive oxygen and nitrogen species which results to cellular damage and pathogenesis of several diseases such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases among others (li et al., 2016). there is a revitalization of interest in the search for natural anti-oxidant that could ameliorate reactive oxygen species which are implicated in numerous disease conditions (ahlem et al., 2015; deepa et al. 2014). in this study, we investigated the antioxidant properties of the extract and fractions of d filix-mas and also elucidated the major compound responsible for its antioxidant activity using in vitro bioassay guided isolation approach. dpph test is a widely acceptable and reproducible assay for screening potential antioxidants (patel et al., 2010). this method involves the ability of anti-oxidants to donate electrons to dpph thereby causing discoloration of the reaction mixture from deep violet color to yellow color (patel et al., 2010). the yellow color change observed in this study substantiates the ability of the extract and fractions of d filix-mas to scavenge free radicals generation by dpph. this further suggests that the constituents present in the extract and fractions could ameliorate oxidative damages by free radicals. reduction of ferric (fe3+) to ferrous (fe2+) ions is a documented method of determining antioxidants with reducing power, capable of breaking free radical chain progression in lipid peroxidation (habibur et al., 2013). from this study, reduction of iron (iii) complex to the ferrous form, iron (ii) could be a possible antioxidant mechanism as a result of the presence of reducing agents in the extract and fractions of d filix-mas. phenolic compounds such as quercetin has been reported to prevent free radicals by forming complex and chelating metal ions such as iron and cupper, thereby preventing auto-oxidative damage to the living system (nimse and pal, 2015). nitric oxide generated following incubation of sodium nitroprusside with phosphate buffer in the presence of oxygen is known to cause toxicity to biomolecules (deepa et al., 2013). the scavenging capacities of the extract and fractions against nitric oxide generation suggest that d filix-mas could prevent nitric oxide generation in various disease conditions. polyphenols such as quercetin has been reported to interact with nitric oxide synthase resulting in modulation of nitric oxide production (hussain et al., 2016). the roles of phenolic compounds from medicinal plants as supplement in the treatment of oxidative stress and free radicals mediated tissue damage cannot the overemphasized (li et al., 2016). flavonoids and phenolic acids from polyphenols have been reported to play a significant role in scavenging free radicals and prevention of oxidative damage to cells. phenolic compounds have capacity to neutralize several forms of oxidizing free radicals due to their electron donating abilities (habibur et al., 2013; ajaghaku et al., 2017). ojo et al., (2013) reported that flavonoids inhibit lipid peroxidation in various biological systems. presence of hydroxyl groups in flavonoids allows them to disconnect free radical chain reactions via the formation of intramolecular hydrogen bonds (monika et al., 2011). erhirhie et al. – antioxidant activities of the leaf extract and fractions … 5 from the forgoing, antioxidant properties elicited by the extract and fractions of d filix-mas could be attributed to presence of flavonoids. this is substantiated by presence of the flavonoid, quercitrin as the major compound in the hplc chromatogram of the extract and fractions of d filix-mas as documented in our earlier study (erhirhie et al., 2019). the ethyl acetate vlc fraction (vlc-e7), where the compound, quercetin-3-o-αl-rhamnopyranoside was isolated exhibited the highest anti-oxidant activity compared to the extract and other fractions. in line with this finding, quercetin-3-o-αl-rhamnopyranoside isolated from other medicinal plants was found to elicit significant antioxidant activity (zhang et al., 2014). it is possible that possible that quercetin-3-o-αlrhamnopyranoside may be the key antioxidant compound of d filix-mas. quercetin, the most widely distributed flavonoid in nature, existing mostly in its glycoside form quercitrin has been reported to possess various properties such as antioxidant, anti-inflammatory, neuroprotective, antiviral, anticancer, hepatoprotective, cardioprotective, antimicrobial and anti-obesity properties (maalik et al., 2014). mir, et al., (2017) reported that flavonoids from tridax procumbens possess antioxidant properties. phenolic compounds, catechin, epicathechin, dihydroquercetin isolated from alchornea floribunda leaf were also found to elicit antioxidant properties (ajaghaku et al., 2017). in this study, the ability of extract and fractions of d filixmas to scavenge generation of free radicals from dpph, nitric oxide as well as its chelating capacity could be attributed to the phenolic compound, quercetin3-o-αl-rhamnopyranoside, which was postulated to be responsible for its anti-inflammatory activity as earlier reported (erhirhie et al., 2019). conclusion the isolated flavonoid, quercetin-3-o-αlrhamnopyranoside from ethyl acetate chromatographed fraction of d filix-mas could be responsible for its antioxidant activity, which validates its folkloric use in the management of rheumatoid arthritis and other free radical mediated diseases. acknowledgments the authors are thankful to prof. dr. peter proksch of the institute of pharmaceutical biology and biotechnology, heinrich-heine university, düsseldolf, germany for making their facilities available for the high performance liquid chromatography (hplc) and nmr analyses that were carried out. we are also thankful to dr. h.a. akinnibosun for assisting in the authentication of the plant specimen used for this study. references ahlem, r., souad, i.b., béatrice, b., valérie, m.l., fathi, m., evelyne, o., jamila, k.c and malika, t.a., 2015, total phenolic, total flavonoid, tannin content, and antioxidant capacity of halimium halimifolium (cistaceae), japs, 5 (01): 052-057. ajaghaku, d.l., obasi, o., umeokoli, b.o., ogbuatu, p., nworu, c.s., ilodigwe, e.e., and okoye, f.b.c., 2017, in vitro and in vivo antioxidant potentials of alchornea floribunda leaf extract, fractions and isolated bioactive compounds, avicenna j phytomed, 7 (1): 80-92. bafor, e.e., omokaro, w.o., uwumarongie, o.h., elvis-offiah, u.b., omoruyi, o, viegelmann, c.v. and edrada-ebel, r., 2017. dryopteris filix-mas (dryopteridaceae) leaves inhibit mouse uterine activity. journal of medicinal plants for economic development, 1(1):1-12. deepa, m. and renuka, d., 2014, potent anti-inflammatory medical plant-a review, int j pharm pharm sci., 6 (4):43-49. erhirhie eo, emeghebo cn, ilodigwe ee, ajaghaku dl, umeokoli bo, eze pm, ngwoke kg, okoye fbc. dryopteris filix-mas (l.) schott ethanolic leaf extract and fractions exhibited profound anti-inflammatory activity. avicenna j phytomed, 2019; 9(4): 396-409. ezeja, m.i., yusuf, n., omeh, s., onoja, o. and ijeoma, h.u., 2015, antiinflammatory and antioxidant activities of the methanolic leaf extract of cissus aralioides, am j pharmacol sci, 3 (1): 1-6. habibur, r., manjula, k., anoosha, t., nagaveni, k., chinna, m.e., and dipankar, b., 2013, in-vitro antioxidant activity of citrulus lanatus seed extracts, asian journal of pharm. clin. res. 6 (3): 152-157. hussain t.,tan, b., yin, y., blachier, f., tossou, m.c.b., rahu n., 2016. oxidative stress and inflammation: what polyphenols can do for us. hindawi publishing corporation oxidative medicine and cellular longevity volume 2016, article id 7432797, 9 pages http://dx.doi.org/10.1155/2016/7432797 khatoon, m., islam, e, islam, r., rahman, a.a., alam, a.h.m.k., khondkar, p., rashid, m. and parvin, s., 2013, estimation of total phenol and in vitro antioxidant activity of albizia procera leaves, bmc res notes. 6:121. 1-7. kumar, s., sandhir, r. and ojh, s., 2014, evaluation of antioxidant activity and total phenol in different varieties of lantana camara leaves, bmc res notes. 7:560.1-7. li j., liu x., shen l., zeng w., qiu g., 2016, natural plant polyphenols for alleviating oxidative damage in man: current status and future perspectives tropical journal of pharmaceutical research; 15 (5): 1089-1098. maalik, a., farhan, a.k., amara, m., adeem, m., saira, a., muhammad, a., sabiha, k., yasir, a. and imran, t., 2014, pharmacological applications of quercetin and its derivatives: a short review, trop j pharm res, 13 (9): 1561-1566. mandal, a. and mondal, a.k., 2014, studies on antimicrobial activities of some selected ferns and lycophytes in eastern india with special emphasis on ethno-medicinal uses, afr. j. plant sci, 5(7):412-420. mir, s.a, jan, z., mir, s., dar, a.m. and chitale, g., 2017, a concise review on biological activity of tridax procumbens linn, organic chem curr res, 6: 177. monika, m., michae s., maegorzta p. and hanna, c., 2011, evaluation of antioxidant potential of flavonoids: an in vitro study, acta pol pharm, 68 (4):611-615. 6 biology, medicine, & natural product chemistry 9 (1), 2020: 1-6 nimse, s.b., pal, d., 2015. free radicals, natural antioxidants, and their reaction mechanisms. royal society of chemistry, 5, 27986-28006. doi: 10.1039/c4ra13315c. ojo, o.a., oloyede, o.i., olarewaju, o.i., ojo, a.b., ajiboye, b.o. and onikanni, s.a., 2013, toxicity studies of the crude aqueous leaves extracts of ocimum gratissimum in albino rats, iosr-jestft, 6 (4):34-39. patel, a., patel, a., patel, n.m., 2010, determination of polyphenols and free radical scavenging activity of tephrosia purpurea linn.leaves (leguminosae), pharmacogn res. 2:152-8. sekendar, a.m., mostafa, k., raihan, m.o., rahman, m.k., hossain, m.a. and alam, m.s., 2012, antioxidant and cytotoxic activities of methanolic extract of dryopteris filixmas (l.) schott leaves, int. j. drug dev. res, 4(2): 223-229. tagarelli, g., tagarelli, a. and piro, a., 2010, folk medicine used to heal malaria in calabria (southern italy), j ethnobiol ethnomed, 6 (27): 1-16. urban, j., tauchen, j., langrova, i. and kokoska, l., 2014, in vitro motility inhibition effect of czech medicinal plant extracts on chabertia ovina adults, j. anim. plant sci, 21(2): 3293-3302. uwumarongie, h.o., enike, m.a. and bafor, e.e., 2016, pharmacognostic evaluation and gastrointestinal activity of dryopteris filix-mas (l.) schott (dryopteridaceae), ew j herbal chempharmacol res, 2(1): 19-25. valentyna, m., iryna, t., t et yan a, d . an d la r ys a, m . , 2 0 1 7 , a review of the medicinal ferns of ukraine, scr. sci. pharm, 4 (1): 46-62. yan, h., yuan, y., xi, z., kun, z., shaohua, c. and zhiyun, d., 2015. curcumin, inflammation, and chronic diseases: how are they linked? molecules, 20, 9183-9213; doi:10.3390/molecules20059183. zhang, y., wang, d., yang, l., zhou, d., zhang, j. 2014, purification and characterization of flavonoids from the leaves of zanthoxylum bungeanum and correlation between their structure and antioxidant activity, plos one, 9(8): e105725. doi: 10.1371/journal.pone.010572. microsoft word 107-117 chemistry | 107 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 glucagon-like peptide-1 levels and related parameters in rheumatoid arthritis patients prone to atherosclerosis noorhan k. shafeeq. dept. of chemistry / college of education for pure science (ibn alhaitham)/baghdad university ban mahmood shaker al-joda dept.of biochemistry /college of medicine /babylon university tamara ala, a dept. of basic sciences /faculty of dentistry/ kufa university received in: 27/april/2016, accepted in: 11/may/2016 abstract the study aimed to estimate the role of glucagon-like peptide-1 (glp-1) and visfatin as a novel pro inflammatory marker in rheumatoid arthritis (ra ) according to the activity scores of disease to assess the possibility of introducing glucagon-like peptide-1 and visfatin in the diagnosis and monitoring of ra patients and to found the correlation of visfatin level with glp-1 and aip in patients prone to atherosclerosis ,fifty healthy individuals as control group (g1) and fifty rheumatoid arthritis (ra) patients (g2) were enrolled in this study with middle age ranged (30 – 40) years and bmi ≥24 kg/m2. esr ,rf, lipid profile, crp,insulin, visfatin and glp-1were determined. results in table (1) revealed highly significant increase in esr,rf,tc,tg,ldl,vldl and aip level in rheumatoid arthritis (g2) comparing to (g1) while significant decrease noticed in (g2) group comparing to (g1). results in table(2) showed highly significant increase in insulin ,crp, visfatin and glp-1 level results in table(3) illustrated correlation relation data and results revealed highly significant ,were r=0.035 for glp-1 with visfatin for (g1), and highly significant , r= -0.089 for (g2) as shown in figure(1-a) and(1-b), the result also highly significant were r= -0.183 for glp-1 with crp for control group and significant ,r= 0.043 for glp-1 with crp for ra group as shown in figure(2-a) and(2-b), and significant, r= -0.056 for glp-1with aip for g1 and no significant, were r= 0.042 for glp-1 with aip for g2 as shown in figure (3-a)and (3-b). conclusion that summarized from this study is glucagon-like peptide-1 and visfatin plays a role in ra pathogenesis that can be considered marker in ra . and may be used as a drug in future for ra patients. keyword:glp-1, rheumatoid arthritis, atherosclerosis. abbreviation: rheumatoid arthritis (ra), esr (erythrocyte sedimentation rate), rf(rheumatoid factor), crp (c-reactive protein) chemistry | 108 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 introduction glucagon-like peptide-1 (glp-1) is a member of the pro-glucagon incretin family involved in the control of zest and satiety ,glp-1 acts during glp-1 receptor (glp-1r), a 463 aminoacid member of the g protein-coupled receptor (gpcr) super family [1]. bioactive glp-1 found in two equipotent molecular forms: glp-17–37 and glp-17–36 amide. glp-1 is rapidly split by dp iv, which results in the production of largely inactive molecular glp-19-36 amide and glp-17-37 forms. in the cardiovascular system, incretins have been recently facilities for the increase of endogenous antioxidant defenses, repression of cardiomyocyte apoptosis, attenuation of endothelial inflammation and dysfunction[2]. in this brief review, we will summarize recent progress about the important role of glp-1 in myocardial protection and signaling pathway. [3]. in pacing-induced heart failure case, the stimulation of glp signaling by glp-1 has also been demonstrated to improve cardiac performance in conscious dogs with dilated cardiomyopathy [4]. recently, a significant number of studies have indicated useful effects of glp-1 on cardiovascular function, which gave justification for the use of glp-1 in the treatment of cardiovascular diseases. [3]. infusions of glp-1 have also been shown to promote post-prandial lipaemic excursions [5]., an distinct risk factor in the development of atherosclerotic cardiovascular disease. glp1 receptors are expressed on cardiac tissue, so, it is possible that the hormone, or its synthetic analogues, may directly mediate a range of cardiac functions. recent research include the investigation of glp-1 involvement in myocardial metabolism, coronary blood flow, pre/post-ischemic conditioning, left ventricular (lv) remodeling and lv performance. studies including animal and human have suggested that glp-1 agonists may have effects on myocardial metabolism, and this may be translated into possible therapeutic benefit on cardiac function. in addition, an animal study suggested that glp-1 may have effects on coronary blood flow and protect against ischemia and reperfusion injury [6]. the rheumatoid arthritis (ra) is a chronic autoimmune disease identified by synovial inflammation, cartilage damage, and bone erosion, [7]. visfatin, known as nicotinamide phosphoribosyltransferase (nampt), was originally described as a cytokine involved in early b-cell development and was later renamed visfatin since it is secreted mainly by visceral fat [8]. a proinflammatory action of visfatin was described to be mediated by the insulin signaling pathway through protein kinase b (pkb ) phosphorylation [9].. studies reported the up regulation of visfatin inactivated ra-sfs in response to proinflammatory stimuli in ra synovium: visfatin was expressed in the lining layer, aggregates of lymphoid, and vessels interstitial . [10]. study demonstrated that heart failure led to death in ra patients [11],which accelerated atherosclerosis than the other subjects [12]. inflammatory processes resemble to the other chronic inflammatory diseases, like atherosclerosis[13]. expression of proinflammatory cytokines and mediators influences of atherosclerosis. aclot formation to thrombus development responsible for myocardial infarction[14]. the study aimed to estimate the role of glucagon-like peptide-1 and visfatin as a novel pro inflammatory marker in rheumatoid arthritis patients according to the active scores of disease to assess the possibility of introducing visfatin in the diagnosis and monitoring of ra patients and to found the correlation of visfatin level with glucagon-like peptide-1 and aip in rheumatoid arthritis patients prone to atherosclerosis. chemistry | 109 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 materials and methods blood collected from fifty healthy individuals as control group (g1) and fifty rheumatoid arthritis (ra) patients (g2) were enrolled in this study with middle aged ranged (30 – 40) years and bmi ≥24 kg/m2. esr was determined in blood using the westergren method. three milliliters of blood was allowed to clot, and was centrifuged 1 h ,.the serum collected was stored until analysis (tc, tg, hdl) were determine according to the procedure of the hospital laboratory. ldl – c and vldl were calculated from friedewald equation: [ldlc(mg/dl)= tc (hdl-c+ vldl-c)] ,vldl-c (mg/dl)=tg/5[15] aip was calculated according to the following equation ( log tg/hdl ). visfatin, was measured by (elisa) kit (phoenix pharmaceuticals inc., burlingame, california, united states) [8]. serum crp was assessed by turbidimetry quantitative method [16].rheumatoid factor (rf) was assessed by turbidimetry quantitative method [17]. insulin and glp-1were determined by using a ready kit based on elisa technique[18]. the results were expressed as mean + sd. differences between the groups were assassed by student’s t-test which p-value of 0.05 and 0.0001 considered as significant and highly significant differences ,respectively .correlation relation was estimated for glp-1 with visfatin ,crp and aip. results and discussion: table(1)represented levels of esr,rf,tc,tg,hdl,ldl,vldl for patients and control groups, results in table (1) revealed highly significant increase in esr,rf,tc,tg,ldl,vldl and aip level in g2 comparing to g1 while significant decreased noticed in hdl levels in g2 comparing to g1. several immune cells and soluble mediators play important role in the pathogenesis of atherosclerosis in ra. [19] ,such as visfatin that is an adipokine synthesis visceral white adipose tissue [4] and represents an additional link between adipose tissue and inflammation[20] jian et al. [21] and nalesnik et al. [22], who observed that esr levels in ra patients was increase more than control group.. rheumatoid factor ( rf) is considered as a nonspecific marker of ra in addition to collagen vascular disease [23]. these data were in agreement with those of novikov et al. [24], who noticed that ra linked the presence of rf. the results were in agreement with those of [khalifa and abdelfattah] [25] and [hui et al]. [26], who showed that the elevation found in rf in ra group when comparing to non ra group. lipid disorder have important role in atherosclerosis, a process which is accelerated by dysfunction characterizing ra. the elevation in changed endothelial cells in ra patients allow the entry of ldl, which is possess in the intima, oxidized with subsequent recruitment of circulating leukocytes within atherosclerotic plaques[27] also, lowers in hdl levels may accelerate atherosclerosis lead to the exerting atheroprotective functions.[28] other study, demonstrated that tc and ldl increase together in ra patients with low levels of hdl. [29] an elevation in the tc/hdl ratio, which display an atherogenic index, is a signification marker for heart disease[30] in this study, the ra patients had an atherogenic index, indicate a higher risk of atherosclerosis. thus, it can be inference that the role of dyslipidaemia in the pathogenesis of subclinical atherosclerosis in ra patients should be seen in the context of other operating mechanisms[19]. moreover, it is potential that other fractions of specific lipoprotein particles as (vldl) and hdl may play a important role in atherogenesis in ra[31]. atherogenesis is accelerated in ra patients[13] .and elevation mortality from acute cardiovascular events[32]. chemistry | 110 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 results in table (2) showed highly significant increase in insulin ,crp, visfatin and glp-1 levels. both glp-1(7-36) amide and the glp-1 receptor agonist, exendin-4 are shown to increase heart rate and blood pressure in both anesthetized and conscious restrained rats, although the mechanisms are controversial [33]. in vitro studies have failed to show any effects of glp-1 on cardiac myocyte contractility. very recently, promising clinical data from shannon’s lab showed that glp-1 infusion improved regional and global function in patients with acute myocardial ischemia and severe systolic dysfunction after successful primary angioplasty [34]. several animal studies and analyses in humans have demonstrated a potential cardioprotective effect of glp-1 ras[35] .there is evidence that the glp-1 protective effect on myocardium may be particularly important in ischemic conditions and may also attenuate other factors causing premature atherosclerosis. [36] other study demonstrated when glp-1 was infused into isolated mouse hearts, cgmp production markedly increased compared with non treated hearts, suggesting a role for this signaling pathway in glp-1 activity. if the signaling mechanisms observed in pancreatic cells are stimulated by glp-1 in vascular and cardiac cells, vascular tone, muscle contractility, cell growth/differentiation, extracellular matrix remodeling, and apoptosis may be affected. [37]. determination of crp and esr ,which used as a marker of inflammation disease have significant role in ra [38]. these results are in agreement with those of al-mesryet al. [39], who found that crp is a protein produced in response to tissue injury, infection, and inflammation. another study revealed that increase visfatin levels in ra patients as a result of alteration in the gene encoding visfatin may be due to the ra triggering factors: a condition that causes elevation in its production autonomously independent of disease activity. [40] another study demonstrated that visfatin have a critical role in atherogenesis and destabilization of plaque [41] results in table (3) illustrated correlation relation data and t-test for glp-1 with visfatin ,crp and aip for g2 and g1 groups . results revealed highly significant ,were r=0.035 for glp-1 with visfatin for g1 and highly significant , r=-0.089 for g2 as shown in figure(1-a) and(1-b) . the result also highly significant , r= -0.183 for glp-1 with crp for g1 and significant , r= 0.043 for glp-1 with crp for g2 as shown in figure(2-a) and (2-b). and significant, r= 0.056 for glp-1 with aip for g1 and no significant, were r= 0.042 for glp-1 with aip for g2 as shown in figure (3-a)and (3-b). gonzalez-gay et al. [40] and senolt et al. [42], illustrated that visfatin levels did not correlate with bmi, age, and duration of disease in patients with active ra when company with control subjects which indicate that adipocytokine production in ra patients due to the processes of disease part of the inflammation systemic and destruction of bone led to suggested a role visfatin in the pathogenesis of ra. the current study also found that there was a positive correlation between visfatin and both crp and esr in ra patients. these observation are in agreement with other study reported that visfatin was correlated with (crp and esr) ,study seen that visfatin has a catabolic function in cartilage and may play significant role in the pathophysiology of arthritis. there is proof for an important function of innate immunity in the pathogenesis of ra [12]. although a study by luk et al. [43] showed that visfatin considered as a novel mediator of in nateimmunity, which led to improved that visfatin is involved in activity of proinflammatory, innateimmunity, and cartilage-catabolic functions in the processes of ra [44]. the conclusion could be drown from this studyglp-1 and visfatin plays a role in the pathogenesis of ra and can be considered as a marker in ra, and may be used as a drug in future for ra patients. chemistry | 111 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 references 1nikolaidis, la; mankad ,s; sokos, gg; miske, g; shah, a; elahi, d; shannon, rp; (2004). effects of glucagon-like peptide-1 in patients with acute myocardialinfarction and left ventricular dysfunction after successful reperfusion. circulation, 109(8):962–965 2erdogdu, o; nathanson, d; sjöholm, a; nyström, t; zhang, q; (2010) .exendin-4 stimulates proliferation of human coronary artery endothelial cells through enos-, pkaand pi3k/akt-dependent pathways and requires glp-1 receptor. mol cell endocrinol, 325:26–35 3zhao, tc.(2013). glucagon-like peptide-1 (glp-1) and protective effects in cardiovascular disease: a new therapeutic approach for myocardial protection. zhao cardiovascular diabetology, 12:90. 4nikolaidis, l.; elahi, d.; shen, y.; shannon, rp: (2005) active metabolite of glp1mediates myocardial glucose uptake and improves ventricularperformance in conscious dogs with dilated cardiomyopathy. am jphysiol heart circ physiol, 289(6):h2401–h2408 5meier, jj.; gethmann ,a; götze, o et al. (2006) glucagon-like peptide 1 abolishes the postprandial rise in triglyceride concentrations and lowers levels of non-esterified fatty acids in humans. diabetologia;3:452–8. 6ban, k; noyan-ashraf, mh; hoefer, j .(2008). cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1receptor-dependent and -independent pathways. circulation ;117:2340–50 7omez, r. g.´; conde, j. ; scotece, m.; g´omez-reino, j. j.; lago, f. and gualillo, o. (2011) “what’s new in our understanding of therole of adipokines in rheumatic diseases?” nature reviews rheumatology, vol. 7, no. 9, pp. 528–536. 8fukuhara, a.; matsuda. m.; m. nishizawa, et al. (2007) “erratum (retracted article): visfatin: a protein secreted by visceral fat that mimics the effects of insulin,” science, vol. 318, no. 5850, p. 565. 9jacques, c.; holzenberger, m. and mladenovic, z. et al. (2012) “proinflammatory actions of visfatin/nicotinamide phosphoribosyltransferase (nampt) involve regulation of insulin signaling pathway and nampt enzymatic activity,” journal of biological chemistry, vol. 287, no. 18, pp. 15100–15108.. 10matsui, h.; tsutsumi, a.; sugihara, m. et al. (2008) “visfatin (pre-b cell colonyenhancing factor) gene expression in patients with rheumatoid arthritis,” annals of the rheumatic diseases, vol. 67, no. 4, pp. 571–572. 11 maradit-kremers, h.; crowson, cs.; nicola, pj; ballman. kv; roger. sj; jacobsen. sj. et al. (2005). increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: a populationbased cohort study. arthritis rheum;52(2):402–11. 12 breland ,um.; hollan, i.; saatvedt, k; almdahl, sm; dama˚ s. jk; yndestad .a. et al. (2010). inflammatory markers in patients with coronary artery disease with and without inflammatory rheumatic disease. rheumatology (oxf);49(6):1118–27. 13bacon, pa.; stevens, rj.; carruthers, dm.; young ,sp.; kitas. gd.(2002) accelerated atherogenesis in autoimmune rheumatic diseases. autoimmun rev;1:338–47. 14-libby, p. (2008). role of inflammation in atherosclerosis associated with rheumatoid arthritis. am j med;121(10 suppl. 1):s21–31 15del rinco.´ n i; o’leary. dh; freeman. gl; escalante. a.( 2007) acceleration of atherosclerosis during the course of rheumatoid arthritis. atherosclerosis;195:354–60 16otsuji ,s; shibata, h.; umeda. m.( 1982). turbidimetric immunoassay of serum creactive protein. clin chem; 28:2121–2124. 17winkles, jw; lunec, j; gray ,l. (1989)automated enhanced latex agglutination assay for rf in serum. clin chem; 35:303–307. chemistry | 112 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 18prasad, sk. (2010).biochemistry of lipids 1st .discovery publishing house pvt. ltd. new delhi-110 002. 19montecucco, f.; mach, f.; (2009).common inflammatory mediatorsorchestrate pathophysiological processes in rheumatoid arthritisand atherosclerosis. rheumatology;48:11–22. 20fantuzzi, g. (2008). adipose tissue, adipokines and inflammation. j body work mov ther;12(2):121–32. 21jian. liu; rui-kai, z.; xue-fang, yu.; yuan, w. (2008). effects of xinfeng capsule on platelet parameters, p-select, platelet ultra-structure and its therapeutic effect on rheumatoid arthritis patients in active phase. china j chinese med;12–16. 22nalesnik, m.; nikolic.´ jm; jandric, s. (2010 ) ada and crp in diagnosing and monitoring of ra. med glas ljek komore zenicko-doboj kantona 2011; 8:163–168 23singh, u. (2010) is rheumatoid factor still a superior test for the diagnosis of ra? rheumatol int; 30:1115–1119. 24novikov, .aa; aleksandrova, en; karateev, .de; luchikhina, el; demidova. nv; cherkasova. mv.et al. (2008). diagnostic value of antibodies to modified citrullinizedvimentin in early rheumatoid arthritis. klin lab diagn; 27–29 25khalifa ,aim.; abdelfattah, a. . (2008)anti ccp2 and anti keratin antibodies in patientswith ra and oa. egyptian soc rheumatol rehabil; 35:1–10. 26 hui, l.; wuqi, s.; yang, l. (2010) .diagnostic value of anti-ccp antibodies in northern chinese han patients with ra and its correlation with disease activity. clin rheumatol; 29:413–417 27hahn, bh.; grossman, j.; chen, w.; mcmahon, m. (2007). the pathogenesis of atherosclerosis in autoimmune rheumatic diseases: roles of inflammation and dyslipidemia. j autoimmun;28:69–75. 28hansson, gk.; libby ,p. (2006). the immune response in atherosclerosis: adouble-edged sword. nat rev immunol;6:508–19. 29gotto, jr. am. (2001). low high-density lipoprotein cholesterol as a risk factor in coronary heart disease: a working group report. circulation;103:2213–8. 30georgiadis, an.; papavasiliou, ec.; lourida, es; alamanos. y; kostara. c; tselepis .ad.et al. (2006). atherogenic lipid profile is afeature characteristic of patients with early rheumatoid arthritis: effect of early treatment–a prospective, controlled study. arthritisres ther;8(3):r82. 30boers, m.; nurmohamed, mt.; doelman, cj.;, lard, lr.; verhoeven, ae.; voskuyl, ae. et al.( 2003). influence of glucocorticoid and disease activity on total and high density lipoprotein cholesterol in patients with rheumatoid arthritis. ann rheum dis;62:842–5. 31linsel-nitschke, p.; samani, nj.; schunkert, h. (2010) .sorting out cholesterol and coronary artery disease. n engl j med;363(25):2462–3. 32chung ,cp; oeser, a; avalos, i; gebretsadik, t; shintani, a; raggi, p. et al. (2006) .utility of the framingham risk score to predict the presence of coronary atherosclerosis in patients with rheumatoid arthritis. arthritis res ther;8:r186 33yamamoto, h.; lee, ce; marcus ,jn; williams, td; overton, jm; lopez ,me; hollenberg ,an; baggio, l; saper, cb; drucker, dj; elmquist, jk.( 2002) glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons. j clin invest, 110(1):43–52. 34sokos, gg; bolukoglu, h; german, j; hentosz, t; magovern, gj ;jr, maher, td; dean, da; bailey, sh; marrone, g; benckart ,dh; elahi, d; shannon, rp. effect ofglucagon-like peptide-1 (glp-1) on glycemic control and left ventricular function in patients undergoing coronary artery bypass grafting. am jcardiol 2007, 100(5):824–829 chemistry | 113 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 35ratner, r.; han, j.; nicewarner, d.; yushmanova, i.; hoogwerf, bj.; shen, l. cardiovascular safety of exenatide bid: an integrated analysis from controlled clinical trials in participants with type 2 diabetes. cardiovasc. diabetol. 10, 22 (2011). 36levantesi, g.; macchia, a; marfisi, rm; franzosi, mg; maggioni ,ap; nicolosi, gl; schweiger, c; tavazzi, l; tognoni, g; valagussa, f; marchioli, r; (2005). on behalf of thegissi-prevenzione investigators: metabolic syndrome and risk ofcardiovascular events after myocardial infarction. j am coll cardiol, 46:277–283. 37ban, k; noyan-ashraf, mh; hoefer, j; bolz, ss; drucker, dj; husain, m; (2008) .cardioprotective andvasodilatory actions of glucagon-like peptide 1receptor are mediated through bothglucagon-like peptide 1 receptor-dependent and-independent pathways.[erratum appears in circulation;118(4):e81]. circulation ;117:2340–2350. 38nam. j; villeneuve. e and emery. p. (2009). the role of biomarkers in the management of patients with ra. curr rheumatol rep; 11:371–377. 39al-mesry .mr;attwa. et; omar. hm. (2003). assessment of cardiovascular risk factors in ra and oa. egypt rheumatol rehabil; 30:323–332. 40gonzalez-gay, ma; vazquez-rodriguez, tr; garcia-unzeuta, a; berja, a; mirandafilloy, ja; de matias, jm. et al. (2010) visfatin is not associated with inflammation or metabolic syndromes in patients with severe rheumatoid arthritis undergoing anti-tna alpha therapy. clin exp rheumatol;28(1):56–62. 41hahn bh, grossman j, chen w, mcmahon m.( 2007).the pathogenesis of atherosclerosis in autoimmune rheumatic diseases: roles of inflammation and dyslipidemia. j autoimmun;28:69–75. 42 senolt .l; krysˇ .tu; fkova. o; hulejova .h. (2011). the level of serum visfatin (pbef) is associated with total number of b cells in patients with rheumatoid arthritis and decreases following b cell depletion therapy. cytokine; 55:116–121 43luk ,t.; malam, z.; marshall, j. (2008). pre-b cell colony enhancing factor (pbef)/ visfatin: a novel media innate immunity. j leukoc biol; 83:804–816 44bao, jp.; hen. wp; wu. ld. (2009) .visfatin; a potential therapeutic target for rheumatoidarthritis. j int med res; 37: chemistry | 114 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 table (1) esr, rf,tc,tg,hdl,ldl,vldl levels in g1 and g2: groups parameters mean± sem g2 mean± sem g1 t-test g1 vs g2 esr(mm/h) 42.35 ± 5.52 12.34±2.28 <0.0001 rf(iu/ml) 135.52 ± 9.14 33.89±9.5 <0.0001 t c (mg/dl) 264.73 ± 32.87 110.85±14.11 <0.0001 tg(mg/dl) 245.14±35.5 98.77±28.6 <0.001 hdl 32.74±3.65 42.64±3.96 <0.001 ldl 178.24±37.87 58.19±18.9 <0.001 vldl 42.14±5.03 25.42±1.57 <0.0001 aip 0.874±0. 98 0.36±0.85 <0.001 ̽˚(s) significant differences which p-value 0.05 ,(hs) high significant differences which p-value 0.001,(ns) no significant differences which p-value 0.05,(crp, c-reactive protein; esr). table (2) glp-1,crp, visfatin, insulin levels for studied groups group variables mean± sem g1 mean± sem g2 t-test g1 vs g2 glp-1 2.24±1.48 0.46±0,07 <0.05 crp(mg/dl) 4.96±0.82 1.76±0.48 <0.0001 visfatin (ng/ml) 63.3±6.5 12.71±1.7 <0.0001 insulin(iu/ml) 15.44±2.43 3.92±0.60 <0.0001 ˚(s) significant differences which p-value 0.05 ,(hs) high significant differences which p-value 0.001,(ns) no significant differences which p-value 0.05 chemistry | 115 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 table (3) correlation coefficient and p-value between glp-1 levels and visfatin.crp and aip. for studied groups. groups parameters g2 g1 r-value p-value r-value p-value glp-1with vis -0.089 <0.0001 0.035 <0.0001 glp1withcrp 0.043 <0.0001 -0.183 <0.0001 glp-1withaip -0.042 0.02 -0.056 <0.05 a b figure (1) correlation between glp-1 and visfatin in g1(a),g2(b) figure (2) correlation between glp-1 and crp in g1(a),g2(b) y = ‐0.6872x + 51.65 r² = 0.0053 0 10 20 30 40 50 60 70 0 2 4 6 v is glp‐1 y = 0.0231x + 5.1374 r² = 0.0026 0 2 4 6 8 0 5 10 15 c r p glp‐1 y = 1.1411x + 14.177 r² = 0.0012 0 5 10 15 20 25 0 0.2 0.4 0.6 0.8 v is glp‐1 y = ‐0.7955x + 2.1855 r² = 0.0123 0 1 2 3 4 0 0.2 0.4 0.6 0.8 c r p glp‐1 chemistry | 116 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 a b figure (3) correlation between glp-1 and crp in g1(a),g2(b) y = 0.005x + 0.8003 r² = 0.0049 0 0.2 0.4 0.6 0.8 1 0 5 10 15 a ip glp‐1 y = ‐0.9469x + 1.9309 r² = 0.2471 0 0.5 1 1.5 2 0 0.2 0.4 0.6 0.8 a ip glp‐1 chemistry | 117 2017عام ) 1(العدد 30مجلة إبن الھيثم للعلوم الصرفة والتطبيقية المجلد ibn al-haitham jour. for pure & appl. sci. vol. 30 (1) 2017 في مرضى الصلةوالمعامالت ذات 1-مستويات الكلوكاكون شبيه الببيتايد بتصلب الشرايين لإلصابةالمعرضين التھاب المفاصل الرثوي فيقش نورھان خالد جامعه بغداد/)ابن الھيثم( الصرفةللعلوم التربيةكليه /قسم الكيمياء هالجودبان محمود شاكر جامعه بابل/بطكليه ال/ الحياتيةقسم الكيمياء تمارا عالء حسين جامعه الكوفة/كليه طب االسنان/األساسيةقسم العلوم 2016/أيار/11:قبل في ،2016/نيسان/27:استلم في الخالصة تھدف ھذه الدراسه الى تحدبد دور الفزفاتين كداله اساسيه لاللتھابات المتواليه في مرضى التھاب المفاصل الرثوي وفقا لدرجات نشاط المرض لتقييم امكانيه استخدام الفزفاتين في تشخيص ومراقبه مرضى التھاب المفاصل الرثوي وايجاد تم جمع نماذج ,في المرضى العرضين لالصابه بتصلب الشرايين glp-1 ,aipمعامالت االرتباط لمستوى الفزفاتين مع بلغت متوسط g2وخمسين شخصا من مرضى التھاب المفاصل الرثوي g1 الدم من خمسين شخصا من االصحاء ظاھريا ومستويات esr,rf,crp,glp-1تم قياس مستويات .2م/كغم ≤24سنه بكتله جسميه ) 40-30(اعمارھم تظھر زياده معنويه عاليه في مستويات ) 1(النتائج في الجدول .الفزفاتين,ناالنسولي,الدھون esr,rf,tc,tg,ldl,vldl,aip قي المجموعهg2 مفارنه بالمجموعه الظابطه g12(اما نتائج الجدول الثاني ( ات العالقات ويوضح الجدول الثالث بيان, crp,glp-1الفزفاتين ,تظھر زياده معنويه عاليه في مستويات االنسولين -ارتباط معنوي ومعامل g1مع الفزفاتين للمجموعه glp-1 لل 0.035االرتباطيه واظھرت النتائج معامل ارتباط معنوي لل 0.183-ارتباط معنوي ومعاملكما اظھرت النتائج , (1-b), (a-1)كما موضح بالشكل g2للمجموعه 0.089 glp-1مع crpللمجموعه g1 لمجموعه المرضى كما موضح بالشكل 0.043ومعامل ارتباط معنوي(2-a) ,(2 -b) لمجموعه 0.042-وغير معنويه 0.056-بالنسبه للمجموعه الظابطه كانت معنويه aipمع glp-1 اما معامل ارتباط دورا فعاال في الحاالت ونستنتج من ھذه الدراسه ان الفزفاتين يلعب , (3 -b), (a-3)المرضى كما موضح بالشكل المرضيه من التھاب المفاصل الرثوي وعالقته ببقيه المعامالت التي تعتبر مؤشرات لالصابه بالجلطه القلبيه ويعتبر مؤشر .raويمكن ان يستخدم مستقبال كعالج لمرض raمرضي قوي لل .تصلب الشرايين,التھاب المفاصل الرثوي ,glp-1 :الكلمات المفتاحيه vol_116_002_sups_a_526722 133..137 upsala journal of medical sciences. 2011; 116: 133–137 original article lumbar radiculopathy caused by foraminal stenosis in rheumatoid arthritis tomoaki koakutsu, naoki morozumi, yutaka koizumi & yushin ishii department of orthopaedic surgery, nishitaga national hospital, sendai, japan abstract study design. case-series study. objective. to describe the clinical presentation, characteristic findings of imaging studies, and treatment of lumbar radiculopathy caused by foraminal stenosis in rheumatoid arthritis. background. lumbar lesions in rheumatoid arthritis are relatively rare, with a limited number of systemic reports. methods. six patients with lumbar radiculopathy caused by foraminal stenosis in rheumatoid arthritis were treated. the patients were all women with a mean age of 69 years and mean rheumatoid arthritis duration of 15 years. the medical records and imaging studies of all patients were reviewed. results. the affected nerve roots were l4 in four patients and l3 in two patients. foraminal stenosis was not demonstrated in magnetic resonance images in four of the six patients. selective radiculography with nerve root block reproduced pain, manifested blocking effect, and demonstrated compression of the nerve root by the superior articular process of the lower vertebra in all patients. conservative treatment was performed on one patient, and surgery was conducted for the rest of the five patients; radiculopathy was improved in all patients. conclusions. lumbar foraminal stenosis is a characteristic pathology of rheumatoid arthritis, and should be kept in mind in the diagnosis of lumbar radiculopathy. selective radiculography is useful in the diagnosis of affected nerve roots. key words: diagnosis, foraminal stenosis, lumbar spine, radiculopathy, rheumatoid arthritis, surgery introduction although cervical lesions are well known as spinal involvement of rheumatoid arthritis (ra), lumbar lesions are relatively rare, with a limited number of systemic reports (1–3). most of the cases which have been reported so far are spinal canal stenosis complicated with neurological symptoms caused by vertebral collapse secondary to vertebral body lesions (4,5) or by segmental instability subsequent to facet joint lesions (6,7); thus very few reports have been published about foraminal stenosis (8). we retrospectively reviewed six cases of lumbar radiculopathy in ra that resulted from foraminal stenosis to describe the clinical presentation, characteristic findings of imaging studies, and treatment of this condition. particularly, we compared the usefulness in the diagnosis of foraminal stenosis between magnetic resonance imaging (mri) and selective radiculography (srg) with nerve root block (9). patients and methods the subjects were six patients with ra who met the diagnostic criteria of the american rheumatism association and who were treated at our hospital in 2003– 2005 for lumbar radiculopathy caused by foraminal stenosis. the patients were all females, aged 57 to 75 years (mean 69 years) with 2 to 28 years of duration correspondence: tomoaki koakutsu, department of orthopaedic surgery, emergency center, tohoku university hospital, 1-1 seiryo-machi, aoba-ku, sendai, miyagi 980-8574, japan. fax: +81-22-717-7492. e-mail: koakutsu@med.tohoku.ac.jp (received 12 may 2010; accepted 18 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.526722 of ra (mean 15 years). according to steinbrocker’s classification (10), ra staging was evaluated as stage 2 in one patient, stage 3 in one patient, and stage 4 in four patients. their functional classification was assessed as class 3 in four patients and class 4 in two patients. mutilating deformities of the limbs were observed in four patients. all hospital and outpatient records as well as the related radiographs (plain x-rays, myelograms, postmyelographic ct scans, mri, and srg) were reviewed to study clinical presentation, characteristic findings of imaging studies, and treatment. lumbar radiculopathy in ra was diagnosed on the basis of neurological examination findings and imaging of the affected nerve roots. severity was rated according to the criteria for evaluation of low back pain proposed by the japanese orthopaedic association (joa score). the therapeutic modality (with or without surgery; surgical procedures) was noted, and recovery rate was determined from preand postoperative joa scores by hirabayashi’s method (11). generally, it is difficult to diagnose ra-specific lumbar lesions from imaging alone, since lesions of spondylosis deformans and/or lesions of osteoporosis associated with ra are also found. however, we defined ra-specific lesions as follows: rheumatoid nodules in the vertebral bodies (4); vertebral collapse without previous trauma (5); disc space narrowing without osteophyte formation; end-plate erosion (2); and segmental instability associated with facet joint destruction (6,7). thus the presence of these lesions had to be confirmed. since coexisting cervical myelopathy may affect neurological diagnosis, the presence of ra cervical lesions (atlantoaxial subluxation, vertical subluxation, and subaxial subluxation) was examined in plain x-rays of the cervical spine. lumbar spinal canal stenosis was assessed by mri or myelography/postmyelographic ct scans. foraminal stenosis was diagnosed on mri (sagittal and transverse sections through the intervertebral foramen) and srg, and their diagnostic usefulness was compared. results all of the patients complained of pain over the buttock to the anterior surface of the thigh. pain was worsened in the sitting and standing positions but was mitigated in the recumbent position. the l2, l3, and l4 dermatomes were suspected to be the responsible nerve roots. however, complications such as cervical myelopathy and articular lesions of ra in the lower limbs made it difficult to identify the responsible nerve roots on the basis of neurological findings alone. from combined neurological and imaging findings, responsible nerve roots were finally identified as l3 and l4 in two and four patients, respectively. radiculopathy was unilateral in five patients and bilateral in one patient. cauda equine syndrome associated with radiculopathy was diagnosed in only one patient. conservative treatment alleviated the symptoms of one patient; five patients were treated by surgical intervention. preoperative joa score was 8.6 ± 6.3 (range 5–13). three patients were treated by posterior decompression for the affected nerve root. a patient with severe low back pain and another patient with segmental instability in multilevels were treated by posterior decompression and posterolateral fusion with instrumentation. although radiculopathy was improved in all patients, one patient was re-operated for radiculopathy that had developed in the contralateral side after the first operation (table i). their clinical courses were monitored postoperatively for 26.0 ± 3.5 months (range 12–48). postoperative joa score was improved to 15.2 ± 7.5 (range 8–20) with a recovery rate of 32.2% ± 3.7% (range 12.5%–52.6%). bone fusion was achieved in all patients who were treated by posterior decompression and posterolateral fusion with instrumentation. plain x-rays of the cervical spine revealed ra lesions (atlantoaxial subluxation, vertical subluxation, and subaxial subluxation) in four of the six patients. plain x-rays of the lumbar spine, ct, and mri found table i. patients’ data. case gender, age (years) duration of ra (years) mutilating classification affected nerve root treatment 1 f, 74 17 + stage iv, class 4 left l4 ! right l4 a decompression 2 f, 69 28 + stage iv, class 4 right l4 decompression 3 f, 57 25 + stage iii, class 3 right l3 decompression and fusion 4 f, 74 2 + stage iv, class 3 right l4 conservative 5 f, 65 26 stage iv, class 3 left l3 decompression and fusion 6 f, 75 3 stage ii, class 3 bilateral l4 decompression a re-operated. 134 t. koakutsu et al. the following ra lesions: vertebral collapse without previous trauma in three patients; disc space narrowing without osteophyte formation in three; end-plate erosion in six; and segmental instability associated with facet joint destruction in four (anterior spondylolisthesis in two; lateral spondylolisthesis in two; includes overlapping lesion). in all patients, severe osteoporosis was evident, although mri revealed no obvious rheumatoid nodules in the vertebral bodies. excluding the one patient with cauda equine syndrome, spinal canal stenosis was mild in the mr images and/or the myelograms. this finding led us to suspect the presence of foraminal stenosis. foraminal stenosis was not demonstrated in mr images (sagittal and transverse sections through the foramen) in four of the six patients. srg showed that all patients had pain radiating towards the sites where pain is usually felt at the time of nerve root puncture (pain reproduction). transiently after nerve root block, the pain dramatically disappeared, leading to clear identification of the affected nerve root. nerve root compression by the superior articular process of the lower vertebra was detected in all patients. representative images are shown in figures 1–3. discussion although the frequency of lumbar lesions in ra is not considered to be as low as previously described, their a d e b c figure 1. a 69-year-old women, with right l4 radiculopathy as revealed by postmyelographic ct scan. a: in the coronal section, bilateral l4 roots were well depicted. b: in the sagittal section, osteoporosis is evident; multiple vertebral collapse and end-plate irregularity are observed despite no history of trauma. c: in the sagittal section through the right foramen, foraminal stenosis is not obvious. d: in the transverse section (l3/4), spinal canal stenosis is very mild. e: in the transverse section (l4/5), prominent l4/5 facet joint destruction and lateral dislocation are noted. lumbar radiculopathy in rheumatoid arthritis 135 natural course has not been elucidated because there have been only a few systemic reports (1–3). this is likely due to the occasional difficulty in differentiation of ra-specific lesions from osteoporotic fractures of the vertebral body and degenerative spondylolisthesis. moreover, complications such as cervical myelopathy and articular lesions of the lower extremities make neurological diagnosis more difficult (2). ra-associated lumbar lesions are roughly divided into vertebral lesions, facet joint lesions, and intervertebral disc lesions. these lesions must be differentiated from spondylosis deformans and osteoporosis lesions. vertebral lesions (defined as rheumatoid nodules of the vertebral bodies) were reported in 1952 by baggenstoss et al. who observed them in autopsy patients (4). facet joint lesions were described by lawrence et al. (6). in the present study, we defined ra lumbar lesions as rheumatoid nodules of the vertebral bodies (4), vertebral collapse without previous trauma (5), disc space narrowing without osteophyte formation, end-plate erosion (2), and segmental instability associated with facet joint destruction (6,7). endplate erosion was obvious in all of the six patients. the occurrence of ra lesions in the vertebral bodies and intervertebral disc (which has no synovial tissue) was attributed to enthesopathy by shichikawa et al. (12). in most of the reported cases of ra lumbar lesions, neurological symptoms are caused by spinal canal stenosis, stemming from vertebral lesions leading to vertebral collapse (4,5) or from facet joint lesions a b figure 3. selective radiculography (srg) (right l4 nerve root). a: posteroanterior view. b: oblique view. the right l4 nerve root is compressed from underneath by the l5 superior articular process (arrow). pain reproduction at the time of nerve puncture and nerve block induction were confirmed. a b figure 2. magnetic resonance imaging (mri). a: in the sagittal section (right l4/5 foramen; t1-weighted image), foraminal stenosis is not obvious (arrow). b: in the transverse section (l4/5 foramen; t2-weighted image) as well, foraminal stenosis is not evident. 136 t. koakutsu et al. leading to segmental instability (6,7). there are very few cases of foraminal stenosis. heywood reported a case of l4 radiculopathy attributable to a l4/5 facet joint lesion (8), yet he did not describe the pathogenesis clearly. myelography and postmyelographic ct scans cannot be used to establish a diagnosis of foraminal stenosis, which can be detected in mri and srg. in four of the six cases of our series, however, mri failed to detect foraminal stenosis. one possible reason is that the nerve root compression is not detected by recumbent mri because the pain was relieved in the recumbent but not the sitting or standing position. in contrast, an invasive examination, srg, detected the nerve root compression attributable to the superior articular process of the lower vertebra in all patients. furthermore, srg was very useful in the functional diagnosis based on pain reproduction at the time of nerve root puncture and blocking effect in ra cases where the neurological diagnosis cannot be easily established. this seemed to be a characteristic feature of ra in terms of arthritis. we assumed that the pathogenesis of foraminal stenosis in the lumbar spine of ra patients is the compression of the nerve root by the superior articular process of the lower vertebra; this is caused by vertical instability of the facet joint when facet joint lesions are associated with disorders in the vertebral body and the intervertebral disc. impaired nerve roots due to foraminal stenosis are most frequently reported to be present at l5 in spondylosis (13), whereas l4 was the most common site for nerve root impairment in ra. with regard to surgical treatment, crawford et al. stated that ra patients who undergo an instrumented lumbar fusion can expect a slightly higher complication rate than patients without ra, which may be related to osteopenia and immunosuppression (3). inaoka et al. performed posterior lumbar interbody fusion on seven ra patients and reported that collapse of graft occurred in one patient, migration of pedicle screw in two, instability of adjacent level in three, and collapse of adjacent vertebra in four (14). fusion surgery might be performed in view of the pathogenesis of lumbar radiculopathy caused by foraminal stenosis in ra, but it was often difficult to employ fusion surgery with instrumentation because most of the patients in our series were in poor general condition or had severe osteoporosis. we performed posterior decompression without fusion in three cases; short-term outcomes were favourable though one patient was re-operated for secondary radiculopathy. symptoms of radiculopathy were reduced by posterior decompression of the affected nerve root with partial resection of superior articular process which served as a compression factor. conclusions lumbar foraminal stenosis is a characteristic pathology of ra, and should be kept in mind in the diagnosis of lumbar radiculopathy. srg is useful in the diagnosis of affected nerve roots. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. yonenobu k, ochi t, fujiwara k, oda t. pathology and management of spinal lesions in rheumatoid arthritis. j jpn orthop assoc. 1996;70:573–82 (in japanese). 2. kawaguchi y, matsuno h, kanamori m, ishihara h, ohmori k, kimura t. radiologic findings of the lumbar spine in patients with rheumatoid arthritis, and a review of pathologic mechanisms. j spinal disord. 2003;16:38–43. 3. crawford ch 3rd, carreon ly, djurasovic m, glassman sd. lumbar fusion outcomes in patients with rheumatoid arthritis. eur spine j. 2008;17:822–5. 4. baggenstoss ah, bickel wh, ward le. rheumatoid granulomatous nodules as destructive lesions of vertebrae. j bone joint surg (am). 1952;34:601–9. 5. fam ag, rubenstein j. rheumatoid burst fracture. arthritis rheum. 1998;41:747–8. 6. lawrence js, sharp j, ball j, bier f. rheumatoid arthritis of the lumbar spine. ann rheum dis. 1964;23:205–13. 7. otani k, kikuchi s, sato k, chiba k. mri findings of pathological lumbar spondylolisthesis due to rheumatoid arthritis; report of a case. rinshoseikeigeka. 1996;31: 1187–90 (in japanese). 8. heywood awb, meyers ol. rheumatoid arthritis of the thoracic and lumbar spine. j bone joint surg (br). 1986;68: 362–8. 9. sato t, hirata s, kanabuchi t. direct oblique approach for puncture of the lumbar and sacral nerve roots. rinshoseikeigeka. 1990;25:221–5 (in japanese). 10. steinbrocker o, traeger ch, batterman rc. therapeutic criteria in rheumatoid arthritis. j am med assoc. 1949;140: 659–62. 11. hirabayashi k, miyakawa j, satomi k, maruyama t, wakano k. operative results and postoperative progression of ossification among patients with ossification of cervical posterior longitudinal ligaments. spine. 1981;6:354–64. 12. shichikawa k, matsui k, oze k, ota h. rheumatoid spondylitis. int orthop. 1978;2:53–60. 13. jenis lg, an hs, gordin r. foraminal stenosis of the lumbar spine; a review of 65 surgical cases. am j orthop. 2001;30: 205–11. 14. inaoka m, tada k, yonenobu k. problems of posterior lumbar interbody fusion (plif) for the rheumatoid sponsylitis of the lumbar spine. arch orthop trauma surg. 2002;122: 73–9. lumbar radiculopathy in rheumatoid arthritis 137 original research 1 sajsm vol. 34 no.1 2022 creative commons attribution 4.0 (cc by 4.0) international license pain and physical activity levels among rheumatoid arthritis patients between the ages of 18 to 50 years in south africa rm wilkinson, bbio; l smith, mphil; s ferreira, mphil department of sport and movement studies in the faculty of health sciences, university of johannesburg, doornfontein campus, p.o. box 524, auckland park, 2006, johannesburg, south africa corresponding author: rm wilkinson (rebecca@wrc.co.za) arthritis is a musculoskeletal disorder which holds the potential of being disabling. [1] arthritis affects people worldwide, with disability and functional limitations being characteristics of the disorder. [1] rheumatoid arthritis is characterised by systemic inflammation, which can result in joint damage, disability and functional limitations. [1] diseasemodifying anti-rheumatic drugs, anti-inflammatories and analgesics are the types of medications commonly prescribed for the management of ra. [1] however, when the systemic inflammation in ra is poorly controlled and patients follow an unhealthy lifestyle, they are at risk for developing various comorbidities. [1, 2, 3] the incidence of cardiovascular events in those diagnosed with ra is estimated to be double that compared to the general population, with cardiovascular disease (cvd) typically developing at an earlier age in this population. [2] obesity further increases the comorbidity risk in those with ra, with obesity and poor body composition related to ra, pharmacology and to physical inactivity. [1, 3] it has been suggested that ra patients are less active due to joint manifestations relating to the disease as well as other ‘general’ barriers, yet regular physical activity is an effective treatment and management tool for ra. [2, 3] currently, exercise and the broad range of treatment options for ra are trumped by medication as the favoured modality; however, when considering longevity, interventions such as exercise become essential. [2, 4] physical activity as therapeutic management has been proven to possess a range of benefits, including the improvement of general health and functional ability, as well as the reduction of associated disability. [2] furthermore, physical activity is suitable for most individuals and can be used in conjunction with prescribed medications, which may allow for a reduced dosage while simultaneously benefitting general health status. [1, 2] however, various international research studies demonstrate that only a small percentage of ra patients are physically active. [1, 5, 6] several countries have conducted research on physical activity participation among ra patients, but with the unpredictable and changing landscape in south africa, as well as its economic development, there is a need to assess the current physical activity levels in a local context, and to identify barriers to physical activity participation. [1, 5, 6] therefore, the purpose of this study was to determine pain and activity levels at work, while travelling or during leisure activities of ra patients in a south african context. the objectives of the study were: (1) determine whether most physical activity is completed during work, travel or recreation; (2) quantify the amount of time spent sedentary on a normal day; (3) determine the correlation between the pain, physical activity level, ambulation and personal grooming; (4) determine self-reported physical activity levels, overall energy levels, strength and endurance; and (5) determine patients’ perceptions on injury, risk and safety of exercise. methods study design this study was cross-sectional in design and quantitative data were collected and analysed. to achieve the aim of the study, a combination of two questionnaires were utilised: the global physical activity questionnaire (gpaq) and the pain background: little epidemiological research on rheumatoid arthritis (ra) has been done in africa, suggesting that it is an uncommon illness. in rural south africa, ra has an overall prevalence of 0.07% and a prevalence of 2.5% in urban areas; therefore, it is not as uncommon as perceived by the lack of research. patient-centred programmes to improve physical function have been lacking and, as a result, the prior assumption was that physical activity should be avoided. objectives: to determine pain and physical activity levels among ra patients between the ages of 18 to 50 years in south africa. methods: a combination of two questionnaires were used, namely, the global physical activity questionnaire (2002) and the pain outcomes questionnaire (2003). the collated questionnaires were distributed by rheumatologists and on social media platforms to ra patients between the ages of 18 to 50 years old living in south africa. this study had a sample size of 105 participants, with participation occurring through the online google forms platform. results: one hundred and five participants with ra were recruited with an average age of 38±9 years. most of the participants were females (93.3%). seventy-two percent of the sample was classified as physically active, where work, leisure and travel activities were considered. no significant correlation between pain and physical activity was evident (r=0.10; p=0.311). results showed significant correlations between pain and personal grooming (r=0.30; p=0.002), pain and ambulation (r=0.60; p=0.000), and pain and stair climbing (r=0.60; p=0.000). conclusion: physical activity has proven to have multiple benefits for those suffering with ra. in this south african sample of ra patients, the majority were classified as physically active, and pain did not affect the activity levels of the involved participants. this study opens further research questions regarding ra prevalence in south africa, and the type and intensity of physical activity that would be beneficial for ra. keywords: inflammatory disease, exercise, physical limitations s afr j sports med 2022;34:1-8. doi: 10.17159/2078-516x/2022/v34i1a11555 mailto:rebecca@wrc.co.za http://dx.doi.org/10.17159/2078-516x/2022/v34i1a11555 https://orcid.org/0000-0002-1446-3458 https://orcid.org/0000-0002-8562-5004 https://orcid.org/0000-0002-9203-8537 original research sajsm vol. 34 no.1 2022 2 outcomes questionnaire (poq). the collated questionnaires were made available on the google forms platform, allowing participants to access and complete them online. selection and description of participants various rheumatologists were contacted to distribute the google forms link to the patients in their practices who met the inclusion criteria of this study. additionally, the link was published to ra support groups and social media platforms by the researcher. a sample of 105 participants complying with the inclusion criteria were recruited by means of purposive sampling. participants were diverse in terms of backgrounds, provinces and treating rheumatologists. inclusion criteria  clinically diagnosed with ra.  between the ages of 18 and 50 years at the time of data collection. the minimum age of 18 years was established to allow the participant to consent independently to participation in the research study. the exclusion of individuals older than 50 years of age was determined due to the relationship between increased age and comorbidities, which could impact physical activity levels and the performance of daily activities.  residents in south africa at the time of data collection.  male or female.  internet access to complete the questionnaire. ethical considerations all participants were informed about the purpose of the research by means of an information letter and were required to provide consent before data collection commenced. the study participants were aware that participation was voluntary and that withdrawal from the study could only take place before submission of the questionnaire. every precaution was taken to protect the privacy of the participants and confidentiality of their personal information was ensured. this study was approved by the institutional research ethics committee (rec-171-2019). questionnaires as mentioned, two combined questionnaires were used to gather subjective data relating to the pain and physical activity levels of the participants. although the questionnaires included demographic questions, no identifying data was gathered and therefore, participation was anonymous. the questions in the gpaq were centred on the participants’ activity levels during work, travel and leisure. the poq was adapted by removing questions that were not relevant to the aims and objectives of this study. therefore, the poq was centred on the participants’ overall pain levels, as well as how pain affects their daily activities. statistical analysis the data collected were quantitative in nature. statistical analysis was completed using the statistical package for social science (spss) version 26.0 and included percentages, means, standard deviations and correlations. the kolmogorovsmirnov test was used to assess the normality of the distribution of the data. to determine the correlations between the variables of interest, the pearson product-moment correlation coefficient was computed because normality of the data was established. a calculation of statistical significance was done yielding 5% as the level of significance. results demographics a total of 105 ra patients took part in the present study, with the demographic results demonstrated in table 1. the mean age of the participants was 38±9, and only 7 participants were of the male gender. the mean age of ra diagnosis was 32 years. eighteen of the 105 participants were not currently seeking treatment for their condition. majority of the participants resided in the gauteng province, with only 36 participants residing elsewhere. global physical activity questionnaire many participants had declared that they were physically active across more than one of the categories presented in table 2. based on the general physical activity guidelines, 29 participants (27.6%) were classified as physically inactive, while 76 participants (72.4%) were classified as physically active. [3] both vigorous and moderate intensity activity was performed at work by 10 (9.5%) and 43 (41%) of the participants table 1. demographic results of the sample (n=105) frequency percentage (%) mean ± sd age (years) 18-29 22 20.9 38 ± 9 30-39 30 28.6 40-50 53 50.5 gender females 98 93.3 males 7 6.7 age of diagnosis (years) 0-17 18 17.1 32 ± 11 18-29 26 24.8 30-40 30 28.6 40-50 31 29.5 seeking treatment yes 87 82.9 no 18 17.1 province of residence gauteng 69 65.7 kwazulunatal 11 10.5 free state 4 3.8 limpopo 1 0.95 northern cape 2 1.9 western cape 15 14.3 north west 2 1.9 mpumalanga 1 0.95 original research 3 sajsm vol. 34 no.1 2022 respectively. the mean time spent being physically active at work at a vigorous intensity was 229 minutes with a mean of four and a half days, while the mean amount of time spent doing moderate intensity activity at work was 175 minutes with a mean of four days. the number of participants in the different categories of physical activity is shown in fig. 1. sixteen participants (15%) performed no physical activity. leisure activity had the highest number of participants and travel the least, with 27 (26%) and six (6%) respectively. nine participants (9%) performed physical activity in all three categories. the participants were required to select the amount of time they spend seated or reclining on a typical day. the categories of choice were 1-2 hours, 3-4 hours, 56 hours and >7 hours, with 12 (11.43%), 24 (22.86%), 42 (40%) and 27 (25.71%) selected respectively. pain outcomes questionnaire a 10-point likert scale was used to respond to the poq questions, results of which can be seen in table 3. out of 10, 5.8 was the mean pain level selected. the interference of their pain with daily activities yielded the following means: walking 5.3, carrying objects 5.9, stair climbing 5.5, and personal grooming 2.7. the participants’ mean rating of their personal physical activity, overall energy and strength and endurance was 4.9, 4.2 and 4.7 respectively. for depressive feelings on the day of the questionnaire, the mean response was 4.5. with regards to the fear of re-injuring themselves and exercise safety, the mean scores were 6.8 and 6.1 respectively, noting that none of the participants selected a score of zero for the last two questions. categories of physical activities and associated mean pain level in table 4, when looking at the average pain levels reported for each of the categories of physical activity, the participants performing activity in both work and travel reported the highest mean pain levels (7.4±1.4 au), followed by the participants performing no physical activity (6.4±2.3 au). the lowest mean pain level reported was by the participants performing physical activity in the travel only category (3.7±2.8 au). correlations table 5 demonstrates the correlations of interest for the present study. the correlation between pain and total physical activity performed was r=0.10 for pearson’s product moment correlations, demonstrating a slight effect of pain on physical activity participation. [7] the correlation between pain and the affected ability to walk was significant (r=0.60, p=0.0001) with a table 2. global physical activity questionnaire data (n=105) category response frequency percentage (%) number of days time (minutes/day) work – vigorous intensity yes 10 9.5 4.5 ± 1.3 229 ± 146 no 72 68.6 unemployed 15 14.3 student 8 7.6 work moderate intensity yes 43 41.0 4.0 ± 1.9 175 ± 138 no 39 37.1 not applicable 23 21.9 travel walking or cycling yes 33 31.4 4.7 ± 2.0 111 ± 157 no 72 68.6 leisure, sports and recreation vigorous intensity yes 37 35.2 3.1 ± 1.4 60 ± 26 no 68 64.8 leisure, sports and recreation moderate intensity yes 56 53.3 2.8 ± 1.4 57 ± 38 no 49 46.7 data expressed as mean ± sd unless indicated otherwise. fig. 1. categories of physical activity completed by the participants (n=105) n u m b e r o f p a r ti c ip a n ts original research sajsm vol. 34 no.1 2022 4 moderate positive correlation. [7] another moderate positive correlation of r=0.60 was found between pain and the affected ability to climb stairs, also demonstrating a significance of p=0.0001. [7] pain and the affected ability to manage personal grooming yielded a fair positive correlation of r=0.30, with significance of p=0.002. [7] discussion the aim of this paper was to determine the relationships between pain and physical activity levels in a south african sample of ra patients between the ages of 18 and 50 years. during the study, the amount of physical activity performed during work, travel or leisure was determined and the amount of time patients are sedentary on a normal day was quantified. in addition, the correlation between pain, physical activity level, personal grooming, ambulation, and stair climbing was determined by means of a self-reported questionnaire. two questionnaires were collated to achieve the above, namely the global physical activity questionnaire and the pain outcomes questionnaire. demographics ra is said to affect 1% of the general population; therefore, it is expected that the sample size in this study would be smaller in comparison to studies centred around more prevalent conditions. [8] the prevalence of ra in south africa ranges from 0.07% to 2.5% in rural and urban areas respectively. [9] the unequal distribution of gender in the study is consistent with numerous studies stating a higher prevalence among the female population. [1, 10] rheumatoid arthritis has been shown to be four to five times more likely in females compared to males below the age of 50 years, but in african countries, the ratio is as large as 6:1 which is similar to that found in the sample in the present study. [10] the prevalence ratio tends to decrease in populations older than 60 years, where the female to male ratio is approximately 2:1. [10] in the present study, the mean age of diagnosis was 32 years, and 41.9% of the sample table 3. pain outcomes questionnaire data (n=105) question mean ± sd max value selected min value selected average pain levels during the last week (0=no pain at all; 10=worst possible pain) 5.8 ± 2.3 10 0 does your pain interfere with your ability to walk (0= not at all; 10= all the time) 5.3 ± 3.1 10 0 does your pain interfere with your ability to carry / handle everyday objects such as bag of groceries or books (0= not at all; 10= all the time) 5.9 ± 2.8 10 0 does your pain interfere with your ability to climb stairs (0= not at all; 10= all the time) 5.5 ± 3.4 10 0 does your pain require you to use a walker, cane, wheelchair or other device (0= not at all; 10= all the time) 0.9 ± 2.2 10 0 does your pain interfere with your ability to manage your personal grooming (combing hair, brushing teeth, etc) (0= not at all; 10= all the time) 2.7 ± 2.8 10 0 how would you rate your physical activity (0= significant limitations with basic activity; 10= can perform vigorous activity without limitations) 4.9 ± 1.9 10 0 how would you rate your overall energy (0= totally worn out; 10= most energy ever) 4.2 ± 1.9 9 1 how would you rate your strength and endurance today (0= very poor; 10= very high) 4.7 ± 2.0 10 1 how would you rate your feelings of depression today (0= not at all depressed; 10= extremely depressed) 4.5 ± 2.5 10 0 how much do you worry about re-injuring yourself if you are more active (0= not at all; 10= all the time) 6.8 ± 2.8 10 1 how safe do you think it is for you to exercise (0= not safe at all; 10= extremely safe) 6.1 ± 2.1 10 1 table 4. the different categories of physical activity and the associated average pain levels category of exercise participation participants (%) pain (au) completely sedentary 15 6.4 ± 2.3 leisure 26 5.2 ± 2.1 work 11 6.3 ± 2.9 travel 6 3.7 ± 2.8 leisure and work 16 5.8 ± 2.5 work and travel 8 7.4 ± 1.4 leisure and travel 9 5.3 ± 1.8 leisure, travel and work 9 6.3 ± 1.2 data expressed as mean ± sd unless indicated otherwise. au, arbitrary units table 5. pearson product moment correlations between variables of interest variables r p-value pain level and total activity time by each participant 0.10 0.31 pain level and the affected ability to walk 0.60 0.0001* pain level and the affected ability to climb stairs 0.60 0.0001* pain level and the affected ability to manage personal grooming 0.30 0.002* * indicates significance (p<0.05). original research 5 sajsm vol. 34 no.1 2022 declared being diagnosed before 30 years of age. research has reported the typical onset age for ra to be between 40 to 50 years; therefore, the mean of the present study is younger than this age. [1] a possible explanation for this is the age limitation for participation in the study, which was 18 to 50 years, and in addition, african samples have reported an average age of onset of 27 years. [11] the large distribution of participants located in the gauteng province is attributed to gauteng having the greatest population density, coupled with the highest internet connectivity in south africa. [12] global physical activity questionnaire (gpaq) when participants were asked about their participation in physical activity, results showed that 15.24% of the sample was sedentary and 72.4% were classified as physically active. results also indicate that although only 15.24% of the sample declared that they were not partaking in any physical activity, a further 12.36% of the participants who were physically active were not performing sufficient levels of physical activity. the classification of sedentariness was determined in consultation with the american college of sports medicine (acsm) guidelines on physical activity for psychological and physiological health benefits. [3] to be classed as physically active one must participate in 150 minutes of moderate intensity activity or 75 minutes of vigorous intensity activity, per week. [3] the results in the present study contrast with previous studies among american and european ra patients, which made use of standardised fitness tests to measure aerobic fitness, physical strength, agility, and endurance, as well as self-report questionnaires to determine physical activity participation, where 69% and 68% of participants, respectively, were classified as inactive. [1, 2] in addition, a study making use of the quest-ra (a quantitative clinical assessment of patients with rheumatoid arthritis seen in standard rheumatology care in 15 countries) was conducted on ra patients across 21 countries, which reported that only 13.8% of the participants were partaking in physical exercise three or more days per week. [6] there is a notable difference between these international studies and the present study, which could be attributed to the international studies collecting responses on exercise participation in isolation, in comparison to the present study which collected responses on physical activity during work, travel and leisure. [3] in 2020, a study conducted across 104 countries revealed that work, household, and travel activity measured with the gpaq are important considerations when evaluating overall physical activity levels, especially in countries that continue to develop economically. [13] these are alternative forms of physical activity that are easily accessible and affordable to individuals who are susceptible to decreased physical activity due to societal and economic barriers. [13] both vigorous and moderate intensity activity was performed by 37% of the participants in the present study. although low impact and moderate intensity activity has been encouraged in this population for safety reasons, there is currently no direct evidence against vigorous intensity exercise participation in those with ra. [3] a pilot study among older ra patients reported that high-intensity interval training performed at levels exceeding that of the current health guidelines (of 150 minutes of moderate intensity activity or 75 minutes of vigorous intensity activity, per week, according to the acsm) is associated with numerous benefits in this population. [14] increased cardiorespiratory fitness and reduced disease activity were among many of the benefits experienced after ten weeks of vigorous intensity activity. [14] evidence also exists that prolonged moderate to vigorous intensity activity reduces inflammatory markers, having a similar effect to medication on the treatment of ra, slows down disease progression, and reduces pain and fatigue. [14] in this study, 14.3% of the sample were unemployed. due to the associated disability and cost incurred through treatment, ra affects patients economically. [4] unemployment is a concern as it impacts the patient’s ability to afford the treatment aiding in the management of their condition. [4] of those who participated, 65.71% spent five or more hours sitting or reclining on a typical day. sedentary behaviour possesses serious health consequences by increasing the risk of both developing and progressing diseases while simultaneously affecting general health. [2, 15] in 13 international studies it was found that those who performed no physical activity and reclined for more than eight hours a day had a similar mortality risk to obese individuals and smokers. [15] it is important to note that prolonged sitting is a risk factor for allcause mortality and may result in a viscous cycle between disease progression and health, therefore; it is essential for health interventions to minimise the amount of time spent seated. [2, 15] pain outcomes questionnaire (poq) the main complaint from ra patients is pain, which is a result of joint inflammation. [1, 10] the mean pain level of the sample in this study was 5.8/10, with 7/20 being the most commonly selected response. a heightened pain response is seen in those with ra, as pain is a major symptom of the condition and generally impacts the modality of treatment. [16] by making use of a 0-to-10-point scale to assess pain, this study only focused on the intensity of pain. other aspects of pain also need to be considered to determine the total impact of pain on the individual. [16] difficulty with daily tasks and reduced quality of life are frequently seen in those with chronic musculoskeletal conditions, which consequently catalyses institutionalisation, dependency, and increased healthcare needs. [4] the responses for this study demonstrated a mean score of 5.3/10, 5.9/10 and 5.5/10 for difficulty walking, carrying everyday objects and climbing stairs, respectively. considering the young and middle ages of the participants in this sample, this is concerning as there are apparent issues with their ability to perform activities of daily living which may intensify with increased age and time should the disease progress. [1] although physical activity offers therapeutic management for ra, there is a consistent concern that exercise participation can or will exacerbate symptoms and joint damage. [8, 17] of the rheumatologists who participated in the iverson et al study, 83% believed that exercise was an effective approach to managing symptoms of ra, yet none deemed themselves original research sajsm vol. 34 no.1 2022 6 knowledgeable enough on exercise participation to correctly advise their patients. [17] when asked to rate their physical activity levels, the most commonly selected response by the participants was 5/10, which is logical considering that 72.4% of the sample were achieving the recommended amount of physical activity in a week. [3] notably, only 19% of the sample selected a seven or more out of 10 for their physical activity. physically active individuals appear to have a greater capability to overcome the associated barriers of participating in physical activity. [8] for patients who are not physically active, rheumatologists advocated a “mind shift” and proposed that positive mental health is achieved prior to the initiation of a physical activity programme. [17] the mean energy levels selected by the participants was 4.2/10, noting that no participant rated their energy level as 10/10. according to the literature, one of the reported symptoms of ra is mental and physical fatigue, hence this finding was not completely unexpected. [1, 8] the mean rating of experiencing symptoms of depression was 4.5/10, with the most commonly selected answer being four. in a 2019 study of ra patients, 55% of the participants reported having mild or worse symptoms of depression and 22% experienced moderate or worse symptoms of depression. [18] of those participants, only 12% were seeking treatment for depression. [18] as mentioned above, the concern and fear of worsening the condition or causing injury when participating in physical activity is one of the most frequently mentioned barriers to physical activity participation in this population. [8, 17] this study enquired about the fear of re-injury with physical activity participation and the mean response was 6.8/10, with the most common response being eight. regarding feelings around the safety of exercise for ra, 80% of the sample selected five or higher out of 10 (with 10 being ‘extremely safe’). of the research studies that have been completed on the safety of physical activity and exercise participation in the ra population, little data has demonstrated that it negatively impacts on the condition. [2, 5, 8] rather, extensive research has advocated for physical activity participation due to its benefits for ra patients. [2, 5, 8] categories of physical activity and mean pain levels additional analysis of the data examined the mean pain levels reported for the different physical activity categories. looking at the different categories, the highest average pain level experienced was in the category of work and travel activity (7.4 au). the mode and intensity of physical activity needs to suite the individual in terms of condition and abilities, as well as being ‘balanced’, with sufficient recovery time. thus, one’s work-related physical activity demands may not be entirely appropriate in this regard. [4] the sedentary participants reported the second highest average pain level (6.4 au), which is consistent with previous literature, as physical activity plays a role in reducing and aiding pain levels experienced in those with ra. [2, 8, 17] the travel only category (involving walking and/or cycling) had the lowest pain level, with an average of 3.7 au. active travel activities, such as walking and cycling, are seen in both highand low-income countries, as beneficial for general health, and can contribute to one’s overall physical activity level. [13] correlations an essential component of ra treatment is the management of pain experienced, with the prospect that effective pain management may promote compliance to therapy as well as fostering physical activity participation. [16] in the present study, it was established that pain had a slight positive effect on the physical activity participation in these participants. [7] including travel, work and leisure activities could explain this unexpected result, as should one experience pain, participants may opt to avoid performing leisurely physical activity; however, should their work or travel require physical activity, they may have no option but to still participate in the activity to complete their tasks. chronic pain can substantially affect an individual’s ability to perform daily tasks. [19] the correlation between pain and affected ability to walk demonstrated that pain does impact the ability to walk through a moderate positive correlation. [7] climbing stairs is another factor in ambulation, which also had a moderate positive correlation with pain. [7] the last correlation explored was between pain and the affected ability to manage personal grooming, which revealed a fair positive correlation. [7] all of these correlations were statistically significant. according to edemekong et al, there are two categories of daily activities. the first category is associated with basic skills that are required to achieve and maintain basic needs, such as eating, grooming, and transferring from one position to another. [20] the second or instrumental category requires more skill, usually mentally and physically, such as transport, cleaning, and shopping. [20] this may explain why grooming is a lower rate disability as it falls within the basic category, can be completed in supine or seated positions, and generally requires less strength and mobility than other daily tasks such as walking and climbing stairs. [20] in a study completed in spain on chronic disease patients, a disability for walking and climbing stairs existed, and when compared to other daily activities, personal grooming was one of the disabilities with a lower rating. [19] although there has been further research on the treatment of ra, it remains a limiting, chronic disease which greatly impacts on the patient’s life, abilities, and morbidity. [4] in contrast to previously mentioned international studies, this study demonstrated that the majority of ra patients were labelled as physically active. [1, 5, 6] according to current literature, participation in physical activity and exercise is both beneficial and safe for these patients, with a wide range of psychological and physiological benefits. [2, 17] some of the concerns surrounding physical activity in this population include the appropriate intensity, frequency and mode of activity, the possibility of causing further joint damage during activity, how joint pain will impact physical activity, and limited knowledge among healthcare professionals in terms of physical activity as therapeutic management. [8, 17] however, addressing these barriers and concerns will assist in encouraging and increasing activity levels in those with ra. [8, 17] preserving one’s physical abilities and decreasing additional health risks are the main original research 7 sajsm vol. 34 no.1 2022 goals for physical activity in the ra population. [2] one does need to be aware of the unique barriers (both physical and mental), motivators and perceptions that may influence physical activity levels among ra patients. [8, 17] study limitations the limitations in this study’s data collection methods include the distribution of the questionnaire using online platforms, resulting in the exclusion of ra patients without internet access. in addition, a small sample size was obtained. the omission of questions regarding the types of treatment sought was a limitation in data collection. by using the two selected questionnaires, only physical activity levels and pain outcomes were assessed. the authors acknowledge that additional questionnaires, such as a quality-of-life questionnaire, may have enhanced this study. recommendations for future research performing a pre-screening assessment before the completion of the questionnaire may assist in excluding participants who have comorbidities that may negatively impact their responses. including a question on the type of treatment that participants are seeking at the time of participation may also be beneficial, as it would allow for the analysis of and correlation between, pain levels and the different treatment modalities. lastly, replacing the pain outcomes questionnaire with the rheumatoid arthritis pain scale questionnaire may provide more accurate results, as the latter questionnaire is specific for ra. conclusion in conclusion, this study demonstrated that in a south african sample, majority of ra patients were classified as physically active. furthermore, and unexpectedly, no significant correlation was found between pain and physical activity levels. this study brings to light the importance of physical activity and exercise in the ra population, while also reminding the relevant healthcare professionals to consider the unique barriers and concerns of such participation among those with ra. conflict of interest and source of funding: the authors declare no conflict of interest and no source of funding. acknowledgements: the authors would like to extend their gratitude to juliana van staden from statkon for her assistance with the data analysis. author contributions: all authors contributed to the design of this research and writing of the article ((i) conception, design, analysis, and interpretation of data; (ii) drafting or critical revision for important intellectual content; and (iii) approval of the version to be published). references 1. ehrman jk, gordon pm, visich ps, et al. clinical exercise physiology. 4th ed. s.l.: human kinetics, 2018:411-437. chapter in book: arthritis 2. cooney jk, law r-j, matschke v, et al. benefits of exercise in rheumatoid arthritis. j aging res 2011; 681640. [doi:10.4061/2011/681640] [pmid: 21403833] 3. metsios gs, lahart im. exercise as medicine in rheumatoid arthritis. mediterr j rheumatol 2015; 26(2):54-61. https://www.mjrheum.org/october-2015/ newsid792/10/ (accessed 22 november 2021) 4. nolte k, janse van rensburg dc. exercise prescription in the management of rheumatoid arthritis. s afr fam pract 2013; 55(4):345-349. [doi:10.1080/20786204.2013.10874374] 5. eurenius e, stenström ch. physical activity, physical fitness and general health perception among individuals with rheumatoid arthritis. arthritis rheum 2005; 53(1):48-55. [doi:10.1002/art.20924] [pmid: 15696555] 6. sokka t, häkkinen a, kautiainen h, et al. physical inactivity in patients with rheumatoid arthritis: data from twenty-one countries in a cross-sectional, international study. arthritis rheum 2008; 59(1):42-50. [doi: 10.1002/art.23255 ] [pmid 18163412] 7. hartling l, hamm m, milne a, et al. validity and inter-reliability testing of quality assessment instruments. rockville md: agency for healthcare research and quality (us) 2012 mar. report no.: 12-ehc039-ef. [pmid: 22536612] table 2, interpretation of fleiss' kappa (κ) (from landis and koch 1977). https://www.ncbi.nlm.nih.gov/books/nbk92295/table/ methods.t2/ (accessed 22 november 2021). 8. veldhuijzen van zanten jj, rouse pc, hale ed, et al. perceived barriers, facilitators and benefits for regular physical activity and exercise in patients with rheumatoid arthritis: a review of the literature. sports med 2015:45(10):1401-1412. [doi: 10.1007/s40279-015-0363-2] [pmid: 26219268] 9. usenbo a, kramer v, young t, et al. prevalence of arthritis in africa: a systemic review and meta-analysis. plos one 2015; 10(8):e0133858. [doi: 10.1371/journal.pone.0133858] [pmid: 26241756] 10. kvien tk, uhlig t, ødegård s, et al. epidemiological aspects of rheumatoid arthritis: the sex ratio. ann n y acad sci 2006;1069:212-222 [doi: 10.1196/annals.1351.019] [pmid: 16855148] 11. adelowo o, mody gm, tikly m, et al. rheumatic diseases in africa. nat rev rheumatol 2021;17 suppl. 1: 363-374. [doi: 10.1038/s41584-021-00603-4] 12. south african market insights. south africa’s population density map. south african market insights 2020. https://www.southafricanmi.com/population-density-map.html (accessed 2 september 2021) 13. strain t, wijndaele k, garcia l, et al. levels of domain-specific physical activity at work, in the household, for travel and for leisure among 327 789 adults from 104 countries. br j sports med 2020; 54(24):1488-1497. [doi: 10.1136/bjsports-2020-102601] [pmid: 33239355] 14. bartlett db, willis lh, slentz ca, et al. ten weeks of highintensity interval walk training is associated with reduced disease activity and improved innate immune function in older adults with rheumatoid arthritis: a pilot study. arthritis res ther 2018; 20(1):127. [doi: 10.1186/s13075-018-1624-x] [pmid: 29898765] 15. laskowski er. what are the risks of sitting to much? mayo clin proc 2020. https://www.mayoclinic.org/healthy-lifestyle/adulthealth/expert-answers/sitting/faq-20058005 (accessed 2 september 2021). 16. singh h, arora s, tanwar v, et al. the validity and sensitivity of the rheumatoid arthritis pain scale on a different ethnic group from indian rheumatoid arthritis patients. arch rheumatol 2019; https://dx.doi.org/10.4061%2f2011%2f681640 https://doi.org/10.1080/20786204.2013.10874374 https://doi.org/10.1002/art.20924 https://doi.org/10.1002/art.23255 https://www.ncbi.nlm.nih.gov/books/nbk92295/ https://doi.org/10.1007/s40279-015-0363-2 https://doi.org/10.1371/journal.pone.0133858 https://doi.org/10.1196/annals.1351.019 https://doi.org/10.1038/s41584-021-00603-4 https://doi.org/10.1136/bjsports-2020-102601 https://doi.org/10.1186/s13075-018-1624-x original research sajsm vol. 34 no.1 2022 8 35(1):90-96. [doi: 10.5606/archrheumatol.2020.7348] [pmid: 32637924] 17. iverson md, scanlon l, frits m, et al. perceptions of physical activity engagement among adults with rheumatoid arthritis and rheumatologists. int j clin rheumtol 2015; 10(2):67-77. [doi: 10.2217/ijr.15.3] [pmid: 26075028] 18. englbrecht m, alten r, aringer m, et al. new insights into the prevalence of depressive symptoms and depression in rheumatoid arthritis – implications from the prospective multicenter vadera ii study. plos one 2019; 14(5): e0217412. [doi: 10.1371/journal.pone.0217412] [pmid: 31136632] 19. valderrama-gama e, damián j, ruigómez a, et al. chronic disease, functional status, and self-ascribed causes of disabilities among noninstitutionalized older people in spain. j gerontol a biol sci med sci 2002; 57(11):m716–m721. [doi: 10.1093/gerona/57.11.m716] [pmid: 12403799] 20. edemekong pf, bomgaars dl, sukumaran s, et al. activities of daily living. in: treasure island (fl): statpearls publishing, 2021 jan [pmid: 29261878] https://pubmed.ncbi.nlm.nih.gov/ 29261878/ (accessed 2 november 2021). https://doi.org/10.5606/archrheumatol.2020.7348 https://dx.doi.org/10.2217%2fijr.15.3 https://doi.org/10.1371/journal.pone.0217412 https://doi.org/10.1093/gerona/57.11.m716 https://pubmed.ncbi.nlm.nih.gov/ ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 elevated levels of sialic acid and lipid-associated sialic acid in plasma of rheumatoid arthritis patients n. s. hamad departme nt of chemistry, college of science, unive rsity of sulaimani abstract the objective of this st udy is to evaluate plasma levels of total sialic acid tsa and lip id –associated sialic acid lsa as a marker of rheumatoid art hritis ar. plasma sialic acid is known as a parameter of inflammation. in the p resent study , in order to exp lore the p otential role of sialic acid in arthritis rheumatoid, p lasma sialic acid levels, p lasma lsa and total p rotein in p atients with arthritis rheumatoid were measured. a total 40 p atients were comp ared with 40 healthy control subjects. plasma tsa, lsa and tp level were determined sp ectrop hotometrically in p lasma samples. plasma sialic acid levels were significantly increased in ra (88.48±14.15 mg/dl, p<0.05) and lsa level were significantly increased in ra (26.37±2.25 mg/dl, p <0.05). by contrast there wasn’t any significant elevation of tp in ra p atients, comp ared with healthy control (tsa,53.31±7.58 mg/dl, p<0.05), (lsa,18.46±1.45 mg/dl) and (tp, 7.39±0.34 g/dl). key word: sialic acid, lip idassociated sialic acid. introduction sialic acid (n-acety lneuraminic acid nana)are acety lated derivatives of neuraminic acid .they are att ached to non reducing residues of the carbohy drate chains of glycoproteins and glycolipids. the suggested biological functions of sialic acid are as following:(a)st abilizing the conformation of glycoproteins and cellular membranes;(b)assisting in cell to cell recognition and interaction ; (c)contributing to membrane transp ort;(d)affecting the function of membrane receptors by p roviding binding sites for ligand ;(e)influencing the function st ability and survival of blood glycoproteins ;and (f) regulating the p ermeability of the basement membrane of glomeruli[1].sialic acid concentration varies p hy siologically with age, but its level may also be influenced by such acondition as inflammation [2].neop last ic tumors or inborn genetic disorder which cause abnormal sialic acid metabolism[3].sialic acid and its derivatives were used in the treatments of several diseases, including neuropathic and inflammatory diseases as well as certain tumors[4].lip id-associated sialic acid(lsa)is a useful adjunct in the management of a variety of malignancies [5]. elevation in blood lsa level was rep orted in p atients with mammary(63%),gastroenteric(65%),p ulmonary(79%)and ovarian(94%) neoplasms as well as those with leukemia(91%),lymphoma(87%),melanoma(84%),and hodgkin disease(91%).[6 – 11].sialic acid level do not app ear to be a good marker for discriminating malignant from non malignant disease of the lung[12]. higher levels of total sialic acid were found in women with metabolic gestation sy ndrome [ 13]and during p eriodontal disease [14]. esr is the most commonly p erformed laboratory test for inflammation in rheumatic disorder reactive protein,transferrin, ceruloplasmin,albumin,α1-antitry p sin were similarly used[15, 16]. .the p resent st udy was undertaken to evaluate the utility of tsa and lsa as an index to measure the disease activity and inflammation in rheumatoid art hritis(ra).tsa, lsa and ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 total protein tp were studied in patients with rheumatoid art hritis .these were comp ared with normal healthy controls. materials and methods chemicals: standard solution for sialic acid 500micro gram/ml concentration was p repared by dissolving 50 mg of st andard n-acety lneuraminic acid in 100ml of distilled water, and on the day of determination, the stock solution was diluted with p hosp hate buffer saline at p h 7.4 to give the following st andard solutions(5,10,15,20,25,30 micro gram/ml)for calibration curve measurement. patie ns: plasma for the measure ment of tsa , lsa and tp levels was obt ained from 40 p atients with rheumatoid arthritis of subjects followed at the central lab in su laimania for the p eriod from dec. 2007 to ap r.2008.the diagnosis of t his case was sp ecified by consultants. plasma preparation: venous blood was collected from fast ing p atients in the early morning. the samples were taken in tubes containing anticoagulant, their p lasma were sep arated and used immediately ,otherwise , plasma samp les were st ored at [-15]until analysis. biochemical methods: 1-total s ialic ac id a-principle the principle of this method dep ends on the formation of chromo gen from the add ition of resorcinol rea gent in to the test tube. the chromogen formed was e xtracted by butyl acetate/methanol r eagent and measured at 580 nm[17]. b-reagents reagent i: resorcinol st ock: made up by dissolving 2 gm of resorcinol in 100 ml of deionized water. this reagent is stable for many months in the refrigerator. reagent ii: resorcinol hcl: 10 ml of the stock solution is added to the mixture of 80 ml of concentrated hcl and 0.25 ml of 0.1m cuso4. the volume is then made up to 100ml with deionized water. this should be p repared at least four hours before use and st able for two weeks in the refrigerator. reagent iii: buty l acetate/methanol: 85ml of buty l acetate is added to 15ml of methanol. c-procedure: the reaction was p erformed in 180×150mm pyrex test tube labeled as test, standard and blank, into which the following reagents were pipettes as follows: blank µl standard µl test µl reagent 20 sample 980 980 980 d.w 20 standard 1000 1000 1000 reagent ii 2000 2000 2 000 reagent iii ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 the tubes were capp ed with glass bulbs and heated for 15 min in a boiling water bath (100c) and then cooled in tap water bath or ice for 10 min.vortexes and centrifuged for 10min at 3000 rp m, the extracted chromophore was read at 580nm. lsa dete rmination: lsa was measured according to the method described by katop odis and his co-worker [18, 19] fifty µl p lasma aliquots were p laced in screw-cap p ed tubes,3ml of cold of (4c)chloroform/methanol(2:1v/v)mixture was added to each tube for total lip id extraction, the tubes were then cap p ed and vortexes for 30 secons,0.5ml of cold water was added to each tube and the tubes were centrifuged for 5 min. at 2500 rp m at room temp. the up p er p hase (aqueous lay er containing lsa) was transferred to another screwcap p ed tube and 50micro ml of p hosp hotungst ic acid (1g/ml) was added to each tube. the tubes were vortexes again and allowed to st and at room temp . for 5min.then the tubes were centrifuged at room temp for 5min.at 2500rp m.after that, the sup ernatants were decanted and the remaining p ellets were redissolved in 1ml of water at 37 ہ c by vigorously vortexing them for 1min and sialic acid content was determined as mentioned for tsa. tp dete rmination: colorimetric method was described by gornall etal[20, 21]. the p eptide bonds of p rotein react with cu 2+ in alkaline solution to form a colored comp lex whose absorbance is p rop ortional to the concentration of the total p rotein in the sp ecimen, and was measured at 550 nm. the biuret reagent contains sodium p otassium tartrate to comp lex cupric ions and maintain their solubility in alkalin solution. re agents: reagent i: sodium hy droxide 370 mmo l/l+ na-k t artrate 10 mmol/l + potassium iodide 3 mmol/l +copp er sulfate 3 mmol/l standard solution –bovine albumin 6 gm/d l. procedure: let st and reagent and sp ecimens were left at room temp erature test µl standard µl blank µl pipette into test tube 1000 1000 1000 reagent 20 standard 20 specimen 20 d.w the above components in the tubes were mixed, left to stand for 10 minutes at room temp erature. record absorbance's at 550nm against reagent blank. calculation result = abs(assay)÷abs(standard) ×standard conc. ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 results the levels of p lasma sialic acid tsa indicate significant st atist ical differences in the rheumatoid arthritis p atients group comp ared to the control group (p<0.05).there is also a significant st atist ical difference (p<0.05) with the resp ect to the levels of p lasma-associated sialic acid between the control group and rheumatoid arthritis p atients group (table-1)(fig3,fig-4).figs (1 and 2) show the p ercent of tsa,lsa and tp in rheumatoid arthritis and control group . tp not changed in this rheumatoid fig-5. table (1) also shows the m ean+sd, variance , p-value, confidence interval ,selectivity and sensitivity for all tests(t sa,lsa and tp). the correlation coefficient equals -0.27, indicating a relatively weak relationship between the variables tsa and lsa (fig -6-a). the correlation coefficient equals 0.25,indicating a relatively weak relationship between the tsa and tp(fig-6-b),where the correlation coefficient equals 0.10 ,indicating a relatively weak relationship between the variables lsa and tp (fig-6-c).the p resent st udy shows that the magnitude of the selectivity varies between (60% for tp )for p atients with ra ,while equals 82.5% for tsa and 85% for lsa for p atients with ra. discussion several st udies were p erformed about the determination of a new marker for the diagnosis of rheumatoid arthritis. the p resent data confirms the p revious evidence that tsa and lsa are significantly higher in p atients with rheumatoid arthritis(ra).ra which is along –term disease that causes inflammation of joints and surrounding tissues. it can also affect other organs .m ost glycoproteins contain mannose ,galactose, fucose, sialic acid and in some instances glucose as the carbohydrate moiety . sp iro, in 1959 established that liver is the major organ involved in the normal sy nthesis of glycoproteins [22].all the tsa and lsa were significantly elevated in the p lasma of p atients with(ra),t p from p atients of ra which showed minimal but st atist ically insignificant elevation . tsa and lsa may ,thus be able to differentiate an inflammatory arthritis from a dehydrative ,non-inflammatory arthritis .our results are sup p orted with p reviously p ublished work [23]. previous st udies were centered largely around the demonst ration of increased levels of carbohy drates of the carbohy drate-p rotein comp lex and carbohy drate – lip id comp lex in p lasma of ra. m ore over, elevated p lasma or serum levels of tsa and / or lsa were observed in many cases (23 25). there was a positive correlation of esr with glycoproteins st udied, the best correlation being with hexosamine followed by sialic acid and other hexose in that order[26].the elevation of p lasma tsa and lsa in ra p atients is of note and we believe our data add to the literature showing changes in tsa and lsa st atus in ra. the mechanisms are unclear and we can only sp eculate as to the reason . in conclusion, the increase of p lasma tsa and lsa levels is associated p ositively with the p resence of inflammation and app ears t o be a consequence of the disease itself, and could be suggested as one of the newly discovered marker for ra. re ference 1schauer r .and kelm s,etc (biochemistry and role of sialic acid .rosenberg a eds. biolojy of the sialic acid 1995,plenum publishin g crop .new y ork01 2novak, j.; tomana, m.;shah, gr.; brow n, r.and mestecky, j. (2005), j. 901.-(10): 89784dent. res., 52-:4735 )1997(clin c hem. clin b iochem,.sg ,rsherki-fang -3 ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 4-witczak,z.j.and nieforth.k. (1997) carbohydrate in drugs design, marcel dekker, new york, 5mahmood ,t.j. and ahmed, s.a. (2008), jmcz, hawler medicinal univ. (in press). :94030 clin c hem., )1984( et al,. k.a ,bhargava-6 42-(2):13713, ann clin lab sci,)1983( .k.m,and sc hwartz .m.a,dinstrian -7 9-(2):40455,,cancer )1985(,et aljones jd and .m.k ,erbil-8 5-(12):527042, ,ecr,cancer )1982(et al .y ,hirshaut.and n ,odiskatop-9 6-(3):16323 j surg oncol,(1983)et al.hj. ,keller.;u ,khanderia-10 11wivedi, c.d.; dixit, m.et al, (1990) cellular and molecular life sciences 94-91:(1)46, :41 bmc plum med)2001( ,et al.i ,gulba.and i ,turgut-21 1335.-: 133125etal (2002), diabetes, .j.a ,reichelt .m,srihana -13 9. –(6): 1 40hygiene, (2006), and j. dent..c .j ,lux –14 15-kulkarni, a.v.and engineer, j.j. (1986)et al, inflammation in rheumatoid 93-(2):8932,ournal of postgraduate med.disorder,j 672-:6646j.rheumatol.,)1979( .p ,.and garbrilw.denko,c-61 11-:60424svenneehlom,l.(1957).biochemica biophysica acta.-71 180.-:17130(1980)res commun chem. path phrmacol.,n.stock cckatopodis8.1 5275-:527042al,caner res (1982) n;hirshaut,etkatopodis-91 751:177j.biol.chem.,)1949( bardawill,et al,and .c.gorna-20 21-tietz, n .w. (1999)text book o clinical c hemistry ,3 rd ed.a.c.burtis,e.r.ashwood,w.b.s ounders;p 477-530 748-:742234 j.biol.c hem)1959(.spiro,r,g-22 23alturfan a. ata .and uslu e. et al, 2007 the tohoku . j.of experimental 248-:241213 medicine, 495-:49450 j.clin.pathol.) 1997( et al, .s ,constable .andm ,rookc -42 9-(3):17410 ,int.j.biol.marker.)1995(lopez,seaz,et al-52 2283-(11):227964, cancer,)2006( m.d,et al. , rudolf voigtmann-62 ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 f ig -4 -l e v e ls o f l s a f ro m p la s m a o f r a a n d c o n tr o l g ro u p 5 3 .1 3 8 8 .4 8 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 n o rm a l rh e u m a to id a rt hr it is lsa mg/dl f ig -3 -l e v e ls o f t s a f ro m p la s m a o f r a a n d c o n tr o l g ro u p 5 3 .1 3 8 8 .4 8 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 no rm a l rh e u m a to id a rt h ri ti s tsa mg/dl ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 fig-1-percent of tsa, lsa and tp from pl asma of control gro up tsa 68% t p 9% lsa 23% fig -2-p ercent o tsa, lsa an d tp from plasma of ra patients tsa 72% l sa 22% tp 6% fi g-5 -le vels o f tp from plas ma o f r a pa teints and c ontrol grou p 7. 35 7.39 7.33 7.34 7.35 7.36 7.37 7.38 7.39 7. 4 normal rh eumatoi art hriti s t p g /d l series1 ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 0 5 10 15 20 25 30 3 6 6.5 7 7.5 8 8.5 9 tp g/dl fi g 6:-correlation between tsa and lsa 0 20 40 60 80 100 120 140 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 l sa mg/dl t s a m g /d l 0 20 40 60 80 100 120 140 6 6.5 7 7.5 8 8.5 9 tp g/dl f ig 6 -c :-c o o re la tio n b e tw e e n l s a a n d t p l s a m g /d ll f ig -6 -b -c o rre la tio n b e tw e en t s a a n d t p t s a m g /d l ibn alhaitham j . fo r pure & appl. sc i vo l. 23 (1) 2010 table (1): data obtained revealing different statistical parameters sensitivity selectivity pvalue confidence –interval variance m ean+_sd mg/dl range m g/dl disease parameter %10 %82.5 p<0.05 %95 200.34 88.48+_14.15 52.65122.33 rheumatoid art hritis n=40 tsa %95 57.48 53.31+_7.58 35.48 69.41 normal n=40 %12.5 %85 p<0.05 %95 5.08 26.37+_2.25 23.0230.85 rheumatoid art hritis n=40 lsa %95 2.11 18.46+_1.45 15.3321.96 normal n=40 %22.5 %60 p<0.05 %95 0.11 7.39+_0.34 g/dl 7.018.02 g/dl rheumatoid art hritis n=40 tp %95 0.11 7.35+_0.33 g/dl 7.048.0 g/dl normal n=40 department of rheumatology, mongi slim hospital, la marsa, tunisia; university of tunis el manar, tunis, tunisia *corresponding author’s e-mail: yasmine.mkhlouf@gmail.com validity of remission criteria in rheumatoid arthritis compared to ultrasound-defined remission kawther ben abdelghani, saoussen miladi, *yasmine makhlouf, alia fazaa, mariem sallemi, leila souebni, kmar ouenniche, selma kassab, selma chekili, kamel ben salem, leith zakraoui, ahmed laatar sultan qaboos university med j, november 2022, vol. 22, iss. 4, pp. 554–560, epub. 7 nov 22 submitted 25 may 21 revision req. 25 jul 21; revision recd. 11 aug 21 accepted 1 sep 21 this work is licensed under a creative commons attribution-noderivatives 4.0 international license. https://doi.org/10.18295/squmj.9.2021.128 clinical & basic research abstract: objectives: remission is the ultimate purpose of treatment in rheumatoid arthritis (ra). however, even when the most stringent composite scores are used, structural damages can occur; hence, ultrasonography (us) appears to be the best way to assess real remission. this study aimed to investigate the validity of different ra remission scores using us as a reference. methods: an analytic diagnostic study, of 30 ra patients in remission (according to the disease activity score in 28 joints [das28]) and a control group with active ra, was conducted between january and october 2018 at mongi slim hospital in tunis, tunisia. among them, patients in remission were identified according to their simple disease activity index (sdai), clinical disease activity index (cdai) and the boolean american college of rheumatology/european league against rheumatism activity index (acr/eular) remission scores. the validity of each activity score for remission was calculated by considering the absence of power doppler (pd) signals as a gold standard. results: all patients were in remission according to the das28, with an average score of 2.03 (1.1–2.6). us examination showed pd signals in 57% of patients. a total of 26 patients were in remission according to the cdai; a doppler signal was detected in 58% of those cases. sdai remission was accomplished in 19 patients, with pd activity in 53% of cases. of the 14 patients in remission according to the boolean acr/eular criteria, synovial hyper-vascularisation was found in 64%. considering true remission as the absence of pd signals, the most sensitive and specific score was the das28 (93% and 68%, respectively). conclusion: considering remission in ra as the absence of vascularised synovitis, the das28 is the most sensitive and most specific score. keywords: rheumatoid arthritis; ultrasonography; doppler; reproducibility of results; tunisia. advances in knowledge various composite outcome measures exist, but structural damage can occur even when the most stringent ones are used. using the most reliable composite score is important to achieve low disease activity and remission as a part of a treat-to-target strategy. finding the most reliable score using ultrasonography as a reference will facilitate the management of patients with rheumatoid arthritis and would help determine further scores that are more stringent. application to patient care considering true remission as the absence of a power doppler (pd) signal, it is important to assess the validity of each disease activity score in obtaining remission. the disease activity score in 28 joints score seems to be the most valid score for assessing remission when the absence of a pd signal is taken as a reference. achieving remission is the ultimate goal of treatment in rheumatoid arthritis (ra). in the past few decades, new therapeutic modalities and strategies have increased the potential to achieve low disease activity and remission by halting the inflammatory process. indeed, a specific strategy of treating early ra, which is adapted to each patient, includes close follow-ups and aims for less disease activity and lower cost, called ‘treat-totarget’ and ‘treat-to-budget’, is now being adopted.1 however, the concept of remission is complex, as there is no consensus on its definition.2 based on clinical and biological criteria, several composite scores are available in daily practice; however, structural damage can occur even when the most stringent ones are used. this is because some patients in clinical remission do not experience an absence of disease activity but exhibit a low level of inflammation that is not easily detectable by clinical examination or reflected in laboratory results. the disease activity score in 28 joints (das28) is the most calculated score used in daily practice. this composite score includes objective, subjective and biological data. other lesser used scores include the simple disease activity index (sdai), the clinical disease activity index (cdai) and the boolean american college of rheumatology/european league against rheumatism activity index (acr/eular) remission criteria. all these composite indices differ when considering remission according to their specific cut-offs. indeed, in the same group of ra patients, the number of those in remission according to the das28 was found to be higher compared to other remission https://creativecommons.org/licenses/by-nd/4.0/ kawther ben abdelghani, saoussen miladi, yasmine makhlouf, alia fazaa, mariem sallemi, leila souebni, kmar ouenniche, selma kassab, selma chekili, kamel ben salem, leith zakraoui and ahmed laatar clinical and basic research | 555 scores such as the sdai.3,4 thus, the das28 may not be considered the most suitable score to diagnose remission. owing to recent technical advances, musculo skeletal ultrasound of the joints now has an important role in the quantification of synovitis. moreover, it can provide important information for the diagnosis, monitoring and management of ra.5 the adoption of this tool as an extension to clinical and biological data may be instrumental in the assessment of remission.6 correspondingly, the present study aimed to invest igate the validity of different ra disease activity scores to assess remission by using ultrasonography (us) as the reference. methods this is an analytic, diagnostic monocentric study carried out in the rheumatology department of mongi slim hospital in tunis, tunisia, between january and october 2018. a total of 30 patients with established ra, meeting the criteria of acr 1987 and in remission according to the eular definition (das28 score ≤2.6), were included.7 the inclusion criteria were ra evolving for more than six months, age at the time of diagnosis greater than 16 years and a state of remission (das28 ≤2.6) diagnosed for at least three months. patients who had a therapeutic adjustment, flare disease or joint steroid injection in the three months prior to the study date were excluded. a control group (active-ra group) was established to compare the validity of the various criteria for remission. it encompassed 37 patients with active established ra (das28 >3.2). at inclusion, clinical data including the age of onset of the disease, duration of morning stiffness, number of night awakenings, visual analogic scale (vas) of pain and patient and physician global assessment (pga and phga) were recorded. a tender and swollen joints count (tjc and sjc) over 66 joints was assessed by the physician who performed the investigation. laboratory markers, including c-reactive protein (crp) and erythrocyte sedimentation rate (esr) levels, were obtained on the same day. immunological assessment of the rheumatoid factor (rf), anticitrullinated peptide antibodies (anti-ccps) and antinuclear antibodies (anas) was collected from the recorded data. the functional impact of the disease was assessed using the health assessment questionnaire. das28 is a composite ra activity score of the tjc, sjc, pga and levels of esr and crp.7 the eular cut-off for disease activity was used and the study group was found to have a das28 score of ≤2.6 and the control group, a das28 score of >3.2. other established scores considered in this study were the sdai, cdai and boolean acr/eular remission criteria.8–10 the cdai includes only clinical parameters: tjc, sjc, pga and phga. the sdai includes all the aforementioned parameters as well as the crp levels. the remission cut-offs considered were cdai ≤2.8, sdai ≤3.3 or the boolean acr/eular remission criteria of, for example, tjc ≤1, sjc ≤1, crp ≤10 mg/l and pga ≤10.11 us of the hands and the wrists was performed for each patient with a delay not exceeding 30 minutes after the clinical examination and the biological sampling. the us examination was performed by a rheumatologist with expertise in musculoskeletal us and at least 10 years of field experience. the operator was blinded to the study group and all other study findings. the equipment used was mylab™ 60 (esaote, genoa, italy) with a 6–18 mhz linear array probe. when using power doppler (pd), the pulse repetition frequency was adjusted at 500–750 hz and the receiver gain was adjusted to eliminate the artefact. overall, 22 joints were scanned bilaterally per patient, including the wrists, metacarpophalangeal joints (mcp) and proximal interphalangeal joints (pip). the wrists and mcp were studied on the dorsal side and pip on their palmar side. the semi-quantitative szkudlarek scale was used in grey-scale (gs) imaging evaluation for synovial hypertrophy (sh) and in pd.11 the sum of grades obtained for each joint and each us mode was established such that it ranged from 0 to 66 for gs and pd. ultrasound detection of erosions was not included in this study, as erosions primarily reflect cumulative lesions related to previous history rather than an ongoing inflammation. to study the validity of the various criteria, the absence of a doppler signal on the sonogram was considered as the gold standard to define us remission. sensitivity, specificity and positive and negative predictive values were calculated and compared between the different remission scores using us remission as a reference. considering us remission, a new threshold for the quantitative scores (das28, cdai, and sdai) was assessed using a receiver operating characteristic (roc) curves. following this, the validity of each score was calculated using the new values. the concordance between the das28 and the other ra activity assessment scores (cdai, sdai and acr/eular remission) was assessed based on the kappa coefficient using us assessment as a reference. validity of remission criteria in rheumatoid arthritis compared to ultrasound-defined remission 556 | squ medical journal, november 2022, volume 22, issue 4 the data were transcribed using excel and analysed using the statistical package for the social sciences (spss), version 12.0 (ibm corp., chicago, illinois, usa). the simple frequencies and relative frequencies (percentages) of the qualitative variables were calculated. similarly, the averages and standard deviations were calculated and the extent of the quantitative variables (extreme values: minimum and maximum) was determined. comparisons of two independent series averages were made using the mann–whitney non-parametric test. the independent series percentage comparisons were made using pearson’s chi-squared test. comparisons of two percentages on paired series were made using mcnemar’s test. the links between the two quantitative variables were studied using spearman’s rank correlation coefficient and the differences were found to be significant: p < 0.05. the agreement between two qualitative variables was measured by cohen’s kappa coefficient. the thresholds for interpreting the kappa coefficient, as determined by landis and koch, were as follows: a κ of 0–0.20 was considered poor, 0.20–0.40 was fair, 0.40–0.60 was moderate, 0.60–0.80 was good and 0.80–1 was excellent. written consent was obtained from the participants. this study was conducted in accordance with the ethical principles of the declaration of helsinki and was approved by the human research ethics committee at mongi slim hospital. results overall, 67 patients with ra were included in the study. the participants of the study group (n = 30) were in remission according to the das28 for a mean period of 16 months (range: 3–72 months). half of the patients were under corticosteroids, with an average dosage of 3.75 mg/day (range: 5–10 mg/ day). conventional disease-modifying anti-rheumatic drugs alone were prescribed for 80% of patients and biotherapies for 20% of patients. the study group and the control group were comparable for demographic data. in the study group, rf and anti-ccp positivity were found in 60% and 66% of patients, respectively. anas were available in 23 patients and positive in 10% of them, with a mean titre of 1/160 (range: 1/80–1/320). in the active ra group (control group; n = 37), rf and anti-ccp positivity were observed in 79.8% and 73.6% of patients, respectively. anas were positive in 6% of the cases [table 1]. table 1: comparison of the demographic and clinical characteristics of 67 patients with rheumatoid arthritis variable mean (range) p value patients (n = 30) controls (n = 37) age in years ± sd 48 ± 8.98 (33–67) 52.4 ± 10.3 (30–70) 0.38 gender ratio 0.20 0.15 0.5 disease duration in years ± sd 8 ± 4.9 (1–23) 10 (0.5–38) 0.12 night awakenings 1 (0–1) 1.23 (0–4) 0.001 morning stiffness in minutes 2 (0–30) 37.7 (0–240) 0.001 tender joint count* 0 (0–1) 7 (0–27) 0.001 swollen joint count* 0.3 (0–9) 6 (0–17) 0.001 vas pain† 6 (0–10) 57 (10–100) 0.012 pga† 3 (0–5) 5 (20–100) 0.025 esr in mm/h ± sd 16.7 ± 10.4 (2–40) 46 ± 25 (15–110) 0.001 crp in mg/l ± sd 3.1 ± 3.7 (0–19) 16.8 ± 15 (5–59) 0.005 das28 ± sd 2.03 (1.1–2.6) 5.2 (3.11–8.6) 0.001 haq 0.12 (0–1) 1.7 (0–2.62) 0.001 sd = standard deviation; vas = visual analogue scale; pga = patient global assessment; esr = erythrocyte sedimentation rate; crp = c-reac tive protein; das28 = disease activity score 28 joints; haq = health assessment questionnaire. *can range from 0–28. †can range from 0–100. figure 1: ultrasound scans of a rheumatoid arthritis patient in clinical remission in power doppler mode showing (a) a persistent power doppler image and (b) an image after the power doppler signal was abolished. kawther ben abdelghani, saoussen miladi, yasmine makhlouf, alia fazaa, mariem sallemi, leila souebni, kmar ouenniche, selma kassab, selma chekili, kamel ben salem, leith zakraoui and ahmed laatar clinical and basic research | 557 with regard to the study group, among the 660 joints studied, sh was detected in 14% of joints and pd signals in 7%. the most affected joints were the wrists. when considering the patient scale, synovitis was present in 80% of patients and pd in 57% of them [figure 1]. in the active ra group, among the 814 joints studied using the ultrasonographic scans, sh was found in 44% and pd in 36% of joints; both tended to occur in the wrists. considering the patient scale, all the patients had sh, with at least one vascularised joint. when comparing the two groups, a significant difference was noted in both the gs and pd modes. grade 0 was more frequent in the study group, while grade 3 was more frequent in the active ra (control) group across both modes [table 2]. the validity of different remission criteria was assessed by considering the absence of any doppler signals on ultrasonographic scans as the gold standard for defining ‘real remission’. the absence of doppler signals was found in 13 patients in remission and in one patient in the active ra group. the das28 was the most sensitive (93%) and the most specific (68%). when considering the state of ‘real remission’, the thresholds that corresponded to the best sensitivity-specificity couple were 3.2 for the das28, 8 for the cdai and 6.5 for the sdai. the validity of the different disease activity scores and each score using new limits according to roc curves are presented in tables 3 and 4, respectively. in gs, synovitis was detected in 80%, 81%, 79% and 78% of patients, using the das28, cdai, sdai and acr/eular remission criteria, respectively. in the pd mode, vascularised synovitis was present in 57%, 58%, 53% and 64% of patients considering the das28, cdai, sdai and acr/eular remission criteria, respectively. there was no significant correlation between any of the remission criteria used and the us findings either in gs or in pd mode [table 5]. among the patients in remission, 26 (87%) were in remission according to the cdai, 19 (63%) were in remission according to the sdai and 14 (47%) were in remission according to the acr/eular criteria. the agreement between das28 remission and cdai was excellent (κ = 0.88), while that between das28 and sdai was good (κ = 0.66). furthermore, the agreement between das28 and acr/eular remission criteria was moderate (κ = 0.47). discussion to the best of the authors’ knowledge, this is the first study that investigates the validity of four different clinical remission scores in ra patients using ultrasonographic remission as a gold standard. in this study, the das28 was the most sensitive score (93%), followed by the cdai (92%), sdai (90%) and acr/eular remission criteria (83%). the das28 was also the most specific (68%), followed by the cdai (65%), sdai (63%) and, finally, acr/eular criteria (59%). only one previous study assessed the validity of the sdai as opposed to that of us in ra patients. table 2: comparison of the ultrasound grades between the study group and the control group grade characteristic n (%) p value study group (n = 660) control group (n = 814) gs grade 0 571 (86) 458 (56) 0.001 grade 1 52 (8) 146 (18) 0.001 grade 2 31 (5) 154 (19) 0.001 grade 3 6 (1) 56 (7) 0.001 pd grade 0 616 (93) 518 (64) 0.001 grade 1 17 (3) 9 (11) 0.001 grade 2 17 (3) 114 (14) 0.001 grade 3 10 (0.1) 93 (11) 0.001 gs = grey scale; pd = power doppler. table 3: reliability of the different scores of remission in the study group remission score % sensitivity specificity ppv npv das28 93 68 43 97 cdai 92 65 37 97 sdai 90 63 33 97 acr/eular 83 59 17 97 ppv = positive predictive value; npv = negative predictive value; das28 = disease activity score 28 joints; cdai = clinical disease activity index; sdai = simplified disease activity index; acr/eular = american college of rheumatology/european league against rheumatism. table 4: validity of the different scores of remission after receiver operating characteristic curve thresholds were applied in the study group remission score threshold % p value sensitivity specificity das28 threshold of 3.2 100 65.4 0.0001 cdai threshold of 8 100 79.1 0.0001 sdai threshold of 6.5 85.7 79.2 0.0001 das28 = disease activity score 28 joints; cdai = clinical disease activity index; sdai = simplified disease activity index. validity of remission criteria in rheumatoid arthritis compared to ultrasound-defined remission 558 | squ medical journal, november 2022, volume 22, issue 4 in that study, balsa et al. showed that when the cut-off was set to 5, the sensitivity of sdai was 65.5%, and the specificity was 55%.12 the specificity of this score was 74.4% when the cut-off was set to 3.3.12 the authors concluded that an sdai ≤3.3 seemed to be a more specific criterion of true remission that is aimed for by different therapeutic strategies than the das28. indeed, when the das28 score was used, a patient could be diagnosed in remission even when they had up to five swollen joints. however, the definition is more stringent with the sdai, which allows for the presence of either two painful or swollen joints or one painful joint and one swollen joint.13 however, based on the present study’s results, it was concluded that the das28 was more specific than the sdai and therefore even better suited to assess true remission. the cdai, which includes only clinical parameters, was found to be more specific than the sdai for assessing remission. the authors did not find any previous study with similar findings. among the multitude of definitions and scores proposed to assess remission, the present study included four, which can be easily used in daily practice. a das28 of ≤2.6 was considered the inclusion criteria, as it is the most used score in daily practice. the concordance between the different ra remission scores compared with the das28 was excellent (0.88) for the cdai, good (0.66) for the sdai and moderate (0.47) for the acr/eular remission criteria. in a study by hmamouchi et al., the agreement of the different scores calculated for patients in remission in the french cohort espoir was moderate between the das28 and the sdai remission (0.54) and poor between the das28 and the acr/eular remission criteria (0.44).14 in another study by chandrashekara and priyanka including 100 ra patients in remission, no agreement was found between the das28 and acr/eular (r = −0.16).15 however, overall, the present study noted good agreement between the different scores; the differences with other studies could be related to patient selection. there is no consensus on the number of joints and sites to be assessed by us to evaluate ra activity. many scores have been proposed for evaluating remission by us. taking into account the conclusions of various authors, the present study evaluated 22 joints: the wrists, the mcp and the pip of both hands. in the current study, 80% of patients had synovitis in gs and 57% had vascularised ones. although heterogeneity was found in published studies, it could be attributed to a different methodology and remission criteria. however, all the studies agreed on the persistence of us activity among patients in remission despite the score used. in a systematic review by ben abdelghani et al. comprising 12 studies of ra in remission, synovitis was detected in 50.7–95% in gs and 14.7–57.4% in pd.16 the detection of synovitis was particularly important, as this infra-clinical activity was responsible for a lownoise evolution during remission.17 importantly, the us scoring system utilized by the current study was the szkudlarek scale instead of the eular as this study was conducted before the validation of the eular score. furthermore, as sh can be seen in many other diseases and even in healthy subjects, the lack of any doppler signal was considered as the real remission state in the present study. no correlation between the das28 and us score was found in the present study either in the gs or pd mode. only one previous study assessed the link between the das28 and us scores; balsa et al. observed a positive correlation between das28 and pd (r = 0.17; p = 0.043).12 the differences between the study by balsa et al. and the present study are probably due to the methodologies used. first, the number of patients in remission included in the two studies was different (74 versus 30). second, inclusion criteria and cut-off values used for assessing remission were different. finally, these differences could mainly be due to the number of joints evaluated in the us. the strength of the present study is that it assessed remission scores that are easily used in current practice. moreover, a control group was included to calculate the validity of the various ra activity scores. however, the current study also suffered from some limitations. the duration of remission, set to three months at inclusion, may be considered insufficient as the persistence of us synovitis was demonstrated in these patients. in the literature, the minimum duration of remission varied in the range of 3–18 months.18 when the duration of remission was prolonged to 12 months, pd was observed in 47.1% of patients.19 another limitation was that the performance of the us examination depended on the technical characteristics of the device and the operator. in the current study, a single operator performed all the us scanning. it would be interesting to perform the us assessments on two separate occasions and by a second sonologist for better validity of the present results. however, the operator in this study was an expert in us and good intraand inter-observer reproducibility of the pd had been confirmed by several studies, making its use by a single operator reliable. another limitation concerns the use of corticosteroids in the remission group; however, the majority of them were treated with low dosages (<7.5 mg/day) and were in remission for 16 months. kawther ben abdelghani, saoussen miladi, yasmine makhlouf, alia fazaa, mariem sallemi, leila souebni, kmar ouenniche, selma kassab, selma chekili, kamel ben salem, leith zakraoui and ahmed laatar clinical and basic research | 559 finally, the absence of the pd in us was considered a state of ‘real remission’ in the present study. this choice was based on the results in the literature, which provided evidence that subclinical disease (a persistent pd signal in clinical remission) is predictive of acute flares in ra and even of future structural damage. it is a very stringent criterion; however, this choice could be discussed as a limitation, as, on the one hand, there is no clear consensus on the definition of ultrasound remission and on the other, the implementation of us in everyday practice as well as in follow-ups has been reconsidered in the recent past. indeed, some authors do not currently support the routine use of us assessment as part of an enhanced treat-to-target strategy recommended by eular.20–22 according to the results of the aiming for remission in rheumatoid arthritis: a randomised trial examining the benefit of ultrasound in a clinical tight control regimen (arctic) trial, the tight control strategy of us did not show additional effect compared to the conventional tight control strategy.22 the results of the targeting synovitis in early rheumatoid arthritis (taser) study are in line with these findings.23 indeed, aiming for a total pd joint count of ≤1 as part of the ultrasonographic treat-to-target strategy led to more intensive treatment with no better clinical or imaging outcomes when compared to the das28-driven strategy (das28 <3.2).24 furthermore, raising the level of requirement in treating ra using us as a reference did not augur well for the treat-to-budget concept. more studies are therefore needed to assess the role of us in evaluating disease activity and tailoring treatment in patients with ra.22 conclusion the das28 and, subsequently, the cdai seem to be the most specific scores for assessing remission in ra. this is because, compared to other composite scores, they appear to provide the closest possible indicator of the absence of inflammatory activity when considering true remission as the absence of pd signals in us. the findings of the present study could be the basis for further research, which should be conducted with a larger sample to draw effective conclusions. a u t h o r s’ c o n t r i b u t i o n sm, ym, ko, sk, sc and lz conceptualised and designed the study. sm and ym collected the data. ka, af, ms, ls, ko, sk, sc, ks, lz and al analysed and interpreted the data. ks performed the statistical analysis. ym performed the literature review and drafted the manuscript. sm edited the manuscript. all authors approved the final version of the manuscript. c o n f l i c t o f i n t e r e s t the authors declare no conflicts of interest. f u n d i n g this work received no funding. references 1. sacristán ja, díaz s, de la torre i, inciarte-mundo j, balsa a. treat-to-target and treat-to-budget in rheumatoid arthritis: measuring the value of individual therapeutic interventions. rheumatol ther 2019; 6:473–7. https://doi.org/10.1007/s4074 4-019-00178-3. 2. paulshus sundlisæter n, olsen ic, aga ab, hammer hb, uhlig t, van der heijde d, et al. predictors of sustained remission in patients with early rheumatoid arthritis treated according to an aggressive treat-to-target protocol. rheumatology (oxford) 2018; 57:2022–31. https://doi.org/10.1093/rheumatology/key202. 3. khanna d, oh m, furst de, ranganath v, gold rh, sharp jt, et al. evaluation of the preliminary definitions of minimal disease activity and remission in an early seropositive rheumatoid arthritis cohort. arthritis rheum 2007; 57:440–7. https://doi. org/10.1002/art.22619. 4. smolen js, aletaha d. activity assessments in rheumatoid arthritis. curr opin rheumatol 2008; 20:306–13. https://doi. org/10.1097/bor.0b013e3282fbd382. 5. wang x, qian g, duan h. diagnostic value of musculoskeletal ultrasound in rheumatoid finger arthritis. j coll physicians surg pak 2020; 30:617–21. https://doi.org/10.29271/jcpsp.2020.06.617. 6. boylan m. should ultrasound be used routinely in the diagnosis of rheumatoid arthritis? ir j med sci 2020; 189:735–48. https:// doi.org/10.1007/s11845-019-02096-3. 7. van der heijde dm, van’t hof m, van riel pl, van de putte lb. development of a disease activity score based on judgment in clinical practice by rheumatologists. j rheumatol 1993; 20:579–81. 8. aletaha d, smolen js. the simplified disease activity index (sdai) and clinical disease activity index (cdai) to monitor patients in standard clinical care. best pract res clin rheumatol 2007; 21:663–75. https://doi.org/10.1016/j.berh.2007.02.004. 9. smolen js, breedveld fc, schiff mh, kalden jr, emery p, eberl g, et al. a simplified disease activity index for rheumatoid arthritis for use in clinical practice. rheumatology (oxford) 2003; 42:244–57. https://doi.org/10.1093/rheumatology/keg072. 10. felson dt, smolen js, wells g, zhang b, van tuyl lh, funovits j, et al. american college of rheumatology/european league against rheumatism provisional definition of remission in rheumatoid arthritis for clinical trials. ann rheum dis 2011; 70:404–13. https://doi.org/10.1136/ard.2011.149765. 11. felson dt, smolen js, wells g, zhang b, van tuyl lh, funovits j, et al. american college of rheumatology/european league against rheumatism provisional definition of remission in rheumatoid arthritis for clinical trials. ann rheum dis 2011; 70:404–13. https://doi.org/10.1136/ard.2011.149765. 12. balsa a, de miguel e, castillo c, peiteado d, martin-mola e. superiority of sdai over das-28 in assessment of remission in rheumatoid arthritis patients using power doppler ultra sonography as a gold standard. rheumatology (oxford) 2010; 49:683–90. https://doi.org/10.1093/rheumatology/kep442. 13. smolen js, aletaha d. activity assessments in rheumatoid arthritis. curr opin rheumatol 2008; 20:306–13. https://doi. org/10.1097/bor.0b013e3282fbd382. 14. hmamouchi i, combe b, fautrel b, rincheval n, lukas c. prevalence and concordance of early and sustained remission assessed by various validated indices in the early arthritis “espoir” cohort. j bone spine 2014; 81:409–15. https://doi. org/10.1016/j.jbspin.2014.02.007. https://doi.org/10.1007/s40744-019-00178-3 https://doi.org/10.1007/s40744-019-00178-3 https://doi.org/10.1093/rheumatology/key202. https://doi.org/10.1002/art.22619 https://doi.org/10.1002/art.22619 https://doi.org/10.1097/bor.0b013e3282fbd382 https://doi.org/10.1097/bor.0b013e3282fbd382 https://doi.org/10.29271/jcpsp.2020.06.617 https://doi.org/10.1007/s11845-019-02096-3 https://doi.org/10.1007/s11845-019-02096-3 https://doi.org/10.1016/j.berh.2007.02.004 https://doi.org/10.1093/rheumatology/keg072 https://doi.org/10.1136/ard.2011.149765 https://doi.org/10.1136/ard.2011.149765. https://doi.org/10.1093/rheumatology/kep442. https://doi.org/10.1097/bor.0b013e3282fbd382 https://doi.org/10.1097/bor.0b013e3282fbd382 https://doi.org/10.1016/j.jbspin.2014.02.007. https://doi.org/10.1016/j.jbspin.2014.02.007. validity of remission criteria in rheumatoid arthritis compared to ultrasound-defined remission 560 | squ medical journal, november 2022, volume 22, issue 4 15. chandrashekara s, priyanka b. remission in rheumatoid arthritis by different criteria does not converge over the inflammatory markers. int j rheum dis 2013; 16:291–6. https://doi.org/10.111 1/1756-185x.12091. 16. ben abdelghani k, miladi s, souabni l, kassab s, chekili s, laatar a, et al. role of ultrasound in assessing remission in rheumatoid arthritis. diagn interv imaging 2015; 96:3–10. https://doi.org/10.1016/j.diii.2014.07.006. 17. vergara f, ruta s, rosa j, marín j, garcía-mónaco r, soriano er. the value of power doppler ultrasound in patients with rheumatoid arthritis in clinical remission: reclassifying disease activity? reumatol clin (engl ed) 2018; 14:202–6. https://doi. org/10.1016/j.reuma.2017.01.007. 18. aletaha d, funovits j, breedveld fc, sharp j, segurado o, smolen js. rheumatoid arthritis joint progression in sustained remission is determined by disease activity levels preceding the period of radiographic assessment. arthritis rheum 2009; 60:1242–9. https://doi.org/10.1002/art.24433. 19. peluso g, michelutti a, bosello s, gremese e, tolusso b, ferraccioli g. clinical and ultrasonographic remission deter mines different chances of relapse in early and long standing rheumatoid arthritis. ann rheum dis 2011; 70:172–5. https:// doi.org/10.1136/ard.2010.129924. 20. nessrine a, siham d, meryem b, samira ef, taoufik h. should the ultrasound of hands be a component of rheumatoid arthritis remission criteria? curr rheumatol rev 2019; 15:312–5. https://doi.org/10.2174/1573397115666181231115233. 21. simpson e, hock e, stevenson m, wong r, dracup n, wailoo a, et al. what is the added value of ultrasound joint examination for monitoring synovitis in rheumatoid arthritis and can it be used to guide treatment decisions? a systematic review and cost-effectiveness analysis. health technol assess 2018; 22:1–258. https://doi.org/10.3310/hta22200. 22. smolen js, landewé rbm, bijlsma jwj, burmester gr, dougados m, kerschbaumer a, et al. eular recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2019 update. ann rheum dis 2020; 79:685–99. https://doi.org/10.1136/ annrheumdis-2019-216655. 23. dale j, stirling a, zhang r, purves d, foley j, sambrook m, et al. targeting ultrasound remission in early rheumatoid arthritis: the results of the taser study, a randomised clinical trial. ann rheum dis 2016; 75:1043–50. https://doi.org/10.1136/ annrheumdis-2015-208941. 24. haavardsholm ea, aga ab, olsen ic, lillegraven s, hammer hb, uhlig t, et al. ultrasound in management of rheumatoid arthritis: arctic randomised controlled strategy trial. bmj 2016; 354:i4205. https://doi.org/10.1136/bmj.i4205. https://doi.org/10.1111/1756-185x.12091 https://doi.org/10.1111/1756-185x.12091 https://doi.org/10.1016/j.diii.2014.07.006 https://doi.org/10.1016/j.reuma.2017.01.007 https://doi.org/10.1016/j.reuma.2017.01.007 https://doi.org/10.1002/art.24433 https://doi.org/10.1136/ard.2010.129924 https://doi.org/10.1136/ard.2010.129924 https://doi.org/10.2174/1573397115666181231115233. https://doi.org/10.3310/hta22200 https://doi.org/10.1136/annrheumdis-2019-216655 https://doi.org/10.1136/annrheumdis-2019-216655 https://doi.org/10.1136/annrheumdis-2015-208941 https://doi.org/10.1136/annrheumdis-2015-208941 https://doi.org/10.1136/bmj.i4205 alfacalcidol modulates oxidative stress parameters in the peripheral blood of patients with active rheumatoid arthritis j. serb. chem. soc. 81 (10) 1127–1139 (2016) udc *alfacalcidol:615.279–188:616.72= jscs–4913 002.77 original scientific paper 1127 alfacalcidol modulates oxidative stress parameters in the peripheral blood of patients with active rheumatoid arthritis tatjana živanović radnić1*, katarina simić-pašalić1, mirjana šefik bukilica1,2, sonja misirlić denčić3, anđelka m. isaković3, tihomir stojković3, nataša petronijević3, nemanja damjanov1,2 and jelena vojinović4 1institute of rheumatology, belgrade, serbia, 2faculty of medicine, university of belgrade, serbia, 3institute of medical and clinical biochemistry, school of medicine, university of belgrade, serbia and 4clinical centre, faculty of medicine, university of niš, serbia (received 6 may, revised 13 may, accepted 16 may 2016) abstract: hormone d and its analogues display immunomodulatory activities providing a beneficial effect in immunoinflammatory diseases. the aim of this study was to assess the effect of alfacalcidol treatment on superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) activity and glutathione (gsh) and malondialdehyde (mda) levels in patients with active ra. sixteen patients with active ra and twenty controls were enrolled in the study. blood samples were taken before and after 12 weeks of alfacalcidol therapy (2 μg/day). oxidative stress parameters were determined spectrophotometrically and by flow cytometry assessment. disease activity was assessed using das28 score. the results revealed that alfacalcidol treatment, significantly (p = 0.04) reduced sod activity and cat activity (p = 0.001) in ra patients. the activity of gpx was significantly lower in ra patients before treatment, compared to controls (p = 0.04). after therapy, gpx activity was restored to control levels, and gsh levels were significantly reduced (p = 0.01). mda levels in patients at the beginning of the study protocol, remained significantly elevated compared to controls (p = 0.01). alfacalcidol treatment decreased mda levels in patients (p = 0.19). furthermore, 12-weeks alfacalcidol therapy, changed the response of ra patients’ pbmc to stimulation preventing the o2 production and mitochondrial membrane depolarisation. after alfacalcidol treatment, significant clinical improvement was observed. keywords: antioxidant activity; disease activity; additional therapy; beneficial; pbmc. * corresponding author. e-mail: tatjanaradnic@yahoo.com doi: 10.2298/jsc160506039z _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1128 živanović radnić et al. introduction one of the most common autoimmune diseases, rheumatoid arthritis (ra) is characterized by the persistent synovitis and systemic inflammation. in the last decade, important advances have been made in the diagnosis, clinical assessment and treatment of ra patients. however, our understanding of the underlying cellular and molecular mechanisms involved in pathogenesis of ra is still incomplete. it was shown that low vitamin d intake is associated with an elevated risk of ra development and furthermore, vitamin d level is associated with ra activity.1–3 the main source of vitamin d is from dietary and supplement intake, with the majority coming from the skin exposure to sunlight, specifically ultraviolet b (uv-b) rays. higher cumulative average uv-b exposure was associated with decreased ra risk in female nurses aged 30–55 years.4 vitamin d from the skin and diet is metabolized in the liver to 25-hydroxyvitamin d (25(oh)d), which is used to determine a patients’ vitamin d status. 25-hydroxyvitamin d is metabolized in the kidneys to its active form 1,25-dihydroxycholecalciferol or calcitriol (vitamin d3) solely biologically active form acting as a steroid hormone.5 vitamin d3, has been shown to be a pleiotropic steroid hormone involved not only in regulation of calcium homeostasis and bone turnover, but also in immunomodulation, having potent anti-inflammatory, antiproliferative, prodifferentiation and antibacterial properties in various cells and tissues.6,7 furthermore, vitamin d3 can be produced by monocytes behaving as a paracrine factor thus having potent actions on all the cellular components of the immune system.8 vitamin d3 exerts its effects via a specific vitamin d receptor (vdr), found in activated lymphocytes, thymocytes and other immunocompetent cells. the significant enrichment of vitamin d response elements at ra associated loci support the hypothesis that vitamin d3 plays a role in the development of ra.9 the contribution of oxidative stress to chronic inflammation of tissues10,11 and its involvement in pathophysiological mechanism of autoimmune diseases12 have been recently recognized. oxidative stress reflects an imbalance between the systemic production of reactive oxygen species (ros) and the antioxidant ability of the organism to readily detoxify the reactive intermediates or to repair the resulting damage. there is a strong evidence supporting the role of oxidative stress in cartilage degradation in experimental arthritis13 as well as in contribution to disease activity in ra patients.14 pro-oxidation environment in ra results in redox imbalance, evidenced by increased production of reactive oxygen, nitrogen and sulphur15 species that amplify the synovial inflammatory-proliferative response.14 however, scarce information is available about the antioxidant properties of vitamin d3 and its analogues, and some data are even controversial.16–18 therefore, the role of vitamin d3 therapy in ra patients deserves to be further explored. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1129 alfacalcidol (1α(oh)d3) is a synthetic vitamin d3 analogue, exerting full biological activity of calcitriol. in a recent study by radovic et al.,19 the alfacalcidol therapy showed a beneficial effect, without any side effects, on the disease activity in 93.3 % of the patients with juvenile idiopathic arthritis (jia), also known as juvenile rheumatoid arthritis (jra), which is the most common form of arthritis in children and adolescents. this study revealed that alfacalcidol therapy induced significant changes in antioxidative enzyme activities. the goal of this study was to examine the effects of alfacalcidol therapy on the superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx) activity, glutathione (gsh) levels in erythrocytes and malondialdehyde (mda) in the plasma of patients with active ra in comparison to the healthy controls. we were also interested in the therapy effect on sensitivity of ra patients peripheral blood mononuclear cells (pbmc) to stimulation. experimental sixteen female ra patients (average age 53±13), in the outpatient clinic of the institute of rheumatology, belgrade, serbia, were consecutively enrolled in the study. controls were twenty ageand sex-matched healthy volunteers selected from the staff. all subjects signed the information consent before participation in the study. the study was conducted in compliance with the declaration of helsinki, international conference on harmonization and good clinical practice guidelines, and was approved by the ethics committee of the institute of rheumatology in belgrade, ethic committee of the university of belgrade, school of medicine, and by medicines and medical devices agency of serbia. inclusion criteria were as follows: established diagnosis of ra (acr/eular criteria 2010),20 at least six months prior to enrolment; methotrexate treatment for 12 weeks or longer with stabile dose of 10-25 mg/week for 8 weeks or longer); active disease defined as 28-joint disease activity score (das28) >3.2. if non-steroidal anti-inflammatory drugs (nsaids) are administered, patients have to be using stable dose of nsaid for at least four weeks. glucocorticoids (systemic and/or local use) were not allowed for at least one month before entering the study. the treatment protocol considered the continuation of previous therapy with the addition of alfacalcidol as oral commercial preparation at the dose of 2 μg daily during 12 weeks. clinical and laboratory assessment of disease activity (serum concentration of c-reactive protein (crp), sedimentation rate (se), number of swollen and tender joints) and safety (regular biochemistry analyses including serum total and ionized calcium concentration and urine calcium concentration as well) of the studied drug were evaluated during regular visits (before start of the therapy, in four-week intervals and after completion of the alfacalcidol therapy). disease activity was assessed using the 28-joint das28.21 blood samples for vitamin d (25(oh)d) levels of our patients were obtained after at least 8 h overnight fasting, before (week 0 = w0) and after 12-week long alphacalcidol therapy (week 12 = w12). the level of vitamin d was determined by electrochemiluminescence assay from roche diagnostics (elecsys 2010). level of vitamin d < 20 ng/ml indicates deficiency, levels 21–29 ng/ml indicate insufficiency and levels ≥ 30 ng/ml indicate sufficiency. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1130 živanović radnić et al. the activity of antioxidant enzymes sod, cat and gpx and the level of gsh were determined in isolated and lysed erythrocytes, while mda was determined in plasma, of the controls and patients, before (w0) and in ra patients after 12-week long (w12) alphacalcidol therapy using manual spectrophotometric method. hemoglobin (hb) concentration in lysed erythrocytes was measured by hematology analyzer. sod activity was assayed as the ability of supernatant obtained after hb precipitation from lysed erythrocytes, to inhibit the radicalmediated autooxidation of epinephrine (using epinephrine hydrochloride, sigma).22 obtained values were expressed as the number of units (u) per gram of hemoglobin in erythrocyte haemolyzate (ghb). the intra-assay and inter-assay cvs were 5.2 and 6.7 %, respectively. cat activity was determined based on the formation of stable complex of hydrogen peroxide with ammonium molybdate whose absorption was measured spectrophotometrically at 405 nm (using hydrogen peroxide solution 30 %, zorka pharma, ammonium molybdate, sigma– –aldrich).23 results were expressed as u/ghb, the intra-assay cv was 3.9 % and the inter -assay cv was 8.5 %. gpx activity in lysed erythrocytes was measured according to previously described spectrophotometric method24 (using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid – hepes, sigma–aldrich; ethylenediaminetetraacetic acid, disodium salt, sigma; l-glutathione reduced, sigma; β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate, sigma; tert-butyl hydroperoxide solution, sigma; glutathione reductase, sigma–aldrich) and presented as u/ghb (the intra-assay cv was 5.7 % and the inter-assay cv was 7.2 %). gsh was determined according to the method by sedlak and lindsay (using the ellman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid),25 dtnb, sigma– aldrich; tris(hydroxymethyl)aminomethane, serva, germany), and obtained values were expressed as μmol/ghb (the intra-assay cv was 3.6 % and the inter-assay cv was 7.3 %). lipid peroxidation, measured as malondialdehyde (mda) level, was determined spectrophotometrically in a reaction with thiobarbituric acid. thiobarbituric acid reacts with mda released from polyunsaturated fatty acids of plasma membrane phospholipids injured by ros, forming a yellow complex whose absorbance was measured at 533 nm (trichloroacetic acid, sigma–aldrich; 2-thiobarbituric acid, sigma).26 results were expressed as nmol of mda per ml (the intra-assay cv was 5.2 % and the inter-assay cv was 7.9 %). mitochondrial production of superoxide anion (o2 -), one of ros, as well as mitochondrial membrane potential were assessed on peripheral blood mononuclear cells (pbmcs) of controls and ra patients (w0 and w12) using flow cytometry. peripheral blood mononuclear cells (pbmcs) were obtained from venous blood of controls and ra patients (on w0 and w12). pbmcs were isolated from heparinized blood by density gradient centrifugation using lymphoprep (axis shield, norway). the pbmc were maintained at 37 °c in a humidified atmosphere with 5% co2, in hepes (20 mm)-buffered rpmi 1640 cell culture medium supplemented with 10 % fetal bovine serum (fbs), 2 mm -glutamine, and 1 % of antibiotic/antimycotic mixture (all from paa, austria). cells (2.5×105 cells/well) were incubated in 24-well plates (sarstedt, nümbrecht, germany). cells were stimulated 30 min after seeding for 4 h, with phorbol 12-myristate 13-acetate (pma) (sigma– –aldrich), final concentration 10 ng/ml and ionomycin (sigma–aldrich), final concentration 1.25 μm. each experiment contained untreated and stimulated (control, patients w0 and patients w12) cells. flow cytometry assessments were performed using facscalibur (bd biosciences, heidelberg, germany) equipped with cellquest pro software for acquisition and analysis. the light-scatter channels were set on linear gains and the fluorescence channels on the logarithmic scale. a minimum of 10,000 cells was analyzed for each condition, adjusting the _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1131 threshold settings so that the cell debris was excluded from the data acquisition. the superoxide anion production was analyzed by measuring the increase in red fluorescence (fl2) intensity emitted by a superoxide-specific fluorochrome, dihydroethidium (dhe; sigma–aldrich), which was incubated with cells (20 mmol/l) for 30 min. obtained results were presented as the increase of mean fluorescence intensity (compared to 1 in non-stimulated cells). mitochondrial membrane potential was assessed using a lipophilic cation jc-1 (r&d systems), which has the property of aggregating upon membrane polarization thus forming an orange-red fluorescent compound. the dye cannot access the transmembrane space if the potential is disturbed, and remains in or reverts to its green monomeric form. the cells were stained with jc-1 as described by the manufacturer, and the green monomer and the red aggregates were detected by flow cytometry. obtained values were expressed as the green/red (fl1/fl2) fluorescence ratio, and the increase corresponded to the extent of mitochondrial depolarization. the results are presented as the increase of fl1/fl2 fluorescence ratio (compared to 1 in non-stimulated cells). statistical analysis was performed using t-test for independent samples, t-test for related samples (for parametric data) and wilcoxon and mann-whiney tests (for nonparametric data). for testing the normality of the distribution parameters, the kolmogorov–smirnov test was used. correlation between parameters was determined using spearman and pearson test. the results are presented as mean±standard deviation (sd, data with normal gaussian distribution) or median (iqr, interquartile range) for non-gaussian distribution data. results at the beginning of the study, vitamin d (25(oh)d) levels in patients were sufficient (w0, 30±11 ng/ml) and remained constant after 12-week treatment (w12, 31±13 ng/ml), indicating that alfacalcidol does not cause significant (p > > 0.05) change in patients’ 25(oh)d level. furthermore, during 12 weeks of alfacacidol treatment and follow-up period no side effects were noticed in patients with ra. serum calcium and ionized calcium levels remained within physiological range of values. although the increase in urine calcium levels was detected in some patients, obtained values remained within physiological range. parameters of antioxidant enzymes (sod, cat and gpx) activity and gsh levels in the erythrocytes and plasma mda levels, of ra patients before and after 12 weeks of alfacalcidol treatment, as well as for control group are presented in table i. the alfacalcidol treatment significantly reduced both sod (w0/w12; p = 0.04) and cat (w0/w12; p = 0.001) activity in erythrocytes of ra patients. while sod activity was reduced to the levels obtained for the healthy control group, cat activity after alfacalcidol treatment was decreased to the levels significantly lower than in the controls (p = 0.0001). however, no significant difference in cat activity in erythrocytes of ra patients before (w0) therapy in comparison to the controls was observed (w0/ctrl; p = 0.37). the activity of gpx was significantly lower in erythrocytes of ra patients before treatment (w0) compared to the controls (p = 0.04). in accordance with lower gpx values, gsh levels were significantly higher in erythrocytes of ra patients (w0) compared to _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1132 živanović radnić et al. the controls (p = 0.03). even though 12-weeks of alfacalcidol therapy restored erythrocytes’ gpx activity to control levels, no significant difference was detected between values obtained for w0 and w12 (p > 0.05). on the other hand, the 12-week long alfacalcidol therapy significantly reduced gsh levels (p = 0.01). in addition to glutathione levels and antioxidant enzyme activity, oxidative stress was further evaluated through measuring the mda levels produced in lipid peroxidation initiated by ros. in the present study, although alfacalcidol treatment decreased mda levels in patients, the difference did not reach a statistical significance (w0/w12; p = 0.19). however, mda levels in patients at the beginning of the study protocol (w0), remained significantly elevated compared to the controls (w0/ctrl; p = 0.01), suggesting the presence of oxidative stress in patients with active disease (table i). table i. the activity of antioxidant enzymes (sod, cat and gpx) and glutathione (gsh) levels in erythrocytes and mda in plasma of ra patients’ before and after twelve weeks of alfacalcidol therapy; sod – superoxide dismutase; cat – catalase; gpx – gluthation peroxidase; gsh – gluthation; mda – malondialdehyde; u – intenational unit of enzyme activity; hb – hemoglobin; ra – rheumatoid arthritis; w0 – before therapy; w12 – after 12 weeks of therapy; values presented are means ± sd or median (iqr); *p < 0.05 compared to w12; **p < 0.01 compared to w12; #p < 0.05 compared to control; ##p < 0.01 compared to control therapy sod u/ghb cat u/ghb gpx u/ghb gsh µmol/ghb mda nmol/ml control 450±110 1300±600 5.2±1.8 4.7±1.2 106±57 ra patients w0 510±100* 1500±800** 3.7±1.6# 6.0±2.1**# 163±30## w12 470±130 258 (302)## 3.9 (0.8) 3.7±1.2 149±42 in order to get further insight into the effect of 12-week long alfacalcidol treatment, we measured the intensity of superoxide anion (o2–) production and mitochondrial membrane potential in controls and patients (w0 and w12) stimulated pbmc. obtained results showed that stimulation caused the increase in dhe fluorescence intensity in control pmbc indicating the o2– production (table ii). furthermore, in patients’ pbmc at the beginning of the study (w0), superoxide production was higher in comparison to the healthy controls, but this difference failed to reach a statistical significance (w0/ctrl; p = 0.09). also, fl1/ /fl2 ratio increased in stimulated controls as well as in patients (w0), suggesting the ability of stimulation to cause depolarization of the inner mitochondrial membrane (table ii). on the other hand, alfacalcidol treatment changed the response of patients (w12) pbmc to stimulation as evidenced by the reduced superoxide production (w0/w12; p = 0.10) and the absence of the inner mitochondrial membrane depolarization (w0/w12; p = 0.001). therefore, these findings suggest the protective influence of alfacalcidol on pbmc sensitivity to oxidative stress induced by stimulation. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1133 table ii. production of superoxide anion (o2-) and change of mitochondrial membrane potential in controls and patients’ stimulated pbmcs before and, in patients, after twelve weeks of alfacalcidol therapy; dhe – dihydroethidium; jc1 – lipophilic cation; pbmc – peripheral blood mononuclear cells; w0 – before therapy; w12 – after 12 weeks of therapy; values presented are means ± sd; *p < 0.05 compared to w12; **p < 0.01 compared to w12; #p < 0.05 compared to control; ##p < 0.01 compared to control therapy dhe jc1 control 1.7±0.5 1.4±0.2 ra patients w0 2.2±0.6 1.3±0.2** w12 1.5±0.5 0.8±0.2 after 12-weeks of alfacalcidol treatment, significant clinical improvement was observed. disease activity score (das28) significantly decreased from 5.8±0.9, before treatment (w0) to 4.3±1.0 (p < 0.01) after (w12) therapy. at the beginning of the study (w0), most of the patients (84 %) were in the group of highactive disease (das28 > 5.1) and 16 % of the patients were with the moderateactive disease (3.2 < das28 < 5.1). there were no patients with the low-active disease (das28 < 3.2). after treatment (w12), only 16 % of the patients remained in the group of high-active disease, 68 % of them were in moderate-active disease, and 16 % of the patients had low-active disease (das28 < 3.2), fig. 1. fig. 1. alfacalcidol therapy causes significant clinical improvement of ra patients regarding disease activity score (das28). after twelve weeks of treatment (w12), 16 % of the patients were in the group of high-active disease (das 28 > 5.1), 68 % were in moderate-active disease (3.2 < das28 < 5.1) while 16 % had low-active disease (das 28 < 3.2). also, crp levels dropped from 29±28 mg/l (w0) to 10±12 mg/l (w12) (p = = 0.02) supporting the clinical improvement evidenced by das28. significant positive correlation (r2 = 0.64, p = 0.02) was observed only between changes in cat activity (δcat) and crp level (δcrp), fig. 2. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1134 živanović radnić et al. fig. 2. alfacalcidol therapy causes significant clinical improvement of ra patients regarding disease activity score (das28). after twelve weeks of treatment (w12), 16 % of the patients were in the group of high-active disease (das28 > 5.1), 68 % were in moderate-active disease (3.2 < das28 < 5.1) while 16 % had low-active disease (das28 < 3.2). discussion herein we report no significant change in patients’ 25(oh)d levels before and after 12-weeks of alfacalcidol treatment. in accordance with this, it is of great importance to emphasize that measurement of 25(oh)d levels, as the only standardized tool to estimate vitamin d status in organism, is actually only the reflection of the balance between food and/or supplement vitamin d diet intake and its utilization in the local tissues as the active d hormone.27 superoxide anion (o2–) is believed to be one of the initiators of free radical production reactions. sod is an enzyme responsible for the conversion of o2–, an important ros, to hydrogen peroxide. in this study, alfacalcidol treatment (12 -weeks), significantly (w0/w12; p = 0.04) reduced sod activity in erythrocytes of ra patients to the levels obtained in the healthy control group. the results of the present study are similar to that published by radovic et al.,19 which showed that 12-weeks of alfacalcidol treatment in patient with juvenile idiopathic arthritis (jia), reduced sod activity to the levels even lower than in healthy control group, confirming role of vitamin d in regulation of cell redox system. one explanation could be the fact that sod production is stimulated through th1 cellular and humoral immunity that are reduced by the alfacalcidol treatment. similarly, cimen et al.28 observed higher sod levels in ra patients than in control group, suggesting that excessive free radical production might be through xanthine–xanthine oxidase system rather than an impaired antioxidant system. likewise, vijaykumar et al.29 reported increased levels of sod and gpx in their study, which could dismutate the excess superoxide radicals that are generated and diffused from the inflammatory sites due to over expression of antioxidant defense system of ra patients. however akyol et al.30 and ozkan et al.31 reported no change in sod levels between ra patients and controls. however our findings are contra_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1135 dictory to the findings of chandankhede et al.32 and desai et al.33 that showed decreased activity of antioxidant enzyme sod in ra patients compared to the controls, but the patients included in their studies were not in the active stage. furthermore, our results showed that 12-weeks long alfaclcidol tretment changed the response of ra patients’ pbmc to stimulation by preventing the o2– production and mitochondrial membrane depolarisation thus supporting the result of reduced sod activity in ra patients erythrocytes after this prolonged treatment. considering that vitamin d is a membrane antioxidant able to inhibit irondependent liposomal lipid peroxidation by stabilizing the cell membranes,34 this result indicates possible protective influence of alfacalcidol on pbmc sensitivity to oxidative stress induction upon stimulation. this could be significant since in our experiments stimulation caused increase in o2– production and depolarisation of mitochondria membrane, not only in patients before treatment (w0), in accordance with the results from bulua et al.,35 but also in healthy control pmbc as well (table ii). cat and gpx are enzymes involved in the neutralization of h2o2. while cat reaction produces oxygen and h2o2, gpx uses gsh oxidized during the reaction that produces water from h2o2. in the present study the activity of cat was higher in erythrocytes of ra patients before treatment compared to the controls. beyond the cell protection against damage caused by ros it was shown that expression of cat in vitro and in vivo affects the expression of genes influencing inflammation.36 interestingly, only for cat we have shown that 12-week therapy with alfacalcidol significantly decreased activity of this enzyme to the levels much lower compared to healthy control. this finding strongly supports alfacalcidol as additional therapy in ra, because cat demonstrated leading role in detoxifying h2o2 within erythrocytes, since this enzyme was strongly elevated in ra synovial fluid.37 significant correlation between changes of cat activity and crp level suggests important role of cat activity in ra pathogenesis. it has been shown that under conditions of severe oxidative stress gpx could be inactivated as o2– can inhibit peroxide function.38 this could explain our finding of gpx decreased activity in erythrocytes of active ra patients before treatment, compared to the controls. also cimen et al.39 found that gpx might not be playing an essential role in rheumatic events. the affinity of gpx for h2o2 is stronger than the affinity of catalase, which makes gpx more efficient at low levels of h2o2 concentrations. decreased activity of gpx reduced the utilization of gsh by erythrocytes and consequently increased levels of gsh. observed decrease of gsh in erythrocytes after alfacalcidol therapy indicated that the patients were trying to recover normal gsh levels as disease improved. in addition to glutathione and antioxidant enzymes, we have also evaluated the presence of lipid peroxidation by measuring the mda levels. our results _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1136 živanović radnić et al. showed that, in patients, mda levels were significantly elevated compared to the controls, but after alfacalcidol therapy patients’ mda levels were lower, in accordance with other studies19,40 suggesting alfacalcidol protective role in prooxidative conditions. after 12-weeks of alfacalcidol treatment, our patients experienced significant clinical improvement without side effects. only one patient showed no improvement but also without disease worsening. our results are in accordance to the study done by andjelkovic et al.8 who showed statistically significant clinical improvement in ra patients treated with alfacalcidol, in both the physician’s overall assessment and in the evaluated parameters of acute phase response. it is well known that all cells of monocyte-macrophage linage have capability to synthesize 1α-hidroxylase and locally produce d-hormone that acts as a potent immunomodulatory molecule, preventing t-cell over-stimulation and decreasing lymphocyte proliferation.41 d3 hormone shifts t-cells from th1 to th2 phenotype, preventing tissue damage associated with th1 responses.42 th17, which produces the proinflammatory cytokine il-17, is thought to play a role in the pathogenesis of autoimmune diseases, including ra. there is evidence suggesting that 1,25(oh)2d3 inhibits the secretion of th17 cells via a number of pathways, including reduction of the expression of th17 stimulatory factor il-6.43 these results indicate that 12-week long alfacalcidol therapy is meaningful approach to improve ra patients’ clinical condition as evidenced by lowering das28 and crp levels. the use of alfacalcidol instead of 1,25(oh)2d3 allows higher dosages to be used with lower risk of hypercalcemia, thus increasing its potential as a therapeutic agent.44 during treatment and follow-up period, in our study, serum and urine calcium as well as ionized calcium levels remained within physiological range. the 25(oh)d form is the most plentiful and stable metabolite of vitamin d in human serum with high affinity to bind serum vitamin d-binding protein and other albumin superfamily in the blood. therefore, the level of 25(oh)d in the serum is the best indicator of vitamin d entering the host, either by coutaneous synthesis or by ingestion in the diet. nevertheless, this form is still not a hormone; rather, it is a prehormonal form of the natural hormone and does not exert almost any biologic activity in the body.45 as a result of the negative feedback regulating the final activation step of 25(oh)d into the active 1,25(oh)2d3 by the kidneys, the oral supplements of plain vitamin d will never lead to increase of hormone d3 to the levels exerting immunomodulatory and/or antioxidative effects.44,46 conclusion the present study demonstrates that twelve weeks of alfacalcidol treatment modulates the activity of antioxidant enzymes and intensity of lipid peroxidation _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1137 that further correlates with significant reduction in disease activity of ra patients. we propose that hormone d could diminish the disease activity by either decreasing ros production or expanding their neutralization by the action of antioxidant enzymes, suggesting the beneficial role of alfacalcidol as the additional therapy in ra. these data could contribute to better understanding and further research of the biological mechanisms of the disease and possibly provide new therapeutic approach. и з в о д утицај алфакалцидола на параметре оксидативног стреса у периферној крви пацијената са активним реуматоидним артритисом татјана живановић раднић1, катарина симић-пашалић1, мирјана шефик букилица1,2, соња мисирлић денчић3, анђелка м. исаковић3, тихомир стојковић3, наташа петронијевић3, немања дамјанов1,2 и јелена војиновић4 1институт за реуматологију, београд, 2медицински факултет универзитета у београду, 3институт за медицинску и клиничку биохемију, медицински факултет универзитета у београду, и 4клинички центар, медицински факултет универзитета у нишу постоје докази да хормон d и његови синтетски аналози испољавају веома важне имуномодулаторне ефекте у аутоимунским болестима. циљ ове студије је био испитивање утицаја дванаестонедељне терапије алфакалцидолом на активност антиоксидативних ензима: супероксид-дисмутазе (sod), каталазе (cат) и глутатион-пероксидазе (gpx) и ниво глутатиона (gsh) и малондиалдехида (mda) у крви болесника са активним реуматоидним артритисом (ra) и здравих контрола. у испитивање је укључено 16 rа пацијената, са активном болешћу и 20 здравих добровољаца. пацијенти су поред своје редовне терапије, добијали терапију алфакалцидолом (2μg/дан), у трајању од 12 недеља. узорци периферне крви су пацијентима узимани пре почетка терапије алфакалцидолом и након 12 недеља. параметри оксидативног стреса испитивани су спектрофотометријски и методом проточне цитометрије. активност болести је процењивана на основу das28 композитног индекса. утврђено је да терапија алфакалцидолом статистички значајно (p = 0,04) смањује активност sod и cат (p = 0,001) у еритроцитима болесника са rа. активност gpx у еритроцитима је била значајно нижа (p = 0,04) код болесника на почетку студије у односу на здраве контроле, док је ниво gsh био значајно виши (p = = 0,03). након терапијског режима, активност gpx у еритроцитима је враћена на ниво активности у групи здравих контрола, а ниво gsh у еритроцитима болесника је статистички значајно смањен (p = 0,01). ниво mda код болесника на почетку истраживања, био је значајно повишен у односу на контролну групу (p = 0,01). третман алфакалцидолом смањио је ниво mda код пацијената (p = 0,19). осим тога, дванаестонедељни третман алфацалцидолом променио је одговор pbmc пацијената на стимулацију, спречавајући о2 производњу и деполаризацију митохондријалне мембране. код болесника је након дванаестонедељног терапијског режима доказано и веома значајно клиничко побољшање. (примљено 6. маја, ревидирано 13. маја, прихваћено 16. маја 2016) references 1. g. g. song, s. c. bae, y. h. lee. clin. rheumatol. 31 (2012) 1733 2. s. m. attar, saudi med. j. 33 (2012) 520 _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1138 živanović radnić et al. 3. p. gatenby, r. lucas, a. swaminathan. curr. opin. rheumatol. 25 (2013) 184 4. e. v. arkema, j. e. hart, k. a. bertrand, f. laden, f. grodstein, b. a. rosner, e. w. karlson, k. h. costenbader. ann. rheum. dis. 72 (2013) 506 5. m. f. holick, n. engl. j. med. 357 (2007) 266 6. j. vojinovic, ann. n.y. acad. sci. 1317 (2014) 47 7. m. r. haussler, c. a. haussler, g. k. whitfield, j. c. hsieh, p. d. thompson, t. k. barthel, l. bartik, j. b. egan, y. wu, j. l. kubicek, c. l. lowmiller, e. w. moffet, r. e. forster, p. w. jurutka, j. steroid biochem. mol. biol. 121 (2010) 88 8. z. andjelkovic, j. vojinovic, n. pejnovic, m. popovic, a. dujic, d. mitrovic, lj. pavlica, d. stefanovic, clin. exp. rheumatol. 17 (1999) 453 9. a. yarwood, p. martin, j. bowes, m. lunt, j. worthington, a. barton, s. eyre, genes immun. 14 (2013) 325 10. i. a. ku, j. b. imboden, p. y. hsue, p. ganz, circ. j. 73 (2009) 977 11. s. pantovic, d. bozovic, g. nikolic, m. martinovic, p. mitrovic, p. m. radulovic, l. isakovic, a. isakovic, i. markovic, j. serb. chem. soc. 80 (2015) 143 12. n. leitinger, subcell. biochem. 49 (2008) 325 13. c. j. wruck, a. fragoulis, a. gurzynski, l. o. brandenburg, y. w. kan, k. chan, j. hassenpflug, s. freitag-wolf, d. varoga, s. lippross, t. pufe, ann. rheum. dis. 70 (2011) 844 14. s. z. hassan, t. a. gheita, s. a. kenawy, a. t. fahim, i. m. el-sorougy, m. s. abdou, int. j. rheum. dis. 14 (2011) 325 15. k. é. szabó-taylor, g. nagy, p. eggleton, p. g. winyard, studies on arthritis and joint disorders, oxidative stress in applied basic research and clinical practice, springer science+business media, berlin, 2013, p. 145 16. x. deng, j. cheng, m. shen. j. endocrinol. invest. 2015, dec 21 [epub ahead of print] 17. s. xu, y. h. chen, z. x. tan, d. d. xie, c. zhang, m. z. xia, h. wang, h. zhao, d. x. xu, d. x. yu. j. steroid biochem. mol. biol. 152 (2015) 133 18. i. g. de medeiros cavalcante, a. s. silva, m. j. costa, d. c. persuhn, c. i. issa, t. l. de luna freire, m. da conceição rodrigues gonçalves, exp. gerontol. 66 (2015) 10 19. j. radovic, d. lazarevic, i. nikolic, j. vojinovic, ann. paediatr. rheum. 1 (2012) 126 20. t. neogi, d. aletaha, a. j. silman, r. l. naden, d. t. felson, r. aggarwal, c. o. bingham, n. s. birnbaum, g. r. burmester, v. p. bykerk, m. d. cohen, b. combe, k. h. costenbader, m. dougados, p. emery, g. ferraccioli, j. m. w. hazes, k. hobbs, t. w. j. huizinga, a. kavanaugh, j. kay, d. khanna, t. k. kvien, t. laing, k. liao, p. mease, h. a. ménard, l. w. moreland, r. nair, t. pincus, s. ringold, j. s. smolen, e. stanislawska-biernat, d. symmons, p. p. tak, k. s. upchurch, j. vencovský, f. wolfe, g. hawker, arthritis rheum. 62 (2010) 2569 21. m. l. l. prevoo, m. a. van’t hof, h. h. kuper, m. a. van leeuwen, l. b. a. van de putte, p. l. c. m. van riel, arthritis rheum. 38 (1995) 44 22. m. sun, s. zigman, anal. biochem. 90 (1978) 81 23. l. góth, clinica chim. acta 196 (1991) 143 24. w. a. gunzler, h. kremers, l. floha, z. klin, chem. klin. biochem. 12 (1974) 444 25. j. sedlak, r. h. lindsay, anal. biochem. 25 (1968) 192 26. s. rehncrona, d. s. smith, b. akesson, e. westerberg, b. k. siesjö, j. neurochem. 34 (1980) 1630 27. m. cutolo c. pizzorni, a. sulli, autoimmun. rev. 11 (2011) 84 28. m. y. cimen, o. b. cimen, m. kacmaz, h. s. ozturk, r. yorgancioglu, i. durak, clin. rheumatol. 19 (2000) 275 _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1139 29. d. vijayakumar, k. suresh, s. manoharan, indian j. clin. biochem. 21 (2006) 104 30. o. akyol, n. isci, i. temel, s. ozgocmen, e. uz, joint bone spine 68 (2001) 311 31. y. ozkan, s. yardym-akaydyn, a. sepici, e. keskin, v. sepici, b. simsek, clin. rheumatol. 26 (2007) 64 32. m. chandankhede, m. gupta, int. j. biol. med. res. 4 (2013) 3088 33. p. b. desai, s. manjunath, s. kadi, k. chetana, j. vanishree, eur. rev. med. pharmacol. sci. 14 (2010) 959 34. x. li, p. fang, j. mai, e. t. choi, h. wang, x. yang, j. hematol. oncol. 6 (2013) 19 35. a. c. bulua, a. simon, r. maddipati, m. pelletier, h. park, k.y. kim, m. n. sack, d. l. kastner, r. m. siegel, j. exp. med. 208 (2011) 519 36. p. y. benhamou, c. moriscot, m. j. richard, o. beatrix, l. badet, f. pattou, j. kerr -conte, j. chroboczek, p. lemarchand, s. halimi, diabetologia 41 (1998) 1093 37. p. biemond, a. j. g. swaak, j. f. koster, arthritis rheum. 27 (1984) 760 38. d. p. neupane, s. majhi, l. chandra, s. rijal, n. baral, indian j. clin. biochem. 23 (2008) 95 39. m. y. cimen, o. b. cimen , m. kaçmaz , h. s. oztürk , r. yorgancioğlu, i. durak, clin. rheumatol. 19 (2000) 275 40. b. y. bao, h. j. ting, j. w. hsu, y. f. lee, int. j. cancer 122 (2008) 2699 41. c. e. hayes, f. e. nashold, k. m. spach, l. b. pedersen, cell. mol. biol. 49 (2003) 277 42. m. hewison, endocrinol. metab. clin. north am. 39 (2010) 365 43. j. h. chang, h. r. cha, d. s. lee, k. y. seo, m. n. kweon, plos one 5 (2010) e12925 44. k. h. w. lau, d. j. baylink, calcif. tissue int. 65 (1999) 295 45. r. nuti, g. bianchi, m. l. brandi, r. caudarella, e. d’erasmo, c. fiore , g. c. isaia, g. luisetto, m. muratore, p. oriente, s. ortolani, rheumatol. int. 26 (2006) 445 46. b. e. c. nordin, a. g. need, h. a. morris, m. horowitz, calcif. tissue int. 65 (1999) 307. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true /preservedicmykvalues true /preserveepsinfo true /preserveflatness true /preservehalftoneinfo false /preserveopicomments true /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /apply /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /cropcolorimages true /colorimageminresolution 300 /colorimageminresolutionpolicy /ok /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 300 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasgrayimages false /cropgrayimages true /grayimageminresolution 300 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 300 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasmonoimages false /cropmonoimages true /monoimageminresolution 1200 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 1200 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects false /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /ara <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> /bgr <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> /chs <feff4f7f75288fd94e9b8bbe5b9a521b5efa7684002000410064006f006200650020005000440046002065876863900275284e8e9ad88d2891cf76845370524d53705237300260a853ef4ee54f7f75280020004100630072006f0062006100740020548c002000410064006f00620065002000520065006100640065007200200035002e003000204ee553ca66f49ad87248672c676562535f00521b5efa768400200050004400460020658768633002> /cht <feff4f7f752890194e9b8a2d7f6e5efa7acb7684002000410064006f006200650020005000440046002065874ef69069752865bc9ad854c18cea76845370524d5370523786557406300260a853ef4ee54f7f75280020004100630072006f0062006100740020548c002000410064006f00620065002000520065006100640065007200200035002e003000204ee553ca66f49ad87248672c4f86958b555f5df25efa7acb76840020005000440046002065874ef63002> /cze <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> /dan <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> /deu <feff00560065007200770065006e00640065006e0020005300690065002000640069006500730065002000450069006e007300740065006c006c0075006e00670065006e0020007a0075006d002000450072007300740065006c006c0065006e00200076006f006e002000410064006f006200650020005000440046002d0044006f006b0075006d0065006e00740065006e002c00200076006f006e002000640065006e0065006e002000530069006500200068006f006300680077006500720074006900670065002000500072006500700072006500730073002d0044007200750063006b0065002000650072007a0065007500670065006e0020006d00f60063006800740065006e002e002000450072007300740065006c006c007400650020005000440046002d0044006f006b0075006d0065006e007400650020006b00f6006e006e0065006e0020006d006900740020004100630072006f00620061007400200075006e0064002000410064006f00620065002000520065006100640065007200200035002e00300020006f0064006500720020006800f600680065007200200067006500f600660066006e00650074002000770065007200640065006e002e> /esp <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> /eti <feff004b00610073007500740061006700650020006e0065006900640020007300e4007400740065006900640020006b00760061006c006900740065006500740073006500200074007200fc006b006900650065006c007300650020007000720069006e00740069006d0069007300650020006a0061006f006b007300200073006f00620069006c0069006b0065002000410064006f006200650020005000440046002d0064006f006b0075006d0065006e00740069006400650020006c006f006f006d006900730065006b0073002e00200020004c006f006f0064007500640020005000440046002d0064006f006b0075006d0065006e00740065002000730061006100740065002000610076006100640061002000700072006f006700720061006d006d006900640065006700610020004100630072006f0062006100740020006e0069006e0067002000410064006f00620065002000520065006100640065007200200035002e00300020006a00610020007500750065006d006100740065002000760065007200730069006f006f006e00690064006500670061002e000d000a> /fra <feff005500740069006c006900730065007a00200063006500730020006f007000740069006f006e00730020006100660069006e00200064006500200063007200e900650072002000640065007300200064006f00630075006d0065006e00740073002000410064006f00620065002000500044004600200070006f0075007200200075006e00650020007100750061006c0069007400e90020006400270069006d007000720065007300730069006f006e00200070007200e9007000720065007300730065002e0020004c0065007300200064006f00630075006d0065006e00740073002000500044004600200063007200e900e90073002000700065007500760065006e0074002000ea0074007200650020006f007500760065007200740073002000640061006e00730020004100630072006f006200610074002c002000610069006e00730069002000710075002700410064006f00620065002000520065006100640065007200200035002e0030002000650074002000760065007200730069006f006e007300200075006c007400e90072006900650075007200650073002e> /gre <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> /heb <feff05d405e905ea05de05e905d5002005d105d405d205d305e805d505ea002005d005dc05d4002005db05d305d9002005dc05d905e605d505e8002005de05e105de05db05d9002000410064006f006200650020005000440046002005d405de05d505ea05d005de05d905dd002005dc05d405d305e405e105ea002005e705d305dd002d05d305e405d505e1002005d005d905db05d505ea05d905ea002e002005de05e105de05db05d90020005000440046002005e905e005d505e605e805d5002005e005d905ea05e005d905dd002005dc05e405ea05d905d705d4002005d105d005de05e605e205d505ea0020004100630072006f006200610074002005d5002d00410064006f00620065002000520065006100640065007200200035002e0030002005d505d205e805e105d005d505ea002005de05ea05e705d305de05d505ea002005d905d505ea05e8002e05d005de05d905dd002005dc002d005000440046002f0058002d0033002c002005e205d905d905e005d5002005d105de05d305e805d905da002005dc05de05e905ea05de05e9002005e905dc0020004100630072006f006200610074002e002005de05e105de05db05d90020005000440046002005e905e005d505e605e805d5002005e005d905ea05e005d905dd002005dc05e405ea05d905d705d4002005d105d005de05e605e205d505ea0020004100630072006f006200610074002005d5002d00410064006f00620065002000520065006100640065007200200035002e0030002005d505d205e805e105d005d505ea002005de05ea05e705d305de05d505ea002005d905d505ea05e8002e> /hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun <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> /ita <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> /jpn <feff9ad854c18cea306a30d730ea30d730ec30b951fa529b7528002000410064006f0062006500200050004400460020658766f8306e4f5c6210306b4f7f75283057307e305930023053306e8a2d5b9a30674f5c62103055308c305f0020005000440046002030d530a130a430eb306f3001004100630072006f0062006100740020304a30883073002000410064006f00620065002000520065006100640065007200200035002e003000204ee5964d3067958b304f30533068304c3067304d307e305930023053306e8a2d5b9a306b306f30d530a930f330c8306e57cb30818fbc307f304c5fc59808306730593002> /kor <feffc7740020c124c815c7440020c0acc6a9d558c5ec0020ace0d488c9c80020c2dcd5d80020c778c1c4c5d00020ac00c7a50020c801d569d55c002000410064006f0062006500200050004400460020bb38c11cb97c0020c791c131d569b2c8b2e4002e0020c774b807ac8c0020c791c131b41c00200050004400460020bb38c11cb2940020004100630072006f0062006100740020bc0f002000410064006f00620065002000520065006100640065007200200035002e00300020c774c0c1c5d0c11c0020c5f40020c2180020c788c2b5b2c8b2e4002e> /lth <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> /lvi <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> /nld (gebruik deze instellingen om adobe pdf-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. de gemaakte pdf-documenten kunnen worden geopend met acrobat en adobe reader 5.0 en hoger.) /nor <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> /pol <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> /ptb <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> /rum <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> /rus <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> /sky <feff0054006900650074006f0020006e006100730074006100760065006e0069006100200070006f0075017e0069007400650020006e00610020007600790074007600e100720061006e0069006500200064006f006b0075006d0065006e0074006f0076002000410064006f006200650020005000440046002c0020006b0074006f007200e90020007300610020006e0061006a006c0065007001610069006500200068006f0064006900610020006e00610020006b00760061006c00690074006e00fa00200074006c0061010d00200061002000700072006500700072006500730073002e00200056007900740076006f00720065006e00e900200064006f006b0075006d0065006e007400790020005000440046002000620075006400650020006d006f017e006e00e90020006f00740076006f00720069016500200076002000700072006f006700720061006d006f006300680020004100630072006f00620061007400200061002000410064006f00620065002000520065006100640065007200200035002e0030002000610020006e006f0076016100ed00630068002e> /slv <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> /suo <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> /sve <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> /tur <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> /ukr <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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice pmmb 2022, 5, 1; a0000277. doi: a10.36877/pmmb.a0000277 http://journals.hh-publisher.com/index.php/pmmb review article exploring the safety and effects of covid-19 vaccination in patients with autoimmune disease kirttna solai vairavan1, loh teng-hern tan1,2, jodi woan-fei law1, priyia pusparajah1, vengadesh letchumanan1* article history 1novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia. kvai0001@student.monash.edu (ksv) loh.teng.hern@monash.edu (lt-ht); jodi.law1@monash.edu (jw-fl); priyia.pusparajah@monash.edu (pp) 2clinical school johor bahru, jeffrey cheah school of medicine and health sciences, monash university malaysia, johor bahru 80100, malaysia *corresponding author: vengadesh letchumanan, novel bacteria and drug discovery research group (nbdd), microbiome and bioresource research strength (mbrs), jeffrey cheah school of medicine and health sciences, monash university malaysia, 47500, selangor darul ehsan, malaysia; vengadesh.letchumanan1@monash.edu (vl) received: 18 september 2022; received in revised form: 3 november 2022; accepted: 10 november 2022; available online: 16 november 2022 abstract: the covid-19 pandemic has quickly become the most significant public health phenomenon, effectively eclipsing the h1n1 and ebola crises that came before it. it can spread rapidly and has caused the death and disability of many worldwide. vaccines are our most effective line of defense against the rapidly spreading and mutating virion. still, there is significant vaccine hesitancy among those with autoimmune conditions who fear the vaccine may cause them more harm than good. this scoping review explores the safety, outcomes, and effects of covid-19 vaccines in autoimmune patients. online databases; pubmed, ovid medline, and scopus were used to search published literature evaluating the effectiveness and side effects of covid-19 vaccines in patients with autoimmune conditions. the search results were limited to 4 distinct autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and myasthenia gravis). thirty-seven studies were retrieved and assessed on the safety, effects, and outcomes of covid-19 vaccination in patients with the chosen autoimmune conditions. overall, the risk of flares and the development of severe side effects after vaccination was low. most autoimmune patients showed a good antibody response to vaccination, especially after the second dose. this review provides a favorable impact of vaccination in patients with autoimmune conditions. keywords: covid-19; vaccines; autoimmune patients; rheumatoid arthritis; systemic lupus erythematosus; psoriasis; myasthenia gravis pmmb 2022, 5, 1; a0000277 2 of 21 1. introduction the covid-19 outbreak, which emerged in china late in 2019, has since quickly spread worldwide to become the most infamous health crisis of our time [1-8]. world health organization (who) declared covid-19 a pandemic caused by severe-acute-respiratorysyndrome-coronavirus-2 (sars-cov-2) [9-11]. covid-19 has caused high morbidity and mortality, socioeconomic burden, and pressure on healthcare systems. all these factors can only be reduced by achieving herd immunity against covid-19 thru vaccination. the virus attacks the host respiratory system and causes flu-like symptoms upon exposure. the disease manifests with a mild respiratory infection, cough, headache, and fatigue at the early stage [12]. still, as the disease progresses, it may lead to acute respiratory failure with severe complications such as multiorgan failure [13-15]. there is a greater risk of infection among the elderly, patients with autoimmune diseases, and immunocompromised patients [15, 16]. given the severity of illness in autoimmune patients, clinicians and healthcare professionals have been advocating the importance of being vaccinated. pharmaceutical companies raced against time to develop covid-19 vaccines. the most common covid-19 vaccine platforms that are currently in use include inactivated virus vaccines (sinovac, covaxin, sinopharm), mrna (pfizer-biontech and moderna), and adenovirus vector (astrazeneca, cansino, sputnik v) [17-19]. these vaccines have produced robust humoral responses and presented good outcomes in most vaccinated populations [20]. vaccinations help to protect the host by creating an antibody response without having the person experience severe illness or post-covid-19 conditions. existing literature suggests that autoimmune patients are at high risk of contracting severe covid-19 and emphasizes the need for vaccination [21, 22]. however, hesitancy has increased in patients with autoimmune diseases since there is limited data on the safety of covid-19 vaccines in autoimmune patients. studies comparing the consequences of different vaccine types between patients and healthy controls are also unavailable [21]. in addition, the disease onset and vaccination can trigger flares. flares are cumbersome to the patient and would thus discourage them from taking the vaccine; the simple possibility is enough to make them wary of the vaccine roll-out. on top of that, many patients with autoimmune conditions are also on immunosuppressants to keep their conditions at bay. this immunosuppressed state may impair their ability to mount an immune response and even increase their risk of vaccine side effects. localized pain on the injection site, fever, body aches, and headaches are among the typical covid-19 vaccine side effects that people have reported worldwide [23]. esquivelvalerio and colleagues studied the impact of six different covid-19 vaccines on patients with autoimmune rheumatic diseases. they found that localized pain, fatigue, headache, and muscle ache are the most prevalent adverse effects in this group of patients [24]. it is also reported hepatitis b, and influenza vaccines trigger the flares of autoimmune diseases by molecular mimicry inducing autoimmunity [25-27]. this review explores the safety, outcomes, and effects of covid-19 vaccines in autoimmune patients. pmmb 2022, 5, 1; a0000277 3 of 21 2. methodology three databases (pubmed, ovid medline, and scopus) were searched using search keywords ‘rheumatoid arthritis, systemic lupus erythematosus, psoriasis, myasthenia gravis, and covid-19 vaccines’ to retrieve studies evaluating the effectiveness and side effects of covid-19 vaccines in patients with autoimmune conditions. this review search was focus to four distinct autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and myasthenia gravis). the included studies were published in english between january 2020 and january 2022. conference proceedings, editorials, dissertations, literature reviews, commentaries, and conference abstracts were excluded. articles were then screened using covidence based on the inclusion and exclusion criteria (table 1). table 1. the inclusion and exclusion criteria. criteria specific criteria inclusion • studies evaluate the effectiveness and side effects of covid-19 vaccines in patients with autoimmune diseases. • population: patients with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and myasthenia gravis. exclusion • studies related other than covid-19 vaccines and autoimmune diseases. • conference proceedings, commentaries, editorials, dissertations, literature reviews, and conference abstracts. 3. results 3.1. search results three electronic databases were chosen to conduct the search process with search terms. the search retrieved 523 studies from pubmed, ovid medline, and scopus. a total of 204 duplicates were removed from the resulting studies, and 319 articles were kept for the title and abstract screening. based on the inclusion and exclusion criteria, 59 full-text articles met the eligibility criteria, and 260 articles were excluded. in the end, 37 articles were chosen and included in this review. the scoping review process is shown in figure 1. pmmb 2022, 5, 1; a0000277 4 of 21 figure 1. the prisma flowchart 3.2. characteristics of included studies of the 37 articles reviewed, the findings were divided into 15 studies on rheumatoid arthritis, 11 studies about psoriasis, seven on systemic lupus erythematosus (sle), and four on myasthenia gravis. 3.2.1. rheumatoid arthritis of the 15 rheumatoid arthritis studies, ten evaluated the administration of mrna vaccines; two discussed mrna vaccines and viral vector vaccines; one looked at mrna vaccines and inactivated vaccines; the remaining two explored inactivated either alone or with viral vector vaccines (table 2). all the study participants were adults (>18 years old). the number of participants varied from 5493 patients in one of the cohort studies to 1 patient in the case report. spinelli et al. [28] and geisen et al. [29] found that the vaccine side effects in those with rheumatoid arthritis were similar to those without autoimmune conditions. local injection site pain was the most common complaint by the patients. furer and colleagues studied the flare of herpes zoster following bnt162b2 mrna covid-19 vaccination in patients with rheumatic diseases. the study found six patients with aiird (autoimmune inflammatory rheumatic diseases) developed mild herpes zoster after the first dose of the vaccine and were given oral antiviral therapy with a resolution of herpes zoster symptoms. five patients completed the second covid-19 vaccine dose without other pmmb 2022, 5, 1; a0000277 5 of 21 adverse effects. they concluded that further studies are required to assess the safety of the mrna-based covid-19 vaccines in patients with aiird and the reactivation of zoster [30]. table 2. summary of studies related to rheumatoid arthritis (ra). study vaccine given population therapies used response cherian et al. [31] chadox1 ncov-19 (viral vector vaccine) or bbv152 (inactivated vaccine) 724 patients with rheumatoid and musculoskeletal disease (rmd). 523 had autoimmune rheumatic disease (aird) and 201 had non-aird in aird group, steroids (n=97) csdmards (n=468) biodmards (n=27) 436 (60.22%) participants had at least one adverse effect (ae). four patients reported a flare of arthritis that resolved in 5 days. geisen et al. [29] biontech/pfizer or moderna (mrna vaccine) 42 healthy controls and 26 patients with chronic inflammatory disease (cid) in cid group, biodmards (n=20) csdmards (n=8) steroids (n=7) sars-cov-2 antibodies and neutralising activity detected in all participants. no severe adverse effects were observed. ferri et al. [32] bnt162b2 and mrna-1273 (mrna vaccines) 478 unselected asd patients (mean age 59 ± 15 years), namely 101 ra, 38 sle, 265 ssc, and a miscellanea of 74 systemic vasculitis. the control group included 502 healthy individuals. in asd group, glucocorticoids, mycophenolatemofetil, rituximab increased prevalence of non-response to vaccines was observed in patients with asd-related interstitial lung disease and those treated with glucocorticoids, mycophenolatemofetil, or rituximab. furer et al. [30] bnt162b2 (mrna vaccination) 491 patients with rheumatoid disease and 99 healthy controls not available prevalence of herpes zoster was 1.2% in patients with the rheumatic disease compared with none in controls. pmmb 2022, 5, 1; a0000277 6 of 21 watad et al. [33] chadox1 ncov-19 (viral vector vaccine), bnt162b2 (mrna vaccine), or mrna-1273 (mrna vaccine) collection of cases (n = 27, 17 flares, and 10 new-onset imid) in patients with imid in 28 days following covid vaccination mixed group including biologics, steroids, and dmards flares were temporally associated with vaccination, but no way to determine causation. iancovici et al. [34] bnt162b2 (mrna vaccine) 12 rheumatoid arthritis patients who were treated with janus kinase inhibitors. 26 healthy controls. janus kinase inhibitors reduced levels of anti-spike antibodies in patients taking janus kinase inhibitors. b cell responsiveness to sars-cov-2 in patients with rheumatoid arthritis was low. simander et al. [35] mrna vaccine 53 patients with ra, 46 patients with spondyloarthropathy and 169 healthy participants dmards (disease-modifying anti-rheumatic drug) seroconversion rates after 1st immunisation was 54% in patients with inflammatory arthritis compared with 98% in control group. seroconversion was 100% in both groups after second dose. seroconversion was reduced in individuals receiving dmard therapy. spinelli et al. [28] mrna vaccine 126 patients with rmd and 85 controls. 70 patients were taking immunosuppressants and/or biologics. 30 patients were taking hydroxychloroquine. 5 patients had confirmed arthritis flares. most common side effect was injection site pain. pmmb 2022, 5, 1; a0000277 7 of 21 li et al. [36] coronavac, bnt162b2 5493 patients with rheumatoid arthritis immunosuppressants, corticosteroids, nsaids, cs/bdmards no significant link between rheumatoid arthritis flares and vaccination. lukaszuk et al. [37] bnt162b2 vaccine (mrna vaccine) 50-year-old female with rheumatoid arthritis methotrexate antibody levels kept increasing but at a lower rate than in patients not receiving immunomodulatory therapies. madelon et al. [38] mrna vaccine 37 patients with rheumatoid arthritis (ra) or multiple sclerosis (ms), 22 healthy controls rituximab in patients with ra and ocrelizumab in patients with ms patients on anti-cd20 treatment can mount potent t-cell responses to mrna covid-19 vaccines. picchiantidiamanti et al. [39] bnt162b2 vaccine (mrna vaccine) 167 controls and 35 rheumatoid arthritis patients ctla-4-inhibitors, il-6 inhibitors sufficient immune responses induced by the covid-19 mrna vaccine were present in majority of ra patients who underwent temporary suspension of immunosuppressive treatment during vaccination. schreiber et al. [40] bnt162b2 and mrna-1273 (mrna vaccines 243 patients with rheumatoid arthritis, spondyloarthropathy or psoriatic arthritis cs/bdmards seventy-two patients (32%) had an insufficient igg response. the median igg level in patients treated with cs/bdmard combination therapy was significantly lower compared to patients without any dmard treatment. pmmb 2022, 5, 1; a0000277 8 of 21 schumacher et al. [41] bnt162b2 (mrna vaccine), mrna1273 (mrna vaccine), astrazeneca/oxford (viral vector vaccine), ad26.cov2.s (viral vector vaccine) 102 patients with idiopathic rheumatic disease on treatment with rituximab rituximab (rtx) • prednisone (n=81) • methotrexate (n=23) 65 patients (64%) showed a negative antibody level after vaccination. seyahi et al. [42] coronavac (inactivated vaccine) 82 hospital workers with rmd and 300 controls. rituximab (rtx), dmards among rmd patients, those using immunosuppressive drugs were significantly less likely to have detectable antibodies than those of treatment. 3.2.2. psoriasis of the 11 psoriasis studies, ten focused on mrna vaccines, and one focused on inactivated vaccines (table 3). one of the above studies included results from patients taking viral vector vaccines, but no studies focused on that subset of vaccines. participants were all adults above 18, and the study population ranged from 1 to 436. five studies focused on the immune response following vaccination, while the remaining 6 explored the risk of exacerbation following anti-covid-19 vaccination. no studies looked specifically into the side effects of vaccination in psoriatic patients. generally, the risk of psoriatic flare following vaccination seems very low, but it can happen. usually, it is not severe and easily managed. the outcomes of vaccination are promising; most patients can mount a detectable immune response even if it is not as significant as that seen in the normal population. methotrexate has shown itself to reduce the immune response produced by the body. table 3. summary of studies related to psoriasis. study vaccine given population therapies used response haberman et al. [43] bnt162b2 (mrna vaccination) patients with imid (n=51). 24 subjects had psoriasis, 22 had rheumatoid arthritis in imid group, methotrexate anticytokine biologics those patients with imid on background methotrexate (n=45) achieve an adequate. pmmb 2022, 5, 1; a0000277 9 of 21 and 5 had other imids. healthy subjects as controls (n=26). a second independent a validation cohort of controls (n=182) and patients with imid (n=31) tnf inhibitors in controls, nil response in only 62.2% of cases. patients with imid on methotrexate do not demonstrate increased cd8+ t-cell activation after vaccination. huang et al. [44] moderna mrna-1273, astrazenecaoxford azd1222 32 unimmunized controls and 51 vaccinated psoriasis patients 49 patients with psoriasis were on biologics. fifteen episodes of exacerbation in the vaccinated group, which is higher than two episodes in the control group. skroza et al. [45] mrna vaccine 436 patients with moderate-severe psoriasis, both male and female, in treatment with biologics biologics. none of the vaccinated patients in concomitant therapy with anti-psoriatic immunomodulating agents developed adverse events (adr). musumeci et al. [46] pfizer mrna bnt162b2 (mrna vaccine) and moderna m1273 (mrna vaccine) 50 patients with stable plaque psoriasis 24 patients were treated with antitnf, 14 with antiil 17, 7 with antiil 12-23 (ustekinumab), and 5 with anti-il 23 (guselkumab). none of the patients experienced any side effects or a psoriatic flare. only one patient treated with infliximab reported an exacerbation of psoriasis after the vaccine. venerito et al. [47] bnt162b2 vaccine (mrna vaccine) 40 patients with psoriasis tnf inhibitors continuing tnfi throughout the vaccination did not hamper immunogenicity. wei et al. [48] moderna mrna-1273 (mrna vaccine) 8 patients biologics 7 patients experienced an acute exacerbation of psoriasis and 1 had newonset psoriasis. pmmb 2022, 5, 1; a0000277 10 of 21 lopez et al. [49] pfizer (mrna vaccine) 58-year-old man with a history of psoriasis 4 days post-second dose of vaccine not available acute exacerbation of psoriasis. mahil et al. [50] bnt162b2 (mrna vaccine) 82 participants after second vaccination. methotrexate (n=14), tnfinhibitors (n=19), il-7 inhibitors (n=14), il-23 inhibitors (n=20) all participants had detectable spike-specific antibodies following the second dose, and all groups demonstrated similar neutralising antibody titres against wild-type, alpha, and delta variants. by contrast, a lower proportion of participants on methotrexate had detectable t-cell responses following the second vaccine dose, compared with controls. mahil et al. [51] bnt162b2 (mrna vaccine) 121 participants were enrolled, 84 with psoriasis receiving immunosuppressive treatment and 17 controls. methotrexate (n=17), tnfinhibitors (n=27), il-7 inhibitors (n=15), il-23 inhibitors (n=25) seroconversion rates were lower in patients receiving immunosuppressants than in controls, with the lowest rate in that receiving methotrexate. onsun et al. [52] coronavac (inactivated vaccine) a 72-year-old male psoriasis patient 4 days post-vaccination not available generalised pustular psoriasis flare. pavlostsky et al. [53] bnt162b2 (mrna vaccine) 51 psoriasis patients on treatment with systemic immune modifiers systemic immune modifiers, especially biologics forty-nine patients had a positive antibody response. 3.3.3. systemic lupus erythematosus (sle) of the 7 sle studies, 3 were case reports, 2 were cohort studies, and the remaining two were case series and case-control studies (table 4). six studies focused on mrna vaccines alone, and only 1 study focused on mrna vaccines and viral vector vaccines. there is a distinct lack of studies looking into the effect of viral vector vaccines and inactivated pmmb 2022, 5, 1; a0000277 11 of 21 vaccines in patients with sle. the results of the studies looking at vaccination outcomes in patients with sle were similar to those found in rheumatoid arthritis and psoriasis. patients with sle could produce an immune response to the vaccine, but it was weaker than that seen in healthy controls. assawaksakul et al. [54] added on the potency of the booster in stimulating a stronger immune response and suggested that the viral vector vaccine may not be as effective as the mrna vaccine in patients with sle. regarding disease exacerbations, multiple studies found that the vaccination triggers sle flares but is not severe enough to warrant hospitalization or emergency care. they have commonly identified side effects of the vaccine, including headache, fatigue, muscle pain, and local injection site pain, similar to that seen in non-autoimmune patients. table 4. summary of studies related to systemic lupus erythematosus (sle). study vaccine given population therapies used response assawasaksakul et al. [54] pfizer/biontech (mrna vaccine) or chadox1 (viral vector vaccine) booster 8 healthcare workers in thailand with known sle who had previously completed the coronavac series (2 doses of inactivated vaccine) mycophenolate mofetil azathioprine calcineurin inhibitor 6 patients had a strong immune response to the booster. 1 patient was heavily immunosuppressed, and the other had taken the viral vector vaccine rather than the mrna vaccine. during the study period of 2 months, no sle flares were recorded. most common side effects reported were injection site pain, fatigue and fever. bartels et al. [55] bnt162b2 (mrna vaccine) 285 subjects with either sle or ra. 128 patients with sle and 154 patients with ra. biologics 5 patients had severe adverse events (1.8%). no patients were hospitalized or died. fatigue, headache, muscle pain and joint pain were the most reported side effects. pmmb 2022, 5, 1; a0000277 12 of 21 hidaka et al. [56] bnt162b2 (mrna vaccine) 53-year-old japanese woman 2 weeks post-second dose of vaccine not available. new onset evans syndrome. izmirly et al. [57] bnt162b2 (mrna vaccine) or mrna-1273 (mrna vaccine) 90 sle patients and 20 healthy controls. hydroxychloroquine prednisone immunosuppressants (ex. azathioprine, mycophenolate mofetil, methotrexate, etc.) sle patients produced significantly lower antibodies compared to healthy controls. the use of any immunosuppressant or prednisone was associated with decreased vaccine response. post-vaccination flares occurred in 11.4% of patients, 1.3% were severe. joseph et al. [58] moderna (mrna vaccine) 54-year-old woman 4 days post-second dose of vaccine mycophenolate mofetil subacute cutaneous lupus erythematosus flare. zavala-flores et al. [59] bnt162b2 (mrna vaccine) 100 patients with sle hydroxychloroquine azathioprine 27 patients experienced sle flares. the predominant type of flare was arthritis, followed by dermal manifestations. pain at the inoculation site was the most common side effect. all side effects were mild. nune et al. [60] bnt162b2 (mrna vaccine) 24-year-old previously well male 2 weeks post-second dose of vaccine nil new onset of sle pmmb 2022, 5, 1; a0000277 13 of 21 3.3.4. myasthenia gravis of the 4 myasthenia gravis studies, 1 was a single center case series, while the remaining 3 were single case reports (table 5). the case series was conducted for one month and looked at 22 participants registered on an mg database. all 3 case reports looked at the elderly population (age>60 years old). the participants of the case series ranged from 25 to 73 years old. three studies looked at the worsening/onset of myasthenia gravis symptoms [6163], whereas one looked at the immune response elicited by vaccination [64]. tagliaferri et al. [62] and chavez et al. [63] reported a myasthenia gravis crisis in a patient following a second vaccination dose. these two articles suggest that covid-19 vaccination may be a potential trigger for myasthenia gravis, especially the second dose. plymate and colleagues showed that immune response was minimal in a myasthenia gravis patient after receiving two doses of the mrna vaccine, but she then demonstrated an excellent response to the third dose given 85 days later [64]. this suggests that patients with myasthenia gravis may require booster shots to mount an adequate immune response. ruan et al. [61] reported a retrospective case series demonstrating the safety of inactivated vaccines in patients with myasthenia gravis, defined as mild/absent worsening of symptoms. table 5. summary of studies related to myasthenia gravis. study vaccine given population therapies used response ruan et al. [61] coronavac vaccine (inactivated vaccine) 22 patients with mg receiving the covid-19 vaccine. 10 have ocular mg and 12 have generalized mg. azathioprine (n=12) mycophenolate mofetil (n=3) steroid and azathioprine (n=3) none (n=4) 20 patients did not have any worsening of mg symptoms. 2 patients reported mild mg symptoms worsening. tagliaferri et al. [62] moderna covid-19 vaccine (mrna vaccine) a 77-year-old man with a fiveyear history of mg 1 week postsecond dose of vaccine prednisone and pyridostigmine myasthenia gravis crisis plymate et al. [64] bnt162b2 covid-19 vaccine (mrna vaccine) a 74-year-old woman diagnosed with generalized mg 44 months 1440 mg mycophenolate sodium and prednisone 11 mg daily. 2 doses of mrna vaccination failed to elicit detectable circulating vaccinespecific igg or ifn-γ t cell responses. pmmb 2022, 5, 1; a0000277 14 of 21 before the initial sars-cov-2 vaccination chavez et al. [63] bnt162b2 covid-19 vaccine (mrna vaccine) 82-year-old man, two days postsecond dose of vaccine nil. new late-onset myasthenia gravis. 4. discussion there is a distinct lack of studies focusing on viral vectors and inactivated vaccines in these four different autoimmune disease patients. each vaccine has a different mechanism of action and, therefore, will have other interactions and effects on the human body, even if they aim to accomplish the same outcome. as such, it isn't easy to conclude the outcomes, safety, and side effects of the vaccines (figure 2). figure 2. the outcome, side-effect and safety of covid-19 vaccine on autoimmune disease patients. 4.1. outcomes overall, most autoimmune patients can mount a significant immune response to the vaccine even if the potency is less than that of healthy participants. several studies also seem to show a reduced response after the first dose but a much better response after the following doses. for example, a study by simander et al. [35], wherein the seroconversion rate after the pmmb 2022, 5, 1; a0000277 15 of 21 first dose was only 54% amongst participants with rheumatoid arthritis as opposed to 98% in the control group, but the seroconversion rates in both groups were 100% after the second dose. a case series by assawaksakul et al. that looks at 8 sle patients found a much stronger immune response in patients following a booster dose of the vaccine [54]. the case report by plymate et al. depicted an older woman who received two doses of the pfizer/biontech vaccine but mounted a negligible immune response following vaccination. she then took a third booster dose 85 days later and showed significant improvement [64]. these studies indicate that patients with autoimmune conditions may benefit more from multiple doses of vaccination than the general population. as mentioned above, taking rituximab and dmards reduces the immune response in patients with rheumatoid arthritis. schreiber et al. [40] identified a 32% non-response rate in patients taking dmards to manage their arthritis. picchianti-diamanti et al. [39] found that suspending the medication during vaccination allowed all patients in their study to develop a strong and sufficient immune response to the vaccine. patients are taking their medication for a reason. while we want them to get the most out of their vaccination, any decision to stop or modify treatment should be discussed and decided with the advice and guidance of a clinician lest they end up with chronic pain or severe flares. rituximab is a form of b-cell-depleting therapy. the study by madelon et al. found that patients taking rituximab could still produce good immune responses [38]. still, the study by schumacher et al. found that 64% of patients taking rituximab were vaccine nonresponders. schumacher’s study had a larger population of 102 participants and found a correlation between the antibody response elicited and the interval since rituximab administration [41]. interval dosing may allow clinicians to optimize vaccine outcomes without stopping the medication that keeps the patient’s arthritis in remission. however, there is still a small risk of non-response, which should be considered. since a booster dose seems to offer some benefit in improving outcomes in patients with sle and myasthenia gravis, it can be considered to optimize vaccine efficacy in patients with rheumatoid arthritis as well, barring any contraindications. prednisone, janus kinase inhibitors, and methotrexate hamper the immune response produced by the vaccine, but they don’t seem to cause non-response. as such, it is still beneficial for patients taking these medications to take the anti-covid-19 vaccine. venerito et al. find that tnf inhibitors do not impair the vaccine's efficacy [47]. a review by wack et al. finds that tnf inhibitors may still cause some impairment compared to healthy controls, and therefore the immune response may still be less robust [65]. interval dosing or suspension of the medication may play a part in all autoimmune patients taking immunosuppressants. still, the decision should be on a case-by-case basis and with the advice and guidance of a clinician. pmmb 2022, 5, 1; a0000277 16 of 21 4.2. safety myasthenia gravis is an autoimmune neuromuscular junction disorder caused by antibodies to the acetylcholine receptor [66]. it causes fatigable weakness, most evident after repeated muscle use or with the day's progression. exacerbations of the condition are usually caused by infection, especially since the advent of this pandemic. since infection with sarscov-2 can cause a worsening, it warrants the suggestion that vaccination against sarscov-2 could also trigger an exacerbation. studies suggest vaccination is unlikely to trigger an exacerbation, with only 2 participants reporting slight worsening symptoms that quickly resolved within a few days [61-63]. more studies with bigger sample sizes must be conducted before establishing a solid link between symptom flares and vaccination. there is also a lack of studies looking into different types of vaccines and their potential link to myasthenia gravis exacerbations. spinelli et al. studied 126 participants, and five rheumatoid arthritis patients had arthritic flare after vaccination [28]. thankfully, the flares were self-limiting and resolved within a few days with some symptomatic management. the study by izmirly et al. looked at the risk of sle flares and found the risk to be about 11.4%, only 1.3% were severe, and none were so severe that they required hospital care [57]. huang and tsai studied 32 unimmunized psoriatic patients and 51 immunized patients. they reported that the immunized group patients presented exacerbations compared to the non-immunized group [44]. the mean psoriatic arthritis severity index (pasi) score also increased from 3.1 to 8 following vaccination. as such, there is undoubtedly some correlation between the vaccine and autoimmune conditions worsening or fluctuating across the board. flares can be triggered by various factors, from infections to stress to poor medication adherence. hence, it is difficult to determine whether a flare has resulted from vaccination, disease progression, or perhaps even a combination. overall, we can see that the risk of disease flares post-covid-19 vaccination is significant. still, the risk of a flare severe enough to require hospitalization or emergency care remains rare and almost exclusive to the odd case report. most of the flares are mild and self-limiting. given the increased risk of morbidity and mortality in patients with comorbidities, it may be better for autoimmune patients to be vaccinated rather than remain unvaccinated. the final decision should be discussed and well-thought-out, considering the patient’s risk factors and disease severity. for patients with mild autoimmune conditions in remission, getting vaccinated is likely the lesser of two evils. 4.3. side effects herpes zoster virus activation was identified as a potential side effect of vaccination in patients with rheumatoid arthritis by furer et al. [30]. this study documents six patients who developed their first episode of herpes zoster shortly after vaccination. the prevalence amounted to roughly 1.2% in patients with autoimmune rheumatic conditions as opposed to none in the healthy controls. herpes zoster viral activation has been reported in connection pmmb 2022, 5, 1; a0000277 17 of 21 to other vaccines, such as trivalent influenza, hepatitis a, and rabies vaccines. potential mechanisms that might explain the link between mrna-covid-19 vaccination and herpes zoster reactivation are related to the stimulation of innate immunity through toll-like receptors (tlrs) by mrna-based vaccines. tlr signaling has been implicated during the reactivation of herpesviruses, a process essential for these viruses to maintain themselves in the host. tlrs are much more highly expressed in patients with rheumatic disease compared to the general population. this might be why the prevalence of herpes zoster activation following vaccination is much higher in this group compared to the controls. hidaka et al. document a case of evans syndrome following covid-19 vaccination [56]. evans syndrome, a rare condition characterized by the coexistence of autoimmune hemolytic anemia (aha) and idiopathic thrombocytic purpura (itp), is associated with sle. interestingly, a case of evans syndrome following the influenza vaccine has also been reported. looking at this, we can conclude that an overactive immune response following vaccination is the most likely trigger causing these sle patients to progress to evans syndrome. it is rare, and there has only been one case so far, but it is worth keeping in mind. geisen et al. discussed side effects in vaccinated patients with chronic autoimmune conditions, but the side effects identified are quite similar to those seen in the general population; headache, malaise, body aches, injection site pain, fever, etc. [29]. a few other studies in this review have come to a similar conclusion. as mentioned, there is a lack of studies looking into specific side effects elicited by vaccination in patients with myasthenia gravis and other autoimmune conditions. it would be essential to establish if there are any condition-specific side effects that clinicians should investigate when counseling these patients on vaccination. 5. conclusions with covid-19 vaccines becoming widely available and the pandemic once again proving itself to be more resilient than the pandemics that came before it, clinicians need to be prepared to discuss the risks and benefits of vaccination with their autoimmune patients. based on the information available, we can deduce that the benefits of vaccination in this population group still outweigh the risks and severe complications are rare if present. neither an autoimmune condition nor any concurrent treatment is a contraindication for the vaccination, but immunosuppressants have shown to reduce the vaccine's efficacy. this can be countered by interval dosing or temporary suspension of the medication, but these are not solutions that would work for everyone. a third vaccination dose to boost immune response appears safe and effective. the decision should be made after viewing each patient as a whole and considering their needs and wants, the severity of their condition, and any limitations they may have. the decision to be vaccinated should not be rushed, especially if patients are hesitant. patients should be monitored for any undesirable side effects and be able to return to the hospital if they face any severe adverse events. it is also essential that more studies should be carried on to understand better the safety and efficacy of different vaccines in various autoimmune conditions. pmmb 2022, 5, 1; a0000277 18 of 21 author contributions: ksv performed the literature search, critical data analysis, and manuscript writing. lt-th, jw-fl, pp, and vl provided critical editing and proofreading. lt-th created the illustrative figure 2 for the manuscript. vl conceptualized this review writing project. acknowledgements: this work was inspired by the jeffrey cheah school of medicine and health sciences “med5101 scholarly intensive placement (sip)”. conflicts of interest: the authors declare no conflict of interest. references 1. letchumanan v, ab mutalib n-s, goh b-h, et al., novel coronavirus 2019-ncov: could this virus become a possible global pandemic. progress in microbes & molecular biology 2020; 3(1). 2. johnson d, ren sec, johnson hd, et al., covid-19: are malaysians embracing or suffering the new normality? progress in microbes & molecular biology 2020; 3(1). 3. tan lt-h, letchumanan v, ser h-l, et al., pmmb covid-19 bulletin: united kingdom (22nd april 2020). progress in microbes & molecular biology 2020; 3(1). 4. loo k-y, letchumanan v, tan lt-h, et al., updated covid-19 condition in australia. progress in microbes & molecular biology 2021; 4(1). 5. lee vs, chong wl, sukumaran sd, et al., computational screening and identifying binding interaction of antiviral and anti-malarial drugs: toward the potential cure for sars-cov-2. progress in drug discovery & biomedical science 2020; 3(1). 6. thye ay-k, law jw-f, pusparajah p, et al., emerging sars-cov-2 variants of concern (vocs): an impending global crisis. biomedicines 2021; 9(10): 1303. 7. thye ay-k, law jw-f, tan lt-h, et al., psychological symptoms in covid-19 patients: insights into pathophysiology and risk factors of long covid-19. biology 2022; 11(1): 61. 8. ser h-l, letchumanan v, law jw-f, et al., pmmb covid-19 bulletin: spain (18th april 2020). progress in microbes & molecular biology 2020; 3(1). 9. loo k-y and letchumanan v, covid-19: malaysia's fight against this deadly virus. progress in microbes & molecular biology 2021; 4(1). 10. ser h-l, tan lt-h, law jw-f, et al., genomic analysis of severe acute respiratory syndrome coronavirus 2 (sarscov-2) strains isolated in malaysia. progress in microbes & molecular biology 2020; 3(1). 11. thye ay-k, pusparajah p, tan lt-h, et al., covid-19: gastrointestinal manifestations and complications. progress in microbes & molecular biology 2021; 4(1). 12. loo ky, law jw-f, tan lth, et al., south africa’s battle against covid-19 pandemic. progress in microbes & molecular biology 2022; 5(1). 13. kaur sp and gupta v, covid-19 vaccine: a comprehensive status report. virus res 2020; 288: 198114. 14. zheng j, sars-cov-2: an emerging coronavirus that causes a global threat. int j biol sci 2020; 16(10): 1678. 15. loo k-y, letchumanan v, ser h-l, et al., covid-19: insights into potential vaccines. microorganisms 2021; 9(3): 605. 16. jordan re, adab p, and cheng k, covid-19: risk factors for severe disease and death. bmj 2020; 368. 17. thye ay-k, tan lt-h, law jwf, et al., covid-19 booster vaccines administration in different countries. progress in microbes & molecular biology 2021; 4(1). 18. thye ay-k, loo k-y, tan kbc, et al., insights into covid-19 delta variant (b. 1.617. 2). progress in microbes & molecular biology 2021; 4(1). pmmb 2022, 5, 1; a0000277 19 of 21 19. thye ay-k, tan lt-h, law jw-f, et al., long covid-19: psychological symptoms in covid-19 and probiotics as an adjunct therapy. progress in microbes & molecular biology 2022; 5(1). 20. dagan n, barda n, kepten e, et al., bnt162b2 mrna covid-19 vaccine in a nationwide mass vaccination setting. new england journal of medicine 2021. 21. boekel l, kummer ly, van dam kp, et al., adverse events after first covid-19 vaccination in patients with autoimmune diseases. the lancet rheumatology 2021; 3(8): e542-e545. 22. gianfrancesco m, hyrich kl, al-adely s, et al., characteristics associated with hospitalisation for covid-19 in people with rheumatic disease: data from the covid-19 global rheumatology alliance physician-reported registry. ann rheum dis 2020; 79(7): 859-866. 23. chen y, xu z, wang p, et al., new-onset autoimmune phenomena post-covid-19 vaccination. immunology 2022; 165(4): 386-401. 24. esquivel-valerio ja, skinner-taylor cm, moreno-arquieta ia, et al., adverse events of six covid-19 vaccines in patients with autoimmune rheumatic diseases: a cross-sectional study. rheumatol int 2021; 41(12): 2105-2108. 25. pellegrino p, carnovale c, pozzi m, et al., on the relationship between human papilloma virus vaccine and autoimmune diseases. autoimmun rev 2014; 13(7): 736-741. 26. segal y and shoenfeld y, vaccine-induced autoimmunity: the role of molecular mimicry and immune crossreaction. cell mol immunol 2018; 15(6): 586-594. 27. wraith dc, goldman m, and lambert p-h, vaccination and autoimmune disease: what is the evidence? the lancet 2003; 362(9396): 1659-1666. 28. spinelli fr, favalli eg, garufi c, et al., low frequency of disease flare in patients with rheumatic musculoskeletal diseases who received sars-cov-2 mrna vaccine. arthritis research & therapy 2022; 24(1): 1-8. 29. geisen um, berner dk, tran f, et al., immunogenicity and safety of anti-sars-cov-2 mrna vaccines in patients with chronic inflammatory conditions and immunosuppressive therapy in a monocentric cohort. ann rheum dis 2021; 80(10): 1306-1311. 30. furer v, zisman d, kibari a, et al., herpes zoster following bnt162b2 mrna covid-19 vaccination in patients with autoimmune inflammatory rheumatic diseases: a case series. rheumatology 2021; 60(si): si90-si95. 31. cherian s, paul a, ahmed s, et al., safety of the chadox1 ncov-19 and the bbv152 vaccines in 724 patients with rheumatic diseases: a post-vaccination cross-sectional survey. rheumatol int 2021; 41(8): 1441-1445. 32. ferri c, ursini f, gragnani l, et al., impaired immunogenicity to covid-19 vaccines in autoimmune systemic diseases. high prevalence of non-response in different patients’ subgroups. j autoimmun 2021; 125: 102744. 33. watad a, de marco g, mahajna h, et al., immune-mediated disease flares or new-onset disease in 27 subjects following mrna/dna sars-cov-2 vaccination. vaccines 2021; 9(5): 435. 34. iancovici l, khateeb d, harel o, et al., rheumatoid arthritis patients treated with janus kinase inhibitors show reduced humoral immune responses following bnt162b2 vaccination. rheumatology 2022; 61(8): 3439-3447. 35. simader e, tobudic s, mandl p, et al., importance of the second sars-cov-2 vaccination dose for achieving serological response in patients with rheumatoid arthritis and seronegative spondyloarthritis. ann rheum dis 2022; 81(3): 416-421. 36. li x, tong x, yeung wwy, et al., two-dose covid-19 vaccination and possible arthritis flare among patients with rheumatoid arthritis in hong kong. ann rheum dis 2022; 81(4): 564-568. 37. lukaszuk k, woclawek-potocka i, jakiel g, et al., humoral response to sars-cov-2 vaccine of a patient receiving methotrexate treatment and implications for the need of monitoring. vaccines 2021; 9(10): 1151. pmmb 2022, 5, 1; a0000277 20 of 21 38. madelon n, lauper k, breville g, et al., robust t-cell responses in anti-cd20-treated patients following covid-19 vaccination: a prospective cohort study. clin infect dis 2022; 75(1): e1037-e1045. 39. picchianti-diamanti a, aiello a, laganà b, et al., immunosuppressivetherapies differently modulate humoral-and t-cell-specific responses to covid-19 mrna vaccine in rheumatoid arthritis patients. front immunol 2021; 12: 740249. 40. schreiber k, graversgaard c, petersen r, et al., reduced humoral response of sars-cov-2 antibodies following vaccination in patients with inflammatory rheumatic diseases—an interim report from a danish prospective cohort study. vaccines 2021; 10(1): 35. 41. schumacher f, mrdenovic n, scheicht d, et al., humoral immunogenicity of covid-19 vaccines in patients with inflammatory rheumatic diseases under treatment with rituximab: a case–control study (covid-19vacrtx). rheumatology 2022. 42. seyahi e, bakhdiyarli g, oztas m, et al., antibody response to inactivated covid-19 vaccine (coronavac) in immune-mediated diseases: a controlled study among hospital workers and elderly. rheumatol int 2021; 41(8): 1429-1440. 43. haberman rh, herati r, simon d, et al., methotrexate hampers immunogenicity to bnt162b2 mrna covid-19 vaccine in immune-mediated inflammatory disease. ann rheum dis 2021; 80(10): 1339-1344. 44. huang y-w and tsai t-f, exacerbation of psoriasis following covid-19 vaccination: report from a single center. frontiers in medicine 2021; 8: 812010. 45. skroza n, bernardini n, tolino e, et al., safety and impact of anti-covid-19 vaccines in psoriatic patients treated with biologics: a real life experience. journal of clinical medicine 2021; 10(15): 3355. 46. musumeci ml, caruso g, trecarichi ac, et al., safety of sars‐cov‐2 vaccines in psoriatic patients treated with biologics: a real life experience. dermatol ther 2022; 35(1): e15177. 47. venerito v, stefanizzi p, fornaro m, et al., immunogenicity of bnt162b2 mrna sars-cov-2 vaccine in patients with psoriatic arthritis on tnf inhibitors. rmd open 2022; 8(1): e001847. 48. wei n, kresch m, elbogen e, et al., new onset and exacerbation of psoriasis after covid-19 vaccination. jaad case reports 2022; 19: 74-77. 49. lopez ed, javed n, upadhyay s, et al. acute exacerbation of psoriasis after covid-19 pfizer vaccination. in baylor university medical center proceedings. 2022. taylor & francis. 50. mahil sk, bechman k, raharja a, et al., humoral and cellular immunogenicity to a second dose of covid-19 vaccine bnt162b2 in people receiving methotrexate or targeted immunosuppression: a longitudinal cohort study. the lancet rheumatology 2022; 4(1): e42-e52. 51. mahil sk, bechman k, raharja a, et al., the effect of methotrexate and targeted immunosuppression on humoral and cellular immune responses to the covid-19 vaccine bnt162b2: a cohort study. the lancet rheumatology 2021; 3(9): e627-e637. 52. onsun n, kaya g, işık bg, et al., a generalized pustular psoriasis flare after coronavac covid-19 vaccination: case report. health promotion perspectives 2021; 11(2): 261. 53. pavlotsky f, segal z, and barzilai a, antibody response to bnt162b2 vaccine in immune modifiers–treated psoriatic patients. journal of psoriasis and psoriatic arthritis 2022; 7(1): 24-28. 54. assawasaksakul t, sathitratanacheewin s, vichaiwattana p, et al., immunogenicity, safety and reactogenicity of a heterogeneous booster following the coronavac inactivated sars-cov-2 vaccine in patients with sle: a case series. rmd open 2021; 7(3): e002019. pmmb 2022, 5, 1; a0000277 21 of 21 55. bartels le, ammitzbøll c, andersen jb, et al., local and systemic reactogenicity of covid-19 vaccine bnt162b2 in patients with systemic lupus erythematosus and rheumatoid arthritis. rheumatol int 2021; 41(11): 1925-1931. 56. hidaka d, ogasawara r, sugimura s, et al., new-onset evans syndrome associated with systemic lupus erythematosus after bnt162b2 mrna covid-19 vaccination. int j hematol 2022; 115(3): 424-427. 57. izmirly pm, kim my, samanovic m, et al., evaluation of immune response and disease status in systemic lupus erythematosus patients following sars–cov‐2 vaccination. arthritis & rheumatology 2022; 74(2): 284-294. 58. joseph ak and chong bf, subacute cutaneous lupus erythematosus flare triggered by covid‐19 vaccine. dermatol ther 2021. 59. zavala-flores e, salcedo-matienzo j, quiroz-alva a, et al., side effects and flares risk after sars-cov-2 vaccination in patients with systemic lupus erythematosus. clin rheumatol 2022; 41(5): 1349-1357. 60. nune a, iyengar kp, ish p, et al., the emergence of new-onset sle following sars-cov-2 vaccination. qjm: an international journal of medicine 2021; 114(10): 739-740. 61. ruan z, tang y, li c, et al., covid-19 vaccination in patients with myasthenia gravis: a single-center case series. vaccines 2021; 9(10): 1112. 62. tagliaferri ar, narvaneni s, and grist w, a case of covid-19 vaccine causing a myasthenia gravis crisis. cureus 2021; 13(6). 63. chavez a and pougnier c, a case of covid-19 vaccine associated new diagnosis myasthenia gravis. j prim care community health 2021; 12: 21501327211051933. 64. plymate lc, pepper g, krist mp, et al., immunogenicity of repeat covid-19 mrna vaccinations in a patient with myasthenia gravis receiving mycophenolate, prednisone, and eculizumab. journal of translational autoimmunity 2021; 4: 100114. 65. wack s, patton t, and ferris lk, covid-19 vaccine safety and efficacy in patients with immune-mediated inflammatory disease: review of available evidence. j am acad dermatol 2021; 85(5): 1274-1284. 66. thye ay-k, law jw-f, tan lt-h, et al., exploring the gut microbiome in myasthenia gravis. nutrients 2022; 14(8): 1647. author(s) shall retain the copyright of their work and grant the journal/publisher right for the first publication with the work simultaneously licensed under: creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). this license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. adaptation and remixing are also permitted.